Subset (5)

F1000Journal · 8.11k rows

Split (1)

data · 8.11k rows

link stringlengths 41 45	date stringlengths 9 9	paper dict	reviews listlengths 1 6	version int64 1 5	main stringlengths 38 42
https://f1000research.com/articles/8-1067/v1	12 Jul 19	{ "type": "Research Article", "title": "Lipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration", "authors": [ "Faith A. Kwa", "Nabeela K. Dulull", "Ute Roessner", "Daniel A. Dias", "Thusitha W. Rupasinghe", "Nabeela K. Dulull", "Ute Roessner", "Daniel A. Dias", "Thusitha W. Rupasinghe" ], "abstract": "Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the ageing population. Without effective treatment strategies that can prevent disease progression, there is an urgent need for novel therapeutic interventions to reduce the burden of vision loss and improve patients’ quality of life. Dysfunctional innate immune responses to oxidative stress observed in AMD can be caused by the formation of oxidised lipids, whilst polyunsaturated fatty acids have shown to increase the risk of AMD and disease progression in affected individuals. Previously, our laboratory has shown that the vegetable-derived isothiocyanate, L-sulforaphane (LSF), can protect human adult pigment epithelial cells from oxidative damage by upregulating gene expression of the oxidative stress enzyme Glutathione-S-Transferase µ1. This study aims to validate the protective effects of LSF on human retinal cells under oxidative stress conditions and to reveal the key players in fatty acid and lipid metabolism that may facilitate this protection. Methods: The in vitro oxidative stress model of AMD was based on the exposure of an adult retinal pigment epithelium-19 cell line to 200µM hydrogen peroxide. Percentage cell proliferation following LSF treatment was measured using tetrazolium salt-based assays. Untargeted fatty acid profiling was performed by gas chromatography-mass spectrometry. Untargeted lipid profiling was performed by liquid chromatography-mass spectrometry. Results: Under hydrogen peroxide-induced oxidative stress conditions, LSF treatment induced dose-dependent cell proliferation. The key fatty acids that were increased by LSF treatment of the retinal cells include oleic acid and eicosatrienoic acid. LSF treatment also increased levels of the lipid classes phosphatidylcholine, cholesteryl ester and oxo-phytodienoic acid but decreased levels of phosphatidylethanolamine lipids. Conclusions: We propose that retinal cells at risk of oxidative damage and apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in the pathogenesis and progression of AMD.", "keywords": [ "Age-related macular degeneration", "fatty acid", "L-Sulforaphane", "lipidomics", "oxidative stress", "retinal pigment epithelium" ], "content": "Introduction\n\nAge-related macular degeneration (AMD) is a major cause of blindness worldwide, especially targeting the ageing population. AMD is categorised into three main stages, namely early, intermediate and late AMD. The early stage is marked by the thickening and inflammation of the Bruch’s membrane, as a result of the accumulation of fatty proteins known as drusen1. The intermediate stage proceeds with an increase in size of these drusen particles, resulting in pressure atrophy on the retinal pigment epithelium (RPE) and thinning of the macula (dry AMD), which results in the deterioration of central vision. In the late stages, the atrophic retinal tissue becomes replaced with granulation tissue consisting of abnormal leaky blood vessels (wet AMD)1. The blood and fluid leak from these blood vessels into the retina; thus, prolonging the chronic inflammatory response and triggering further oxidative damage. Many factors contribute to AMD. One dominant factor is the increasing age of the retina, where the RPE becomes damaged due to a progressively impaired DNA repair system that fails to repair oxidative damage from prolonged exposure to visible light, ultraviolet A and reactive oxygen species (ROS) over time2. Cigarette smoking is another factor that contributes to the production of ROS and oxidative damage on the RPE layer. Many studies have shown a link between excessive cigarette smoking and AMD3,4. For the retina to maintain its normal physiological functions, a well-balanced diet is also necessary. Poor nutrition in the elderly influences the progression of AMD. Studies by Rochtchina et al. (2007) and Gopinath et al. (2013) showed that a deficiency of Vitamin B12 is linked to an increased risk of AMD5,6. Despite recent evaluations of stem cell–derived therapeutic approaches in Phase I clinical trials, such novel methods require long-term use of immunosuppressive drugs, which may lead to other medical implications7. Conventional therapies include FDA-approved anti-angiogenic agents, thermal laser photocoagulation or intravitreal injection of medications to limit neovascularisation. However, each of these treatments resulted in the development of atrophic scars and haemorrhage in patients8. In view of the above, there is still no cure for AMD and available therapies aim mainly to reduce patients’ symptoms and target the late stages of the disease. Therefore, further studies must be carried out to find an effective preventative measure, especially in targeting early stages of the disease before the onset of vision loss.\n\nThe mentioned pathological features of AMD are known to be regulated by genes such as Vascular Endothelium Growth Factor A (VEGFA) and Glutathione-S-Transferase µ1 (GSTM1). VEGFA upregulation is associated with neovascularisation, and a decrease in GSTM1 expression is associated with an increased susceptibility to oxidative damage8,9. Previously, imbalanced levels of fatty acids responsible for the abnormal function of the retina were associated with AMD progression. There are five major fatty acids in the human retina, namely, docosahexaenoic acid, arachidonic acid, stearic acid, oleic acid and palmitic acid, which are all classified as long chain polyunsaturated fatty acids (LC-PUFAs)10. It was reported that a deficiency in docosahexaenoic acid and arachidonic acid interfere in neurological and visual signalling pathways, and intake of these LC-PUFAs increased the risk of AMD10,11. In addition, other studies found that ROS produced during oxidative stress can damage the essential PUFAs in the retina and generate toxic lipid peroxidation end products (i.e. reactive aldehydes 4-hydroxynoneal and 4-hydroxyhexenal); thus, exacerbating the chronic-inflammatory damage in the retina. These accumulated aldehydes can in turn, inhibit redox enzyme reactions, DNA and RNA synthesis and biosynthesis of proteins12. PUFAs are an important substrate for redox enzymes such as glutathione S transferases (GSTs) during oxidative stress-mediated lipid peroxidation and healthy fatty acid (FA) levels are crucial for the efficient removal of ROS from the retina13,14. Dysfunctional innate immune responses to oxidative stress observed in AMD are also reported to be attributed to the formation of oxidized lipids15. Therefore, lipid and fatty acid pathways remain vital in maintaining a healthy environment in the retina. Furthermore, patients with AMD were reported to have low levels of other metabolites, such as glucose, lactate, glutamine and albumin, suggesting the possible role of a dysregulated metabolome in this disease16,17. As such, the pathogenesis of AMD is likely to involve the abnormal expression of VEGFA, GSTM1 and imbalanced levels of selective metabolites, such as fatty acids. This prompts the investigation of new and potential therapeutic agents that can alleviate the aberrant gene expression via chromatin remodelling processes and restore normal levels of metabolites in the retina.\n\nHere, we propose the use of L-Sulforaphane (LSF), a naturally occurring isothiocyanate found in many cruciferous vegetables like broccoli in the treatment of AMD18. LSF has been shown to have epigenetic properties in solid tumours by enhancing the acetylation of histones, resulting in an ‘opened’ chromatin state, which triggers the transcription of genes involved in cell death and restores the apoptotic potential of cancer cells19,20. These anti-carcinogenic effects have also been associated with downregulation of the pro-inflammatory marker, hypoxia inducing factor 1-α, and VEGF while increasing redox enzyme activities21,22. Such antioxidant properties could be useful for the treatment of the AMD. Whilst it has the potential to induce cell death in malignant cancer cells, it can protect retinal tissue from photoreceptor degeneration under oxidative stress conditions23. This protection is mediated via the induction of phase II detoxification enzyme NAD(P)H:quinoxidoreductase and transcriptional activation of antioxidant response element; thus elevating glutathione levels in the retinal cells23. Hence, the action of LSF is unique and seems to be disease specific. This characteristic enables LSF to be considered a potential drug candidate in targeted therapy.\n\nIn 2018, our laboratory reported the ability of LSF at micro molar concentrations (3µM and 5µM) to protect human retinal pigment epithelium from cell death and promoted the regeneration of these cells under oxidative stress conditions24. This preliminary study involving gas chromatography mass spectrometry (GC-MS) analytical methods revealed that LSF treatment induced changes in the levels of FAs, such as nonanoic acid and 9,12,15- (Z-Z-Z)-Octodectrienoic acid, and upregulated the levels of GSTM1 gene expression24. However, many of these significant changes were observed with the 5µM LSF treatment. These findings have warranted the current study to further examine lipids and fatty acids that may regulate the protective and antioxidant effects of LSF. In the current study, dose response data using LSF concentrations of 3-30µM validate the previously reported protective and regenerative properties of this compound against oxidative stress, where a dose-dependent increase in cell proliferation is observed and then plateaus at a concentration higher than 20µM. For the first time, we report the use of a lipidomic approach using liquid chromatography with triple-quadrupole mass spectrometry (LC-QqQ-MS) to analyse human retinal pigment epithelial (ARPE-19) cells pre-treated with 5 and 20µM LSF under oxidative stress conditions. The total pool of FAs affected by the treatment will be confirmed by gas chromatography-mass spectrometry (GC-MS) and used to putatively identify lipid classes. We hypothesize that LSF can increase the levels of lipids containing unsaturated FAs while decreasing levels of lipids with saturated FAs for the protection of ARPE-19 cells against oxidative damage.\n\n\nMethods\n\nThe Adult Retinal Pigment Epithelium-19 (ARPE-19) cell line was purchased from the American Type Cell Collection (USA). The cells were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM/F12) containing 200mM L-glutamine and 15mM HEPES (Life Technologies, USA). The culture media was further supplemented with 10% foetal calf serum (FCS; Sigma Aldrich, USA) and 1% penicillin-streptomycin 10,000 U/ml (Life Technologies, USA). The ARPE-19 cells were sustained at 37°C in an atmosphere of 95% air and 5% CO2, and phenotypic characteristics of these cells were validated in our previous publication24.\n\nThe ARPE-19 cells were starved in a serum-deprived DMEM/F12 media containing 1% FCS and 1% penicillin-streptomycin for 24 hours. For the CellTiter 96 AQueous One Solution Cell Proliferation (MTS) Assay, the cells were exposed to 0.025% dimethyl sulfoxide (DMSO; Sigma Aldrich, USA) as the drug vehicle control or 3µM LSF, 5µM LSF, 10µM LSF, 20µM LSF or 30µM LSF for 24 hours. For the GC-MS/LC-MS analysis, the cells were exposed to 0.025% DMSO, 5µM LSF or 20µM LSF for 24 hours. The negative control for all analyses was untreated cells that were incubated in serum-deprived DMEM/F12 media. After 24 hours incubation, the treatments were discarded from all the wells and the cells were incubated with 200µM hydrogen peroxide (H2O2; Sigma Aldrich, USA) for two hours. Untreated cells or LSF-treated cells incubated in Hanks Balanced Salt Solution (HBSS; Sigma Aldrich, USA) for two hours were used as the negative control for oxidative stress. Subsequently, the H2O2 or HBSS was removed and the cells were allowed to recover for 24 hours in serum-deprived DMEM/F12 media before either the MTS assay or the GC-MS/LC-MS analysis were carried out24.\n\nThe ARPE-19 cells were trypsinised using 0.25% trypsin EDTA (Life Technologies, USA) and centrifuged at 200 g for three minutes, before being seeded at a density of 105 cells/mL in 100µL of complete cell culture media in 96-well flat-bottom plates and treated with the agents described above. Each well contained 10,000 cells. To assess the effects of LSF treatment in the presence or absence of oxidative stress on cell proliferation, the MTS assay (catalogue number G3580; Promega, USA) was carried out according to the manufacturer’s protocol and as previously described24. A volume of 20µL MTS reagent was added to the cells in each well and plate was incubated for four hours at 37°C and in an atmosphere of 95% air and 5% CO2. Absorbance readings (at 490nm) of drug-treated cells were normalised to the untreated control. As per the manufacturer’s protocol, % cell proliferation = (Absorbance drug treatment – Absorbance blank) / (Absorbance untreated – Absorbance blank) x 100%. The percentage of cell proliferation was calculated as the mean of results from three independent experiments with three technical replicates per experiment.\n\nThe ARPE-19 cells were seeded at a density of 1.5x106 per well in 6-well plates and conditioned as indicated above. The cells were removed using 0.25% trypsin EDTA, followed by centrifugation at 200 g for three minutes. The cell pellets were resuspended in ice-cold 1X phosphate-buffered saline (pH 7.4) and transferred to microcentrifuge tubes. These tubes were centrifuged twice at 200 g for three minutes and after each spin, the pellets were resuspended in ice-cold PBS (washing step). The tubes were spun a third time, the supernatant was removed and the pellets were frozen at -80°C to be used for the extraction. Four replicates of each control and treated samples were performed.\n\nUpon cell harvesting, each cell pellet was washed with 200 μL of water by vigorous vortexing for 19 seconds. A volume of 250 μL of methanol and 0.01% butylated hydroxytoluene (v/v) mixture was added to the cell pellets and to the internal standard, 10 µM d7-cholesterol. The samples were then frozen for five minutes in liquid nitrogen, followed by sonication for another five minutes at room temperature at 100 rpm. The freeze-sonication steps were then repeated three times to lyse the cell pellets. The lysed cells were then vortexed vigorously for one minute. A volume of 500 μL of chloroform was added to the lysate and was mixed for 30 minutes at room temperature using a shaker. Next, the samples were centrifuged at 14,100g, 5°C for 15 minutes. The supernatant from each sample was transferred into respective clean 1.5 mL Eppendorf tubes (Tube A). A mixture containing 500 μL of chloroform:methanol (2:1) (v/v) was added to the cell pellets as the second extraction step. The samples were vortexed for 30 seconds and shaken for 15 minutes at room temperature before centrifugation at 16,100g, 0°C for 15 minutes. The supernatant from the second extraction was then combined into the supernatant in the respective Tube As. The combined supernatant for each sample was dried down under a stream of nitrogen. Each dried lipid extract was resuspended in 200 μL of butanol:methanol (1:1) (v/v) with 10 mM ammonium formate for LC-MS analysis25. Additionally, a 30 μL aliquot was transferred into a glass insert and dried in vacuo for subsequent fatty acid methyl ester (FAME) analysis on the GC-MS. All samples were stored in the dark in bags containing silica beads prior to GC-MS and LC-MS analysis.\n\nThe dried ARPE-19 cell extracts were resuspended in chloroform:methanol (2:1 v/v) (25µL) containing 60 μM of the internal standard (13C-labelled myristic acid), followed by the addition of the derivatizing agent (5µL) (catalogue number 11370591, Meth-Prep II™, Grace Davison Discovery, Deerfield, IL, US,). Each sample was subsequently incubated at 37oC for 30 min, then held for 10 min at room temperature. Then, 1 μL of the derivatised ARPE-19 cell extract was injected onto the GC-MS system consisting of a Gerstel 2.5.2 autosampler (catalogue number G7368A), a 7890A Agilent gas chromatograph (catalogue number G3440B), and a 5975C Agilent quadrupole MS (catalogue number G7042A) (Agilent Technologies, Santa Clara, US). The FAME analysis was carried out using a 30 m column with a 0.25 μm film thickness, 0.25 mm inner diameter and a 10 m guard column (catalogue number CP8944, Agilent J&W Scientific VF-5MS GC Column). The following parameters were set for GC-MS FAME analysis: injection port temperature (250°C), MS transfer line (280°C), ion source temperature (230°C) and quadrupole (150ºC). The carrier gas used for the analyses was helium (UHP 5.0) at a flow rate of 1.0 mL/min. For the FAME analysis, the temperature program used was: start at injection (50°C), hold for one min followed by a 15°C.min-1 oven temperature ramp to 230°C, hold for three min followed by a 10°C.min-1 oven temperature ramp to 325°C and a final three min heating at 325°C. Mass spectra were recorded at two scans/s with a 50–600 m/z scanning range26.\n\nLipid analysis using LC-MS was carried out as published previously26. Briefly, to separate the lipids, 5 µL aliquots per sample were injected onto a 50 mm × 2.1 mm × 2.7 µm Ascentis Express RP Amide column (catalogue number 53911-U, Supelco, Sigma, St Louis, USA) at 35°C using an Agilent LC 1200 (Mulgrave, Australia).\n\nLipid detection was carried out using Agilent 6410 triple quad (catalogue number, 6410, Mulgrave, Australia) in electrospray ionisation (ESI) mode. Lipid species were identified based on the lipid class using precursor ion and Neutral loss scanning techniques as discussed previously26. Diacylglycerol and triacylglycerol species were identified based on the neutral loss of fatty acyl moiety.\n\nIdentified lipid species were quantified via multiple reaction monitoring (MRM) with a 20 ms dwell time for the simultaneous measurements of ~20 to 50 compounds and the chromatographic peak width of 30 sec to 45 secs. A minimum of 12 to 16 data points was collected across the peak. Optimised parameters for capillary, fragmented, and collision voltages were 4000 V, 140 - 380, and 15–60 V, respectively. The collision gas used was nitrogen at 7 Lmin-1.\n\nLipid standards (Avanti Polar Lipids, Alabaster, USA) were prepared by combining equal volumes of individual lipid stock solutions. Calibration curves were constructed from calibration solutions ranging from 0.1 to 10 µM by least squares linear regression, following the serial dilutions of the lipid standards. Reverse phase peak area of the analyte was plotted against the concentration of the lipid in the reference standards. The concentration of each lipid species in the cell extract sample was estimated by using the regression model to convert normalized peak area to lipid concentration. Detected lipid species were annotated as lipid class (sum of carbon atoms in the two fatty acid chains: sum of double bonds in the fatty acid chains).\n\nSignificant changes in cell proliferation and levels of total fatty acids or lipid species were validated by one-way analysis of variance and the post-hoc Bonferroni/Fisher tests and paired t-test. The GC-MS was plotted using MetaboAnalyst Software Version 2 (USA)27. The LC-MS ESI-MRM data was processed using Agilent Mass Hunter Quantitative Analysis software (Version 6) (Mulgrave, Australia) and plotted using MetaboAnalyst Software Version 2 (USA).\n\n\nResults\n\nThe drug vehicle control (0.025% DMSO) did not affect the percentage of proliferation regardless of exposure to oxidative stress stimulus, H2O2 (Figure 1; all p values > 0.05)28. In the absence of H2O2, 3 µM - 30 µM LSF treatments did not have a significant impact on cell proliferation (Figure 1A; all p values > 0.05). In contrast, a dose-dependent increase in the proliferation of LSF-treated cells was observed at doses of 3µM to 20 µM under H2O2 conditions (Figure 1B; all p-values < 0.0001). Increasing the dose to 30µM LSF did not induce any further increase in cell proliferation (Figure 1B: vs 20µM, p value > 0.9999). These results validate the ability of LSF to protect ARPE-19 cells against oxidative stress by stimulating the regeneration of these cells. Henceforth, GC-MS and LC-MS analyses were performed on cells treated with the lowest and highest doses of LSF that resulted in significant increases in cell proliferation (i.e. p < 0.0001) compared to the untreated cells as the control group. Since there were no significant differences in cell proliferation between 20 µM and 30 µM LSF treatment groups, 30 µM LSF was not included in the GC-MS and LC-MS analyses.\n\nEffects of LSF on ARPE-19 cell proliferation in (A) without or (B) with H2O2. The proliferative effects of vehicle control (0.025% DMSO) and 3 µM - 30 µM LSF on cells were determined. The cells were treated with 0.025% DMSO or LSF for 24 hours prior to exposure with 200 µM H2O2 for two hours. The mean absorbance values for each treatment group are presented as a percentage of that of their respective untreated controls (CA) in the absence or presence of H2O2. n= 3; not significant (ns): p > 0.05, * p < 0.01 and ** p < 0.0001). [CA, cells alone; DMSO, dimethyl sulfoxide; H2O2, hydrogen peroxide; LSF, L-Sulforaphane].\n\nIn the absence of oxidative stress, 5µM LSF increased the levels of the fatty acid, cis-oleic acid (18:1) by 1.23x108 times while 20µM LSF increased level of trans-oleic acid by 7.42x107 times, cis-oleic acid by 2.81x108 times and eicosatrienoic acid (ETA) (20:3) by 2.53x109 times (Figure 2 and Figure 3; all p values < 0.001). In the presence of oxidative stress, there were no significant differences in the fatty acid levels between the control, 5µM LSF and 20µM LSF treatment groups and untreated controls (all p values > 0.05; see Underlying data)28.\n\nTotal fatty acid levels in (A) 5µM or (B) 20µM LSF-treated ARPE-19 cells without H2O2. Four replicate samples for each treatment group were compared with that of the untreated controls. Data in red and blue indicate an increase and decrease in FA levels, respectively. Total fatty acids (FACs) highlighted in red boxes show statistically significant changes in levels between treated groups and untreated controls (p values < 0.001). [5_LSF: 5µM LSF; 20_LSF: 20µM LSF. FA, fatty acid; FAC, total fatty acid; H2O2, hydrogen peroxide; LSF, L-Sulforaphane; UN, untreated].\n\nFour replicate LSF samples were compared with that of the untreated controls. Sample variation is depicted by the error bars. The y-axis values are automatically generated as arbitrary units by MetaboAnalyst software. [FAC, total fatty acid; FAC 18:1 n9c, cis-oleic acid; FAC 18:1 n9t, trans-oleic acid; FAC 20:3, eicosatrienoic acid; H2O2, hydrogen peroxide; LSF, L-Sulforaphane].\n\nIn the absence of oxidative stress, no changes in lipid levels between LSF-treated cells and the untreated control were reported (all p values > 0.05; see Underlying data)28. In the presence of oxidative stress, significant changes (all p values < 0.001) were observed in the 20µM LSF treatment groups. This study showed that LSF treatment increased levels of phosphatidylcholine (PC 33:3) by 2.33-fold and cholesteryl ester (CE 18:2 and CE 20:2) lipids containing unsaturated FAs by 3.490-fold and 5.498 fold respectively, and oxo-phytodienoic acid (oPDA 34:3-PC 16:0) by 2.445-fold. However, LSF treatment decreased levels of phosphatidylethanolamine (PE 34:0) consisting of saturated FAs by 0.394-fold (Figure 4B and Figure 5) was also observed. Other PE lipids containing unsaturated FAs (PE 38:5) were also decreased by 0.292-fold. In contrast, treatment with 5µM LSF did not result in any statistically significant changes in lipid levels (all p values > 0.05; Figure 4A).\n\nLipid levels in (A) 5µM LSF or (B) 20µM LSF-treated ARPE-19 cells exposed to H2O2. Four replicate samples across the treatment groups were compared with that of the untreated [UN] controls. Data in red and blue indicate an increase and decrease in lipid levels, respectively. Lipids highlighted in red boxes show statistically significant changes in levels between treated groups and untreated controls (p values < 0.001). [CE, cholesteryl ester; oPDA, oxo-phytodienoic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; 5LSF, 5µM LSF; 20LSF, 20µM LSF; H2O2, hydrogen peroxide; LSF, L-Sulforaphane].\n\nFour replicate LSF samples were compared with that of the untreated controls. Sample variation is depicted by error bars. The y-axis values are automatically generated as arbitrary units by the Agilent Mass Hunter Quantitative Analysis software. [CE, cholesteryl ester; oPDA, oxo-phytodienoic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; H2O2, hydrogen peroxide; LSF, L-Sulforaphane].\n\n\nDiscussion\n\nIn 2019, the World Health Organisation has classified AMD as one of the top 10 priority eye diseases and leading cause of blindness in the ageing population29. Therefore, without a current cure, there is an urgent need for better prevention, treatment and management strategies to reduce the burden of vision loss and improve patients’ quality of life. Oxidative stress and abnormal neovascularization are processes known to promote the pathological changes observed in the retina of AMD patients. The underlying molecular mechanisms triggering these processes involve aberrant downregulation of GSTM1 and upregulation of VEGFA8,9. More recently, deficient levels of dietary PUFAs have shown to increase the risk of AMD and disease progression in affected individuals30. Previously, our laboratory has shown that the cruciferous vegetable-derived compound, LSF, can protect human adult pigment epithelial cells from oxidative damage by upregulating GSTM1 expression and modulating levels of selected PUFAs24. Here, we validated the protective effects of LSF on human retinal cells under oxidative stress conditions and revealed the key fatty acids and lipids that may facilitate this protection.\n\nA dose-dependent increase in cell proliferation was observed in LSF-treated ARPE-19 cells exposed to H2O2-induced oxidative damage but no changes in cell proliferation were detected in the absence of stress. This finding demonstrates that LSF is not harmful at the investigated micromolar doses when oxidative stress is absent but can induce regeneration of retinal cells in an oxidative stress environment. Thus, LSF may be beneficial in the treatment of AMD without causing unwanted cellular toxicity and downstream side effects.\n\nFatty acids are freed from the triglyceride state by a process called lipolysis. During this process, glycerol is removed from the triglycerides by lipases to release free fatty acids31. The free fatty acids are then broken down to acetyl-coA in the mitochondria in the presence of nicotinamide adenine dinucleotide and the reduced form of flavin adenine dinucleotide to generate energy in a reaction known as beta oxidation31. Many free fatty acids are key components of phospholipids, which stabilise the cell membranes of various cells including those of the retina. These phospholipids are cleaved into several metabolites, such as 1-palmitoyl-2-oleoyl-glycerol, which consists of side-chains derived from palmitic acid and oleic acid31. Patients with AMD have demonstrated dysregulated levels of such fatty acids, which may contribute in the impairment of the retinal pigment epithelial cells seen in this disease32.\n\nTo determine the types of total fatty acids possibly implicated in LSF’s impact on ARPE-19 cells, GC-MS was performed. We showed that LSF treatment increased the levels of trans- and cis-oleic acid and ETA. Oleic acid is one of the most abundant monosaturated fatty acids (MUFAs) of the omega-9 fatty acid family, while common omega-3 PUFAs include ETA, eicosapentaenoic acid and docosahexaenoic acid, found in fish oil33. These fatty acids contribute to several biological processes, including visual pathways signalling in the retina, anti-inflammatory properties and protection against metabolic diseases. The benefits of a high dietary intake of omega-3 and omega-9 fatty acids in alleviating the risk of AMD by about 30% to 40% and neovascularisation have been extensively reviewed by van Leeuwen et al. (2018)30. The action of LSF appears to be cell-type specific. Pasko et al. (2018) revealed that the pro-apoptotic effect of LSF on hepatocellular carcinoma and colorectal cancer cell lines was correlated with increased levels of oleic acid found in the cancer cells34. This is in contrast to our findings, where no toxicity was seen in LSF-treated ARPE-19 cell line despite increased oleic acid levels. The lack of harmful effects and the evident protective effects of LSF on human retinal cells shown here can be mirrored by findings from an association study that demonstrated a correlation between a high MUFA diet and significantly reduced risk of AMD35. This protective effect of MUFAs against AMD may involve anti-atherogenic pathways, as discussed by Parekh et al. (2009)35.\n\nMany studies have shown that an increased dietary intake of the selected omega-3 PUFAs lowers the risk of dementia, improves cognition and aids age-related degenerative disorders36. Connor et al. used a hypoxia-induced animal model of retinopathy to show that an omega-3 PUFA diet suppressed retinal expression of the inflammatory cytokine tumour necrosis factor (TNF)-α and macrophage-induced inflammatory responses in retinal cells10,37. This anti-inflammatory phenomenon promoted a suppression of neovascularisation of comparable magnitude to that induced by VEGF inhibitors37,38. Interestingly, AMD patients demonstrated lower levels of oleic acid and omega-3 PUFAs in their red blood cells compared to their age-matched healthy controls32. Furthermore, a good distribution of omega 3-PUFAs in the retina is said to be protective against photo-sensitised oxidation and peroxidation of lipids (e.g. 7-ketocholesterol) in the eyes of aging adults15,32. Oxidised lipids can induce the migration and activation of retinal microglia into an M1 pro-inflammatory phenotype, which triggers the expression of pro-angiogenic cytokines and subsequent choroid neovascularisation seen in advanced AMD. Therefore, the findings from these reports support the potential use of LSF as a naturally-occurring enhancer of omega-3 levels in RPE cells to protect RPE cells from inflammation and abnormal neovascularisation observed in AMD patients and with possibly less risk of side effects caused by conventional VEGF inhibitors39. The direct relationship between the action of LSF, omega-3 PUFAs and anti-oxidative pathways has yet to be elucidated but it is known that omega-3 PUFAs, when oxidised, can protect cells against free radical superoxide and H2O2 by activating the nuclear factor erythroid-derived-2 like-2 (Nrf2) pathway40. It has been reported that ageing impairs Nrf2 responses to oxidative stress41. As discussed in our recent publication, LSF acts as a potent Nrf-2 activator, which further promotes its use as a therapeutic agent in chronic inflammatory conditions such as AMD24,42. Future studies arising from our GC-MS data may include investigations into the possible synergistic effects of LSF and omega-3 PUFA combination treatment on the suppression of oxidative stress, neovascularisation and VEGF expression in RPE cells and choroid-derived endothelial cells.\n\nTo identify the lipid classes that are affected by LSF treatment of ARPE-19 cells, LC-MS was performed. In the presence of oxidative stress, LSF treatment decreased levels of PE lipids but increased levels of levels CE, oPDA and PC lipids. Lipofuscin, a type of pigment granule, accumulates in the ageing retina as a result of light-associated vitamin A recycling43. A major component of lipofuscin is A2E, which has the capacity to destabilise cell membranes of RPE cells and compromise their viability. The creation of A2E within retinal cells involves condensation reactions between PE lipids and all-trans-retinal44. The photo-oxidation of such lipids in RPE cells can be initiated via sensitisation of A2E, triggered by blue light exposure over time. Consequently, H2O2 is generated and complement is activated via C3-dependent pathways, leading to oxidative stress, inflammation and apoptosis45. This supports the use of H2O2 as an ideal stimulant of both photo-oxidation and oxidative stress seen in the ageing retina of AMD patients and validates our in vitro model reported here. Other studies have shown that phytochemicals including anthocyanin and LSF can reduce A2E photo-oxidation and confer RPE cell protection by increasing expression of oxidative pathway phase II enzyme NAD(P)H:quinone reductase46. This is aligns with our previous findings where we showed that LSF treatment of ARPE-19 cells can confer protection against H2O2-induced oxidative stress by upregulating another phase II enzyme, GSTM124. In this present study, we demonstrate that LSF treatment of ARPE-19 cells in the presence of H2O2 can downregulate levels of PE lipids (i.e. PE 34:0 and PE 38:5). Since PE lipids are precursors of A2E, we propose that retinal cells experiencing oxidative stress can benefit from LSF treatment, since this compound can reduce PE levels and, consequently, a smaller amount of PE lipids is available for the biosynthesis of A2E, which may attenuate the risk of photo-oxidation leading to retinal cell death.\n\nIn patients with early AMD, pathological observations include the accumulation of drusen particles containing lipoproteins in the Bruch’s membrane, accompanied by apoptosis of RPE cells. The RPE is responsible for controlling lipoprotein uptake into the retina and their distribution to photoreceptors for the replacement of shed membrane disks. These lipoproteins mainly consist of CEs but when these lipids are oxidised, they become cytotoxic to retinal cells47. The levels of CEs can also be upregulated by oxidative stress stimuli, and treatment of ARPE-19 cells with lipoproteins containing oxidised lipids can increase levels of CEs consisting of oleic acid47. Here, we report that LSF upregulates levels of CEs containing omega 6-PUFAs linoleic acid (18:2) and eicosadienoic acid (20:2) in the presence of H2O2. Since H2O2 is an oxidative stress stimulus, it is possible that the increased CE levels we observe in LSF-treated cells may be attributed, to some extent, to the exposure of cells to H2O2. It is noteworthy that omega 6-PUFAs are more prone to lipid peroxidation due to the increased risk of attacks to their double bonds by reactive oxygen species and because accumulation of peroxidised lipids in retinal cell membranes over time can trigger AMD progression35. However, the relationship between LSF-induced mechanisms and oxidised/peroxidised lipids is not well known. Hence, a future study stemming from this work may include evaluating the oxidation/peroxidation status of lipids in LSF-treated ARPE-19 cells using well-established assays.\n\nThe vast majority of phospholipids that make up the membranes of cells in the retina are PC lipids, with omega-3 PUFAs making up about 20% of the fatty acids in this lipid class45. Perhaps, the upregulation of ETA fatty acids resulting from LSF treatment observed here is reflected in the elevated levels of PC 33:3 lipids. PC and CE lipids are commonly found in drusen particles but they also accumulate in the Bruch’s membrane in normal healthy eyes throughout adulthood48. Lipid accumulation in the Bruch’s membrane eventually forms a “lipid wall” that prevents the normal exchange of oxygen and nutrients between the RPE and the choroid15. In addition, the higher the content of PC and CE lipids in the Bruch’s membrane, the higher the risk of lipid peroxidation and oxidation, complement activation, inflammation and generation of toxic metabolites with age. If these lipids are retained at higher levels in the RPE cells, there is a lower tendency for lipids to be shed into the Bruch’s membrane or accumulate in drusen particles; thus, lowering the risk of toxic metabolite production and apoptosis48. Since LSF treatment can increase the levels of PCs and CEs in ARPE-19 cells in the current study, this suggests that this compound may have the potential to restore or maintain healthy levels of such lipids within the retinal cells by interfering with the biosynthesis or transportation of major drusen components. Genome-wide association studies have identified risk variants in genes (e.g. ATP-binding cassette transporter, cholesteryl ester transfer protein, apolipoprotein E4, etc.) that regulate lipid metabolism and transportation that may confer a protective status against AMD pathophysiology49. Thus, investigating the changes in the expression of such genes may help to further dissect the lipid pathways responsible for the LSF-mediated regeneration of RPE cells under oxidative stress conditions.\n\nLipids are major components of plant stress hormones. An example is oPDA, which is the key precursor of the oxylipin stress hormone, jasmonate. oPDA lipids can activate genes involved in oxidative stress pathways and a correlation between oPDA signalling and decreased hydrogen peroxide levels has been reported in plants50,51. Interestingly, administrating the stress hormone jasmonate to broccoli sprouts increased levels of LSF52. This suggests that LSF may be a by-product of a compensatory mechanism found in plants that maintains cellular redox homeostasis in stressful environments. Additionally, oPDA treatment of human neuroblastoma cells can prevent harmful effects from oxidative stress and apoptosis by activating the Nrf2 pathway53. The redox activity of oPDA is also evidenced in its capacity to regulate the expression of GST genes54. Taken together, oPDA behaves as a Nrf2 activator like LSF. Therefore, LSF’s antioxidant effects on ARPE-19 cells shown here may either involve: 1) independent activation of Nrf2; 2) an upregulation of oPDA, which in turn triggers the Nrf2 pathway; or 3) a synergistic activation of this pathway mediated by the combined action of LSF and oPDA signalling.\n\nAlthough we attempt to discuss the possible relationship between the observations arisen from the total fatty acid analysis (GC-MS) and lipidomic data (LC-MS), drawing a correlation between fatty acid data and the LC-MS lipid profile in this study proved to be challenging, since the methods used here could not explicitly identify the source of the fatty acids (i.e. free/circulating or conjugated to lipids) implicated in LSF’s protection of the ARPE-19 cell line. Despite this limitation, this study revealed the ability of LSF to alter levels of selected fatty acids and lipid classes involved in mechanisms that can promote AMD processes in human RPE cells.\n\nIn conclusion, we propose that RPE cells at risk of apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in downstream pathways of photo-oxidation, inflammation and oxidative stress for the generation of a protective state against the ageing process and AMD progression. This work warrants future investigations, such as trialling LSF treatment in co-culture models of ARPE-19 and choroid-derived cells, and animal models of AMD. Performing high throughput transcriptomics methods will also help to identify key genes that mediate LSF’s effects on fatty acid and lipid metabolism, biosynthesis and translocation in RPE cells under AMD-like pathological conditions55. These further studies will facilitate the design of targeted therapies that can be co-administered with LSF for the prevention of AMD progression.\n\n\nData availability\n\nHarvard Dataverse: Lipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration. https://doi.org/10.7910/DVN/C9VCBX28\n\nThis project contains the following underlying data:\n\n- GCMS Fatty Acid Analysis Data.tab (raw fatty acid analysis data)\n\n- LCMS Lipid Analysis Data.tab (raw lipid analysis data)\n\n- MTS Raw Data_Kwa.tab (raw cell proliferation assay data)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis work was funded by the School of Health and Biomedical Sciences, Royal Melbourne Institute of Technology University, Australia.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Narin Osman (Discipline of Human Biosciences, RMIT University, Australia) for purchasing the ARPE-19 cell line.\n\n\nReferences\n\nAmbati J, Fowler BJ: Mechanisms of age-related macular degeneration. Neuron. 2012; 75(1): 26–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNita M, Grzybowski A: The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxid Med Cell Longev. 2016; 2016: 3164734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan JC, Thurlby DA, Shahid H, et al.: Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisation. Br J Ophthalmol. 2006; 90(1): 75–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThornton J, Edwards R, Mitchell P, et al.: Smoking and age-related macular degeneration: a review of association. Eye (Lond). 2005; 19(9): 935–44. PubMed Abstract \| Publisher Full Text\n\nRochtchina E, Wang JJ, Flood VM, et al.: Elevated serum homocysteine, low serum vitamin B12, folate, and age-related macular degeneration: the Blue Mountains Eye Study. Am J Ophthalmol. 2007; 143(2): 344–6. PubMed Abstract \| Publisher Full Text\n\nGopinath B, Flood VM, Rochtchina E, et al.: Homocysteine, folate, vitamin B-12, and 10-y incidence of age-related macular degeneration. Am J Clin Nutr. 2013; 98(1): 129–35. PubMed Abstract \| Publisher Full Text\n\nda Cruz L, Fynes K, Georgiadis O, et al.: Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. Nat Biotechnol. 2018; 36(4): 328–37. PubMed Abstract \| Publisher Full Text\n\nHuo X, Li Y, Jiang Y, et al.: Inhibition of ocular neovascularization by co-inhibition of VEGF-A and PLGF. Cell Physiol Biochem. 2015; 35(5): 1787–96. PubMed Abstract \| Publisher Full Text\n\nKovacs K, Marra KV, Yu G, et al.: Angiogenic and Inflammatory Vitreous Biomarkers Associated With Increasing Levels of Retinal Ischemia. Invest Ophthalmol Vis Sci. 2015; 56(11): 6523–30. PubMed Abstract \| Publisher Full Text\n\nLiu A, Chang J, Lin Y, et al.: Long-chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration. J Lipid Res. 2010; 51(11): 3217–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanGiovanni JP, Chew EY, Agrón E, et al.: The relationship of dietary omega-3 long-chain polyunsaturated fatty acid intake with incident age-related macular degeneration: AREDS report no. 23. Arch Ophthalmol. 2008; 126(9): 1274–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanito M, Brush RS, Elliott MH, et al.: High levels of retinal membrane docosahexaenoic acid increase susceptibility to stress-induced degeneration. J Lipid Res. 2009; 50(5): 807–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMukherjee PK, Marcheselli VL, Serhan CN, et al.: Neuroprotectin D1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress. Proc Natl Acad Sci U S A. 2004; 101(22): 8491–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelmastro-Greenwood M, Freeman BA, Wendell SG: Redox-dependent anti-inflammatory signaling actions of unsaturated fatty acids. Annu Rev Physiol. 2014; 76: 79–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanda JT, Cano M, Wang L, et al.: Lipids, oxidized lipids, oxidation-specific epitopes, and Age-related Macular Degeneration. Biochim Biophys Acta Mol Cell Biol Lipids. 2017; 1862(4): 430–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsborn M, Park Y, Parks MB, et al.: Metabolome-wide association study of neovascular age-related macular degeneration. PLoS One. 2013; 8(8): e72737. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPushpoth A, Fitzpatrick M, Young S, et al.: Metabolomic analysis in patients with age related macular degeneration. Invest Ophthalmol Vis Sci. 2013; 54: 3662. Reference Source\n\nVanduchova A, Anzenbacher P, Anzenbacherova E: Isothiocyanate from Broccoli, Sulforaphane, and Its Properties. J Med Food. 2019; 22(2): 121–6. PubMed Abstract \| Publisher Full Text\n\nMyzak MC, Tong P, Dashwood WM, et al.: Sulforaphane retards the growth of human PC-3 xenografts and inhibits HDAC activity in human subjects. Exp Biol Med (Maywood). 2007; 232(2): 227–34. PubMed Abstract \| Free Full Text\n\nPledgie-Tracy A, Sobolewski MD, Davidson NE: Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Mol Cancer Ther. 2007; 6(3): 1013–21. PubMed Abstract \| Publisher Full Text\n\nYao H, Wang H, Zhang Z, et al.: Sulforaphane inhibited expression of hypoxia-inducible factor-1alpha in human tongue squamous cancer cells and prostate cancer cells. Int J Cancer. 2008; 123(6): 1255–61. PubMed Abstract \| Publisher Full Text\n\nXiao D, Powolny AA, Antosiewicz J, et al.: Cellular responses to cancer chemopreventive agent D,L-sulforaphane in human prostate cancer cells are initiated by mitochondrial reactive oxygen species. Pharm Res. 2009; 26(7): 1729–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe L, Yu T, Li Y, et al.: Sulforaphane enhances the ability of human retinal pigment epithelial cell against oxidative stress, and its effect on gene expression profile evaluated by microarray analysis. Oxid Med Cell Longev. 2013; 2013: 413024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDulull NK, Dias DA, Thrimawithana TR, et al.: L-Sulforaphane Confers Protection Against Oxidative Stress in an In Vitro Model of Age-Related Macular Degeneration. Curr Mol Pharmacol. 2018; 11(3): 237–53. PubMed Abstract \| Publisher Full Text\n\nBotte CY, Yamaryo-Botté Y, Rupasinghe TW, et al.: Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites. Proc Natl Acad Sci U S A. 2013; 110(18): 7506–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlmstead IL, Hill DR, Dias DA, et al.: A quantitative analysis of microalgal lipids for optimization of biodiesel and omega-3 production. Biotechnol Bioeng. 2013; 110(8): 2096–104. PubMed Abstract \| Publisher Full Text\n\nXia J, Mandal R, Sinelnikov IV, et al.: MetaboAnalyst 2.0--a comprehensive server for metabolomic data analysis. Nucleic Acids Res. 2012; 40(Web Server issue): W127–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwa F: Lipidomics reveal the protective effects of a vegetable-derived isothiocyanate against retinal degeneration. Harvard Dataverse, V1, UNF:6:uPFsboNxg85nYicAP1V4gQ== [fileUNF], 2019. http://www.doi.org/10.7910/DVN/C9VCBX\n\nOrganization WH: Priority Eye Disease. World Health Organization; 2019; [cited 2019 6 Feburary]. Reference Source\n\nvan Leeuwen EM, Emri E, Merle BMJ, et al.: A new perspective on lipid research in age-related macular degeneration. Prog Retin Eye Res. 2018; 67: 56–86. PubMed Abstract \| Publisher Full Text\n\nKlausner RD, Kleinfeld AM, Hoover RL, et al.: Lipid domains in membranes. Evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. J Biol Chem. 1980; 255(4): 1286–95. PubMed Abstract\n\nOuchi M, Ikeda T, Nakamura K, et al.: A novel relation of fatty acid with age-related macular degeneration. Ophthalmologica. 2002; 216(5): 363–7. PubMed Abstract \| Publisher Full Text\n\nLim JH, Gerhart-Hines Z, Dominy JE, et al.: Oleic acid stimulates complete oxidation of fatty acids through protein kinase A-dependent activation of SIRT1-PGC1α complex. J Biol Chem. 2013; 288(10): 7117–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasko P, Tyszka-Czochara M, Galanty A, et al.: Comparative Study of Predominant Phytochemical Compounds and Proapoptotic Potential of Broccoli Sprouts and Florets. Plant Foods Hum Nutr. 2018; 73(2): 95–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParekh N, Voland RP, Moeller SM, et al.: Association between dietary fat intake and age-related macular degeneration in the Carotenoids in Age-Related Eye Disease Study (CAREDS): an ancillary study of the Women's Health Initiative. Arch Ophthalmol. 2009; 127(11): 1483–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDangour AD, Allen E, Elbourne D, et al.: Effect of 2-y n-3 long-chain polyunsaturated fatty acid supplementation on cognitive function in older people: a randomized, double-blind, controlled trial. Am J Clin Nutr. 2010; 91(6): 1725–32. PubMed Abstract \| Publisher Full Text\n\nConnor KM, SanGiovanni JP, Lofqvist C, et al.: Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med. 2007; 13(7): 868–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAiello LP, Pierce EA, Foley ED, et al.: Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A. 1995; 92(23): 10457–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalavarjani KG, Nguyen QD: Adverse events and complications associated with intravitreal injection of anti-VEGF agents: a review of literature. Eye (Lond). 2013; 27(7): 787–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMajkova Z, Layne J, Sunkara M, et al.: Omega-3 fatty acid oxidation products prevent vascular endothelial cell activation by coplanar polychlorinated biphenyls. Toxicol Appl Pharmacol. 2011; 251(1): 41–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSachdeva MM, Cano M, Handa JT: Nrf2 signaling is impaired in the aging RPE given an oxidative insult. Exp Eye Res. 2014; 119: 111–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoughton CA, Fassett RG, Coombes JS: Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician's Expectation Be Matched by the Reality? Oxid Med Cell Longev. 2016; 2016: 7857186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSparrow JR, Fishkin N, Zhou J, et al.: A2E, a byproduct of the visual cycle. Vision Res. 2003; 43(28): 2983–90. PubMed Abstract \| Publisher Full Text\n\nLiu J, Itagaki Y, Ben-Shabat S, et al.: The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane. J Biol Chem. 2000; 275(38): 29354–60. PubMed Abstract \| Publisher Full Text\n\nSparrow JR, Wu Y, Kim CY, et al.: Phospholipid meets all-trans-retinal: the making of RPE bisretinoids. J Lipid Res. 2010; 51(2): 247–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJang YP, Zhou J, Nakanishi K, et al.: Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells. Photochem Photobiol. 2005; 81(3): 529–36. PubMed Abstract \| Free Full Text\n\nYamada Y, Tian J, Yang Y, et al.: Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells. J Neurochem. 2008; 105(4): 1187–97. PubMed Abstract \| Publisher Full Text\n\nWang L, Clark ME, Crossman DK, et al.: Abundant lipid and protein components of drusen. PLoS One. 2010; 5(4): e10329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen W, Stambolian D, Edwards AO, et al.: Genetic variants near TIMP3 and high-density lipoprotein-associated loci influence susceptibility to age-related macular degeneration. Proc Natl Acad Sci U S A. 2010; 107(16): 7401–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLópez-Cruz J, Óscar CS, Emma FC, et al.: Absence of Cu-Zn superoxide dismutase BCSOD1 reduces Botrytis cinerea virulence in Arabidopsis and tomato plants, revealing interplay among reactive oxygen species, callose and signalling pathways. Mol Plant Pathol. 2017; 18(1): 16–31. PubMed Abstract \| Publisher Full Text\n\nSham A, Al-Azzawi A, Al-Ameri S, et al.: Transcriptome analysis reveals genes commonly induced by Botrytis cinerea infection, cold, drought and oxidative stresses in Arabidopsis. PLoS One. 2014; 9(11): e113718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo L, Wang P, Gu Z, et al.: Proteomic analysis of broccoli sprouts by iTRAQ in response to jasmonic acid. J Plant Physiol. 2017; 218: 16–25. PubMed Abstract \| Publisher Full Text\n\nTaki-Nakano N, Ohzeki H, Kotera J, et al.: Cytoprotective effects of 12-oxo phytodienoic acid, a plant-derived oxylipin jasmonate, on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells. Biochim Biophys Acta. 2014; 1840(12): 3413–22. PubMed Abstract \| Publisher Full Text\n\nPark SW, Li W, Viehhauser A, et al.: Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis. Proc Natl Acad Sci U S A. 2013; 110(23): 9559–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDulull NK, Thrimawithana TR, Kwa FAA: Mimicking the ocular environment for the study of inflammatory posterior eye disorders. Drug Discov Today. 2017; 22(2): 440–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "52230", "date": "08 Aug 2019", "name": "Chris Barlow", "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\nOverview:\nThis paper seeks to build on previous work published by this group which has demonstrated that pre-treatment of ARPE-19 cells with L-Sulforaphane (LSF) leads to protection against oxidative damage as assessed using a cell proliferation assay. In particular, here the authors seek to identify any changes in fatty acids and more complex lipids following pre-treatment with LSF and then H2O2. The authors report that no fatty acids (total fatty acid analysis) were significantly altered under conditions of oxidative stress. However, they report extremely large fold-changes for cis-oleic, trans-oleic and eicosatrienoic acids upon treatment with LSF in the absence of oxidative stress. For the lipidomic portion of the study the authors report that several lipids showed statistically significant differences under oxidative stress following pre-treatment with LSF. Unfortunately, the paper suffers from problems associated with data analysis making it unsuitable for indexing at this time.\nFatty acid analysis:\nOn page 5 the authors report very large fold-changes on the order of 107 to 109 for oleic (cis and trans) and ETA upon treatment with LSF in the absence of oxidative stress. Upon inspection of the underlying data I found that for all four of the \"Untreated –\" samples, the intensity for these fatty acids was 1x10-7 for both cis- and trans- oleic acid and 1x10-9 for ETA (see table here). It would appear that these fatty acids were not detected in these samples and subsequently values have been imputed automatically. While imputation is common in metabolomic analysis it is not valid to report a fold-change or p-value where all values for the control group have been imputed.\nLipidomic Analysis:\nHere the authors report that several lipids were significantly different (p < 0.001) upon pre-treatment with 20 µM LSF relative to the untreated controls under conditions of oxidative stress. Careful examination of the underlying data suggests that a couple of errors may have been made in data processing. Specifically, the fold-change for PC 33:3 was 1.946 and not 2.33 as reported. Similarly, PE 34:0 and PE 38:5 had fold-changes of 0.723 and 0.684 respectively and not the reported 0.394 and 0.292 reported in the paper. I have reproduced the relevant data from the underlying data in the table here for clarity. I’m also confused about the statistical analysis. Using a two-tailed t-test none of the lipids reported as significant had a p-value < 0.001 as reported in the text, more generally it is unclear how the authors have dealt with the issue of multiple comparisons. The experimental section of lists several statistical tests but it is unclear which test was used for each analysis. Greater clarification as to how the statistical analysis was performed is necessary.\nExperimental Design:\nThe authors have used n = 4 for each group. While n = 4 is probably fine for using assays with a single metric such as the proliferation assay it is probably insufficient for lipidomic analysis. Indeed, the lipid data seems to be highly variable. For example, the total amount of lipid seems to vary substantially more than I would have expected. A crude measure of this variation is reflected in the median intensity of all the lipid measured for each sample which I have plotted here. From the experimental section on page 4 it appears that cells were seeded at a density of 1.5 x 106 before being conditioned as described in the “Cell treatment prior to analysis” section. If I’m reading this correctly then there was no adjustment for the number of cells following the conditioning but immediately before lipid extraction. Were an equivalent number of cells extracted (as opposed to seeded) in each sample, and if so, do the authors have any insight into why such a high degree of variability was observed in the lipidomics data? Similarly, I assume that treatment with hydrogen peroxide resulted in some cell death, what steps were taken to ensure that dead cells were not being extracted along with the live cells? Finally, I would suggest that some important comparisons have not been analysed. Presumably the hypothesis is that LSF treatment leads to changes in lipid profile which are then somehow protective against oxidative damage. I would suggest that the authors need to compare the lipid profiles of the LSF 20 µM – against the untreated – group. This should capture lipidomic differences associated with LSF treatment without the confounding effect of H2O2 treatment.\n\nAdditional notes and suggestions:\nOn page 3 the authors state that all five major fatty acids in the human retina are long chain polyunsaturated fatty acids (PUFAs). This is incorrect, docosahexaenoic and arachidonic acids are PUFAs, oleic acid is monounsaturated and stearic and palmitic are saturated.\n\nFigures 2 and 4: The annotations are too small to read. The graphs need to be re-drawn with a larger font.\n\nFigure 3 and 5: The authors state that the y-axis values are automatically generated as arbitrary units by the software used. The authors need to clearly state how the data is being processed. (In this case it seems likely that the data is being log2 transformed.) Is a box and whisker plot appropriate for four data points?\n\nPage 5: The text here indicates that lipid quantitation was performed using external calibration curves however no concentrations are given throughout the text or underlying data.\n\nIn the “LCMS Lipid Analysis Data.xlsx” file from the underlying data, five lipids are included in duplicate; PC 31:1, PC 33:3, PC 33:2, PC 34:4 and PC 37:4.\nConclusion:\nDue to the problems with the data analysis outlined above I’m unable to recommend this paper for indexing. If the authors are able to address these problems however, this paper should be considered as a fresh submission.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "4950", "date": "16 Oct 2019", "name": "Faith Kwa", "role": "Author Response", "response": "Rebuttal to Reviewer 1 Chris Barlow’s comments. Please find our response below. Overview: This paper seeks to build on previous work published by this group which has demonstrated that pre-treatment of ARPE-19 cells with L-Sulforaphane (LSF) leads to protection against oxidative damage as assessed using a cell proliferation assay. In particular, here the authors seek to identify any changes in fatty acids and more complex lipids following pre-treatment with LSF and then H2O2. The authors report that no fatty acids (total fatty acid analysis) were significantly altered under conditions of oxidative stress. However, they report extremely large fold-changes for cis-oleic, trans-oleic and eicosatrienoic acids upon treatment with LSF in the absence of oxidative stress. For the lipidomic portion of the study the authors report that several lipids showed statistically significant differences under oxidative stress following pre-treatment with LSF. Unfortunately, the paper suffers from problems associated with data analysis making it unsuitable for indexing at this time. Response: We thank Reviewer 1’s concise summary of the work presented in our manuscript. However, we have to respectfully disagree that the data analysis using one-way ANOVA and paired t-test is not appropriate for the nature of the work here. The data was always analysed at the significance level of p<0.05 but where p values generated were indeed less than 0.001, we have indicated this as p<0.001. We have now clearly indicated a statistically significant level of p<0.05 in the Methods section. This significance level and both statistical tests used in the fatty acid and lipid data here are standard statistical packages recommended by MetaboAnalyst Version 2 which we have already referenced in Reference 27. For the fatty acids where fold-changes for cis-oleic, trans-oleic and eicosatrienoic acids upon treatment with LSF in the absence of oxidative stress were reported, the untreated groups had an amount of fatty acids that were below the level of detection and hence for the purpose of performing univariate statistics, the missing values were imputed in alignment with common practice in metabolomics analysis as acknowledged by the Reviewer below. Taking the reviewer’s comments into consideration, we have edited the Result section to indicate that the levels of these fatty acids were only detected in the respective LSF treatment groups but not in the untreated controls. We have also removed any mention of fold changes in fatty acid levels in the manuscript and in Figure 3 but indicated that a comparison in the detection levels between untreated and treated groups. We have also removed any references to p values when reporting fatty acid data. On page 5 the authors report very large fold-changes on the order of 107 to 109 for oleic (cis and trans) and ETA upon treatment with LSF in the absence of oxidative stress. Upon inspection of the underlying data I found that for all four of the \"Untreated –\" samples, the intensity for these fatty acids was 1x10-7 for both cis- and trans- oleic acid and 1x10-9 for ETA (see table here). It would appear that these fatty acids were not detected in these samples and subsequently values have been imputed automatically. While imputation is common in metabolomic analysis it is not valid to report a fold-change or p-value where all values for the control group have been imputed. Response: Kindly see our response to the previous comment. Lipidomic Analysis: Here the authors report that several lipids were significantly different (p < 0.001) upon pre-treatment with 20 µM LSF relative to the untreated controls under conditions of oxidative stress. Careful examination of the underlying data suggests that a couple of errors may have been made in data processing. Specifically, the fold-change for PC 33:3 was 1.946 and not 2.33 as reported. Similarly, PE 34:0 and PE 38:5 had fold-changes of 0.723 and 0.684 respectively and not the reported 0.394 and 0.292 reported in the paper. I have reproduced the relevant data from the underlying data in the table here for clarity. I’m also confused about the statistical analysis. Using a two-tailed t-test none of the lipids reported as significant had a p-value < 0.001 as reported in the text, more generally it is unclear how the authors have dealt with the issue of multiple comparisons. The experimental section of lists several statistical tests but it is unclear which test was used for each analysis. Greater clarification as to how the statistical analysis was performed is necessary. Response: We thank Reviewer 1 for pointing out the typographical errors which have been amended in the revised version of the manuscript. Regarding the statistical analyses, we used one-way ANOVA to analyse the GCMS and LCMS data presented in the heat maps that illustrates how the expression of fatty acids or lipids differ with each treatment or oxidative stress/normal conditions. Therefore, a multiple comparison was made determine the effect of LSF treatment and oxidative stress across the various groups. In contrast, a paired t test was used to analyse the GCMS and LCMS data represented in the box plots. This was a direct comparison between the fold change levels seen in the untreated control and those in the groups treated with 20 µM LSF. We did not use the two-tailed t-test. We would also like to clarify that we used the statistical significance level of p<0.05 and not p <0.001 in MetaboAnalyst Version 2. The revised manuscript has been corrected to indicate where p values are less than 0.05 instead of 0.001. Experimental Design: The authors have used n = 4 for each group. While n = 4 is probably fine for using assays with a single metric such as the proliferation assay it is probably insufficient for lipidomic analysis. Indeed, the lipid data seems to be highly variable. For example, the total amount of lipid seems to vary substantially more than I would have expected. A crude measure of this variation is reflected in the median intensity of all the lipid measured for each sample which I have plotted here. Response: Due to the complexity of the study, it is challenging to generate a higher number of replicates for this study. A minimum of 1.5 million cells were seeded for each of the four replicates per control and treatment groups (i.e. a minimum of 36 million cells used in the metabolomics investigations). There are many papers reporting lipidomic/ LCMS studies which analyse data from less than 4 repeat experiments. These include recent articles published in Q1 journals such as Oncology Reports and PLOS Biology. Examples can be found in the links below: https://www.spandidos-publications.com/10.3892/or.2018.6510 https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2002214 From the experimental section on page 4 it appears that cells were seeded at a density of 1.5 x 106 before being conditioned as described in the “Cell treatment prior to analysis” section. If I’m reading this correctly then there was no adjustment for the number of cells following the conditioning but immediately before lipid extraction. Were an equivalent number of cells extracted (as opposed to seeded) in each sample, and if so, do the authors have any insight into why such a high degree of variability was observed in the lipidomics data? Similarly, I assume that treatment with hydrogen peroxide resulted in some cell death, what steps were taken to ensure that dead cells were not being extracted along with the live cells? Response: Although not apparent in our Methods section of the manuscript, during cell harvesting prior to lipid extraction, we washed the wells containing the adherent cells with PBS (pH 7.4) to remove any dead cells and cell debris. Following trypsinisation of the adherent cells per well, we performed a live cell count using the trypan blue exclusion method. Furthermore, the data has been normalised to both the number of cells per sample and the median of the reverse phase peak area response using the MetaboAnalyst Software Version 2 which will reduce any impact of variation by any differences in cell numbers. We have added these details in the revised version of the manuscript. Finally, I would suggest that some important comparisons have not been analysed. Presumably the hypothesis is that LSF treatment leads to changes in lipid profile which are then somehow protective against oxidative damage. I would suggest that the authors need to compare the lipid profiles of the LSF 20 µM – against the untreated – group. This should capture lipidomic differences associated with LSF treatment without the confounding effect of H2O2 treatment. Response: We thank Reviewer 1 for his recommendation. We have indeed compared fatty acid and lipid levels between LSF 20 µM and untreated groups in the presence and absence of H2O2. This is already stated in the manuscript in the first line of the relevant paragraph: “In the absence of oxidative stress, no changes in lipid levels between LSF-treated cells and the untreated control were reported (all p values > 0.05; see Underlying data)”. However, we only had presented figures where significant differences were established using the statistical tests described. Additional notes and suggestions:•On page 3 the authors state that all five major fatty acids in the human retina are long chain polyunsaturated fatty acids (PUFAs). This is incorrect, docosahexaenoic and arachidonic acids are PUFAs, oleic acid is monounsaturated and stearic and palmitic are saturated. Response: We have amended this statement to “There are five major fatty acids in the human retina, namely, docosahexaenoic acid (DHA), arachidonic acid (ACA), stearic acid, oleic acid and palmitic acid. Both DHA and ACA are classified as long chain polyunsaturated fatty acids (LC-PUFAs).” •Figures 2 and 4: The annotations are too small to read. The graphs need to be re-drawn with a larger font. Response: We have enlarged the font in these figures in the revised version of the manuscript. •Figure 3 and 5: The authors state that the y-axis values are automatically generated as arbitrary units by the software used. The authors need to clearly state how the data is being processed. (In this case it seems likely that the data is being log2 transformed.) Is a box and whisker plot appropriate for four data points?Response: All the data has been normalised to the median of the reverse phase peak area response and log2 transformed and number of cells per sample. An auto-scale has also been applied. These statements have been added to the Methods section of the revised manuscript. A box and whisker plot is one of the standard ways to present data generated by Metaboanalyst 2.0. •Page 5: The text here indicates that lipid quantitation was performed using external calibration curves however no concentrations are given throughout the text or underlying data.Response: All the figures were generated using reverse phase peak area response of each lipid species rather than the absolute concentration. We did not have standards for some of the lipid classes and therefore, to be consistent, we have used such responses to make comparison between the untreated and treated groups. We have amended the Methods section by removing the use of calibration curves and added the statement “The data was generated using the reverse phase peak area response of each lipid species rather than the absolute concentrations”. •In the “LCMS Lipid Analysis Data.xlsx” file from the underlying data, five lipids are included in duplicate; PC 31:1, PC 33:3, PC 33:2, PC 34:4 and PC 37:4. Response: We have removed the duplicated columns and reuploaded the underlying data onto the Version 2 of the Harvard Dataverse link. Conclusion: Due to the problems with the data analysis outlined above I’m unable to recommend this paper for indexing. If the authors are able to address these problems however, this paper should be considered as a fresh submission. Response: We believe that our revised manuscript following the inclusion of the recommended edits deserves another peer-review and approval for publication in F1000 Research. The data has been statistically validated and does support the conclusions made in this manuscript." } ] } ]	1	https://f1000research.com/articles/8-1067
https://f1000research.com/articles/9-1197/v1	05 Oct 20	{ "type": "Opinion Article", "title": "Potential therapeutic manipulations of the CXCR3 chemokine axis for the treatment of inflammatory fibrosing diseases", "authors": [ "Morgan K. Groover", "Jillian M. Richmond", "Morgan K. Groover" ], "abstract": "Chemokines play important roles in homeostasis and inflammatory processes. While their roles in leukocyte recruitment are well-appreciated, chemokines play additional roles in the body, including mediating or regulating angiogenesis, tumor metastasis and wound healing. In this opinion article, we focus on the role of CXCR3 and its ligands in fibrotic processes. We emphasize differences of the effects of each ligand, CXCL9, CXCL10 and CXCL11, on fibroblasts in different tissues of the body. We include discussions of differences in signaling pathways that may account for protective or pro-fibrotic effects of each ligand in different experimental models and ex vivo analysis of human tissues. Our goal is to highlight potential reasons why there are disparate findings in different models, and to suggest ways in which this chemokine axis could be manipulated for the treatment of fibrosis.", "keywords": [ "CXCR3", "CXCL9", "CXCL10", "CXCL11", "fibrosis", "fibroblast", "pericyte", "endothelial cell" ], "content": "Introduction: understanding CXCR3’s typical and atypical functions\n\nChemokine receptors are a subgroup of class A G-protein coupled receptors (GPCRs) that are relatively conserved across eukaryotes1. They bind to chemokine ligands, a special class of 8–10kDa chemotactic cytokines, which are classified based on their amino acid structure (e.g. CC, CXC, or CX3C)2. With a few exceptions, most ligand-receptor relationships are promiscuous, meaning that a single chemokine receptor has multiple ligands and a single chemokine can bind to multiple receptors. As of now, there are 18 known chemokine receptors with Gαi-dependent chemotactic activity, and 5 atypical (non-chemotactic, recycling or scavenging) chemokine receptors in humans. Many chemokines are considered inflammatory, as they recruit leukocytes during inflammatory responses. However, there are also homeostatic chemokines that are important for immune cell maturation, tissue development, and angiogenesis. Homeostatic chemokines often exhibit tissue tropism, providing signals for recirculating immune cells, paracrine signals for cells that comprise tissues, and even tumor growth and metastasis3.\n\nCXCR3 is typically considered to be an inflammatory chemokine receptor because it is expressed by leukocytes that migrate towards interferon-induced ligands to sites of tissue inflammation4. However, CXCR3 is also expressed on non-hematopoietic cells including endothelial cells, where it plays roles in promoting or inhibiting angiogenesis, and fibroblasts, in which it mediates wound healing responses.\n\nThere are several examples of diseases where inflammation precedes or is admixed with fibrosis, including infectious diseases (e.g. schistosomiasis, tuberculosis), cancers (e.g. pancreatic cancer, post-irradiation breast cancer) and autoimmune diseases (e.g. hepatitis, pulmonary fibrosis in scleroderma and skin fibrosis in morphea). Hallmarks of inflammatory fibrosis include infiltration of leukocytes; activation of endothelium; fibroblast activation, migration, proliferation and differentiation; production of collagen and other extracellular matrix proteins; and increased collagen bundle thickness and disorganization5. Data from our lab and others have demonstrated that the CXCR3 chemokine axis can mediate protective or pro-fibrotic signals depending upon the context of the involved organs. In this opinion article, we will discuss potential reasons for disparate findings, and provide our opinions about how this system can be targeted therapeutically for the treatment of fibrosis.\n\n\nCXCR3 signaling pathways in leukocytes\n\nCXCR3 has four extracellular domains that bind its ligands (CXCL9, CXCL10, and CXCL11), and four intracellular domains that mediate the receptor’s different functions. The differential involvement of CXCR3 receptor domains in ligand binding and subsequent differences in downstream signaling contribute to the complex nature of this chemokine system, which has been mapped out in leukocytes using mutational constructs and competition binding assays. Like many other GPCRs, CXCR3-mediated chemotaxis is pertussis-toxin sensitive. However, CXCR3 activates several other pathways in addition to Gα subunit proteins, which we will review below.\n\nCXCR3 binding and activation requires ligand interactions with at least one sulfated tyrosine in the N terminus and an interaction with amino acid residue R216 in the second extracellular loop6. The proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation, but not CXCL9 activation. R216 in the second extracellular domain plays no role in CXCL10 or CXCL11 binding or ligand-mediated internalization, but this residue is necessary to activate chemotaxis by all three CXCR3 ligands7. Both the DRY site, which encompasses the R216, and the CXCR3 carboxyl terminus are essential for CXCL9-, CXCL10-, and CXCL11-induced chemotaxis, calcium mobilization, and Erk phosphorylation (Figure 1A).\n\n(A) Major CXCR3 signaling pathways in leukocytes. CXCL9, CXCL10 or CXC11 bind to CXCR3 to mediate chemotaxis and T cell skewing. Different domains facilitate ligand binding, with the N terminus of CXCR3 facilitating binding of CXCL10 and CXC11. R216 in the second extracellular loop (green star) is required for chemotactic responses for all 3 ligands. All three ligands can induce calcium flux, pERK, and pAKT, though CXCL9/10 require Gαi2 for pERK and β-arrestin2 for pAkt. CXCL11 can activate PLC and PI3K/AKT to mediate migration independent of Gαi. Internalization of CXCR3 induced by CXCL9/10 requires the C terminus, whereas CXC11 requires the 3rd intracellular loop. CXCL9/10 can activate STAT1/5 to enforce Tbet/RORγT expression, whereas CXCL11 activates STAT3/6 to enforce GATA3 expression. (B) CXCR3-dependent and independent signaling pathways in endothelial cells. CXCL10 activates cAMP, PKA and MEK in dermal endothelial cells to inhibit m-calpain and dampen angiogenesis. In cardiac microvascular endothelial cells, CXCL10 activates the p38/FAK pathway to induce migration, but not proliferation. CXCL10 also exerts effects on endothelial cells independently of CXCR3, but in a manner that requires GAG binding. (C) CXCR3 signaling in pericytes. Pericytes activate Src, Ras/ERK and PI3K/AKT pathways downstream of CXCR3, which mediate chemotactic responses. Kidney pericytes exhibit increased proliferation downstream of CXCL9/10, which is ERK-dependent (inhibited by PD98059). Interferons inhibit proliferation of pancreatic stellate cells via STAT1, though it is unclear whether this response is via CXCR3 ligands. (D) Fibroblast responses to CXCR3 ligands. Intestinal myofibroblasts exhibit calcium flux and phosphorylation of PKB, ERK, p90RSK induced by all three ligands. All three ligands induce actin polymerization in a Rho-associated coiled coil-forming protein kinase (ROCK)-dependent manner that is independent of PI3K and Gαi. Signaling in skin dermal fibroblasts has not been fully mapped, though CXCR3 deficiency leads to hypertrophic and hypercellular scarring in mice via increased extracellular matrix proteins, including tenascin C, fibronectin, type I & III collagen, MMP9 and decorin. Color key: CXCL9 = red; CXCL10 = blue; CXCL11 = yellow; CXCL9/10 = purple; CXCL9/10/11 = green.\n\nCXCR3 ligands selectively activate different receptor internalization pathways via β-arrestin and Gαi family members. Differences in signaling pathway activation by each ligand is called biased agonism8,9. CXCL10-induced receptor internalization relies on the CXCR3 carboxyl terminus, dynamin and β-arrestin110. CXCL11 is the most potent inducer of CXCR3 internalization11, and dominant negative dynamin and β arrestin 1 are unable to impede internalization10. The third intracytoplasmic loop is required for maximal CXCL11-induced internalization (Figure 1A).\n\nIn T cells, Gαi2 is required for mediating CXCR3 ligand signaling, whereas Gαi3 limits activation of this signaling pathway12. Western blotting of peripheral blood leukocytes stimulated with CXCR3 ligands demonstrated that CXCL11 and, to a lesser extent, CXCL9/10 induce dose- and time-dependent phosphorylation of p44/42 MAPK (ERK) and Akt that is prevented by pertussis toxin treatment (inhibition of Gα subunit binding)13. However, inhibition of MEK/ERK (U0126 or PD98059) does not prevent CXCL11-mediated chemotaxis, whereas PLC inhibition (U73122), PI3K (wortmannin) or, to a lesser extent, AKT inhibition (LY294002) does abrogate or reduce human T cell migration, respectively. Akt activation in human T cells was recently reported to be dependent upon β-arrestin214.\n\nThere is increasing evidence that chemokine receptors can mediate JAK/STAT signaling, which has typically been attributed to common gamma chain cytokine signaling15. While JAK activation downstream of CXCR3 has not yet been studied, STAT activation in response to incubation with CXCL9/10/11 has been assessed in T cell cultures: addition of recombinant CXCL9/10 activates STAT1/STAT5 to promote Th1 and/or Th17 differentiation via Tbet/RORγT expression, whereas CXCL11 activates STAT3/STAT6 via GATA3 expression to augment regulatory function16.\n\n\nCXCR3 signaling pathways in vascular endothelial cells, smooth muscle cells, pericytes and fibroblasts\n\nCXCR3 is also expressed by some non-hematopoietic cells, including endothelial cells, smooth muscle cells and fibroblast subsets. In endothelial cells, CXCR3 ligands mediate pro- or anti-angiogenic signals depending upon the model and tissue of origin. CXCL10 inhibits VEGF-induced dermal endothelial cell motility and tube formation in vitro via cAMP, PKA and MEK inhibition of m-calpain; this likely serves as a way to inhibit angiogenesis late in the wound healing process17. CXCL10 exhibits angiostatic properties in non-small cell lung cancer and idiopathic pulmonary fibrosis18,19. CXCL10 is also able to induce angiostatic effects by binding glycosaminoglycans (GAGs) independently of CXCR320. However, CXCL10 induces migration, but not proliferation, of rat cardiac microvascular endothelial cells via the p38/FAK pathway21 and rat and mouse liver endothelial cell progenitors through an unknown pathway22 (Figure 1B).\n\nPericytes are contractile cells on capillaries and post-capillary venules in tissues that play integral roles in tissue healing and remodeling23. Examples include hepatic stellate cells in the liver, glomerular mesangial cells in the kidney and pancreatic stellate cells. Pericytes express CXCR3, and activate the Src, Ras/ERK, and PI3K/Akt pathways downstream of CXCR3 ligand binding24. Inhibitor studies indicate that Src and subsequent Ras/ERK activation are required for chemotaxis of hepatic pericytes, which is in direct contrast to observations in T cells. PI3K/Akt also plays an important role in hepatic pericyte migration as evidenced by abrogation of CXCL10 migration in the presence of wortmannin or LY294002. In addition to chemotaxis, kidney pericytes exhibit increased proliferation downstream of CXCL9/10, which is ERK-dependent (inhibited by PD98059). Unlike hepatic pericytes, kidney pericytes exhibit a second wave of ERK phosphorylation following incubation with CXCL10. CXCR3 signaling in pancreatic stellate cells has not been as extensively mapped, but they respond to PDGF by activating Src-JAK2-STAT325. Interferons (IFNs), which drive expression of CXCR3 ligands, inhibit proliferation of pancreatic stellate cells via STAT126 (Figure 1C).\n\nCXCR3 plays a homeostatic role in wound healing and re-epithelialization responses by fibroblasts27–29. CXCR3 deficiency leads to hypertrophic and hypercellular scarring in mice that have experienced skin trauma30,31. While CXCR3-mediated signaling in skin fibroblasts has not been well-characterized, the consequence of loss of signaling during wound healing includes increases in extracellular matrix proteins including tenascin C, fibronectin, type I & III collagen, MMP9 and decorin 180 days post-wounding compared to WT controls31.\n\nStudies of CXCR3 signaling downstream of CXCL9 and CXCL10 in intestinal myofibroblasts have shown modest differences in signaling, including CXCL9/10-induced calcium flux at 10min versus 8min for CXCL11, and prolonged phosphorylation of PKB, ERK, p90RSK induced by all 3 ligands as compared to shorter phosphorylation time in peripheral blood leukocytes (e.g. 2-20min versus 1-2min)32. All three ligands induce actin polymerization in a Rho-associated coiled coil-forming protein kinase (ROCK)-dependent manner that is independent of PI3K and Gαi (Figure 1D). Further detailed signaling pathway analyses for CXCL9/10/11 signaling are warranted in non-hematopoietic cells from different organs.\n\n\nPost-translational modifications, proteolytic processing and potential alternate receptors for the CXCR3 chemokine axis\n\nPost-translational modifications of the CXCR3 chemokine axis modulates the function and signaling ability of the ligands and their receptor. CXCL10/11 have heparin binding sites that allow it to be presented on endothelium33. CXCL10 presentation by the endothelium requires oligomerization34. CXCL10/11 may be citrullinated by peptidylarginine deiminase, which inhibits their ability to induce chemotaxis and calcium flux and reduces their ability to bind heparin35. CXCR3 itself requires tyrosine sulfation to bind to its ligands and mediate chemotaxis6.\n\nCXCL9/10/11 are cleaved/truncated by CD26, and CXCL11 is cleaved by CD1336,37. The CD26 truncations of CXCR3 ligands retain angiostatic activity while losing CXCR3-mediated signaling38. The C’ terminus of CXCL9 can inhibit neutrophil migration via competition with CXCL8-mediated binding to heparin, heparan sulfate, and cellular GAGs, which normally facilitate adhesion to vessels and subsequent transmigration39,40. CD13 is expressed by endothelial and epithelial cells as well as fibroblasts in angiogenic tissue, but not normal tissue41. Truncation of just the first two amino acids in CXCL11 by CD13 abrogates Akt and ERK phosphorylation and greatly reduces calcium flux to prevent migration of CXCR3-transfected CHO cells36. Truncation of the first six amino acids still retains angiostatic activity as assessed by scratch assay of endothelial cell cultures.\n\nThere are two other isoforms of CXCR3: CXCR3-B which binds to CXCL4 and mediates angiostatic effects in cultured human endothelial cells42; and CXCR3-alt which binds CXCL1143. Of note, C57BL/6 (B6) mice do not express CXCR3-B20. The roles of CXCR3-B and CXCR3-alt in fibrosis have not been studied. CXCR3 can also crosstalk with CXCR4 and CXCR7 via CXCL11 and CXCL1244. CXCR4 mediates profibrotic effects in the liver, while CXCR7 mediates more homeostatic regenerative responses45. CXCL9 can induce heterologous desensitization of CXCR4 to its ligand CXCL1246. Notably, autoantibodies against CXCR3 and CXCR4 correlate with increased lung and skin disease severity in scleroderma patients, though it is unclear exactly how these impact signaling47,48. CXCL11 binds to CXCR7, which is expressed on activated endothelial cells, tumor cell lines and fetal liver cells49. CXCL11 ligation by CXCR7, which has an affinity of 2-5nM, does not induce calcium flux or migration; rather it promotes survival and adhesion. CXCR7 has been proposed to be a scavenger receptor for CXCL1150. CXCR7 can attenuate TGFβ signaling in the lung, though the role of CXCL11 in this process has not been studied51,52.\n\n\nProfibrotic roles of CXCR3 ligands\n\nCXCR3 and its ligands are reported to promote fibrosis in certain disease models and organs. An important caveat to bear in mind when assessing B6 mouse models is that CXCL11 is not expressed in this strain due to a null mutation. However, CXCL9 and CXCL10 knockout mice were generated using 129 oocytes and were backcrossed to B6. Therefore, WT B6 mice express CXCL9 and CXCL10, CXCL9-/- mice express CXCL10 and CXCL11, and CXCL10-/- mice express CXCL9 and CXCL11. This means that while CXCL11 cannot be directly assessed in B6 models, insights about its function can be gleaned by comparing CXCL9-/-, CXCL10-/- and WT B6 mice.\n\nThe nephrotoxic serum nephritis model of inflammatory kidney disease, which exhibits tubulointerstitial fibrosis, is dependent on CXCR3 and CXCL9, but not CXCL10, as determined by histopathology and loss of renal function53. CXCR3-/- and CXCL9-/- mice had fewer intrarenal activated T cells and macrophages, as well as fewer IgG glomerular deposits and antigen-specific IgG in serum. These data suggest that CXCR3 and CXCL9 initiate nephritis through cell-mediated events, which ultimately promote tubulointerstitial fibrosis. CXCL10-/- animals developed kidney disease similar to WT controls, indicating that any potential antifibrotic role of CXCL11 in the kidney is potentially nullified by profibrotic effects of CXCL9. Similarly, any potential profibrotic role of CXCL11 in CXCL9-/- mice may be nullified by antifibrotic effects of CXCL10. However, a Balb/c mouse model of unilateral ureteral obstruction-induced renal tubulointerstitial fibrosis was exacerbated by JAK inhibition, and STAT3 played a protective role54. Several factors may contribute to the disparate findings between these models, namely whether the process is immune-mediated or obstructive nephropathy, which other signals are being disrupted by JAK inhibition, and whether all three CXCR3 ligands are present to balance pro- versus anti-fibrotic signaling (Table 1).\n\nTable 1 Complete References:\n\n1. Lin C-F, Su C-J, Liu J-H, Chen S-T, Huang H-L, Pan S-L. Potential Effects of CXCL9 and CCL20 on Cardiac Fibrosis in Patients with Myocardial Infarction and Isoproterenol-Treated Rats. J Clin Med Res [Internet]. 2019 May 11;8(5). Available from: http://dx.doi.org/10.3390/jcm8050659\n\n2. Faé KC, Palacios SA, Nogueira LG, Oshiro SE, Demarchi LMF, Bilate AMB, et al. CXCL9/Mig mediates T cells recruitment to valvular tissue lesions of chronic rheumatic heart disease patients. Inflammation [Internet]. 2013 Aug;36(4):800–11. Available from: http://dx.doi.org/10.1007/s10753-013-9606-2\n\n3. Nogueira LG, Santos RHB, Ianni BM, Fiorelli AI, Mairena EC, Benvenuti LA, et al. Myocardial chemokine expression and intensity of myocarditis in Chagas cardiomyopathy are controlled by polymorphisms in CXCL9 and CXCL10. PLoS Negl Trop Dis [Internet]. 2012 Oct 25;6(10):e1867. Available from: http://dx.doi.org/10.1371/journal.pntd.0001867\n\n4. Menke J, Zeller GC, Kikawada E, Means TK, Huang XR, Lan HY, et al. CXCL9, but not CXCL10, promotes CXCR3-dependent immune-mediated kidney disease. J Am Soc Nephrol [Internet]. 2008 Jun;19(6):1177–89. Available from: http://dx.doi.org/10.1681/ASN.2007111179\n\n5. Sahin H, Borkham-Kamphorst E, Kuppe C, Zaldivar MM, Grouls C, Al-samman M, et al. Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice. Hepatology [Internet]. 2012 May 19;55(5):1610–9. Available from: http://doi.wiley.com/10.1002/hep.25545\n\n6. Pineda-Tenor D, Berenguer J, García-Álvarez M, Guzmán-Fulgencio M, Carrero A, Aldámiz-Echevarria T, et al. Single Nucleotide Polymorphisms of CXCL9-11 Chemokines Are Associated With Liver Fibrosis in HIV/HCV-Coinfected Patients. JAIDS Journal of Acquired Immune Deficiency Syndromes [Internet]. 2015 Apr 1 [cited 2020 Sep 23];68(4):386. Available from: https://journals.lww.com/jaids/fulltext/2015/04010/Single_Nucleotide_Polymorphisms_of_CXCL9_11.3.aspx?casa_token=2kMFCv_y5KcAAAAA:HJXjuc13C4IdQ0jXRz84X8bBYfKwrt3RWyPB1FpyLCOBTq2l4yTRsbYdS8OG9T0O0-hh-nBzVTb-_you33IXjJo\n\n7. Jiménez-Sousa MÁ, Gómez-Moreno AZ, Pineda-Tenor D, Medrano LM, Sánchez-Ruano JJ, Fernández-Rodríguez A, et al. CXCL9-11 polymorphisms are associated with liver fibrosis in patients with chronic hepatitis C: a cross-sectional study. Clin Transl Med [Internet]. 2017 Jul 28;6(1):26. Available from: https://doi.org/10.1186/s40169-017-0156-3\n\n8. Wasmuth HE, Lammert F, Zaldivar MM, Weiskirchen R, Hellerbrand C, Scholten D, et al. Antifibrotic effects of CXCL9 and its receptor CXCR3 in livers of mice and humans. Gastroenterology [Internet]. 2009 Jul;137(1):309–19, 319.e1–3. Available from: http://dx.doi.org/10.1053/j.gastro.2009.03.053\n\n9. Berres M-L, Asmacher S, Lehmann J, Jansen C, Görtzen J, Klein S, et al. CXCL9 is a prognostic marker in patients with liver cirrhosis receiving transjugular intrahepatic portosystemic shunt. J Hepatol [Internet]. 2015 Feb;62(2):332–9. Available from: http://dx.doi.org/10.1016/j.jhep.2014.09.032\n\n10. Shen J, Gao J, Chen C, Lu H, Hu G, Shen J, et al. Antifibrotic role of chemokine CXCL9 in experimental chronic pancreatitis induced by trinitrobenzene sulfonic acid in rats. Cytokine [Internet]. 2013 Oct;64(1):382–94. Available from: http://dx.doi.org/10.1016/j.cyto.2013.05.012\n\n11. O’Brien JC, Rainwater YB, Malviya N, Cyrus N, Auer-Hackenberg L, Hynan LS, et al. Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea. J Invest Dermatol [Internet]. 2017 Aug;137(8):1663–70. Available from: http://dx.doi.org/10.1016/j.jid.2017.04.008\n\n12. Mertens JS, de Jong EMGJ, Pandit A, Seyger MMB, Hoppenreijs EPAH, Thurlings RM, et al. Regarding “Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea.” J Invest Dermatol [Internet]. ncbi.nlm.nih.gov; 2018 May;138(5):1212–5. Available from: http://dx.doi.org/10.1016/j.jid.2017.11.032\n\n13. Hasegawa M, Fujimoto M, Matsushita T, Hamaguchi Y, Takehara K, Sato S. Serum chemokine and cytokine levels as indicators of disease activity in patients with systemic sclerosis. Clin Rheumatol [Internet]. 2011 Feb;30(2):231–7. Available from: http://dx.doi.org/10.1007/s10067-010-1610-4\n\n14. Rabquer BJ, Tsou P-S, Hou Y, Thirunavukkarasu E, Haines GK 3rd, Impens AJ, et al. Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis. Arthritis Res Ther [Internet]. 2011 Feb 8;13(1):R18. Available from: http://dx.doi.org/10.1186/ar3242\n\n15. Liu X, Mayes MD, Tan FK, Wu M, Reveille JD, Harper BE, et al. Correlation of interferon-inducible chemokine plasma levels with disease severity in systemic sclerosis. Arthritis & Rheumatism [Internet]. 2013;65(1):226–35. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/art.37742\n\n16. Hintermann E, Bayer M, Pfeilschifter JM, Luster AD, Christen U. CXCL10 promotes liver fibrosis by prevention of NK cell mediated hepatic stellate cell inactivation. J Autoimmun [Internet]. 2010 Dec;35(4):424–35. Available from: http://dx.doi.org/10.1016/j.jaut.2010.09.003\n\n17. Tager AM, Kradin RL, LaCamera P, Bercury SD, Campanella GSV, Leary CP, et al. Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10. Am J Respir Cell Mol Biol [Internet]. 2004 Oct;31(4):395–404. Available from: http://dx.doi.org/10.1165/rcmb.2004-0175OC\n\n18. Jiang D, Liang J, Campanella GS, Guo R, Yu S, Xie T, et al. Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4. J Clin Invest [Internet]. 2010 Jun;120(6):2049–57. Available from: http://dx.doi.org/10.1172/JCI38644\n\n19. Cardarelli S, Facco M, Fittà C, Del Rosso A. CXCL11 in bronchoalveolar lavage fluid and pulmonary function decline in systemic sclerosis. Clinical and [Internet]. 2012; Available from: https://www.academia.edu/download/45798612/CXCL11_in_bronchoalveolar_lavage_fluid_a20160520-15769-1pwsbty.pdf\n\n20. Burdick MD, Murray LA, Keane MP, Xue YY, Zisman DA, Belperio JA, et al. CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling. Am J Respir Crit Care Med [Internet]. 2005 Feb 1;171(3):261–8. Available from: http://dx.doi.org/10.1164/rccm.200409-1164OC\n\n21. Sulpice E, Contreres J-O, Lacour J, Bryckaert M, Tobelem G. Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms. Eur J Biochem [Internet]. 2004 Aug;271(16):3310–8. Available from: http://dx.doi.org/10.1111/j.1432-1033.2004.04263.x\n\nMorphea, or localized scleroderma, is an inflammatory fibrosing disease of the dermis and underlying tissue. Several studies have identified CXCR3 ligands as positively correlating with disease severity and activity in patients55–57. Systemic sclerosis, or scleroderma, also exhibits upregulation of CXCR3 ligands that correlates with disease severity58. Preliminary studies from our laboratory support a pro-fibrotic role of CXCL9 in the skin: CXCL9-/- mice are protected from bleomycin-induced skin fibrosis, and in vitro treatment of mouse and human fibroblasts with CXCL9 induces transcription of collagen 1a1 (col1a1); these data are available on a preprint server and are currently undergoing peer review59.\n\nCXCL10 has pro-fibrotic effects in the liver, where it prevents NK cells from inactivating hepatic stellate cells60. CXCL10-/- mice and WT mice treated with anti-CXCL10 antibody are protected from carbon tetrachloride-induced liver fibrosis. Hepatic stellate cells upregulate CXCR3 in response to carbon tetrachloride, and CXCL10 induces their migration but not proliferation. CXCL10 also mediates T and B cell aggregates in lymphoid tissue, which are essentially absent in CXCL10-/- mice (Table 1).\n\n\nAntifibrotic roles of CXCR3 ligands\n\nWhile CXCL9 has pro-fibrotic effects in renal and skin tissue, CXCL9 has direct angiostatic and antifibrotic effects in experimental models of pancreas and liver fibrosis. In the trinitrobenzene sulfonic acid (TNBS) induced-pancreatitis rat model, administration of anti-CXCL9 antibody worsened fibrosis, whereas administration of recombinant CXCL9 improved fibrosis, as assessed by trichrome staining and hydroxyproline assay61 (Table 1). In vitro stimulation of pancreatic stellate cells with CXCL9 downregulated TGFβ1 and col1a1 production by confocal microscopy. Of note, antibody and recombinant CXCL9 were administered subcutaneously (s.c.) to rats in this model. We hypothesize that this route of administration may have pulled inflammatory infiltrates away from the gastrointestinal (GI) tract and towards the skin, considering there was 1.5ng/mL CXCL9 in serum and approximately 30µg was administered s.c. daily (assuming average weight of 300g/rat at a dose of 100 μg/kg body weight).\n\nIn the carbon tetrachloride-induced liver fibrosis model, CXCR3-/- mice exhibited augmented liver damage at 24h62. Follow-up studies from the same laboratory used mice treated exogenously with CXCL9, which reduced the severity of liver fibrosis as assessed by Sirius red staining, hydroxyproline assay, and α-SMA expression63 (Table 1). In vivo CXCL9 treatment also inhibited angiogenesis as assessed by CD31 staining and ultrasound visualization of liver perfusion. However, CXCL9 treatment did not impact the number of Th1-polarized, IFN-γ-positive cells in the liver amongst treatment groups. Treatment of endothelial cells in vitro with CXCL9 was able to inhibit VEGF-mediated proliferation and migration via PLCγ, JNK and ERK. In vitro treatment of hepatic stellate cells reduced TGFβ and col1a1 by protein and RNA64.\n\nWhile CXCL10 has profibrotic effects in the liver, CXCL10 limits lung fibrosis in the murine model of bleomycin-induced pulmonary fibrosis (Table 1). CXCR3-/- and CXCL10-/- mice display exaggerated pulmonary fibrosis after bleomycin administration, and transgenic mice overexpressing CXCL10 are protected from bleomycin-induced mortality65,66. Bleomycin did not alter the T cell cytokine milieu in CXCL10-/- mice, weakening the support for the idea that CXCL10 might limit fibrosis by skewing T cell polarization to the Th1 phenotype as demonstrated in hepatitis models. CXCL10 also did not decrease lung tissue-derived angiogenic activity and von Willebrand Factor expression after bleomycin delivery, despite that angiogenesis is considered a rate-limiting step in the development of pulmonary fibrosis. CXCR3 mRNA, but not protein, was detected in lung fibroblasts. Rather, direct interaction of the heparin-binding domain of CXCL10 and syndecan-4 on the lung interstitial compartment inhibits fibroblast recruitment, TGFβ signaling and subsequent fibrosis67,68. Similar findings were reported in myocardium, which required CXCL10 fibroblast responses through proteoglycans69, and urethral fibrosis, in which CXCL10 signaling interfered with profibrotic TGFβ signaling70.\n\nSimilar to CXCL10, CXCL11 attenuates lung fibrosis in the bleomycin mouse model and inhibits angiogenesis in the corneal micropocket assay71 (Table 1). A double-blind, placebo controlled study of 330 idiopathic pulmonary patients treated with subcutaneous IFN-γ 1b treatment exhibited increased CXCL11 in bronchoalveolar lavage fluid and plasma, with concomitant decreased elastin72. The pro- and anti-fibrotic roles of the CXCR3 ligands in different organs are summarized in Table 2.\n\n\nPotential therapeutic manipulations of CXCR3 for the treatment of fibrosis\n\nTo select how to manipulate CXCR3 and/or its ligands for the treatment of fibrosis, it is our opinion that the suspected cell-of-origin in the fibrotic response and the level of angiogenesis during fibrogenesis need to be assessed. Based on the evidence discussed above, we hypothesize that fibrosing disorders primarily mediated by pericyte-type cells that require ERK signaling and exhibit more angiogenesis as a disease feature would be more dependent upon CXCL10, and fibrosing disorders primarily mediated by fibroblast or myofibroblast-type cells that require AKT and JAK signaling and exhibit less vascular involvement would be more dependent upon CXCL9. For example, hepatic fibrosis has prominent vascular changes and is driven by hepatic stellate cells and CXCL10, whereas morphea has a low incidence of vascular changes and is driven by fibroblasts/myofibroblasts and CXCL9. GI organs, in which fibrosis is driven by pericytes, also generally seem to use CXCL9 for protective responses, whereas lung and skin, in which fibrosis is driven by fibroblast subsets, use CXCL10 for protective responses. Nuances in the signaling pathways, the relative chemokine responsiveness, as well as potential coreceptors, will need to be addressed in future studies. Technologies such as single cell RNA sequencing and proteomics may ultimately help resolve the heterogeneity of chemokine receptor and coreceptor expression, as well as preferential signaling pathway usage.\n\nThe first potential class of small molecules that could be used to disrupt CXCR3-mediated inflammatory fibrosis are JAK inhibitors. We and others have shown that JAK inhibitors prevent fibrosis in mice73–75, and demonstrated efficacy in our case studies of human morphea patients who were recalcitrant to standard therapies73,76. In our study of intradermal bleomycin injection in mice and human morphea tissue, we observed p-STAT1 and p-STAT3 activation in both immune infiltrates and cells with fibroblast morphology73. Notably, STAT1, STAT3 and STAT5 have predicted binding sites in the collagen 1a1 (col1a1) promoter and enhancer regions (GeneCards), which may account for our observation that JAK inhibitors were able to suppress col1a1 transcription by human and mouse fibroblasts in vitro73. We also noted that the JAK 1/2 inhibitor ruxolitinib yielded a slightly better p value than the JAK 3>>1>2 inhibitor tofacitinib for inhibition of dermal thickening in the intradermal bleomycin mouse model. These data are in agreement with previously published studies examining JAK2 as a driver of fibrosis in scleroderma fibroblasts and a bleomycin mouse model74. Zhang et al demonstrated that following long-term selective inhibition of JAK2, JAK2 may be transphosphorylated by JAK1 to mediate fibrosis77, supporting the use of a combination JAK1/2 inhibitor for treatment of fibrosis. It is interesting to note that ruxolitinib was originally FDA approved for myelofibrosis78, and patients receiving ruxolitinib therapy often resolve fibrosis79. While this is encouraging for potential repurposing of ruxolitinib for other fibrosing diseases, we would caution that careful tapering and monitoring is needed to prevent potential rebound effects80,81. Cessation of ruxolitinib can cause hyperphosphorylation of JAK2, increasing inflammation and subsequent fibrosis82. Selecting a JAK1/2 inhibitor with a longer half-life, such as baricitinib83, might be a safer option for patients who are tapering.\n\nThe second potential class of therapeutics would be agonist peptides to mimic the antifibrotic role of CXCL10 for lung fibrosis. As suggested by Tager and Jiang et al, maintaining heparin binding but excluding CXCR3 binding would mitigate potential toxicities related to T cell recruitment65,67. CXCL10-based therapeutics might also prove useful for improving lung fibrosis and function in patients recovering from infectious lung disease, particularly Sars-CoV2 infection/COVID-19 disease84. Smith et al recently reported biased agonists of CXCR3 that can differentially mediate inflammation and migration of immune cells which they examined in the context of contact hypersensitivity in skin14, providing a basis for the feasibility of this approach.\n\nThe third potential class of therapeutics would be agents that inhibit the pro-fibrotic signaling events mediated by CXCR3 ligands, such as CXCL9 in Th1/IFNγ-driven kidney disease or morphea. These approaches could include anti-CXCL9 blocking/neutralizing antibodies, CXCL9 siRNA, or antagonistic peptide ligands. Of note, antibody neutralization of CXCL10 for treatment of hepatic fibrosis may be challenging, as CXCL10 antibodies neutralize the free form and not endothelial-bound chemokine85. Similar challenges may arise when attempting to neutralize CXCL9 with antibody, as would anti-drug antibody responses.\n\nA fourth class of therapeutics could leverage the cell- or organ-specific context of chemokine expression. For example, stimulating γδ T-cells to produce CXCL10 in the lung could have therapeutic benefits in pulmonary fibrotic disease. Inhibiting macrophage production of CXCL9 in the skin could prevent collagen deposition in morphea. Drawing immune infiltrates away from the pancreas and towards the skin could reset the fibrotic process, as in the TNBS-induced rat model. Agents are in development to target specific cell types, such as antibody-drug conjugates86 some with cleavable linkers87, bispecific antibodies88,89 and nanoparticles90, which can be preferentially phagocytosed by antigen presenting cells of the immune system. These could be leveraged to achieve the aforementioned goals of stimulating CXCL10 or inhibiting CXCL9 production by key cell types. Different drug delivery routes and systems may also help accomplish the goal of drawing immune cells away from the pancreas or other organs, with cutaneous administration via creams, injections or microneedle patches91 helping to achieve a high local concentration.\n\nA fifth class of therapeutics could leverage existing enzymatic cleavage of CXCR3 ligands. Recombinant peptides lacking amino-terminal amino acids can exert angiostatic effects, while inhibiting CXCR3-mediated migration. Administration of bioactive CD26 and/or CD13, or inhibitors of these enzymes, may modulate fibrotic processes in specific organs or diseases. CD26 inhibitors have been reported to reduce or prevent fibrosis in models of myocardial fibrosis, lung fibrosis and kidney fibrosis92–94, and a CD13 inhibitor improved fibrosis in a mouse model of silica-induced lung fibrosis95.\n\nLast, combination therapies targeting both prevention of inflammation and fibrosis in addition to promoting tissue remodeling will likely provide the best therapeutic outcome for fibrosis patients96. Tissue remodeling will ultimately allow for breakdown of fibrotic plaques and better disease outcomes, which could be achieved through agonists or inducers of matrix metalloproteinases (MMPs) or antagonists of tissue inhibitors of MMPs (TIMPs).\n\n\nConclusion\n\nThe differential impact of CXCR3 and its ligands on tissues depends on disparate signaling pathways involving multiple cell types and potential coreceptors; the nuances of which should be addressed in future research involving single cell RNA sequencing and proteomics. As we have examined the known fibrotic and antifibrotic roles of CXCR3 and its ligands, we suggest that future therapeutic options should be centered around the suspected cell-of-origin in the fibrotic response, tissue-specific signaling factors and the degree of angiogenesis is during fibrosis. Likely, a combination of these therapies will have the best potential to ameliorate symptoms of patients with fibrosing diseases.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe thank M. Tuzova and C. Garelli for insightful comments.\n\n\nReferences\n\nProudfoot AEI: Chemokine receptors: multifaceted therapeutic targets. Nat Rev Immunol. 2002; 2(2): 106–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffith JW, Sokol CL, Luster AD: Chemokines and chemokine receptors: positioning cells for host defense and immunity. Annu Rev Immunol. 2014; 32: 659–702. PubMed Abstract \| Publisher Full Text\n\nZlotnik A, Burkhardt AM, Homey B: Homeostatic chemokine receptors and organ-specific metastasis. Nat Rev Immunol. 2011; 11(9): 597–606. PubMed Abstract \| Publisher Full Text\n\nGroom JR, Luster AD: CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol Cell Biol. 2011; 89(2): 207–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWynn TA: Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 2007; 117(3): 524–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColvin RA, Campanella GSV, Manice LA, et al.: CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis. Mol Cell Biol. 2006; 26(15): 5838–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXanthou G, Williams TJ, Pease JE: Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation. Eur J Immunol. 2003; 33(10): 2927–36. PubMed Abstract \| Publisher Full Text\n\nZidar DA, Violin JD, Whalen EJ, et al.: Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands. Proc Natl Acad Sci U S A. 2009; 106(24): 9649–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajagopal S, Bassoni DL, Campbell JJ, et al.: Biased agonism as a mechanism for differential signaling by chemokine receptors. J Biol Chem. 2013; 288(49): 35039–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColvin RA, Campanella GSV, Sun J, et al.: Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11 function. J Biol Chem. 2004; 279(29): 30219–27. PubMed Abstract \| Publisher Full Text\n\nSauty A, Colvin RA, Wagner L, et al.: CXCR3 internalization following T cell-endothelial cell contact: preferential role of IFN-inducible T cell alpha chemoattractant (CXCL11). J Immunol. 2001; 167(12): 7084–93. PubMed Abstract \| Publisher Full Text\n\nThompson BD, Jin Y, Wu KH, et al.: Inhibition of G alpha i2 activation by G alpha i3 in CXCR3-mediated signaling. J Biol Chem. 2007; 282(13): 9547–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmit MJ, Verdijk P, van der Raaij-Helmer EMH: CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase. Blood. 2003; 102(6): 1959–65. PubMed Abstract \| Publisher Full Text\n\nSmith JS, Nicholson LT, Suwanpradid J, et al.: Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation. Sci Signal. 2018; 11(555): eaaq1075. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoriano SF, Serrano A, Hernanz-Falcó n P, et al.: Chemokines integrate JAK/STAT and G-protein pathways during chemotaxis and calcium flux responses. Eur J Immunol. 2003; 33(5): 1328–33. PubMed Abstract \| Publisher Full Text\n\nZohar Y, Wildbaum G, Novak R, et al.: CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis. J Clin Invest. 2014; 124(5): 2009–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBodnar RJ, Yates CC, Wells A: IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain. Circ Res. 2006; 98(5): 617–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArenberg DA, Kunkel SL, Polverini PJ, et al.: Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J Exp Med. 1996; 184(3): 981–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeane MP, Arenberg DA, Lynch JP 3rd, et al.: The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol. 1997; 159(3): 1437–43. PubMed Abstract\n\nCampanella GSV, Colvin RA, Luster AD: CXCL10 can inhibit endothelial cell proliferation independently of CXCR3. PLoS One. 2010; 5(9): e12700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia JB, Mao CZ, Chen ZY, et al.: The CXCL10/CXCR3 axis promotes cardiac microvascular endothelial cell migration via the p38/FAK pathway in a proliferation-independent manner. Exp Mol Pathol. 2016; 100(2): 257–65. PubMed Abstract \| Publisher Full Text\n\nLing CC, Ng KTP, Shao Y, et al.: Post-transplant endothelial progenitor cell mobilization via CXCL10/CXCR3 signaling promotes liver tumor growth. J Hepatol. 2014; 60(1): 103–9. PubMed Abstract \| Publisher Full Text\n\nAttwell D, Mishra A, Hall CN, et al.: What is a pericyte? J Cereb Blood Flow Metab. 2016; 36(2): 451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonacchi A, Romagnani P, Romanelli RG: Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J Biol Chem. 2001; 276(13): 9945–54. PubMed Abstract \| Publisher Full Text\n\nMasamune A, Satoh M, Kikuta K, et al.: Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells. World J Gastroenterol. 2005; 11(22): 3385–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaumert JT, Sparmann G, Emmrich J, et al.: Inhibitory effects of interferons on pancreatic stellate cell activation. World J Gastroenterol. 2006; 12(6): 896–901. PubMed Abstract \| Free Full Text\n\nKroeze KL, Boink MA, Sampat-Sardjoepersad SC, et al.: Autocrine regulation of re-epithelialization after wounding by chemokine receptors CCR1, CCR10, CXCR1, CXCR2, and CXCR3. J Invest Dermatol. 2012; 132(1): 216–25. PubMed Abstract \| Publisher Full Text\n\nYates CC, Whaley D, Hooda S, et al.: Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3. Wound Repair Regen. 2009; 17(1): 34–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuen AC, Wells A: The Beginning of the End: CXCR3 Signaling in Late-Stage Wound Healing. Adv Wound Care (New Rochelle). 2012; 1(6): 244–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates CC, Whaley D, Kulasekeran P, et al.: Delayed and deficient dermal maturation in mice lacking the CXCR3 ELR-negative CXC chemokine receptor. Am J Pathol. 2007; 171(2): 484–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates CC, Krishna P, Whaley D, et al.: Lack of CXC chemokine receptor 3 signaling leads to hypertrophic and hypercellular scarring. Am J Pathol. 2010; 176(4): 1743–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKouroumalis A, Nibbs RJ, Aptel H, et al.: The chemokines CXCL9, CXCL10, and CXCL11 differentially stimulate G alpha i-independent signaling and actin responses in human intestinal myofibroblasts. J Immunol. 2005 [cited 2019 Feb 15]; 175(8): 5403–11. PubMed Abstract \| Publisher Full Text\n\nCampanella GSV, Lee EMJ, Sun J, et al.: CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). J Biol Chem. 2003; 278(19): 17066–74. PubMed Abstract \| Publisher Full Text\n\nCampanella GSV, Grimm J, Manice LA, et al.: Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity. J Immunol. 2006; 177(10): 6991–8. PubMed Abstract \| Publisher Full Text\n\nLoos T, Mortier A, Gouwy M, et al.: Citrullination of CXCL10 and CXCL11 by peptidylarginine deiminase: a naturally occurring posttranslational modification of chemokines and new dimension of immunoregulation. Blood. 2008; 112(7): 2648–56. PubMed Abstract \| Publisher Full Text\n\nProost P, Mortier A, Loos T: Proteolytic processing of CXCL11 by CD13/aminopeptidase N impairs CXCR3 and CXCR7 binding and signaling and reduces lymphocyte and endothelial cell migration. Blood. 2007; 110(1): 37–44. PubMed Abstract \| Publisher Full Text\n\nLambeir AM, Proost P, Durinx C, et al.: Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family. J Biol Chem. 2001; 276(32): 29839–45. PubMed Abstract \| Publisher Full Text\n\nProost P, Schutyser E, Menten P, et al.: Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties. Blood. 2001; 98(13): 3554–61. PubMed Abstract \| Publisher Full Text\n\nVanheule V, Janssens R, Boff D, et al.: The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74-103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice. J Biol Chem. 2015; 290(35): 21292–304. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVanheule V, Boff D, Mortier A, et al.: CXCL9-Derived Peptides Differentially Inhibit Neutrophil Migration In Vivo through Interference with Glycosaminoglycan Interactions. Front Immunol. 2017; 8: 530. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasqualini R, Koivunen E, Kain R, et al.: Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res. 2000; 60(3): 722–7. PubMed Abstract \| Free Full Text\n\nLasagni L, Francalanci M, Annunziato F: An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4. J Exp Med. 2003; 197(11): 1537–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhlert JE, Addison CA, Burdick MD, et al.: Identification and partial characterization of a variant of human CXCR3 generated by posttranscriptional exon skipping. J Immunol. 2004; 173(10): 6234–40. PubMed Abstract \| Publisher Full Text\n\nSingh AK, Arya RK, Trivedi AK, et al.: Chemokine receptor trio: CXCR3, CXCR4 and CXCR7 crosstalk via CXCL11 and CXCL12. Cytokine Growth Factor Rev. 2013; 24(1): 41–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing BS, Cao Z, Lis R, et al.: Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature. 2014; 505(7481): 97–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiegold O, Ogrissek N, Richter C, et al.: CXCL9 causes heterologous desensitization of CXCL12-mediated memory T lymphocyte activation. J Immunol. 2013; 190(7): 3696–705. PubMed Abstract \| Publisher Full Text\n\nWeigold F, Günther J, Pfeiffenberger M, et al.: Antibodies against chemokine receptors CXCR3 and CXCR4 predict progressive deterioration of lung function in patients with systemic sclerosis. Arthritis Res Ther. 2018; 20(1): 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRecke A, Regensburger AK, Weigold F, et al.: Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3. Front Immunol. 2018; 9: 428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurns JM, Summers BC, Wang Y, et al.: A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor developmen. J Exp Med. 2006; 203(9): 2201–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaumann U, Cameroni E, Pruenster M, et al.: CXCR7 functions as a scavenger for CXCL12 and CXCL11. PLoS One. 2010; 5(2): e9175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuan S, Zhou J: CXCR7 attenuates the TGF-β-induced endothelial-to-mesenchymal transition and pulmonary fibrosis. Mol Biosyst. 2017; 13(10): 2116–24. PubMed Abstract \| Publisher Full Text\n\nCao Z, Lis R, Ginsberg M, et al.: Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis. Nat Med. 2016; 22(2): 154–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMenke J, Zeller GC, Kikawada E, et al.: CXCL9, but not CXCL10, promotes CXCR3-dependent immune-mediated kidney disease. J Am Soc Nephrol. 2008; 19(6): 1177–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoike K, Ueda S, Yamagishi SI, et al.: Protective role of JAK/STAT signaling against renal fibrosis in mice with unilateral ureteral obstruction. Clin Immunol. 2014; 150(1): 78–87. PubMed Abstract \| Publisher Full Text\n\nO’Brien JC, Rainwater YB, Malviya N, et al.: Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea. J Invest Dermatol. 2017; 137(8): 1663–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagee KE, Kelsey CE, Kurzinski KL, et al.: Interferon-gamma inducible protein-10 as a potential biomarker in localized scleroderma. Arthritis Res Ther. 2013; 15(6): R188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMertens JS, de Jong A, Pandit A, et al.: Regarding \"Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea\". J Invest Dermatol. 2018; 138(5): 1212–5. PubMed Abstract \| Publisher Full Text\n\nRabquer BJ, Tsou PS, Hou Y, et al.: Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis. Arthritis Res Ther. 2011; 13(1): R18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichmond JM, Patel D, Watanabe T, et al.: An acute bleomycin inflammatory and fibrotic mouse model of morphea is dependent upon CXCL9 and CXCR3. medRxiv. 2019. Publisher Full Text\n\nHintermann E, Bayer M, Pfeilschifter JM, et al.: CXCL10 promotes liver fibrosis by prevention of NK cell mediated hepatic stellate cell inactivation. J Autoimmun. 2010; 35(4): 424–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen J, Gao J, Chen C, et al.: Antifibrotic role of chemokine CXCL9 in experimental chronic pancreatitis induced by trinitrobenzene sulfonic acid in rats. Cytokine. 2013; 64(1): 382–94. PubMed Abstract \| Publisher Full Text\n\nZaldivar MM, Berres ML, Sahin H, et al.: The chemokine receptor CXCR3 limits injury after acute toxic liver damage. Lab Invest. 2012; 92(5): 724–34. Publisher Full Text\n\nSahin H, Borkham-Kamphorst E, Kuppe C, et al.: Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice. Hepatology. 2012; 55(5): 1610–9. PubMed Abstract \| Publisher Full Text\n\nWasmuth HE, Lammert F, Zaldivar MM, et al.: Antifibrotic effects of CXCL9 and its receptor CXCR3 in livers of mice and humans. Gastroenterology. 2009; 137(1): 309–19, 319.e1-3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTager AM, Kradin RL, LaCamera P, et al.: Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10. Am J Respir Cell Mol Biol. 2004; 31(4): 395–404. PubMed Abstract \| Publisher Full Text\n\nJiang D, Liang J, Hodge J, et al.: Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J Clin Invest. 2004; 114(2): 291–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang D, Liang J, Campanella GS, et al.: Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4. J Clin Invest. 2010; 120(6): 2049–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanino Y, Wang X, Nikaido T, et al.: Syndecan-4 Inhibits the Development of Pulmonary Fibrosis by Attenuating TGF-β Signaling. Int J Mol Sci. 2019; 20(20): 4989. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaxena A, Bujak M, Frunza O, et al.: CXCR3-independent actions of the CXC chemokine CXCL10 in the infarcted myocardium and in isolated cardiac fibroblasts are mediated through proteoglycans. Cardiovasc Res. 2014; 103(2): 217–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXie H, Feng C, Fu Q, et al.: Crosstalk between TGF-β1 and CXCR3 signaling during urethral fibrosis. Mol Cell Biochem. 2014; 394(1–2): 283–90. PubMed Abstract \| Publisher Full Text\n\nBurdick MD, Murray LA, Keane MP, et al.: CXCL11 attenuates bleomycin-induced pulmonary fibrosis via inhibition of vascular remodeling. Am J Respir Crit Care Med. 2005; 171(3): 261–8. PubMed Abstract \| Publisher Full Text\n\nStrieter RM, Starko KM, Enelow RI, et al.: Effects of interferon-gamma 1b on biomarker expression in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2004; 170(2): 133–40. PubMed Abstract \| Publisher Full Text\n\nDamsky W, Patel D, Garelli CJ, et al.: Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea. J Invest Dermatol. 2020; 140(7): 1446–1449.e4. PubMed Abstract \| Publisher Full Text\n\nDees C, Tomcik M, Palumbo-Zerr K, et al.: JAK-2 as a novel mediator of the profibrotic effects of transforming growth factor β in systemic sclerosis. Arthritis Rheum. 2012; 64(9): 3006–15. PubMed Abstract \| Publisher Full Text\n\nWang W, Bhattacharyya S, Marangoni RG, et al.: The JAK/STAT pathway is activated in systemic sclerosis and is effectively targeted by tofacitinib. J Scleroderma Relat Disord. 2020; 5(1): 40–50. Publisher Full Text\n\nKim SR, Charos A, Damsky W, et al.: Treatment of generalized deep morphea and eosinophilic fasciitis with the Janus kinase inhibitor tofacitinib. JAAD Case Rep. 2018; 4(5): 443–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Liang R, Chen CW, et al.: JAK1-dependent transphosphorylation of JAK2 limits the antifibrotic effects of selective JAK2 inhibitors on long-term treatment. Ann Rheum Dis. 2017; 76(8): 1467–75. PubMed Abstract \| Publisher Full Text\n\nDeisseroth A, Kaminskas E, Grillo J, et al.: U.S. Food and Drug Administration approval: ruxolitinib for the treatment of patients with intermediate and high-risk myelofibrosis. Clin Cancer Res. 2012; 18(12): 3212–7. PubMed Abstract \| Publisher Full Text\n\nMolica M, Serrao A, Saracino R, et al.: Disappearance of fibrosis in secondary myelofibrosis after ruxolitinib treatment: new endpoint to achieve? Ann Hematol. 2014; 93(11): 1951–2. PubMed Abstract \| Publisher Full Text\n\nTefferi A, Pardanani A: Serious adverse events during ruxolitinib treatment discontinuation in patients with myelofibrosis. Mayo Clin Proc. 2011; 86(12): 1188–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColtro G, Mannelli F, Guglielmelli P, et al.: A life-threatening ruxolitinib discontinuation syndrome. Am J Hematol. 2017; 92(8): 833–8. PubMed Abstract \| Publisher Full Text\n\nTvorogov D, Thomas D, Liau NPD, et al.: Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis. Sci Adv. 2018; 4(11): eaat3834. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarkham A: Baricitinib: First Global Approval. Drugs. 2017; 77(6): 697–704. PubMed Abstract \| Publisher Full Text\n\nAckermann M, Verleden SE, Kuehnel M, et al.: Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19. N Engl J Med. 2020; 383(2): 120–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonvin P, Gueneau F, Buatois V, et al.: Antibody Neutralization of CXCL10 in Vivo Is Dependent on Binding to Free and Not Endothelial-bound Chemokine: IMPLICATIONS FOR THE DESIGN OF A NEW GENERATION OF ANTI-CHEMOKINE THERAPEUTIC ANTIBODIES. J Biol Chem. 2017; 292(10): 4185–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZolot RS, Basu S, Million RP: Antibody-drug conjugates. Nat Rev Drug Discov. 2013; 12(4): 259–60. PubMed Abstract \| Publisher Full Text\n\nBargh JD, Isidro-Llobet A, Parker JS, et al.: Cleavable linkers in antibody-drug conjugates. Chem Soc Rev. 2019; 48(16): 4361–74. PubMed Abstract \| Publisher Full Text\n\nWilhelm S, Tavares AJ, Dai Q, et al.: Analysis of nanoparticle delivery to tumours. Nat Rev Mater. 2016; 1(5): 16014. Publisher Full Text\n\nLabrijn AF, Janmaat ML, Reichert JM, et al.: Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 2019; 18(8): 585–608. PubMed Abstract \| Publisher Full Text\n\nProw TW, Grice JE, Lin LL, et al.: Nanoparticles and microparticles for skin drug delivery. Adv Drug Deliv Rev. 2011; 63(6): 470–91. PubMed Abstract \| Publisher Full Text\n\nPrausnitz MR: Microneedles for transdermal drug delivery. Adv Drug Deliv Rev. 2004; 56(5): 581–7. PubMed Abstract \| Publisher Full Text\n\nHirakawa H, Zempo H, Ogawa M, et al.: A DPP-4 inhibitor suppresses fibrosis and inflammation on experimental autoimmune myocarditis in mice. PLoS One. 2015; 10(3): e0119360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanasaki K, Shi S, Kanasaki M, et al.: Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen. Diabetes. 2014; 63(6): 2120–31. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Qi Y: Vildagliptin, a CD26/DPP4 inhibitor, ameliorates bleomycin-induced pulmonary fibrosis via regulating the extracellular matrix. Int Immunopharmacol. 2020; 87: 106774. PubMed Abstract \| Publisher Full Text\n\nKühlmann UC, Chwieralski CE, van den Brule S, et al.: Modulation of cytokine production and silica-induced lung fibrosis by inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases. Life Sci. 2009; 84(1–2): 1–11. PubMed Abstract \| Publisher Full Text\n\nWynn TA, Ramalingam TR: Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med. 2012; 18(7): 1028–40. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "72473", "date": "16 Oct 2020", "name": "Christian D. Sadik", "expertise": [ "Reviewer Expertise My areas of research are innate immunity", "immomediators", "immunopharmacology", "and autoimmune and autoinflammatory diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review focuses on the role of CXCR3 and its ligands in fibrotic processes. It starts with an excellent overview on the complex receptor pharmacology of CXCR3 and continues to exhaustively summarize and discuss the role of CXCR3 in the fibrotic processes in different organs.\nThe review is very illustrative including excellent figures. The current state of knowledge on CXCR3 and its role in fibrotic processes is comprehensively summarized and discussed in a balanced manner. All in the article are supported by the literature, which is adequately cited.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6051", "date": "20 Oct 2020", "name": "Jillian Richmond", "role": "Author Response", "response": "We thank Dr. Sadik for taking the time to review our opinion article." } ] }, { "id": "72472", "date": "22 Oct 2020", "name": "Patricia A. Pioli", "expertise": [ "Reviewer Expertise macrophage activation in fibrotic disease", "including systemic sclerosis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report provides an excellent summary of the role of CXCR3 and associated ligands (CXCL9, 10 and 11) in fibrosis. Significantly, these mediators have been reported to have both pro- and anti-fibrotic effects, which potentially confounds their importance in the regulation of fibrotic activation. One of the strengths of this report is that it provides an explanation for these ostensibly opposing effects by emphasizing the importance of cell and tissue context and the role of angiogenesis. Indeed, rather than serving redundant functions, it is likely that the relative contribution of these chemokines is dictated by the extent of vascularization and the cell type responsible for mediating tissue damage. As suggested by the authors, the use of combination therapies is likely to generate the most therapeutic benefit to patients, and the last section of this review does an excellent job of summarizing treatment targets. Although beyond the scope of this current article, future reviews may investigate the utility of specifically targeting immune subpopulations as sources of these chemokines.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6057", "date": "26 Oct 2020", "name": "Jillian Richmond", "role": "Author Response", "response": "We thank Dr. Pioli for taking the time to review our opinion article. We agree that future studies/reviews could focus on targeting specific immune cell populations in the context of different fibrosing disorders." } ] } ]	1	https://f1000research.com/articles/9-1197
https://f1000research.com/articles/9-1192/v1	02 Oct 20	{ "type": "Research Article", "title": "The challenges of theory-software translation", "authors": [ "Caroline Jay", "Robert Haines", "Daniel S. Katz", "Jeffrey C. Carver", "Sandra Gesing", "Steven R. Brandt", "James Howison", "Anshu Dubey", "James C. Phillips", "Hui Wan", "Matthew J. Turk", "Robert Haines", "Daniel S. Katz", "Jeffrey C. Carver", "Sandra Gesing", "Steven R. Brandt", "James Howison", "Anshu Dubey", "James C. Phillips", "Hui Wan", "Matthew J. Turk" ], "abstract": "Background: Software is now ubiquitous within research. In addition to the general challenges common to all software development projects, research software must also represent, manipulate, and provide data for complex theoretical constructs. Ensuring this process of theory-software translation is robust is essential to maintaining the integrity of the science resulting from it, and yet there has been little formal recognition or exploration of the challenges associated with it. Methods: We thematically analyse the outputs of the discussion sessions at the Theory-Software Translation Workshop 2019, where academic researchers and research software engineers from a variety of domains, and with particular expertise in high performance computing, explored the process of translating between scientific theory and software. Results: We identify a wide range of challenges to implementing scientific theory in research software and using the resulting data and models for the advancement of knowledge. We categorise these within the emergent themes of design, infrastructure, and culture, and map them to associated research questions. Conclusions: Systematically investigating how software is constructed and its outputs used within science has the potential to improve the robustness of research software and accelerate progress in its development. We propose that this issue be examined within a new research area of theory-software translation, which would aim to significantly advance both knowledge and scientific practice.", "keywords": [ "scientific software", "research software engineering", "research software", "scientific computing", "high performance computing", "scientific software development" ], "content": "Introduction\n\nSoftware has transformed scientific practice, creating new forms of analysis and representation, and enabling research or thinking that was not previously possible. The growing use of computation has also added significantly to the complexity of conducting research.\n\nThe process of representing, precisely, a scientific entity, method, or system in software is extremely challenging. Having sufficient accuracy is paramount: the more the implementation deviates from the concept it is intended to represent, the lower the value of the resulting knowledge. Verification and validation—commonly referred to as V&V—have the potential to give a level of confidence that the software is both a correct representation of the theory and free of defects1. Verifying the accuracy of a scientific artefact is difficult, however, as there may not be an oracle against which to test it. The artefact may also pass a set of tests, yet still contain errors that have been introduced, unnoticed, during the engineering process. Validation of the artefact—being sure it is a true representation of the theory—is even more challenging.\n\nThe fact that we are able to design and build computational systems does not mean we fully understand them or what they do, or that they do exactly what we want them to do; the proliferation of defects found during the lifetime of any software system illustrates how difficult it is to accurately predict how software will behave at runtime. Huge progress towards system reliability has been made through formal approaches to software verification, and comprehensive tooling exists to assist with many aspects of programming, from detecting code smells (e.g., duplicated code, highly coupled entities, or high cyclomatic complexity), to monitoring test coverage. In spite of this, defects remain a significant problem.\n\nIn addition to addressing the general difficulties common to all software development projects, research software must represent, manipulate, and provide data for complex theoretical constructs. Such a construct may take many forms: an equation, a heuristic, a method, a model; here we encapsulate all of these, and others, in the term theory. In the process of mapping a theory to a programmatic or software-based implementation, defects may occur at a number of points:\n\nThe science is wrong: The theory itself may contain defects, which are discovered through the process of trying to represent it computationally.\n\nThe software is wrong: The way in which the code is written may contain defects—although it is possible to translate from theory to implementation, the chosen form is not appropriate.\n\nThe translation process incurs loss or ambiguity: whilst it may be straightforward to represent a theory verbally or mathematically, it may be difficult to represent it computationally— “Mathematics provides a framework for dealing precisely with ‘what is.’ Computation provides a framework for dealing precisely with ‘how to”’2.\n\nAll of these situations, and the last in particular, interact to make the process of conducting computational research complicated and defect-prone, resulting in a human-machine translation gap.\n\nWithin science, any form of defect or unreliability is highly problematic: if the software does not behave as desired or anticipated, the results may not stand. At present, we lack any formal means of explaining how and why an implementation differs from a concept in unanticipated ways. Whilst a mismatch may be due to obvious limitations of the representation (e.g., ﬂoating point rounding errors), the way in which an implementation is constructed can also result in inaccuracies that were not apparent at the time they were created. Understanding these issues, via empirical research, is vital to ensuring the accuracy and validity of research software.\n\nCompounding the difficulties of formally translating between theory and software are the many cultural and organisational factors that add further challenges to the process of building research software and using its results to advance science. These include the bespoke and highly dynamic nature of research software, the funding model, the academic hierarchy and career structure (in particular the difference in status accorded to domain and software specialists), the difficulties communicating in large scientific teams, and the pressures exerted by the current publication model3.\n\nIn this paper, we provide evidence to motivate the systematic investigation of the Theory-Software Translation process. We achieve this through analysis of the discussion sessions that occurred at the Theory-Software Translation Workshop held in New Orleans4 in February 2019, which explored in depth the process of both instantiating theory in software—for example, implementing a mathematical model in code as part of a simulation—and using the outputs of software—such as the behavior of a simulation—to advance knowledge.\n\nIn the Methods section, we describe the workshop format and goals, its participants, and the process used to collect and systematically analyse data from the discussion sessions. In the Results section, we present the themes that emerged from the analysis, and map these to potential research questions. In the Discussion section, we compare our findings to those of other work in this area, in particular the earlier Code/Theory workshop that took place in the UK3. We conclude by summarising the case for theory-software translation research, and proposing future activities that will lead towards establishing it as a new and fruitful domain.\n\n\nMethods\n\nThe workshop report5 contains a full description of the event, including the agenda, participant list, talk titles and supplementary materials. Below, we describe the key details relevant to the analysis reported here.\n\nThe workshop started with an introduction from the organisers, which was followed by talks from the participants on their background and interest in the topic. The main part of the workshop consisted of a series of breakouts. The first, where participants were pre-allocated to groups to ensure each group had people with a mix of backgrounds, focused on defining the overall challenges of theory-software translation. Following a feedback session, and noting the themes that were starting to emerge, the organisers divided the next set of breakouts into groups that considered training and culture, software design, software stack and tools, and miscellaneous (to catch any issues falling outside the first three). Participants self-selected to join one of these groups for one session, and then moved to a different group for the next session. In each case, participants were asked to discuss the topic, list challenges, identify current successes, and indicate how we could make progress. A final plenary session considered the prospects for Theory-Software Translation as a research area, and considered next steps.\n\nWorkshop participants comprised 20 experts in the field of High Performance Computing and researchers interested in the process of research software engineering. Nine participants were employed at the time at US national laboratories, and 11 at US or UK universities. Three participants were academics whose main focus was studying the process of research software engineering. The rest were involved in research software engineering or the management thereof, with an interest in the idea of theory-software translation, and a desire to improve the process of research software engineering. A full list of participants and their talk materials can be found in the workshop report and on the website4,5.\n\nDuring the breakout sessions, groups kept a record of their conversation, transcribing as much of the discussion as possible, and then summarising key points at the top of the document. There were three breakout sessions, each with four groups, resulting in 12 discussion documents. The first breakout session was split into two parts: in the first part, people wrote notes; in the second, the topic document was given to a different group who added to and commented (using the ‘add comment’ functionality) on the contents. Because common topics arose across groups and sessions, the breakout notes were analysed as a single corpus. Two of the authors (CJ and RH) performed a thematic analysis, with CJ coding the full set of discussion notes and generating initial themes, RH reviewing these and cross-checking with the discussion notes, and both iteratively refining the final set. Formal analysis software was not used for this process; instead the text was collated into a single document, and then parts were grouped together and labelled under subheadings, following familiarisation with the data.\n\nFollowing this initial analysis, all workshop attendees were invited to review and comment on the results, and 13 subsequently endorsed the output as authors of the workshop report5. None of the participants raised any issues with regard to the results of the analysis, or expressed a view that they were unrepresentative. The final stage of the analysis is presented in this paper, in which the authors (a self-selecting subset of the workshop attendees) have collectively further refined the sub-themes within this document via working on the manuscript draft together, providing more detail and adding examples.\n\nThe discussion documents are collectively owned by the workshop participants. During the workshop, it was agreed verbally that these would be analysed and written up in a report, which all participants would be invited to author. This model of collective data ownership and knowledge production had previously been used successfully in the Code/Theory Workshop3.\n\n\nResults\n\nThree overarching themes emerged during the analysis, aligning to the challenges of design, infrastructure, and culture. We explain these below and describe the key areas of research identified within each, framed as open questions.\n\nParticipants considered design in terms of software design, research design, and the way in which the two interact. Topics covered included the extent to which it is possible to separate concerns, whether theory should be ‘readable’ from software, and potential techniques for evaluating and improving the design process.\n\nCan/should we separate concerns? In an era of growing complexity in research models and questions, translating scientific theories to software in a reliable way is becoming increasingly difficult. One perspective on the process is that of moving from ‘science’ to ‘equations to be solved’ to ‘computational algorithms/numerical analysis’ to ‘computer science/software engineering’. (See Babuska and Oden1 for a formal description of this process and these domains.) Each of these is a discipline in its own right, and each is complex. There was a view that it is not realistic for every scientist to understand all of these, and thus an informed ‘separation of concerns’ is crucial. Considering these parts of the process independently also allows each individual in a research team to focus on the aspect(s) for which they are most qualified.\n\nAn alternative view was that concerns cannot always be separated within computational research, from both a theoretical and a practical perspective. At present, a paper and a code are separate things, but the boundaries are blurring. Jupyter notebooks are an example of documentation interspersed with executable code, but this approach is unlikely to be sufficient or scalable on its own. If the boundaries between publication and code increasingly overlap, then it becomes difficult to see where the theory ends and the software begins. Simply documenting the code by commenting it with the theory increases the maintenance cost of the code and risks the two becoming out of sync. Code marked up with the wrong theory is worse than useless, even dangerous, so it is important to be able to verify that the code and theory are consistent. Ince et al.6 argue for the necessity of source code provision along with papers, citing research showing poor effectiveness of specifications in producing equivalency across implementations7.\n\nAnother example of how boundaries are becoming blurred by the introduction of computational methods, is the fact that code and theory are increasingly developed alongside each other. Although it is natural to think (and is most often indeed the case) that one needs to formulate the equations and then apply computational algorithms to obtain the numerical solutions, the formulation of the equations can be affected by the choice of computational method. For example, the equations representing the physics behind a wave will be written for different quantities and hence take different forms, depending on whether a wave pattern is numerically described by a collection of discrete values sampled at selected locations, or the superposition of a number of Fourier modes.\n\nShould theory be readable from software? There was considerable discussion about the extent to which it is possible to write research software in a way that maintains the essence and readability of the underlying theory. Software is highly complex, and can unintentionally obfuscate the theory it contains, particularly when it is optimized for high performance. Preserving a balance between readability (in terms of how easy it is to understand the code) and performance can be difficult. Optimizing code often makes it harder to understand, potentially obfuscating the theory that the software represents, and making it more difficult to reproduce, maintain and modify.\n\nIn an ideal project, mathematical concepts are contained in software components, offering reuse, support for testing, and a clear map to and from the underlying theory. Modular representation of theory is likely to be more readable and testable, but it would be interesting to investigate whether there are areas where this approach is not suitable.\n\nA number of questions emerged from this discussion: Is there a particular design process that should be used for embedding theory within software such that it is readable? To what extent is it necessary for someone reading the software to understand the underpinning theory? When software is assembled from many components, each having their own theoretical foundations, what does this mean for conveying the overall theory underlying the whole? Is there value in an unoptimized, understandable version of a simulation serving as a reference implementation?\n\nTo facilitate theory-software translation in practical terms, domain norms and expertise may need to be taken into account. An example of this can be found in the US Department Of Energy’s effort to develop a new version of its Earth system model8 for cutting-edge computational platforms. The final production code will be written in C++ using Kokkos9 for performance and portability. Because most climate scientists are trained in Fortran, a two-step approach is being used: the domain scientists develop their code in Fortran, then the computational scientists and software engineers take the Fortran code, translate it to C++, and then work on HPC performance. What are the benefits and trade-offs of introducing these further translation steps into the software development process?\n\nWhat are the effects of automation in programming? In the future, code generators may offer a route to translating theory to software. This approach could do a better job of preserving information during implementation and lead to a higher order transformation, due to higher order input. It may allow for timely cross-code validation, where different theory comparisons are made, as it is less human-resource-intensive. This may also be a way to reduce human error (for example, one of the General Relativity solvers10 in the relativistic astrophysics Einstein Toolkit11 uses a Mathematica-based code generator called Kranc12, as this work would otherwise be repetitive and error prone), although it should be noted that code generators, being software themselves, may also introduce defects. Recording the provenance of the code is important in understanding how theory is ultimately arrived at through software outputs. Does using a code generator obfuscate that provenance, or make it clearer?\n\nHow can we evaluate the design process? There are many ways of expressing theory in software. Gathering evidence for what works well would help to inform and refine the software design process. One approach to empirically examining software design is model inter-comparison, which is the process of comparing the results of different implementations of the same underlying theory, such as different climate models, and trying to understand the reasons for, and sources of, similarities and differences in model outputs.\n\nThis is a technically challenging endeavour, and how to do it remains an open research question, but the results could provide an understanding of the efficiency and effectiveness of different implementations, and open up opportunities for code adaptation and reuse. Could we adapt existing codes to new paradigms? Domain specific languages (DSLs) are generally community specific at present. Could we make progress through merging or integrating them, at least where we can be reasonably certain that the models that they are representing are comparable? There is an explosion of tools and services across all domains. How can we tell if they are reliable? Would being able to compare them across domains help with the verification and validation of these tools?\n\nHow can we better link domain science and computer science? There appears to be a disconnect between computer science research and its deployment in scientific discovery; improving the linkage could lead to better science, and more efficient use of computing resources. There are many areas that require computer science research: new languages; more ﬂexible operators; code generation; code transformation; test generation. Theory-software translation research was recognised as having the potential to expose and contribute to these challenges.\n\nTheory-software translation is not solely about mapping scientific constructs to algorithms, but rooted in and affected by a wider software and hardware infrastructure. This part of the discussion gave consideration to verification and validation, sustainability and portability, and how the uncertainty introduced by infrastructure might be recognised and measured.\n\nHow should we verify results arrived at through computation? There is currently no formally established, efficient means of verifying a software simulation, and as such this is an area that requires further attention. Where there is unexpected behavior in a simulation, both software and data provenance are crucial to knowing whether it is caused by a defect or highlights a discovery. Where there is a defect, how can we tell where it lies? Is it in the theory, or the mathematics, or the code? Knowledge is required, not just of the code and the theory, but of the full software stack, including the sequence of dependencies, and how the code is compiled or interpreted.\n\nThe number of potential inputs to most codes is much larger than can be tested in its entirety. A further barrier to comprehensive test coverage is presented by the way in which some applications are configured—both at build-time and run-time. In large, ﬂexible codes features, methods and algorithms can be switched on or off, or swapped; how do we test all of these permutations and combinations of configurations to ensure that they do not interact with each other in unexpected ways? Is there a way we can express theory as a set of tests for code to pass, and ultimately automate test generation from theory specification?\n\nHow should we address reproducibility and sustainability? The importance of reproducibility within research is becoming increasingly recognised. The extent to which true reproducibility is possible in computational science is not clear, due to portability problems, continually changing technology and ‘software collapse’, where software stops working due to changes in underlying layers13. Nevertheless, it was seen as important to strive to get as close as possible to this ideal, and also to work out practical ways of achieving something that approximates this. Having different teams trying to reproduce results, through multiple people running the same codes, could be useful in terms of verification and building knowledge.\n\nWhilst sustaining software for reproducibility is difficult and resource intensive, paradoxically, software almost always lives longer than planned, as (for example) adding features to a prototype is quicker and cheaper than engineering a new and robust code from scratch. What are the implications of this for theory-software translation? What are the effects on the software’s integrity, the way new theory must subsequently be implemented, and the results it produces? What are the issues caused by technical debt?\n\nWhat are the constraints posed by platforms and architectures? Scientific software is generally going to be utilized on multiple generations of computational architectures, and the original developers of the software typically do not (and cannot) take this into account. Changing hardware impedes both portability and reproducibility. Build systems and supporting infrastructure also require maintenance, and any updates to these also have the potential to introduce defects. Where concepts or operations require workarounds to implement on current hardware—such as the representation of real numbers14—the view was that we should ideally aim to change the hardware, rather than restrict the theory, while accepting that this is rarely possible.\n\nWe should also remain mindful that hardware, as well as software, can be an error source, as code that functions correctly on one platform may not on another, unbeknownst to the programmer.\n\nMeasuring uncertainty in theory-software translation Whilst theory is often exact, code has tolerances and approximations. Recognising this was seen as an important part of understanding and improving theory-software translation. One suggestion was to frame this issue in terms of implementation decisions introducing uncertainty. Rather than assuming, ‘this output is correct,’ would it be better to state, ‘there is x% chance some error has been introduced along the way, according to the architecture/code size etc., and therefore we should interpret the result accordingly?’ Could we develop diagnostics that verify the ‘health’ of the simulation, such that we could estimate the potential for defects caused by issues with code quality or age? There is also loss when moving between different stages of theory-software translation (theory, equations, algorithms, software). How can we measure this, and understand its effects?\n\nThe environment in which theory-software translation takes place was recognised as a key inﬂuence on the process. Discussion relating to this topic covered collaboration, expectations, research environments and use of software engineering process.\n\nHow can we foster a culture of collaboration? Computational science, particularly that conducted in large projects, is necessarily interdisciplinary. The heavily domain-contextual specification of the problem and the deep technical knowledge required to implement solutions can lead to an initial communication barrier between domain scientists and computer/computational scientists. Embedding software engineers and applied mathematicians in research teams is a good way of facilitating communication, and there was discussion about what more could be done. One question was whether explicitly recognising the idea that software is a translation of theory might change the communication process. Could conversations across different roles be improved using this approach? The US Department of Energy’s Scientific Discovery Through Advanced Computing program15 is an example of interdisciplinary efforts that directly engage computer scientists and applied mathematicians with the scientists of targeted application domains, with promising results.\n\nImplementing theory in code was viewed as different from implementing non-research software, especially where the requirements are concerned. A key issue was that it may not be possible to separate specification from design, a situation analogous to building an aircraft in ﬂight. Given this, there is a lack of clarity about the best way to approach requirements engineering within research projects.\n\nIt was viewed as crucial to emphasise that software engineering is a core intellectual contribution to the research, not just a service. Close interaction between an application scientist and an applied mathematician can be helpful in designing the appropriate mathematical/numerical method. The discussion about the ‘separation of concerns’ within research software design extends to research software teams. Separating concerns too strictly may lead to different people concentrating on their own tasks, with their own goals and motivations, neglecting the overall picture. On the other hand, focusing on a particular aspect can provide better abstractions and more performant solutions. How do we balance these two pressures?\n\nWhat are the external expectations of the reliability of the software? Validation, which was discussed extensively from a technical perspective, was also considered from an administrative/organisational perspective. Software may need to be considered as a scientific instrument that needs to be validated and/or calibrated. A current example of this is that in the UK, any software that collects patient symptom data, that can be used to access medical advice, or that can be used to assist with a diagnosis, must be developed as a ‘medical device’16. Might there be a requirement to think of software as an instrument that meets formal standards in other research settings17? Would this make results more reliable, or would it stiﬂe creativity? Can we expect complete ‘precision’? If not, should there be ‘guards’ or ‘contracts’ to detail this?\n\nSoftware is not an oracle. There needs to be an improved understanding of which parts of a software tool can be treated as a black box and taken on faith, and which cannot. Without this understanding, software may be used in ways it is not designed for and so give spurious results. Software can be ﬂexible, and because of this, be used in domains for which it was not originally intended, and may not be appropriate; in this case, it should be validated within the new domain before any results are published.\n\nHow does the research environment affect the translationprocess? There is a perception that academic researchers are under pressure to publish at all costs, diminishing the attention paid to good software engineering practices, which are perceived as slowing down the research and publication process. Valuing software as a deliverable in its own right was viewed as an important part of improving its quality and availability. Citing software (via, e.g., the Journal of Open Source Software18 or by more direct citations to the software19) is another part of this process. Considering software explicitly as an output of research, and systematically assessing the impact of research software20,21, remains relatively unusual, and there is still work to be done in understanding how to achieve this.\n\nThere was a view that funding bodies should be involved in discussions regarding theory-software translation. Many of the costs of software development, maintenance, and evolution are hidden; they need to be articulated, and be part of an open, ongoing conversation. The cost of developing software is often underestimated by principal investigators and funding bodies. A lot of time is spent porting software to new hardware, but it is difficult to obtain funding for this, with a negative impact on the quality of the software as a result.\n\nWhat is the best way to embed software engineering skills in science? Often the people writing scientific code are graduate students or researchers who do not have a background in software engineering22,23. Data Carpentry/Software Carpentry was viewed as a good start, but not sufficient. Instilling the necessity of thinking about theory-software translation in graduate students right from the start would help to avoid the need to continually fix poorly-written and poorly-designed code. While this lack of training is a specific problem, software development training is a general challenge, because academic supervisors do not necessarily see the value of it, or even know about it themselves. Awareness that training exists, and a belief in the necessity of undertaking the training, is critical. There is potential for technical training to be conceptualised as a hierarchy, covering: the issues of theory-software translation at an abstract level; the principles of translating between theory and software at a process level; and in-depth expertise in the implementation of theory-software translation at the developer level (with possible specialization).\n\nTraining in communication was also seen as essential, and should go both ways: all members of a research team need to be proficient in cross-disciplinary communication. Being able to communicate scientific requirements to software developers is essential. Being a careful and skeptical user of simulation outputs is also essential, and this requires an understanding of the workings and limitations of the software method, such that the outputs are viewed through the appropriate lens.\n\n\nDiscussion\n\nThe material gathered from the workshop revealed a number of areas where theory-software translation research could significantly advance both knowledge and scientific practice.\n\nSupporting evidence for the identified themes comes from the Code/Theory workshop organised by authors CJ and RH in the UK, which examined the challenges faced by practising research software engineers and data scientists3. The primary themes that emerged at the UK event related to designing software, sustaining software, and communication issues between domain scientists and software engineers, all of which map to our themes of research software design, infrastructure, and culture. The UK workshop focused on identifying practical solutions to these issues, with suggestions including improving communication, tools and training, and raising the profile of software in research, such that people understood its complexity and intellectual contribution. This last issue was also identified as a priority in a survey of Software Sustainability Institute Fellows, who comprise people who have been recognised for their contribution to the research software engineering community24.\n\nAnother activity that has provided insight into this area is the United States Research Software Sustainability Institute (URSSI) conceptualization project, funded by the US National Science Foundation. As part of this effort, the researchers conducted a survey of research software developers and users25. Based on the 1,194 responses, they made some observations relevant to the results we report here. First, relating to our Design and Culture themes, the majority of respondents said they had not received software development training. Furthermore, only half of the respondents indicated sufficient training was available and only 25% said there was sufficient time for training. Second, regarding Culture, the vast majority of respondents indicated that the level of funding for research software was “insufficient and creates barriers to their work”. A wide-scale survey providing further strong evidence for the utility of theory-software translation research explicitly examined problems within research software engineering, providing a taxonomy of ‘pains’ covering technical, scientific and social issues26. This work highlights that many common software engineering challenges, such as requirements gathering, communication difficulties and debugging, are particularly challenging— and often qualitatively different—within science, due to the nature of the research process, and the environment in which the work is conducted.\n\nTheory-software translation research has the potential to contribute to the evidence base for research software infrastructure strategy and practice at a local (individual/group), institutional (organization), national, and international level. It could also identify cross-cutting challenges for computational research, and advance the techniques we use to perform such research. We anticipate that a better understanding of the theory-software translation process will lead to more robust and accurate research software.\n\nThe results of our analysis demonstrate that research software is not merely used to perform a task, but to understand a problem and advance knowledge. While current software engineering research outputs and methods are relevant to addressing these challenges, theory-software translation research would involve tackling new problems that are rooted within the scientific domain. We summarise key, emergent areas for research as follows:\n\nThe translation process moves from theory to algorithm to software (and vice-versa). Information is lost in moving from one domain to another, as the way in which ideas are represented changes. Can we quantify or explain this loss/difference, and articulate the trade-offs resulting from translation?\n\nHow does incorporating theory in software (e.g., a simulation) differ from standard requirements engineering? The development of software and theory happen together. While requirements changes are generally constrained for typical software, theory can change much more dramatically, resulting in not just an addition, but a fundamental divergence from what the software was initially designed to do.\n\nHow do we understand the results of a simulation, and translate this back to the underlying theory? How should real world data be used in the verification process?\n\nHow do we go from viable theory to validated, verified code in a time-efficient way? Can theory be expressed as a set of tests?\n\nThere is a distinction between theory, model, numerics, and code, and there are difficulties mapping between them. There can be errors in any part of the mapping process that may affect the resulting science. How can we detect and handle these errors?\n\nIs a true separation of concerns (theory, and its implementation in software) possible? What are the implications for how we write scientific software?\n\nShould theory be recoverable from software (where software includes documentation)? Can theory today be represented solely in papers, or is it really also in the code? How can we help people to read and understand it?\n\nThe increase in model complexity and sophistication of questions that models are designed to answer makes translating them into software increasingly difficult. How do we determine the appropriate level of complexity? Is it possible to optimise for complexity reduction, as well as performance?\n\nHow can we define functional reproducibility? Long-term reproducibility is likely to remain out of reach due to software collapse13. Can we create ways of representing theory that will persist and remain usable, so as to increase software sustainability and prolong the period of reproducibility? Is there a way of providing a reference implementation that can be used as a blueprint for the theory?\n\nCan we identify the range of practices currently used, and gather empirical data concerning their efficacy, to result in evidence-based best practice?\n\nWhat are the benefits and implications of the increasing use of automation in research software engineering?\n\nSoftware is unlikely to ever be 100% ‘correct’. Can we measure how well software represents theory, and estimate the error that might have been introduced during the translation process? Can we develop ways to quantify uncertainty for software functioning such that appropriate probabilities can be applied to results?\n\n\nConclusions\n\nThe discussion sessions at the theory-software translation workshop identified a series of challenges in building and using scientific software. Related research questions map to the areas of research software design, infrastructure, and culture. Whilst the distinctions between these categories are not absolute, they provide a means of structuring our understanding of the theory-software translation process, and advocating for its improvement via research.\n\n\nData availability\n\nThe workshop documents have all been retained in their original form, but they are highly identifying (including the names of all individuals who contributed to and commented on them, as well as personal views and experiences). Formal IRB approval was not sought for the study, as we instead used the collective ownership model described in the Methods section. We do not have explicit permission from workshop participants to share the original documents.\n\nEmails between the authors, and iterative refinement of the manuscript draft on the Overleaf platform also formed part of the analysis process, and could be viewed as contributing additional data/metadata. We would be happy to answer questions on the data and analysis process. All queries should be directed to the corresponding author, CJ. The validity of the results is guaranteed in that they are co-produced and represent the cumulative, collective reﬂections of the authors, as they see them, based on the workshop discussions. The workshop report4 can be considered as a published, interim stage in the analysis process of moving from the discussion documents to this final output.", "appendix": "References\n\nBabuska I, Tinsley Oden J: Verification and validation in computational engineering and science: basic concepts. Comput Methods Appl Mech Eng. 2004; 19(3): 4057–4066. Publisher Full Text\n\nAbelson H, Sussman GJ, Sussman J: Structure and Interpretation of Computer Programs. MIT Press, 2nd edition, 1996. Reference Source\n\nJay C, Haines R, Vigo M, et al.: Identifying the challenges of code/theory translation: report from the code/theory 2017 workshop. Res Ideas Outcomes. 2017; 3: e13236. Publisher Full Text\n\nTheory-Software Translation Workshop - US Edition in New Orleans, LA (February 4-5, 2019). Accessed: 2020-06-21. Reference Source\n\nJay C, Haines R, Katz DS, et al.: Theory-software translation: Research challenges and future directions. 2019. Reference Source\n\nInce DC, Hatton L, Graham-Cumming J: The case for open computer programs. Nature. 2012; 482(7386): 485–488. PubMed Abstract \| Publisher Full Text\n\nvan der Meulen MJP, Revilla MA: The Effectiveness of Software Diversity in a Large Population of Programs. IEEE T Software Eng. 2008; 34(6): 753–764. Publisher Full Text\n\nE3SM Project: Energy Exascale Earth System Model (E3SM). [Computer Software]. Accessed: 2020-06-23. Publisher Full Text\n\nKokkos: The C++Performance Portability Programming Model. Accessed: 2020-06-21. Reference Source\n\nBrown D, Diener P, Sarbach O, et al.: Turduckening black holes: An analytical and computational study. Phys Rev D. 2009; 79: 044023. Publisher Full Text\n\nBabiuc-Hamilton M, Brandt SR, Diener P, et al.: The Einstein Toolkit. Zenodo. 2019. Publisher Full Text\n\nHusa S, Hinder I, Lechner C: Kranc: a mathematica package to generate numerical codes for tensorial evolution equations. Comput Phys Commun. 2006; 174(12): 983–1004. Publisher Full Text\n\nHinsen K: Dealing with software collapse. Comput Sci Eng. 2019; 21(3): 104–108. Publisher Full Text\n\nIEEE Standard for Floating-Point Arithmetic - 754-2019. Accessed: 2020-06-19. Reference Source\n\nUS Department of Energy, Office of Science: Scientific Discovery Through Advanced Computing (SciDAC). Accessed: 2020-06-23. Reference Source\n\nMedicines and Healthcare products Regulatory Agency: Medical devices: software applications (apps). 2018. Reference Source\n\nCarver JC: Software engineering for science. Comput Sci Eng. 2016; 18(2): 4–5. Publisher Full Text\n\nJournal of Open Source Software. Accessed: 2020-06-21. Reference Source\n\nSmith AM, Katz DS, Kyle E, et al.: Software citation principles. PeerJ Comput Sci. 2016; 2: e86. Publisher Full Text\n\nHowison J, Deelman E, McLennan MJ, et al.: Understanding the scientific software ecosystem and its impact: Current and future measures. Res Eval. 2015; 24(4): 454–470. Publisher Full Text\n\nDubey A, Tzeferacos P, Lamb D: The dividends of investing in computational software design: a case study. Int J High Perform Comput Appl. 2018. Publisher Full Text\n\nHeaton D, Carver JC: Claims about the use of software engineering practices in science: A systematic literature review. Inf Softw Technol. 2015; 67: 207–219. Publisher Full Text\n\nCarver J, Heaton D, Hochstein L, et al.: Self-perceptions about software engineering: A survey of scientists and engineers. Comput Sci Eng. 2013; 15(1): 7–11. Publisher Full Text\n\nSufi S, Jay C: Raising the status of software in research: A survey-based evaluation of the software sustainability institute fellowship programme [version 1; peer review: 3 approved with reservations]. F1000Res. 2018; 7: 1599. Publisher Full Text\n\nCarver J: URSSI Conceptualization Survey Results. 2019; Accessed: 2020-06-15. Reference Source\n\nWiese I, Polato I, Pinto G: Naming the pain in developing scientific software. IEEE Softw. 2020; 37(4): 75–82. Publisher Full Text" }	[ { "id": "72416", "date": "14 Oct 2020", "name": "Konrad Hinsen", "expertise": [ "Reviewer Expertise scientific computing", "statistical physics", "biomolecular simulation", "protein dynamics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a structured summary of the discussion sessions of a recent workshop on theory-software translation, followed by carefully crafted arguments for establishing theory-software translation as a new field of research. It represents a significant effort at making progress on an important question, but I don't consider it a \"research article\" for the reasons outlined below, and therefore I can approve it only with reservations. My recommendation to the authors is to relabel their work as an opinion article.\nA research article should start with a research question, and my first task as a reviewer would be to judge if the method design is appropriate to answer this question. Indeed, a question in the review form is \"Is the study design appropriate and is the work technically sound?\" In the absence of a research question, I cannot answer it meaningfully. One can imagine an underlying research question such as \"Which issues do practicing scientists and engineers see with the current state of theory-software translation?\", and then the method design could be evaluated critically (for example, I would criticize the overrepresentation of HPC practitioners, and the complete absence of theoretical scientists not involved with software development). But then, the call for action in the final sections would be inappropriate and would have to be replaced by something like \"We find that practitioners believe that...\". Put differently, you can study science policy or shape science policy, but you cannot do both at the same time. My remaining comments on this article assume that its main intention is to shape science policy.\nThe authors state the goal of the proposed new research area of theory-software translation as \"investigating how software is constructed and its outputs used within science\". This indicates a strong focus on the software end of the bridge being envisaged. In my opinion, it is also necessary to investigate how theoretical work is being performed in science, how computers have impacted the practices of theoreticians, and how their use of computers could be improved in the future. This would lead to a stronger focus on non-numerical computational tools, for example, computer algebra systems, which I expect to be important in improving theory-software translation. It would also help with answering some of the questions raised in the workshop. The answer to \"Should theory be readable from software?\" is clearly \"yes\" for computer algebra applications. And that means that if we succeed in creating formally verifiable correspondences between computer algebra and high-performance simulation software, the readability of the latter is no longer an issue.\nIt would also be useful to separate the challenges and goals of theory-software translation research into short-term and long-term. Short-term work, which is what the article seems to concentrate on, must take into account the constraints of existing technology that cannot be overcome quickly. Long-term work can be more ambitious and envisage completely new software and tool stacks designed specifically for supporting scientific research. Importantly, long-term research is necessary to detect if short-term efforts are likely to create additional technical debt that then becomes an obstacle to more substantial progress.\nThere is one sentence in the article that I strongly disagree with: \"Whilst theory is often exact, code has tolerances and approximations.\" (page 6). Approximations are an important aspect of all theoretical work in the natural sciences. Exact theory can only exist in what Herbert Simon called the \"sciences of the articifial\". In particular, the approximations made in simulation and data analysis, for example, numerical approximations, clearly belong to the domain of theory, not software. The best evidence is that such questions are discussed in research articles on numerical algorithms, independently of any concrete implementation. In the final list of research questions for the new field, the one starting with \"There is a distinction between theory, model, numerics, and code\" is related to this point and suggests that the authors are mainly considering the scientific disciplines represented in HPC applications. There are disciplines that do not have theories as frameworks for their models or use models that are not numerical.\nThe section on \"Measuring uncertainty in theory-software translation\" (page 6) overlooks the fundamental lack of robustness in software. Unlike in many other domains of engineering, there is no way to ensure that small mistakes have small consequences (see https://dx.doi.org/10.1109/MCSE.2016.67 for a detailed discussion)1. As a consequence, the \"x% chance some error has been introduced along the way\" is of little practical relevance because it permits no conclusion about the possible magnitude of the resulting error in the output produced by the software. Long-term work on theory-software translation should therefore investigate paths to higher robustness. For example, do we really need Turing-complete languages to implement scientific models? Maybe a less powerful medium could help reduce the impact of errors.\nFinally, a minor technical point: \"such as the representation of real numbers\" (page 6) suggests that real numbers can be represented in a computer, whereas the root issue with precision-related problems is that a finite-size representation for arbitrary real numbers doesn't exist. The largest subset of the real numbers that are representable in a computer is the set of computable numbers, which were first studied by Alan Turing in his seminal paper introducing the Turing machine.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72420", "date": "26 Oct 2020", "name": "Mike Heroux", "expertise": [ "Reviewer Expertise Scientific software", "high-performance computing", "mathematical modeling and simulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article represents a summary and analysis of the output from the 2019 Theory-Software Translation workshop held in New Orleans, LA, USA in February 2019.\n\nThe authors summarize the purpose and format of the workshop and then present an analysis of the workshop breakout session content, folding the content into a holistic discussion that characterizes the challenges of theory-software translation, key research questions, and some discussion of potential solution strategies.\n\nOverall, the article is very well written. The authors are some of the leaders in this field, which is reflected in the scope of discussion. I have just a few comments and suggestions:\nWhile this article is about theory-software translation, did the topic of model-free approaches come up during the workshop or subsequent discussions? For example, some scientific teams are successfully using machine learning (ML) approaches, where prediction and insight may come from an inference engine constructed from raw data with no explicit mathematical model. These approaches are increasingly integrated into more established theoretical frameworks, with promising impact. If there were no comments in the workshop, then omitting the topic from this article is appropriate.\n\nIn the introduction, you state that “Validation of the artefact—being sure it is a true representation of the theory—is even more challenging.” Is this a sufficiently expansive definition of validation? The terse definition of validation as “Doing things right.” is often interpreted as meaning that the computational results are consistent with physical experiments, not just the theory that the software encodes.\n\nYou state that the workshop discussion notes were coded. To a software person who is not familiar with text coding, this term may be unfamiliar, and may indeed be confused by their familiarity with software coding. A brief footnote, parenthetic explanation, or similar explanation could improve the accessibility of the paper, since the coding activity was important for producing the article content.\n\nAt the end of page 4, you state, “Ince et al. argue for the necessity of source code provision along with papers, citing research showing poor effectiveness of specifications in producing equivalency across implementations.” I think this sentence could be expanded and better explained for the reader. In particular, what are the definitions of provision, specification, and equivalency? I think I understand the statement, but a simple example might help the reader.\n\nWhile I have no concerns about the validity of the content in this article, the unavailability of the raw workshop discussion notes and the informal nature of the coding process used on the combined discussion notes text, prohibits rigorous reproducibility of the results. The omission is clearly acknowledged and explained, so I do not view it as a critical problem. Perhaps future approaches could be designed to permit availability of raw content by anonymizing attribution or getting ARB approval. Automated, or carefully documented coding would be useful too.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "72412", "date": "10 Nov 2020", "name": "Mozhgan Kabiri Chimeh", "expertise": [ "Reviewer Expertise High-performance computing. Simulation acceleration. Optimization and tuning of scientific applications." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract and title reflect the content of the article. The motivation and contribution of the paper are clear. It’s well written. A good range of papers were included in the related work paper.\nVery good, comprehensive workshop report that can be used as a good base for future similar events for further discussion.\nMy only negative feedback is for the workshop itself. Looking at the speakers' list, I can hardly see a diverse and inclusive list! That is something that is missing really, and I hope in future events and workshops as such could be kept as diverse and inclusive as possible to give stronger meaning to the results and conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1192
https://f1000research.com/articles/9-1191/v1	01 Oct 20	{ "type": "Method Article", "title": "Improving the design of an online course with virtual focus group feedback", "authors": [ "Celine Young", "Olivia M. Chesniak", "Denise Drane", "Henry Campa III", "Noah Green", "Robin Greenler", "Jessica Middlemis Maher", "Richard McGee", "Antonio Nunez", "Bennett B. Goldberg", "Sarah Chobot Hokanson", "Celine Young", "Olivia M. Chesniak", "Denise Drane", "Henry Campa III", "Noah Green", "Robin Greenler", "Jessica Middlemis Maher", "Richard McGee", "Antonio Nunez", "Bennett B. Goldberg" ], "abstract": "Virtual focus groups played a significant role in guiding the design and development of an innovative professional development program for postdoctoral scholars, called The Postdoc Academy. The primary goal of obtaining feedback from postdocs was to ensure the program content is relevant, approachable, and inclusive for participants of all backgrounds, career stages, professional aspirations, and disciplines. The data collected in 13 focus groups shaped the final content and structure for the Massive Open Online Course (MOOC), ‘The Postdoc Academy: Succeeding as a Postdoc’. Evaluation of participant experience in a post-focus group survey suggests that engaging a target audience is an effective approach to obtain participant feedback and engage learners in the material. Content and activities modified by this feedback were highly rated by course participants in self-reported post-module evaluations. This article describes a method on how structured virtual focus groups of diverse future course participants can provide valuable feedback on developing the content and structure of professional development programming.", "keywords": [ "virtual focus groups", "massive open online course", "evaluation", "instructional design", "postdoctoral scholars", "professional development" ], "content": "Introduction\n\nProfessional development is critical for postdoctoral scholars (postdocs) to develop skills beyond the technical focus of their research (Davis, 2009; Gibbs et al., 2015). A recent study highlights the lack of postdoc training in precisely the skills necessary for success in the current job market, including career preparation, leadership, and collaboration (Alund et al., 2020). To address this training gap, our team created online and supplemental in-person professional development programming for postdocs, based on the National Postdoctoral Association core competencies (NPA Core Competencies, n.d.). The Postdoc Academy implements an effective model for online professional development incorporating active learning, participant reflection, and skill building (Hokanson et al., 2019). The goal of the Postdoc Academy is to create a comprehensive and flexible professional development program targeting the needs of postdocs that develops transferable skills to enable their success in a diverse set of careers. Although the program is based in the United States, the program is open to postdocs from all countries. To date, more than 60 countries have been represented by the program registrants.\n\nPrior to the launch of a Massive Open Online Course (MOOC) titled ‘The Postdoc Academy: Succeeding as a Postdoc’, the program team implemented virtual focus groups to guide program design, development, and implementation. The goals of the postdoc focus groups were to: 1) align course content and design with the needs of the postdoc community; 2) ensure course content is relevant to postdocs of different disciplines, professional aspirations, career stages, and backgrounds; and 3) integrate the voices, experiences, and stories of postdocs in the content. Focus groups as a data collection approach have been used for many years. Recently, researchers have investigated using virtual focus groups as an effective approach to obtain feedback (Forrestal et al., 2015; Lobe, 2017; Matthews et al., 2018; Tates et al., 2009). Compared to in-person focus groups, virtual focus groups have been shown to have equivalent participation rates, participant satisfaction, and consistent numbers of unique ideas generated and relevant comments from participants (Underhill & Olmsted, 2003).\n\nWith similar outcomes as in-person focus groups, virtual focus groups have additional advantages. Virtual focus groups can reach a more diverse population compared to in-person focus groups. Based on the target audience of the postdoctoral community, the program team wanted to increase the likelihood of achieving a representative sample. Participants engage with the content online and are isolated in the virtual environment of the focus groups, often working through activities in silence, partially simulating an experience similar to the online course. Forrestal et al. (2015) and Krueger (1994) suggest that, since participants can turn off their video and choose their display name, a sense of privacy exists in virtual focus groups that allows for more open discussion of personal or sensitive topics. Another potential benefit is the lower cost compared to in-person focus groups, since no expenses for travel, room, or food were incurred.\n\nThis article will share the process used by the Postdoc Academy to facilitate virtual focus groups and how engaging a target audience is an effective approach to obtain participant feedback and engage learners in professional development programming.\n\n\nMethods\n\nBetween April and July 2019, virtual focus groups were conducted with participants from the program’s intended audience: postdocs. The content reviewed was part of a MOOC, titled the Postdoc Academy: Succeeding as a Postdoc (see the edX course and http://www.postdocacademy.org/explore/). Modules within the MOOC include: 1) finding success as a postdoc; 2) building an actionable career plan; 3) developing resilience; and 4) working effectively in an intercultural environment. Over the course of three weeks, three focus groups were held for each of the four content modules, with the exception of one module (Developing Resilience) that had an additional focus group to review significant modifications implemented based on the initial focus group feedback. Each session was capped at approximately ten participants. On average, focus groups had seven participants, which is consistent with focus group best practices to include six to eight participants in each session (Sweet, 2001).\n\nAs inclusivity and transparency are central to the goals of the program, we prioritized these themes in developing this feedback group approach. Social presence, or a sense of community, is critical to maximize participation and foster open discussion in an online environment (Akyol & Garrison, 2008; Gunawardena & Zittle, 1997). Transparency is another key factor to promote affinity in virtual communities (Dalsgaard & Paulsen, 2009). We aimed to promote a sense of transparency in the focus groups such that participants understood the goals and process of content creation, as well as the rationale for focus group design.\n\nSocial media (Facebook, Twitter, and Instagram) were used to market the virtual focus groups to the postdoc community. In addition, newsletters from the Postdoc Academy, the four collaborating institutions, and the program partners (postdoc associations, postdoc offices, international offices, and professional societies) shared marketing information. Participants were allowed to participate in one session per module. The only criteria for focus group participation was status as a postdoc and focus group registration and attendance.\n\nParticipants were sent an email reminder for the focus group with logistics and the list of questions that would be asked. They completed a pre-session survey that served to collect demographic information for National Institutes of Health reporting, such as gender and ethnicity, time in postdoc position (in years), discipline, and institution.\n\nAll virtual focus groups were hosted on the Zoom platform, chosen as an institutionally supported platform and its ease of use. Each focus group was scheduled for two hours and facilitated by two program team members, joining from separate rooms and devices. The lead facilitator reviewed the module content using screen share, guided the discussion, and moderated live polling during the session. The secondary facilitator took notes and responded to participant questions and comments in the chat window.\n\nIntroductions: At the beginning of the focus group, each facilitator introduced themselves, with the lead facilitator then calling on each participant to introduce themselves.\n\nVideo optional: While the facilitators had their video turned on, it was communicated to participants this was not a requirement.\n\nTechnical check: The facilitators checked for technical difficulties.\n\nProgram introduction: The lead facilitator shared PowerPoint slides on their screen to provide an overview of the program and the module being discussed. At the time of focus group facilitation, the course content was primarily in Google Docs and Google Slides. Because of this, the participants were asked to be mindful that this work is still in progress and the final course on the edX platform may look different.\n\nTypes of feedback: The participants also were informed that there are no right or wrong answers to the discussion prompts and live poll questions, and that the program team is looking for positive and negative constructive feedback.\n\nData use and management: The lead facilitator then informed participants that the session would be recorded. Participants were also informed of how data would be used. The data collected would be anonymized and used for evaluation and improvement of content, as well as conference presentations or manuscripts in support of the pedagogical approach.\n\nConfidentiality: Participants were asked to maintain confidentiality of the content that was reviewed during the session.\n\nBegin recording. The recording of the focus group then began, and facilitators shared how the session would be facilitated. Participants were encouraged to follow along using an outline of the module content that contained links to video scripts, activities, and discussion prompts. The two formats of obtaining feedback were also discussed: anonymous live polling questions and open discussion questions. The participants also received a worksheet to add additional written feedback after the session (see extended data (Young et al., 2020)).\n\nContent review and discussion\n\nModule content review: The lead facilitator provided an overview of the module. The learning objectives, context or framing of the content, and main components of the module were shared. The facilitators used incremental content sharing (approximately 10 minutes at a time) with group discussions interspersed. In doing so, the format of interaction was consistently varied throughout the virtual focus group, which may have contributed to high participant engagement.\n\nDiscussion and polling: Live polling and group discussion questions were used to prompt discussion in the focus groups (see extended data (Young et al., 2020)). On average, each session used six live poll questions and 12 discussion prompts. The multiple-choice poll questions and discussion prompts were drafted by each module development team.\n\nFacilitation methods: Facilitators asked open-ended questions, one at a time, and asked for clarification when a response was unclear. Participants spoke in turn without reliance on hand-raising features. Comments shared in the chat feature were highlighted by facilitators verbally. Facilitators remained neutral and avoided leading questions. If a participant was not participating, the facilitator would call on the participant by name to share their answer.\n\nConcluding the session. After all of the discussion prompts had been asked, the lead facilitator closed the session with a few reminders. First, participants were reminded of the worksheet that they could use to provide additional written feedback. Second, participants were informed of how they would receive an Amazon gift card for their participation. Finally, participants were thanked for their time and contributions to the program.\n\nAfter the session, participants were emailed within two days to remind them of the worksheet for providing written feedback and to update them on the status of their gift card. The electronic Amazon gift card ($75 each) was emailed to participants within one week of participating in the focus group.\n\nAfter each focus group, the facilitators analyzed the live polling results and each added additional notes about the discussion prompts to capture the thoughts and inputs of both facilitators. Once focus groups were completed for each module, the facilitators summarized the findings and shared them with the module development team. Each module development team reviewed and discussed the feedback to modify content within the online course.\n\nWithin two weeks of the conclusion of the focus group series, a Qualtrics survey was emailed to participants to obtain feedback on their virtual focus group experience. This anonymous survey contained 19 multiple choice and free response questions and took an estimated 7 minutes to complete. Upon survey completion, participants had the opportunity to enter a raffle for an additional $75 Amazon gift card. Data were anonymized and quantitative results analyzed by frequency. Thematic analysis of open-response questions was conducted, using Braun and Clark’s framework for thematic data analysis (Braun & Clark, 2006; Clark & Braun, 2013).\n\nThe focus groups in this article were determined to not be human subjects research by the Boston University IRB (#4046X), thus written consent was not required. An IRB (#5419X) from Boston University has been obtained to perform research on the course data. This article focuses on the methods of the focus groups, rather than creating generalizable knowledge from the course data.\n\n\nResults\n\nAverage number of participants in each focus group was seven, and ranged from 4 – 11. All focus group participants were postdocs at a United States-based institution of higher education (eight institutions represented). A total of 53 postdocs participated in focus groups, with approximately half of respondents (53%) attending one focus group, and 47% attending more than one session. Of those who filled out the pre-focus group survey (94% response rate, 49 out of 53 participants), the majority of respondents (92%, 45 out of 49 respondents) were in their first 3 years as a postdoc. Most respondents (78%, 38 out of 49 respondents) were classified as STEM, 69% (34 out of 49 respondents) identified as female and 31% (15 out of 49 respondents) identified as male (see Table 1). One respondent participated in all four module focus groups, as well as the additional focus group for “Developing Resilience”.\n\nThree types of feedback to evaluate the efficacy of virtual focus groups were collected: 1) post-focus group survey; 2) facilitator reflections; and 3) post-module and post-course surveys after the online course. Based on all three sources of feedback, significant changes were made to the course content. In evaluation of the online course, course participants were highly satisfied with the course.\n\nResults of the post-focus group survey indicate that focus group participants had an overall positive experience. We asked participants about their experience in virtual focus groups, using a 4-point Likert scale from strongly agree to strongly disagree. Of the 30 survey respondents, 33% (10) strongly agreed and 67% (20) agreed that the online format was effective for learning about the content. Next, 33% (10) strongly agreed and 67% (20) agreed that the content presented in the focus groups was suitable for people from different cultural backgrounds. Finally, 60% (18) strongly agreed and 40% (12) agreed that their contributions to the discussion were valued.\n\nTo demonstrate the types of feedback we received in focus groups and the changes made to the online course based on that feedback, we will use the module titled “Developing Resilience” because major modifications were made to the course content after virtual focus groups (Table 2). One example of changes to the online course was based on the feedback received when focus group participants were asked if the module content is approachable and relatable for a diverse audience. The feedback indicated that the content seemed to be American-centric. One participant commented “...we need to see lots of different people from different backgrounds, foreign and domestic.” Since the feedback suggested that the content is approachable but not relatable for all postdocs, the program team revised the module content by: 1) incorporating the stories and perspectives of international postdocs; 2) discussing how international postdocs might face barriers to resilience compared to domestic postdocs; and 3) highlighting strategies that are used in other cultures to build resilience.\n\nAnother example of modifying course content based on focus group feedback was in alignment of course content with the learning objectives. The program team identified a learning objective to support participant resilience across the work-life continuum, and the feedback from focus group participants indicated that the course content was not aligned. One commented, “...it felt more like work resilience than work-life resilience or work and life resilience.” Generally, participants felt that the content was more representative of professional resilience than resilience that spans the personal and professional aspects of life. With this feedback, the program team revised content to emphasize that resilience spans personal and professional components of one’s life by: 1) incorporating literature on recovery internal and external to the workplace; 2) sharing interviews of individuals discussing resilience in personal and professional contexts; and 3) highlighting strategies to build resilience beyond work.\n\nConsidering the extent of feedback during the focus groups that reviewed “Developing Resilience”, the same group of participants were invited to an additional focus group (n=4) to ensure that the revised content was relatable, engaging, and informative for participants. Participants confirmed that the implemented revisions improved the course content. This was an important step in the iterative feedback process before launching and evaluating the online course.\n\nEvaluation of the online course with post-module and post-course surveys indicated that the changes made based on the focus group feedback were well-received. Not all focus group participants took the online course and course surveys, therefore, the results reported in Table 3 are a summary from all course participants who completed course surveys (Underlying data (Young et al., 2020)). Overall, 89% of survey respondents (94 out of 103) were satisfied with the course. Additionally, 91% of survey respondents (84 out of 92) indicated that they would recommend the course to a friend. Table 3 displays the evaluation results for “Developing Resilience”. The survey items are aligned with the learning objectives and short-term outcomes identified for the module. These results indicate that the online course content, including the major modifications made to “Developing Resilience” based on focus group feedback, was well-received by course participants.\n\nRespondents self-reported agreement or disagreement on a 5-point Likert Item. N=number of survey respondents.\n\nIn addition to evaluating the efficacy of virtual focus groups, we also wanted to evaluate the experience of focus group participants. Figure 1 displays the reasons postdocs said they were motivated to participate in the focus groups. Participants were able to indicate more than one reason. An intrinsic motivation to contribute intellectually as described in the literature is altruism, the enjoyment of helping others (Kankanhalli et al., 2005). One of the most frequent survey responses was to provide feedback to guide program development, which suggests altruism may be a motivator in these virtual focus groups.\n\nParticipants could select more than one motivator.\n\nTo gain insight into the experience of focus group participants and improve the experience for future sessions, the Critical Incident Questionnaire was used (Brookfield, 1995). As displayed in Table 4, participants indicated they were most engaged while interacting with other postdocs and when offering specific feedback. While the half of the survey respondents (53%, 10 out of 19 respondents) indicated there was not a time when they were not engaged, others indicated they were least engaged while reviewing the material and while slides were being presented.\n\n\nDiscussion\n\nIn summary, we developed and implemented a model of high-engagement virtual focus groups, received quality feedback, made significant changes to the course content, and the resulting content was well-received by course participants. We evaluated the experience of participants to ensure a well-defined process for facilitating virtual focus groups and ensure that participants felt valued for their contributions. From this work, multiple recommendations for improving professional development programming emerged.\n\nVirtual focus groups are an effective, low-cost method to receive quality feedback and ensure programming is engaging, inclusive, and relevant to the target audience. Creating an inclusive and transparent environment for focus group participants can lead to higher engagement. Reviewing the content in small amounts and creating space for open discussion are two key takeaways. We envision this process would be beneficial for those who support graduate and postdoctoral professional development.\n\nTo improve upon the process described here, the program team plans to expand recruitment to reach an audience that more accurately represents the target audience, the postdoc population. To do so, we will invite postdocs at non-academic institutions (government labs, industry, agencies). Additionally, we will consider including other key stakeholders in addition to future participants, such as faculty and staff who support postdocs in separate focus groups to gain insight to other perspectives.\n\nAlthough there are many benefits to virtual focus groups, there are limitations for implementing this process that should be considered. One limitation can be the technical barriers with the web conferencing service used to host the focus groups (Kite & Phongsavan, 2017). It’s critical that facilitators consider the pros and cons of different platforms, including strategies to enhance privacy and security, and test the web conferencing service. Additionally, we suggest providing detailed instructions on how to access the platform and allow additional time in each session to ensure there are no technical difficulties.\n\nAnother limitation can be the limited number of participants who were included in the virtual focus groups. The postdocs who participated in the focus groups might have a higher level of engagement in professional development activities relative to the postdoc population. Thus, the feedback received and the resulting course content may cater to course participants with similar identities and experiences as the focus group participants. To be more representative of the course participants, the focus groups could be held in a number of different environments. Additionally, not all focus group participants completed the online course, which limits analysis of the changes made based on focus group feedback.\n\nIn conclusion, structured virtual focus groups of diverse future participants provided valuable feedback on the content and structure of an online course for postdocs. Evaluating the experience of focus group participants provided insights into the benefits of using this approach and has helped improve the process for the future. Facilitators must consider the benefits and potential limitations when planning, implementing, and evaluating virtual focus groups as a method of soliciting input on a professional development program.\n\n\nData availability\n\nThe transcripts of the focus groups and facilitator reflections contain identifying information about the participants. In order to keep the focus group participants anonymous, the focus group transcripts and facilitator reflections are not included in the underlying data. Upon request, deidentified transcripts can be provided.\n\nHarvard Dataverse: Improving the design of an online course with virtual focus group feedback. https://doi.org/10.7910/DVN/ZBWSHS (Young et al., 2020)\n\nThis project contains the following underlying data:\n\nDeidentified Postdoc Academy Feedback Session Survey.tab\n\nPost-Module and Post-Course Survey Results.tab\n\nHarvard Dataverse: Improving the design of an online course with virtual focus group feedback. https://doi.org/10.7910/DVN/ZBWSHS (Young et al., 2020)\n\nThis project contains the following extended data:\n\nExtended data.pdf (contains Appendix A: Example of Focus Group Questions and Appendix B: Example of Live Poll Results).\n\nPostdoc Academy Feedback Session Worksheet.pdf (Sent to participants prior to focus group.)\n\nPostdoc_Academy_Feedback_Session_Survey.pdf (Blank survey sent to participants within 2 weeks of completion of focus groups.)\n\nPostdoc_Academy_Post-Module_4_Survey.pdf (Completed by course participants at the end of Module 4 within “The Postdoc Academy: Succeeding as a Postdoc”.)\n\nPostdoc_Academy_Post-Course_Survey.pdf (Completed by course participants at the end of “The Postdoc Academy: Succeeding as a Postdoc”.)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAkyol Z, Garrison DR: The Development of a Community of Inquiry over Time in an Online Course: Understanding the Progression and Integration of Social, Cognitive and Teaching Presence. Journal of Asynchronous Learning Networks. 2008; 12(3-4). Reference Source\n\nAlund M, Emery N, Jarrett BJM, et al.: Academic ecosystems must evolve to support a sustainable postdoc workforce. Nat Ecol Evol. 2020; 4: 777–781. PubMed Abstract \| Publisher Full Text\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual Res Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nBrookfield SD: Becoming a Critically Reflective Teacher. San Francisco: Jossey-Bass. 1995. Reference Source\n\nClarke V, Braun V: Teaching thematic analysis: Overcoming challenges and developing strategies for effective learning. Psychologist. 2013; 26(2): 120–123. Reference Source\n\nDalsgaard C, Paulsen MF: Transparency in Cooperative Online Education. The International Review of Research in Open and Distributed Learning. 2009; 10(3). Publisher Full Text\n\nDavis G: Improving the Postdoctoral Experience: An Empirical Approach. Science and Engineering Careers in the United States: An Analysis of Markets and Employment. In: R.B. Freeman and D.L. Goroff (Eds.). Chicago: University of Chicago Press. 2009; 99–127. Reference Source\n\nForrestal SG, D’Angelo AV, Vogel LK: Considerations for and Lessons Learned from Online, Synchronous Focus Groups. Survey Practice. 2015; 8(3): 1–8. Publisher Full Text\n\nGibbs KD, McGready J, Griffin K: Career Development among American Biomedical Postdocs. CBE Life Sci Educ. 2015; 14(4): ar44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGunawardena CN, Zittle FJ: Social presence as a predictor of satisfaction within a computer‐mediated conferencing environment. Am J Distance Educ. 1997; 11(3): 8–26. Publisher Full Text\n\nHokanson SC, Grannan S, Greenler R, et al.: A Study of Synchronous, Online Professional Development Workshops for Graduate Students and Postdocs Reveals the Value of Reflection and Community Building. Innovative Higher Education. 2019; 44: 385–398. Publisher Full Text\n\nKankanhalli A, Tan BCY, Kwok-Kee W: Contributing knowledge to electronic knowledge repositories: an empirical investigation. MIS Quarterly. 2005; 29(1): 113–143. Publisher Full Text\n\nKite J, Phongsavan P: Insights for conducting real-time focus groups online using a web conferencing service [version 1; peer review: 2 approved with reservations]. F1000Res. 2017; 9(6): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrueger RA: Focus Groups: A Practical Guide for Applied Research. Thousand Oaks, CA: SAGE Publications. 1994.\n\nLobe B: Best Practices for Synchronous Online Focus Groups. In: R. Barbour and D. Morgan (Eds.) A New Era in Focus Group Research. London: Palgrave Macmillan. 2017; 227–250. Publisher Full Text\n\nMatthews KL, Baird M, Duchesne G: Using Online Meeting Software to Facilitate Geographically Dispersed Focus Groups for Health Workforce Research. Quantitative Health Research. 2018; 28(10): 1621–1628. Publisher Full Text\n\nNPA Core Competencies. (n.d.). Reference Source\n\nSweet C: Designing and conducting virtual focus groups. Quantitative Market Research. 2001; 4(3): 130–135. Publisher Full Text\n\nTates K, Zwaanswijk M, Otten R, et al.: Online focus groups as a tool to collect data in hard-to-include populations: examples from paediatric oncology. BMC Med Res Methodol. 2009; 9(15). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnderhill C, Olmsted MG: An Experimental Comparison of Computer-Mediated and Face-to-Face Focus Groups. Soc Sci Comput Rev. 2003; 21(4): 506–512. Publisher Full Text\n\nYoung C, Chesniak OM, Drane D, et al.: Improving the design of an online course with virtual focus group feedback. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/ZBWSHS" }	[ { "id": "92657", "date": "09 Sep 2021", "name": "Uranchimeg Tudevdagva", "expertise": [ "Reviewer Expertise e-learning", "evaluation model", "evaluation of e-learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper aims to figure out how positive constructive feedback from participants can influence and improve the offering of professional development programs.\nData collected from 13 focus groups which are reasonable for analyses and conclusions. In total 53 postdoc researchers took part in the offered course. For data collection, authors are using an online survey with support of a promotion method: Amazon card for free, which I found interesting, but not really usual for scientific data collection.\nTechnical writing of the paper is excellent. Transparency of used methods for evaluation and activities with focus groups is well done. It is easy to read and follow sections to understand the content of work.\nBased on collected data authors did analyze professional development programs and did some change in program with the aim to improve it. In summary, virtual groups for studying professional development programs have many advantages for participants.\nMost positive part of the paper is several ways to collect fundamental data from target groups. And excellent comments on data analysis. But some data is not really available for free, if the reader wants to follow all details.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "115651", "date": "18 Jan 2022", "name": "Cender Udai Quispe-Juli", "expertise": [ "Reviewer Expertise Online Courses", "Medical Education", "Digital Health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe bibliography reviewed is not up-to-date and gives the impression that a systematic review of previous research was not done. The argument developed in the introduction is not enough to support the idea that it is a new method, the investigation could be better justified.\nWithin the limitations, it must be recognized that the conduct of the participants may vary in digital environments, conditioning their participation and objectivity. It should also be recognized that focus group feedback is not the only method that could be used to improve an online course. Other indicators such as eye-tracking responses, cue points and hotspots within the platform where the course is located could be very helpful.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1191
https://f1000research.com/articles/8-1126/v1	18 Jul 19	{ "type": "Research Note", "title": "Anti-pathogenic potential of a classical ayurvedic formulation- Triphala", "authors": [ "Hinal Patel", "Foram Patel", "Vinit Jani", "Neha Jha", "Afsa Ansari", "Bhumika Paliwal", "Bharatsingh Rathod", "Dhruvi Patel", "Pooja Patel", "Vijay Kothari", "Hinal Patel", "Foram Patel", "Vinit Jani", "Neha Jha", "Afsa Ansari", "Bhumika Paliwal", "Bharatsingh Rathod", "Dhruvi Patel", "Pooja Patel" ], "abstract": "A classical ayurvedic polyherbal formulation namely Triphala was assessed for its anti-pathogenic potential against five different pathogenic bacteria. Virulence of four of them towards the model host Caenorhabditis elegans was attenuated (by 18-45%) owing to pre-treatment with Triphala (≤20 µg/ml). Triphala could also exert significant therapeutic effect on worms already infected with Chromobacterium violaceum, Serratia marcescens or Staphylococcus aureus. Prophylactic use of Triphala allowed worms to score 14-41% better survival in face of subsequent pathogen challenge. Repeated exposure to this formulation induced resistance in S. marcescens, but not in P. aeruginosa. It also exerted a post-extract effect (PEE) on three of the test pathogens. Triphala was able to modulate production of quorum sensing (QS)-regulated pigments in three of the multidrug-resistant gram-negative test bacteria. Haemolytic activity of S. aureus was heavily inhibited under the influence of this formulation. P. aeruginosa's lysozyme-susceptibility was found to increase by ~25-43% upon Triphala-pretreatment. These results validate therapeutic potential of one of the most widely used polyherbal ayurvedic formulations called Triphala.", "keywords": [ "Antimicrobial resistance (AMR)", "Quorum Sensing (QS)", "Triphala", "Polyherbal", "Post Extract Effect (PEE)", "Anti-virulence", "Lysozyme" ], "content": "Background\n\nAntibiotic-resistant bacterial infections are among the most serious public-health threats. Since the emergence and spread of antimicrobial resistance (AMR) is shrinking the utility spectrum of conventional bactericidal antibiotics, there is an urgent need for discovery and development of novel anti-virulence formulations. Traditional Medicine (TM) systems like Ayurveda offer several sophisticated formulations for a variety of disease conditions. One such classical ayurvedic formulation with a long history of safe use is Triphala. Triphala is a polyherbal formulation containing three myrobalans fruits i.e. Phyllanthus emblica, Terminalia chebula, and Terminalia bellerica (Patwardhan et al., 2015). Triphala is prescribed as a general health promoter, for management of metabolic disorders, dental and skin problems, and for wound management. It has been reported to be active against bacterial pathogens of urinary tract (Bag et al., 2013), and as an anticaries agent for control of gum infections (Bhattacharjee et al., 2015; Prakash & Shelke, 2014). Though many popular formulations like Triphala have been used historically in TM and as a household remedy, their validation through modern scientific methods is necessary for their wider acceptance in modern medicine (Kothari, 2018). This study aimed to investigate the anti-pathogenic efficacy of Triphala against five different pathogenic bacteria.\n\n\nMethods\n\nTriphala formulation (TF) (Emami Ltd; batch no. EM0029; Proportion of 3 constituent plant species: 1:1:1) was purchased from a local market. For assay purpose, 150 mg of this formulation was suspended in 5 ml of DMSO (Merck, Mumbai), followed by vortexing for 15 min. Then it was centrifuged at 8,000 rpm for 30 min at ambient temperature, and resulting supernatant was collected in a sterile glass vial (15 ml; Borosil) and stored under refrigeration till further use. Remaining pellet was subjected to drying in an oven at 70-80°C until the solvent was completely evaporated, followed by weighing of the dried plant material. Subtracting the latter from the initial weight of 150 mg, the concentration of test formulation in supernatant was calculated to be 22.94 mg/ml. This way the whole formulation was found to contain 70% DMSO soluble fraction, which was used for our experiments.\n\nChromobacterium violaceum (MTCC 2656), Serratia marcescens (MTCC 97), Staphylococcus aureus (MTCC 737), and Streptococcus pyogenes (MTCC 1924) were procured from Microbial Type Culture Collection (MTCC), Chandigarh. Pseudomonas aeruginosa was available in our institutional culture collection. All the three gram-negative bacteria used in this study were multidrug resistant, and their antibiogram has previously been reported by us (Joshi et al., 2019; Patel et al., 2019a). Additionally, C. violaceum and S. marcescens strains mentioned here have been reported by us as beta-lactamase producers (Sarvaiya & Kothari, 2017).\n\nIn vivo efficacy of Triphala against bacterial infections was tested in the nematode host Caenorhabditis elegans (N2-Bristol strain; maintained at the Institute of Science, Nirma University). Maintenance and synchronization of the worm population was done as previously described in Joshi (2019). Worms were maintained on NGM [Nematode Growing Medium; 2.5 g/L peptone (HiMedia, RM001-500G), 3 g/L NaCl (HiMedia, MB023-500G), 1 M CaCl2 (HiMedia, GRM534-500G), 1 M MgSO4 (Merck, 1.93645.0521), 5 mg/mL cholesterol (HiMedia, TC101-5G), 1 M phosphate buffer of pH 6, 17 g/L agar-agar (HiMedia, GRM666-500G)] agar plate with E. coli OP50 (LabTIE B.V. OP50 V.2; batch # 002, JR Rosmalen, the Netherlands) as food. For synchronization of the worm population, adult worms from a 4–5 days old NGM plate were first washed with distilled water, and then treated with 1 mL of bleaching solution [1N NaOH (HiMedia MB095-100G) + 4% NaOCl (Merck 61842010001730) + water in 1:1:3 proportion], followed by centrifugation (22°C; 1,500 rpm) for 1 min. Eggs in the resultant pellet were washed with sterile distilled water, and then transferred onto a new NGM plate seeded with E. coli OP50. L3-L4 stage worms appearing on this plate after 2–3 days of incubation at 22°C were used for further experiments.\n\nThree different types of in vivo assays were done as under, employing the methodology described in reference cited in parenthesis following the assay name:\n\nAnti-infective assay (Patel et al., 2018a): Triphala exposed-pathogenic bacteria were allowed to infect C. elegans (L3-L4 stage), and their capacity to kill the worm population was compared with their Triphala-unexposed counterparts, over a period of 5 days.\n\nProphylactic assay (Patel et al., 2018b): Triphala-fed worms were challenged with pathogenic bacteria (previously not exposed to the test formulation), and their ability to survive in face of pathogen challenge was compared with their Triphala-unfed counterparts. C. elegans worms maintained on NGM were kept unfed for 24 h prior to being used for experiments. These worms were then fed with TF by mixing required concentration of this formulation (100 µL) with M9 medium (800 µL) and placed in a 24-well plate (non-treated polystyrene plates, sterile; HiMediaTPG24-1X50NO) containing 10 worms per well. Duration of exposure of worms to TF was kept to 96 h, followed by addition of pathogenic bacteria (100 µL of bacterial suspension with OD764= 1.50 measured with Agilent Cary 60 UV-Vis spectrophotometer). Appropriate controls i.e. worms previously not exposed to TF, but exposed to pathogenic bacteria; worms exposed neither to TF nor bacteria; and worms exposed to TF, but not to bacterial pathogens, were also included in the experiment. Incubation was carried out at 22°C. Number of dead vs. live worms were counted every day for 5 days by putting the plate (with lid) under a light microscope (4X; Catalyst Biotech CatScope CS-U207T). Straight worms were considered to be dead. Plates were gently tapped to confirm lack of movement in the apparently-dead worms. On the last day of the experiment, when plates could be opened, their death was also confirmed by touching them with a straight wire, wherein no movement was taken as confirmation of death.\n\nTriphala as a post-infection therapy (Patel et al., 2019b): Worms already infected with pathogenic bacteria (not previously exposed to the test formulation) were treated with Triphala to see whether the test formulation can exert any therapeutic effect on already infected worms. Assay methods remained the same as described in previous section, except that TF was added into assay wells after allowing bacteria either for 6 h or 24 h to establish infection.\n\nCatechin (Sigma Aldrich; C1251-5G) and standard antibiotics (HiMedia; Ampicillin CMS645-1G; Gentamicin TC026-1G; Chloramphenicol CMS218-5G; Vancomycin CMS217-500MG) were used as positive controls. Catechin was employed at 100 µg/ml, whereas different sub-MIC concentrations (0.1–5 µg/ml) of the antibiotics were used against different organisms as per their susceptibility.\n\nVideos of some of the in vivo assays were captured on the fifth day of the experiment, using an inverted microscope (Nikon Eclipse Ti) under 4X objective lens, wherein 100 µl of the liquid content from 24-well assay plate was transferred onto a large cover slip for observation and video capturing [see extended data (Patel et al., 2019c)].\n\nAfter confirming the in vivo anti-pathogenic efficacy of the Triphala formulation, we performed following in vitro assays to gain insight into interaction of this formulation with the pathogenic bacteria, as per the methodology described in respective references mentioned in the parenthesis:\n\na. Broth dilution assay (Joshi et al., 2016) to investigate effect of Triphala on bacterial growth and quorum sensing (QS)-regulated pigment production: C. violaceum, and S. marcescens were inoculated in nutrient broth (HiMedia MV002-500G) supplemented with TF. Media used for S. aureus and P. aeruginosa were Staphylococcus broth (HiMedia M578-500G) and Pseudomonas broth [10 g/l potassium sulfate (SRL 44277), 1.4 g/l magnesium chloride (Merck 1.9366.30521), 16 g/l peptone (HiMedia RM001-500G)] respectively. S. pyogenes was grown in BHI (brain heart infusion; HiMedia MV210-500G) broth. Following incubation, cell density was measured at 750 nm (Biorad 680). Pigment from these culture broths were extracted as previously described by us in Joshi et al. (2016). Cell pellets of C. violaceum, S. marcescens, and S. aureus were dissolved in DMSO [(Merck 1.07046.2521), acidified methanol [4 ml HCl (HiMedia AS003-500ML) into 96 ml of methanol], and methanol (Merck 1.94516.2521) respectively. This allowed their pigments to be extracted in the solvent applied. In case of P. aeruginosa, pigment extraction was achieved by mixing chloroform (Merck 1.67024.0521) with culture broth (2:1 ratio). Quantification of each pigment was done at the wavelength nearest to its λmax, available in the microplate reader (Biorad 680) used by us.\n\nb. Effect of Triphala on biofilm formation and its possible potential to eradicate pre-formed biofilm was assessed through crystal violet assay (Patel et al., 2013); and its effect on biofilm viability was assessed through MTT assay (Trafny et al., 2013). For the crystal violet assay, the biofilm-containing tubes (after discarding the inside liquid) were washed with PBS in order to remove all nonadherent bacteria and air-dried for 15 min. Then, each of the washed tubes was stained with 1.5 mL of aqueous crystal violet solution (0.4%; SRL 54862-9) for 30 min. Afterwards, each tube was washed twice with 2 ml of sterile distilled water and immediately destained with 1500 μL of ethanol (95%). After 45 min of destaining, 1 mL of destaining solution was transferred into separate tubes and read at 570 nm (Biorad 680). For the MTT assay, the biofilm-containing tubes (after discarding the inside liquid) were washed with PBS in order to remove all nonadherent bacteria and air-dried for 15 min. Then, 900 µL of minimal media was added into each tube, followed by addition of 100 μL of 0.3% MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HiMedia MB186-100MG). After 2 h incubation at 37°C, resulting purple formazan derivatives were dissolved in DMSO and measured at 570 nm (Biorad 680).\n\nc. Effect of Triphala on haemolytic potential of the test pathogens (Neun et al., 2015): Small volume of human blood required for this assay was obtained from the authors, who each gave their written informed consent. The use of this blood was approved by the Institutional Ethics Committee of the Institute of Science, Nirma University (approval no: EC/NU/18/IS/4). Blood collection was executed by one of the authors (AA) on three different times in heparinized vials. OD750 of the overnight grown (in presence or absence of TF) culture was standardized to 1.00. Cell-free supernatant was prepared by centrifugation at 15,300 g for 10 min. 10 μl of human blood was incubated with this cell-free supernatant for 2 h at 37°C, followed by centrifugation at 800 g for 15 min. OD of the supernatant was read at 490 nm, to quantify the amount of hemoglobin released. 1% Triton X-100 (CDH, New Delhi; CDH030632) was used as a positive control. Phosphate buffer saline was used as a negative control.\n\nd. Effect of Triphala on lysozyme-susceptibility of test pathogens: Bacterial cells were first inoculated in a TF-containing media for 24 h, and then the cell pellet was separated by centrifugation [15,000 rpm (21130 g) for 15 min] to be resuspended into phosphate buffer saline (PBS; pH 7.4), so as to attain OD750=1 (Biorad 680). 200 µl of this bacterial suspension was mixed with lysozyme (750 µg/ml; Sigma Aldrich L6876-1G) prepared in PBS, and then incubated for 24 h at appropriate temp for each organism. At the end of incubation OD750 was measured.\n\nBifidobacterium bifidum (NCDC 255), Enterococcus faecium (NCIM 5366), and Lactobacillus plantarum (MTCC 2621) were grown in Lactobacillus MRS broth (HiMedia GM369-500G) containing TF, in screw capped tubes at 37°C for 22-24 h. For B. bifidum, this medium was supplemented with 0.05% cysteine (HiMedia PCT0305-25G). Effect of TF (10–100 µg/ml) on these bacteria was interpreted by comparing their cell density [OD655 measured with microplate reader (Biorad 680)] in TF-supplemented media to that in TF-free media.\n\nAll the experiments were performed in triplicate, and measurements are reported as mean ± standard deviation (SD) of 3 independent experiments. Statistical significance of the data was evaluated by applying t-test using Microsoft Excel® 2013. p values ≤0.05 were considered to be statistically significant. Trial version of GraphPad Prism 7 was used to make Kaplan-Meier survival curve for worms.\n\n\nResults\n\nAnti-infective assay. When all the five pathogens were pre-treated with Triphala (0.5-100 µg/ml) before being allowed to attack C.elegans, Triphala formulation (TF) was able to attenuate virulence of all test pathogens except S. pyogenes at ≤20 µg/ml [Figure 1; underlying data (Patel et al., 2019c)]. Worms challenged with TF-treated pathogens demonstrated 18–45.50% better survival than those challenged with TF-unexposed pathogens. Effect of catechin and standard antibiotics (both used as positive controls) on bacterial virulence is shown in Figure 2 [underlying data (Patel et al., 2019c)].\n\nTF-treatment attenuated virulence of four of the test pathogens towards C. elegans. DMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of any of the bacterium towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. (A) TF at 0.5 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, and 100 µg/ml allowed 32%±5.10, 29%*±9.60, 41.9%±2.40, 38.4%±7.10, 39.9%±9.40, and 41.4%±7.10 better survival of the worm population respectively, when challenged with S. marcescens. Also see videos a-b submitted as part of extended data. (B) TF at 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml allowed 18%±5.01, 40%±7.01, 45.5%±0, and 45%±5.00 better survival of the worm population respectively, when challenged with P. aeruginosa. (C) TF at 20 µg/ml, 50 µg/ml and 100 µg/ml allowed 31.5%±2.35, 41.5%±2.35and 44.5%±14.14 better survival of the worm population respectively, when challenged with S. aureus. (D) TF-treatment did not attenuate virulence of S. pyogenes towards the worms. (E) TF at 0.5µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml allowed 31%±5.77, 29.5%±8.81, 29%±1.92, 29%±1.92, 32.3%±1.92, and 32.3%±5.09 better survival of the worm population respectively, when challenged with C. violaceum. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nCatechin was able to reduce virulence of different test bacteria towards C. elegans by 50–100% (p≤0.05). Various standard antibiotics at sub-MIC level could reduce bacterial virulence by 55–100% (p≤0.05).\n\nAfter confirming the anti-infective activity of TF, we investigated (as described in Patel et al., 2019a) whether this formulation exerts any post-extract effect (PEE - https://doi.org/10.32388/359873) on the test pathogens i.e. whether the virulence-attenuating effect suffered by the parent bacterial culture is retained even in their daughter population never receiving any direct exposure to TF. When the TF-treated bacteria were subsequently subcultured on TF-free media, their daughter populations were unable to exert virulence at par with that of control (DMSO-treated parent culture). In case of P. aeruginosa and S. aureus, this PEE lasted up to the second subculturing, whereas in the case of S. marcescens PEE lasted until first subculturing [Figure 3; underlying data (Patel et al., 2019c)].\n\nTF-treatment reduces the virulence of all the test pathogens towards C. elegans even after subculturing of cells in TF-free media. DMSO present in the ‘vehicle control’ at 0.5% v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. (A) S. marcescens obtained after first subculturing on TF-free media were able to kill 38%±4.71 lesser worms than control; (B) P. aeruginosa and obtained after first and second subculturing on TF-free media were able to kill 18.5%±2.35 and 15.5%±2.35 lesser worms respectively, than control; (C) S. aureus obtained after first and second subculturing on TF-free media were able to kill 31.5%*±2.35 and 23%±0 lesser worms respectively, than control. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nTF as a post-infection therapy. To test the therapeutic efficacy of TF in already-infected worm population, we first allowed different pathogenic bacteria (not previously exposed to TF) to infect C. elegans either for 6 h or 24 h, and then exposed the infected worms to two different concentrations of TF. TF failed to exert any therapeutic effect on worms infected with P. aeruginosa. However, it could exert significant (p≤0.05) therapeutic effect on worms infected with C. violaceum or S. marcescens. Against S. aureus, TF was effective only if the worms were given TF-treatment early (i.e. 6 hour-post infection) [Figure 4; underlying data (Patel et al., 2019c)]. TF could also not rescue the worms if they already had a mixed infection (S. aureus and P. aeruginosa).\n\nDMSO (0.5%v/v) did not affect survival of pre-infected worms. DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. A1–D1: TF employed 6 h post-infection A2–D2: TF employed 24 h post-infection (A1) TF at 10 µg/ml, 50 µg/ml used as therapy for already S. marcescens infected C. elegans after 6 h incubation conferred 31.5%±2.35 and 28.5%±2.35 survival benefit, respectively; (A2) TF at 10 µg/ml, 50 µg/ml used as therapy for already S. marcescens infected C. elegans after 24 h incubation conferred 11.5%±2.35 and 13%* survival benefit, respectively; (B1–B2):TF could not rescue P. aeruginosa-infected worms when tested as post-infection remedy. (C1) TF at 20 µg/ml, 50 µg/ml used as therapy for already S. aureus infected C. elegans after 6 h incubation conferred 11.5%*±2.35 and 21.5%±2.35 survival benefit, respectively; (C2) TF could not rescue S. aureus infected worms when tested as post-infection remedy. (D1) TF at 10 µg/ml, 50 µg/ml used as therapy for already C. violaceum infected C. elegans after 6 h incubation conferred 18.5%±2.35 and 28.5%±2.35 survival benefit, respectively; (D2) TF at 10 µg/ml, 50 µg/ml used as therapy for already C. violaceum infected C. elegans after 24 h incubation conferred 22%±2.35 and 28.5%±2.35 survival benefit, respectively. (E) TF could not rescue the worms in face of mix-culture infection by S. aureus and P. aeruginosa, when tested as post-infection remedy. Survival benefit refers to the difference between number of worms surviving in experimental and control wells. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nProphylactic potential of TF. To investigate whether previous feeding with TF can make the worm population tolerate subsequent challenge with pathogenic bacteria (not treated with TF) better; worms were first maintained in a TF-containing M9 buffer for 96 h, and then challenged with different bacterial pathogens. Such TF-fed worms scored 14.50–41.50% better survival in face of pathogen challenge [Figure 5; underlying data (Patel et al., 2019c)]. However, TF did not confer any prophylactic benefit on the worm population against mix-culture challenge of P. aeruginosa and S. aureus. Since prophylactic activity of any formulation can be said to stem mainly from its effect on the host, we also compared whether TF imparts any extension of longevity on the worm. Worms fed with TF (10-20 µg/ml) registered marginally better survival up to 11 days (Figure 6).\n\nPre-treatment of worms with DMSO (0.5%v/v) did not alter their susceptibility to subsequent challenge with pathogenic bacteria. DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. Among positive controls, catechin (100 μg/ml) pre-treatment conferred 23%±0 protection on worm population against subsequent S. marcescens, P. aeruginosa, S. aureus, and C. violaceum challenge; (A) Ampicillin (5 μg/ml) pre-treatment conferred 26%±0 protection on worm populations against subsequent S. marcescens challenge; TF (100 µg/ml) pre-treatment conferred 18%±2.35 protection on fifth day worm population against subsequent S. marcescens challenge; (B) Gentamicin (0.5 μg/ml) pre-treatment conferred 26% protection on worm populations against subsequent P. aeruginosa challenge; TF pretreatment at 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, conferred 18%±0, 33%±2.35, 38%±2.35, 41.5%±0 and 34.5%±2.35 protection on worm population against subsequent P. aeruginosa challenge (C) Vancomycin (0.1 μg/ml) pre-treatment conferred 26% protection on worm population, TF pretreatment at 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, conferred 14.5%±2.35, 13%±4.71, 11.5%±2.35, 21.5%±2.35, and 33%±0 protection on worm population against S. aureus (D) Chloramphenicol (0.5 μg/ml) pre-treatment conferred 26% protection on worm population against C. violaceum, TF pre-treatment at 0.5 µg/ml, 5 µg/ml, 10 µg/ml, 20 µg/ml, 50 µg/ml, 100 µg/ml, conferred 14.5%±0, 19.5%±4.71, 14.5%±2.35, 28%±7.07, 34.5%± 2.35 and 35.5%±0 protection on worm population against C. violaceum. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nWorms fed with TF (10 µg/ml and 20 µg/ml) scored 11.5%±1.20, and 16%±0.96 better survival on 11th day. All worms (not fed with TF) in control were dead by the 11th day. DMSO (0.5%v/v) and TF at tested concentration had no effect on worm longevity. p≤0.001; TF: Triphala Formulation.\n\nRepeated exposure of test pathogens to TF. Since one of the major challenges with even the most potent antimicrobial agents/formulations is development of resistance against them by the pathogen populations, we tested whether repeated exposure of the test pathogens to TF can induce any resistance in them. For this, we subcultured two of the gram-negative test pathogens (P. aeruginosa and S. marcescens) in TF (50 µg/ml)-containing broth for 10 subsequent times, and then the 'TF-habituated' cultures thus obtained were tested for their virulence towards the nematode host. Repeated TF-exposure was found to induce resistance in S. marcescens [Figure 7A; underlying data (Patel et al., 2019c)]. Though TF-habituated P. aeruginosa could kill more worms than its counterpart receiving single TF-exposure, it still could not kill as many worms as TF-unexposed P. aeruginosa [Figure 7B; underlying data (Patel et al., 2019c)]. These results indicate that it may be difficult for the pathogenic bacteria to develop complete resistance against polyherbal formulations like Triphala, but not impossible. Though our previous results on other polyherbal formulations (Joshi et al., 2019; Patel et al., 2019a) and multicomponent crude plant extracts (Joshi, 2019) have indicated that the probability of pathogens developing resistance against multi-component anti-virulence preparations is low, such a probability can certainly not be ruled out (Kalia et al., 2014; Singh, 2014)\n\nDMSO present in the ‘vehicle control’ at 0.5%v/v did not affect virulence of the bacterium towards C. elegans; DMSO (0.5%v/v) and TF at tested concentrations showed no toxicity towards the worm. (A) C. elegans challenged with TF (50 μg/ml) treated S. marcescens conferred 35%±2.35 survival benefit. C. elegans could not conferred survival benefit when challenged with S. marcescens which subcultured 5 times or 10 times on TF containing media could not conferred survival benefit. (B) P. aeruginosa obtained after 5 and 10 subculturings in TF (50 μg/ml)-containing media were able to kill 21.5%±2.35 and 21%±7.07 lesser worms respectively, as compared to control (DMSO-treated) bacterial population. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nSince TF showed significant in vivo anti-pathogenic potential in the C. elegans model, we performed various in vitro experiments to gain insight into its interaction with the target pathogens. TF was able to modulate production of quorum sensing (QS)-regulated pigments in all the three gram-negative bacteria [Figure 8; underlying data (Patel et al., 2019c)]. It did so with S. marcescens without affecting its growth, which is characteristic of an ideal anti-virulence agent i.e. attenuation of virulence without exerting any selection pressure on the susceptible pathogen. However, TF did exert a growth-inhibitory effect on P. aeruginosa, wherein its IC50 was observed to be near 50 µg/ml. The quorum modulatory effect of TF on pigment production in P. aeruginosa was observed not to be amenable to be described by a linear dose-response curve. It seems to fall within the realm of hormesis (Calabrese, 2004). For example, production of both pigments was not affected maximally at the highest test concentration. Pyocyanin production was inhibited more at 0.5 µg/ml than at 20 µg/ml. Effect of TF on pyoverdine production followed a linear threshold model within concentration range of 0.5–50 µg/ml, but it took an inverted-U shape over 20-100 µg/ml. Though the exact mechanism responsible for a non-linear dose-response relationship is not known, it may be the varying magnitude of bacterial adaptive response at different concentrations of the test agent, which generates such non-linear response curves (Lushchak, 2014).\n\nBacterial cell density was quantified as OD750. DMSO (0.5%v/v) in the vehicle control did not affect growth and pigment production in any of the test pathogens. (A) S. marcescens: OD of prodigiosin was measured at 490 nm, and Prodigiosin Unit was calculated as the ratio OD490/OD750 (an indication of prodigiosin production per unit of growth). Catechin (100 µg/ml) inhibited prodigiosin production by 13.05%±0.10 without affecting bacterial growth; Ampicillin (5 µg/ml) inhibited growth and prodigiosin production by 8.48%*±0.02 and 40.60%±0.23, respectively. (B) P. aeruginosa: OD of pyoverdine and pyocyanin was measured at 405 nm and at 490 nm. Pyoverdine Unit and Pyocyanin Unit was calculated as the ratio OD405/OD750 and OD490/OD750 (an indication of pyoverdine and pyocyanin production per unit of growth). Catechin (100 µg/ml) inhibited, pyoverdine and pyocyanin production by 3.85%± 0.38 and 12.74%± 2.60 without affecting bacterial growth; Gentamicin (0.5 µg/ml) inhibited, pyoverdine, pyocyanin production by 10.53%±2.07 and 57.93%±6.47 without affecting bacterial growth; (C) S. aureus: OD of staphyloxanthin was measured at 450 nm, and Staphyloxanthin Unit was calculated as the ratio OD450/OD750 (an indication of staphyloxanthin production per unit of growth). Catechin (100 µg/ml) and vancomycin (0.1 µg/ml) did not affect growth as well as staphyloxanthin pigment production. (D) C. violaceum: OD of violacein was measured at 570 nm, and Violacein Unit was calculated as the ratio OD570/OD750 (an indication of violacein production per unit of growth). Catechin (100 µg/ml) not affect growth as well as violacein pigment productions; Chloramphenicol (0.5 µg/ml) inhibited growth by 40.31%±0.44 without affecting violacein pigment production. (E) S. pyogenes: TF and catechin (100 µg/ml) did not affect the growth when measured as OD655; Chloramphenicol (0.5 µg/ml) inhibited growth by 7.56%*±3.46. p<0.05, p<0.01, p<0.001; TF: Triphala Formulation; QS: Quorum Sensing.\n\nWe also tested the effect of TF on two important virulence traits of the bacterial pathogens i.e. haemolytic activity, and biofilm. Though TF could not curb haemolytic activity of any of the gram-negative bacteria, this activity of S. aureus was heavily inhibited under the influence of TF [Figure 9; underlying data (Patel et al., 2019c)]. While P. aeruginosa biofilm was not affected by TF, TF was able to reduce biofilm formation by S. marcescens, and S. aureus. When TF was applied on pre-formed bacterial biofilms, it seemed to enhance synthesis of the biofilm matrix material (quantified thorough crystal violet assay), and also the metabolic activity (measured in terms of organism's ability to reduce MTT) of the bacterial biofilm [Figure 10; underlying data (Patel et al., 2019c)]. It may be speculated that TF-treatment induces stress in the bacterial population residing in biofilm form, and this causes the bacteria to mount stress-response. Slow metabolism is a general characteristic of bacterial biofilms (Singh et al., 2017), but TF seems to have forced the biofilms of two of our test bacteria to enhance the rate of their metabolic activity, as well as synthesis/secretion of biofilm matrix components (e.g. polysaccharides, proteins, and extracellular DNA). Enhanced production of exopolysaccharide and e-DNA is believed to occur in stressed bacterial populations (Chang et al., 2007; Zatorska et al., 2018). Sub-inhibitory concentrations of beta-lactam antibiotics have been reported to induce extracellular DNA release and biofilm formation in some S. aureus strains (Kaplan et al., 2012).\n\nTF had no effect on hemolytic activity of S. marcescens (A), P. aeruginosa (B), and C. violaceum (D). However, it curbed haemolytic potential of S. aureus notably. Hemoglobin released as a result of haemolysis was quantified as OD490; 1% triton (OD490 = 1.2), and PBS (pH 7.4) were used as positive and negative control respectively; p<0.05, *p<0.001; TF: Triphala Formulation; PBS: Phosphate Buffer Saline.\n\nCrystal violet assay was performed to quantify biofilm formation or eradication, wherein amount of this dye retained by the biofilm was read at 570 nm after extracting it in ethanol. MTT assay was performed to quantify biofilm viability (metabolic activity), wherein change in colour of the MTT dye owing to bacterial metabolism was read at 570 nm. Catechin (100 µg/ml) for all test bacteria, ampicillin (5 µg/ml) for S. marcescens, gentamicin (0.5 µg/ml) for P. aeruginosa, and vancomycin(0.1 µg/ml) for S. aureus were used as positive controls.\n\n(A)Effect of TF on S. marcescens biofilm. Catechin inhibited S. marcescens biofilm formation by (29.80±9.38)%, eradicated pre-formed biofilm by (49.60 ±12.00)%, and reduced metabolic activity of biofilm by (86.20 ±0.88)% *. Ampicillin inhibited all three by 29.25* ±7.30, 54.23* ±3.67, 86.11* ±0.21 for S. marcescens. (B) Effect of TF on P. aeruginosa biofilm. Catechin inhibited P. aeruginosa biofilm formation by (22.18±1.16)%*, eradicated pre-formed biofilm by (30.54 ±3.50)%, and enhanced metabolic activity of biofilm by (32.27 ± 4.74)%*. Gentamicin did not affect biofilm formation, eradicated pre-formed biofilm by (23.27±4.91)%, reduced metabolic activity of biofilm by (123.99 ±26.81)% . (C) Effect of TF on S. aureus biofilm. Catechin inhibited S. aureus biofilm formation by (26.24±7.35)%, enhanced pre-formed biofilm by (22.54 ±10.90)%, and enhanced metabolic activity of biofilm by (177.71 ± 16.49)% . Vancomycin inhibited biofilm formation by (41.53±5.49)% , eradicate pre-formed biofilm by (42.37±11.21)%, enhanced metabolic activity of biofilm by (70.90 ±5.10)% . p<0.05, p<0.01, p<0.001; TF: Triphala Formulation.\n\nDuring the host-pathogen interaction, host defense mechanisms play a determinant role in deciding the outcome of this interaction. Since lysozyme is an important component of the innate defense machinery of human immune system against invading microbes (Herbert et al., 2007), we also studied whether TF can have any effect on susceptibility of the test pathogens to lysozyme. TF-treated cells of S. marcescens and S. aureus were found to suffer marginal (albeit statistically significant; p≤0.05) increase in their susceptibility to lysis by lysozyme. P. aeruginosa's lysozyme-susceptibility was found to increase heavily (by ~25-43%) upon TF-pretreatment [Figure 11; underlying data (Patel et al., 2019c)].\n\n(A) S. aureus; (B) S. marcescens; (C) P. aeruginosa p≤0.05 ***p≤0.001.\n\nMost conventional antibiotics suffer from an inherent limitation of not being selectively inhibitory to pathogenic bacteria, and they simultaneously inhibit resident bacterial members of the human microbiome; which may lead to gut dysbiosis (Wipperman et al., 2017). Thus an ideal anti-pathogenic formulation should exert anti-pathogenic effects without inhibiting indigenous members of human microbiome. We tested TF's effect on three such bacteria (Enterococcus faecium, Bifidobacterium bifidum, and Lactobacillus plantarum) which are part of human microbiome, and also used as probiotic strains. Though TF did not exert any prebiotic potential by promoting growth of the probiotic bacteria, it also had no negative effect on them [Extended data: Figure S1 (Patel et al., 2019c)].\n\n\nConclusion\n\nThis study has found the classical Triphala formulation to possess significant anti-infective potential against various gram-positive and gram-negative pathogenic bacteria. It was also found to be efficacious as a post-infection therapy as well as a prophylactic measure against bacterial infection. Triphala can be said to possess a broad-spectrum of anti-pathogenic activity, which seems to partly arise from its ability to interfere with bacterial quorum-sensing. Its prophylactic efficacy indicates that it is not only exerting inhibitory effect on the susceptible bacteria, but also beneficial effect on the host worm, and thus can be described as a combination of immunomodulatory and anti-pathogenic activities in one formulation. Exerting such combined efficacy without displaying any negative effect on beneficial members of human microbiome are key attributes for 21st century antimicrobials (Laxminarayan et al., 2013). Further investigation for elucidating the molecular mechanisms associated with the biological effects of Triphala are warranted, with special emphasis on its role in combating AMR. Such traditional medicine polyherbal formulations need not necessarily be thought of as replacement of conventional antibiotic treatments, but more realistically as adjunctive therapies boosting our efforts to tackle AMR effectively.\n\n\nData availability\n\nFigshare: Anti-pathogenic potential of a classical ayurvedic formulation- Triphala. https://doi.org/10.6084/m9.figshare.8052143.v2 (Patel et al., 2019c)\n\nRaw data_Figures 1-11_S1.rar\n\nFigshare: Anti-pathogenic potential of a classical ayurvedic formulation- Triphala. https://doi.org/10.6084/m9.figshare.8052143.v2 (Patel et al., 2019c)\n\nThis project contains the following extended data:\n\nVideo (a).avi (Video of C. elegans challenged with S. marcescens)\n\nVideo (b).avi (Video of C. elegans exposed to TF-treated S. marcescens)\n\nFigure S1.jpg (effect of TF treatment on probiotic bacterial strains)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis work was supported by Nirma Education & Research Foundation (NERF).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nAuthors thank Nirma Education & Research Foundation (NERF), Ahmedabad for financial and infrastructural support; and Virupakshi Soppina (IIT-Gn) for imaging of C. elegans.\n\n\nReferences\n\nBag A, Bhattacharyya SK, Pal NK, et al.: Antibacterial potential of hydroalcoholic extracts of triphala components against multidrug-resistant uropathogenic bacteria--a preliminary report. Indian J Exp Biol. 2013; 51(9): 709–714. PubMed Abstract\n\nBhattacharjee R, Nekkanti S, Kumar NG, et al.: Efficacy of triphala mouth rinse (aqueous extracts) on dental plaque and gingivitis in children. J Investig Clin Dent. 2015; 6(3): 206–210. PubMed Abstract \| Publisher Full Text\n\nCalabrese EJ: Hormesis: from marginalization to mainstream: a case for hormesis as the default dose-response model in risk assessment. Toxicol Appl Pharmacol. 2004; 197(2): 125–136. PubMed Abstract \| Publisher Full Text\n\nChang WS, van de Mortel M, Nielsen L, et al.: Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under water-limiting conditions. J Bacteriol. 2007; 189(22): 8290–8299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerbert S, Bera A, Nerz C, et al.: Molecular basis of resistance to muramidase and cationic antimicrobial peptide activity of lysozyme in staphylococci. PLoS Pathog. 2007; 3(7): e102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoshi C: Investigation on anti-pathogenic potential of Panchvalkal and Punica grantum peel extract against certain human-pathogenic bacteria. Doctoral thesis. Nirma University, Ahmedabad, India. 2019. Reference Source\n\nJoshi C, Kothari V, Patel P: Importance of Selecting Appropriate Wavelength, While Quantifying Growth and Production of Quorum Sensing Regulated Pigments in Bacteria. Recent Pat Biotechnol. 2016; 10(2): 145–152.. PubMed Abstract \| Publisher Full Text\n\nJoshi C, Patel P, Palep H, et al.: Validation of the anti-infective potential of a polyherbal 'Panchvalkal' preparation, and elucidation of the molecular basis underlining its efficacy against Pseudomonas aeruginosa. BMC Complement Altern Med. 2019; 19(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalia VC, Wood TK, Kumar P: Evolution of resistance to quorum-sensing inhibitors. Microb Ecol. 2014; 68(1): 13–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaplan JB, Izano EA, PrernaGopal MT, et al.: Low levels of β-lactam antibiotics induce extracellular DNA release and biofilm formation in Staphylococcus aureus. mBio. 2012; 3(4): e00198–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKothari V: Validation of Traditional Medicinal Practices through Modern Scientific Tools and Techniques. Curr Pharmacogenomics Person Med. 2018; 16(1): 3–3. Publisher Full Text\n\nLaxminarayan R, Duse A, Wattal C, et al.: Antibiotic resistance-the need for global solutions. Lancet Infect Dis. 2013; 13(12): 1057–1098. PubMed Abstract \| Publisher Full Text\n\nLushchak VI: Dissection of the hormetic curve: analysis of components and mechanisms. Dose-Response. 2014; 12(3): 466–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeun BW, Ilinskaya AN, Dobrovolskaia MA: Analysis of hemolytic properties of nanoparticles. NCL method ITA-1 Version, 1. 2015. Reference Source\n\nPatel P, Joshi C, Funde S, et al.: Prophylactic potential of a Panchgavya formulation against certain pathogenic bacteria [version 1; peer review: 3 approved]. F1000Res. 2018b; 7: 1612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel P, Joshi C, Kothari V: Antipathogenic Potential of a Polyherbal Wound-Care Formulation (Herboheal) against Certain Wound-Infective Gram-Negative Bacteria. Adv Pharmacol Sci. 2019a; 2019: 1739868. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel P, Joshi C, Kothari V: Anti-Pathogenic Efficacy and Molecular Targets of a Polyherbal Wound- Care Formulation (Herboheal) Against Staphylococcus aureus. Infect Disord Drug Targets. 2019b; 19(2): 193–206. PubMed Abstract \| Publisher Full Text\n\nPatel P, Joshi C, Palep H, et al.: Anti-infective potential of a quorum modulatory polyherbal extract (Panchvalkal) against certain pathogenic bacteria. J Ayurveda Integr Med. 2018a. Publisher Full Text\n\nPatel H, Patel F, Jani V, et al.: Anti-pathogenic potential of a classical ayurvedic formulation- Triphala. figshare. Dataset. 2019c. http://www.doi.org/10.6084/m9.figshare.8052143.v2\n\nPatel I, Patel V, Thakkar A, et al.: Tamarindus indica (Cesalpiniaceae), and Syzygium cumini (Myrtaceae) seed extracts can kill multidrug resistant Streptococcus mutans in biofilm. J Nat Med. 2013; 13(2): 81–94. Reference Source\n\nPatwardhan B, Mutalik G, Tillu G: Integrative approaches for health: biomedical research, ayurveda and yoga. Academic Press (Chapter 9), 2015; 241–242. Publisher Full Text\n\nPrakash S, Shelke AU: Role of Triphala in dentistry. J Indian Soc Periodontol. 2014; 18(2): 132–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarvaiya N, Kothari V: Audible Sound in Form of Music Can Influence Microbial Growth, Metabolism and Antibiotic Susceptibility. J Appl Biotechnol Bioeng. 2017; 2(6): 212–219. Publisher Full Text\n\nSingh BR: Multiple-herbal-antimicrobial-resistance (MHAR) in microbes of animals, birds, fish, food, lizard and water origin. In Proceedings of International conference and 28th Annual convention of IAVMI-2014 on Challenges and opportunities in animal health at the face of globalization and climate change, Department of Veterinary Microbiology and Immunology, DUVASU, Mathura, India. 2014; 1: 26–29. Reference Source\n\nSingh S, Singh SK, Chowdhury I, et al.: Understanding the Mechanism of Bacterial Biofilms Resistance to Antimicrobial Agents. Open Microbiol J. 2017; 11: 53–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrafny EA, Lewandowski R, Zawistowska-Marciniak I, et al.: Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J Microbiol Biotechnol. 2013; 29(9): 1635–1643. PubMed Abstract \| Publisher Full Text\n\nWipperman MF, Fitzgerald DW, Juste MAJ, et al.: Antibiotic treatment for Tuberculosis induces a profound dysbiosis of the microbiome that persists long after therapy is completed. Sci Rep. 2017; 7(1): 10767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZatorska B, Arciola CR, Haffner N, et al.: Bacterial Extracellular DNA Production Is Associated with Outcome of Prosthetic Joint Infections. Biomed Res Int. 2018; 2018: 1067413. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "51849", "date": "15 Aug 2019", "name": "Shibabrata Pattanayak", "expertise": [ "Reviewer Expertise Ethno- Pharmacology", "Immunology", "Bacteriology", "Virology", "Biochemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFollowing remarks are to be considered. A.Test formulation Triphala formulation (TF) (Emami Ltd; batch no. EM0029; Proportion of 3 constituent plant species: 1:1:1) was purchased from a local market. For assay purpose, 150 mg of this formulation was suspended in 5 ml of DMSO (Merck, Mumbai), followed by vortexing for 15 min. Then it was centrifuged at 8,000 rpm for 30 min at ambient temperature, and resulting supernatant was collected in a sterile glass vial (15 ml; Borosil) and stored under refrigeration till further use. Remaining pellet was subjected to drying in an oven at 70-80°C until the solvent was completely evaporated, followed by weighing of the dried plant material. Subtracting the latter from the initial weight of 150 mg, the concentration of test formulation in supernatant was calculated to be 22.94 mg/ml. This way the whole formulation was found to contain 70% DMSO soluble fraction, which was used for our experiments.\n\nRemark DMSO is having some effect on at least some microorganisms. What concentration of DMSO was used? Please add report / reference of non – germicidal effect of DMSO at the concentration used in your experiment. You may consult the following articles:\nKirkwood ZI, Millar BC, Downey DG, Moore JE. Antimicrobial effect of dimethyl sulfoxide and N, N- Dimethylformamide on Mycobacterium abscessus: Implications for antimicrobial susceptibility testing. Int J Mycobacteriol 2018;7:134-136.1\n\nMi H, Wang D, Xue Y, Zhang Z, Niu J, Hong Y, Drlica K, Zhao X. 2016. Dimethyl sulfoxide protects Escherichia coli from rapid antimicrobial-mediated killing. Antimicrob Agents Chemother 60:5054 –5058. doi:10.1128/AAC.03003-15.2\n\nAshraf S. Hassan (2014) The Antibacterial Activity of Dimethyl Sulfoxide (DMSO) with and without of Some Ligand Complexes of the Transitional Metal Ions of Ethyl Coumarin against Bacteria Isolate from Burn and Wound Infection. Journal of Natural Sciences Research. Vol.4, No.19, 106-111.3\n\nHoward C. Ansel, William P. Norred, Ivan L. Roth (1969) Antimicrobial activity of dimethyl sulfoxide against Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. J Pharmaceutical sciences 58(7): 836-839.4\n\nB. Some portions are appeared unclear. Please modify the sentences:\n\nPage 2, Column 2 Paragraph 4.\nProphylactic assay (Patel et al., 2018b): …..These worms were then fed with TF by mixing required concentration of this formulation (100 μL) with M9 medium (800 μL) and placed in a 24-well plate (non-treated polystyrene plates….\n\nRemark:\n\nIs it the DMSO –TRIFALA mixed supernatant?\n\nPage 4 Column 1 Paragraph 4. …..at par with that of control (DMSO treated parent culture). –\nRemark: Point is not clear.\n\nOverall Comment:\n\nThe research work is very good. The writing style is also good. After the mentioned modifications, the article may be accepted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5956", "date": "01 Oct 2020", "name": "Vijay Kothari", "role": "Author Response", "response": "We thank both the referees for devoting their valued time in reviewing our manuscript. Our comment-wise response to their comments is as under:Response to comments from Dr. S Pattanayak: DMSO was used in our experiments at 0.5%v/v. This information along with the fact that this much DMSO did not affect growth / pigment production / virulence of the bacterial strains employed in this study has already been there in legends of all the figures. For example, absence of in vitro effect of DMSO on growth and pigment production has been reported in legend of Figure-8. Legend of Figure-1 includes the statement that DMSO (0.5%v/v) affected neither bacterial virulence, nor did it exert any toxicity toward the host worms. Referee has very logically raised this concern, as DMSO at higher concentrations have been reported to affect bacterial growth by multiple researchers including our group [doi: 10.1016/S1995-7645(14)60233-9;] and those cited in the referee report. However, since we have already included the ‘vehicle control’ in all our experiments; we did not cite any separate references for that. DMSO at the concentration (0.5%v/v) used in this study has earlier been reported by us to exert no effect on bacterial growth and pigment production: https://shodhganga.inflibnet.ac.in/bitstream/10603/245830/14/17_annexure.pdf 2. Yes, it is Triphala dissolved in DMSO, which was fed to the worm population during prophylactic assay. The sentence “These worms were then……………….” has been modified.3. For the sake of brevity, we have modified the sentence: “………..at par with………….”. Here what we could demonstrate is that effect of TF remains even on the ‘daughter cells’ of the TF-exposed ‘parent bacterial cells’. It should be noted that these daughter cells themselves were never directly exposed to the test formulation, and still their virulence is attenuated owing to their parent population’s exposure to TF. To make the concept of ‘post-extract effect’ (PEE) clear to the readers, we have already provided a link [https://doi.org/10.32388/359873] in the same paragraph, which takes the reader to the definition of PEE. We hope that the revised version along with clarifications provided by us will be able to earn referee’s and editor’s approval." } ] }, { "id": "56187", "date": "02 Sep 2020", "name": "Felipe Alves de Almeida", "expertise": [ "Reviewer Expertise Microbiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is interesting, but some adjustments are needed. Initially, the quality of the figures should be improved. One point that concerns me is the interpretations of the results of the in vitro experiments, because when the concentration of TF influences the growth of bacteria, nothing can be said about its anti-quorum sensing effect. The other comments are marked in a document in PDF format which can be found here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5957", "date": "01 Oct 2020", "name": "Vijay Kothari", "role": "Author Response", "response": "Response to Referees We thank both the referees for devoting their valued time in reviewing our manuscript. Our comment-wise response to their comments is as under: Response to general comments from Dr. Felipe Alves de Almeida: Quality of the figures: We have set the resolution of all figures at 1000 dpi (which also satisfies journal’s criteria of minimum 600 dpi), and are unable to improve them further. The referee has pointed to a very logical issue with respect to interpretation of the in vitro results that if the test formulation affects bacterial growth, then how can we judge its effect on pigment production as quorum-modulatory effect? To safeguard against this, we have already plotted ‘Pigment Unit’, and not the absolute pigment OD. ‘Pigment Unit’ are OD of pigment divided by cell density. Since this is a quantification of pigment produced per unit of growth, it nullifies any variation in pigment production due to changes in cell density in experimental vs. control tubes. Calculation of these ‘Pigment Units’ for individual pigments has already been mentioned in the legend of Figure-8. To further clarify this point, we have added this sentence to the figure-8 legend: “Pigment Units were calculated to nullify the effect of any change in cell density on pigment production”. On the same line, for other in vitro assays too (e.g. lysozyme susceptibility, haemolysis assay, etc.), before performing the assay we had equalized cell density of the control and experimental cultures, to ensure that TF’s effect (if any) on bacterial growth does not interfere with interpretation of our results. This OD equalization step has already been mentioned in the ‘Methods’ section. Response to comments from Dr. Felipe Alves de Almeida marked within the annotated manuscript: The referee has asked for why have we chosen 750 nm for quantifying cell density. Please note that the bacteria being studied here are pigmented ones, and their pigments can interfere in measuring cell density within the visible range of spectrum (400-700 nm). In our earlier study [Joshi et al. (2016); DOI: 10.2174/1872208310666160414102848; which we have already cited in the current paper], we have shown 764 nm as the more appropriate one for quantifying cell density of pigmented bacteria, as compared to the conventionally used wavelengths e.g. 600 nm. Since the wavelength nearest to 764 nm available in the instrument used by us was 750 nm, we used that. We had earlier also confirmed that OD of the bacterial suspension used by us do not statistically differ at 764 nm vs. 750 nm. Appropriateness of 764 nm for studying C. violaceum has also been reported by Gallardo et al. AMB Express 2014, 4:4 [http://www.amb-express.com/content/4/1/4]. In the ‘Results’ section under ‘in vivo assays’, the referee has raised a question that why different concentrations of TF used for pathogens. In response to that we would like to state that all pathogens were challenged with identical concentrations of TF in the range of 0.5-100 microgram/mL. However in figures corresponding to these results, we have showed only non-overlapping lines. Data regarding other concentrations has been mentioned in the figure legends. If we include lines pertaining to all concentrations in the graph, then it becomes very fuzzy and difficult to understand. Though referee’s suggestion of merging Figure-2 with Figure-1 is fully logical, we are unable to implement it, as that will cause multiple lines in Figure-1 to overlap making it fuzzy and difficult to understand. For the same reason, we were not able to put lines for all TF concentrations in the graphs of Figure-1, and had to include that data in figure legend. Referee has suggested for including the details of Post-Extract Effect (PEE) in ‘Method’ section. However that was not done as the method is not different than that described for ‘anti-infective assay’ except that in the PEE assay the host worm was challenged with daughter cells of the TF-treated parent cells; and this small piece of information has already been mentioned in the ‘Results’ section while first mentioning PEE. The referee has asked for data of PEE against C. violaceum. However PEE assay was done only with the pathogenic bacteria which are considered as serious threats, i.e. whose pathogenic/ antibiotic-resistant strains are listed as priority pathogens by CDC/ WHO. The referee has raised a query about the difference in survival % of unchallenged/ unfed worms between figure 5 and figure 6. It should be noted that prior to the prophylactic assay, whose results are depicted in Figure-5, worms were kept unfed for 5 days; whereas prior to longevity assay (results depicted in Figure-6) the worms were kept unfed for 3 days. Hence this additional 2-dyas of non-availability of food in the prophylactic assay explains lower survival of worms in ‘control’ wells in Figure-5. As suggested by the referee, we have replaced ‘Triphala formulation’ with ‘TF’ at multiple places. Similarly at many places, he has suggested removal of parentheses, that has also been implemented. Figure-8 has been revised as per his suggestion. Figure-10: Referee has raised query about accuracy of the % values. We thank him for his fine observation, as investigating this has made us locate errors in some % values as well as bar heights. All those errors have been corrected, and we are submitting revised figure. All other minor corrections/ modifications marked by the referee in the PDF file have also been either implemented, OR we have provided appropriate response/ explanation by inserting ‘comments’ in the word file of revised version. We hope that the revised version along with clarifications provided by us will be able to earn referee’s and editor’s approval." } ] } ]	1	https://f1000research.com/articles/8-1126
https://f1000research.com/articles/9-1032/v1	24 Aug 20	{ "type": "Research Article", "title": "A cross-sectional observational study investigating the association between sedges (swamp grasses, Cyperaceae) and the prevalence of immature malaria vectors in aquatic habitats along the shore of Lake Victoria, western Kenya", "authors": [ "Getachew E. Bokore", "Paul Ouma", "Patrick O. Onyango", "Tullu Bukhari", "Ulrike Fillinger", "Paul Ouma", "Patrick O. Onyango", "Tullu Bukhari", "Ulrike Fillinger" ], "abstract": "Background: Strategies that involve manipulations of the odour-orientation of gravid malaria vectors could lead to novel attract-and-kill interventions. Recent work has highlighted the potential involvement of graminoid plants in luring vectors to oviposition sites. This study aimed to analyse the association between water-indicating graminoid plants (Cyperaceae, sedges), other abiotic and biotic factors and the presence and abundance of early instar Anopheles larvae in aquatic habitats as a proxy indicator for oviposition. Methods: A cross-sectional survey of 110 aquatic habitats along the shores of Lake Victoria was done during the rainy season. Habitats were sampled for mosquito larvae using the sweep-net method and habitat characteristics recorded. Results: Anopheles arabiensis was the dominant species identified from aquatic habitats. Larvae of the secondary malaria vectors such as Anopheles coustani, An. rufipes and An. maculipalpis were found only in habitats covered with graminoids, whereas An. arabiensis, An. ziemanni and An. pharoensis were found in both habitats with and without graminoid plants. The hypothesis that sedges might be positively associated with the presence and abundance of early instar Anopheles larvae could not be confirmed. The dominant graminoid plants in the habitats were Panicum repens, Cynodon dactylon in the Poaceae family and Cyperus rotundus in the Cyperaceae family. All of these habitats supported abundant immature vector populations. The presence of early instar larvae was significantly and positively associated with swamp habitat types (OR=22, 95% CI=6-86, P<0.001) and abundance of late Anopheles larvae (OR=359, CI=33-3941, P<0.001), whilst the association was negative with tadpole presence (OR=0.1, CI=0.0.01-0.5, P=0.008). Conclusions: Early instar malaria vectors were abundant in habitats densely vegetated with graminoid plants in the study area but specific preference for any of the graminoids could not be detected. In search for oviposition cues, it might be useful to screen for chemical volatiles released from all dominant plant species.", "keywords": [ "Anopheles", "oviposition", "larval ecology", "malaria", "vector control", "vegetation", "graminoid plants" ], "content": "Background\n\nMalaria, despite increased control efforts, is still among the leading human diseases in Africa. In 2018, 213 million people were infected and 380,000 died1. The majority of people in sub-Saharan Africa live in poverty and in areas with suitable conditions for the proliferation of malaria vectors2. The major malaria vectors are in the Anopheles gambiae and An. funestus species complexes, but a number of less efficient, so-called secondary vectors also contribute to malaria transmission3,4.\n\nWith growing physiological and behavioural resistance of malaria vectors to insecticides5–7, research efforts are geared towards additional, non-insecticidal vector control strategies8,9. Manipulations of the odour-orientation of adult vectors could lead to novel attract-and-kill interventions10–12. The gravid female searching for a suitable oviposition site is a desirable target for control. This strategy is specifically important as a single gravid mosquito may lay between 50 to 150 eggs13, hence killing a single gravid mosquito affects the growth of the population. Understanding the cues for habitat selection is of paramount importance for the development of such a tool. Recent work has highlighted the potential involvement of graminoid plants in luring vectors to oviposition sites14. It has, for example, been shown that Anopheles mosquitoes respond to volatile chemical compounds that emanate from rice plants15.\n\nMalaria vector mosquitoes lay their eggs in standing water and grass-like (graminoid) plants that often dominate wetlands associated with high Anopheles larval densities16–18. Some graminoid plants, similar to lowland rice (Oryza sativa), are well adapted to wetlands. Most species in the sedge family, also known as swamp grasses, (Cyperaceae) are wetland indicators. One sedge species, Cyperus rotundus, was recently associated with the discovery of the oviposition attractant cedrol19 but its connection to the sesquiterpene compound was not clearly understood.\n\nWe considered it plausible to hypothesize that there might be an association between chemical cues released by water-indicating plants that are used by gravid malaria vectors in search of suitable oviposition sites. Therefore, we implemented this study driven by the hypothesis that sedges (Cyperaceae) are associated with the presence and abundance of early instar Anopheles larvae, as a proxy indicator for oviposition, in western Kenya.\n\nSwamp habitats are very common along lakeshores and serve as permanent or semi-permanent breeding sites for malaria vectors20. Studies support that the abundance of Anopheles larvae are associated with habitats surrounded by grass-like plants14. In the current study, we investigated: (1) the distribution of graminoid plants associated with aquatic habitats along the shores of Lake Victoria in western Kenya, (2) the association of the graminoid plants with the occurrence and abundance of Anopheles larvae, and (3) the association of abiotic and biotic factors in aquatic habitats with the occurrence and abundance of Anopheles larvae.\n\n\nMethods\n\nThis study was conducted on Rusinga Island (0°21′ and 0°26 south, 34°13′ and 34°07′ east) along the shore of Lake Victoria in Homa Bay County, western Kenya21 (Figure 1). The area is endemic for malaria and the estimated prevalence of malaria in the population of Rusinga Island is around 10%18,22,23. Rusinga Island is only around 100 metres away from the mainland and connected via a bridge. The island has an area of 44 km2 with altitude ranging from 1100 m to 1300 m above sea level and a population size of about 25,00024. The daily average temperatures of Rusinga Island range from 16°C to 34°C and peak in dry seasons24. The area experiences bimodal rainy seasons with long rains from March to June and short rains from November to December. Malaria transmission peaks following the end of the long rainy season in June/July25. The field survey was implemented between May and June 2018, towards the end of the long rainy season.\n\nHabitat surveys were done along stretches of 700 m long and 300 m wide (clusters of approx. 0.2 km2). A total of 13 sampling clusters were selected around the lake shores of Rusinga Island (Figure 1B). The areas were selected with the help of Google Earth, aiming at a homogeneous distribution around the island. Inaccessible areas with steep rocks at the shoreline were excluded. Within each sampling cluster all aquatic habitats’ locations were recorded using a smartphone, a unique identifier allocated and sampled as outlined below.\n\nThe aquatic habitat types were categorized as either swamp, puddle, fishpond, drainage/trench or artificial pit. The perimeter of every habitat was estimated by walking in large steps around the habitat. Water turbidity was measured using a turbidity meter (TRB 355IR, WTW Germany) and water pH and temperature were measured using a portable multi-parameter probe (Multi, WTW Germany).\n\nEvery aquatic habitat was inspected for the presence of larvae using the sweep-net method as described by Ndenga and others26. The sweep-net (40 cm × 15 cm × 30 cm) was made from fine cotton cloth with a 150 cm long handle. It was chosen for sampling due to its better efficiency in sampling the diverse aquatic fauna including freshly hatched mosquito larvae and mosquito pupae than the standard dipper26,27. A dipper was used for sampling when the habitat was too small to be sampled by a sweep-net. Sampling of mosquito larvae using either sampling tools was randomly done at different points of the habitats since different species of malaria vectors prefer different conditions and vegetations. The duration of sweeping was dependent on the perimeter of the habitat. About 10 minutes were taken to sweep habitats with perimeters exceeding 10 metres, while 5 minutes were taken to sweep habitats <10 m in perimeter. All sweeps were emptied into white trays and mosquito immature stages were counted separately for the two encountered genera, Anopheles and Culex. Culex and Anopheles larvae were identified morphologically. Culex larvae possess siphon on the posterior part of their abdomen for breathing through at the interface of the water surface during resting whereas Anopheles larvae have no siphon and rest horizontal to the water body28. The larvae were grouped as early (1st and 2nd instar) and late (3rd and 4th instar) instars based on their body size. In addition, macroinvertebrates sampled from a habitat were grouped as Odonata (dragonfly and damselfly larvae), Coleoptera (water beetle larvae and adults), Heteroptera (Notonectidae, Naucoridae and Nepidae), fish and tadpoles. All late instar Anopheles larvae and mosquito pupae were transferred to water bottles (1 L) containing habitat water and taken to the International Centre of Insect Physiology and Ecology-Thomas Odhiambo Campus (icipe-TOC) for rearing to adults. Rearing of the field collected larvae was done in 1 L plastic rectangle food mate (H67 × W126 × L184 mm, Kenpoly manufacturer, Nairobi, Kenya). Larvae were fed with a pinch of ground dry cat food (Nestlé Purina PetCare Company, Nairobi, Kenya) once daily. The emerged adults were killed in a -20°C refrigerator, sorted by genera and all Anopheles adults stored in Eppendorf tubes (1.5 ml) at -71°C until they were identified morphologically using printed keys29 and molecularly using polymerase chain reaction (PCR) followed by gel-electrophoresis30. Randomly selected mosquito samples were used for molecular identification. Polymerase chain reaction was implemented for the amplification of the ribosomal internal transcribed spacer (ITS2) gene using primers31. Positive controls of An. gambiae s.s. and An. arabiensis (from Mbita insectary colony) were analyzed with the samples from field. Extraction of genomic DNA was done for each mosquito separately using Tissue Kit (Quagen, GmbH Hilden, Germany). The PCR in a 10 µl (per sample) was prepared by mixing PCR mix of 2 µl of 5XHot Firepol Blended Master Mix (Ready to Load), primers (0.5 µM each), DNA template (2 µl) and nuclease-free water (5 µl). The thermal recycling conditions involved initial denaturation for 5 min at 95°C, after which 30 cycles of denaturation followed for 30 s at 94°C, annealing for 30 s at 50°C, extension for 30 s at 72°C and final extension for 5 min at 72°C. We used Kyratec Thermal Cycler (SC300T-R2, Australia) for the thermal reactions. Agarose gel-electrophoresis (2.0%) stained with 2 µl ethidium bromide against a 100 bp DNA ladder (Bioline, A Maridian Life Science@ Company, UK) and a positive control was conducted to identify the species.\n\nVegetation coverage, vegetation types and the dominant vegetation type were recorded separately for habitat edge and water surface. Habitat edge was defined as the area along the waterline, approximately 10 cm inside and/or outside the water. Vegetation coverage was estimated visually as the proportion of the habitats covered with vegetations and categorized as (1) 1–25% (2) 25–50% (3) 50–75% (4) 75–100%. Graminoid plants across the edge and inside water were recorded as Poaceae, Cyperaceae, Juncaceae, Typhaceae. The graminoid plants were identified to family using morphology of their leaves (two or three-ranked; open or closed sheaths), and their stem type (three-sided or round; hollow or solid) using Revuelta32. Furthermore, herbaceous (not woody and non-graminoid plants) were collectively recorded as forbs. The presence of water plants and algae in the aquatic habitats was also recorded. The percent coverage of water plants and algae on the water surface was visually determined as above. For each habitat, the dominant type of vegetation was identified and recorded. Full specimens of all dominant graminoid plants found in the aquatic habitats were collected and planted at icipe-TOC for further identification33,34.\n\nGeneralised estimating equations (GEE) with Poisson distribution fitted to a log function and exchangeable correlation matrix were used to test for associations between biological and environmental factors and the abundance of early instar Anopheles larvae. The cluster ID in which habitats were located was included in the model as repeated measurement. A GEE model was also used to analyse associations between factors and the presence of early instar Anopheles larvae. Here we included the presence of early instar Anopheles larvae as dependent variable in the model with binomial distribution fitted to a logit function and exchangeable correlation matrix to analyse its association with biotic and abiotic factors of the habitats (independent variables). The presence and abundance of early instar Anopheles larvae (rather than eggs which are difficult to identify from field samples) were used as dependent variable as a proxy for oviposition. This is based on recent work confirming that early instar density correlates with the abundance of females selecting a habitat for oviposition35. The statistical outputs were reported as incidence rate ratios (RR) for the abundance of the first instar larvae and odds ratios (OR) for the presence with their 95% confidence intervals (95% CI). R statistical software version 3.5.136 was used for the analyses.\n\nThis field survey was largely descriptive and observational and had no human study participants. Habitat surveys on privately owned lands were made after seeking consent from the landowners and were implemented in their presence.\n\n\nResults\n\nA total of 110 aquatic habitats were identified during the survey37. As expected, given the targeted areas within 300 metres of the lake shore, the most prevalent aquatic habitat types were swamps (65.5%, n=72) defined as permanent or semi-permanent water-logged sections of land with tall graminoid vegetation and/or floating plants (Figure 2A). The water sources of these were largely groundwater supplemented by rainwater. Other habitats (see Figure 2B, Figure 3C, 3D and 3E) included ponds formerly used for breeding fish but abandoned at the survey time (11%, n=12), rainfed puddles (9%, n=10), drainages (9%, n=10) and artificial pits (5.5%, n=6). Given that all non-swamp habitats were few in number, they were pooled for statistical analysis and the swamp habitats used as the reference group (Figure 3). Early instar Anopheles larvae were found frequently during the survey in the habitat types: artificial holes (n=6, 100%), drainages (n=9, 90%), ponds (n=5, 42%), puddles (n=7, 70%) and swamps (n=61, 85%). The majority of these habitat types were characterized by possessing graminoid plants: graminoids dominated the vegetation in 100% of the swamps, in 83% of the ponds, in 80% of the puddles, in 70% of the drainages, and in 50% of the artificial pits.\n\n(A) Swamp, (B) Fishpond, (C) Puddle, (D) Drainage, (E) Artificial pit.\n\nAll the swamp habitats were boarded by graminoid plants along the water edges and had a high surface coverage. Similarly, 84% (32/38) of non-swamp habitats had graminoids along their edges and 76% (29/38) had graminoids at their surfaces. Unexpectedly, swamp grasses were not the most frequently found graminoid plants in the survey. Representatives of the Cyperaceae family were found only in 39% of the aquatic habitats sampled. Among the Poaceae family, torpedo grass (Panicum repens) and Bermuda grass (Cynodon dactylon) were the dominant species (Figure 4).\n\n(A) Panicum repens (Poaceae), (B) Cynodon dactylon (Poaceae) and (C) Cyperus rotundus (Cyperaceae).\n\nOf the surveyed habitats, 42 (38%) were found covered by P. repens along their edges and 47 (43%) of the habitats at their surfaces. Cynodon dactylon was found covering the habitats both along the edges in 35 (32%) habitats and surfaces in 25 (23%) habitats (Table 1). Overall, graminoid plants dominated in 96 habitats whilst forbs dominated only in five habitats during the survey. Nine habitats had no vegetations at their surface and five of them were colonized by early instar Anopheles larvae. We found water plants in 26 (24%) out of the 110 habitats and most (n=20, 77%) of them in swamp habitats. Filamentous algae were recorded in 21 habitats. Contrary to our hypothesis, there was no significant association between the presence or abundance of early instar Anopheles larvae and the dominant graminoid plant present in a habitat (Table 1).\n\nSelected as reference based on initial hypothesis and earlier association of Cyperus rotundus with oviposition. OR= odds ratio, RR= rate ratio, CI= confidence interval.\n\nA total of 14,145 early and late instar Anopheles larvae and 402 pupae were collected. Out of those, 4,650 emerged into adults and were morphologically identified (Table 2). Anopheles gambaie s.l. represented 96% of all Anopheles specimen collected. Molecular identification was done for a random sample of 10% of the An. gambiae s.l. (n=480) and revealed 100% An. arabiensis.\n\nAnopheles coustani, An. rufipes and An. maculipalpis were found only in aquatic habitats covered with graminoid plants, whereas An. arabiensis, An. ziemanni and An. pharoensis were found in both habitats with and without graminoid plants. These six species of Anopheles mosquitoes were recorded in swamp habitats. All of these Anopheles species were found in aquatic habitats with dense graminoid vegetation (50–100%) (Table 3). However, only three species of Anopheles mosquitoes (An. arabiensis, An. ziemanni, and An. pharoensis) were collected in habitats sparsely (1–25%) covered by graminoids.\n\n Molecular identification of a random sample of 10% of the An. gambiae s.l. revealed 100% An. arabiensis.\n\nCI= confidence interval.\n\nAquatic habitats populated with Panicum repens and forbs at their edges had all the six Anopheles species identified (Table 4a). Megaloprotachne albescens was found dominant in six out of 110 habitats surveyed but was found to have all the six different species of Anopheles (Table 4b). Anopheles arabiensis, An. coustani, and An. pharoensis were coexisting with all the graminoid types and forbs found dominating along the surfaces of the habitats.\n\n(a)\n\nCI= confidence interval.\n\n(b)\n\nCI= confidence interval.\n\nThe presence of early instar Anopheles larvae in habitats was significantly and positively associated with swamp-type habitats (OR=22, 95%CI=6-86, P<0.001), presence of late instar Anopheles larvae (OR=359, CI=33-3941, P<0.001), and presence of Culex larvae (OR=17, 95%CI=3-107, P=0.002) (Table 5). In habitats containing pupae the odds of finding early instar Anopheles larvae was lower (OR=0.08, CI=0.01-0.42, P=0.003) than habitats without pupae. Notably, the majority of habitats with Anopheles larvae were also well colonised by other invertebrates, many of which are considered predators of mosquitoes, such as Odonatan, Notonecta, and Coleoptera larvae. However, the presence of early instar Anopheles was only significantly and negatively associated with presence of tadpoles (OR=0.09, 0.01-0.53, P=0.003). Correspondingly, the abundance of early instar Anopheles larvae significantly decreased with the presence of tadpoles (RR=0.5, CI=0.2-0.9, P=0.03). Similarly, larval abundance was negatively associated with presence of Odonata (RR=0.5, CI=0.3-0.9, P=0.019) and presence of Coleoptera (RR=0.4, CI=0.2-0.8, P=0.004). There was no significant association between the presence and abundance of early instar Anopheles larvae and habitat size, habitat depth, distance to the nearest house, water pH, water turbidity, biofilm, debris, algae, and water plants.\n\nOR= odds ratio, RR= rate ratio, CI= confidence interval.\n\n\nDiscussion\n\nThe work presented here was done with the aim of identifying graminoid plants for further behavioural and chemical ecology studies due to their association with habitats used by gravid malaria vectors for egg-laying. However, the presence of early instar Anopheles larvae in the majority of the surveyed habitats and the presence and high coverage of various graminoid plants did not allow us to analyse any statistically significant association. All the habitats surveyed provided excellent oviposition sites and favourable conditions for the development of immature stages based on the high and consistent number of early instar larvae as a proxy for oviposition and the associated high abundance of late instar larvae as an indicator for survival. The study, as implemented, did not allow us to infer specific plant-based factors with oviposition. Generally, the association between graminoid plants and Anopheles breeding sites as well as the presence and increased densities of Anopheles larvae in both temporary and permanent aquatic habitats have been shown before16,17. It has been suggested that vegetation can protect mosquito immature stages from being washed off by running water38 and from predation39,40. Our study has several limitations that might be responsible for the negative results. The timing of the survey towards the end of the rainy season meant that all potential habitats were flooded and vegetation thrived. Habitats for oviposition were not a limiting factor and likely easy to identify without major cues for orientation. This might have been different if the survey had been implemented during the dry season. Furthermore, this survey was limited to locations close to the lake shores, biasing the study towards swampy habitats. Lastly, due to high water levels during the peak rainy season, a number of habitats with swamp graminoids of the families Cyperaceae, Typhaceae, and Juncaceae were impossible to access, hence could not be sampled. This might also explain why only very few secondary malaria vector species and no Anopheles funestus were sampled, even though An. funestus is the major vector that was encountered in houses in the study area41–43.\n\nNot many strong associations were found with early Anopheles larvae presence or abundance and other observed factors that would allow conclusions on oviposition preferences. However, the odds of finding early instars increased when late instar Anopheles larvae were present as opposed to when they were absent, potentially indicating that Anopheles arabiensis females oviposit in habitats containing late instar conspecific larvae as an indicator of suitable development conditions. This contrasts with experimental studies on An. coluzzii44,45, where it has been suggested that late instar conspecific larvae repel gravid females potentially due to the risk of cannibalism46.\n\nThe presence and abundance of early instar Anopheles larvae was negatively associated with the presence of tadpoles. It was previously shown that rainwater conditioned with tadpoles repelled gravid An. gambiae from oviposition in the laboratory47. Mature tadpoles can prey on larvae48 and might compete for resources in aquatic habitats49. Our field survey indicated that early instar larvae were cohabiting with predatory invertebrates in most habitats. Whilst Anopheles larvae might be reduced by these organisms, as suggested by the negative association between Anopheles density and the presence of Odonata and Coleoptera, gravid females nevertheless did not avoid these habitats for oviposition. This finding agrees with studies elsewhere that have shown strong associations between the presence of anopheline larvae and high invertebrate diversity50.\n\nSix species of Anopheles mosquitoes were identified from the samples which have all been reported in previous studies in western Kenya18,51,52. Anopheles arabiensis was the predominant malaria vector from both vegetated and non-vegetated aquatic habitats in the study area during the peak rainy season. Anopheles arabiensis has historically been the predominant vector species on Rusinga Island53. In recent years however, An. funestus predominates indoor vector collections41,43,54, but larvae were not found during our survey. Breeding sites preferred by this mosquito species were inaccessible by the field team due to the large volumes of water in the lake after the long rains; An. funestus prefer breeding habitats that are covered by tall vegetations37,55,56.\n\n\nConclusions\n\nOur results did not support the hypothesis and nullified the aim of the research to identify graminoid plant species positively associated with malaria vector oviposition. Our results did not support our initial hypothesis and did not allow us to identify any association between Anopheles larvae and specific graminoid plants. However, Panicum repens, Cynodon dactylon, and Cyperus rotundus were the predominant graminoid plants found in the aquatic habitats. The habitats covered by this vegetation were abundantly colonized by early instar Anopheles larvae even though no specific preference for any of these could be detected, likely due to study limitations. We recommend further studies on the identification of oviposition cues from graminoid plants during the dry seasons when habitats are limited and water-levels low enough to provide access to most of them. Furthermore, it might be warranted to implement bioassays in the laboratory with the here identified grass-like plants, which will allow more standardised comparisons and sufficient replication.\n\n\nData availability\n\nHarvard Dataverse: Association between graminoids and the prevalence of immature malaria. https://doi.org/10.7910/DVN/NAT0YY57.\n\nThis project contains the following underlying data:\n\n- Bokore et al. 2020_F1000Research_All_Collected_Data.csv\n\n- Bokore et al. 2020_F1000Research_Data_used_for_final_analysis.csv\n\n- Bokore et al. 2020_F1000Research_Variable_Codes.csv\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe are grateful to the residents of Rusinga Island for allowing us to collect the field data in their farms. The administrative support of Ibrahim Kiche is highly appreciated.\n\n\nReferences\n\nWHO: World Malaria Report 2019. Geneva: World Health Organization. 2019; 1–232. Reference Source\n\nMuema JM, Bargul JL, Njeru SN, et al.: Prospects for malaria control through manipulation of mosquito larval habitats and olfactory-mediated behavioural responses using plant-derived compounds. Parasit Vectors. 2017; 10(1): 184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinka ME, Bangs MJ, Manguin S, et al.: A global map of dominant malaria vectors. Parasit Vectors. 2012; 5: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoetzee M, Hunt RH, Wilkerson R, et al.: Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. 2013; 3619(3): 246–74. Reference Source\n\nRanson H, Lissenden N: Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation that Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016; 32(3): 187–96. PubMed Abstract \| Publisher Full Text\n\nColeman M, Hemingway J, Gleave KA, et al.: Developing global maps of insecticide resistance risk to improve vector control. Malar J. 2017; 16(1): 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCook J, Tomlinson S, Kleinschmidt I, et al.: Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: Trends in pyrethroid resistance during a WHO-coordinated multi-country prospective study. Parasit Vectors. 2018; 11(1): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell TL, Beebe NW, Cooper RD, et al.: Successful malaria elimination strategies require interventions that target changing vector behaviours. Malar J. 2013; 12(1): 56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakken W, Knols BG: Malaria vector control: current and future strategies. Trends Parasitol. 2009; 25(3): 101–4. PubMed Abstract \| Publisher Full Text\n\nLindh JM, Okal MN, Herrera-Varela M, et al.: Discovery of an oviposition attractant for gravid malaria vectors of the Anopheles gambiae species complex. Malar J. 2015; 14(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEneh LK, Fillinger U, Borg Karlson AK, et al.: Anopheles arabiensis oviposition site selection in response to habitat persistence and associated physicochemical parameters, bacteria and volatile profiles. Med Vet Entomol. 2019; 33(1): 56–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMbare O, Lindsay SW, Fillinger U: Testing a pyriproxyfen auto-dissemination station attractive to gravid Anopheles gambiae sensu stricto for the development of a novel attract-release -and-kill strategy for malaria vector control. BMC Infect Dis. 2019; 19(1): 800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLyimo Edith O, Takken W: Effects of adult body size on fecundity and the pre-gravid rate of Anopheles gambiae females in Tanzania. Med Vet Entomol. 1993; 7(4): 328–32. PubMed Abstract \| Publisher Full Text\n\nAsmare Y, Hill SR, Hopkins RJ, et al.: The role of grass volatiles on oviposition site selection by Anopheles arabiensis and Anopheles coluzzii. Malar J. 2017; 16(1): 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWondwosen B, Birgersson G, Seyoum E, et al.: Rice volatiles lure gravid malaria mosquitoes, Anopheles arabiensis. Sci Rep. 2016; 6(1): 37930. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFillinger U, Sonye G, Killeen GF, et al.: The practical importance of permanent and semipermanent habitats for controlling aquatic stages of Anopheles gambiae sensu lato mosquitoes : operational observations from a rural town in western Kenya. Trop Med Int Health. 2004; 9(12): 1274–89. PubMed Abstract \| Publisher Full Text\n\nImbahale SS, Paaijmans KP, Mukabana WR, et al.: A longitudinal study on Anopheles mosquito larval abundance in distinct geographical and environmental settings in western Kenya. Malar J. 2011; 10(1): 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinakawa N, Dida GO, Sonye GO, et al.: Malaria Vectors in Lake Victoria and Adjacent Habitats in Western Kenya. PLoS One. 2012; 7(3): e32725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEneh LK, Saijo H, Borg-Karlson AK, et al.: Cedrol, a malaria mosquito oviposition attractant is produced by fungi isolated from rhizomes of the grass Cyperus rotundus. Malar J. 2016; 15(1): 478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinakawa N, Sonye G, Dida GO, et al.: Recent reduction in the water level of Lake Victoria has created more habitats for Anopheles funestus. Malar J. 2008; 7(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoogle Earth: Satellite view of Lake Victoria region: Study sites view of Rusinga Islands along the shore of Lake Victoria. Reference Source\n\nNoor AM, Gething PW, Alegana VA, et al.: The risks of malaria infection in Kenya in 2009. BMC Infect Dis. 2009; 9(1): 180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlanga EA, Okombo L, Irungu LW, et al.: Parasites and vectors of malaria on Rusinga Island, Western Kenya. Parasit Vectors. 2015; 8(1): 250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoman T, di Pasquale A, Onoka K, et al.: Profile: The Rusinga Health and Demographic Surveillance System, Western Kenya. Int J Epidemiol. 2016; 45(3): 718–27. PubMed Abstract \| Publisher Full Text\n\nKagaya W, Gitaka J, Chan CW, et al.: Malaria resurgence after significant reduction by mass drug administration on Ngodhe Island, Kenya. Sci Rep. 2019; 9(1): 19060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNdenga BA, Simbauni JA, Mbugi JP, et al.: Productivity of Malaria Vectors from Different Habitat Types in the Western Kenya Highlands. Amdam GV editor. PLoS One. 2011; 6(4): e19473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Klerk AR, Wepener V: The influence of biotope and sampling method on the assessment of the invertebrate community structure in endorheic reed pans in South Africa. African J Aquat Sci. 2011; 36(1): 67–74. Publisher Full Text\n\nSnell AE: Identification keys to larval and adult female mosquitoes (Diptera: Culicidae) of New Zealand. New Zeal J Zool. 2005; 32(2): 99–110. Publisher Full Text\n\nGillies MT, Coetzee M: A Supplement to the Anophelinae of Africa South of the Sahara. Publ South African Inst Med Res. 1987; 55(55): 63. Reference Source\n\nScott JA, Brogdon WG, Collins FH: Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am J Trop Med Hyg. 1993; 49(4): 520–9. PubMed Abstract \| Publisher Full Text\n\nZianni MR, Nikbakhtzadeh MR, Jackson BT, et al.: Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis. J Biomol Tech. 2013; 24(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRevuelta EA: Introduction to Florida’s Graminoids. 2019; 1–44. Reference Source\n\nFish L, Mashau AC, Moeaha MJ, et al.: Identification guide to southern African grasses. An identification manual with keys, descriptions and distributions. In Pretoria: South African National Biodiversity Institute; 2015; 1–30. Reference Source\n\nMuyekho FN, Barrion AT, Khan ZR: A Primer on grass identification and their uses in Kenya. 2004; 86. Reference Source\n\nOdero JO, Fillinger U, Rippon EJ, et al.: Using sibship reconstructions to understand the relationship between larval habitat productivity and oviposition behaviour in Kenyan Anopheles arabiensis. Malar J. 2019; 18(1): 286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. Vienna, Austria; 2018. Reference Source\n\nDia I, Guelbeogo MW, Ayala D: Advances and perspectives in the study of the malaria mosquito Anopheles funestus. In: Anopheles mosquitoes - New insights into malaria vectors. InTech; 2013. Publisher Full Text\n\nPaaijmans KP, Wandago MO, Githeko AK, et al.: Unexpected high losses of Anopheles gambiae larvae due to rainfall. Carter D, editor. PLoS One. 2007; 2(11): e1146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutuku F, Bayoh M, Hightower A, et al.: A supervised land cover classification of a western Kenya lowland endemic for human malaria: associations of land cover with larval Anopheles habitats. Int J Health Geogr. 2009; 8(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinakawa N, Sonye G, Mogi M, et al.: Habitat characteristics of Anopheles gambiae s.s. larvae in a Kenyan highland. Med Vet Entomol. 2004; 18(3): 301–5. PubMed Abstract \| Publisher Full Text\n\nMcCann RS, Ochomo E, Bayoh MN, et al.: Reemergence of Anopheles funestus as a Vector of Plasmodium falciparum in Western Kenya after Long-Term Implementation of Insecticide-Treated Bed Nets. Am J Trop Med Hyg. 2014; 90(4): 597–604. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathenge EM, Misiani GO, Oulo DO, et al.: Comparative performance of the Mbita trap, CDC light trap and the human landing catch in the sampling of Anopheles arabiensis, An. funestus and culicine species in a rice irrigation in western Kenya. Malar J. 2005; 4: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoman T, Hiscox A, Mweresa CK, et al.: The effect of mass mosquito trapping on malaria transmission and disease burden (SolarMal): a stepped-wedge cluster-randomised trial. Lancet. 2016; 388(10050): 1193–201. PubMed Abstract \| Publisher Full Text\n\nSchoelitsz B, Mwingira V, Mboera LEG, et al.: Chemical mediation of oviposition by Anopheles mosquitoes: a push-pull system driven by volatiles associated with larval stages. J Chem Ecol. 2020; 46(4): 397–409. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMwingira VS, Spitzen J, Mboera LEG, et al.: The influence of larval stage and density on oviposition site-selection behavior of the Afrotropical malaria mosquito Anopheles coluzzii (Diptera: Culicidae). Reisen W, editor. J Med Entomol. 2020; 57(3): 657–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoenraadt CJM, Takken W: Cannibalism and predation among larvae of the Anopheles gambiae complex. Med Vet Entomol. 2003; 17(1): 61–6. PubMed Abstract \| Publisher Full Text\n\nMunga S, Minakawa N, Zhou G, et al.: Effects of larval competitors and predators on oviposition site selection of Anopheles gambiae sensu stricto. J Med Entomol. 2006; 43(2): 221–4. PubMed Abstract \| Publisher Full Text\n\nKweka EJ, Zhou G, Gilbreath TM 3rd, et al.: Predation efficiency of Anopheles gambiae larvae by aquatic predators in western Kenya highlands. Parasit Vectors. 2011; 4: 128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMokany A, Shine R: Competition between tadpoles and mosquito larvae. Oecologia. 2003; 135(4): 615–20. PubMed Abstract \| Publisher Full Text\n\nFillinger U, Sombroek H, Majambere S, et al.: Identifying the most productive breeding sites for malaria mosquitoes in The Gambia. Malar J. 2009; 8(1): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKweka EJ, Munga S, Himeidan Y, et al.: Assessment of mosquito larval productivity among different land use types for targeted malaria vector control in the western Kenya highlands. Parasit Vectors. 2015; 8(1): 356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSt Laurent B, Cooke M, Krishnankutty SM, et al.: Molecular characterization reveals diverse and unknown malaria vectors in the western Kenyan highlands. Am J Trop Med Hyg. 2016; 94(2): 327–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinakawa N, Mutero CM, Githure JI, et al.: Spatial distribution and habitat characterization of anopheline mosquito larvae in Western Kenya. Am J Trop Med Hyg. 1999; 61(6): 1010–6. PubMed Abstract \| Publisher Full Text\n\nMachani MG, Ochomo E, Amimo F, et al.: Resting behaviour of malaria vectors in highland and lowland sites of western Kenya: Implication on malaria vector control measures. Frischknecht F editor. PLoS One. 2020; 15(2): e0224718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGimnig JE, Ombok M, Kamau L, et al.: Characteristics of larval anopheline (Diptera: Culicidae) habitats in Western Kenya. J Med Entomol. 2001; 38(2): 282–8. PubMed Abstract \| Publisher Full Text\n\nTakken W, Knols BGJ: Olfaction in vector-host interactions. Ecology and Control of Vector-borne Diseases. The Netherlands: Wageningen Academic Publishers; 2010; 2: 438. Publisher Full Text\n\nBokore G: Association between graminoids and the prevalence of immature malaria. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/NAT0YY" }	[ { "id": "70203", "date": "08 Sep 2020", "name": "Sisay Dugassa", "expertise": [ "Reviewer Expertise Medical Entomologist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPresentation of the work is very clear with a well designed methodology. Moreover, the analyses and interpretation of the results were properly presented. Importantly, the authors analyzed the association of early instar larvae of Anopheles with graminoid plants and reported that “Anopheles coustani, An. rufipes and An. maculipalpis were found only in aquatic habitats covered with graminoid plants, whereas An. arabiensis, An. ziemanni and An. pharoensis were found in both habitats with and without graminoid plants”. Moreover, they analyzed the correlation between the dominant graminoid plant species and early instar larvae and indicted the correlation is not species dependent. Such results are very important for future work in this area of research. However, there might be less importance of graminoid plants for some Anopheles species such as An. arabiensis, An. ziemanni and An. pharoensis. Therefore, authors should clearly indicate the potential importance of other factors than the plant species for the availability and density of the larvae (at least for An. arabiensis, An. ziemanni and An. pharoensis) in their conclusion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "70204", "date": "14 Sep 2020", "name": "Eric Ochomo", "expertise": [ "Reviewer Expertise Medical Entomology", "epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important article that attempts to associate the presence of sedges and the presence of immature malaria vectors along the shores on Lake Victoria on the Rusianga Island. The article is quite informative and can generate a tool that can be instrumental in the fight against malaria. I agree with most aspects of it but would like to suggest some improvements that can make it even look better. Below are some of the areas I noted that needs adjustment.\nExploitation of the oviposition cues can be very important in implementing the attract and kill mosquito control technique. The authors should identify and discuss more chemical cues that would potentially attract gravid females to lay eggs in the graminoid other than cedrol present in Cyperus rotundus in the other members of Cyperaceae family identified in this article.\n\nThe method used to estimate the perimeter of individual habitats is subject to errors incase two different people are involved since one person’s step cannot be exactly be the same to another ones. I suggest a verifiable method ought to have been used in evaluating habitat sizes.\n\nThe author indicates that he performed test for abiotic factors like turbidity, pH and temperature only. I think that the parameters were not the only abiotic factors that would influence the distribution of immature stages of malaria vectors. Abiotic factors like Dissolved oxygen (DO), salinity atmospheric pressure could also influence the abundance and distribution of mosquito larvae in the water and ought to have been evaluated.\n\nIn establishing the coverage of the various graminoid plants in the larval habitats by visually assigning percentages in a look and see manner, I think this is subject to error too in reporting the coverage of each Cyperaceae member. They are supposed to use a more objective method of estimating the abundance of the sedges.\n\nThere is a contradiction in reporting the result for species found in the habitats densely covered with graminoid vegetation and those that are found in habitats which are sparsely covered by graminoid vegetation and I am not sure if this was an error.\n\nThe article could use a thorough review for grammatical errors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5983", "date": "01 Oct 2020", "name": "Getachew Bokore", "role": "Author Response", "response": "We appreciate the constructive comments of the reviewer. Below we provide a point by point response. 1. Exploitation of the oviposition cues can be very important in implementing the attract and kill mosquito control technique. The authors should identify and discuss more chemical cues that would potentially attract gravid females to lay eggs in the graminoid other than cedrol present in Cyperus rotundus in the other members of Cyperaceae family identified in this article. We included some discussion on this as suggested. 2. The method used to estimate the perimeter of individual habitats is subject to errors incase two different people are involved since one person’s step cannot be exactly be the same to another ones. I suggest a verifiable method ought to have been used in evaluating habitat sizes. We clarified in the methods that the perimeter was estimated always by the same person. The perimeter was a relative estimate rather than a precise measure which we did not consider necessary in context of our study. 3. The author indicates that he performed test for abiotic factors like turbidity, pH and temperature only. I think that the parameters were not the only abiotic factors that would influence the distribution of immature stages of malaria vectors. Abiotic factors like Dissolved oxygen (DO), salinity atmospheric pressure could also influence the abundance and distribution of mosquito larvae in the water and ought to have been evaluated. We included a justification for the selection of the measures in the method section. All habitats surveyed were very similar in their characteristics, hence no major variation was expected; however, it cannot be excluded and we aimed to interpret the work carefully within its discussed limitations. 4. In establishing the coverage of the various graminoid plants in the larval habitats by visually assigning percentages in a look and see manner, I think this is subject to error too in reporting the coverage of each Cyperaceae member. They are supposed to use a more objective method of estimating the abundance of the sedges. Indeed for an ecological mapping of plant cover, there are various methods available for sampling, for example, the quadrant method and similar approaches. We explored and piloted some of these methods prior to the survey, however did not find them very informative or feasible given the nature of habitats. The most common way to measure cover is the visual estimation method. Visual estimation is popular because it is fast, requires no specialized equipment, and can be adapted to plants of various growth forms. Again, we have clarified that the estimation was done by a single person for relative comparability across sites. In the light of our findings, we would not expect that a different method would have led to a different conclusion. 5. There is a contradiction in reporting the result for species found in the habitats densely covered with graminoid vegetation and those that are found in habitats which are sparsely covered by graminoid vegetation and I am not sure if this was an error. We were not able to locate the contradiction, possibly this was a misunderstanding? 6. The article could use a thorough review for grammatical errors. We have gone through the article and corrected the English for errors." } ] } ]	1	https://f1000research.com/articles/9-1032
https://f1000research.com/articles/9-1188/v1	01 Oct 20	{ "type": "Brief Report", "title": "Journal- or article-based citation measure? A study of academic promotion at a Swiss university", "authors": [ "Nicole Steck", "Lukas Stalder", "Matthias Egger", "Nicole Steck", "Matthias Egger" ], "abstract": "In academia, decisions on promotions are influenced by the citation impact of the works published by the candidates. The Medical Faculty of the University of Bern used a measure based on the journal impact factor (JIF) for this purpose: the JIF of the papers submitted for promotion should rank in the upper third of journals in the relevant discipline (JIF rank >0.66). The San Francisco Declaration on Research Assessment (DORA) aims to eliminate the use of journal-based metrics in academic promotion. We examined whether the JIF rank could be replaced with the relative citation ratio (RCR), an article-level measure of citation impact developed by the National Institutes of Health (NIH). An RCR percentile >0.66 corresponds to the upper third of citation impact of articles from NIH-sponsored research. We examined 1525 publications submitted by 64 candidates for academic promotion at University of Bern. There was only a moderate correlation between the JIF rank and RCR percentile (Pearson correlation coefficient 0.34, 95% CI 0.29-0.38). Among the 1,199 articles (78.6%) published in journals ranking >0.66 for the JIF, less than half (509, 42.5%) were in the upper third of the RCR percentile. Conversely, among the 326 articles published in journals ranking <0.66 regarding the JIF, 72 (22.1%) ranked in the upper third of the RCR percentile. Our study demonstrates that the rank of the JIF is a bad proxy measure for the actual citation impact of individual articles. The Medical Faculty of University of Bern has signed DORA and replaced the JIF rank with the RCR percentile to assess the citation impact of papers submitted for academic promotion.", "keywords": [ "Relative Citation Ratio", "Journal Impact Factor", "DORA Declaration" ], "content": "Introduction\n\nIn academia, decisions on promotion to senior positions are influenced by the work published by the candidate. The assessment of publication lists should be systematic, using standardized criteria, and straightforward1. Bibliometric measures such as the journal impact factor (JIF) or its rank within a given field meet this requirement. The JIF or its rank reflect citations to all articles published in the journal, rather than citations to the article submitted by the candidate. Of note, it was initially created as a tool to help librarians identify the journals they should subscribe to, and not as a measure of the scientific quality or impact of research2. In 2013, the San Francisco Declaration on Research Assessment (DORA) was published with the aim to improve the way research output is evaluated2. Research should be assessed on its own merits rather than based on the journal in which it is published. The first general recommendation of DORA says “Do not use journal-based metrics, such as Journal Impact Factors, as a surrogate measure of the quality of individual research articles, to assess an individual scientist’s contributions, or in hiring, promotion, or funding decisions.”2. As of May 2020, more than 1,900 organizations and over 15,000 individuals have signed the DORA declaration.\n\nThere is thus growing consensus that the JIF is not a good measure to assess individual research papers. Efforts have been underway for several years to find a practical measure by which the citation impact of papers can be individually evaluated3–7. In 2016 Hutchins et al.8, presented the Relative Citation Ratio (RCR), an article-level measure of citation impact which compares the citations to the article of interest with the articles in the network of co-cited articles8. The RCR is benchmarked to National Institutes of Health (NIH)-funded research9: an article with an RCR equal to 1.0 is at the median for NIH-funded articles in this year10. The NIH provides access to the RCR and percentile of papers indexed in the PubMed bibliometric database on a website11. Several studies used the RCR to assess the citation impact of researchers, for example, vascular surgeons within the NIH12, biomedical scientists in one country13 or papers from scientific publications produced by the United States Food and Drug Administration (FDA)14.\n\nThe University of Bern signed the DORA declaration in January 2016. Therefore, the Medical Faculty decided to review its practice for internal promotion, where the assessment of citation impact was based on the rank of the JIF15. In 2018, a working group of the Medical Faculty examined whether the RCR could replace the ranking of the JIF as a decision-making aid for hiring, tenure and promotion decisions. The present study aimed to investigate the effects of switching from journal-based JIF-ranking to the RCR in the assessment of the papers submitted by candidates.\n\n\nMethods\n\nIn medical faculties in Switzerland, the habilitation degree and promotion to associate professor are essential steps in an academic career. The habilitation degree was introduced in the first half of the 19th century to ensure the quality of academic teaching and research at German universities. Today the habilitation is a post-doctoral qualification, which is required for independent teaching and supervision of doctoral students and to obtain an associate or full professorship in many European countries, including Switzerland16. At the University of Bern, the degree is conferred based on an application which includes a list of papers and a summary of the work highlighting the applicant’s contributions in research and teaching. An academic committee reviews the application, and the candidate presents and discusses his/her research at a faculty meeting. A similar process is followed for promotion to associate professor.\n\nUntil 2019, the faculty used the rank of the JIF15 for its assessment of applications for promotion to habilitation or associate professorships, and also The Journal Citation Reports (JCR)15 rank journals based on the JIF within subject categories, for example, oncology, surgery or nursing. Per university regulations17, candidates for habilitations needed at least ten original articles with a JIF rank in the upper third of the relevant discipline, and among the ten papers four as first or last author. The successful habilitation is a prerequisite for promotion to associate professor. The guidelines for promotion to associate professor required at least six additional original papers published in journals of the upper third of the JIF-based ranking, with at least three as first or last author18.\n\nThe Dean’s office of the Medical Faculty of the University of Bern compiled the publication lists submitted by a randomly selected 34 candidates for habilitation and 30 candidates for associate professor in 2017 and 2018. For each paper, we recorded the JIF of that year and its ranking in the corresponding field. The data were obtained from the JCR of Clarivate Analytics15. The Relative Citation Ratio (RCR) and the RCR percentile were obtained from the iCite tool11. Since the results for the papers submitted by candidates for the habilitation degree and for associate professorship were similar, we combined the data in the analysis.\n\nWe assessed the papers submitted by the candidates and the number of first- or last-author papers. We calculated the number with a JIF ranking in the upper third (the cutoff defined in the regulations) and the number with an RCR percentile >66%. To visualize the distribution of RCR percentile and JIF ranking by candidate, we used beam plots19 and kernel density estimation (Epanechnikov distribution, bandwith=5.0). We examined the relation between RCR percentiles and JIF rank in scatterplots and calculated the Pearson correlation coefficient and its confidence interval.\n\nThe calculation of the RCR and its percentile requires the article of interest to be cited so that these citations can be compared with those received by the articles in the co-citation network8. Papers in the second year after publication or more recent papers with five or more citations receive a provisional RCR11. We included both articles with definitive and provisional RCRs in the main analysis. For each candidate with articles from both categories, we calculated the difference between the articles with definitive RCR and all articles, including provisional RCRs, and combined the differences using random-effects meta-analysis. All statistical analyses were done in Stata version 15 (StataCorp, College Station, TX, USA).\n\n\nResults\n\nThe 64 candidates submitted 1,903 original articles, including 801 (42.1%) first- or last-author papers. A total of 134 papers (7.0%) had no JIF, and 328 (17.2%) had no RCR; 378 papers had to be excluded. A total of 1,525 articles were included in the analyses, including 625 (41.0%) first- or last-author papers and 223 (14.6%) articles with a provisional RCR. At the time of the download of the bibliometric data (12 September 2018) the total number of citations to the 1,525 articles was 45,119.\n\nThe beam plot in Figure 1 shows, row-wise for each candidate, the JIF ranking and RCR percentile of all 64 candidates. The kernel density estimation at the bottom of Figure 1 shows the differences in distribution of JIF ranking and RCR percentiles. As expected from the university regulations, the majority of papers were in the upper third of the JIF rank for all candidates. In contrast, they were more evenly distributed across percentiles of the RCR. Of note, some candidates had few or no articles above the 66th percentile of the RCR. Overall, 1,199 (78.6%) of 1,525 papers had a JIF rank in the upper third, and 581 (38.1%) had an RCR percentile above 66. Among the 625 first- and last-author papers, 489 (78.2%) had a JIF rank in the upper third, and 233 (37.3%) had an RCR percentile above 66. The beam plot and the kernel density estimation for first- and last-author papers was similar to the plot for all papers (see Extended data: Figure S120).\n\nJIF rank (left panel) and RCR percentile (right panel) are shown for each article submitted by candidates for habilitation (1-34) and associate professorship (35-64). Each candidate corresponds to one row. Kernel density estimation (epanechnikov, bandwith=5.0) for JIF rank (left panel) and RCR percentile (right panel) are shown below. The broken lines show rank 0.66 (left panel) and RCR percentile 66 (right panel).\n\nFigure 2 shows a scatterplot of the RCR percentile against the JIF ranks of all 1,525 papers submitted by applicants for habilitation and candidates for the associate professorship with data on both indicators. The correlation coefficient was 0.34 (95% CI 0.29 to 0.38). The scatter plot is divided into for quadrants by cutoffs 0.66 (for JIF rank) and 66 (for RCR percentile). Among the articles published in journals with a JIF ranking in the upper third, 57.5% (690 of 1,199) did not have an RCR percentile above 66 (blue quadrant in Figure 2). Conversely, 22.8% (72 of 326) of articles published in journals with a JIF rank in the lower two thirds (<0.66) had an RCR percentile above 66 (pink quadrant in Figure 2). The results for first- and last-author papers were similar: the correlation coefficient was 0.31 (95% CI 0.24 to 0.38), and the percentages of papers in the blue and pink quadrants were 59.1% (289 of 489) and 24.3% (33 of 136), respectively (Extended data: Figure S220).\n\nPublications are shown as point or cross for candidates for habilitation and associate professorship, respectively. Cutoffs of 0.66 for the JIF rank (as per university regulations) and 66 for the RCR percentile define four quadrants. The pink top-left quadrant shows the publications that have an RCR percentile >66 but were published in a journal with a JIF ranking <0.66. The blue quadrant shows the papers published in a journal with a JIF rank >0.66 but had an RCR percentile <66.\n\nIn total 57 candidates had both definitive and provisional RCRs (Figure 3). The meta-analysis of the differences between definitive and all RCRs across candidates gave an overall weighted mean difference of -0.04 (95% CI -0.13 to 0.04). There was thus no evidence of a systematic bias due to provisional RCRs, and no heterogeneity between candidates (I squared 0.0%).\n\nFor each candidate submitting articles with definitive and provisional RCRs the weighted mean difference (WMD, filled diamonds) and its 95% confidence interval (horizontal line) were calculated. The estimates were combined in a random-effects meta-analysis model. The empty diamond at the bottom shows the combined estimates from the meta-analysis of all candidates.\n\n\nDiscussion\n\nThis analysis of papers submitted to promotion committees at a Swiss university illustrates that the rank of the journal’s impact factor within its discipline is a bad proxy measure of the citation impact of individual articles. Many articles published in higher impact journals were cited less than their companion papers in the co-citation network. Whereas the majority of the papers submitted by candidates for the habilitation or an associate professorship were, by university regulation, published in journals with a JIF that ranked in the upper third of its field, only about 40% of these papers had an RCR percentile in the upper third. Furthermore, 20–25% of the papers that did not meet the requirement for the JIF rank (below 0.66) were, in fact, more impactful than their peers in the co-citation network. Our study thus confirms the findings of the RCR developer’s analysis of 80,000 papers: “Though journals with the highest impact factors have the highest median RCR, influential papers can be found in virtually all journals”8. Unsurprisingly, the correlation between the journal-based measure, the JIF, and the article-based measure, the RCR, was weak.\n\nSeveral previous studies used the RCR to assess the citation impact of different groups of researchers12–14. To the best of our knowledge, this is the first study investigating differences between a journal-based metric and an article-based metric of citation impact in the context of academic promotion. We used a large “real-world” dataset of over 1,500 papers submitted by candidates for academic promotion at a large Swiss medical faculty. Our results provide further empirical evidence supporting the San Francisco Declaration on Research Assessment (DORA)2, and challenges the practices at a Swiss university that relied inappropriately on the JIF. The results indicate that moving from a journal-based measure to an article-based measure is feasible. Indeed, the regulations of the Medical Faculty at the University of Bern have since been revised. The new regulations state that the assessment of candidates must follow the DORA principles. The committee analysing the papers should examine the novelty of the research question, the suitability of the methods, the interpretation of results and their relevance to the field. The evaluation should be based on the scientific content of the work. Article-based impact measures or qualitative indicators for the impact of the research (e.g. influence on policy and practice) may complement the assessment. Specifically, the regulations state that two or more of the papers with first or last authorship should have an RCR of 1 or higher. Also, the regulations explicitly state that “the journal and its impact factor will not be considered”21. The regulations for associate professor and titular professor were revised in the same spirit. They also refer to DORA2.\n\nThe RCR is based on citations and shares all the limitations of using citations as a proxy for impact. For example, the number of citations is influenced by factors unrelated to the quality of the research. The impact outside academia, including for political decision-making, is not well captured by citations22–24. Furthermore, unlike the JIF, the RCR requires time to allow citations to the article of interest to appear in the literature, which may limit its use in the context of academic promotion. In our study, only 328 of 1,903 (17.2%) had to be excluded because no RCR was available. Furthermore, within candidates, the inclusion of provisional RCRs of recent papers did not influence their mean RCR, indicating that provisional RCRs can be included in assessments. Of note, the developers of the RCR have shown that the RCR is usually very stable after one year11. The RCR is based on Medline and therefore not suitable for assessing non-biomedical literature10, for example, on medical ethics or teaching.\n\nMoreover, when using the RCR, it should not be forgotten that the reference is the papers financed by the NIH8. While an RCR of 1.0 corresponds to the median of the citations of NIH-funded articles, the median of all papers has an RCR of around 0.3711, subject to annual fluctuations. Another important point of criticism regarding the RCR is that “papers may be penalized rather than rewarded for receiving interdisciplinary citations”25. If a paper from a low-citation field is published in a journal from a high-citation field, this could reduce its RCR25. However, a comparison with other bibliometric indicators did not support this criticism26. Also, the developers of the RCR found a good agreement between metric and expert reviewer scores8.\n\nIn conclusion, we hope that our study will serve as a model to other researchers who, in the spirit of the DORA2 intend to challenge research assessment practices at medical and other faculties that rely inappropriately on Journal Impact Factors and contribute to promoting best practice that focuses on the value and influence of specific research outputs.\n\n\nData availability\n\nThe original raw data is composed of personal data of individuals applying for academic promotion and can therefore not be shared. The data without the identifying variables is available:\n\nOpen Science Framework: Journal- or article-based citation measure? A study of academic promotion at a Swiss university, https://doi.org/10.17605/OSF.IO/H7SKN20.\n\nOpen Science Framework: Journal- or article-based citation measure? A study of academic promotion at a Swiss university, https://doi.org/10.17605/OSF.IO/H7SKN20.\n\nThis project includes the following extended data:\n\n- Figure S1. Beam plots and Kernel density estimation of JIF rank and RCR percentile of first- and last-author publications submitted by candidates for academic promotion at the University of Bern. JIF (left panel) and RCR percentile (right panel) are shown for each article submitted by candidates for habilitation (1-34) and associate professorship (35-64) as first or last author. Each candidate corresponds to one row. Kernel density estimation (epanechnikov, bandwith=5.0) for JIF rank (left panel) and RCR percentile (right panel) are shown below. The broken lines show rank 0.66 (left panel) and RCR percentile 66 (right panel).\n\n- Figure S2. Scatter plot of RCR percentile against JIF rank of publications submitted by candidates for promotion at the University of Bern as first or last authors. Publications are shown as point or cross for candidates for habilitation and associate professorship, respectively. Cutoffs of 0.66 for the JIF rank (as per university regulations) and 66 for the RCR percentile define four quadrants. The pink top-left quadrant shows the publications that have an RCR percentile >66 but were published in a journal with a JIF ranking <0.66. The blue quadrant shows the papers published in a journal with a JIF rank >0.66 but had an RCR percentile <66.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank the Dean’s office of the Medical Faculty of the University of Bern for providing the data for the study. We are grateful to the working group of the Medical Faculty at University of Bern (Martin Bachmann, Thomas Geiser, Andrea Huwiler) for helpful discussions of this study. We thank Stephen Curry and Michael Hill for their constructive comments on a previous version of the manuscript.\n\n\nReferences\n\nAdler R, Ewing J, Taylor P: Citation Statistics: A Report from the International Mathematical Union (IMU) in Cooperation with the International Council of Industrial and Applied Mathematics (ICIAM) and the Institute of Mathematical Statistics (IMS). Statistical Science. 2009; 24(1): 1–14. Reference Source\n\nDORA – San Francisco Declaration on Research Assessment (DORA). [Accessed 3 July 2020]. Reference Source\n\nMoed HF, Burger WJM, Frankfort JG, et al.: The use of bibliometric data for the measurement of university research performance. Res Policy. 1985; 14(3): 131–149. Publisher Full Text\n\nZitt M, Small H: Modifying the journal impact factor by fractional citation weighting: The audience factor. J Am Soc Inf Sci Technol. 2008; 59(11): 1856–1860. Publisher Full Text\n\nBornmann L, Leydesdorff L: The validation of (advanced) bibliometric indicators through peer assessments: A comparative study using data from InCites and F1000. Journal of Informetrics. 2013; 7(2): 286–291. Publisher Full Text\n\nWaltman L, Yan E, van Eck NJ: A recursive field-normalized bibliometric performance indicator: An application to the field of library and information science. Scientometrics. 2011; 89(1): 301–314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaltman L, van Eck NJ, van Leeuwen TN, et al.: Towards a new crown indicator: Some theoretical considerations. Journal of Informetrics. 2011; 5(1): 37–47. Publisher Full Text\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. Vaux DL editor. PLoS Biol. 2016; 14(9): e1002541. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIan Hutchins B, Baker KL, Davis MT, et al.: The NIH open citation collection: A public access, broad coverage resource. PLoS Biol. 2019; 17(10): e3000385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSurkis A, Spore S: The relative citation ratio: What is it and why should medical librarians care? J Med Libr Assoc. 2018; 106(4): 508–513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNIH Office of Portfolio Analysis. NIH-Website iCite. Reference Source\n\nDavis FM, Obi AT, Gallagher KA, et al.: Accessing the academic influence of vascular surgeons within the National Institutes of Health iCite database. J Vasc Surg. Mosby Inc.; 2020; 71(5): 1741–1748.e2. PubMed Abstract \| Publisher Full Text\n\nSpiroski M: Relative citation ratio of top twenty Macedonian biomedical scientists in pubmed: A new metric that uses citation rates to measure influence at the article level. Open Access Maced J Med Sci. 2016; 4(2): 187–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider JA, Miklos AC, Onken J, et al.: An Analysis of Recent FDA Oncology Scientific Publications. Oncologist. 2020; 25(3): 266–270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJournal Impact Factor - Journal Citation Reports. In: Web of Science Group. [cited 6 Jun 2020]. Reference Source\n\nWeineck SB, Koelblinger D, Kiesslich T: [Medical habilitation in German-speaking countries : Quantitative assessment of content and elaboration of habilitation guidelines]. Chirurg. 2015; 86(4): 355–365. PubMed Abstract \| Publisher Full Text\n\nMedizinische Fakultät der Universität Bern: Habilitationsreglement der Medizinischen Fakultät der Universität Bern. 2016. Reference Source\n\nMedizinische Fakultät der Universität Bern. Assoziierte Professur1 Richtlinien, Anforderungen, Verfahren, 1997.\n\nBornmann L, Marx W: Distributions instead of single numbers: Percentiles and beam plots for the assessment of single researchers. J Assoc Inf Sci Technol. 2014; 65(1): 206–208. Publisher Full Text\n\nSteck N, Stalder L, Egger M: Journal- or article-based citation measure? A study of academic promotion at a Swiss university. 2020. http://www.doi.org/10.17605/OSF.IO/H7SKN\n\nMedizinische Fakultät der Universität Bern: Habilitationsreglement der Medizinischen Fakultät der Universität Bern. 2019; 8–8. Reference Source\n\nRavenscroft J, Liakata M, Clare A, et al.: Measuring scientific impact beyond academia: An assessment of existing impact metrics and proposed improvements. PLoS One. 2017; 12(3): e0173152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHogan AM, Winter DC: Changing the Rules of the Game: How Do We Measure Success in Social Media? Clin Colon Rectal Surg. 2017; 30(4): 259–263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiverani M, Hawkins B, Parkhurst JO: Political and Institutional Influences on the Use of Evidence in Public Health Policy. A Systematic Review. PLoS One. 2013; 8(10): e77404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaltman L: NIH’s new citation metric: A step forward in quantifying scientific impact? [cited 28 Apr 2020]. Reference Source\n\nBornmann L, Haunschild R: Relative Citation Ratio (RCR): An empirical attempt to study a new field-normalized bibliometric indicator. J Assoc Inf Sci Technol. 2017; 68(4): 1064–1067. Publisher Full Text" }	[ { "id": "72322", "date": "14 Oct 2020", "name": "Sandra L. Schmid", "expertise": [ "Reviewer Expertise I am a cell biologist and served as Chair of the Departments of Cell Biology at The Scripps Research Institute and UT Southwestern for a total of ~20 years", "hiring and promoting faculty. I was also a co-author and original signator of the DORA Proclamation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 straight-forward comparison of the article-specific bibliometric RCR with the journal specific metric JIF. The results clearly support the conclusion that the JIFG is NOT a reliable metric of the impact of individual papers published in the journal and therefore should NOT be used as a tool to assess promotion and tenure. This is a very important, yet not unexpected, finding. It should be widely distributed and acted upon, just as the University of Bern has altered its policies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "72318", "date": "19 Oct 2020", "name": "Bernd Pulverer", "expertise": [ "Reviewer Expertise Molecular Biology", "Scientific Publishing in biomedical sciences. Co-author of DORA declaration" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 valuable analysis underlying an important research assessment policy change at the University of Bern: a switch from assessing the top third research papers published by candidates for academic promotion as classified by the Clarivate 'Journal Impact factor' to the 'Relative Citation Ratio' (RCR), which uses a subject/citation network weighed algorithm. The analysis is based on 1,525 paper by 64 individuals and represents a sufficiently large dataset to yield meaningful results. The article concludes that RCR and JIF based rankings of papers do not show a significant correlation. This is important, but the authors should be careful not to conclude from the presented data that RCR is necessarily a good metric for academic performance. I suggest the authors consider enhancing the study by addressing the following points:\nMajor:\nThe papers analyzed are from 2017/18 and the analysis from 2018. I suggest to re-run the citation counts for 2020. This will ensure that citation rates to most medical papers have peaked and will allow the authors to extend the comparison between JIF and RCR to a third important metric: actual citations to the papers analyzed (I suggest using a fixed time window of e.g. 18 months).\n\nGiven the dramatic differences in the assessment by RCR vs. JIF and the policy change by Bern, it would be helpful to others considering adopting this policy change to specify how many candidates would have been reclassified in the academic assessment as a result of this change.\n\nThe RCR is presented without any critical discussion as the clear article-level indicator of choice. Please discuss limitations at least briefly e.g. the citation network is heavily based on reference lists, which can in principle be 'gamed' by authors to increase RCR rankings.\n\nOn p. 5 the authors conclude that candidates were more evenly distributed across percentiles using the RCR. This may not affect habilitation and academic performance evaluation, but it may affect hiring, where rank lists are used, since candidate ranking appear less pronounced.\n\nMinor:\nThe Abstract refers to a 'moderate correlation', while p.7 refers to a 'weak' correlation. fig shows a very weak correlation at best.\n\nIt would be helpful to specify in more detail what disciplines were covered and if any differences were noted between subject areas in the analyzed dataset.\n\nJIF and JIF rank within a Clarivate attributed field are treated as similarly problematic: I suggest to note that field ranking is even more problematic as the journals included are v. patchy e.g. general interest journals are not included.\n\nIt would be useful to briefly describe how the faculty plans to implement this significant policy change, especially during the transition period. Equally, to describe in more detail if other attributes are considered such as teaching quality (since 'habilitation' is described as a degree to 'ensure the quality of academic teaching and research' (p. 3).\n\nV. minor: add a comma for 1,525, in the abstract; add 'in principle' to line 6, p. 3; add 'the decl. states that' to line 14, p.3; change 'had no JIF' to 'were publ. in jnl. without JIF' & add 'Thus, after RCR: p. 4, l. 6.\n\nAdditional suggestions for further-reaching analyses:\n\nCandidates may chose different journals to publish if their assessment is based on RCR; a retrospective analysis if the journal profile of the faculty changes in the future would be fascinating.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72324", "date": "29 Oct 2020", "name": "Leonhard Held", "expertise": [ "Reviewer Expertise LH: Biostatistics", "Reproducibility and Replicability", "Bayesian Biostatistics", "Infectious Disease Epidemiology EF: Biostatistics", "Reproducibility", "Scientific Rigor and Science Policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments This is an interesting paper that compares journal- with article-based citation measures for the promotion of Swiss academics. The results from this analysis seem to have been used as a basis to change the current system for academic proportion at the University of Berne. In the following we outline some aspects of the present study that could be improved to further justify its use for decision making. Some additional discussion of other promotional criteria related to teaching, academic service (such as peer review) and more general contributions for the benefit of society (e.g. software, policy briefs, open data etc) could also be added. A consistent use of either “paper” or “article” would add more clarity to the paper.\nSpecific comments Introduction, 2nd paragraph:\n“The RCR is benchmarked to National Institutes of Health (NIH)-funded research: an article with an RCR equal to 1.0 is at the median for NIH-funded articles in this year.” Change “this year” to “the same year”\n\nMethods, Academic promotion, 2nd paragraph\n“Until 2019, the faculty used the rank of the JIF for its assessment of applications for promotion to habilitation or associate professorships, [...]” Surely the applications have been assessed in other ways too and the JIF has only been used to quantify the publication output of the candidates?\n\n“Per university regulations, candidates for habilitations needed at least ten original articles” Change “habilitations” to “habilitation”\n\n“The successful habilitation is a prerequisite for promotion to associate professor.” This has been mentioned in the paragraph above and can be deleted.\n\nStudy sample and data sources\n“The Dean’s office of the Medical Faculty of the University of Bern compiled the publication lists submitted by a randomly selected 34 candidates for habilitation and 30 candidates for associate professor in 2017 and 2018.” Change “submitted by a randomly selected” to “submitted by randomly selected”. This paragraph suggests that the study sample was not just a simple convenience sample. More details are needed on how this random sample has been obtained: how large was the underlying source population, why the sample size of 64 and how exactly was the sampling done?\n\n“For each paper, we recorded the JIF of that year and its ranking in the corresponding field.” Change “that year” to “the corresponding year”\n\n“Since the results for the papers submitted by candidates for the habilitation degree and for associate professorship were similar, we combined the data in the analysis.” What does similar mean here exactly?\n\nAnalysis The following issues need attention:\nDoes a study protocol (with pre data collection analysis plan) exist?\n\nThe data provided on OSF is not easy to read in - please separate explanation of variables from the actual data file, preferably not in Excel.\n\nFor “Promotion=AssocProf” the variables (“First_author”, “Last_author”) sometimes have the values (1, .) resp. (., 1) where “.” presumably means “missing”. What is the difference to (1, 0) resp. (0, 1) for “Promotion=Habil”? If a candidate is a first author then she/he cannot be a last author and vice versa.\n\nThe code of the analysis should be made available to make the analysis reproducible.\n\nWould it be possible and useful to follow the STROBE reporting guidelines on observational studies to some extent?\n\nFigure 1: The use of kernel smoothing along with the beam plots has the disadvantage that it extends beyond the domain boundaries (0-1). Also the y-scale of the left plot (kernel density estimation of JIF rank) is wrong, as the density does not integrate to unity. A simple histogram would provide the same information and is methodologically more appropriate.\n\nOne of the key results of the paper is the weak positive correlation between JIF rank and RIC percentile. However, in view of the scatter plot in Figure 2 a Spearman or Kendall rank correlation seems more appropriate than a Pearson correlation.\n\nFor the meta analysis it is written “For each candidate with articles from both categories, we calculated the difference between the articles with definitive RCR and all articles, including provisional RCRs, and combined the differences using randomeffects meta analysis.” This seems to indicate that for each candidate an average RCR of all articles with definitive RCR has been subtracted from an average RCR of all definitive and provisional articles. In Figure 3 it is mentioned that a weighted mean difference has been used. This needs to be clarified in more detail. Moreover, it needs to be made clearer to what purpose the meta analysis is used. All methods for which results are provided in the Results section should be described in the methods section, specifically the I-squared and the corresponding test. Figure 3 would profit from an indication which side corresponds to higher RCR for articles including only definitive RCR and which side for higher RCR for articles including definitive and provisional RCR.\n\nWe are also wondering if the Hartung-Knapp or the traditional DerSimonian-Laird method is used for random effects meta-analysis, Hartung-Knapp is known to perform better, see http://www.biomedcentral.com/1471-2288/14/25.\n\nIn the Results Section we learn that missing values are present. A discussion in the Methods Section on the treatment of missing values is needed. Specifically, an explanation why no imputation methods have been considered and some discussion of possible bias this may have caused.\n\nResults, 1st paragraph\n378 papers had to be excluded: how many authors does this affect and on average how many papers per author?\n\nThe relation between journal rank and article RCR\nA more consistent section header would be “The relation between JIF rank and article RCR percentile”.\n\n1st paragraph\n“The beam plot in Figure 1 shows, row-wise for each candidate, the JIF ranking and RCR percentile of all 64 candidates.” The plot shows the JIF ranking and RCR of all papers of a candidate not of the candidates themselves.\n\n2nd paragraph\n72 divided by 326 is 22.1% not 22.8%.\n\nDefinitive versus provisional RCRs\n“In total 57 candidates had both definitive and provisional RCRs (Figure 3).” Again, it is not the candidates but their papers that have RCRs.\n\nDiscussion, 2nd paragraph\n“Our results provide further empirical evidence supporting the San Francisco Declaration on Research Assessment (DORA), and challenges the practices at a Swiss university that relied inappropriately on the JIF.” Change “challenges” to “challenge”, “practices” to “former practices” We think this paragraph could be made clearer how exactly the results of this study influenced the faculty board in their decision to change practices. Was the study done before the decision and was the decision conditional on its results?\n\n3rd paragraph\n“For example, the number of citations is influenced by factors unrelated to the quality of the research.” Can you give a reference for this statement?\n\n“The RCR is based on Medline and therefore not suitable for assessing non-biomedical literature, for example, on medical ethics or teaching.” It would be better to stick with the term “Pubmed” and not use Medline, this will be confusing to readers not familiar with biomedical research.\n\n4th paragraph\n“Moreover, when using the RCR, it should not be forgotten that the reference is the papers financed by the NIH.” Change “the reference is the papers” to “the reference are the papers”.\n\nLast paragraph\nYou imply that the change in Berne was due to the initiative of researchers. Is this the case or was it a management decision? Hence the public you want to address with this article are not individual researchers but researchers who are now in managerial positions, right?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1188
https://f1000research.com/articles/9-1187/v1	01 Oct 20	{ "type": "Research Article", "title": "A framework for building and maintain trust in remote and virtual teams", "authors": [ "Zaheera Soomar" ], "abstract": "Trust is an important concept in assessing and measuring business behaviour from an organisational performance and culture lens, and has become a source of competitive advantage for organisations especially within the knowledge economy. Studies show that organizations with a high level of trust have increased employee morale, more productive workers, and lower staff turnover. Most organisations factor and measure trust as part of keeping a pulse on their organisational culture and design their initiatives around building and maintaining trust. While it is not impossible to build trust virtually, it certainly is harder and requires a different set of considerations. There has been a big shift by organizations catering for more remote and flexible work conditions over the past decade with the “virtual team” becoming the norm. The recent impacts of the COVID-19 pandemic have forced most, if not all, organizations to move in that direction faster than planned. With this movement to more remote working conditions, that are likely to have longer-term impacts, companies will be faced with challenges that virtual teams typically face in establishing and maintaining trust. This paper sought to highlight a framework that organisations, with remote and virtual teams, can use as a guideline to build and maintain trust. The framework suggests that trust is reliant on components from three key areas, namely 1) Foundational, 2) Organisational and 3) Individual. Components related to external aspects that contribute to trust, such as laws, reputation and society, have not been factored in. It is acknowledged that this will play a role in organisational and team trust but has been excluded from the scope of this research.", "keywords": [ "trust", "remote", "virtual", "organizational culture" ], "content": "F1000 Research Statement of Endorsement\n\nGiovani da Silveira confirms that the author has an appropriate level of expertise to conduct this research, and confirms that the submission is of an acceptable scientific standard. Giovani da Silveira declares they have no competing interests. Affiliation: Haskayne School of Business, University of Calgary.\n\n\nIntroduction\n\nTrust is an important concept in assessing and measuring business behaviour from an organisational performance and culture lens (Bachmann & Inkpen, 2011), and has become a source of competitive advantage for organisations especially within the knowledge economy (Barney & Hansen, 1994; Zanini, 2007 as cited in Pučėtaitė et al., 2015). Studies show that organizations with a high level of trust have increased employee morale, more productive workers, and lower staff turnover (Wichtner-Zoia, 2014). Most organisations factor and measure trust as part of keeping a pulse on their organisational culture and design their initiatives around building and maintaining trust. Many of these initiatives focused on building trust, rely on individuals having established face to face encounters (Nydegger & Nydegger, 1986). While it is not impossible to build trust virtually, it certainly is harder and requires a different set of considerations (Greenberg et al., 2007).\n\nThere has been a big shift by organizations catering for more remote and flexible work conditions over the past decade with the “virtual team” becoming the norm (Ford et al., 2017). Some of this has been driven by new generation talent requirements and cost reduction measures, but the recent impacts of the COVID-19 pandemic have forced most, if not all, organizations to move in that direction faster than planned. As of March 2020, most organisations have found themselves having to adapt overnight to cater for remote and flexible teams. While organisations are currently dealing with the immediate impacts of COVID-19, the longer-term repercussions are yet to kick in. The World Economic Forum (WEF) has indicated that the longer-term impacts could be worse than the 2008 economic recession (WEF, 2020). As a result of this and with growing pressure on organisations to offer higher levels of safety and precaution at work, organizations are likely to consider continuing their remote based working conditions beyond 2020 (WEF, 2020). Some tech giants have already indicated this, such as Twitter, Google, Facebook.\n\nWith this movement to more remote working conditions, which are likely to have longer-term impacts, companies will be faced with challenges that virtual teams typically face in establishing and maintaining trust (Owens & Khazanchi, 2018). This will require different considerations and actions, as compared to in-person teams, and this paper seeks to highlight a framework that organisations, with remote and virtual teams can use as a guideline to build and maintain trust.\n\n\nDefinition of trust\n\nTrust is an intrinsically relational construct (Bachmann et al., 2015) and described as “an attitude, or ‘state of mind’ that an individual develops over time in the face of experiences with other relevant individual actors” (Bachmann & Inkpen, 2011). Trust between two or a set of individuals is premised on risk-taking to rely on one another (Bachmann & Inkpen, 2011) where one party is willing to be vulnerable to the actions of another with limited control (Breuer et al., 2016), but trusting that the trustee will act in the best interest and perform an action that is important to the trusting party (Ford et al., 2017). Most current literature describes a trustworthy person as honest, able and caring, and factors in components of integrity, ability and benevolence (Greenberg et al., 2007).\n\nAccording to business ethics literature, trust is fundamental in all relationships (Brien 1998; Castaldo et al., 2010; Hosmer, 1995; Swift, 2001 as cited in Kujala et al., 2016), generates supportive behaviour, can save transaction costs and contributes to overall efficacy within an organisation. It is therefore fundamental for an organisation and its long-term success (Kujala et al., 2016).\n\nTrust can be both interpersonal, as well as inter-organisational, and is a requirement for building successful organisational relationships where the levels of trust accumulate over time, ultimately maturing and strengthening the relationship (Camén et al., 2011). Trust within a broader organisation context is critical for minimizing uncertainty, managing risks and operating costs, building and enhancing employee productivity and commitment, supporting business transactions and facilitating effective market participation. Therefore, a loss of trust can result in detrimental internal and external organisational performance (Bachmann et al., 2015; Pučėtaitė et al., 2015). The value of organisational trust has also been attributed to an organisation’s ability to reduce transaction costs, leading to new ideas and fostering their innovation capabilities (Bachmann & Zaheer, 2006, as cited in Bachmann & Inkpen, 2011).\n\nRegardless of the evidence and knowledge of the contribution of trust to organisational performance, many organisations still focus on an individual’s performance as the biggest metric attributable to company performance. As a result, organisations spend an enormous amount of time and resources improving and measuring performance but not nearly enough on trustworthiness, even though a loss or lack of trust can have far bigger repercussions (Sinek, 2020). An organisation is far better off with mediocre performers that inspire high levels of trust rather than high-performing employees with a low level of trust, as they are likely to impact the overall organisational culture, broader trust levels and ultimately organisational performance (Sinek, 2020).\n\nAs with organisational performance, there are many factors that contribute to team effectiveness; however, trust is known to be one of they key contributors (Ford et al., 2017) as it’s proven to be positively correlated with team effectiveness (Breuer et al., 2016). Clark, Clark, & Crossley’s (2010) research (as cited in Ford et al., 2017) shows that teams with a high degree of trust are “more proactive, more focused on task output, more optimistic, more frequently initiate interactions, and provide more substantive, productive feedback”. This is particularly important in high performing and meritocratic cultures where a lack of trust can limit team members willingness to share information or be collaborative with each other, impeding the team and certain individuals’ performances (Owens & Khazanchi, 2018).\n\nDespite the importance of trust in organisational and team relationships, there is a need to manage trust balances, i.e. too high or low is detrimental and organisations should aim to strike the right balance (Bachmann et al., 2015). High levels of trust within teams contribute to productive working relationships but coupled with strong ties, it can also account for false organisational unity, which can result in group think, negligent risk management, low levels of innovation and exclusion of different yet competent others (Kujala et al., 2016). In order to maintain the ethical culture and behaviour within and organisation, both levels of trust and distrust can co-exist as multi-dimensional and dynamic constructs (Kujala et al., 2016). Given that trust can have negative consequences, at an interpersonal and organisational level, it is still imperative for society as a whole to operate on a surplus of trust (Bachmann et al., 2015).\n\nThe foundation of trust is generally premised on values, standards and principles between two or more parties with the expectation of mutual fairness and honesty (Pučėtaitė et al., 2015). Trust typically develops in two ways. The first is called affective trust and is founded on emotional connections established through a mutual relationship, centered around care and concern for each other, resulting in a social bond. The second is called cognitive trust, which is based more on the rationale or evaluation of one’s performance reliability and competence (Greenberg et al., 2007).\n\nThere are many elements that are important to establishing trust between two or more individuals. These include leadership role-modelling, integrity, benevolence, fairness, and inclusiveness (Tuason, 2007). However, inclusiveness is subject to one’s propensity to trust, which is based on his/her past experiences, beliefs, values, and feelings. This influences how vulnerable that individual is willing to be and therefore each person’s capacity and propensity to trust is different (Tuason, 2007). Trust that develops based on personal face-to-face experiences is called interaction-based trust (Bachmann & Inkpen, 2011), whereas trust based on one’s personal traits or previous knowledge about them is called interpersonal trust (Camén et al., 2011). Trust can also be established through a third party who acts as a broker by being a proxy for the unknown counterpart, known as trust transferability (Bachmann et al., 2015). This requires the third party to have a trusted relationship with each of the individuals who do not know each other, and act as a guarantor to develop the trust (Bachmann & Inkpen, 2011). Institutional based trust is relationally embedded within the context of the institutional environment and focuses more on the favourable assumption of future behaviour within the context of that environment (Bachmann & Inkpen, 2011). When organisations need to make decisions swiftly or require rebuilding of teams, business units or the organisation itself, often during times in a crisis, institutional trust is essential (Bachmann & Inkpen, 2011).\n\nWhen new teams are formed, swift trust is established and based on initial judgements of trustworthiness, centered around personality traits, stereotyping, initial interactions and team trust (Ford et al., 2017). Team trust is an accumulation of the trust shared amongst team members based on a shared set of expectations and their willingness to be vulnerable to the actions of the rest of the team without having full control of other team members (Breuer et al., 2016). Following this initial swift trust, cognitive trust forms during the early stages of a team’s life and is based on the team’s competence (i.e. ability to accomplish the task) and the perception of others’ integrity through interactions with the team. As the team’s life grows, the dependency of cognitive trust related to competence reduces as team members get to know each other more. At this point, affective trust becomes more important, which is based on the continued assessment of integrity and benevolence (Greenberg et al., 2007).\n\nWithin organisations, trust is formed at the company-level (inter-organisational) and not directly linked to the individual or their interpersonal relationships. Therefore, it is important to differentiate between trusting a person and trusting an organisation. For this reason, it is important that an organisation acts in a trustworthy way, in addition to the individuals it employs (Camén et al., 2011).\n\nInstitutions and organisations are made up of commonly accepted practices, behaviours and rules, which guide employees on their behaviour and actions (Bachmann & Inkpen, 2011). Many scholars believe that institutions play a critical role in helping to establish trust in inter-organisational relationships (Bachmann & Inkpen, 2011). This can be done through established routines and practices that help facilitate communication, channel interactions between individuals, as well as through third party guarantor relationships (Bachmann & Inkpen, 2011).\n\nInstitutional-based trust is very similar to interaction-based trust, however, may be seen as weaker since interaction-based trust is generated based on intensive face-to-face encounters (Bachmann & Inkpen, 2011).\n\nRemote or virtual teams can be described as a set of two or more individuals who are organisationally or geographically dispersed that are unable to physically work together on a day to day basis and rely on technology and communication platforms to accomplish their common goal (Townsend, DeMarie, & Hendrickson, 1998 as cited in Ford et al., 2017). Traditional definitions of remote or virtual teams focused on the differences between face-to-face and virtual, while current literature focuses on teams along that continuum with a combination of both aspects (Fiol & O’Connor, 2005 as cited in Ford et al., 2017).\n\nRemote and virtual teams have become a common phenomenon within organisations over the past two decades (Breuer et al., 2016) as a result of trying to solve two common problems, namely 1) how to organise a set of individuals based on their expertise that cross traditional organisational design clusters, and 2) how to address location specific needs without replicating the team in each location (Ford et al., 2017). Today most large organisations are likely to have remote or virtual teams that sit along that continuum (Breuer et al., 2016) with leaders managing individuals that they might not have met and rather are only connected with through technology (Carrison, 2017).\n\nThere are many benefits of remote and virtual teams, due to their geographic and organisational dispersion such as flexibility to draw on knowledge, diversity of language, culture and perspective, a variety of cross functional skills and better coverage of time zones. This enables organisations to meet the demands of today’s hypercompetitive global environment (Greenberg et al., 2007). As a result, organisations are increasingly adopting new and innovative technologies for communication and collaboration to enhance performance within these team (Greenberg et al., 2007).\n\nHowever, remote and virtual teams add other associated challenges, in addition to the ones experienced by face-to-face teams. These include increased complexity, reduced inclusion, and barriers on language, culture and working styles (Ford et al., 2017) as a result of different location, time zones, cultural norms and multiple reporting lines (Nydegger & Nydegger, 1986). Despite there being a large set of literature available on how to manage in-person teams, there is far less literature, best practices and understanding for managing remote or virtual teams effectively (Ford et al., 2017).\n\nThe role, status, and importance that each team member brings and employs in a virtual team depends largely on the value created and brought to that group. This is truer for virtual teams than face-to-face where the measure of performance in virtual teams tends to be higher (Nydegger & Nydegger, 1986).\n\nHandy (1995) (as cited in Nydegger & Nydegger, 1986) stated that trust cannot be established in virtual teams. However, it has been proven that trust can be established in such teams but the process and speed of establishing trust is different and requires a different set of initiatives and actions (Nydegger & Nydegger, 1986). The process required for establishing and sustaining trust in remote and virtual teams is complex (Greenberg et al., 2007), as trust is very fragile in such teams and there are limited opportunities that present themselves to establish and build trust upon (Nydegger & Nydegger, 1986).\n\nIn traditional teams, trust develops from a history of face-to-face interactions between individuals that allow for interpersonal relationships to be established and result in the formation of affective trust (Greenberg et al., 2007). With remote and virtual teams, there are far less or no opportunities to build trust from a basis of face-to-face relationships, at least not from the get-go. Remote and virtual teams do establish an initial swift trust based primarily on external signals (roles, reputation, rules) and intrinsic reasons that are necessary for the team to immediately start working together (Greenberg et al., 2007). This is, however, based on their own dispositional trust, linked to their propensity to trust, and is less focused on an assessment of characteristics of other team members. This initial swift trust is therefore very fragile and if not built on or harnessed further, it can dissipate requiring teams to rebuild through new routes (Greenberg et al., 2007). Beyond this initial swift trust, there are further challenges in converting this trust into affective trust. Remote and virtual environments present very few opportunities for individuals to observe the subtle nuance, non-verbal cues and informally interact, typically through corridor chats and coffee breaks, with each other (Ford et al., 2017). Therefore it is difficult for individuals to create bonds of cohesion with fellow teammates that lead to assessments of benevolence (Greenberg et al., 2007).\n\nIt is established that trust in teams is central for effective teamwork and this seems to be well accepted amongst practitioners, particularly in virtual teams (Breuer et al., 2016). Individuals within teams need to be able to trust their leaders, each other and the organisations as a whole in order to be effective, particularly in virtual teams where there is less opportunity to mitigate for these challenges (Ford et al., 2017). A lack of or reduced trust within teams impacts individual and team performance, results in lower employee support and ultimately increases employee turnover (Nydegger & Nydegger, 1986).\n\nMost, if not all, remote and virtual teams rely on technology as the basis for communication and collaboration amongst team members, which has resulted in the growing presence of electronically mediated teamwork (Breuer et al., 2016). Despite the sophistication and constant innovation of technology and various collaboration and communication platforms, virtual teams often fail to meet their envisioned potential (Greenberg et al., 2007). Many scientists and practitioners have stressed the importance of trust as a big contributor to team effectiveness and success in electronically mediated collaboration as this often comes with feelings of uncertainty and perceived risks, which are present at much higher levels than face-to-face teams (Breuer et al., 2016). This is partly because the traditional social and cultural norms that exist in face-to-face teams are not available for influence by team members operating remotely or virtually and impacts the ability to establish cooperative behaviour or build familiarity with each other that often reduces these feelings of uncertainty or perceived risks (Greenberg et al., 2007). Another common challenge posed by electronically mediated teamwork is free-riding and lack of commitment, as team members don’t have to “face” each other, which makes it therefore even more critical to have a foundation of trust (Ford et al., 2017).\n\nRemote and virtual teams are here to stay, and likely to increase substantially in our current economic climate with degrees of virtuality varying across organisation. A foundation of trust is key in such teams and even more so as technological capabilities and platforms advance (Ford et al., 2017).\n\n\nFramework – antecedents of employee trust in remote and virtual teams\n\nThis framework indicates the key components and experiences of trust within remote and virtual teams. The framework suggests that trust is reliant on components from three key areas, namely 1) Foundational, 2) Organisational, and 3) Individual. The following section goes into depth on each of the three keys areas, and their components, supported by Figure 1 to show the relationship between them.\n\nThis framework is developed from a literature review as well as the author’s own experience. The framework is positioned simply for understanding and application purposes. However, the researcher acknowledges that complexities and overlap exist amongst the various elements.\n\nThe literature review encompassed a search of articles across various databases, using terms such as “remote work”, “remote teams”, “virtual team”, “culture”, “remote culture”, “remote working environment”, “virtual culture”, “communication in remote and virtual teams”, “technology in remote and virtual teams”, “trust”, “trust in teams”, “building trust”, “trust in organizations”, “trust in remote teams”, “virtual trust”, “trust in virtual teams”. Due to the recent shifts towards remote work and virtual teams, there is not an exhaustive list of articles available on this topic. After a review, a total of 22 articles (included within the reference list of this article) were maintained that were deemed relevant to the scope of the research. A qualitative analysis was done on all articles. This was done by coding the articles into categories that emerged. Once all categories were identified, the researcher grouped the categories into themes to build the framework.\n\nThe proposed foundational components consist of 1) Communication, 2) Transparency and accountability, and 3) Ethical culture. These areas are essential to any organisation, regardless of where they sit on the continuum of remote/virtual teams vs face-to-face teams. All three components are critical for building and maintaining trust between individuals and within teams and organisations, but even more so within a virtual and remote team environment. The lack of a strong foundation within organisations will constantly impair the ability to build and maintain trust regardless of other initiatives and elements in place.\n\nCommunication. Trust is paramount to all relationships, personal and business, and it is based on how, when and what is being communicated within that relationship (Denton, 2012 as cited in Owens & Khazanchi, 2018), which includes knowledge sharing related to the tasks or goals at hand (Owens & Khazanchi, 2018). There is a lot of evidence and literature that proves team effectiveness is heavily correlated with effective communication, regardless of the structure of the team (i.e. face-to-face vs remote or virtual) (Nydegger & Nydegger, 1986).\n\nProfessor Mark Mortensen (HBR, 2015) cautioned that the “social distance”, separating virtual team members from corporate headquarters, can create an us vs. them mentality, and that the way to combat team-alienation is to “reinforce what’s shared: the team’s purpose.” This kind of reinforcement depends on consistent communication (Carrison, 2017). The traditional forms and rules of communication in a face-to-face environment need revisiting though when dealing with remote and virtual teams to cater for the complexities of these teams and factor in elements such as time zone differences, the associated delays and feeling of contribution and inclusiveness (Greenberg et al., 2007). Given that the forms, structure and rules of communication might differ when dealing with virtual and remote teams, it is expected that the interpersonal dynamics resulting from these communications will be different too (Nydegger & Nydegger, 1986), which can have an impact on levels of trust.\n\nRemote and virtual teams rely heavily on technology for effective communication; however, this can be very constraining (Greenberg et al., 2007). Regardless of the actual technology (hardware or software) used, success is reliant more on the quality of the information and how it is being communicated (Nydegger & Nydegger, 1986). Electronically mediated communication does not cater for the same levels of empathy, emotion and physical reaction that one can deliver in a face-to-face setting. This limits the communicator’s ability to read non-verbal cues that signal acceptance, support, behaviour and general attitude (Greenberg et al., 2007). Therefore, it is critical to consider the message, the audience and the potential modes (when, how) when communicating to remote and virtual teams and even more so when dealing with a combination of both face-to-face and remote/virtual teams to ensure the experience felt by the receivers are similar (Greenberg et al., 2007). Electronically mediated communication also impacts trust levels, as these forms of communications are often recorded, shared and stored (such as call recordings and chat histories), which can limit individuals’ willingness and ability to be honest or speak freely (Breuer et al., 2016). It is therefore suggested that leaders encourage social conversation, apart from task/goal related conversation, to build stronger connections and cater for trust building opportunities (Greenberg et al., 2007).\n\nTransparency and accountability. Transparent organisations share information that is accurate, timely and relevant amongst its employees and stakeholders, allowing these individuals to build an understanding, reflect and make informed decisions (Bachmann et al., 2015). This includes transparency around employees’ ethical and unethical behaviour, the associated implications and the perception thereof (Pučėtaitė et al., 2015), which goes beyond sharing of information and ensures accountability too (Bachmann et al., 2015). Transparency and accountability extend into teams, and leaders must find ways to be transparent with each other (Ford et al., 2017). Organisations and teams that foster transparency and accountability build cognitive trust (Bachmann et al., 2015) through logic and rational understanding of actions and their associated impact.\n\nEthical culture. Ethical organisations are transparent, accountable and ensure proper internal communication around breaches of ethical principles and values (Pučėtaitė et al., 2015). When these ethical values are embedded into an organisation’s routines and procedures, this allows the organisation to safeguard against unethical behaviour. This is critical but needs to be coupled with the role modelling of the leadership team to really build and strengthen the ethical culture of an organisation. A strong ethical culture can then serve as a compass for all employees to do the right thing in every circumstance (Bachmann et al., 2015).\n\nDuring early stages of remote/virtual team and organisation setup, while swift trust is emerging through initial interactions, an organisation’s ethical culture can be enabling by both strengthening the initial swift trust and serve in developing the organisational trust (Pučėtaitė et al., 2015). The impact of this enabling function is related to the person/organisation fit and is defined by the comparison between the individuals and organisations ethical values. Previous studies show that higher correlation between these values leads to a higher person/organisation fit and ultimately stronger levels of trust (Pučėtaitė et al., 2015).\n\nOrganisational structures and strategies can vary over time. An organisation takes its cues from its employees and stakeholders on what is working and what needs adjusting. When organisations consider and respond to these cues, it sends signals to its employees that it cares, and this builds trust amongst employees and the organisation. Given that trust is critical in effective virtual teams, focusing on areas and strategies that consistently builds trust amongst employees and the organisation, is critical for having remote or virtual teams (Ford et al., 2017).\n\nThe proposed organisational components are centred around considerations and provisions made or controlled by the organisation and/or the top-level organisational leadership. These components are generally applicable to the whole organisation. They are 1) Systems, policies, and procedures, 2) Technology, and 3) Rewards and incentives. This section will not focus on external components related to trust such as laws, reputation, and community norms (outlined in Bachmann et al., 2015).\n\nSystems, policies and procedures. Organisational systems, policies and procedures help frame how employees can make decisions and take action based on accepted ethical norms and principles. When employee’s role model these norms and principles by following the policies and procedures, they indicate to fellow employees and team members that they can be relied on. This allows others to predict one’s behaviour and action leading to increased levels of trust (Pučėtaitė et al., 2015).\n\nThere are a number of recognized policies and procedure practices that help build trust in remote and virtual teams over the life cycle of a team. At the start of a team or when a new team member joins, it is very important to have the right training and onboarding systems in place. It is critical to include additional information for virtual team onboarding, such as working styles, team norms, team member backgrounds, qualifications and task roles that will create a sense of inclusion and belonging for the new team member, which is generally acquired in face-to-face engagements (Ford et al., 2017). Another common strategy for managing trust within virtual organisations is to have clear policies, process, contracts and codes of conduct to make explicit what is acceptable vs unacceptable behaviour (Bachmann et al., 2015). While this is true for face-to-face teams too, these teams tend to build an understanding of acceptable and unacceptable behaviour by watching leaders and others in the organisation and then following suit. This military practice, quoted by Lieutenant General David Lindsay Morrison, which states “The standard you walk past, is the standard you accept”, is often adopted in face-to-face organisational settings. For virtual teams, this is not always possible and therefore it is important for organisations to clearly articulate these behavioural expectations into documents such as a code of conduct, to make it easier for virtual team members to consistently embody them. Organisations should also constrain unacceptable behaviour and incentivise acceptable and trustworthy behaviour in order to develop this understanding and reduce the likelihood of violation (Bachmann et al., 2015). Another well used tool is a contract, which is generally seen as a complementary control mechanism to create trust (Camén et al., 2011). Contracts are used to define the relationship, agree on key principles and accepted practices, outline each parties’ contribution and act as a communication tool to reduce risk and uncertainty (Camén et al., 2011). Effective systems, policies and procedures can also be used as a substitute for direct leadership in teams where physical team presence is dispersed. This can be done in the way of guidelines, documents on “what good looks like” and training guides (Ford et al., 2017).\n\nWhile systems, policies and procedures are setup to aid in the establishment of trust, they can also hinder trust. It is important for organisations to ensure that their systems, policies and procedures are fit-for-purpose and are not seen as too strict, overly structured or inflexible, which can be demotivating to employees and ultimately impact trust (Bachmann et al., 2015).\n\nTechnology. Virtual teams depend on having the appropriate communication technology to connect, support and deliver on their goals individually and as a team. This technology is the main form of connection between these members, and while the quality of the information is more important for building trust, the technology is an important enabler for this. Organisations who recognise and understand this send cues to their employees by investing in and equipping them with quality technology (hardware and software) to enable and enhance their ability to deliver and build trust-based relationships (Ford et al., 2017).\n\nRewards and incentives. It is very easy for virtual team members to believe that out of sight leads to out of mind in relation to leadership. This can be very challenging and demotivating for remote and virtual team members, where they often feel like their work is not seen or valued. It is important for leaders to communicate to virtual teams that their work is not only seen but valued and recognised. This is important in maintaining trust between virtual teams and organisational leaders and signals that their careers are protected (Ford et al., 2017).\n\nThere is a high dependency and reliance on information sharing within virtual teams and therefore an organisation with virtual teams’ reward structure should focus on team performance and cooperative rewards, which will encourage and foster team trust. Individual competitive reward structures negatively influence individual’s willingness to share information, which affects the way team members perceive each other’s behaviour and their integrity levels (Greenberg et al., 2007).\n\nThe proposed individual components are centred around considerations that are within an individual, leaders or team’s ability to influence and control. These can vary across an organisation from team to team or within relationships and can be tailored to the type, structure of the team or relationship, the outcomes or goals required, or just general preferences between individuals. The components are 1) Selection of team and the 2) Relationship between line manager and employee.\n\nSelection of team. Leading and managing a team comes with its challenges and complexities, especially with different generations making up the workforce. It is far more challenging leading teams and individuals who are physically based in different offices or locations around the world and who seldom see each other (Ford et al., 2017). These management setups have increased considerably over the past decade with the number of remote and virtual teams being catered for in organisations, and this has created an escalating interest in developing best practices on how to manage such teams.\n\nLiterature has shown that managers of such teams have to take steps to create a foundation of trust, prior to team members joining the team (Greenberg et al., 2007) and the team members selected to join need to have a predisposition to trust each other in order to perform collaboratively (Ford et al., 2017). This requires a new set of skills and capabilities from individuals, especially managers, and organisations are having to increasingly factor in training and development for those that require this new skill (Ford et al., 2017). Professor Erin Meyer, writing for Forbes (Carrison, 2017)., believes that the type of skills needed by leaders and manages in face-to-face teams differ considerably to those in remote and virtual teams and are sometimes the opposite. Virtual teams require high levels of coordination where a leader should provide clearly defined direction to his/her team and remove all ambiguity from the process (Carrison, 2017). Leaders of virtual teams need to also pay more attention to drawing in the human needs of their team members, as this can easily get lost in an electronically mediated team. They need to find alternate methods to cater for and interpret non-verbal cues and cater for and foster differences that are respected across the team (Ford et al., 2017).\n\nOrganisations who recognise and understand the different type of leadership needed for these teams send cues to these teams that build trust by intentionally investing in the selection and preparation of such leaders (Ford et al., 2017).\n\nRelationship with line manager and employee. People don’t leave a company, as the saying goes, they quit their manager. We have all heard this quote many times and it has been proven in multiple employee and market surveys across different regions. An employee’s perception of an organisation is determined by the employee’s perception of the quality of the manager-employee relationship (Pučėtaitė et al., 2015). The quality of this manager-employee relationship is critical for employee-manager trust (Tuason, 2007) and extends beyond, onto the organisation.\n\nSuccessful managers of virtual teams build trust by giving advice and guidance instead of dictating and micromanaging. They focus on giving plentiful feedback and concentrate on building confidence amongst their teams (Macaulay & Cook, 2011). Employees judge a manager by the consistency between what he/she says and does. The more consistent this is, the higher the sense of integrity the employee holds for that manager, thus increasing levels of trust (Tuason, 2007). Employee trust is also increased when an employee is given greater control of the outcomes by being included in the decision-making process. This also signals an increase in manager trust as they have shown willingness to share the control and responsibly (Tuason, 2007). A manager’s efforts to loosen or share controls is somewhat reflective of their efforts to show and build trust, however, can be challenging when trying promote subordinate cooperation and balance the tension between controls and trust (Long, 2018).\n\n\nLimitations\n\nThe framework is limited to the literature covered, which was based on trust within organisations and virtual/remote teams. Further research into each of the framework components is limited and is required to substantiate or identify any missing components. Lastly, components related to external aspects that contribute to trust, such as laws, reputation and society, have not been factored in. The researcher acknowledges that this will play a role in organisational and team trust but has been excluded from the scope of this research\n\n\nConclusions\n\nAs more organizations are being forced to consider remote, virtual and flexi working options, it will become important for them to consider the impact this has on their culture and specifically around building trust within the organization and teams. The aim of this research was not to provide an exhaustive list of all categories that would encompass a framework of trust within remote and virtual teams. The researcher acknowledged that there is complexity and overlap that is likely to exist amongst the categories and additional elements might exist. However, the purpose of this research was to develop a simple framework that allows organizations to realise and consider multiple aspects across the elements (foundational, organizational and individual) when building and maintaining trust amongst remote and virtual teams.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nBachmann R, Gillespie N, Priem R: Repairing Trust in Organizations and Institutions: Toward a Conceptual Framework. Organ Stud. 2015; 36(9): 1123–1142. Publisher Full Text\n\nBachmann R, Inkpen AC: Understanding institutional-based trust building processes in inter-organizational relationships. Organ Stud. 2011; 32(2): 281–301. Publisher Full Text\n\nBreuer C, Hüffmeier J, Hertel G: Does trust matter more in virtual teams? A meta-analysis of trust and team effectiveness considering virtuality and documentation as moderators. J Appl Psychol. 2016; 101(8): 1151–1177. PubMed Abstract \| Publisher Full Text\n\nCamén C, Gottfridsson P, Rundh B: To trust or not to trust?: Formal contracts and the building of long-term relationships. Manage Decis. 2011; 49(3): 365–383. Publisher Full Text\n\nCarrison DAN: In managing virtual teams, email is a key. Industrial Management. 2017; 59(1): 6. Reference Source\n\nFord RC, Piccolo, RF, Ford LR: Strategies for building effective virtual teams: Trust is key. Bus Horiz. 2017; 60(1): 25–34. Publisher Full Text\n\nGreenberg PS, Greenberg RH, Antonucci YL: Creating and sustaining trust in virtual teams. Bus Horiz. 2007; 50(4): 325–333. Publisher Full Text\n\nKujala J, Lehtimäki H, Pučėtaitė R: Trust and Distrust Constructing Unity and Fragmentation of Organisational Culture. J Bus Ethics. 2016; 139(4): 701–716. Publisher Full Text\n\nLong CP: To control and build trust: How managers use organizational controls and trust-building activities to motivate subordinate cooperation. Accounting, Organizations and Society. 2018; 70: 69–91. Publisher Full Text\n\nMacaulay S, Cook S: Building trust into your organisation. Training Journal. 2011; 17–20. Reference Source\n\nMortensen M: A First-Time Manager’s Guide to Leading Virtual Teams. Harvard Business Review Digital Articles. 2015; 2–5. Reference Source\n\nNydegger R, Nydegger L: Challenges in managing virtual teams. Preventing School Failure. 1986; 8(3): 49–51. Reference Source\n\nOwens D, Khazanchi D: Exploring the impact of technology capabilities on trust in virtual teams. American Journal of Business. 2018; 33(4): 157–178. Publisher Full Text\n\nPučėtaitė R, Novelskaitė A, Markūnaitė L: The mediating role of leadership relationship in building organisational trust on ethical culture of an organisation. Economics and Sociology. 2015; 8(3): 11–31. Publisher Full Text\n\nSinek S: Simon Sinek: How to Deal With \" Toxic \" Team Members. 2019-2021. 2020. Reference Source\n\nTuason IJ: The Relationship between Trust and Organizational Culture Change. E 9th Annual International \\rConference on Business: Accounting, \\rFinance, Management & Marketing, \\r, (July 2011), 2007; 1008. Publisher Full Text\n\nWEF: COVID-19 Risks Outlook A Preliminary Mapping and Its Implications. 2020. Reference Source\n\nWichtner-Zoia Y: https://www.canr.msu.edu/news/understanding_the_importance_of_trust_in_the_workplace. 2014." }	[ { "id": "76728", "date": "15 Apr 2021", "name": "Mohammad Alsharo", "expertise": [ "Reviewer Expertise Virtual teams", "social computing", "and health information systems." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 discusses not a new but interesting topic, trust in virtual teams. The author introduces an interesting argument and the paper itself is well written and well organized. Up until the methodology section the paper was easy to read and easy to follow. However, I noticed that although the topic dates back to the mid 1990s the author focused on recent research in supporting her argument.\nThere is a major limitation in this paper which is the methodology which brings questions more than the answers it provides. Consequently, I strongly believe that the methodology should be revised to provide support for the proposed framework. The major weakness of the manuscript is clearly written in the limitations. In the author’s words “The framework is limited to the literature covered”. I do not think the literature is satisfactorily covered to reach the conclusion of the manuscript. Please pay attention to the following concerns I have in the methodology:\n“This framework is developed from a literature review as well as the author’s own experience. “\nHow is the author’s experience relevant to the methodology? This is not obvious in the paper and I do not think is relevant in a literature review.\n“The literature review encompassed a search of articles across various databases: “\nCan you specify which databases were utilized and what is the literature review process was like? Did the author undergo a systematic review? How many research papers were initially retrieved before deciding that 22 are relevant? What were the search criteria in terms of language, publication years, research method…etc. I would like to refer the author to a recent article which I believe would benefit this paper in terms of literature review methodology and virtual teams literature: Alaiad, A., Alnsour, Y., & Alsharo, M. (2019). Virtual teams: Thematic taxonomy, constructs model, and future research directions1. From this research paper’s abstract: “This paper reports on a systematic examination of the literature on virtual teams through which we provide a thorough review, analysis, and synthesis of research published in the past 10 years”\n“Due to the recent shifts towards remote work and virtual teams, there is not an exhaustive list of articles available on this topic.”\nI do not understand the logic of this sentence, how come that due to recent shift towards remote work leads to no exhaustive list of articles available on this topic? I think there exist an exhaustive literature on trust in virtual teams context. In fact, throughout the paper I noticed that many important articles relevant to the topic are not cited, for example:\nR.C. Mayer, J.H. Davis, F.D. Schoorman, An integrative model of organizational trust2. Zaheer, V. Perrone, B. McEvily, Does trust matter? Exploring the effects of inter-organizational and interpersonal trust on performance3. K. Dirks, D. Ferrin, The role of trust in organizational settings4. S. Jarvenpaa, D. Leidner, Communication and Trust in Global Virtual Teams5. S. Jarvenpaa, T. Shaw, D. Staples, Toward a contextualized theories of trust: the role of trust in global virtual teams6.\nTaking into consideration that several highly cited published research papers relevant to the theme and goal of this research manuscript are not discussed or cited, I do not think the literature review conducted here is satisfactory to reach with high confidence the conclusion which the author argue. I do not disagree with the argument, but the weakness in the methodology hinders the findings.\nFoundational components title is mentioned twice in the paper. In page 6 where the author actually argues foundational components and in page 7 where I believe it should be organisational not foundational\nOverall, I recommend a major revision for the methodology section in order to approve this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1187
https://f1000research.com/articles/8-1825/v1	30 Oct 19	{ "type": "Research Article", "title": "Evaluation of cropping method for perennial ratoon rice (SALIBU)", "authors": [ "Masato Oda", "Huu Chiem Nguyen", "Van Thao Huynh", "Huu Chiem Nguyen", "Van Thao Huynh" ], "abstract": "Background: Generally, the yield of ratoon rice is at most 50% of the main crop. However, a cropping method “SALIBU” achieved more yield than the main crop and could be used for the cultivation of perennial cropping. Although the SALIBU method is implementing 10 additional management practices to conventional method, the effect of each management practice is unclear. Therefore, we aimed to evaluate the effect size and the robustness of each management practice. Methodology: We evaluated the effect size using an L16 orthogonal array design pot experiment. For the robustness, we duplicated the experiment under both standard and checked whether the practice shows the same effect size. The bad conditions were low plant density, no fertilization, continuous flooding water management, and late harvesting. Results: The ratoon rice yield was proportional to the number of ratoon tillers used as in conventionally produced ratoon rice. Late cutting was most affected to the number of ratoon tillers. Importantly, this effect was reversed; the effect was positive under bad conditions, but was negative under standard conditions. Furthermore, late irrigation, a recommended management practice, had a robust negative effect on ratoon tillers and yield under both the conditions. Positive large effects were shown in the bad condition only. Discussion: Our results show that the SALIBU cropping method includes practices with unstable, negative, or minimal effects. The practices have unstable effects should be clarifying the interaction with the condition. The practices that have negative effects should exclude. Using practice on small effect size should depend on a cost-benefit analysis. Conclusions: SALIBU will be acceptable to the Mekong Delta triple cropping rice cultivation without the additional practice of original SALIBU cropping method. However, further work is needed to clarify the interaction between late cutting and the cultivation condition, and on the negative effect of late irrigation.", "keywords": [ "The Mekong Delta", "Triple rice cropping", "Methane mitigation", "Row input", "Sustainable", "Taguchi method", "Effect size" ], "content": "Introduction\n\nRice is usually an annual crop but can be renewed using the ratoon cropping method. Perennial cropping of rice requires less labor and water, while reducing climate risk and greenhouse gas emissions. Perennial rice cropping is a traditional cropping method but is rarely used because of the low yield. However, the additional yield achieved after harvesting the main crop has allowed its successful commercial application in the southern region of the United States of America and parts of southern China (Sacks, 2013). Most studies on ratoon rice have focused on additional yield, and the yield is at most 50% of the main crop (Negalur et al., 2017). A previous study reported a yield of up to 90% of the main crop using a special variety (PR23), but the fluctuations in yield were very large (Zhang et al., 2014).\n\nRecently, a breakthrough in increasing yield was achieved in Indonesia. Rice has limited growth period during winter; therefore, rapid growth is the key to success of ratoon cropping. Moreover, ratoon cropping from higher nodes of rice is important, because the carbon can be accessible to the main crop culm (Balasubramanian et al., 1992). However, because tropical regions have no winter, the method used can be different. Fitri et al., (2019) looked at an updated method of traditional perennial rice cropping developed by Mr. Erdiman in 2010. The method was named “SALIBU,” a portmanteau of the Indonesian words “SALIN” (replication) and “IBU” (mother). Using SALIBU, the same or higher yield than that of the main crop was achieved. The mechanisms inducing the high yield have not been well studied, but a possible reason is that lower nodes can extend new roots and improve nutrient uptake from the soil (Yamaoka et al., 2017). The implementation of SALIBU has been successful in different areas and at different elevations and groundwater levels in Indonesia (Fitri et al., 2019), and has recently spread to Myanmar (Yamaoka et al., 2017).\n\nIn some cases, the yield was less than 50% of the main crop yield (Hidayati et al., 2018). To adapt the SALIBU cropping method to areas with different growing conditions, we should evaluate the performance of each management practice, and modify the practices to suit the conditions. We aim to adapt the SALIBU method to direct seeding triple-cropping of rice in the Mekong Delta. Here, we found that the most effective (positive) management practice under poor conditions had an adverse effect (negative) under standard conditions. Furthermore, we found that the practice has a robust negative effect on the yields under both poor and standard conditions.\n\n\nMethods\n\nEvaluating each practice of a cropping method under different conditions is difficult because of the huge number of possible combinations. We have summarized the management practices of SALIBU method (Yamaoka et al., 2017) into four practices. We allocated those practices to two levels of an L16 orthogonal array (Taguchi, 1986) and conducted a pot experiment. To test robustness, the experiment was replicated under standard conditions and poor conditions, namely low plant density, no fertilization, continuous flood water management, and late harvesting: these conditions are known to reduce the yield of ratoon cropping of rice (Negalur et al., 2017). We analyzed the effect of each of the four practices on ratoon tillers and yield. Then, we evaluated the robustness of the effect of practices between the two conditions.\n\nThe pot experiment was conducted in a fine net house at Can Tho University (Can Tho city, Vietnam) from December 2018 to June 2019. We used 38 cm × 58 cm wide and 30 cm high containers. All containers were filled up to 20 cm with paddy soil. The soil was collected from topsoil (about 25 cm) of a paddy field at TL2 Hamlet, Thuan Hung village, Thot Not district, Can Tho city, Vietnam, just after natural flooding of the Mekong River and used on the day it was collected. The soil was well mixed in advance. Germinated seeds (Jasmin 85 variety from Can Tho University, popular in the Mekong Delta) were used. Jasmin is an Indica and has characteristics unsuitable for ratoon cropping of rice (Negalur et al., 2017). These disadvantages will amplify the effects of the practices. We used urea (46% N), single superphosphate (16% P2O5), and potassium chloride (61% K2O) as fertlizers; the applied amount of those contents (kg ha-1) used for each treatment are given in the following section.\n\nSALIBU management consists of nine special management practices in addition to the conventional cropping management practice of rice transplanting (Yamaoka et al., 2017). The practice of early harvesting (physiological maturity; 25% green color husk) is conventional in Mekong Delta triple-cropping cultivation. The rest of the practices are as follows. (1) Pre-fertilization: 25 kg ha−1 N and 46.75 kg ha−1 P2O5 at seven days before harvesting. (2) Cutting twice: all rice was harvested 25 cm above the ground, then cut again beneath the first node above ground on day seven (or day zero for control plants) after harvesting (rice straws were returned to the ground). The recommendation is to cut 3–5 cm above ground; we kept only the node below ground. (3) Late irrigation: irrigation was started on day 14 (or day seven for control plants) after harvesting (the water table was about 5 cm until irrigation started). (4) Adjusting: the practice consisted of (a) hand weeding, (b) dividing hills into two or three tillers and replanting to fill the space, (c) pushing the rice plants into the soil if the root came up on soil surface, (d) removing excess plants to keep original plant density, and (e) draining from day 29 to 43 after harvesting (though (e) is not “adjusting”, it is technically inseparable because “adjusting” requires draining). We did the pot experiment using an L16 orthogonal array design. We set the pots randomly in the fine net house.\n\nThe standard conditions were based on the standard of direct seeding triple rice cropping in the Mekong Delta: the plant density was 230 kg ha−1 dry weight (about 173 seeds per pot), fertilizer was applied three times on day seven (27.6 kg ha-1 N, 45.2 kg ha-1 P2O5, 3.68 kg ha–1 K2O), 20 (36.7 kg ha-1 N), and 42 (27.6 kg ha-1 N, 3.68 kg ha-1 K2O) after seeding, with alternate wet and dry water management (15 to 5 cm; from seven days after seeding to 10 days before harvesting).\n\nThe poor conditions were as follows: low plant density (nine plants per pot), no fertilization (except the pre-fertilization treatment), continuous flooding water management (0 to 5 cm, from seven days after seeding to 10 days before harvesting), and late harvesting (seeded 10 days before the standard condition plants and harvesting on the same day of harvesting as the standard conditions). These conditions are known to negatively affect ratoon cropping of rice (Negalur et al., 2017).\n\nWe recorded the number of plants and ratoon tillers at the harvesting time. We immediately oven-dried the sample then weighed the grain and straw. We analyzed the effect of the practices using the mean value and Cohens’ d effect size using the following formula (Cohen, 1992):\n\nThe p value of the significance test is affected by the sample size and cannot be used to assess the effect. Measuring effect sizes allows for evaluation involving variance and is not affected by the sample size. Data were processed using Microsoft Excel 2016.\n\n\nResults\n\nWe examined the SALIBU management practices using an L16 orthogonal array design pot experiment and duplicated the experiment under standard and poor conditions. The ratoon rice yield was proportional to straw biomass, and the straw biomass was proportional to the number of ratoon tillers. Cutting twice had the highest effect, and the effect was reversed between the standard and poor conditions. Furthermore, late irrigation had a robust negative effect (Oda et al., 2019).\n\nThe ratoon rice yield was proportional to straw biomass. The harvest index under poor conditions was higher than that under standard conditions (Figure 1). Importantly, straw biomass was proportional to the number of ratoon tillers under both conditions (Figure 2). The ratoon rice yield is determined by the number of ratoon tillers, and the relationship between the number of ratoon tillers and the yield is consistent with those reported in a previous study (Hidayati et al., 2018). The number of ratoon tillers was also in proportion to the number of plants under poor conditions (Figure 3), although it is important to note that under poor conditions, half of the pots had no ratoons.\n\nDW, dry weight.\n\nWe examined the effect of management practices such as pre-fertilization, cutting twice, late irrigation, and adjusting on the number of ratoon tillers.\n\nImportantly, the effect of cutting twice was positive under poor conditions but was negative under standard conditions. In other words, there is an interaction between the practice and the condition. The average cutting heights (length of the first node) of the standard condition plants were 5.5 cm (cutting twice) and 4.0 cm (harvesting time), and those of the poor conditions were 6.8 cm and 3.0 cm, respectively. The extensions of nodes were smaller under standard conditions than those under poor conditions. There is no consensus about the ideal cutting height (Negalur et al., 2017), although previous studies were not carried out using the SALIBU method.\n\nFurthermore, late irrigation had a negative effect on the number of ratoon tillers under both conditions (Table 1). This might be a drawback of the pot experiment method due to decreased percolation; however, this is unlikely because the SALIBU method is successful in the lowlands (Fitri et al., 2019).\n\nThe mean value of the practices, n = 8.\n\nEffect sizes provide an evaluation involving variance and are not affected by the sample size. The effect sizes of Cohen’s d < 0.2, 0.5, 0.8, and 1.2, and d > 2.0 correspond to small, medium, large, very large, and huge, respectively (Cohen, 1992: Sawilowsky, 2009). Figure 4 shows the relationship of the effect sizes between the conditions. The effect on ratoon tillers (Figure 4, left) and on yield (Figure 4, right) was similar but the effect on tillers was high under poor conditions. Pre-fertilization, cutting twice, and late irrigation had medium to large effect sizes. When the effect is near the 1:1 line, the effect is independent of the condition and is robust. A non-robust effect signifies an interaction between the practice and the conditions. Positive large effects were shown under poor conditions only.\n\nPF, pre-fertilization; TC, cutting twice; LI, late irrigation; Ad, Adjusting and mid-term draining. Robust practices show similar effect sizes. Unstable practices, shown by differing effect sizes, have interactions with conditions.\n\n\nDiscussion\n\nThe results of the effect size analysis show that the SALIBU cropping method includes practices that are unstable, negative, or small. Improving these practices could improve the method.\n\nFor the effects of SALIBU management on ratoon tillers, we found an interaction between cutting twice and the cultivation conditions (standard and poor). However, the poor conditions consisted of four factors, which are as follows: low plant density, no fertilization, continuous flooding water management, and late harvesting. Therefore, which of the factors interacts with cutting twice should be clarified.\n\nLate irrigation has a robust negative effect. We can erase the negative effect by simply removing the practice. On the other hand, early irrigation may have a positive effect. In this way, an agricultural cropping method may include negative management practices if the effects are not evaluated. Our method is useful for screening positive practices in cropping methods.\n\nAdjusting has a robust small effect and therefore, implementation should depend on a cost-benefit analysis. In contrast, pre-fertilization has a small effect under standard conditions, but has a large effect under poor conditions. The difference shows an interaction between the practice and the condition; however, this is reasonable because the plants under poor conditions were unfertilized.\n\nSALIBU is an excellent cropping method; however, its adaptability is unclear. Although evaluating each practice in a cropping method under different conditions is difficult because of the huge number of potential combinations, we overcame this difficulty by using an orthogonal array design pot experiment and duplicating the experiment under standard and poor conditions. Our results show that the SALIBU cropping method includes practices with unstable, negative, or small effect sizes. Improving the use of these practices could improve the method. Practices with unstable effects should be used when known to have a positive effect under a specific condition. Negative effects can be excluded by excluding the practice. Small effect practices should be used depending on the outcome of a cost-benefit analysis. Perennial ratoon rice cropping will be possible for Mekong Delta triple rice cropping without the nine special management practices of the original SALIBU cropping method, because most of the effects of practices under standard conditions are small or negative.\n\n\nConclusions\n\nWe examined the management practices of the SALIBU ratoon rice cropping method. Cutting twice has a large effect on ratoon tillers and the effect reverses depending on the cultivation condition. Furthermore, late irrigation has a robust negative effect on the yield. Perennial ratoon rice cropping will be possible for the Mekong Delta triple rice cropping with the sole practice of harvesting rice near the ground; however, further work should be conducted with regard to perennial cropping of ratoon rice. The use of the orthogonal array design under different conditions is useful for future studies.\n\n\nData availability\n\nFigshare: Salibu Effect. https://doi.org/10.6084/m9.figshare.9937928.v1 (Oda et al., 2019)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe thank the students who supported this work.\n\n\nReferences\n\nBalasubramanian R, Balakrishnan K, Manoharan S: Influence of Stubble Thickness, Carbohydrate Content and Leaf Senescence on Ratoon Rice. J Agron Crop Sci. 1992; 168(1): 10–12. Publisher Full Text\n\nCohen J: A power primer. Psychol Bull. 1992; 112(1): 155–159. PubMed Abstract \| Publisher Full Text\n\nFitri R, Erdiman, Kusnadi N, et al.: SALIBU technology in Indonesia: an alternative for efficient use of agricultural resources to achieve sustainable food security. Paddy Water Environ. 2019; 17(3): 403–410. Publisher Full Text\n\nHidayati N, Triadiati T, Anas I: Rooting System of Rice Cultivated Under System of Rice Intensification (SRI) Method Which Improving Rice Yield. HAYATI Journal of Biosciences. 2018; 25(2). Reference Source\n\nNegalur RB, Yadahalli GS, Chittapur BM, et al.: Ratoon Rice: A Climate and Resource Smart Technology. International journal of current microbiology and applied sciences. 2017; 6(5): 1638–1653. Publisher Full Text\n\nOda M, Nguyen HC, Huynh VT: Salibu Effect. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9937928.v1\n\nSacks EJ: Perennial rice: challenges and opportunities. In: Batello, C. ed. Perennial Crops for food security. FAO. 2013; 16–26. Reference Source\n\nSawilowsky SS: New effect size rules of thumb. J Mod Appl Stat Methods. 2009; 8(2): 597–599. Publisher Full Text\n\nTaguchi G: Introduction to quality engineering: designing quality into products and processes. 1986; [Accessed: 18 March 2014]. . Reference Source\n\nYamaoka K, Htay KM, Erdiman, et al.: Increasing Water Productivity through Applying TropicalPerennial Rice Cropping System (SALIBU Technology) in CDZ, Myanmar. In: The 23rd ICID Congress: Towards A New Green Revolution 2.0. International Commission on Irrigation and Drainage (ICID). 2017. Reference Source\n\nZhang S, Wang W, Zhang J, et al.: The Progression of Perennial Rice Breeding and Genetics. In: Perennial Crops for Food Security Proseedings of the FAO Expert Workshop. Food and Agriculture Organization of the United Nations (FAO). 2014. Reference Source" }	[ { "id": "55968", "date": "02 Dec 2019", "name": "Triadiati Antono", "expertise": [ "Reviewer Expertise Plant physiology and plant ecophysiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 not yet clearly and accurately presented, nor does it cite the current literature. The literature review is limited, especially about salibu (definition, advantages, yield). The problems are not clear yet. What is the importance of this research (please use Pertanika et al., 20181 as reference for example, because the article states the advantages.)\n\nBecause the methods (the treatments) are not clear and analysis does not use tools, the methods are not provided to allow replication by others.\n\nBecause the statistical analysis does not explain the tools used, it will be difficult to interpret easily\n\nResults: did not need to state references.\n\nResults: please just write down the results of the study, without mentioning the references.\n\nFig 1-3, Tab 1: please use statistical analysis to explain the results.\n\nDiscussion: explain the reasons for the results, use the references to discuss and compare research results. Please, use references for discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5106", "date": "23 Dec 2019", "name": "Masato Oda", "role": "Author Response", "response": "Thank you for your helpful comments. Our work has two aspects, evaluating SALIBU method, and adopting SAIBU to Mekong Delta Triple cropping. The former is means and the latter is the aim. We changed the title and improved the abstract and introduction. Thank you so much for introducing the correct citation. We referred the paper but mistakenly typed another paper wrote by the same authors in the same year. We corrected that. Thanks again. We guess that you mention about a table of the treatments. That is provided in \"figshare\". We added the link to the methods section. Please kindly point specifically if that is not enough. For the tools, We show \"Microsoft Excel 2016\" and \"the formula of the Cohens’ d effect size\". That is enough to replicate our work. We provided the raw data by \"figshare\" and show the link. This is the regulation of F1000Research. For the location of citation, F1000Research has no regulations. Citations in the result section are commonly seen. For example, a famous book, “Science Research Writing: A Guide for Non-Native Speakers of English”, recommends no references should use unless essential in the discussion section. We added a statistical analysis to Figures 1-3 and Table 1." } ] }, { "id": "56890", "date": "09 Dec 2019", "name": "Le Thi Hoa Sen", "expertise": [ "Reviewer Expertise Adaptation to climate change in agriculture production." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract well conveys the research objectives, methods, key research results, discussion and conclusions. However, it needs to be shorter and more precise.\n\nThe study found very significant results for rice producers in three-crop rice-producing areas or in lowland areas that are vulnerable to natural disasters. However, the results will be more convincing if the collected data is analyzed more deeply, concerning the causes of fluctuating results. For instance, under poor conditions the effect of cutting twice was positive but negative under standard condition, why? What might be the causes? It needs further analyses of the results.\nIn addition, to be more practical and to validate research findings, experiments should be carried out one more time in the net house or in the real conditions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5107", "date": "23 Dec 2019", "name": "Masato Oda", "role": "Author Response", "response": "Thank you for your helpful comments. We shortened the abstract and made it more precise. We also changed the title too.About the cutting twice, we are conducting an additional experiment. That is a portion of developing SALIBU for Mekong Delta triple cropping. We started the field experiment with suitable varieties." }, { "c_id": "5863", "date": "30 Sep 2020", "name": "Masato Oda", "role": "Author Response", "response": "We added the explanation for the Taguchi method as follows.\"The Taguchi method is a popular method to test the robustness of technologies in actual condition by artificial condition.\"" } ] } ]	1	https://f1000research.com/articles/8-1825
https://f1000research.com/articles/9-1183/v1	29 Sep 20	{ "type": "Systematic Review", "title": "The impact of environmental risk factors on delirium and benefits of noise and light modifications: a scoping review", "authors": [ "Haleh Hashemighouchani", "Julie Cupka", "Jessica Lipori", "Matthew M. Ruppert", "Elizabeth Ingersent", "Tezcan Ozrazgat-Baslanti", "Parisa Rashidi", "Azra Bihorac", "Haleh Hashemighouchani", "Julie Cupka", "Jessica Lipori", "Matthew M. Ruppert", "Elizabeth Ingersent", "Tezcan Ozrazgat-Baslanti", "Parisa Rashidi" ], "abstract": "Background: To explore existing literature on the association between environmental risk factors and delirium, and to investigate the effectiveness of environmental modifications on prevention or management of delirium. Methods: This is a scoping review of peer-reviewed studies in PubMed and the reference lists of reviewed articles. Observational studies reporting the effect of noise, light, and circadian rhythm on delirium and interventional studies assessing delirium in modified environments were reviewed. Results: 37 studies were included, 21 of which evaluated the impact of environment on delirium and 16 studied possible solutions to mitigate those impacts. Mixed findings of the reviewed studies yielded inconclusive results; a clearly delineated association between high noise levels, abnormal amounts of light exposure, and sleep disruption with delirium could not be established. The environmental interventions targeted reducing noise exposure, improving daytime and mitigating night-time light exposure to follow circadian rhythm, and promoting sleep. The overall evidence supporting effectiveness of environmental interventions was also of a low confidence; however, quiet-time protocols, earplugs, and bright light therapy showed a benefit for prevention or management of delirium. Conclusions: Environmental modifications are non-invasive, risk-free, and low-cost strategies that may be beneficial in preventing and managing delirium, especially when used as part of a multi-component plan. However, given the limited evidence-based conclusions, further high-quality and larger studies focusing on environmental modifications and delirium outcomes are strongly recommended.", "keywords": [ "delirium", "environmental intervention", "noise", "light", "circadian", "scoping review" ], "content": "Introduction\n\nDelirium is a multifactorial, acute, confusional state characterized by disturbance of consciousness and cognition; it is particularly common in the intensive care unit (ICU) with incidence of 19 to 87% with higher rates in mechanically ventilated patients1–3. ICU delirium is associated with adverse outcomes, including prolonged mechanical ventilation, increased risk of long-term cognitive dysfunction, prolonged hospitalization, higher cost of care, and increased mortality4–7. While the pathophysiology of delirium is poorly understood, there are multiple factors associated with increased risk of delirium including age, education, pre-existing conditions such as hypertension, neurological or psychological disorders, illness severity, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, sensory impairment, and use of analgesics, sedatives, and polypharmacy8–12. The ICU environment may be a modifiable risk factor for delirium. Decreased natural daylight, night-time light exposure, excessive noise, immobilization, and isolation are potential delirium risk factors in ICU13–15.\n\nICU noise levels are above the World Health Organization’s (WHO) recommendations, which suggest 30 A-weighted decibels (dBA) for background noise, a maximum of 35 dBA for treatment and observation areas, and a maximum of 40 dBA at night16–18. Patients interviewed post-ICU discharge report noise as an overall stressor and contributor to loss of sleep19,20. Another common environmental disturbance for ICUs is non-cycling light sources. Disruptions in normal amounts of blue light (460–480 nm) hitting the retina affect neurological processes responsible for melatonin release15. Constant delivery of these wavelengths may cause abnormal suppression of melatonin, altering circadian cycles15. Although the ICU does not lend itself to quietude, it is feasible to employ noise-reducing techniques and light modifications that synchronize circadian rhythm, facilitating recovery.\n\nThe prevalence of delirium-associated adverse health effects and the multitude of risk factors in the ICU make delirium prevention and management essential. Current strategies include pharmacological, non-pharmacological, and multi-component interventions geared towards decreasing delirium incidence and duration. Pharmacological interventions focus on haloperidol and dexmedetomidine, with limited research into ramelteon, melatonin, and ziprasidone21–24. The largest clinical trial to date on haloperidol or ziprasidone in delirious patients failed to show significant clinical benefit23, and current literature does not support use of anti-psychotic agents, benzodiazepines, or melatonin in delirium management21–25. Given the lack of evidence supporting pharmacological measures, research into efficacy of non-pharmacological techniques is crucial. Implementing effective delirium management strategies shows promise in decreasing morbidity, mortality, length of stay, and resource burden in the ICU2. The purpose of this scoping review is to examine the extent and nature of available literature, and highlight areas requiring further inquiry regarding these questions: “How do environmental noise, light, and disrupted circadian rhythms affect delirium?” and “How do existing environmental interventions such as noise reduction, light modifications, and sleep promotion help prevent or manage delirium?”\n\n\nMethods\n\nThis review was conducted according to the methods of Arksey and O’Malley26 and Levac et al.27, and reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Extension for Scoping Reviews (extended data)28,29. The aim of this review is to map existing literature identifying modifiable environmental risk factors for delirium, and assess the role of non-pharmacological noise, light, and circadian rhythm interventions for delirium prevention and management.\n\nStudies were identified by searching PubMed for articles relating to our questions. Search results were restricted to the English language and peer-reviewed studies, with no restriction on year of publication. The search was last executed on November 20, 2019 in order to cover recent publications. Search queries were generated using the following combination of keywords: [“delirium” AND “noise OR sound OR light OR circadian”]. The search was applied with no field tags to maximize results.\n\nAfter compiling research results and removing duplicates, HH and JL screened titles and abstracts to retrieve articles for eligibility. Articles on pediatric populations, animal subjects, case reports, or where the full-text was unavailable were excluded. Additional studies were identified through hand-searches and searching the reference list of reviewed articles. Disagreements on study eligibility were resolved by involving a third reviewer and a discussion between the reviewers. HH, JC, and JL reviewed the full text of eligible articles and extracted data using a pre-designed worksheet reviewed and tested by the team before data charting (Table 1). Elements of the data charting worksheet included study design, setting, sample size, aim, detailed methodology, characteristics of intervention and control groups, measured outcomes, diagnostic tools, main conclusions, and study strengths and limitations. Disagreements were resolved by discussion.\n\nWe included observational studies analyzing the association between noise levels, light exposure or disrupted circadian rhythm and delirium, and interventional studies assessing the effectiveness of modified noise or light exposure or improved circadian rhythm on delirium. Articles were excluded if environmental intervention was an element of a multi-component non-pharmacological bundle, not the main focus. In the initial full text review and data charting, we reviewed all interventional articles reporting results on delirium or the environmental risk factors of delirium, including noise or light levels, and quality/quantity of sleep. We acknowledge these outcomes are modifiable risk factors linked to delirium prevention or management; however, we excluded articles without results linked to delirium.\n\n\nResults\n\nThe electronic database search retrieved 457 articles, which were screened by title and abstract, resulting in 166 studies for full-text review. Hand-search and the searching of reference lists added 28 additional articles. During the full-text review of these 194 articles, 157 were excluded. In total 37 studies were included: 21 assessed association between environmental risk factors and delirium13,19,20,30–47, and 16 reported on delirium after an environmental intervention7,14,15,18,48–59 (Figure 1).\n\nIncluded studies were conducted between 1997 to 2019, in the USA19,31,37,39,43,46,58,59, the Netherlands14,32,48,50,51, Japan38,41,52,53, France33,36,57, Belgium13,49, Denmark15,34, Italy42,47, Sweden18,20, Canada35, China45, India40, Israel30, Singapore55, South Korea54, Thailand56, Turkey44, and the UK7. Of these, 31 studies were conducted among critically ill patients while five reviewed general hospital populations30,35,41,44,54, and one a geriatric monitoring unit for acute delirium care55. Among the 37 reviewed articles, all observational association studies and 12 interventional studies reported delirium incidence, while two interventional studies measured delirium prevalence18,58. Delirium severity was assessed in three interventional studies48,54,55. Three articles reviewed delirium duration7,48,51.\n\nMost studies assessed delirium using the Confusion Assessment Method for the ICU (CAM-ICU)7,15,18,19,34,36,37,39,40,42,45–48,51,56–59; other identification methods included the validated Dutch CAM-ICU32, Confusion Assessment Method (CAM)41,50,55, Intensive Care Delirium Screening Checklist (ICDSC)14,33,43, Neelon and Champagne Confusion Scale (NEECHAM)13,49, non-validated52 and validated53 Japanese NEECHAM, Delirium Observation Screening Scale (DOSS)50, behavioral observations based on the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition (DSM-III)35, 3rd edition-revised (DSM-III-R)38, and 4th Edition (DSM-IV)20,41,44, and behavioral observations based on International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) criteria30. One study used both retrospective chart review and site-specific pre-specified criteria based on new and rapid onset of disturbed consciousness and/or perceptual disturbances31. Studies assessed delirium severity by non-validated Delirium Severity Index (DSI)48, Delirium Rating Scale (DRS)54, Delirium Rating Scale-Revised-98 (DRS-R-98)55, and Memorial Delirium Assessment Scale (MDAS)54. Details, including study design, setting, sample size, methodology, outcomes, and findings with statistics are summarized in Table 2 to Table 4 for observational studies reporting on environmental risk factors, and Table 5 to Table 7 for environmental intervention studies.\n\nOf the observational studies, two analyzed for an association between delirium and noise19,20, five for light13,30–33, 12 for sleep34–45, and two evaluated multiple factors46,47. Study populations ranged from 7 to 6660 participants, and the majority were done in an ICU (17 of 21)13,19,31–34,36–40,42,43,45–47. The remaining studies did not specify a ward30,35,41,44. Study details and statistical results are in Table 2–Table 4.\n\nNoise. Although ICU noise is a suggested predictor for delirium development, two of the three investigating studies found no significant association between ICU noise levels and delirium development19,20 (Table 2). One study assessed A-weighted sound levels with subjective patient reports on ICU noise20. They found no correlation between A-weighted equivalent continuous (LAeq) or maximum (LAmax) noise pressure levels and delirium, while patients’ responses about ICU sounds spread evenly over a spectrum from scary to non-disturbing20. In comparison, Knauert et al.19 evaluated equivalent continuous sound pressure level (Leq) and peak sound occurrences for both A-weighted and C-weighted measurements, finding no correlation with delirium development. There are no industry-standard recommendations for C-weighted levels, but LAeq and LAmax values from both studies were higher than recommended by the WHO17,19,20. In contrast, a study by Davoudi et al.46 assessing the associations between delirium and multiple environmental factors, found average night-time sound pressure levels were significantly higher for patients with delirium46. However, they did not provide exact decibel measurements, likely because they were reporting preliminary findings for a larger cohort study unpublished at the time of this review46.\n\nAbbreviations: CAM-ICU: Confusion Assessment Method for the Intensive Care Unit; CI: confidence interval; dB: decibel; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th Edition; hrs: Hours; ICU: intensive care unit; Leq: equivalent continuous sound pressure level; MICU: medical intensive care unit; RASS: Richmond Agitation and Sedation Scale; RRR: relative risk ratio; SICU: surgical intensive care unit; X2: chi-squared test.\n\nLight. Abnormal lighting cycles are another suggested contributor to delirium60. Seven reviewed studies considered exposure to natural sunlight and relationships with delirium13,30–33,46,47 (Table 3). There were two approaches to analysis: effects of windows on delirium incidence13,31 ,33,46,47 and association with admission season46,47. Findings were mixed, suggesting no easily provable relationship between natural light exposure and delirium occurrence. Two window and one seasonal study found no statistical association between delirium and windows or season of admission/duration of preadmission sunlight exposure, respectively31–33. Kohn et al.31 compared windowed versus non-windowed rooms in the medical ICU, and natural versus industrial window views in the surgical ICU.31. They also investigated impact of half-sized versus full-sized windows, finding no association between delirium incidence and any of these factors31. Similarly, Smonig et al. found no difference in delirium incidence between patients admitted to windowed versus non-windowed rooms while proving windowed rooms retained natural circadian light variations and non-windowed rooms did not33. In the seasonal study, Simons et al. investigated the effect of admission season on delirium incidence and found no correlation32. A simultaneous assessment found no correlation between preadmission cumulative sunlight exposure and delirium incidence for three photoperiods (7, 28, and 60 days pre-hospital admission)32.\n\nIn comparison to studies showing no association between natural sunlight exposure and delirium occurrence, three window studies and one seasonal study found a significant correlation13,30,46,47. In the window studies, Simeone et al.47 associated the lack of natural sunlight with delirium while Van Rompaey et al.13 found an absence of visible daylight led to higher risk of delirium. Davoudi et al.46 examined the pervasive sensing of ICU patients, finding that the measured light intensity in windowed rooms was significantly different between patients with and without delirium46. Additionally, a study on seasonal impact on delirium diagnosis by Balan et al. found a higher incidence of delirium among patients admitted in winter compared to summer30.\n\nAbbreviations: CAM-ICU: Confusion Assessment Method for the Intensive Care Unit; CI: confidence interval; dB: decibel; df: degrees of freedom; GCS: Glasgow Coma Scale; hrs: hours; ICD-9-CM: International Classification of Diseases, Ninth Revision, Clinical Modification; ICDSC: Intensive Care Delirium Screening Checklist; ICU: intensive care unit; MICU: medical intensive care unit; MV: mechanical ventilation; NEECHAM: Neelon and Champagne Confusion Scale; OR: odds ratio; RASS: Richmond Agitation and Sedation Scale; RRR: relative risk ratio; SICU: surgical intensive care unit; X2: chi-squared test.\n\nSleep. Disrupted sleep-wake cycles are associated with altered mental states in hospitalized patients, and are connected with delirium61. In this review, 14 studies19,34–38,40–47 assessed sleep and delirium with two main methodologies: objective measurements of physiological sleep phases and subjective reports by staff or patient. Five studies objectively measured sleep quality using overnight polysomnography (PSG) or a Zeo wireless sleep monitor19,34,36,42,43, while eight assessed staff reports of behavioral observations and/or self-reports by patients34,35,37,38,41,45–47. One study compared both methods33, and two did not specify the measurement method.40,44 (Table 4).\n\nSimilar to the articles on natural light exposure, association studies for sleep and delirium have mixed findings, but lean towards disrupted sleep being a delirium predictor. Six of 14 studies found no relationship between sleep and delirium: two PSG studies19,34, three using subjective measures34, and one with an unspecified method37,40,46. One study found no difference in the rate of delirium between patients with typical and atypical sleep on PSG39, while another by Boesen et al. also found no difference in atypical PSG results between patients who did or did not develop delirium34. They compared PSG results with clinical behavioral observations and were only able to ascertain that the more pathological the patient and electroencephalogram findings, the less association with observed sleep34. A study using the Richards-Campbell Sleep Questionnaire (RCSQ) found no significant correlation between perceived sleep quality and delirium, nor any significant relationship when asking how disruptive noise was to sleep37. The study by Davoudi et al.46 used the Freedman Sleep Questionnaire and found no correlation between overall sleep quality and delirium, although they noted patients with delirium were more likely to have difficulty falling asleep and find night-time lighting disruptive46. The last study did not detail their methodology, but found delirium was not significantly related to sleep deprivation40.\n\nOf the nine studies showing statistical correlation between sleep and delirium, three used electronic sleep monitoring36,42,43, five subjective survey measures35,41,45,47,62 and one did not specify the methodology used44. One study found atypical sleep on PSG was significantly tied to increased delirium, while another PSG study found delirium was associated with severe rapid eye movement (REM) reduction36,42. A third study used a novel sleep monitoring device and found a relationship between a lack of REM sleep and delirium43. Their results must be taken in the context of the device being commercially unavailable (Zeo wireless sleep monitor), and the authors not reporting statistical analyses. Among the remaining positive correlational studies, two had patients self-report sleep satisfaction and quality and both saw significantly poorer responses when comparing patients who developed delirium with those who did not35,45. Two studies involved nursing staff observing clinical behaviors and found sleep disturbances were positively linked to higher likelihood of developing delirium38,41.Two studies found an association between delirium incidence and sleep deprivation (methodology not specified)44, and between sleeping disorders and delirium development47.\n\nAbbreviations: AASM: American Academy of Sleep Medicine; CABG: coronary artery bypass graft; CAM: Confusion Assessment Method; CAM-ICU: Confusion Assessment Method for the Intensive Care Unit; CBO: clinical behavioral observation; CI: confidence interval; CNS: central nervous system; dB: decibel; DSM-III: Diagnostic and Statistical Manual of Mental Disorders, 3rd Edition; DSM-III-R: Diagnostic and Statistical Manual of Mental Disorders, 3rd Edition, Revised; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th Edition; ECG: electrocardiography; EEG: electroencephalography; EMG: electromyography; EOG: electrooculography; GCS: Glasgow Coma Scale; HCU: high intensive care unit; hrs: hours; Hz: Hertz; ICDSC: Intensive Care Delirium Screening Checklist; ICU: intensive care unit; MICU: medical intensive care unit; MMSE: Mini-Mental State Examination; MV: mechanical ventilation; NIV: non-invasive ventilation; OR: odds ratio; PSG: polysomnography; RASS: Richmond Agitation and Sedation Scale; RCSQ: Richards-Campbell Sleep Questionnaire; REM: rapid eye movement; RN: registered nurse; RRR: relative risk ratio; SAS: Riker Sedation-Agitation Scale; SICU: surgical intensive care unit X2: chi-squared test.\n\nIn total, 16 studies evaluated the effects of a modified environment on delirium prevention or management7,14,15,18,48–59 (Table 5–Table 7). Half were randomized control trials (RCT)18,49,51–54,56,57, while half used different study designs including: before-after7,14,48,59, retrospective cohort15,50, and prospective cohort55,58. Sample sizes varied from 11 to 748. Interventions focused on controlling environmental risk factors, including noise and light exposure, disrupted circadian rhythm, and sleep (Figure 2). We categorized these interventions into four modification types: architectural design18,48, environmental noise14,49, environmental light15,50–57, and environmental modification bundles with noise and light components7,57–59. A summary of environmental interventions on delirium and reported statistical results are presented in Table 8. The interventional articles with results on delirium modifiable risk factors such as noise, light, and sleep were excluded if they did not assess delirium as an outcome. Table 9 represents list of these excluded studies.\n\nArchitectural design. Two studies18,47 explored a modified ICU design. One study altered the acoustics of the ICU18, whereas the other used a multi-aspect architectural design intervention48. Results were mixed, but subtly suggest the benefit of architectural designs that consider acoustic features (Table 5). Zaal et al.48 assessed patient outcomes in a multi-bed ICU room with less natural light and more noise exposure versus a private room with improved daylight and reduced noise by sound absorbers, glass sliding doors, optimized alarms, and remotely controlled monitors. There was no effect on delirium incidence or severity, but they found a reduction of delirious days in the study group by 0.4 (95% confidence interval (CI) 0.1–0.7, p = 0.005). Another quasi-randomized feasibility study18 conducted noise reduction by refurbishing an ICU room. They installed a wall-to-wall drop ceiling, low frequency sound absorbers, and used a visually plain design. Implementing the noise reduction strategies was deemed feasible, requiring improvements in noise measurements and delirium assessments. Given the small sample size (n=31) and feasibility nature of the study, no further statistical analysis of outcomes was performed; Delirium developed in 33% (2/6) versus 25% (5/25) of study versus control patients. There was a slight reduction in noise reverberation and increase in speech clarity in the modified room, though sound levels remained higher than the WHO recommendations17.\n\nAbbreviations: CAM-ICU: Confusion Assessment Method for the ICU, DSI: Delirium Severity Index, GCS: Glasgow Coma Scale/Score, hrs: hours, ICU: intensive care unit, RASS: Richmond Agitation and Sedation Scale, RCT: Randomized control trial.\n\n1 Only outcomes of interest including delirium related outcomes, sleep quality, sound pressure levels, and light intensity levels, has listed in this table.\n\n2 Details of measured noise and light, such as devices, location, and frequency has not been discussed in detail in this table.\n\nNoise modification. In this review, there were two approaches to mitigate patient exposure to excessive sound. One was to reduce source noise by utilizing behavioral strategies and device/alarm optimization. The other was noise abatement by earplugs. No studies investigated the impacts of behavioral modification on delirium as an independent intervention, but this strategy was used as part of an environmental modification bundle in four studies7,14,58,59. Earplugs were mostly a component of an environmental bundle7,14,57,58, though one study evaluated the effect of earplugs as a single-component intervention49. One article implemented a combination of behavioral strategies and earplugs to reduce excessive noise14. There were mixed findings across studies with noise modification component(s), but results suggest behavioral strategies and earplugs together might help delirium prevention, particularly as part of a multi-disciplinary program targeting environmental risk factors. However, the implementation of sustained behavioral changes and tolerability of earplugs remain challenges57 (Table 6).\n\nVan de Pol et al.14 analyzed the impact of noise reduction on 421 non-delirious ICU patients in an interrupted time series before-after study. They used earplugs and behavioral strategies, including limited bedside conversations, lowered voices, grouped care activities, optimized alarm settings, and closed doors. Reported noise levels were still higher than the WHO limit post-intervention17, however there was a significant decrease in delirium incidence by 3.7% per time interval (p = 0.02), and reduction in sleep medication usage (p < 0.0001) in the study group. Perceived night-time noise was improved, but with no effect on sleep quality or use of delirium medication. Van Rompaey et al. show associations between environmental noise, sleep perception, and delirium49. They conducted a randomized control trial on 136 non-delirious ICU patients and found use of earplugs (from 2200 to 0600) reduced risk of confusion or delirium by 53% (hazard ratio 0.47, 95% CI 0.27–0.82) and improved sleep perception.\n\nOur full-text review and data extraction appraised articles studying single-component noise control strategies, such as behavioral programs65–69, earplugs or noise cancelling headphones71–74, and headphones equipped with an alarm filtering system70; however these were not included since they reviewed the impact of interventions on the level of noise or quality of sleep, but delirium was not reported as an outcome (Table 9).\n\nAbbreviations: CAM-ICU: Confusion Assessment Method for the ICU, dB: decibels, GCS: Glasgow Coma Scale/Score, hrs: hours, ICDSC: Intensive Care Delirium Screening Checklist, ICU: intensive care unit, K: Kelvin, lx: Lux, MV: mechanically ventilated, NEECHAM: Neelon and Champagne Confusion Scale, PSG: Polysomnography, RASS: Richmond Agitation and Sedation Scale, RCSQ: Richards-Campbell Sleep Questionnaire, RCT: Randomized control trial.\n\n1 Only outcomes of interest including delirium related outcomes, sleep quality, sound pressure levels, and light intensity levels, has listed in this table.\n\n2 Details of measured noise and light, such as devices, location, and frequency has not been discussed in detail in this table.\n\nLight modification. Light interventions were implemented in an attempt to realign circadian rhythms by reducing night-time exposure and/or improving natural or artificial daylight exposure (Table 7).\n\nAbbreviations: BLT: bright light therapy, CAM: Confusion Assessment Method, CAM-ICU: Confusion Assessment Method for the ICU, CCU: coronary care unit, CMMSE: Chinese Mini–Mental State Examination, dB: decibels, DOSS: Dutch version of the Delirium Observation Screening, DRS: Delirium Rating Scale, DRS-98: Delirium rating scale-R98, DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, GMU: Geriatric Monitoring Unit (A specialized delirium management unit), HELP: Hospital Elder Life Program (standardized protocols to manage cognitive impairment, sleep deprivation, immobility, visual impairment, hearing impairment, and dehydration), hrs: hours, ICU: intensive care unit, ISI: Insomnia Severity Index, K: Kelvin, LED: light-emitting diode, lx: Lux, MDAS: Memorial Delirium Assessment Scale, MV: mechanically ventilated, NEECHAM: Neelon and Champagne Confusion Scale, PSG: Polysomnography, RASS: Richmond Agitation and Sedation Scale, RCSQ: Richards-Campbell Sleep Questionnaire, RCT: Randomized control trial.\n\n1 Only outcomes of interest including delirium related outcomes, sleep quality, sound pressure levels, and light intensity levels, has listed in this table.\n\n2 Details of measured noise and light, such as devices, location, and frequency has not been discussed in detail in this table.\n\nReduction of nocturnal light exposure\n\nThe included articles in this review, studied eye masks7,57,59 and overnight light dimming7,58,59 as part of an environmental modification bundle to reduce night-time light exposure. However, the effects of less nocturnal light exposure on delirium, was not evaluated as a single intervention.\n\nImproving natural daylight exposure\n\nThree observational studies31,33,87 and one before-after study48 investigated improved natural lighting via windows. They compared patient outcomes in rooms with a window or larger-sized windows versus windowless or smaller-sized windows, respectively. No observational studies suggested association between improved natural lighting and delirium31,33,87. Zaal et al.48 demonstrated reduction in delirium duration, comparing patients in private rooms with more natural light versus less bright multi-bed rooms; however, there were no differences in delirium incidence or severity between groups.\n\nImproving artificial daylight exposure\n\nEight studies examined effect of improved daylight exposure via artificial lighting, of which three used an artificial circadian lighting system15,50,51, and five used bright light therapy (BLT)52–56. No study implementing artificial dynamic or circadian lighting revealed significant effects on delirium. BLT studies had mixed results; three studies significantly improved delirium prevention or management, while the other two showed a non-significant tendency to reduce delirium rates.\n\nA retrospective cohort study of 183 non-sedated ICU patients by Estrup et al.15 used a circadian lighting system from 0700 to 2300 which varied in intensity and color temperature. During the morning, light intensity was greatest, up to 4000 lux (lx), and the amount of blue light strongest. As the day progressed, light intensity decreased and color temperature shifted towards warmer tones until no blue light was present. There was no improvement in delirium incidence, and no association between circadian lighting and delirium incidence (odds ratio (OR) 1.14; 95% CI 0.55, 2.37; p = 0.73). Pustjens et al.50 retrospectively studied a cohort of 748 non-sedated patients. They implemented a dynamic lighting system consisting of two ceiling-mounted light-emitting diode (LED) panels which delivered variable intensities of light (peak of 750 lx) with a color temperature between 2700 and 6500 Kelvin (K). There was no effect on delirium incidence. Another RCT by Simons et al.51 measured the effects of a dynamic lighting application (DLA) in 734 ICU patients. DLA was administered through ceiling-mounted fluorescent lights which delivered a variety of bluish-white light from 0700 to 2230 with a maximum intensity of 1700 lx and a maximum temperature of 4300 K between 0900 and 1600, except between 1130 and 1330 when light intensity was 300 lx. This study was terminated early, but preliminary analysis demonstrated delirium incidence of 38% versus 33% in control versus study patients, with no significant improvement on delirium incidence or duration.\n\nFour studies investigated the use of BLT as a single-component intervention to prevent52,53,56 or treat54 delirium, while one study used BLT as an element of a multi-component bundle to manage delirium55. BLT consisted of exposure to high intensity light (2000 to 10000 lx) for one to four hours daily. Three studies used a peak intensity of 5000 lx52,53,56. Taguchi et al.52 conducted a randomization pilot study on 11 post-operative patients, utilizing a daily light intensity of 5000 lx from 0730 to 0930 for days 2 through 5 post-surgery. Delirium assessment scores decreased on day 3 of BLT (p = 0.014), but there was no significant effect on overall delirium incidence (16% versus 40% study versus control group, p = 0.42). In another RCT, Ono et al.53 applied BLT on 22 post-operative patients, for two hours from 0730 to 0930 for four days. Light intensity started at 2500 lx, increasing to 5000 lx, then decreasing to 2500 lx. There was a non-significant tendency towards lower rates of delirium in the study group (1 of 10 patients) versus control group (5 of 12 patients), while BLT significantly reduced the amount of activity during sleep on days 4 and 5. Potharajaroen et al.56 studied 62 post-operative patients by implementing BLT at 5000 lx from 0900 to 1100. Eleven of 31 control patients versus 2 of 31 patients in the intervention group developed delirium. There was a significant association between BLT and decreased delirium incidence (OR 0.12, 95% CI 0.03–0.54, p = 0.005). A study by Yang et al.54 on 36 delirious patients used a higher light intensity (10000 lx) over a shorter period (0700 to 0800). This study investigated the use of BLT as an adjunctive treatment of delirium with risperidone. They found a significant decrease in delirium severity in patients receiving BLT in addition to risperidone (DRS 23.9 ± 4.9 versus 20.6 ± 3.6 in control versus study group, p = 0.03). Chong et al.55 studied 228 delirious elderly patients admitted to a delirium management unit. They incorporated lower intensity BLT as part of their multi-component program, and exposed patients to 2000 to 3000 lx of light for four hours from 1800 to 2200 daily. They reported significant improvement in total sleep time and functional outcomes during treatment of delirious patients.\n\nIntervention bundles (combination of light and noise modification)\n\nEarplugs and eye mask\n\nOne reviewed study explored effects of earplugs and an eye mask on delirium57, while two others used earplugs and an eye mask as part of their interventional bundle7,59. All three noted decreased incidence of delirium, but observed different effects on sleep quality. Demoule et al.57 conducted an RCT on 43 non-sedated ICU patients to investigate the impact of sleeping with earplugs and an eye mask from 2200 to 0800 on patient outcomes. They found no improvement in delirium incidence, duration or architecture of sleep in the study group, regardless of patient compliance using the equipment. Although compliant study subjects experienced improved sleep with longer N3 (deeper sleep) duration and a lower number of prolonged awakenings, there was no significant change in delirium incidence. There were several articles in our initial screening reporting improved perceived noise or sleep quality with the use of earplugs and eye masks, however those were excluded since none reported results on delirium78–77 (Table 9).\n\nQuiet time, and sleep promotion bundles\n\nQuiet time is a specific amount of time during which modifiable noise and light is actively reduced. Our review included three studies installing quiet time as the single interventional element58 or as a part of a sleep promotion bundle7,59. Core elements of quiet time were behavioral strategies, minimized bedside activity by clustering care, reduced volume of devices/alarms, and dimmed lights7,58,59. The study that implemented daytime quiet time failed to show significant effects on delirium58, while two sleep promotion studies decreased delirium incidence using nocturnal quiet time combined with components such as earplugs, eye masks, and pharmacological targets7,59. Although the multi-component sleep promotion trials decreased delirium incidence, effectiveness of the separate components is unclear.\n\nMcAndrew et al.58 applied quiet time from 1400 to 1600 among 72 mechanically ventilated ICU patients. In the 24 hours after starting quiet time, there was no increase in delirium rate and 19% of delirious patients improved to a negative CAM-ICU status. However, there was no significant effect on delirium in their analysis. Quiet time did lead to moderately improved sleep quality and less frequently administered sedatives which helped remove patients from mechanical ventilation. A pre-post research by Patel et al.7 studied a nocturnal multidisciplinary environmental sleep promotion program in 338 non-delirious, non-sedated ICU patients. Their program included nocturnal quiet time with earplugs, eye mask, patient orientation, early mobilization, and sedation targets. The study group showed significant reduction in delirium incidence (by 33% p < 0.001), and a decrease in delirium duration (3.4 ± 1.4 versus 1.2 ± 0.9 days, p = 0.021). Sleep quality and night-time light and noise levels were also improved in the study group, however reported noise levels were still higher than the WHO limits17. They additionally reported a significant association between sleep efficiency and a lower risk of developing delirium (OR 0.90, 95% CI 0.84–0.97). A larger pre-post study (n=300) by Kamdar et al.59 initiated a multi-faceted sleep promotion protocol consisting of three additive stages: 1) nightly quiet time and realignment of circadian rhythm, 2) sleeping with earplugs, eye masks, and soothing music, and 3) pharmacological targets to reduce sedatives. They reported decreased delirium incidence (OR = 0.46, 95% CI 0.23–0.89, p = 0.02) and perceived night-time noise in the study group, but no improvements in sleep quality.\n\nAbbreviations: OR: Odds Ratio, CI: Confidence Interval, DSI: Delirium Severity Index, SD: Standard Deviation, HR: Hazard Ratio, NEECHAM: Neelon and Champagne Confusion Scale, BLT: Bright light therapy, DRS: Delirium Rating Scale, MDAS: Memorial Delirium.\n\n1 No statistical analysis was done\n\n2 No significant effect\n\n3 Decreased\n\nAbbreviations: CTICU: cardiothoracic Intensive care unit, dBA: A-weighted decibel, ICU: intensive care unit, ICU-CS; post cardiac surgery intensive care unit, LED; light-emitting diode, MICU: medical intensive care unit, NICU; neurology intensive care unit, NSICU; neurosurgical intensive care unit, PACU; post-anaesthesia care unit, RCT: randomized control trial, SICU: surgical intensive care unit.\n\n1 This table does not provide complete summary of characteristics and findings of these excluded studies. The purpose of this table is only to present a list of excluded studies investigating impact of environmental interventions on delirium modifiable risk factors. These studies were excluded from this review since no delirium outcome was reported.\n\n\nDiscussion\n\nIn this scoping review, the existing literature was searched for studies on the impact of environmental risk factors and interventions on delirium: 21 studies were retrieved reporting the effects of environmental risk factors on delirium and 16 studies reported experiments on possible solutions to modify the environment. Small sample sizes, heterogeneous study methods, and inconsistent results among reviewed studies proved the need for expanding research on the impacts of environmental risk factors and efficacy of mitigations related to delirium.\n\nICUs are high-tech environments with round-the-clock activities that have a negative impact on patients’ experience and clinical outcomes due to excessive noise, light, and disturbed sleep and circadian rhythm13,48,49.\n\nNoise. The WHO set recommendations for hospitals not to exceed an average of 30 dBA or a maximum of 35 dBA in treatment areas (maximum of 40 dBA at night)17. A 2016 study by Hu et al. found average sound levels of 62.8 dB, with a mean level of 59.6 dB between 0000–0700, when investigating sound in various ICUs88. Consistently, five reviewed articles measuring ICU sound pressure with or without noise modification interventions reported levels exceeding the WHO recommendations14,18–20.\n\nA 2009 WHO report set night-time noise guidelines and reported on relationships between night-time noise, sleep, and health. According to the report, excessive night-time noise (above 35 dB) disturbs sleep, provokes annoyance and agitation, reduces cognition, impairs communication and comprehension of surroundings, and contributes to psychiatric disorders. The combination of sleep disruption, decreased cognitive function, and lowered comprehension of surroundings associated with high noise levels may contribute to acute confusion and delirium89,90. In our review, two of three observational studies investigating the association between high noise levels and ICU delirium found that high noise levels had no significant effect on delirium incidence19,20. This result is surprising as it has been suspected that noise levels exceeding a normal threshold have detrimental effects on patient recovery, especially with regard to sleep and mental status. It is worth considering the difficulty in assessing the true effect of high noise levels in these two studies. First, there is no available baseline research to compare delirium incidence in high noise level ICUs versus those with statistically lower decibel values. It is possible the threshold for adverse effects is lower or higher than the most recently investigated decibel levels. In addition, Knauert et al.19 mentioned a limitation for their study in the inadequate statistical power to detect differences in decibel level between patient comparisons. For the study by Johansson et al.20, their results need to be taken in context of using a non-validated delirium diagnosis protocol.\n\nLight. During the daytime, normal light intensity is around 10000 lx and recommended night-time light levels conducive to sleep are below 30 lx60. Natural fluctuation of light levels throughout the day contributes to the natural sleep-wake cycle by triggering the release and suppression of melatonin. Alteration of the sleep-wake cycle and a lack of daylight schedule have been shown to be associated with psychiatric diseases60. Daytime light levels in the ICU are below normal daylight levels and above the threshold for sleep disruption at night60. In a study by Hu et al. light intensity was measured over 24 hours near windows, in the center of rooms, and at the eye level of mechanically ventilated patients. Average light intensity at these locations were 425 lx, 191 lx, and 388 lx respectively over 24 hours and 84 lx, 103 lx, and 87 lx between 2401 and 075988. Minimal variation in daytime and night-time light levels disrupts the natural sleep-cycle and may contribute to patients becoming unable to distinguish day from night.\n\nAbnormal natural light cycles are cited in recent literature as a potential modifiable risk factor for delirium management60. Seven studies analyzing the impact of natural light on delirium incidence suggest this element of the ICU lacks a definitive causative relationship with development of the condition. Most of these studies enrolled critically ill patients whose condition gives them a higher likelihood of having consistently closed eyes compared to the general hospital population. It should be considered for future research that these patients’ retinas may not receive the same strength light stimulus as other populations, suggesting the need for ICU-specific lighting strategies. For the two seasonal studies, one found delirium was diagnosed significantly more in the winter than summer30, while the other found exhaustive evidence ruling out a link between delirium and pre-hospital photoperiod exposure year-round32. These findings suggest there are factors aside from seasonal light exposure affecting delirium. Additionally, of the three studies with a positive correlation between exposure to natural daylight or season of admission, the two natural daylight studies had vague descriptions of their measurements of patient’s exposure to natural or artificial light13,47. It is hard to assess whether the patient could have received benefits when the proximity of the stimulus to the patient is unclear.\n\nAs with excessive noise levels, further research into abnormal natural lighting cycles is necessary to delineate any threshold for adverse effects to patients’ well-being.\n\nSleep. Similar to our findings regarding effects of noise and light levels on delirium, reviewed articles on sleep showed mixed results for both forms of measure (electronic sleep monitoring and subjective reports). Recent literature states sleep is disturbed in ICU patients regardless of delirium19,42, and this concern is supported by the fact that unmeasurable sleep was found in non-delirious patients in included PSG studies. It is hard to compare results of included wireless monitoring studies, since different methodologies were used for each study, with different devices, leads, and levels of adherence to American Academy of Sleep Medicine standards. Similarly, it is difficult to compare findings from objective sleep monitoring protocols and subjective survey methods, and these need separate consideration. A major concern in analyzing subject sleep quality in delirious patients is patients with an altered mental state and/or confusion may not answer consistently or truthfully, and measures must be taken to assess whether answers are a correct representation of their condition.\n\nNoise modification. The negative impact of patient exposure to noise led to several studies focusing on noise pollution in the clinical environment. Mitigated exposure to noise levels might promote patient outcomes and staff satisfaction58,91. Noise reduction or abatement strategies include architectural features, behavioral alterations, alarm optimization, earplugs, headphones, and noise cancelling devices. Whilst these strategies have been studied in relation with improved noise levels and sleep promotion (Table 9), further research is required to make evidence-based recommendations for the effect of noise reduction on delirium prevention and treatment.\n\nImplementing ICU designs with acoustic features such as sound absorbers, reversible drawers to open both inside and outside the room, or room designs with the ability to locate alarmed devices or transfer alarms away from the patient, might improve exposure to noise and benefit delirium management48,92,64. Zaal et al. demonstrated a lower delirium duration by modifying ICU design with acoustic considerations, however there was no change in delirium incidence rate48. These strategies require major renovation or early construction planning, and further research is required to confirm cost-effectiveness and clinical benefits.\n\nStaff and family conversations and care-activities are significant sources of ICU noise pollution16,65,66. Although behavioral modification might be ineffective as a single-component intervention67, low-cost adjustments such as limited bedside conversation, lowered voices, clustered care-activities, minimized TV and overhead use and volume, use of vibrating pagers, and visual noise-warning devices may be necessary to achieve better results in sound reduction7,14,65,66,68,69, sleep improvement66, and decreased delirium14. To be successful, continuous awareness, education of staff on the impact of excessive noise exposure, and routine monitoring of implemented strategies is crucial7. Technologies that help staff and visitors recognize excessive noise might complement implementation of behavioral strategies. Visual noise-warning devices display colored warnings at higher levels of noise and can be an effective, sustained noise reduction strategy68,69. Use of noise-warning systems has a greater impact on the reduction of ambient noise compared with peak noise levels68,69. This is likely a result of change in staff behavior after visual warning while having no effect on medical equipment or alarms.\n\nAlarms are a significant source of ICU noise pollution16,65, and a large portion are considered false positives93. Studies show modifying ICU alarms by lowering volume, optimizing device settings, and filtering false alarms may reduce disturbing alarm noise94–80. Schlesinger and colleagues equipped wearable earbuds with a frequency-selective silencing device, which could successfully filter ICU alarms while allowing patients to hear and communicate effectively without experiencing the negative consequences of audible alarms70. Optimization of alarms was used as an element of a noise reduction bundle and sleep promotion studies of this scoping review7,14,59.\n\nAbating environmental noise by earplugs or headphones appears feasible and effective to reduce noise and improve sleep in the ICU49,57,71–74. Here, one study failed to prove benefits of using earplugs and eye masks during sleep on delirium57, while another earplug trial decreased risk of confusion, and delayed initiation of cognitive disturbances with no significant effect on incidence of delirium49. Given the potential effectiveness and low costs, this method is frequently used in multi-component interventions7,59; however, non-compliancy is an issue in earplugs studies57. A recent meta-analysis91 reported a 13.1% (95% CI, 7.8–25.4) rate of non-compliancy due to intolerance, anxiety, or accidental removal of earplugs. Headphones with active noise cancellation technology might improve patient outcomes by mitigating exposure to noise. Gallacher et al. modeled an experiment by embedding sound meters in the auditory meatus of polystyrene model heads located near patients’ beds in a cardiac ICU74. They demonstrated a significant reduction in overall noise exposure and exposure to high intensity noises using noise cancelling headphones.\n\nDespite inconsistent results of the reviewed studies on efficacy of noise modifications on delirium, this review suggests considering physical design features and multi-component noise reduction programs may benefit delirium prevention or management. This is consistent with current recommendations suggesting multi-component interventions to achieve adequate noise reduction91; Van de Pol et al.14 reduced delirium incidence by implementing a noise reduction program consisting of behavioral strategies, device optimization, and earplugs. However, there is a need for high-quality randomized control trials with larger sample sizes to evaluate efficacy, sustainability, and long-term effects of noise modification interventions with a focus on delirium.\n\nLight modification. Optimized circadian rhythm needs bright days and dark nights. Various light modification strategies have been proposed to follow circadian rhythms. These are categorized as such: decreasing night-time light exposure, and increasing daylight.\n\nRound-the-clock ICU activities make nigh-time light reduction challenging to maintain a level of light sufficient for providing care, but not disturbing sleep. Dimming lights as part of quiet time strategies is effective to mitigate intensity of light during quiet time hours, however, this may cause variation in perceived light and consequently cause sleep disturbance80. Possible solutions are clustering care-activities to reduce bedside interruptions7 and use of portable lighting pods with less blue wavelength during the night79. Whilst the trial of sleep masks and earplugs by Demoule at al.57 failed to show benefit to delirium, eye masks are effective in promoting sleep by light abatement7,78,76,77. However, poor compliance in the use of eye masks due to accidental removal, or anxiety/claustrophobia, and the risk of sensory deprivation in mechanically ventilated patients, remains challenging57.\n\nEnvironmental modification to increase daylight exposure is possible through the architectural considerations of promoting natural lighting or utilizing artificial illumination. Research into whether windows allow enough light to promote sleep-wake cycles and prevent delirium, and whether seasonal light levels contribute to delirium, has been conducted with inconclusive findings31,33,87. From our results, the greatest interventional effect on delirium was from bright light therapy.\n\nOur review included five studies on BLT, three reporting a significant effect on delirium incidence or severity52,54,56 with sleep promoted in four studies53–56. BLT has the greatest effect between 2500 and 10000 lx for 30 to 60 minutes, with a shorter duration for greater intensities of light, when administered either at twilight or dawn to obtain a circadian effect61. The BLT in this review applied 2000 -10000 lx of illuminance for between one and four hours. The use of 2000 lx was effective in improving sleep quantity and functional status during management of delirium as part of a bundle. The use of 5000 lx was associated with decreased delirium incidence in two of three studies and the use of 10000 lx, as an adjunctive treatment with risperidone, was associated with a decrease in delirium severity54. While BLT may help regulate sleep-wake cycles and prevent/treat delirium, research into melatonin secretion and circadian rhythms suggests periods of darkness play as large a role as daytime light levels in promoting sleep and preventing delirium60,95. The importance of light and darkness prompts a need for research into effects of dynamic lighting systems. This review included three studies focused on dynamic lighting among sedated and non-sedated patients, using lighting systems which produced cooler blue light in the mornings and shifted towards warmer tones as the day progressed. The lighting systems produced different levels of intensity throughout the day, reaching a peak of between 750 and 4000 lx and a minimum level of 0 lx. None of these studies showed significant effects on delirium15,50,51; however, they used peak light levels below normal daytime levels.\n\nMaintaining a circadian rhythm, by nocturnal darkness and BLT, as a low-cost, low-risk, easy-to-apply intervention can help improve patient outcomes. Research is required to investigate the use of dynamic lighting with higher peak light intensities or the combination of dynamic lighting and BLT. Additionally, there is a need for defining effective characteristics of light modification strategies for sedated and non-sedated patients. Sedated patients may have disrupted circadian rhythm of melatonin96, and application of light therapies might have limited retina stimuli when eyes are closed. Studies comparing efficacy of light modifications on prevention or treatment of delirium among these two groups of patients, with application of different intensity levels of light in closed-eyes patients, might be of benefit.\n\nIntervention bundles (light and noise modification). There is a growing interest in using quiet time interventions to promote sleep. Quiet time protocols have successfully reduced sound pressure, improved sleep quality, and reduced the use of sedatives80,81–83, but effects of quiet time on delirium development needs further research. McAndrew et al. implemented a daily quiet time protocol in ICU patients and reported inconclusive results on delirium scores and moderate improvement in sleep perception58. Two neurocritical ICU studies have implemented a two hour quiet time during day and night81,82. A significant improvement in subjective sleep and increased staff satisfaction was achieved81,82. They reported decreased light by 75–85% and noise by 15%, with results being more significant during day-shift quiet time; this might be due to overall lower levels of nocturnal light and noise82.\n\nSleep promotion protocols utilize noise and light control strategies with other components, such as patient orientation, early mobilization, medication optimization, and sedation targets to improve sleep in quality and quantity. Here we included two sleep promotion studies reporting results on delirium, however future research is needed to evaluate which component of sleep promotions are effective in reducing delirium. Patel et al.7 significantly improved sleep quality and reduced delirium incidence by implementing a non-pharmacological multidisciplinary sleep program. They raised protocol compliance to > 90% by ongoing education, signage and posters, monitoring, and spot-checking program quality by experienced nurse champions. Interestingly, a large sleep promotion study by Kamdar et al., decreased delirium incidence while there was no effect on sleep59. It is not clear if improvements in delirium are attributable to sleep, emphasizing the need for future studies focused on single interventions or single components of multifaceted interventions with regard to delirium results.\n\nThe main strength of this review is synthesizing results of both observational association studies and interventional studies. This approach details a broader picture of the current state of this research field and bridges the gap between establishing correlational relationships and continuation of experimental trials. A major limitation of this review is the narrow search method. By searching one database (Pubmed) and the included articles’ reference lists, there is likely additional literature available to expand our findings, however the authors did a hand search within related journals, Embase, and Google Scholar databases to include existing interventional research articles. Another limitation was that the generated data from reviewed studies did not have full details, and quality of evidence was not evaluated among studies; however, this review was intended to be a literature mapping with limited description of relevant publications.\n\n\nConclusions\n\nThis review of studies investigating the association between delirium and either high noise levels, abnormal amounts of natural daylight, and/or sleep disruptions did not reveal a clear relationship between delirium and these variables. It is recommended to perform additional research into more comprehensive, but related, risk factors to find a stronger predictor. Additional research could include analyses of specific noise sources or a comparison between overcast, rainy, and sunny times. This review revealed the need for further research targeting the effectiveness of environmental interventions on delirium. Current literature lacks randomized control trials with larger sample sizes to evaluate the efficacy of intervention on delirium and its long-term outcomes. Another knowledge gap is the lack of adequate conclusive research on single-component interventions. The interventional bundle studies lead to uncertainty about which component impacts the result. Given the low-cost and non-invasive nature of environmental modifications and their potential beneficial role in reduction of modifiable risk factors, it is recommended to implement these interventions in current practice, especially as multi-component bundles.\n\n\nData availability\n\nAll data underlying the results are part of the article and no additional source data are required.\n\nOSF: PRISMA-ScR checklist for ‘The Impact of Environmental Risk Factors on Delirium and Benefits of Noise and Light Modifications: A Scoping Review’. https://doi.org/10.17605/OSF.IO/NHWKA29\n\nData are available under the terms of the ‘Creative Commons Zero \"No rights reserved\" data waiver’ (CC0 1.0 Public domain dedication).", "appendix": "References\n\nLin WL, Chen YF, Wang J: Factors Associated With the Development of Delirium in Elderly Patients in Intensive Care Units. J Nurs Res. 2015; 23(4): 322–9. PubMed Abstract \| Publisher Full Text\n\nKalabalik J, Brunetti L, El-Srougy R: Intensive care unit delirium: a review of the literature. J Pharm Pract. 2014; 27(2): 195–207. PubMed Abstract \| Publisher Full Text\n\nPage VJ, Ely EW, Gates S, et al.: Effect of intravenous haloperidol on the duration of delirium and coma in critically ill patients (Hope-ICU): a randomised, double-blind, placebo-controlled trial. Lancet Respir Med. 2013; 1(7): 515–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalluh JI, Wang H, Schneider EB, et al.: Outcome of delirium in critically ill patients: systematic review and meta-analysis. BMJ. 2015; 350: h2538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWitlox J, Eurelings LS, de Jonghe JF, et al.: Delirium in elderly patients and the risk of postdischarge mortality, institutionalization, and dementia: a meta-analysis. JAMA. 2010; 304(4): 443–51. PubMed Abstract \| Publisher Full Text\n\nMehta S, Cook D, Devlin JW, et al.: Prevalence, risk factors, and outcomes of delirium in mechanically ventilated adults. Crit Care Med. 2015; 43(3): 557–66. PubMed Abstract \| Publisher Full Text\n\nPatel J, Baldwin J, Bunting P, et al.: The effect of a multicomponent multidisciplinary bundle of interventions on sleep and delirium in medical and surgical intensive care patients. Anaesthesia. 2014; 69(6): 540–9. PubMed Abstract \| Publisher Full Text\n\nMicek ST, Anand NJ, Laible BR, et al.: Delirium as detected by the CAM-ICU predicts restraint use among mechanically ventilated medical patients. Crit Care Med. 2005; 33(6): 1260–5. PubMed Abstract \| Publisher Full Text\n\nAhmed S, Leurent B, Sampson EL: Risk factors for incident delirium among older people in acute hospital medical units: a systematic review and meta-analysis. Age Ageing. 2014; 43(3): 326–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFong TG, Tulebaev SR, Inouye SK: Delirium in elderly adults: diagnosis, prevention and treatment. Nat Rev Neurol. 2009; 5(4): 210–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuppert MM, Lipori J, Patel S, et al.: ICU Delirium Prediction Models: A Systematic Review. arXiv e-prints. 2019; [arXiv: 1911.02548 ]. Reference Source\n\nDavoudi A, Ebadi A, Rashidi P, et al.: Delirium Prediction using Machine Learning Models on Preoperative Electronic Health Records Data. Proc IEEE Int Symp Bioinformatics Bioeng. 2017; 2017: 568–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Rompaey B, Elseviers MM, Schuurmans MJ, et al.: Risk factors for delirium in intensive care patients: a prospective cohort study. Crit Care. 2009; 13(3): R77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan de Pol I, van Iterson M, Maaskant J: Effect of nocturnal sound reduction on the incidence of delirium in intensive care unit patients: An interrupted time series analysis. Intensive Crit Care Nurs. 2017; 41: 18–25. PubMed Abstract \| Publisher Full Text\n\nEstrup S, Kjer CKW, Poulsen LM, et al.: Delirium and effect of circadian light in the intensive care unit: a retrospective cohort study. Acta Anaesthesiol Scand. 2018; 62(3): 367–75. PubMed Abstract \| Publisher Full Text\n\nDarbyshire JL, Young JD: An investigation of sound levels on intensive care units with reference to the WHO guidelines. Crit Care. 2013; 17(5): R187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerglund B, Lindvall T, Schwela DH, et al.: Guidelines for community noise. Geneva: World Health Organization; 1999. Reference Source\n\nJohansson L, Lindahl B, Knutsson S, et al.: Evaluation of a sound environment intervention in an ICU: A feasibility study. Aust Crit Care. 2018; 31(2): 59–70. PubMed Abstract \| Publisher Full Text\n\nKnauert M, Jeon S, Murphy TE, et al.: Comparing average levels and peak occurrence of overnight sound in the medical intensive care unit on A-weighted and C-weighted decibel scales. J Crit Care. 2016; 36: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohansson L, Bergbom I, Waye KP, et al.: The sound environment in an ICU patient room--a content analysis of sound levels and patient experiences. Intensive Crit Care Nurs. 2012; 28(5): 269–79. PubMed Abstract \| Publisher Full Text\n\nThom RP, Bui MP, Rosner B, et al.: A Comparison of Early, Late, and No Treatment of Intensive Care Unit Delirium With Antipsychotics: A Retrospective Cohort Study. Prim Care Companion CNS Disord. 2018; 20(6): 18m02320. PubMed Abstract \| Publisher Full Text\n\nKnauert MP, Pisani MA: Dexmedetomidine for hyperactive delirium: worth further study. J Thorac Dis. 2016; 8(9): E999–E1002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGirard TD, Exline MC, Carson SS, et al.: Haloperidol and Ziprasidone for Treatment of Delirium in Critical Illness. N Engl J Med. 2018; 379(26): 2506–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen S, Shi L, Liang F, et al.: Exogenous Melatonin for Delirium Prevention: a Meta-analysis of Randomized Controlled Trials. Mol Neurobiol. 2016; 53(6): 4046–53. PubMed Abstract \| Publisher Full Text\n\nArora RC, Cunningham C: Losing Sleep Over Delirium. Crit Care Med. 2018; 46(6): 1036–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArksey H, O'Malley L: Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nLevac D, Colquhoun H, O'Brien KK: Scoping studies: advancing the methodology. Implement Sci. 2010; 5: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018; 169(7): 467–73. PubMed Abstract \| Publisher Full Text\n\nBihorac A: The Impact of Environmental Risk Factors on Delirium and Benefits of Noise and Light Modifications: A Scoping Review. OSF. 2020. http://www.doi.org/10.17605/OSF.IO/NHWKA\n\nBalan S, Leibovitz A, Freedman L, et al.: Seasonal variation in the incidence of delirium among the patients of a geriatric hospital. Arch Gerontol Geriatr. 2001; 33(3): 287–93. PubMed Abstract \| Publisher Full Text\n\nKohn R, Harhay MO, Cooney E, et al.: Do windows or natural views affect outcomes or costs among patients in ICUs? Crit Care Med. 2013; 41(7): 1645–55. PubMed Abstract \| Publisher Full Text\n\nSimons KS, Workum JD, Slooter AJ, et al.: Effect of preadmission sunlight exposure on intensive care unit-acquired delirium: a multicenter study. J Crit Care. 2014; 29(2): 283–6. PubMed Abstract \| Publisher Full Text\n\nSmonig R, Magalhaes E, Bouadma L, et al.: Impact of natural light exposure on delirium burden in adult patients receiving invasive mechanical ventilation in the ICU: a prospective study. Ann Intensive Care. 2019; 9(1): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoesen HC, Andersen JH, Bendtsen AO, et al.: Sleep and delirium in unsedated patients in the intensive care unit. Acta Anaesthesiol Scand. 2016; 60(1): 59–68. PubMed Abstract \| Publisher Full Text\n\nBowman AM: Sleep satisfaction, perceived pain and acute confusion in elderly clients undergoing orthopaedic procedures. J Adv Nurs. 1997; 26(3): 550–64. PubMed Abstract \| Publisher Full Text\n\nDrouot X, Roche-Campo F, Thille A, et al.: A new classification for sleep analysis in critically ill patients. Sleep Med. 2011; 13(1): 7–14. PubMed Abstract \| Publisher Full Text\n\nKamdar BB, Niessen T, Colantuoni E, et al.: Delirium transitions in the medical ICU: exploring the role of sleep quality and other factors. Crit Care Med. 2015; 43(1): 135–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaneko T, Takahashi S, Naka T, et al.: Postoperative delirium following gastrointestinal surgery in elderly patients. Surg Today. 1997; 27(2): 107–11. PubMed Abstract \| Publisher Full Text\n\nKnauert MP, Yaggi HK, Redeker NS, et al.: Feasibility study of unattended polysomnography in medical intensive care unit patients. Heart Lung. 2014; 43(5): 445–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar AK, Jayant A, Arya VK, et al.: Delirium after cardiac surgery: A pilot study from a single tertiary referral center. Ann Card Anaesth. 2017; 20(1): 76–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShigeta H, Yasui A, Nimura Y, et al.: Postoperative delirium and melatonin levels in elderly patients. Am J Surg. 2001; 182(5): 449–54. PubMed Abstract \| Publisher Full Text\n\nTrompeo AC, Vidi Y, Locane MD, et al.: Sleep disturbances in the critically ill patients: role of delirium and sedative agents. Minerva Anestesiol. 2011; 77(6): 604–12. PubMed Abstract\n\nWhitcomb JJ, Morgan M, Irvin T, et al.: A pilot study on delirium in the intensive care unit: a creative inquiry project with undergraduate nursing students. Dimens Crit Care Nurs. 2013; 32(5): 266–70. PubMed Abstract \| Publisher Full Text\n\nYildizeli B, Ozyurtkan MO, Batirel HF, et al.: Factors associated with postoperative delirium after thoracic surgery. Ann Thorac Surg. 2005; 79(3): 1004–9. PubMed Abstract \| Publisher Full Text\n\nZhang WY, Wu WL, Gu JJ, et al.: Risk factors for postoperative delirium in patients after coronary artery bypass grafting: A prospective cohort study. J Crit Care. 2015; 30(3): 606–12. PubMed Abstract \| Publisher Full Text\n\nDavoudi A, Malhotra KR, Shickel B, et al.: Intelligent ICU for Autonomous Patient Monitoring Using Pervasive Sensing and Deep Learning. Sci Rep. 2019; 9(1): 8020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimeone S, Pucciarelli G, Perrone M, et al.: Delirium in ICU patients following cardiac surgery: An observational study. J Clin Nurs. 2018; 27(9–10): 1994–2002. PubMed Abstract \| Publisher Full Text\n\nZaal IJ, Spruyt CF, Peelen LM, et al.: Intensive care unit environment may affect the course of delirium. Intensive Care Med. 2013; 39(3): 481–8. PubMed Abstract \| Publisher Full Text\n\nVan Rompaey B, Elseviers MM, Van Drom W, et al.: The effect of earplugs during the night on the onset of delirium and sleep perception: a randomized controlled trial in intensive care patients. Crit Care. 2012; 16(3): R73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPustjens T, Schoutens AM, Janssen L, et al.: Effect of dynamic light at the coronary care unit on the length of hospital stay and development of delirium: a retrospective cohort study. J Geriatr Cardiol. 2018; 15(9): 567–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimons KS, Laheij RJ, van den Boogaard M, et al.: Dynamic light application therapy to reduce the incidence and duration of delirium in intensive-care patients: a randomised controlled trial. Lancet Respir Med. 2016; 4(3): 194–202. PubMed Abstract \| Publisher Full Text\n\nTaguchi T, Yano M, Kido Y: Influence of bright light therapy on postoperative patients: a pilot study. Intensive Crit Care Nurs. 2007; 23(5): 289–97. PubMed Abstract \| Publisher Full Text\n\nOno H, Taguchi T, Kido Y, et al.: The usefulness of bright light therapy for patients after oesophagectomy. Intensive Crit Care Nurs. 2011; 27(3): 158–66. PubMed Abstract \| Publisher Full Text\n\nYang J, Choi W, Ko YH, et al.: Bright light therapy as an adjunctive treatment with risperidone in patients with delirium: a randomized, open, parallel group study. Gen Hosp Psychiatry. 2012; 34(5): 546–51. PubMed Abstract \| Publisher Full Text\n\nChong MS, Tan KT, Tay L, et al.: Bright light therapy as part of a multicomponent management program improves sleep and functional outcomes in delirious older hospitalized adults. Clin Interv Aging. 2013; 8: 565–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPotharajaroen S, Tangwongchai S, Tayjasanant T, et al.: Bright light and oxygen therapies decrease delirium risk in critically ill surgical patients by targeting sleep and acid-base disturbances. Psychiatry Res. 2018; 261: 21–7. PubMed Abstract \| Publisher Full Text\n\nDemoule A, Carreira S, Lavault S, et al.: Impact of earplugs and eye mask on sleep in critically ill patients: a prospective randomized study. Crit Care. 2017; 21(1): 284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcAndrew NS, Leske J, Guttormson J, et al.: Quiet time for mechanically ventilated patients in the medical intensive care unit. Intensive Crit Care Nurs. 2016; 35: 22–7. PubMed Abstract \| Publisher Full Text\n\nKamdar BB, King LM, Collop NA, et al.: The effect of a quality improvement intervention on perceived sleep quality and cognition in a medical ICU. Crit Care Med. 2013; 41(3): 800–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuther R, McLeod A: The effect of chronotherapy on delirium in critical care - a systematic review. Nurs Crit Care. 2018; 23(6): 283–90. PubMed Abstract \| Publisher Full Text\n\nMadrid-Navarro CJ, Sanchez-Galvez R, Martinez-Nicolas A, et al.: Disruption of Circadian Rhythms and Delirium, Sleep Impairment and Sepsis in Critically ill Patients. Potential Therapeutic Implications for Increased Light-Dark Contrast and Melatonin Therapy in an ICU Environment. Curr Pharm Des. 2015; 21(24): 3453–68. PubMed Abstract \| Publisher Full Text\n\nKonkani A, Oakley B, Penprase B: Reducing hospital ICU noise: a behavior-based approach. J Healthc Eng. 2014; 5(2): 229–46. PubMed Abstract \| Publisher Full Text\n\nGabor JY, Cooper AB, Crombach SA, et al.: Contribution of the intensive care unit environment to sleep disruption in mechanically ventilated patients and healthy subjects. Am J Respir Crit Care Med. 2003; 167(5): 708–15. PubMed Abstract \| Publisher Full Text\n\nLuetz A, Weiss B, Penzel T, et al.: Feasibility of noise reduction by a modification in ICU environment. Physiol Meas. 2016; 37(7): 1041–55. PubMed Abstract \| Publisher Full Text\n\nKahn DM, Cook TE, Carlisle CC, et al.: Identification and modification of environmental noise in an ICU setting. Chest. 1998; 114(2): 535–40. PubMed Abstract \| Publisher Full Text\n\nMonsé MG, Edéll-Gustafsson UM: Noise and sleep disturbance factors before and after implementation of a behavioural modification programme. Intensive Crit Care Nurs. 2005; 21(4): 208–19. PubMed Abstract \| Publisher Full Text\n\nCrawford KJ, Barnes LA, Peters TM, et al.: Identifying determinants of noise in a medical intensive care unit. J Occup Environ Hyg. 2018; 15(12): 810–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuisasola-Rabes M, Sola-Enriquez B, Velez-Pereira AM, et al.: Effectiveness of a visual noise warning system on noise levels in a surgical ICU: A quality improvement programme. Eur J Anaesthesiol. 2019; 36(11): 857–62. PubMed Abstract \| Publisher Full Text\n\nPlummer NR, Herbert A, Blundell JE, et al.: SoundEar noise warning devices cause a sustained reduction in ambient noise in adult critical care. J Intensive Care Soc. 2018; 20(2): 106–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchlesinger J, Reynolds E, Sweyer B, et al.: Frequency-Selective Silencing Device for Digital Filtering of Audible Medical Alarm Sounds to Enhance ICU Patient Recovery. 2017. Publisher Full Text\n\nWallace CJ, Robins J, Alvord LS, et al.: The effect of earplugs on sleep measures during exposure to simulated intensive care unit noise. Am J Crit Care. 1999; 8(4): 210–9. PubMed Abstract \| Publisher Full Text\n\nScotto CJ, McClusky C, Spillan S, et al.: Earplugs improve patients' subjective experience of sleep in critical care. Nurs Crit Care. 2009; 14(4): 180–4. PubMed Abstract \| Publisher Full Text\n\nLitton E, Elliott R, Ferrier J, et al.: Quality sleep using earplugs in the intensive care unit: the QUIET pilot randomised controlled trial. Crit Care Resusc. 2017; 19(2): 128–33. PubMed Abstract\n\nGallacher S, Enki D, Stevens S, et al.: An experimental model to measure the ability of headphones with active noise control to reduce patient’s exposure to noise in an intensive care unit. Intensive Care Med Exp. 2017; 5(1): 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichardson A, Allsop M, Coghill E, et al.: Earplugs and eye masks: do they improve critical care patients' sleep? Nurs Crit Care. 2007; 12(6): 278–86. PubMed Abstract \| Publisher Full Text\n\nHu RF, Jiang XY, Zeng YM, et al.: Effects of earplugs and eye masks on nocturnal sleep, melatonin and cortisol in a simulated intensive care unit environment. Crit Care. 2010; 14(2): R66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones C, Dawson D: Eye masks and earplugs improve patient's perception of sleep. Nurs Crit Care. 2012; 17(5): 247–54. PubMed Abstract \| Publisher Full Text\n\nLe Guen M, Nicolas-Robin A, Lebard C, et al.: Earplugs and eye masks vs routine care prevent sleep impairment in post-anaesthesia care unit: a randomized study. Br J Anaesth. 2014; 112(1): 89–95. PubMed Abstract \| Publisher Full Text\n\nAlbala L, Bober T, Hale G, et al.: Effect on nurse and patient experience: overnight use of blue-depleted illumination. BMJ Open Qual. 2019; 8(3): e000692. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalder B, Francioli D, Meyer JJ, et al.: Effects of guidelines implementation in a surgical intensive care unit to control nighttime light and noise levels. Crit Care Med. 2000; 28(7): 2242–7. PubMed Abstract \| Publisher Full Text\n\nOlson DM, Borel CO, Laskowitz DT, et al.: Quiet time: a nursing intervention to promote sleep in neurocritical care units. Am J Crit Care. 2001; 10(2): 74–8. PubMed Abstract \| Publisher Full Text\n\nDennis CM, Lee R, Woodard EK, et al.: Benefits of quiet time for neuro-intensive care patients. J Neurosci Nurs. 2010; 42(4): 217–24. PubMed Abstract \| Publisher Full Text\n\nBartick MC, Thai X, Schmidt T, et al.: Decrease in as-needed sedative use by limiting nighttime sleep disruptions from hospital staff. J Hosp Med. 2010; 5(3): E20–4. PubMed Abstract \| Publisher Full Text\n\nLi SY, Wang TJ, Vivienne Wu SF, et al.: Efficacy of controlling night-time noise and activities to improve patients' sleep quality in a surgical intensive care unit. J Clin Nurs. 2011; 20(3–4): 396–407. PubMed Abstract \| Publisher Full Text\n\nBoyko Y, Jennum P, Nikolic M, et al.: Sleep in intensive care unit: The role of environment. J Crit Care. 2017; 37: 99–105. PubMed Abstract \| Publisher Full Text\n\nGoeren D, John S, Meskill K, et al.: Quiet Time: A Noise Reduction Initiative in a Neurosurgical Intensive Care Unit. Crit Care Nurse. 2018; 38(4): 38–44. PubMed Abstract \| Publisher Full Text\n\nArenson BG, MacDonald LA, Grocott HP, et al.: Effect of intensive care unit environment on in-hospital delirium after cardiac surgery. J Thorac Cardiovasc Surg. 2013; 146(1): 172–8. PubMed Abstract \| Publisher Full Text\n\nHu RF, Hegadoren KM, Wang XY, et al.: An investigation of light and sound levels on intensive care units in China. Aust Crit Care. 2016; 29(2): 62–7. PubMed Abstract \| Publisher Full Text\n\nInouye SK, Bogardus Jr ST, Charpentier PA, et al.: A multicomponent intervention to prevent delirium in hospitalized older patients. N Engl J Med. 1999; 340(9): 669–76. PubMed Abstract \| Publisher Full Text\n\nInouye SK, Viscoli CM, Horwitz RI, et al.: A predictive model for delirium in hospitalized elderly medical patients based on admission characteristics. Ann Intern Med. 1993; 119(6): 474–81. PubMed Abstract \| Publisher Full Text\n\nDelaney L, Litton E, Van Haren F: The effectiveness of noise interventions in the ICU. Curr Opin Anaesthesiol. 2019; 32(2): 144–149. PubMed Abstract \| Publisher Full Text\n\nUlrich RS, Zimring C, Zhu X, et al.: A review of the research literature on evidence-based healthcare design. HERD. 2008; 1(3): 61–125. PubMed Abstract \| Publisher Full Text\n\nLutter NO, Urankar S, Kroeber S: False alarm rates of three third-generation pulse oximeters in PACU, ICU and IABP patients. Anesth Analg. 2002; 94(1 Suppl): S69–75. PubMed Abstract\n\nKonkani A, Oakley B, Bauld TJ: Reducing hospital noise: a review of medical device alarm management. Biomed Instrum Technol. 2012; 46(6): 478–87. PubMed Abstract \| Publisher Full Text\n\nBarroso A, Simons K, de Jager P: Metrics of circadian lighting for clinical investigations. Lighting Research & Technology. 2013; 46(6): 637–49. Publisher Full Text\n\nOlofsson K, Alling C, Lundberg D, et al.: Abolished circadian rhythm of melatonin secretion in sedated and artificially ventilated intensive care patients. Acta Anaesthesiol Scand. 2004; 48(6): 679–84. PubMed Abstract \| Publisher Full Text" }	[ { "id": "79721", "date": "09 Mar 2021", "name": "Ari Ercole", "expertise": [ "Reviewer Expertise Intensive care. Interest in wearable technology and monitoring." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a systematic scoping review of the literature studying the impact of environmental risk factors on delirium. This is a potentially important area. Delirium is a problem in healthcare and whilst environmental factors have been implicated, they have received relatively little systematic/robust analysis to date. The delirium field, whilst a popular area of research, is complicated by difficulties in defining robust diagnostic criteria, disease heterogeneity and a lack of effective treatments. A better understanding of and the impact of environmental interventions is potentially important for hospital design, service configuration and care and the authors' attempts to synthesise the available literature is both welcome and timely.\nIn summary, this is a nice piece of work that has been well carried out. The key message is that the current literature is heterogeneous in terms of methodology and quality. The question I am left with, however, is how best to improve this moving forwards and if I have a minor criticism it is that the authors could be more definitive in making suggestions as to how, in their opinion having appraised the literature, methodological quality can be improved in the future? Where do they see the key gaps? If a study was being designed into, for example, an environmental intervention, can the authors comment on what they think the most important confounders are and how future researchers can reduce study heterogeneity? Can the authors draw conclusions as to what a ‘gold standard’ for an environmental intervention study might look like?\nI think a discussion (a minor revision) around this would enhance this piece of work and make it an important resource for the future.\nMinor points:\nThe search methodology is well described and reporting guidelines followed appropriately. All settings are included in the search but the majority of the references pertain to the ICU and, indeed, the authors describe this as a key area of interest in the introduction. I wonder, therefore, whether non-ICU setting papers should be excluded? In particular are the data of reference 55 (geriatric monitoring unit for acute delirium care) transferable to other settings? If all settings are retained, please justify the inclusion of non-ICU settings or discuss the likely limitations of this further. The abstract and title do not state the setting explicitly: Whatever the authors chose in the end, it should be reflected in the abstract at least.\n\nThe search terms seem reasonable. The manuscript states “HH and JL screened titles”: Presumably this was done independently / each covered all titles? Similarly, was the extraction duplicated (or a sub-sample)? These are not limitations but should be explicitly stated.\n\nThe authors searched Pubmed only and explicitly comment on this limitation. They state that a hand-search with other databases was performed- can they comment (broadly) that this did not change the number of abstracts substantially / to what extent they found other literature? I do not think it necessary to expand to other databases systematically as the authors have, I think, proved their point well: The literature is fundamentally heterogeneous in terms of methodology, outcomes, interventions, etc.\n\nThe search is now a little out of date, but I do not think that updating it will significantly alter the main conclusions.\n\nI would like to see some assessment of the quality of the evidence presented / risk of bias. I do not suggest this needs to be too detailed but it would be helpful for the reader to be able to quickly survey which (if any!) of the cited references were of high methodological quality or, alternatively, it would be powerful if the authors can make a statement to the contrary. Perhaps a modified/simplified implementation of the Cochrane risk of bias assessment?\n\nDelirium is unlikely to be a single entity and a heterogeneous range of assessment tools were used (although CAM-ICU was the most frequently employed). Can the authors also comment on how these tools were applied (I imagine the timing and staff conducting the assessments were very heterogeneous)?\n\nA major area that seems to be missed in much of the literature is a robust definition or characterisation of diurnal environmental factors or interventions, particularly for sound. It seems reasonable that this is important in establishing day-night cycles yet only a subset (generally light-based studies) of references address this explicitly. The authors may wish to discuss this further as it would be important in designing better studies in the future.\n\nPlease provide a brief summary (i.e. critique) of the differences between A- and C- spectral weighting as clinical readers are likely to be unfamiliar with this and critique these choices. Which do the authors feel is the most appropriate moving forwards? As a minor point, perhaps replace ‘Leq’ with ‘LAeq / LCeq’ to standardise the terminology for reference 19. Continuous sound measurements should also give the continuous sounds duration over which the averaging is performed (e.g. LAeq, 10mins)- was this uniform across studies that used such measurements (this is not a limitation of the review but perhaps yet another heterogeneity in the literature)?\n\nICU studied unlikely to be comparable (SICU vs MICU vs General)? Were any neurosciences ICUs (likely different with regular neuro-obs)? Confounded by non-pharmacological (re-orientation) and pharmacological intervention policy?\n\nThe authors state that “One study found atypical sleep on PSG was significantly tied to increased delirium”. As a minor point, it is probably better to use the word ‘associated’ rather than ‘tied’ as causation is very difficult to establish here given the definition of delirium.\n\nTo what extent do the authors think that the results in the references they found are generalisable across institutions? Crucially, ICU (and hospital design/operation) varies substantially around the world with differences in side-room use etc. To what extent is this defined in the literature that the authors found?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "88467", "date": "16 Jul 2021", "name": "Annmarie Hosie", "expertise": [ "Reviewer Expertise Palliative care", "delirium (including multicomponent interventions)", "nursing", "evidence synthesis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review your manuscript, 'The impact of environmental risk factors on delirium and benefits of noise and light modifications: a scoping review'.\nOverall, this is a clearly written manuscript reporting a large volume of data and demonstrating excellent attention to detail. Thank you also for aligning your report with the appropriate reporting guideline.\n\nHowever, there are some areas requiring attention before the manuscript is ready for indexing, as follows:\nPlease provide the rationale for using a scoping review methodology, that aligns with those identified in the literature (see: Tricco et al., 20181)\n\nThe abstract and included articles are not limited to discussion/studies in the ICU, but the introduction focuses solely on delirium in the ICU. This discrepancy needs to be addressed, either by revising the introduction or revising the abstract and the inclusion criteria for included studies.\n\nWhy was only one database (PubMed) searched, given the broad intent of a scoping review?\n\nPlease state the eligibility criteria for included articles/studies.\n\nGiven the aim of the study (i.e., to map the literature and assess the role of …), a rationale for why literature reporting on-pharmacological noise, light, and circadian rhythm strategies within multicomponent interventions were excluded is required.\n\nThe method of data charting and synthesis should be stated.\n\nThe final box in the PRISMA flow chart should state ‘scoping review’ not ‘systematic review’.\n\nTable 9, the summary of excluded studies (excluded because delirium was not measured as an outcome) is unnecessary and confusing, especially as the manuscript is already very data-dense. Given also that it is not customary to include summaries of excluded articles in reviews, I highly recommend removing this information.\n\nThe discussion seems to focus too much on the effectiveness or otherwise of the interventions and too little on what is required to build the evidence for these interventions going forward. It also seems overly long, especially following the extensive results. My suggestion is to revise the discussion for greater focus, conciseness, and direction for future research.\n\nThe last sentence of the conclusion is not justified, given the inconclusiveness of the effectiveness of the examined interventions, the lack of risk of bias appraisal (which is in line with the scoping review methodology), and the fact that studies in which the examined interventions were part of multicomponent interventions were excluded.\n\nI have some reservations about the helpfulness of examining environmental risk factors and interventions in isolation from other interventions targeted at a wider range of risk factors, given there is extensive evidence of the multitude of risk factors for delirium. This is a point you have likely considered, so it would be good if the decision to focus only on environmental risk factors and interventions was acknowledged and justified in more depth.\n\nLastly, the search needs to be updated as it is now almost two years old.\n\nI hope these suggestions are helpful in revising your manuscript and wish you well in your ongoing work in this area.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1183
https://f1000research.com/articles/9-1182/v1	29 Sep 20	{ "type": "Systematic Review", "title": "Nurse practitioners and physician assistants working in ambulance care: A systematic review", "authors": [ "Risco van Vliet", "Remco Ebben", "Nicolette Diets", "Thomas Pelgrim", "Jorik Loef", "Lilian Vloet", "Remco Ebben", "Nicolette Diets", "Thomas Pelgrim", "Jorik Loef", "Lilian Vloet" ], "abstract": "Background: This review aims to describe the activities of nurse practitioners (NPs) and physician assistants (PAs) working in ambulance care, and the effect of these activities on patient outcomes, process of care, provider outcomes, and costs. Methods: PubMed, MEDLINE (EBSCO), EMBASE (OVID), Web of Science, the Cochrane Library (Cochrane Database of Systematic Review), CINAHL Plus, and the reference lists of the included articles were systematically searched in November 2019. All types of peer-reviewed designs on the three topics were included. Pairs of independent reviewers performed the selection process, the quality assessment, and the data extraction. Results: Four studies of moderate to poor quality were included. Activities in medical, communication and collaboration skills were found. The effects of these activities were found in process of care and resource use outcomes, focusing on non-conveyance rates, referral and consultation, on-scene time, or follow-up contact Conclusions: This review shows that there is limited evidence on activities of NPs and PAs in ambulance care. Results show that NPs and PAs in ambulance care perform activities that can be categorized into the Canadian Medical Education Directives for Specialists (CanMED) roles of Medical Expert, Communicator, and Collaborator. The effects of NPs and PAs are minimally reported in relation to process of care and resource use, focusing on non-conveyance rates, referral and consultation, on-scene time, or follow-up contact. No evidence on patient outcomes of the substitution of NPs and PAs in ambulance care exists. PROSPERO registration: CRD42017067505 (07/07/2017)", "keywords": [ "nurse-practitioners", "physician-assistants", "ambulance care", "patient outcomes", "implementation", "emergency medical services" ], "content": "Background\n\nAmbulance utilisation has increased in the Western world over the past 20 years, potentially compromising access, quality, safety, and patient outcomes1,2. Population ageing, changes in social support and accessibility, increasing community health awareness, patients presenting themselves with higher complexity and comorbidities, repeated ambulance care requests, and ambulance care request for primary healthcare problems have been described as associated factors for this increase1,3–6. The pressure to reduce costs and the potentially negative effects of this increase of ambulance utilisation have led to the redefinition of the roles of professionals in prehospital care1,2. With the impending rise in demand for health services, an effective utilization of the workforce is paramount to ensure high-quality yet cost-effective health service delivery7. This can be done by optimising triage and ambulance allocation, but also by introducing other types of healthcare professionals in the ambulance domain. A possible solution to improve the balance between the increasing demand for care and the decreasing supply of medical healthcare workers is enhancing the role of allied healthcare workers, such as nurse practitioners (NPs) or physician assistants (PAs)8 .\n\nThe first NPs and PAs in the Dutch healthcare system made their appearance in 2001 and 2004. NPs are situated in the nursing domain and perform broadening activities in the medical domain within selected groups of patients and simultaneously on deepening activities in the nursing domain. PAs focus on broadening and deepening activities in the medical domain, within their medical specialty.\n\nSeveral reviews about the implementation of NPs and PAs have been performed9–12. These reviews have revealed not only a higher quality of care but also an increase in patient satisfaction and that NPs and PAs have the potential to reduce doctors’ workloads and direct health care costs. However, this research has been limited to long-term care facilities and primary health care; there currently is no evidence pertaining to what activities NPs or PAs in ambulance care perform and what the effects of these activities are.\n\n\nAims\n\nTherefore, this review has two aims. First, to describe the activities of nurse practitioners and physician assistants working in ambulance care. Second to describe the effects of these activities on patient outcomes, process of care, provider outcomes, and costs.\n\n\nMethods\n\nThis study is a systematic literature review reported according to the steps of Cochrane Handbook13 and reported to conform with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. For background and an extensive method section, see the study protocol14.\n\nThe Cochrane Database for Systematic Reviews and PROSPERO were inspected for similar reviews or protocols. No (pending) review was identified, so systematic searches were performed in PubMed, MEDLINE (EBSCO), EMBASE (OVID), Web of Science, the Cochrane Library (Cochrane Database of Systematic Reviews), and CINAHL Plus in November 2019.\n\nSearch strategies were developed to represent terms for ambulance care and NPs or PAs. Full search strategies per database are provided as extended data15.\n\nSearches were not restricted by year of publication. All types of peer-reviewed systematic reviews, and quantitative or qualitative designs in real clinical practice or simulation situations on NPs or PAs working in ambulance care for all kinds of patients were included. Conference abstracts, narrative reviews, editorials, personal communications, and unpublished studies were excluded.\n\nStudies were included if they (a) described activities of professionals with a master’s degree in ambulance care (NPs or PAs) and/or described the effects of the NP or PA on patient outcomes, process of care, provider outcomes and costs.\n\nDue to the heterogeneity of the names that are used for the emergency medical service (EMS) professional worldwide16, we began with a broad search. Some terms covered a variety of different professionals; for example, the education level of the emergency care practitioner (ECP) can be that of a paramedic or a nurse. We explicitly searched for professionals with a master’s degree and excluded studies where this was not present.\n\nTwo reviewers (RvV and ND) independently screened the title and abstract of each potentially relevant study. Differences between the reviewers were resolved through discussion. Couples of two independent reviewers (RvV, RE, ND, JL, and LV) screened the full texts of the remaining articles. In addition, two reviewers (RvV and RE) screened the reference lists of the included articles.\n\nTo assess the quality of observational studies, we used a tool developed for evaluating primary research papers in a variety of fields17. Couples of independent researchers (RvV, RE, ND, JL, and LV) performed this assessment. Differences between two reviewers were resolved through discussion; in cases of doubt, a third reviewer from another couple made the final decision.\n\nCouples of independent researchers (RvV, RE, ND, JL, and LV) extracted the data. Due to the heterogeneity of study designs, settings, countries, care providers, interventions, and outcome measures, a meta-analysis was not possible; the results of this systematic review are therefore presented in tabular form.\n\n\nResults\n\nThe initial search identified 1283 unique records; 68 articles were included for full-text screening (see Figure 1), from which we included four articles for data extraction. A list of excluded articles and the reasons for their exclusion (n = 64) is provided as extended data18. Common reasons for exclusion were a non-master educational level, the lack of peer review, and the wrong setting (not prehospital ambulance care).\n\nDesigns of the included studies comprised of one cross sectional19, one retrospective observational20, one action research21, and one retrospective descriptive review22 (Table 1). Two studies were performed in the UK, one in the Netherlands and one in the USA. All these studies extracted the data from ambulance run records or patient records. The focus of three of these studies19,21,22 was primarily on ambulance care, where the retrospective observational study20 had a broader perspective of home care, ambulance care, and emergency care. One study19 compared the PA with a registered nurse (RN), two compared the NP with other EMS professionals (e.g., paramedics)20,21 and one solely described the NP22 without comparison.\n\nAbbreviations: APRU Advanced Provider Response Unit, ECP Emergency care practitioner, ED Emergency department, EMS Emergency medical services, NP Nurse practitioner, PA Physician assistant, RN Registered nurse\n\nThe cross sectional study19 is of moderate quality due to the representativeness of results, and small population. The other three studies20–22 are of poor quality.\n\nActivities by a NP or PA (Table 2). Two articles reported on activities NPs or PAs perform in ambulance care19,20, these activities were related to medical skills, communication and collaboration. For medical skills, the usage of the SCEBS methodology and overall care, assessment, investigation, management, and quality of record registration were described19,20. For communication, the provision of medical advice and for collaboration referral to the ED or GP were described19.\n\nAbbreviations: ECG electrocardiograph, ED emergency department, EMS emergency medical services, GCS/AVPU Glasgow coma scale/ Alert Voice Pain Unresponsive, GP general practitioner, SCEBS Somatic complaints, Cognitions, Emotions, Behaviour and Social functioning of the patient, NP nurse practitioner, PA physician assistant\n\nNone of the included studies reported on patient outcomes, care provider outcomes, or costs; the studies did report on process of care and resource use.\n\nAll four studies19–22 reported on process of care outcomes. The outcomes used included the proportion of non-conveyance, the number of referrals in non-conveyance patients, the number of consultations, the length of on-scene treatment, the follow-up contact of non-conveyance patients, diagnostic measurements, adherence to guidelines and protocols and, number of performed interventions. One study reported on resource use.\n\nAbbreviations: EMS emergency medical services, GP general practitioner,NP nurse practitioner, PA physician assistant, RN registered nurse\n\nNon-conveyance (n=3). Three studies reported on non-conveyance19,21,22 and showed non-conveyance rates ranging from 20% –50% for PAs. Non-conveyance rates for the NP were not described.\n\nReferral and consultation (n=1). One study19 found that PAs refer 50% of their patients to another health care professional (e.g., a GP or an emergency department (ED)) while RNs referred 73%. Furthermore, PAs consulted other health care professionals (e.g., a GP, an emergency physician, or a medical specialist) significantly more often compared to RNs.\n\nOn-scene time (n=2). One study found no significant difference between PAs and RNs regarding the length of on-scene treatment time19. Another study described22 an average length of treatment time on scene of 21.47min, but made no comparison with other EMS professionals.\n\nFollow-up contacts (n=1). Follow-up contact after the completion of prehospital EMS care also indicated no significant differences between PAs and RNs19.\n\nResource use (n=1). One study22 found in 107 cases other EMS resources were released from the scene and put back in service while the NP attended the patient, (by default, two units respond to a call). 18 high utilizers of 911 were connected with a social work organization, and 12 of 18 (66.7%) decreased their use of EMS in the 90-days following.\n\n\nDiscussion\n\nThis review aimed to describe which activities NPs or PAs deploy in ambulance care, and if there were effects on patient outcomes, process of care, provider outcomes, and costs.\n\nThe results indicate that little is known on the activities PAs and NPs deployed in ambulance care. This can be explained by the relatively young professions these professionals represent. The activities that were identified can be categorized using the Canadian Medical Education Directives for Specialists (CanMED) framework. The CanMEDS system is a widely used instrument to describe medical professionals activities and forms the basis of the education of NPs and PAs24. A competent professional seamlessly integrates all seven competencies CanMEDS roles24 (Medical Expert (the integrating role), Communicator, Collaborator, Leader, Health Advocate, Scholar and Professional). However, the activities found in this review can be categorized into the medical professional, communicator and collaborator. This is remarkable because the full NP and PA profiles includes seven CanMEDS roles. There are several reasons why all seven roles are not reported on. First, it is possible that not all seven roles are applicable in ambulance care, or are not visible in the primary process of ambulance care. Also, PAs and NPs have only recently integrated into the ambulance care system, a clear job description or interpretation of their duties may be lacking. Developing a systematic description of the roles and competences of NPs or PAs in ambulance care would therefore be useful.\n\nAlthough there are differences in education between NPs and PAs, there also seems to be a large degree of overlap in the tasks that NPs and PAs perform in practice25; for instance, both professionals perform tasks that are part of the medical process, such as, drafting and evaluating treatment plans, and carrying out interventions25.\n\nResults shows that little is known on the effects of the activities of NPs and PAs in ambulance care. Some effects found can be linked to process of care and resource use. We found no effects on patient outcomes or care provider outcomes. Reviews12,25,26 in other health care settings revealed an increase in quality of care and patient satisfaction. Evidence in primary care26, elderly care3, and out-of-hours primary care27 suggests that the substitution of NPs or PAs is feasible with at least the maintenance of quality and no increase in costs.\n\nAlthough we have found no description on the effect on costs, Walsh et al.21 assumed that the substitution of NPs could produce substantial savings for the EMS and relieve the burden on hard-pressed ambulance and ED. Bloemhoff et al.19 recommended further exploration into the costs.\n\nFurther research is necessary to draw any conclusions on the effects of the substitution of NPs and PAs in ambulance care for multiple outcomes. This should be addressed by using the six dimensions of quality of healthcare: 1- effectiveness, 2- efficiency, 3- patient safety, 4- accessibility, 5- timeliness and 6- target population directed26. Measuring these outcomes within all phases of the ambulance process (from initial call, to handover or referral) will gain more insight in the effects of PAs and NPs in ambulance care.\n\n\nStrengths and limitations\n\nA strength of this systematic review is that the search began with a broad strategy for six online databases, following the quality standards from the Cochrane Handbook13 and reported to conform with the PRISMA statement28.\n\nOne limitation of this review lies in the fact that our broad search strategy produced only four studies that described NPs or PAs working in ambulance care. Within these studies, the settings are completely different which made it impossible to perform a meta-analysis. Due to the diversity of the professionals working in ambulance care worldwide, it was difficult to identify the educational level of the professionals. Another limitation concerns the quality assessment tools for quantitative and qualitative designs a variety of these tools exists without a clear evidence base13.\n\n\nConclusion\n\nThis review shows that there is limited evidence on activities of NPs and PAs in ambulance care. Results show that NPs and PAs in ambulance care perform activities that can be categorized into the CanMED roles of Medical Expert, Communicator, and Collaborator. The effects of NPs and PAs are minimally reported in relation to process of care and resource use, focusing on non-conveyance rates, referral and consultation, on-scene time, or follow-up contact. There is no evidence on patient outcomes of the substitution of NPs and PAs in ambulance care. Further research is necessary to provide insight into these effects.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Appendix 1 search strategies.docx. https://doi.org/10.6084/m9.figshare.12949730.v115\n\nFigshare: Appendix 2 Reason full text exclusion.docx.\n\nhttps://doi.org/10.6084/m9.figshare.12949736.v118\n\nFigshare: Appendix 3 Quality of quantitative studies (n=4).docx. https://doi.org/10.6084/m9.figshare.12949748.v123\n\nFigshare: PRISMA checklist for ‘Nurse practitioners and physician assistants working in ambulance care: A systematic review’ https://doi.org/10.6084/m9.figshare.12949766.v129\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nLowthian JA, Cameron PA, Stoelwinder JU, et al.: Increasing utilisation of emergency ambulances. Aust Health Rev. 2011; 35(1): 63–69. PubMed Abstract \| Publisher Full Text\n\nde Hoon S, Jansen T, Verheij R: De Verpleegkundig Specialist: Een Alternatief Om de Ambulancezorg Te Ontlasten? Een Draagvlakmeting Onder Zorgverleners in Ambulanceregio Zuid-Holland Zuid. 2017. Reference Source\n\nLovink MH, Persoon A, Koopmans RTCM, et al.: Effects of substituting nurse practitioners, physician assistants or nurses for physicians concerning healthcare for the ageing population: a systematic literature review. J Adv Nurs. 2017; 73(9): 2084–2102. PubMed Abstract \| Publisher Full Text\n\nJones CMC, Wasserman EB, Li T, et al.: The Effect of Older Age on EMS Use for Transportation to an Emergency Department. Prehosp Disaster Med. Cambridge University Press; 2017: 32(3); 261–268. PubMed Abstract \| Publisher Full Text\n\nEdwards MJ, Bassett G, Sinden L, et al.: Frequent callers to the ambulance service: patient profiling and impact of case management on patient utilisation of the ambulance service. Emerg Med J. 2015; 32(5): 392–396. PubMed Abstract \| Publisher Full Text\n\nBooker MJ, Purdy S, Shaw ARG: Seeking ambulance treatment for ‘primary care’ problems: a qualitative systematic review of patient, carer and professional perspectives. BMJ Open. 2017; 7(8): e016832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOzcan S, Taranto Y, Hornby P: Shaping the health future in Turkey: a new role for human resource planning. Int J Health Plann Manage. 1995; 10(4): 305–319. PubMed Abstract \| Publisher Full Text\n\nCooper RA: New directions for nurse practitioners and physician assistants in the era of physician shortages. Acad Med. 2007; 82(9): 827–828. PubMed Abstract \| Publisher Full Text\n\nWoo BFY, Lee JXY, Tam WWS: The impact of the advanced practice nursing role on quality of care, clinical outcomes, patient satisfaction, and cost in the emergency and critical care settings: a systematic review. Hum Resour Health. 2017; 15(1): 63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKmietowicz Z, Anderson E: Primary care trusts should give local people a voice, says report. BMJ. 2002; 324(7338): 633a. Publisher Full Text \| Free Full Text\n\nLaurant M, Reeves D, Hermens R, et al.: Substitution of doctors by nurses in primary care. Cochrane Database Syst Rev. 2005; 18(2): CD001271. PubMed Abstract \| Publisher Full Text\n\nMartin-Misener R, Harbman P, Donald F, et al.: Cost-effectiveness of nurse practitioners in primary and specialised ambulatory care: systematic review. BMJ Open. 2015; 5(6): e007167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggens JGS: Cochrane Handbook for Systematic Review of interventions Version 5.1.0. The Cochrane Collaboration. 2011. Reference Source\n\nNHS National Institure for Health Research: PROSPERO, Inrenational prospective register of systematic reviews. The Cochrane Collaboration. 2017. Reference Source\n\nvan Vliet R: Appendix 1 search strategies.docx. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12949730.v1\n\nPulcini J, Jelic M, Gul R, et al.: An international survey on advanced practice nursing education, practice, and regulation. J Nurs Scholarsh. 2010; 42(1): 31–39. PubMed Abstract \| Publisher Full Text\n\nKmet LM, Cook LS, Lee RS: Standard Quality Assessment Criteria for Evaluating Primary Research Papers from a Variety of Fields. 2004. Publisher Full Text\n\nvan Vliet R: Appendix 2 Reason full text exclusion.docx. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12949736.v1\n\nBloemhoff A, Schoonhoven L, de Kreek AJL, et al.: Solo emergency care by a physician assistant versus an ambulance nurse: a cross-sectional document study. Scand J Trauma Resusc Emerg Med. 2016; 24(1): 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Hara R, O’Keeffe C, Mason S, et al.: Quality and safety of care provided by emergency care practitioners. Emerg Med J. 2012; 29(4): 327–332. PubMed Abstract \| Publisher Full Text\n\nWalsh M, Little S: Study of a nurse practitioner working in a paramedic role. Emerg Nurse. 2001; 9(6): 11–14. PubMed Abstract \| Publisher Full Text\n\nSanko S, Kashani S, Ito T, et al.: Advanced Practice Providers in the Field: Implementation of the Los Angeles Fire Department Advanced Provider Response Unit. Prehosp Emerg Care. 2020; 24(5): 693–703. PubMed Abstract \| Publisher Full Text\n\nvan Vliet R: Appendix 3 Quality of quantitative studies (n=4).docx. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12949748.v1\n\nFrank J, Snell L, Sherbino J: CanMEDS 2015 Physician Competency Framework. Ottawa: Royal College of Physicians and Surgeons of Canada. CanMEDS 2015 Physician Competency Fram Ottawa R Coll Physicians Surg Canada. 2015; 1–30. Reference Source\n\nWijers M: Een studie naar functieprofielen, taken en verantwoordelijkheden van Physician Assistants en Verpleegkundig Specialisten. 2014; 1–146. Reference Source\n\nLovink MH, Persoon A, van Vught AJAH, et al.: Substituting physicians with nurse practitioners, physician assistants or nurses in nursing homes: protocol for a realist evaluation case study. BMJ Open. 2017; 7(6): e015134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Der Biezen M, Adang E, Van Der Burgt R, et al.: The impact of substituting general practitioners with nurse practitioners on resource use, production and health-care costs during out-of-hours: a quasi-experimental study. BMC Fam Pract. 2016; 17(1): 132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiberati A, Altman DG, Tetzlaff J, et al.: The PRISMA Statement for Reporting Systematic Reviews and Meta-Analyses of Studies That Evaluate Health Care Interventions: Explanation and Elaboration. PLoS Med. 2009; 6(7): e1000100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Vliet R: Prisma statement. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12949766.v1" }	[ { "id": "72226", "date": "23 Oct 2020", "name": "Elena Lopatina", "expertise": [ "Reviewer Expertise Models of care", "quality of care", "non-physician care providers" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting systematic review. The review explores activities of nurse practitioners and physician assistants working in ambulance care and effects of those activities in terms of patient and provider outcomes, processes of care, and costs. The fact that only 4 studies were identified and none of the identified studies reported on patient or care providers outcomes, or costs highlights the need for more research in this area.\nTwo minor comments appeared while reviewing the manuscript:\nIntroduction, paragraph 3: NPs’ roles and activities as well as outcomes of those activities have also been explored in the context of inpatient and outpatient specialized care. Also, it would be useful to provide references to showcase previous research on NPs’ and PAs’ activities in various settings.\n\nAuthors limited their search to care providers with a Master degree only, thus excluding 22 articles during the full-text review. While authors recognize the existing differences in the level of training of NPs and PAs working in ambulatory care, it would be helpful to explain the rationale for focusing on providers with a Master degree only. Would authors expect roles and activities to differ between providers with a Master degree and without it? Given the low number of articles included and a relatively large number of full-texts excluded based on this criteria, one would wonder if it would be useful to take a broader approach.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "72764", "date": "22 Dec 2020", "name": "Lisette Schoonhoven", "expertise": [ "Reviewer Expertise Nursing Science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript. The topic presented is very timely given the strains healthcare is under and the future developments in the Western population. This review is performed well and the fact that only 4 papers were identified which only reported on limited outcomes signals the need for further research in this area.\n\nAfter reading the paper I do have some questions and comments.\nThe abstract does not state why it is important to do this review. Perhaps this is due to a restriction in number of words.\n\nIn the background the authors mention broadening activities and deepening activities for NPs and PAs. Examples of these activities could have added clarity for the reader who is not familiar with these positions.\n\nThe results on effects of the activities seem to focus on a comparison with RN's. While I understand this is the focus of some of the papers included, this is not the focus of this review and does not answer the research question.\n\nGiven that there are only 4 studies describing NPs and PAs in ambulance care, I wonder if other studies are needed first before focusing on quality outcomes. For example a study on where and how are these professionals are employed and a focus on their CanMEDS roles. Why are these professionals the answer to the problems mentioned in the introduction? The authors could have discussed this more thoroughly.\n\nThe limitations mention the quality assessment tools used. This comes as a bit of a surprise. Also, given that only quantitative designs were found (according to table 1) I am not sure how the lack of an evidence base for the tools for assessing qualitative designs can be a limitation in this paper.\n\nOverall, I wonder if doing a systematic review limited to scientific papers should have been the design of choice for this review. Perhaps also looking at policy documents etc could have provided a richer overview of the positioning of these professionals in the field of ambulance care. This could have been mentioned in the discussion.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "6234", "date": "23 Dec 2020", "name": "Risco van Vliet", "role": "Author Response", "response": "Dear Lisette Schoonhoven Thank you for your valuable review of our manuscript and the critical questions you have asked. Before we started this review, we searched for available information that provides insight into NPs and PAs working in ambulance care. We found very little and what was written was often a personal story and not judged by a peer reviewer. We wanted to tackle this in a scientific way and expected that little had been written but were surprised by the low inclusions. Because of this, you can indeed question whether this is the correct design to answer this research question. Perhaps we would have emphasized this more strongly in the discussion. After evaluating the process and based on the information found, we find a reason to continue scientific research into the role of NPS and PAs in ambulance care and to support initiatives and distant developments with solid research." } ] }, { "id": "75887", "date": "06 Jan 2021", "name": "Barbara Todd", "expertise": [ "Reviewer Expertise NP and PA models of care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review and comment on this manuscript. As care models continue to emerge, it is improved to report on outcomes of care related to NP and PA provided care. I have a few comments.\nThe abstract could be more robust in the background section to provide the context and necessity for this review.\n\nThere are limited studies as pointed out by the authors and concern with comparison of PA to RN rather than PA to NP care. I suspect there would be differences dependent on RN scope in some of the regions.\n\nIs there enough evidence to put forward a systematic review, obviously more research is needed on PA and NP care in ambulance services.\n\nIt would have been helpful to describe utilization of NPs and PAs in other care delivery models.\n\nWhat was the acuity of the patients managed by NP and PA, how does that compare to others delivering the same care. I think this would have provided rich context to the manuscript.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1182
https://f1000research.com/articles/9-1121/v2	28 Sep 20	{ "type": "Research Article", "title": "Socio-demographic and lifestyle factors associated with understanding fast food consumption among adults in Cambodia", "authors": [ "Wongsa Laohasiriwong", "Sim Samphors", "Pall Chamroen", "Rebecca S. Dewey", "Thiwakorn Rachutorn", "Vong Pisey", "Wongsa Laohasiriwong", "Pall Chamroen", "Rebecca S. Dewey", "Thiwakorn Rachutorn", "Vong Pisey" ], "abstract": "Background: Over the past decades, fast food has been rapidly gaining popularity and availability worldwide. Its consequential impact on human health is among the highest in terms of non-communicable diseases. Therefore, this study aimed to investigate the level of understanding of fast food consumption among adults in Phnom Penh, the capital city of Cambodia. Methods: A cross-sectional analytical study aimed to investigate the level of understanding of factors associated with fast food consumption, among adults in Phnom Penh. Multi-stage random sampling was used to select 749 respondents from 12 communes of five districts in Phnom Penh. A structured questionnaire was used to assess the level of understanding of fast food consumption, and associated factors. Data were analyzed using descriptive statistics, together with bivariate and multivariable logistic regression. Crude odds ratios (CORs) and adjusted odds ratios (AORs) with 95% confident intervals (CI) were calculated to show the strength of associations. Results: The understanding of factors associated with fast food consumption was poor in 52.07% (95% CI: 48.48-55.66), fair in 22.70% (95% CI: 19.69-25.70) and good in 25.23% (95% CI: 22.12-28.35) of those surveyed. After adjusting for other covariates, unsatisfactory levels of knowledge around fast food consumption were found to be significantly associated with not taking regular exercise (AOR = 1.53; 95% CI: 1.15-2.25; p<0.001) and sleeping less than eight hours per night (AOR = 1.64; 95% CI: 1.09-2.12; p=0.014). Conclusion: Health promotion and disease prevention should be conducted among at-risk populations in order to raise the level of understanding of factors around fast food consumption.", "keywords": [ "Fast food consumption", "level of knowledge", "lifestyle", "Cambodia" ], "content": "Introduction\n\nFast food is rapidly gaining popularity the world over. In past decades, both the demand for and availability of fast food have been increasing worldwide1. Specifically, the net worth of the fast food industry increases measurably from day to day, and fast food menus are becoming more and more extensive2. Many individuals and families now routinely consume takeaway food at home, with 25% of all food expenditure being on takeaway food3. The popularity of fast-food restaurants is also on the rise. In 2001, the USA had approximately 220,000 fast food outlets, with the revenues from the sales reaching over $125 billion USD4. Globally, in 2013, the market share of fast food was approximately $447.1 billion USD, and increased by 4.4% between 2013 and 2019 to reach $617.6 billion USD5. The story is no different for at least five of the 10 Association of Southeast Asian Nations (ASEAN) countries. The fast food industry in Thailand is worth approximately $700 million USD, and between $300 million and $500 million in each of Cambodia, Vietnam, Laos and Myanmar6. With over 31,000 restaurants worldwide, McDonald’s has branches in 126 countries on six continents7. Furthermore, many other fast food chains and independent restaurants are in existence all over the world. Burger King operates more than 11,100 restaurants in 65 countries8, and KFC operates across 25 countries.\n\nAn individual who consumes fast food once week is at a 20% higher risk of developing coronary heart disease compared to an individual who never eats fast food9. This increases to a 50% higher risk if that individual consumes fast food two or three times per week, and increases to an 80% chance of developing coronary heart disease if an individual consumes fast food more than three times per week. Consuming fast food more than twice a week is associated with a 27% risk of developing type 2 diabetes9. Consequently, the World Health Organization recommends keeping fast food consumption to a minimum10. In the USA, 80% of the population consume fast food, compared to 67%, 63%, and 56% in New Zealand, Australia and the UK, respectively11. Fast food consumption among adolescents varies greatly worldwide. For example, 13% of Mexican adolescents consume fast food12, whereas the rate in Brazilian, Canadian and Australian adolescents is 20%13, 30%14 and 89.9%15, respectively. Fast food consumption also varies greatly across ASEAN countries, such as Malaysia (59%), Thailand (58%), Vietnam (53.6%), and Indonesia (9.5%)16,17.\n\nCambodia currently enjoys a vigorous rate of economic growth. This growth is thought of as stable, with rates of 7.1% in 2014, 7% in 2015, and similarly from 2016 onwards. Much of this growth has been attributed to the garment manufacture sector, together with the service and construction industries. The stability of growth is thought to be because of recouping costs from local demand and high exports of garments manufactured in the country, balancing out the stagnation experienced in the agriculture industry and the less dramatic growth in tourism. Poverty in Cambodia is on the decrease. In 2012, the poverty rate was 7.7%, representing approximately 3 million people below the poverty line, and over 8.1 million people being near poverty. Approximately 90% of those living in poverty reside in the countryside. In 2009, the World Bank estimated that the level of poverty in Cambodia had reduced sufficiently so as to reach the Millennium Development Goal18.\n\nDue to the increases in population and economic power, along with significant growth of the fast-food sector, in an analysis of the causes of death in Cambodia, cardiovascular diseases now represent the leading cause of death of all non-communicable diseases. Non-communicable diseases account for 52% of the total number of deaths in Cambodia. Of these, cardiovascular diseases represent 24%, cancers 13%, chronic respiratory diseases 4%, diabetes 2% and the final 9% by other non-communicable diseases. Adult risk factors for cardiovascular disease include raised blood pressure (19.4% in males and 14.9% in females)19, and 5.4% of the urban population were reported as living with diabetes in 201020. Hence, the rapid increase in popularity of fast food constitutes a significant public health concern, requiring investigation. Little is known about the understanding of factors surrounding fast food consumption among adults in Phnom Penh, Cambodia.\n\n\nMethods\n\nThe study design was reviewed and approved by the human research ethics committee at the Khon Kaen University (Reference No. HE582071). All participants gave written informed consent prior to the commencement of the questionnaire interview.\n\nA cross sectional study was conducted using a structured questionnaire administered by in-person interview. Interviews were conducted between March and July, 2018. The sample comprised 749 individual residents of Phnom Penh, selected using a multi-stage random sampling technique.\n\nMulti-stage random sampling was used to select the sample of 749 study participants. Five districts were randomly selected using a lottery method by which the researcher gives each district a number, drawing numbers from the box randomly to choose the five samples from the total of 12 districts within Phnom Penh. Then, either two or three communes were randomly selected from each selected district, to a total of 12 communes. The sample size for this study was calculated using an established formula21 and the estimated sample size was 749. A systematic random sampling procedure was used to select 749 households from a list of all households in a given commune, which were provided by each commune chief, out of a total 44,436 households. All households in the 12 communes were labelled with a number and every 10 households were selected until the target was fulfilled with 749 households. Finally, one household member aged 18–59 was randomly selected from each household (unless the household had only one member).\n\nThe inclusion criteria for the study were that participants were between 18 and 59 years of age, and that they were able to understand the questionnaire. Residents with any serious health problems at time of data collection (causing them to be bedbound), diarrhea (defecating more than three times per day), pregnancy, mental illness, deformity or lower limb amputation were excluded from participating in the study.\n\nThe questionnaire was designed specifically to answer the research question, and was informed by literature review. A pilot study (n = 30) was conducted to assess content validity. One district in Phnom Penh municipality was selected for the pilot study that was different from the five districts used for the main study. Some items that were found to be difficult to understand were changed after pilot testing. Following the results of the pilot study, the questionnaire content was approved by five experts in the field of nutrition. The experts were sent the questionnaires and had three weeks to check its content. Experts recommended few changes to the questionnaire following their assessment. Internal consistency was assessed using Cronbach’s alpha, and found to have a reliability coefficient of 0.857.\n\nThe questionnaire had three parts (see Extended data)22: part 1 covered demographic and socioeconomic characteristics (gender, age, marital status, educational attainment, occupation, number of family members, personal monthly income); part 2 covered lifestyle and behavioral factors (tobacco use, alcohol consumption, food habits, soft drink consumption, physical activity and sedentary behavior); and part 3 covered the understanding of factors associated with fast food consumption. Prior to participant interviews, the questionnaire was translated into Khmer by an independent translator\n\nA field team of three research assistants underwent one-day training to standardize their knowledge of the study objectives and administration of the questionnaire. Researchers used the face-to-face questionnaire to interview the participants at their home in their free time after work. Data was collected after informing participants about the objectives of the study, benefits and assuring confidentiality to those who was eligible for this study. Some participants refused to participate, the reasons being that they were tired after work, or that they did not want to provide information (due to perceptions of privacy/security). The raw data of the 749 respondents were recorded into MS-Excel for database management before in-depth analysis.\n\nKnowledge of fast food consumption was the dependent variable used in the study, and took levels poor, fair and good. Participants were asked seven questions relating to fast food to determine their knowledge, answering true or false to each statement. The correct answer was given a score of 1, and the incorrect answer given a score of 0. The minimum total score was 0, and the maximum was 7. Responses were categorized using Bloom’s cut off with poor knowledge being assigned to scores <60%, fair knowledge for scores between 60 and 79%, and good knowledge for scores ≥80%.\n\nAge, household income and expenditure, and the number of individuals in the household were coded as continuous variables. Gender (male or female), marital status (single, married or divorced/widowed/separated), level of education (no formal education, primary, secondary, high school, associated degree, bachelor’s degree, master’s degree, or doctoral degree and higher), occupation (farmer, unemployed, non-governmental organization employee, self-employed, student, government officer, home maker, unskilled worker, or other), people they were cohabiting with (spouse, parents, relatives, none, friend, or other), smoking (yes, no), alcohol consumption (yes, no), vegetable intake (standard portion size of rice spoons/day), fruit intake (portions/day), number of episodes of exercise per week, hours of screen time per day, and hours of sleep per night were coded as categorical variables.\n\nData were imported to Stata version 13 (College Station, Texas, USA) for analysis. Continuous and categorical data were inspected using descriptive statistics to determine the frequencies and percentages (categorical variables) and means, medians and standard deviations (continuous variables) of each socio-economic, demographic and lifestyle characteristic collected. Bivariate logistic regression was used to estimate the association between each socioeconomic and lifestyle factor and the outcome measure of understanding of fast food consumption. Crude odds ratios (CORs) were computed using 95% confidence intervals (CIs) and variables with statistical significance of p<0.25 were entered into the final model. In the final model, multivariate logistic regression was used to estimate the association between socioeconomic lifestyle factors and understanding of fast food consumption. Adjusted odds ratios (AORs) were then computed using 95% CIs. Significance was considered at p<0.05.\n\n\nResults\n\nA total of 749 participants from five districts and 12 communes were recruited for this study. The socio-demographic characteristics of respondents are summarized in Table 1. Participants were 50.20 % female with an average age of 32 ± 11 years23. The relationship status of participants was 53.94% married, 43.52% single, and 2.54% divorced/widowed/separated. Regarding educational attainment, 31.91% of participants had completed high school, 26.44% had completed Bachelor’s degrees, whereas 15.22% had only completed primary school. The most common occupations were private company workers (28.97%), self-employed (21.36 %) and students (20.16%), with only 0.67% reporting to be farmers and 0.93% unemployed. Nearly half of the sample lived with their spouse (47.93%), a quarter lived with parents (25.37%), 12.95% lived with other relatives, and only 13.89% of participants lived with fewer than three family members. Monthly earnings ranged from $40 to $5,100 US with a median of $300 ± 687 USD. Monthly expenditure ranged from $20 to $3,750 USD with median $200 ± 394 USD.\n\nSD, standard deviation; USD, United States Dollars.\n\nOnly 10.41% of participants smoked, and 54.34% had consumed alcohol in the past 12 months. Most participants (65.95%) ate at least 6 portions of vegetables per day, 21.76 % ate between 4 and 6 spoonfuls of vegetables per day, and only 12.28% ate less than 4 spoonfuls. Similarly with fruit, only 44.33% of respondents ate fewer than three portions of fruit portions per day, 20.03% ate between three and five portions per day, and 35.65% ate at least five portions. 54.21% of respondents consumed fewer than six spoonfuls of meat per day, and 45.79% consumed at least six spoonfuls of meat per day. With respect to physical exercise and sedentary behavior, 50.07% of the respondents exercised at least weekly. In addition, 73.56% of respondents had at least two hours of screen time a day and 56.21% of the sample slept more than eight hours each night (Table 2).\n\nSD, standard deviation.\n\nIn their responses to seven questions related to knowledge about fast food consumption, there were four questions which respondents answered correctly at least half the time, and the remaining three questions were answered correctly less than half the time. For example, the question “Milk tea such as pearl tea is good for health because it contains both carbohydrate and dairy milk” was answered correctly as false 38.72% of the time; “Cola drinks containing high carbohydrates could help digestion” was answered correctly as false 45.66% of the time, and “Fast food such as hamburgers and pizza contain fiber which is good for your digestive system” was answered correctly as false 49.93% of the time. Descriptive statistics for all questions are given in Table 3.\n\nBased on respondents’ answers to the questions in Table 3, a level of knowledge was assigned as one of three levels; good, fair, and poor, according to Bloom’s cut-off point. The range of possible scores was from 0 to 7, and the mean sample score was 4.20 (±1.70). Boundary criteria used were a score of less than 60% signified poor knowledge, a score of between 60 and 79% signified fair knowledge, and a score of 80% or higher signified good knowledge. In the sample of respondents, 52.07% had poor knowledge, 22.70% had fair knowledge, and 25.23% had good knowledge (Table 4).\n\nSD, standard deviation; CI, confidence interval.\n\nKnowledge levels poor and fair were deemed unsatisfactory for the purpose of performing logistic regression to determine the predictive factors of knowledge level. The overall prevalence of poor and fair knowledge of fast food consumption was 74.76% (95%CI: 71.64-77.88%). In a simple bivariate logistic regression, the factors gender, marital status, weekly exercise and nightly hours of sleep were found to be associated with having poor or fair knowledge of fast food consumption (p<0.25) and were taken forward as factors in the final model (Table 5).\n\nOR, odds ratio; CI, confidence interval.\n\nThe final model using a multiple logistic regression, found only two factors to be associated with poor and fair knowledge of fast food consumption. These were weekly exercise and nightly hours of sleep. Those who did not exercise at least once a week (AOR: 1.53, 95%CI: 1.15-2.25; p <0.001) and those who slept for less than eight hours per night (AOR: 1.45, 95%CI: 1.09-2.12; p =0.014) were more likely to have poor or fair (“unsatisfactory”) knowledge of fast food consumption (Table 6).\n\nOR, odds ratio; CI, confidence interval.\n\n\nDiscussion\n\nIn a random sample of 749 participants across Phnom Penh, the prevalence of unsatisfactory knowledge of fast food consumption was 74.76% (95% CI: 71.64%-77.88%). 52.07% of the sample (95% CI: 48.48%-55.66%) had poor knowledge, 22.70% (95% CI: 19.69%-25.70%) had fair knowledge and 25.54% had good knowledge (95% CI: 25.23%-28.35%). This result is contradictory to the findings of a previous study on the knowledge of fast food consumption in Jakarta, Indonesia24, which showed 73.2% of the sample to have a good level of knowledge, 25.3% moderate and only 1.6% low. The two samples represent two different geographical areas and target populations; and further used different methodologies (i.e. different thresholds for discrete knowledge levels). However, the findings of the present study are in agreement with those of a study conducted in Karnataka, India, which reported 31.9% of respondents as having inadequate levels of knowledge around fast food, 41.9% moderate knowledge and only 26.2% to be adequate25.\n\nIn the present study, the two factors found to be associated with unsatisfactory levels of knowledge about fast food consumption were not taking weekly exercise and sleeping less than eight hours a night. Individuals who did not do any exercise were 1.53 times more likely to have unsatisfactory knowledge of fast food consumption compared to those who did (95% CI: 1.15-2.25, p<0.001). It is likely that having a poor general understanding of health and wellbeing would be associated with low levels of exercise, and with having the wrong perceptions and assumptions about fast food consumption26. Finally, those who slept less than eight hours per night were 1.64 times more likely to have unsatisfactory knowledge of fast food compared to those who slept at least eight hours per night (95% CI: 1.09-2.12, p=0.014). This further reinforces the argument that general understanding of health and wellbeing reflects strongly on individual factors. It is likely that for those who did not understand the impact of fast food on a person’s health also did not understand the risk factors of getting insufficient amounts of sleep27.\n\nFirstly, the participants in this investigation were from a capital city and these results may not be representative of the whole population of Cambodia; if other provinces or cities were included, the results might be different. Secondly, as the current study was a cross-sectional analytical study, we could not infer causality. Lastly, all information collected in the study was based on self-reporting; there might be methodological bias due to under- and over-reporting of certain behaviors.\n\n\nConclusion\n\nThe prevalence of poor and fair knowledge of fast food consumption in Cambodia has become a recent concern for public health. This study found that insufficient levels of exercise and not getting enough sleep were both predictors of inadequate understanding around the impact of fast food on health, in a sample of 749 individuals in Phnom Penh. Based on these research findings, we recommend taking measures to improve public understanding of the impact of fast food consumption on health. Cooperation from all stakeholders within each Cambodian government ministry is needed to promote and raise awareness of fast food consumption within the population.\n\n\nData availability\n\nFigshare: Socio-demographic and Lifestyle factors associated with Understanding Fast Food Consumption among Adults in Cambodia. https://doi.org/10.6084/m9.figshare.12894740.v123\n\nThis project contains the following underlying data:\n\n- Knowledge of fast food consumption.xlsx\n\n- Code Book (Knowledge of fast food consumption).docx\n\nFigshare: Socio-demographic and Lifestyle factors associated with Understanding Fast Food Consumption among Adults in Cambodia. https://doi.org/10.6084/m9.figshare.12894644.v122\n\nThis project contains the following extended data:\n\n- Questionnaire in English.docx\n\n- Questionnaire in khmer.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBowman SA, Vinyard BT: Fast food consumption of U.S. adults: impact on energy and nutrient intakes and overweight status. J Am Coll Nutr. 2004; 23(2): 163–8. PubMed Abstract \| Publisher Full Text\n\nAkroush MN, Abu-ElSamen AA, Samawi GA, et al.: Internal marketing and service quality in restaurants. Marketing Intelligence & Planning. 2013; 31(4): 304–36. Publisher Full Text\n\nCohen DA, Bhatia R: Nutrition Standards for Away-from-home Foods in the United States. Obes Rev. 2012; 13(7): 618–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaeratakul S, Ferdinand DP, Champagne CM, et al.: Fast-food consumption among US adults and children: Dietary and nutrient intake profile. J Am Diet Assoc. 2003; 103(10): 1332–8. PubMed Abstract \| Publisher Full Text\n\nThird Party Logistics: Global Fast Food Market To Grow Through 2019 As Developing Markets Urbanize. 2015. Reference Source\n\nCasico: Market Analysis for fast food chain in Cambodia, Vietman, Loa and Myanmar. 2014. Reference Source\n\nFast Food’s Founding Father: Fast Food Factory. Reference Source\n\nBurgerking: Take a break from boredom. Reference Source\n\nOdegaard AO, Koh WP, Yuan JM, et al.: Western-style fast food intake and cardiometabolic risk in an Eastern country. Circulation. 2012; 126(2): 182–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Diet, nutrition and the prevention of chronic diseases. 2003. [cited 2016 August 23]. Reference Source\n\nNezakati H, Kuan YL, Asgari O: Factors Influencing Customer Loyalty Towards Fast Food Restaurant. International Research Symposium in Service Management. 2011; 10: 12. Reference Source\n\nOrtiz-Hernandez L, Gomez-Tello BL: Food consumption in Mexican adolescents. Rev Panam Salud Publica. 2008; 24(2): 127–35. PubMed Abstract \| Publisher Full Text\n\nVargas IC, Sichieri R, Sandre-Pereira G, et al.: Evaluation of an obesity prevention program in adolescents of public schools. Rev Saude Publica. 2011; 45(1): 59–68. PubMed Abstract \| Publisher Full Text\n\nGarriguet D: Canadians' eating habits. Health Rep. 2007; 18(2): 17–32. PubMed Abstract\n\nAustralian Government DoHaA: Australian national children’s nutrition and physical activity survey. Australia: Australian Government, Department of Health and Ageing. 2007. Reference Source\n\nresearch. WSm: Comparative report on Fast Food Study in Thailand, Indonesia and Vietnam in 2015. 2015. Reference Source\n\nAbbas MY, Bajunid AFI, Mohamed Thani SK, et al.: ASLI QoL2014 (Annual Serial Landmark International Conference on Quality of Life) / AQoL 2014 Istanbul (ABRA International Conference on Quality of Life), Istanbul Technical University, Istanbul, Turkey, 26 - 28 December 2014Trend on Fast Food Consumption in Relation to Obesity among Selangor Urban Community. Procedia - Social and Behavioral Sciences. 2015; 202: 1–530. Reference Source\n\nThe World Bank: Working for a World Free of Poverty. 2016. [cited 2016 August 24]. Reference Source\n\nAn Y, Yi S, Fitzpatrick A, et al.: Appropriate body mass index and waist circumference cutoff for overweight and central obesity among adults in Cambodia. PLoS One. 2013; 8(10): e77897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCambodia NCDs. 2014. Reference Source\n\nHsieh FY, Bloch DA, Larsen MD: A simple method of sample size calculation for linear and logistic regression. Stat Med. 1998; 17(14): 1623–34. PubMed Abstract \| Publisher Full Text\n\nSim S, Pall C, Dewey RS, et al.: Socio-demographic and Lifestyle factors associated with Understanding Fast Food Consumption among Adults in Cambodia. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12894644.v1\n\nSim S, Pall C, Pisey V: Socio-demographic and Lifestyle factors associated with Understanding Fast Food Consumption among Adults in Cambodia. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12894740.v1\n\nFatikhani DA, Setiawan A: The relationship between the level of knowledge regarding fast food and the dietary habits among adolescents in Jakarta, Indonesia. Enferm Clin. 2019; 29 Suppl 2: 172–175. PubMed Abstract \| Publisher Full Text\n\nKhongrangjem T, Dsouza SM, Prabhu P, et al.: A study to assess the knowledge and practice of fast food consumption among Pre-University students in Udupi Taluk, Karnataka, India. Clin Epidemiol Glob Health. 2018; 6(4): 172–5. Publisher Full Text\n\nLuo X, Xu X, Chen H, et al.: Food safety related knowledge, attitudes, and practices (KAP) among the students from nursing, education and medical college in Chongqing, China. Food Control. 2019; 95: 181–188. Publisher Full Text\n\nBou-Mitri C, Mahmoud D, El Gerges N, et al.: Food safety knowledge, attitudes and practices of food handlers in lebanese hospitals: A cross-sectional study. Food Control. 2018; 94: 78–84. Publisher Full Text" }	[ { "id": "78503", "date": "12 Feb 2021", "name": "Magdalena Czlapka-Matyasik", "expertise": [ "Reviewer Expertise Public health nutrition" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI commend the authors to contribute to this body of literature regarding the socio-demographic and lifestyle factors associated with understanding fast food consumption in Cambodia adults.\nThe paper brings quantified information about the factors influenced by understanding fast food consumption. The authors revealed that poor and fair knowledge, insufficient exercise levels, and not getting enough sleep were predictors of inadequate understanding of the impact of fast food on health. Such conclusions do not bring entirely new knowledge to the literature, on this matter. Across the whole world population, the problems related to fast food consumption have been discussed. Nevertheless, I consider the work to be original, well designed and contribute knowledge to this field of public health research.\nMy suggestions concern:\nIn the introduction, the authors indicate the system of fast-food restaurants; it would be more attractive to explain, how it was developed in Cambodia directly?\n\nWhat would be very interesting is information concerning the real take away or fast food intake in those groups. It must or might be in direct relation to this matter?\n\nMy main concern about the validation of the \"Level of knowledge of fast food consumption\": Could the authors explain the procedure? How were the questions selected and validated?\n\nI regret that the authors discussed the interesting results in such a concise way.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "121244", "date": "02 Mar 2022", "name": "Wanshui Yang", "expertise": [ "Reviewer Expertise Nutritional epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript investigated the level of understanding of fast-food consumption among adults in Cambodia. The authors found that unsatisfactory levels of knowledge around fast food consumption were significantly associated with not taking regular exercise and sleeping less than eight hours per night. The results are interesting, but I have several comments.\nThe authors should introduce the recent development of fast-food sector in Cambodia in the introduction part.\n\nIs there an analysis of fast-food intake among these participants?\n\nInterestingly, the authors found that not taking regular exercise and sleeping less than eight hours per night were associated with unsatisfactory levels of knowledge around fast food consumption. However, they were not well explained in the discussion part. In other words, the discussion part is a little too concise.\n\nIn addition, the education levels were not associated with the knowledge of fast-food consumption in the present study. I would like authors to discuss it and, at least, mention its possible causes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	2	https://f1000research.com/articles/9-1121
https://f1000research.com/articles/9-1078/v1	01 Sep 20	{ "type": "Review", "title": "Immunopathology of galectin-3: an increasingly promising target in COVID-19", "authors": [ "John L. Caniglia", "Swapna Asuthkar", "Andrew J. Tsung", "Maheedhara R. Guda", "Kiran K. Velpula", "John L. Caniglia", "Swapna Asuthkar", "Andrew J. Tsung", "Maheedhara R. Guda" ], "abstract": "The pandemic brought on by the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has become a global health crisis, with over 22 million confirmed cases and 777,000 fatalities due to coronavirus disease 2019 (COVID-19) reported worldwide. The major cause of fatality in infected patients, now referred to as the “Cytokine Storm Syndrome” (CSS), is a direct result of aberrant immune activation following SARS-CoV2 infection and results in excess release of inflammatory cytokines, such as interleukin (IL)-1, tumor necrosis factor α (TNF-α), and IL-6, by macrophages, monocytes, and dendritic cells. Single cell analysis has also shown significantly elevated levels of galectin 3 (Gal-3) in macrophages, monocytes, and dendritic cells in patients with severe COVID-19 as compared to mild disease. Inhibition of Gal-3 reduces the release of IL-1, IL-6, and TNF-α from macrophages in vitro, and as such may hold promise in reducing the incidence of CSS. In addition, Gal-3 inhibition shows promise in reducing transforming growth factor ß (TGF-ß) mediated pulmonary fibrosis, likely to be a major consequence in survivors of severe COVID-19. Finally, a key domain in the spike protein of SARS-CoV2 has been shown to bind N-acetylneuraminic acid (Neu5Ac), a process that may be essential to cell entry by the virus. This Neu5Ac-binding domain shares striking morphological, sequence, and functional similarities with human Gal-3. Here we provide an updated review of the literature linking Gal-3 to COVID-19 pathogenesis. Dually targeting galectins and the Neu5Ac-binding domain of SARS-CoV2 shows tentative promise in several stages of the disease: preventing viral entry, modulating the host immune response, and reducing the post-infectious incidence of pulmonary fibrosis.", "keywords": [ "COVID-19", "galectin", "cytokines", "ARDS", "fibrosis", "sialic acid", "galectin-3" ], "content": "Introduction\n\nGalectin 3 (Gal-3) is an animal lectin that exhibits pleiotropic effects throughout the body, including the modulation of apoptosis, cell migration and adhesion, angiogenesis, tumorigenesis, and post-injury remodeling (Chen & Kuo, 2016; Elola et al., 2018; Nangia-Makker et al., 2018). Recent discoveries have begun to shed light on its role in viral infections as well (Wang et al., 2019). In particular, Gal-3 has been shown during infection to induce a dysregulated pattern expression of pro-inflammatory cytokine expression via the JAK/STAT1, ERK, and AKT signaling pathways (Nita-Lazar et al., 2015). The cytokine profile observed includes tumor necrosis factor α (TNFα), interleukin (IL)-1β, and IL-6, among others (Nita-Lazar et al., 2015). Gal-3 is also a known agonist of toll like receptor 4 (TLR4) and nuclear factor kappa beta (NF-kB) dependent pathways, which are well characterized and potent inducers of inflammation during infection (Yip et al., 2017; Zhou et al., 2018). Patients suffering from severe coronavirus disease 2019 (COVID-19) show highly elevated levels of Gal-3, TNFα, IL-1β, and IL-6, as compared to those with moderate disease (De Biasi et al., 2020; Wang et al., 2020a). Inhibition of Gal-3 significantly reduces the levels of these cytokines, and so may show promise in reducing inflammatory sequelae associated with COVID-19 (De Biasi et al., 2020; Kalfaoglu et al., 2020; Liu et al., 2020).\n\nThe continued lack of an effective standard of care for treating patients with COVID-19 has brought on an urgent need to identify effective therapies. In a prior review article, we had discussed promising indications for Gal-3 targeted therapy in the treatment of COVID-19, with the goal of inspiring further research on the topic (Caniglia et al., 2020). In recent months, however, a substantial amount of new evidence has emerged that further links Gal-3 to severe COVID-19 infection. As such, the authors see a need to achieve two aims in this review: highlighting novel discoveries to expand upon previously discussed treatment indications, and to detail a further potential role for anti-galectin therapy in reducing post-infectious pulmonary fibrosis. This article may be particularly useful for immunologists studying COVID-19, as well as any researchers with a structural or functional focus on galectins.\n\n\nSARS-CoV2: host cell attachment and entry\n\nA critical step prior to viral infection is the entry of the virus into host cells, a process that in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is mediated by the S1 subunit of the spike protein (Blaas, 2016; Zhai et al., 2020). Within coronaviridae, it is commonplace to refer to the S1 protein as consisting of two distinct regions: the C-terminal domain (CTD) and N-terminal domain (NTD) (Li, 2016). In most cases, the CTD binds peptide receptors and the NTD binds sugar receptors (Li, 2016). The main entry mechanism of SARS-CoV2 has been shown to be via the CTD binding to angiotensin converting enzyme receptor 2 (ACE2) receptors (Wang et al., 2020b). Until recently, the role of the NTD has been largely overlooked. A study from Baker et al. has shown evidence that SARS-CoV2 also binds N-acetylneuraminic acid (Neu5Ac), with this interaction being mediated by the NTD of the S1 subunit (Baker et al., 2020). This is the first in vitro evidence of this occurring, although several prior bioinformatics and modeling studies have hypothesized that a Neu5Ac binding site exists, with one suggesting its affinity for Neu5Ac (0.88) is only slightly lower than that of influenza hemagglutinin (0.94) (Alban et al., 2020; Behloul et al., 2020; Fantini et al., 2020; Kim, 2020; Milanetti et al., 2020; Robson, 2020). Binding of sialic acids by the NTD is the main entry mechanism in several other coronaviruses known to infect humans, most notably members of the bovine coronavirus family (Li, 2015). Additionally, the closely related middle eastern respiratory syndrome coronavirus (MERS-CoV) has been shown to exhibit a dual attachment model similar to SARS-CoV2, where the CTD binds a peptide receptor and the NTD binds sialic acids (Li et al., 2017). Depletion of sialic acids with neuraminidase inhibitors prevented MERS-CoV infection of Calu-3 human airway cells, indicating that NTD-targeted therapies may be effective in preventing cell entry by coronaviruses possessing this function (Li et al., 2017). The dual mechanism by which SARS-CoV2 may enter host cells is seen in Figure 1.\n\nEvidence has shown that a pocket in the NTD of SARS-CoV2 is capable of binding N-acetylneuraminic acid (Neu5Ac). This strongly supports a dual attachment model for SARS-CoV2, where NTD-Neu5Ac interactions facilitate initial host cell recognition by the virus and stabilize its entry via ACE2 receptors\n\nThe binding of Neu5Ac may also explain the greater infectivity of SARS-CoV2 as compared to SARS-CoV (Alban et al., 2020). While the CTD of SARS-CoV2 has been shown to exhibit higher affinity for ACE2 receptors than that of SARS-CoV, this is likely insufficient to fully explain the marked disparity in transmissibility (Tai et al., 2020). The NTD of SARS-CoV2 has been rigorously analyzed and compared to both human galectins and the NTD of other coronaviruses (Behloul et al., 2020). Behloul et al. found that while SARS-CoV2 and SARS-CoV share 74.75% similarity in the CTD, they exhibit just 52.69% similarity in the NTD region (Behloul et al., 2020). This is particularly noteworthy when viewed together with the findings that despite SARS-CoV2 being able to bind Neu5Ac in vitro, the same domain on SARS-CoV did not exhibit this ability (Baker et al., 2020). Modeling studies comparing the NTD of SARS-CoV2 and SARS-CoV have led to the same conclusion (Behloul et al., 2020). The far greater abundance of Neu5Ac in the human body as compared to ACE2 receptors, particularly at common viral entry points such as the nasopharynx and oral mucosa, may explain the high transmissibility of SARS-CoV2 (Barnard et al., 2019).\n\nSeveral studies to date have referred to the “galectin fold” present on the NTD of coronaviruses (Behloul et al., 2020; Li, 2016; Li et al., 2017; Peng et al., 2011; Peng et al., 2012; Tortorici et al., 2019). The structures are so similar, in fact, that it is hypothesized that coronaviruses incorporated a host galectin gene into their genome (and then the NTD) at some point in their evolution (Li, 2015). Structural analysis comparing the SARS-CoV2 NTD to Gal-3 resulted in a Z-score of 6 (p < 0.00001), indicating a high degree of similarity between the structures (Behloul et al., 2020). In fact, human Gal-3 was shown to be equally similar to SARS-CoV2 NTD as the NTD of NL63-CoV and infectious bronchitis coronavirus, accounting for both sequence and structure (Behloul et al., 2020). Given the high degree of structural and promising sequence similarity (12%) of the NTD with Gal-3, it may be possible that existing Gal-3 inhibitors possess dual-binding capabilities (Behloul et al., 2020). Such a mechanism shows promise in reducing viral entry to host cells (Milanetti et al., 2020).\n\n\nGal-3 in severe infection: promoting immunologic sequelae of COVID-19\n\nThe major cause of death in patients infected with SARS-CoV and MERS-CoV infection was found to be the “Cytokine Storm Syndrome” (CSS), and this is likely to be the case in COVID-19 as well (Channappanavar & Perlman, 2017; Zhang et al., 2020). CSS develops due to hyper-activation of macrophages, monocytes, and dendritic cells, which are stimulated to release a variety of inflammatory mediators including IL-1, IL-6, and TNF-α (Zhang et al., 2020). This in turn leads to systemic organ dysfunction that may result in death (England et al., 2020). Notably, a study of nearly 4,000 patients has found the levels of IL-1, IL-6, and TNF-α to be significantly elevated in the sera of patients suffering from severe COVID-19 as compared to those with mild disease (Wang et al., 2020a). This data speaks to the urgency of identifying therapeutics to reduce the incidence of CSS (Wang et al., 2020a).\n\nThere is a plethora of evidence that makes Gal-3 a promising target to achieve this aim. First, the most concerning sequelae of CSS is evolution to acute respiratory distress syndrome (ARDS), a condition which often leads to respiratory failure despite proactive measures such as mechanical ventilation and intubation (Vabret et al., 2020). Elevated serum levels of Gal-3 are significantly associated with worse outcomes and lower survival in patients suffering from ARDS (Xu et al., 2017). Additionally, significantly elevated levels of Gal-3 have been shown in the serum of patients suffering from severe COVID-19 as compared to those with mild disease (De Biasi et al., 2020). On a cellular level, Gal-3 was shown to be most elevated in immune cells during severe COVID-19 (Kalfaoglu et al., 2020) The highest levels of Gal-3 were seen in infected macrophages, monocytes, and dendritic cells, the very cells responsible for initiating CSS (Liu et al., 2020). A pathway through which Gal-3 may contribute to the development of CSS is detailed in Figure 2.\n\nDuring severe SARS-CoV2 infection, increased concentrations of Gal-3 are observed in macrophages, monocytes, and dendritic cells. When secreted, Gal-3 can then agonize TLR4 receptors on their surfaces and induce the release of inflammatory cytokines such as IL-1, IL-6, and TNF-α. This process also results in the secretion of further Gal-3, resulting in a positive feedback loop that may contribute to the development of CSS.\n\nSeveral studies to date have shown the effects of anti-Gal-3 therapy on cytokine release. Significant reductions in IL-1, IL-6, and TNF-α secretion by dendritic cells has been observed upon silencing of Gal-3 (Chen et al., 2015). In models of traumatic brain injury and spinal cord injury, treatment with anti-Gal-3 antibodies and the Gal-3 inhibitor GB1107, respectively, both led to significant reductions in the systemic levels of IL-1, IL-6, and TNF-α (Ren et al., 2019; Yip et al., 2017). Lastly, in mice infected with H5N1 influenza virus, Gal-3 K/O led to a significant reduction of IL-1ß secretion by macrophages and improved survival rate as compared to controls (Chen et al., 2018). These findings are due to Gal-3’s known role as an alarmin of the innate immune system, triggering the release of inflammatory cytokine, such as TNF-α and IL-6 from monocyte-derived cells during infection or other inflammatory insults (Mishra et al., 2013; Yip et al., 2017). The enhanced secretion of cytokines likely occurs through TLR4/NF-kB mediated pathways (Yip et al., 2017; Zhou et al., 2018). With all this information taken together, Gal-3 inhibition shows promise in reducing the incidence and symptoms of CSS.\n\n\nGal-3 post-infection: pathologic fibrosis\n\nIt is well known that persistent viral infections are a risk factor for the subsequent development of pulmonary fibrosis (Sheng et al., 2020). A study found that tests for SARS-CoV2 RNA in the serum of infected individuals did not become negative until a median of 24 days post-symptom onset, with some individuals remaining positive even greater than a month from the beginning of symptoms (Gombar et al., 2020). This indicates that for some, COVID-19 infection may run a particularly long course. Findings such as this have led to the question of whether or not anti-fibrotic therapy would be beneficial for such patients (George et al:, 2020).\n\nIn SARS-CoV infection, particularly in patients who suffered from ARDS, marked pulmonary fibrosis was found in a cohort of patients following prolonged infection (Ye et al., 2007). Though long term outcomes remain to be seen, lung tissue in the acute phase of COVID-19 shows similar changes (Xu et al., 2020a). Following a 24 hour incubation of SARS-CoV2, human airway cells showed upregulation of ACE2, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), fibronectin (FN), and transforming growth factor ß (TGF-ß), a molecular signature highly similar to that of patients with diagnosed pulmonary fibrosis (Xu et al., 2020a). It is believed that a large number of COVID-19 patients will go on to develop pulmonary fibrosis, and that these changes are mediated by a number of cytokines including TGF- ß, IL-1, IL-6, and TNF-α (Delpino & Quarleri, 2020).\n\nThe role of Gal-3 as a mediator of lung fibrosis has long been studied since the discovery that its levels are elevated in alveolar macrophages following lung injury (Kasper & Hughes, 1996; Nishi et al., 2007). Higher levels of Gal-3 have now been extensively associated with the development of restrictive lung diseases (Ho et al., 2016). Following cellular stress, the secretion of Gal-3 by macrophages upregulates TGF-ß receptors on fibroblasts and myofibroblasts (Henderson et al., 2008). This in turn activates these cells, initiating the formation of granulation tissue (via collagen deposition) that is eventually remodeled to a fibrous scar (Henderson et al., 2008; Mackinnon et al., 2012). This Gal-3 mediated pathway is widespread throughout the body and fundamental to the development of fibrotic change in the liver, kidneys, and heart as well (Hara et al., 2020). Gal-3 mediated fibrosis often has deleterious effects; for example, pathologic scar formation is the likely explanation for serum Gal-3’s utility as an independent predictor of mortality and heart failure post-myocardial infarction (Asleh et al., 2019). The mechanism by which Gal-3 may contribute to post-infectious pulmonary fibrosis in COVID-19 patients can be seen in Figure 3.\n\nDuring SARS-CoV2 infection, transcriptional upregulation of VEGF, TGF-ß, and fibronectin (FN) is seen in the pulmonary epithelium, creating a pro-fibrotic microenvironment. Secretion of Gal-3 by macrophages contributes to fibrosis by increasing the expression of TGF-ß receptors on the surface of fibroblasts. The fibroblasts and myofibroblasts are then activated by TGF-ß mediated signaling, stimulating the deposition of extracellular matrix and collagen that leads to fibrotic damage. Cytokines induced by Gal-3 expression such as IL-1, IL-6, and TNF-α further accelerate this process.\n\nGal-3 inhibitors show promise in limiting fibrotic change following lung injury. In a model of adenovirus induced lung injury, Gal-3 K/O mice showed significant reductions in lung fibrosis and ß-catenin activation, indicating the beneficial effects were mediated via interruption of TGF-ß signaling (Mackinnon et al., 2012). Treatment with the drug TD139 showed significant reductions in these parameters as well following bleomycin-induced pulmonary fibrosis (Mackinnon et al., 2012). This drug (now referred to as GB0139) was well tolerated in phase I/IIa trials in the treatment of idiopathic pulmonary fibrosis (IPF) and is now in phase IIb trials (Saito et al., 2019). An additional indication for this drug may be in reducing the post-viral development of pulmonary fibrosis (Mackinnon et al., 2012). The drug TD139 has recently begun phase II trials for the treatment of COVID-19, the first clinical trial of a galectin inhibitor in COVID-19 to date.\n\n\nConclusions and future directions\n\nIn summary, Gal-3 is a lectin that exhibits a pleiotropic role in mediating the acute and chronic consequences of infection and inflammation. Multiple studies have shown Gal-3 to be highly upregulated in patients suffering from severe COVID-19 (De Biasi et al., 2020; Kalfaoglu et al., 2020; Liu et al., 2020). On a cellular level, Gal-3 is most highly expressed in monocytes, macrophages, and dendritic cells during severe COVID-19 infection (Liu et al., 2020). CSS complicated by the development of ARDS is the major cause of fatality in COVID-19 patients (Xu et al., 2020b; Zhai et al., 2020; Zhang et al., 2020). This process is chiefly mediated by the release of IL-1, IL-6, and TNF-α from macrophages, monocytes, and dendritic cells (Zhang et al., 2020). Gal-3 inhibition has been shown to reduce the release of these cytokines from immune cells (Chen et al., 2015; Ren et al., 2019; Yip et al., 2017). Additionally, high Gal-3 is directly associated with worse outcomes and lower survival in ARDS patients (Xu et al., 2017).\n\nA key domain in the spike protein exhibits a high degree of morphological and sequence similarity to human Gal-3 (Behloul et al., 2020). This NTD has been shown to bind Neu5Ac in vitro, an interaction that likely explains the high infectivity of SARS-CoV2 and may be essential for cell entry (Alban et al., 2020; Barnard et al., 2019; Baker et al., 2020). Inhibitors of Gal-3 that target regions of structural overlap with the NTD may possess dual binding capabilities, exhibiting a novel mechanism by which to inhibit viral entry (Milanetti et al., 2020).\n\nLastly, pulmonary fibrosis has been observed following SARS-CoV infection and is likely to be a major complication in survivors of COVID-19 that is cytokine-mediated (Delpino & Quarleri, 2020; Xu et al., 2020b; Ye et al., 2007). Among other mediators, elevated levels of TGF-ß have been observed following SARS-CoV2 infection (Xu et al., 2020a). Gal-3 secreted by macrophages during injury promotes the upregulation of TGF-ß receptors, leading to fibroblast activation and collagen deposition (Delpino & Quarleri, 2020). Gal-3 inhibition has been shown to reduce adenovirus-induced lung fibrosis, and an inhibitor is currently in Phase IIb clinical trials for IPF treatment (Mackinnon et al., 2012; Saito et al., 2019). The indications for targeting Gal-3 in the treatment of COVID-19 are widespread. Processes directly mediated or affected by Gal-3 have been shown to be deleterious in several stages of the disease process. As such, Gal-3 represents a highly promising target for COVID-19 treatment that should urgently be investigated.\n\n\nLiterature search methodology\n\nThis review consists of original studies that provided information about SARS-CoV2, Gal-3, or Gal-3 inhibitors. Compiled results from both in vivo, in vitro, and clinical studies were used for analysis. Studies with only an abstract or no full-text available were excluded from the review.\n\nTo retrieve primary literature, electronic searches were performed on PubMed and Google Scholar. A list of search terms can be seen in Table 1.\n\nTo minimize the risk of error, all authors involved assessed the cited studies for quality. To discuss important claims in the article, including that SARS-CoV2 binds sialic acids with the S1-NTD, that Gal-3 is upregulated in human immune cells, and Gal-3 inhibitors’ ability to reduce fibrosis, multiple sources were included. Additionally, the use of open-ended searches ensured that an accurate profile of results was obtained on the topics discussed.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nThe authors thank Mark Linder Walk for the Mind, Illinois Neurological Institute, OSF foundation, Peoria, IL, and KB Strong Foundation, Washington, IL for their support. The authors thank Erika Sung for help in the formatting the manuscript.\n\n\nReferences\n\nAlban TJ, Bayik D, Otvos B, et al.: Glioblastoma Myeloid-Derived Suppressor Cell Subsets Express Differential Macrophage Migration Inhibitory Factor Receptor Profiles That Can Be Targeted to Reduce Immune Suppression. Front Immunol. 2020; 11: 1191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsleh R, Enriquez-Sarano M, Jaffe AS, et al.: Galectin-3 Levels and Outcomes After Myocardial Infarction: A Population-Based Study. J Am Coll Cardiol. 2019; 73(18): 2286–2295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker AN, Richards SJ, Guy CS, et al.: The SARS-COV-2 Spike Protein Binds Sialic Acids, and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device. ChemRxiv. Preprint. 2020. Publisher Full Text\n\nBarnard KN, Wasik BR, LaClair JR, et al.: Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses. mBio. 2019; 10(6): e02490–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehloul N, Baha S, Shi R, et al.: Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein. Virus Res. 2020; 286: 198058. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaas D: Viral entry pathways: the example of common cold viruses. Wien Med Wochenschr. 2016; 166(7–8): 211–226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaniglia JL, Guda MR, Asuthkar S, et al.: A potential role for Galectin-3 inhibitors in the treatment of COVID-19. PeerJ. 2020; 8: e9392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChannappanavar R, Perlman S: Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. Semin Immunopathol. 2017; 39(5): 529–539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen SC, Kuo PL: The Role of Galectin-3 in the Kidneys. Int J Mol Sci. 2016; 17(4): 565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen SS, Sun LW, Brickner H, et al.: Downregulating galectin-3 inhibits proinflammatory cytokine production by human monocyte-derived dendritic cells via RNA interference. Cell Immunol. 2015; 294(1): 44–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen YJ, Wang SF, Weng IC, et al.: Galectin-3 Enhances Avian H5N1 Influenza A Virus-Induced Pulmonary Inflammation by Promoting NLRP3 Inflammasome Activation. Am J Pathol. 2018; 188(4): 1031–1042. PubMed Abstract \| Publisher Full Text\n\nDe Biasi S, Meschiari M, Gibellini L, et al.: Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia. Nat Commun. 2020; 11(1): 3434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelpino MV, Quarleri J: SARS-CoV-2 Pathogenesis: Imbalance in the Renin-Angiotensin System Favors Lung Fibrosis. Front Cell Infect Microbiol. 2020; 10: 340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElola MT, Ferragut F, Mendez-Huergo SP, et al.: Galectins: Multitask signaling molecules linking fibroblast, endothelial and immune cell programs in the tumor microenvironment. Cell Immunol. 2018; 333: 34–45. PubMed Abstract \| Publisher Full Text\n\nEngland JT, Abdulla A, Biggs CM, et al.: Weathering the COVID-19 storm: Lessons from hematologic cytokine syndromes. Blood Rev. 2020; 100707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFantini J, Di Scala C, Chahinian H, et al.: Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int J Antimicrob Agents. 2020; 55(5): 105960. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeorge PM, Wells AU, Jenkins RG: Pulmonary fibrosis and COVID-19: the potential role for antifibrotic therapy. Lancet Respir Med. 2020; 8(8): 807–815. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGombar S, Chang M, Hogan CA, et al.: Persistent detection of SARS-CoV-2 RNA in patients and healthcare workers with COVID-19. J Clin Virol. 2020; 129: 104477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHara A, Niwa M, Noguchi K, et al.: Galectin-3 as a Next-Generation Biomarker for Detecting Early Stage of Various Diseases. Biomolecules. 2020; 10(3): 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenderson NC, Mackinnon AC, Farnworth SL, et al.: Galectin-3 expression and secretion links macrophages to the promotion of renal fibrosis. Am J Pathol. 2008; 172(2): 288–298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo JE, Gao W, Levy D, et al.: Galectin-3 Is Associated with Restrictive Lung Disease and Interstitial Lung Abnormalities. Am J Respir Crit Care Med. 2016; 194(1): 77–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalfaoglu B, Almeida-Santos J, Adele Tye C, et al.: T-cell hyperactivation and paralysis in severe COVID-19 infection revealed by single-cell analysis. BioRxiv. Preprint. 2020. Publisher Full Text\n\nKasper M, Hughes RC: Immunocytochemical evidence for a modulation of galectin 3 (Mac-2), a carbohydrate binding protein, in pulmonary fibrosis. J Pathol. 1996; 179(3): 309–316. PubMed Abstract \| Publisher Full Text\n\nKim CH: SARS-CoV-2 Evolutionary Adaptation toward Host Entry and Recognition of Receptor O-Acetyl Sialylation in Virus-Host Interaction. Int J Mol Sci. 2020; 21(12): 4549. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F: Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol. 2016; 3(1): 237–261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F: Receptor recognition mechanisms of coronaviruses: a decade of structural studies. J Virol. 2015; 89(4): 1954–1964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi W, Hulswit RJG, Widjaja I, et al.: Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein. Proc Natl Acad Sci U S A. 2017; 114(40): E8508–E8517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Zhu A, He J, et al.: Single-cell analysis reveals macrophage-driven T-cell dysfuntion in severe COVID-19 patients. MedRxiv. Preprint. 2020. Publisher Full Text\n\nMackinnon AC, Gibbons MA, Farnworth SL, et al.: Regulation of transforming growth factor-β1-driven lung fibrosis by galectin-3. Am J Respir Crit Care Med. 2012; 185(5): 537–546. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilanetti E, Miotto M, Di Rienzo L, et al.: In-silico evidence for two receptors based strategy of SARS-CoV2. BioRxiv. Preprint. 2020. Publisher Full Text\n\nMishra BB, Li Q, Steichen AL, et al.: Galectin-3 functions as an alarmin: pathogenic role for sepsis development in murine respiratory tularemia. PLoS One. 2013; 8(3): e59616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNangia-Makker P, Hogan V, Raz A: Galectin-3 and cancer stemness. Glycobiology. 2018; 28(4): 172–181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNishi Y, Sano H, Kawashima T, et al.: Role of galectin-3 in human pulmonary fibrosis. Allergol Int. 2007; 56(1): 57–65. PubMed Abstract \| Publisher Full Text\n\nNita-Lazar M, Banerjee A, Feng C, et al.: Galectins regulate the inflammatory response in airway epithelial cells exposed to microbial neuraminidase by modulating the expression of SOCS1 and RIG1. Mol Immunol. 2015; 68(2 Pt A): 194–202. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng G, Sun D, Rajashankar KR, et al.: Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor. Proc Natl Acad Sci U S A. 2011; 108(26): 10696–10701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng G, Xu L, Lin YL, et al.: Crystal structure of bovine coronavirus spike protein lectin domain. J Biol Chem. 2012; 287(50): 41931–41938. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRen Z, Liang W, Sheng J, et al.: Gal-3 is a potential biomarker for spinal cord injury and Gal-3 deficiency attenuates neuroinflammation through ROS/TXNIP/NLRP3 signaling pathway. Biosci Rep. 2019; 39(12): BSR20192368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobson B: Bioinformatics studies on a function of the SARS-CoV-2 spike glycoprotein as the binding of host sialic acid glycans. Comput Biol Med. 2020; 122: 103849. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaito S, Alkhatib A, Kolls JK, et al.: Pharmacotherapy and adjunctive treatment for idiopathic pulmonary fibrosis (IPF). J Thorac Dis. 2019; 11(Suppl 14): S1740–S1754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheng G, Chen P, Wei Y, et al.: Viral Infection Increases the Risk of Idiopathic Pulmonary Fibrosis: A Meta-Analysis. Chest. 2020; 157(5): 1175–1187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTai W, He L, Zhang X, et al.: Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020; 17(6): 613–620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTortorici MA, Walls AC, Lang Y, et al.: Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol. 2019; 26(6): 481–489. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniversity of Edinburgh: Rapid Experimental Medicine for COVID-19 (DEFINE). In: ClinicalTrials.gov. [cited 2020 Aug 6]. Reference Source\n\nVabret N, Britton GJ, Gruber C, et al.: Immunology of COVID-19: Current State of the Science. Immunity. 2020; 52(6): 910–941. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Jiang M, Chen X, et al.: Cytokine storm and leukocyte changes in mild versus severe SARS-CoV-2 infection: Review of 3939 COVID-19 patients in China and emerging pathogenesis and therapy concepts. J Leukoc Biol. 2020a; 108(1): 17–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Q, Zhang Y, Wu L, et al.: Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell. 2020b; 181(4): 894–904.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang WH, Lin CY, Chang MR, et al.: The role of galectins in virus infection - A systemic literature review. J Microbiol Immunol Infect. 2019; S1684-1182(19)30149-5. PubMed Abstract \| Publisher Full Text\n\nXu J, Xu X, Jiang L, et al.: SARS-CoV-2 induces transcriptional signatures in human lung epithelial cells that promote lung fibrosis. Respir Res. 2020a; 21(1): 182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu Z, Li X, Huang Y, et al.: The Predictive Value of Plasma Galectin-3 for Ards Severity and Clinical Outcome. Shock. 2017; 47(3): 331–336. PubMed Abstract \| Publisher Full Text\n\nXu Z, Shi L, Wang Y, et al.: Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med. 2020b; 8(4): 420–422. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe J, Zhang B, Xu J, et al.: Molecular pathology in the lungs of severe acute respiratory syndrome patients. Am J Pathol. 2007; 170(2): 538–545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYip PK, Carrillo-Jimenez A, King P, et al.: Galectin-3 released in response to traumatic brain injury acts as an alarmin orchestrating brain immune response and promoting neurodegeneration. Sci Rep. 2017; 7: 41689. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhai P, Ding Y, Wu X, et al.: The epidemiology, diagnosis and treatment of COVID-19. Int J Antimicrob Agents. 2020; 55(5): 105955. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Wu Z, Li JW, et al.: Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality. Int J Antimicrob Agents. 2020; 55(5): 105954. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou W, Chen X, Hu Q, et al.: Galectin-3 activates TLR4/NF-kappaB signaling to promote lung adenocarcinoma cell proliferation through activating lncRNA-NEAT1 expression. BMC Cancer. 2018; 18(1): 580. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70959", "date": "08 Sep 2020", "name": "Talia H Swartz", "expertise": [ "Reviewer Expertise SARS-CoV-2 infection", "COVID-19", "viral pathogenesis", "inflammatory signaling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 here describe the role of Galectin 3 in mediating inflammatory cytokine signaling as a possible source of disease pathogenesis in SARS-CoV-2 infection. The review is well written and describes the relevant literature supporting the role of Gal-3 in COVID-19.\n\nThe following suggestions would improve the strength of the work:\nThe authors have published a similar article entitled: A potential role for Galectin-3 inhibitors in the treatment of COVID-19 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301894/[ref-1]; the title of this current article should clearly reflect how these two works are non-overlapping; the authors describe that this is an update of the prior work.\n\nThe authors write: “Recent discoveries have begun to shed light on its role in viral infections (Wang et al. 2019) but should go into further detail about what role(s) it plays; there is a literature on HIV (Wang 2014, Okamoto 2019, Fogel 1999).\n\nThe introduction provides very little information about Galectin 3 besides that it is an animal lectin and exerts pleiotropic effects. Some more description should be provided as to the function of this molecule, its tissue expression, and any literature about epigenetics as it pertains to infection and inflammation.\n\nOn page 3, the authors state: “Several studies to date have referred to the “galectin fold” present on the NTD of coronaviruses (Behloul et al., 2020; Li, 2016; Li et al., 2017; Peng et al., 2011; Peng et al., 2012; Tortorici et al., 2019). The structures are so similar, in fact, that it is hypothesized that coronaviruses incorporated a host galectin gene into their genome (and then the NTD) at some point in their evolution (Li, 2015).” The structures of ‘what’ are so similar? NTD to Gal-3? This should be more explicitly defined. This should refer back to Figure 1 in Caniglia PeerJ 2020.\n\nOn page 4, the authors state “Notably, a study of nearly 4,000 patients has found the levels of IL-1, IL-6, and TNF-α to be significantly elevated in the sera of patients suffering from severe COVID-19 as compared to those with mild disease (Wang et al., 2020a).” The authors should additionally cite Del Valle et al. Nature Medicine 2020 that noted similar findings in 1500 patients.2\n\nFigure 1 does not add richly to this work and perhaps could be a panel combined with Figure 2.\n\nThe authors state “Several studies to date have shown the effects of anti-Gal-3 therapy on cytokine release.” These studied should be cited.\n\nFigure 2 figure legend should address the tissue sites where Gal-3 is produced in macrophages, monocytes, and dendritic cells. Is it lung? Plasma?\n\nThe authors note on p. 5 “A study found that tests for SARS-CoV2 RNA in the serum of infected individuals did not become negative until a median of 24 days post-symptom onset, with some individuals remaining positive even greater than a month from the beginning of symptoms (Gombar et al., 2020). This indicates that for some, COVID-19 infection may run a particularly long course. Findings such as this have led to the question of whether or not anti fibrotic therapy would be beneficial for such patients (George et al., 2020).” The persistence of SARS-CoV-2 RNA should not be equated with replication competent virus; there is significant literature to suggest residual nucleic acid that does not represent infectious virus. This should not be equated with long term infection or increased risk of fibrotic disease and these patients should not be treated with anti-fibrotic therapy for that reason. Severe lung injury from ARDS would be much more plausible an explanation for fibrotic lung disease than persistent SARS-CoV2 RNA.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "5921", "date": "28 Sep 2020", "name": "Kiran Velpula", "role": "Author Response", "response": "Response to Reviewer: Talia Swartz, MD, PhD The authors here describe the role of Galectin 3 in mediating inflammatory cytokine signaling as a possible source of disease pathogenesis in SARS-CoV-2 infection. The review is well written and describes the relevant literature supporting the role of Gal-3 in COVID-19. The following suggestions would improve the strength of the work: The authors have published a similar article entitled: A potential role for Galectin-3 inhibitors in the treatment of COVID-19 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301894/[ref-1]; the title of this current article should clearly reflect how these two works are non-overlapping; the authors describe that this is an update of the prior work. The reviewer makes a great point. With the title of this work we are trying to convey a distinction in so far as the prior article explicitly argued for the repurposing of a drug toward COVID-19 treatment. In this article, we are discussing the pathologic effects Gal-3 may exert in severe COVID-19, hence the title. The authors write: “Recent discoveries have begun to shed light on its role in viral infections (Wang et al. 2019) but should go into further detail about what role(s) it plays; there is a literature on HIV (Wang 2014, Okamoto 2019, Fogel 1999). Thank you for providing the additional references. We have added a couple sentences to more explicitly detail the roles of Gal-3 in viral infection and have incorporated the findings of Okamato et. al into multiple sections of the review. The introduction provides very little information about Galectin 3 besides that it is an animal lectin and exerts pleiotropic effects. Some more description should be provided as to the function of this molecule, its tissue expression, and any literature about epigenetics as it pertains to infection and inflammation. The reviewer makes an excellent point. We have added additional sources (Diaz Alvarez et. al 2017; Sato et. al 2009) to provide further introduction to the role of galectins in infections and to reference the cell types Gal-3 is most highly expressed in. On page 3, the authors state: “Several studies to date have referred to the “galectin fold” present on the NTD of coronaviruses (Behloul et al., 2020; Li, 2016; Li et al., 2017; Peng et al., 2011; Peng et al., 2012; Tortorici et al., 2019). The structures are so similar, in fact, that it is hypothesized that coronaviruses incorporated a host galectin gene into their genome (and then the NTD) at some point in their evolution (Li, 2015).” The structures of ‘what’ are so similar? NTD to Gal-3? This should be more explicitly defined. This should refer back to Figure 1 in Caniglia PeerJ 2020. We have revised the sentence to more explicitly define the structural similarities of Gal-3 and S1-NTD of coronavirus spike proteins. We have also added an additional reference of Caniglia et. al. On page 4, the authors state “Notably, a study of nearly 4,000 patients has found the levels of IL-1, IL-6, and TNF-α to be significantly elevated in the sera of patients suffering from severe COVID-19 as compared to those with mild disease (Wang et al., 2020a).” The authors should additionally cite Del Valle et al. Nature Medicine 2020 that noted similar findings in 1500 patients.2 Thank you for providing the additional citation. We will certainly add these findings to the manuscript. Figure 1 does not add richly to this work and perhaps could be a panel combined with Figure 2. Thank you for this comment. We believe Figure 1 to be essential as it details the likely role of the galectin-like S1-NTD in COVID-19 infection. To further highlight the importance of this figure and the NTD, we have added a recent publication (Chi et. al, 2020) which shows a neutralizing antibody against the NTD inhibits viral entry. It then follows that if a drug such as a galectin inhibitor is able to also bind this region of the NTD, it may also inhibit cell entry by SARS-CoV2. The authors state “Several studies to date have shown the effects of anti-Gal-3 therapy on cytokine release.” These studied should be cited. We have added the appropriate sources to support this statement. Figure 2 figure legend should address the tissue sites where Gal-3 is produced in macrophages, monocytes, and dendritic cells. Is it lung? Plasma? The reviewer makes an excellent point. We have updated the figure legend to show that Gal-3 is produced in circulating immune cells in the plasma. The authors note on p. 5 “A study found that tests for SARS-CoV2 RNA in the serum of infected individuals did not become negative until a median of 24 days post-symptom onset, with some individuals remaining positive even greater than a month from the beginning of symptoms (Gombar et al., 2020). This indicates that for some, COVID-19 infection may run a particularly long course. Findings such as this have led to the question of whether or not anti fibrotic therapy would be beneficial for such patients (George et al., 2020).” The persistence of SARS-CoV-2 RNA should not be equated with replication competent virus; there is significant literature to suggest residual nucleic acid that does not represent infectious virus. This should not be equated with long term infection or increased risk of fibrotic disease and these patients should not be treated with anti-fibrotic therapy for that reason. Severe lung injury from ARDS would be much more plausible an explanation for fibrotic lung disease than persistent SARS-CoV2 RNA. The reviewer makes an excellent point here. We have added multiple sources to this section that we believe better characterize the chronic inflammatory signature some COVID-19 patients report months after the initial infection. We have also included an additional citation of an excellent commentary on the concern for development of post-infectious IPF in COVID-19 patients." } ] }, { "id": "70657", "date": "16 Sep 2020", "name": "Thirunavukkarasu Velusamy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review article is clear, concise, and well structured.\n\nThe article highlights the novel discoveries by linking the pathogenesis of COVID-19 with immunopathologic effects of Gal-3. Dual targeting of Neu5Ac-binding domain of SARS-CoV2 and galectin-3 using galectin-3inhibitors could be a very effective approach in reducing the spread, cytokine storm, and post-infection pulmonary fibrosis.\n\nThe quality of the figures in the article is good and clearly explains the concept discussed.\n\nThe authors are requested to address the following specific comments mentioned below: 1) The authors have mentioned Gal-3 inhibitors could target Neu5Ac-binding domain, thereby reducing viral entry to host cells. Since ACE2 receptors serve as the main entry mechanism ofSARS-CoV2, To what extent Gal-3 inhibitors alone can provide mitigatory effects?. Also, could ACE2 inhibitors be used as adjuvants along with Gal-3 inhibitors?. What are the possibilities?. These need to be explained.\n2) The authors are advised to cite the article Garcia-Revilla, J. et al., 2020in their manuscript. The article discusses similar concepts mentioned in the current review by the authors. Therefore, citing it could provide more support to the authors claim.\n\nOverall the article is well-written and recommended for publication once the minor corrections have been addressed.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "5962", "date": "28 Sep 2020", "name": "Kiran Velpula", "role": "Author Response", "response": "Response to Reviewer: Thirunavukkarasu Velusamy The review article is clear, concise, and well structured. We appreciate the above comment. Thank you. The article highlights the novel discoveries by linking the pathogenesis of COVID-19 with immunopathologic effects of Gal-3. Dual targeting of Neu5Ac-binding domain of SARS-CoV2 and galectin-3 using galectin-3inhibitors could be a very effective approach in reducing the spread, cytokine storm, and post-infection pulmonary fibrosis. The quality of the figures in the article is good and clearly explains the concept discussed. The authors are requested to address the following specific comments mentioned below: 1) The authors have mentioned Gal-3 inhibitors could target Neu5Ac-binding domain, thereby reducing viral entry to host cells. Since ACE2 receptors serve as the main entry mechanism ofSARS-CoV2, To what extent Gal-3 inhibitors alone can provide mitigatory effects?. Also, could ACE2 inhibitors be used as adjuvants along with Gal-3 inhibitors?. What are the possibilities?. These need to be explained The reviewer brings up a great point. To address this question, we have added a citation that shows the detection of a neutralizing antibody against the NTD of SARS-CoV2 S1 protein. This antibody is effective at completely neutralizing SARS-CoV2 cell entry even in the absence of other antibodies that bind the receptor binding domain or ACE2 receptors. With this in mind, it follows that a drug such as a galectin inhibitor targeting the NTD may be effective as a standalone therapy. The reviewer brings up a good point regarding combination therapy, in that combination of NTD / CTD targeted therapies may be a better long term treatment strategy given the mutagenicity of the virus' spike protein. 2) The authors are advised to cite the article Garcia-Revilla, J. et al., 2020 in their manuscript. The article discusses similar concepts mentioned in the current review by the authors. Therefore, citing it could provide more support to the authors claim. Thank you for providing this citation. We are certainly glad to see other laboratories participating in this field of research. However, given that this article is also a review article and not primary research, we do not see a role for it to be cited in our current review article, which aims to summarize primary research findings regarding galectin-3 and COVID-19. Overall the article is well-written and recommended for publication once the minor corrections have been addressed." } ] } ]	1	https://f1000research.com/articles/9-1078
https://f1000research.com/articles/9-1175/v1	28 Sep 20	{ "type": "Data Note", "title": "RNA-Seq analysis of genes affected by Cyclophilin A/DIAGEOTROPICA (DGT) in tomato root development", "authors": [ "Maria G. Ivanchenko", "Olivia R. Ozguc", "Stephanie R. Bollmann", "Valerie N. Fraser", "Molly Megraw", "Maria G. Ivanchenko", "Olivia R. Ozguc", "Stephanie R. Bollmann", "Valerie N. Fraser" ], "abstract": "Cyclophilin A/DIAGEOTROPICA (DGT) has been linked to auxin-regulated development in tomato and appears to affect multiple developmental pathways. Loss of DGT function results in a pleiotropic phenotype that is strongest in the roots, including shortened roots with no lateral branching. Here, we present an RNA-Seq dataset comparing the gene expression profiles of wildtype (‘Ailsa Craig’) and dgt tissues from three spatially separated developmental stages of the tomato root tip, with three replicates for each tissue and genotype. We also identify differentially expressed genes, provide an initial comparison of genes affected in each genotype and tissue, and provide the pipeline used to analyze the data. Further analysis of this dataset can be used to gain insight into the effects of DGT on various root developmental pathways in tomato.", "keywords": [ "Tomato", "Root", "Development", "Diageotropica", "DGT", "RNA-Seq", "Gene expression" ], "content": "Introduction\n\nThe tomato (Solanum lycopersicum) cyclophilin DIAGEOTROPICA (DGT) has been linked to auxin-regulated development through identification of the gene affected by the diageotropica (dgt) mutation1,2. Tomato dgt mutants are auxin-resistant and display a pleiotropic phenotype that includes slow gravitropic response, lack of lateral root initiation, altered vascular development, reduced ethylene production in response to auxin, reduced apical dominance, impaired shoot and root growth, reduced fertility, and impaired fruit growth3–11. DGT likely interacts with auxin transport and signaling in a complex manner. For instance, DGT has been shown to negatively regulate auxin efflux via PIN-FORMED (PIN) transporters by altering subcellular localization of PINs and expression of some PIN genes9. DGT, in turn, is downregulated by auxin at the root tip8, suggesting functional feedback between DGT, auxin, and PINs. Through targeted gene expression quantification with RT-PCR and northern blots, DGT has been demonstrated to affect expression of a number of other auxin-related genes in addition to PIN genes. The dgt mutation reduces auxin-induced expression of genes encoding certain 1-aminocyclopropane-1-carboxylic acid synthases (ACCSs; key ethylene biosynthesis regulatory enzymes), SMALL AUXIN UPREGULATED RNA (SAUR) genes, and several members of the auxin-regulated Aux/IAA gene family in a tissue- and developmental stage-specific manner4,6,10–14. However, full transcriptomic profiling of tomato dgt mutants in different developmental zones has not been performed. Given the complex role of DGT in auxin pathways, an exploration into the widespread effects of DGT on the transcriptome may provide valuable insights into its potentially extensive and multifaceted role in development.\n\nIn this study, we perform a global analysis of gene expression in dgt roots that compares the root meristem, elongation zone, and differentiation zone in wildtype (‘Ailsa Craig’) and dgt tomato plants. The root tip provides an excellent system for studying development-related plant gene expression because cell division, elongation, and maturation are not only temporally but also spatially separated in this growth region; this allows for anatomical dissection and analysis of specific developmental zones, such as the meristem, elongation zone, and differentiation zone. The root tip is also most appropriate for this study because the dgt phenotype is the strongest in the root tip and has been characterized morphologically in the root tip7,8. We have previously performed histological analyses of tomato root tips including the meristem, elongation zone, and maturation zone, and demonstrated a decrease in length and number of cells of the dgt meristem and elongation zone8, whereas the initiation of lateral root primordia was abolished in the dgt root maturation zone7–9. Here, we present an RNA-Seq dataset containing raw reads and abundance estimates for three replicates in each zone and genotype, the pipeline used for analysis, and an initial exploration of expressed and differentially expressed genes in each developmental region that can be used to guide future investigations.\n\n\nMaterials and methods\n\nThree biological replicates for each tissue and genotype were performed. Seven-day old tomato (Solanum lycopersicum) wildtype (WT) and dgt1-1 plants in the ‘Ailsa Craig’ background7 were used. Seeds were sterilized in 20% commercial bleach for 30 min and rinsed four times for 10 min with sterile water. Sterilized seeds were vernalized at 4°C for two days to ensure even germination and then planted on media containing 0.2× Murashige and Skoog basal medium with vitamins (PhytoTechnology), 1% sucrose, 10 mM MES buffer pH 5.7, and 0.8% agar. Seeds were germinated in Magenta boxes (16 seeds per box) in a growth chamber at 21°C under long day (16h light, 8h dark) conditions and light intensity as in Ivanchenko et al. (2013). Root samples were dissected under DIC optics at 4x objective as in Ivanchenko et al. (2006). On average, 50–100 root portions were collected per biological replicate. The meristem was dissected between the root tip and the root portion where tissue becomes more transparent. The elongation zone was collected from the proximal meristem border and the first hair bulge, and approximately 1 cm portions were collected above the first hair bulge for the differentiation zone.\n\nTissue samples were collected in Plant RNA Reagent (Life Technologies) on ice, and total RNA was prepared using the RNeasy Mini kit (Qiagen) according to manufacturer’s recommendations. The RNA pellets were dried in 1.7 mL centrifuge tubes and solubilized in 178 µL 1X RNA Secure Reagent (Ambion) preheated at 65°C, and incubated at 65°C for 10 min, mixing by pipetting a few times. Then 20 µL 10X DNase I buffer and 2 µL RNase-free DNase I (Ambion) were added to each tube, and tubes incubated for 10 min at 37°C. 700 µL RLT buffer was added (to which 7 µL 2-Mercaptoethanol was freshly added) to each sample, which were then mixed by vortexing. 500 µL ethanol was then added and samples were mixed again by vortexing. Each sample (2 X 700 µl) was applied to an RNeasy Mini spin column from the RNeasy kit and RNA cleanup performed following the manufacturer’s instructions. Each sample was eluted with 30 µL nuclease-free ultrapure H2O, and the RNA concentrations were measure using a NanoDrop 1000 spectrophotometer (Thermo Fisher).\n\nRNA-Seq library preparation and sequencing were performed at the Oregon State University Center for Genome Research and Biocomputing. Libraries were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) and sequenced as single-end 51 bp reads on the Illumina HiSeq 2000 using a total of two lanes.\n\nSequencing produced 5-8 FASTQ files per replicate, which were merged into a single FASTQ file per replicate. Reference transcripts were extracted and preprocessed from the NCBI Heinz 1706 genome sequence15 using RSEM’s16 (RNA-Seq by Expectation Maximization, version 1.3.1) rsem-prepare-reference function. Using RSEM, raw sequence reads were then aligned to the reference transcript sequences and estimated transcripts per million (TPM) values were calculated using default parameters. Resulting gene-level estimates from biological replicates were merged into a single input matrix and EBSeq17 (version 1.26.0) was then used to test for differential expression between dgt and WT for each root-tip zone. Raw RNA-Seq reads, abundance estimates, and differential expression analysis are publicly available in the Sequence Read Archive (SRA) and Gene Expression Omnibus (GEO), see Data availability.\n\nAs a preliminary analysis to guide future investigations, a basic comparison of presence/absence calls and differential gene expression is presented. Expression analysis was performed using TPM and differential expression analysis was performed using the posterior probability that the gene is differentially expressed (PPDE) and posterior fold change (postFC). Genes with an average of TPM > 2 across biological replicates were compared between zones and genotypes (Figure 1; data summarized in Data File 118). Following EBSeq analysis, genes were filtered for PPDE = 1 to identify those which were differentially expressed. Of the differentially expressed genes, those with postFC (dgt over WT) > 2 were considered upregulated in dgt and those with postFC < 0.5 were considered downregulated in dgt. Upregulated and downregulated genes were compared between zones and genotypes (Figure 2; data summarized in Data File 219). Principal component analysis (PCA) of the data was performed using TPM values of each gene and was calculated using the scikit-learn20 PCA function with default parameters (Figure 3).\n\n(A) Number of genes expressed in each zone of dgt roots, transcripts per million (TPM) > 2.0. (B) Number of genes expressed in each zone of WT roots, TPM > 2.0. (C) Number of genes expressed exclusively in the differentiation zone with TPM > 2.0, compared between WT and dgt. (D) Number of genes expressed exclusively in the elongation zone with TPM > 2.0, compared between WT and dgt. (E) Number of genes expressed exclusively in the meristem with TPM > 2.0, compared between WT and dgt.\n\n(A) Differentially expressed genes downregulated in each zone with postFC < 0.5, posterior probability that the gene is differentially expressed (PPDE) = 1 in dgt vs WT. (B) Differentially expressed genes upregulated in each zone with postFC > 2, PPDE = 1 in dgt vs. WT.\n\n(A) PCA of sequenced samples in differentiation zone (B) PCA of sequenced samples in elongation zone (C) PCA of sequenced samples in meristem.\n\n\nConclusion\n\nIt is clear from the outcomes in Figure 1 and Figure 2 that reduced function of DGT has sweeping effects on the transcriptome in all three developmental zones examined in this study, supporting the concept that DGT very likely plays important and potentially complex roles in multiple developmental pathways. Additionally, PCA using TPM values for each sample demonstrated that while there was some variance within the replicate pools, replicates from different genotypes were distinctly separate from each other (Figure 3). Further functional genomics studies are needed to narrow down the most likely direct interactions with DGT, leading to the identification of specific functional roles. We hope that this dataset will be of value to the community in future studies in this area.\n\n\nData availability\n\nHeinz 1706 genome available from Assembly, Accession number GCF_000188115.4: https://www.ncbi.nlm.nih.gov/assembly/GCF_000188115.4/\n\nRaw RNA-Seq reads and expression estimates provided by RSEM on Gene Expression Omnibus, Accession number GSE156398: https://identifiers.org/geo:GSE156398\n\nFigshare: Data File 1: Genes referenced in figure 1. https://doi.org/10.6084/m9.figshare.12891773.v118.\n\nFigshare: Data File 2: Genes referenced in figure 2. https://doi.org/10.6084/m9.figshare.12891941.v119.\n\n\nSoftware availability\n\nSource code available from: https://github.com/ozguco/tomato_dgt_RNASeq\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.402939921\n\nLicense: GNU General Public License v3.0", "appendix": "Acknowledgements\n\nWe thank Jordan Holdaway for her work on the initial raw data processing scripts.\n\n\nReferences\n\nOh K, Hardeman K, Ivanchenko MG, et al.: Fine mapping in tomato using microsynteny with the Arabidopsis genome: the Diageotropica (Dgt) locus. Genome Biol. 2002; 3(9): research0049. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOh K, Ivanchenko MG, White TJ, et al.: The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling. Planta. 2006; 224(1): 133–144. PubMed Abstract \| Publisher Full Text\n\nZobel RW: Some Physiological Characteristics of the Ethylene-requiring Tomato Mutant Diageotropica. Plant Physiol. 1973; 52(4): 385–389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMito N, Bennett AB: The diageotropica Mutation and Synthetic Auxins Differentially Affect the Expression of Auxin-Regulated Genes in Tomato. Plant Physiol. 1995; 109(1): 293–297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRice MS, Lomax TL: The auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway. Planta. 2000; 210(6): 906–913. PubMed Abstract \| Publisher Full Text\n\nBalbi V, Lomax TL: Regulation of Early Tomato Fruit Development by the Diageotropica Gene. Plant Physiol. 2003; 131(1): 186–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIvanchenko MG, Coffeen WC, Lomax TL, et al.: Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle. Plant J. 2006; 46(3): 436–447. PubMed Abstract \| Publisher Full Text\n\nIvanchenko MG, den Os D, Monshausen GB, et al.: Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth. Ann Bot. 2013; 112(6): 1107–1116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIvanchenko MG, Zhu J, Wang B, et al.: The cyclophilin A DIAGEOTROPICA gene affects auxin transport in both root and shoot to control lateral root formation. Development. 2015; 142(4): 712–721. PubMed Abstract \| Publisher Full Text\n\nBradford KJ, Yang SF: Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica. Plant Physiol. 1980; 65(2): 327–330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelly MO, Bradford KJ: Insensitivity of the diageotropica tomato mutant to auxin. Plant Physiol. 1986; 82(3): 713–717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNebenführ A, White TJ, Lomax TL: The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato. Plant Mol Biol. 2000; 44(1): 73–84. PubMed Abstract \| Publisher Full Text\n\nCoenen C, Christian M, Lüthen H, et al.: Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato. Plant Physiol. 2003; 131(4): 1692–1704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVidoz ML, Loreti E, Mensuali A, et al.: Hormonal interplay during adventitious root formation in flooded tomato plants. Plant J. 2010; 63(4): 551–562. PubMed Abstract \| Publisher Full Text\n\nTomato Genome Consortium: The tomato genome sequence provides insights into fleshy fruit evolution. Nature. 2012; 485(7400): 635–641. 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\nLeng N, Kendziorski C: EBSeq: An R package for gene and isoform differential expression analysis of RNA-seq data. R package version 1.28.0. 2020. Reference Source\n\nOzguc O: Data File 1: Genes referenced in figure 1. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12891773.v1\n\nOzguc O: Data File 2: Genes referenced in figure 2. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12891941.v1\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine Learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nozguco:ozguco/tomato_dgt_RNASeq: First release (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4029399" }	[ { "id": "72160", "date": "12 Oct 2020", "name": "Gustavo Rodríguez-Alonso", "expertise": [ "Reviewer Expertise JD: Plant development", "root development", "root growth and branching", "lateral root development. GR-A: Transcriptomics and evolutionary biology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Maria Ivanchenko et al. “RNA-Seq analysis of genes affected by Cyclophilin A/DIAGEOTROPICA (DGT) in tomato root development” presents the results of RNAseq analysis of the roots of a tomato mutant affected in Cyclophilin A/DIAGEOTROPICA (DGT) vs wild type. The authors compare three developmental zones in the root tip of mutant and wild type seedlings and identify differentially expressed genes. Considering the pleiotropic effect of the mutation and its relation to auxin signaling and transport, this work is potentially important to understand abnormalities in the root development in the dgt mutant and the role of DGT in root development. Considering that the formation of lateral root primordia is impaired in dgt mutants, this analysis can also be useful in the identification of candidate genes involved in lateral root initiation.\nAll the raw data are publicly available, the same as the scripts used for the RNA-seq analysis. A minor suggestion: the authors should explicitly state whether any pre-processing steps were performed on the raw sequencing reads, such as removal of low-quality bases, adapter filtering, or trimming. Regarding the PCA analysis, one of the WT replicates does not group closely with the other WT samples. It could be useful if the authors provide some insight on why this occurs and discuss the effect of this on TPM variation among replicates.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "72162", "date": "16 Oct 2020", "name": "Aurélien Bailly", "expertise": [ "Reviewer Expertise Plant Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIvanchenko and colleagues describe here a transcriptomics data set profiling gene expression in root tips of the tomato mutant dgt and matching wildtype. As for several plant immunophilins, loss of function of DIAGETROPICA triggers dramatic structural and behavioural phenotypes in tomato. The link between immunophilins and hormonal control has been extensively recognised; however, the detailed molecular mechanisms behind them remain elusive.\nThe interest in this RNAseq dataset lies within the dissection of the root into the three standard spatio-temporal zones of development. Gaining insight into the effect of the dgt mutation in the transcriptomes of the meristematic, elongation and mature zones of the root would potentially lead to the isolation of long-sought components of root growth and development machineries.\nThe work presented here is technically sound and used state of the art transcripts-reading methods. Data deposition was correctly performed and gene lists produced after alignments and differential expression analysis through regular scripts are clear and readily exploitable. Although the level of variation between replicates, as mentioned by authors, do vary in the different root zones samples, I trust that they separate enough in PCA to yield interesting outcomes. In addition, the relatively reduced number of differentially expressed genes filtered in this analysis will allow for a rapid identification of proteins associated with the dgt phenotype, perhaps outside of the classical pool of auxin-related genes.\nI would express only two regrets: 1) the DGT protein was shown to hold a conserved auxin signalling function down to moss, but it is not mentioned here; and 2) other immunophilins present overlapping roles with DGT, a paradigm worth mentioning somewhere in the introduction.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1175
https://f1000research.com/articles/7-1220/v1	08 Aug 18	{ "type": "Software Tool Article", "title": "sPop: Age-structured discrete-time population dynamics model in C, Python, and R", "authors": [ "Kamil Erguler" ], "abstract": "This article describes the sPop packages implementing the deterministic and stochastic versions of an age-structured discrete-time population dynamics model. The packages enable mechanistic modelling of a population by monitoring the age and development stage of each individual. Survival and development are included as the main effectors and they progress at a user-defined pace: follow a fixed-rate, delay for a given time, or progress at an age-dependent manner. The model is implemented in C, Python, and R with a uniform design to ease usage and facilitate adoption. Early versions of the model were previously employed for investigating climate-driven population dynamics of the tiger mosquito and the chikungunya disease spread by this vector. The sPop packages presented in this article enable the use of the model in a range of applications extending from vector-borne diseases towards any age-structured population including plant and animal populations, microbial dynamics, host-pathogen interactions, infectious diseases, and other time-delayed epidemiological processes.", "keywords": [ "deterministic", "stochastic", "vector", "population", "model", "age-specific", "survival", "development", "dynamic", "difference equations", "C", "Python", "R" ], "content": "Introduction: The age-structured population dynamics model\n\nPopulations become heterogenous as individuals age and their physical/biochemical characteristics change. This often introduces time delays and has a strong non-linear impact on dynamics1. As mosquito vectors develop, they react differently to environmental factors2. An infection requires a minimum incubation period before it is ready to be transmitted3. Age dependent population stratification should be accounted for in mathematical models to improve population projections and aid in conservation and control.\n\nIncorporating age dependency in mathematical models can be challenging due to the need to keep track of the age of each individual in a population. A common work-around is to introduce predetermined intermittent development stages to account for the different characteristics of each stage and the time it takes to pass from one to another. This approach has been used in various modelling frameworks including deterministic, stochastic, as well as discrete- and continuous-time models4–7. Although intermittent development stages are capable of representing age-structured populations to a certain extent, a large number of age classes are required for accuracy. Consequently, model development becomes a non-trivial task.\n\nNumerous packages including popbio8, demogR9, and bayesPop10 have been implemented to facilitate modelling and analysis of age- and stage-structured projection matrix models. As a viable alternative, Kettle and Nutter implemented an R package for age-structured population dynamics, StagePop, which offers true time delays in continuous time domain using deterministic delay differential equations11.\n\nHere, I present an alternative age-structured population dynamics model based on the population dynamics and disease-spread models described in Erguler et al. 201612 and 201713. The model stems from the canonical projection matrix approach and is based on discrete-time difference equations. At present, three implementations of the model exists (the sPop packages) for three programming languages, C, Python, and R. The sPop packages provide a flexible number of age and development categories, include both deterministic and stochastic dynamics, and offer high-speed simulations to facilitate parameter inference.\n\nThe following section describes the theory behind the model and presents the use of each implementation with a commonly encountered case. The same case is modelled with each sPop package to emphasise the nuances in their usage. The Use Cases section concludes with a presentation of the sPop implementations of a short list of well-known mathematical models from a range of disciplines.\n\n\nModels and software\n\nThe sPop packages help to incorporate age-structured species in discrete-time population dynamics and epidemiological models. Survival (𝒮) and development (𝒟) are assumed to act upon each individual in a sequential manner, where development proceeds only if survival is guaranteed for the duration of an iteration, ᴛ . Survival and development may (i) progress at a fixed propensity, (ii) delay for a given number of iterations, (iii) follow a gamma-distributed (or negative binomial-distributed) life-time, or (iv) halt for a given time period. Here, I adopt the term propensity to refer to either the rate of a deterministic process or the probability of a stochastic one. Although propensity is assumed constant in each iteration, it can be redefined as a user-supplied function, which may depend on age (tα), number of completed development cycles (tπ), the degree of completion of the present development cycle (tδ), and any other environmental factors.\n\nEach individual is allowed to stay in the population given that neither death (𝒳 where Pr(𝒳 ) = 1−Pr(𝒮 )) or development occurs during an iteration. In a deterministic setup, a certain fraction of the population will die (r𝒳) or survive and complete development (r𝒟) every ᴛ. In a stochastic setup, the number of individuals to die or develop is chosen from a binomial distribution, ℬ(n, p), where p is the daily probability of death (p𝒳) or development (p𝒟) and n is the size of the population. In most experimental setups, development is observed on the condition that individuals survive; therefore, this condition is implicit in the definition of development propensity, i.e. p𝒟 = Pr(D\|S) per individual per ᴛ.\n\nAt each iteration, tα and tδ are incrementally adjusted to keep track of the age and the degree of development, respectively, for each individual. When a batch of individuals complete development, the indicator tπ is adjusted, tδ is reset to zero, and the batch is removed from the population. When modelling periodic development processes, such as the gonotrophic cycle, the batch can be reintroduced to the population for the subsequent cycle of development.\n\nWhen propensity is fixed for either 𝒳 or 𝒟, the time required for the completion of life or development follows a geometric distribution where the size of the population (n) can be described with\n\nnt = nt−1 (1 − r𝒳) (1 − r𝒟),\n\nfor the deterministic case, or with\n\n\n\nfor the stochastic case, where n0 is a user-defined initial condition.\n\nWhen modelling age dependency, the default assumption is that survival is a function of age, tα, and development is a function of the degree of development, tδ. The duration of each process can be a fixed number of iterations or can be described by a discrete negative binomial distribution or a continuous gamma distribution. In case of a negative binomial or gamma distribution, the daily propensity of death or development can be calculated as the ratio of the probability that the process is completed between iteration ᴛd and ᴛd+1 to the total probability of surviving or developing for at least ᴛd days. For instance, the daily probability of death in a stochastic setup can be written as\n\n\n\nwhere x represents the iteration when death occurs, and F(·) is the cumulative distribution function of either the negative binomial distribution or the gamma distribution. The parameters of each distribution are determined from the desired mean (µ) and standard deviation (σ) of the number of iterations to complete the process. An advantage of the gamma distribution over the negative binomial is its flexibility in accommodating various combinations of mean and standard deviation. However, the negative binomial distribution is restrictive over the minimum allowed standard deviation for a given mean.\n\n\nImplementation\n\nThe R implementation of sPop is available on CRAN as the albopictus package (v.0.4) and on the GitHub repository https://doi.org/10.5281/zenodo.132509514. The C and Python implementations are available as part of the albopictus package (v.1.9.3) on PyPI and the GitHub repository https://doi.org/10.5281/zenodo.132511115. The packages are implemented for R version 3.3.3 and Python version 3.7.0.\n\nThis section is reserved for outlining the use of each implementation to model the same theoretical population where both development and survival are age-dependent and gamma-distributed. In addition, the population exhibits a periodic development process with a mean duration of 50 hours and a standard deviation of 10 hours, and survival is a function of the number of development cycles,\n\n\n\nBefore we begin modelling, we load the albopictus package in R, and define the survival function as described in Equation 1.\n\n\n\nThe function returns a data.frame with a desired mean and standard deviation. Next, we initiate a population by calling the initiation routine of the spop class.\n\n\n\nWith this line, we construct a stochastic population model with the gamma distribution as the basis of survival and development. Setting prob to nbinom selects the negative binomial distribution instead.\n\nIn order to introduce the first batch of individuals, we use the add method.\n\n\n\nBy default, age, development cycle, and the duration of development will be set to zero for all individuals. These can be customised by supplying additional fields to the data.frame: age to set age, devcycle to set the number of development cycles, and development to set the number of iterations the current development cycle has taken.\n\nWe can directly access the population structure of the spop class to inspect the number of individuals grouped with respect to age, development cycle, and the degree of development. Here, we will use these information to calculate the mean and standard deviation of expected lifetime for each age-development group.\n\n\n\nThe following step iterates the population for one time-unit by using the iterate method.\n\n\n\nBy defining dev_mean and dev_sd, we opt to use the gamma distribution to describe the probability of development. Setting dev_sd to zero results in the gamma distribution being discarded and a fixed number of iterations (indicated by dev_mean) being assigned for development. Instead, setting dev instead of dev_mean and dev_sd results in a daily constant development probability. Same principles apply for the survival process, where we provide the mean and the standard deviation of the gamma-distribution for each age-development group as calculated by the death function. After each iteration, the age and degree of development of the population are updated and the total number of individuals completing development is recorded together with the detailed account of the corresponding age-development groups. We access these data using the developed and devtable methods, respectively. In addition, the dead method returns the number of dead individuals following an iteration.\n\n\n\nIn this example, we assume a periodic development process; therefore, we introduce all the individuals completing development back to the population using the add method.\n\nIn addition to iterate, the perturbate method performs the same procedure to iterate the population for a day, however, leaves age and development unchanged.\n\nThis method can be used for modelling external influences such as harvesting and population control, and also emmigration.\n\nFinally, we read the total size of the population using the size method.\n\n\n\nThe Python implementation of the population dynamics model can be imported from the albopictus package.\n\n\n\nWe begin by declaring the survival function as in Equation 1.\n\n\n\nUnlike the R implementation, the population structure is stored in a numpy.ndarray with the following order of columns: age, development cycle, degree of development, and number. Although the initiation step is similar to the R implementation, a two-dimensional list or a numpy.ndarray should be supplied to intriduce batches of individuals to a population.\n\n\n\nBy using the add method above, we introduce 1000 individuals with zero age, development cycle, and degree of development. The population structure is directly accessible, which enables us to calculate a different mean and standard deviation for the gamma-distributed development of each age-development group.\n\n\n\nNext, we iterate the population for one time-unit using the iterate method.\n\n\n\nIn order to read the total number of individuals completing development, the detailed account of the corresponding age-development groups, and the total size of the population, we access the developed, devtable, and size attributes of the spop class. Please note that these attributes are overwritten each time the iterate method is called. Here, we record the total number of developed individuals and the population size, and reintroduce the developed individuals to the population for the next round of development.\n\n\n\nThe Python implementation of the iterate method accepts an additional logical indicator pause to prevent updating age and development. If this parameter is supplied and if it is false, the iterate method acts as the perturbate method of the R implementation.\n\nThe C implementation of the sPop package is further optimised for speed. The source code resides in the albopictus package of Python, and it needs to be compiled with the GNU Scientific Library (version 2.1 or later). We begin by locating the package directory and compiling three source files into the object code. Assuming that the file name of our model is test_spop.c, we produce the executable with the following.\n\n\n\nwhere $pkgdir is a bash variable holding the package directory. In order to use the package, we need to include the following header files in test_spop.c.\n\n\n\nThe first header file defines the routines required for random number generation, and the second one defines the routines for the gamma and negative binomial distributions. The last header file defines the spop population structure and the associated functions for initialisation, modification, and garbage collection.\n\nEach age-development group is stored in the individual_st data structure,\n\n\n\nwhere the age, development cycle, degree of development, and the number of individuals in each age-development group are stored in age, devcycle, development, and number variables in the same order. The sdnum is a union data structure holding an unsigned int for a stochastic population or a double for a deterministic population. The spop data structure holds an array of individuals (individuals), population size (size), the number of dead and developed individuals following an iteration (dead and developed, respectively), a detailed account of developed individuals (devtable), an indicator for the probability distribution of age dependence (gamma_mode), a logical indicator for a stochastic or a deterministic model (stochastic), and two counters to manage the dynamic size of individuals (ncat and cat).\n\n\n\nFollowing the procedure in previous sections, we begin implementing the model in test_spop.c by declaring the survival function in Equation 1.\n\n\n\nPlease note that the C implementation handles a single age-development group at a time; therefore, the survival function is redesigned accordingly.\n\nNext, we initiate a stochastic model with the gamma distribution as the basis of survival and development using the spop_init function with the first parameter set to a logical true.\n\n\n\nThe macro MODE_GAMMA_HASH refers to the optimised implementation of the gamma distribution. Alternatively, MODE_NBINOM_RAW and MODE_GAMMA_RAW refer to the unoptimised implementations of the binomial and the gamma distributions. Optimisation involves recording previously-used values in a hash table for reuse, however, is memory intensive and should be used with caution. Faster more efficient implementations of the probability distributions are the main concern for future releases.\n\nHaving initiated vec, we introduce 1000 individuals of zero age with the spop_add function.\n\n\n\nspop_add accepts parameters in the following order:\n\n1. spop s: the spop data structure\n\n2. unsigned int age: the age of individuals\n\n3. unsigned int devcycle: the number of development cycles passed\n\n4. unsigned int development: the degree of development of individuals\n\n5. sdnum number: the number of individuals (unsigned int or double)\n\nIn order to iterate the population for one time interval, we use the spop_iterate function.\n\n\n\nspop_iterate accepts the following parameters in the given order:\n\n1. spop s: the spop data structure\n\n2. double dev_prob: fixed daily development probability (priority over the other development-related parameters)\n\n3. double dev_mean: mean development time (gamma or negative binomial)\n\n4. double dev_sd: standard deviation of the development time\n\n5. iter_func dev_fun: development function (similar to the death function above)\n\n6. double death_prob: fixed daily death probability (priority over the other survival-related parameters)\n\n7. double death_mean: mean time of death (gamma or negative binomial)\n\n8. double death_sd: standard deviation of the time of death\n\n9. iter_func death_fun: survival function\n\n10. unsigned char pause: logical indicator to prevent updating age and development\n\nFollowing each iteration, the list of age-development groups that completed their development is stored in the devtable variable of the spop data structure. In order to reintroduce these individuals back to the population, we use the spop_popadd function.\n\n\n\nIt is possible to obtain a summary output of the population structure by using the spop_print function, which takes the spop data structure as the only parameter. spop can be recycled by emptying its contents with the spop_empty function.\n\n\n\nFinally, in order to clear the memory used by vec, we supply its address to the spop_destroy function.\n\n\n\nAll three implementations of the model are given in Supplementary File 1–Supplementary File 3 (test_spop.R, test_spop.py, and test_spop.c. The resulting distribution of the number of individuals completing a development cycle during the first 20 days of simulation is given in Figure 1.\n\nThick solid line indicates the mean trajectory from the deterministic simulation, while the thin solid line and the dotted lines indicate the median and the 95% range of the stochastic simulation output. The timestep for each iteration is one hour.\n\nFive cycles of development are clearly seen from the figure, while the population survives for less than 15 days with the survival function defined in Equation 1. Blending of development cycles is apparent and progressive due to the uncertainty in the duration of development (50 hours on average with a standard deviation of 10 hours).\n\n\nUse cases\n\nThis section describes how the Python implementation of sPop can be used to model some of the well-known population dynamics models. These models can also be constructed in R and C by following the guideline presented in the previous section.\n\nWe begin with Nicholson’s Blowflies, a classic example of time-delayed stage-structured population model11,16. The model comprises five distinct life-stages and exhibits stable quasi-cyclic oscillations. Although, originally the model was constructed using continuous time-delay equations, we will demonstrate that the sPop model adheres well to the observed dynamics and the implementation presented in the StagePop package11. Furthermore, we will present a stochastic version of the model, which helps to improve our understanding of the observed variation.\n\nBoth the deterministic and stochastic versions of the model are presented in Supplementary File 4 (case_studies.py). The adaptation assumes fixed daily survival and strict development durations, values of which are the same as the original model (Figure 2 in Gurney et al. 198316). A scaling factor is introduced to calculate hourly instead of daily propensities to improve accuracy on a par with the continuous-time simulations.\n\nIn (a), the deterministic trajectories of eggs (dashed line) and mature adults (solid line) are shown for the corresponding generations. In (b), five stochastic trajectories of mature adults are shown for the same duration.\n\nAs a result, the output of the model is almost identical to the output of the original model (compare Figure 2(a) with the Figure 3a of Gurney et al. 198316). The six peaks shown between generations 100 and 300 are matched by the stochastic version of the model (Figure2(b)). However, the amplitude of the oscillations and the relative heights of the minor cycles in each oscillation change drastically at successive generations. Similar fluctuations were also reported by Nicholson (1954)17 in a laboratory culture of the Australian sheep blowfly (also presented in Gurney et al. 198316 Figure 1).\n\nIn (a), the Nicholson-Bailey model (dashed lines) is compared with its age-structured version (solid lines) where parasites choose host in an age-dependent manner. The number of parasites is scaled down to 25% to aid visualisation. In (b), the effect of age-dependent host survival on stability is shown. Mortality rate is given in the inset with respect to age (the number of generations). Dashed line: age-independent mortality; solid green: life expectancy of precisely 10 generations; solid red, teal, and purple: gamma-distributed life expectancy with mean 10 and standard deviation 1.25, 2.5, and 5 generations, respectively.\n\nAnother classic example of age dependency in population dynamics was proposed by 18 as a variation of the host-parasite interaction model of 19. The Nicholson-Bailey model considers dynamics in discrete generations where parasites traverse a given area in search of a host. As a result, the number of parasites in the subsequent generation corresponds to the number of hosts parasitised. Hastings introduced age-structure to the host population and assumed that only juvenile hosts are targeted by parasites.\n\nBoth the original Nicholson-Bailey model and its age-structured version are implemented in Supplementary File 4 (case_studies.py). As shown in Figure 3(a), the original model without age dependency (dashed lines) exhibits oscillations with increasing amplitude around an unstable steady state. The model output matches Figure 10 in Nicholson (1935)19. Introducing age-structure with a fixed survival rate for host results in the stabilisation of dynamics as reported by Hastings (1984)18 and seen in Figure 3(a) (solidlines).\n\nHastings (1984)18 also discusses the disruptive effect of age-dependent host survival in stability. This is evident in Figure 3(b), where the survival imbalance between young and old individuals drives the dynamics away from the steady state, eventually rendering it unstable. When the age-dependent mortality curve is close to being horizontal, the dynamics closely resembles the Hastings’ model with no age limit (solid lines in Figure 3(a) and the dashed line in Figure 3(b)). When mortality is considerably higher in older individuals (red line in Figure 3(b)) or a strict age limit is introduced (green line in Figure 3(b)), the stability is lost and the amplitude of oscillations increase in time.\n\nThe final case we study is the severe outbreak of bubonic plague in Eyam (Sheffield, UK) in 1965–196620. The outbreak was initially modelled by Raggett et al. 198220, and later, a simple deterministic susceptible-infectious-removed (SIR) epidemic model was developed by Brauer et al. 200121 to study the outbreak.\n\nAn age-structured version of the SIR epidemic model where the infectious stage duration is modelled with a gamma distribution is presented in Supplementary File 4 (case_studies.py). As shown in Figure 4, the number of infectious cases with respect to the number of susceptible individuals, the S-I plane, (Figure 4(a)) and the time trajectory of the outbreak (Figure 4(b)) closely follow the data presented in Brauer et al. 200121 Table 9.1.\n\nIn (a), the simulated numbers of infectious versus susceptible individuals are shown together with the outbreak data. In (b), the number of susceptibles and infectious cases are plotted with respect to time for the duration of the outbreak. Model simulations (solid lines) are compared with the outbreak data (dots).\n\nFigure 5 demonstrates the effect of age-structure on outbreak dynamics by comparing model output with different characteristics of the infectious period. The blue lines indicate the trajectory matching the Great Plague in Eyam. Please note that the corresponding infectious period is only slightly time-dependent where there is a minor difference between the mortalities of newly infected and long-time infectious individuals (plotted in the inset). If the rate of exit from the infectious stage is completely independent from the duration spent in the stage (as is the case with canonical SIR models), the outbreak duration increases (the red lines). In the opposite scenario, where the length of the infectious case is precise, the entire outbreak resolves rapidly as seen in the green trajectory. Since time-dependence has a significant impact on outbreak trajectory, using realistic mathematical formulations to infer outbreak parameters, such as the incubation period and the rate of infection, becomes a critical step in developing predictive epidemic models.\n\nThe outbreak trajectory shown in Figure 4(b) (blue lines) is compared with alternative forms of mortality. Red lines indicate time-independence where the gamma distributed infectious period has σ = µ. Green lines indicate that the infectious period has a precise length of µ = 11.71 days (σ = 0).\n\n\nSummary\n\nThe sPop packages are designed to facilitate developing time-delayed age-structured population dynamics models in discrete time domain. The underlying population dynamics model incorporates survival, development, and migration, and it can accommodate both deterministic and stochastic dynamics. In order to promote applications, three versions of the model were implemented: the R and Python implementations are aimed at educational and introductory level use, while the C implementation offers further optimisation and high-speed simulations. This paper demonstrated that the model is capable of representing age-structured population dynamics from a range of disciplines including insect populations, host-parasite/prey-predator interactions, and infectious disease epidemiology. Future research concerns optimising to limit simulation times and implementing the model in continuous time domain for improved accuracy.\n\n\nSoftware and data availability\n\nR implementation of sPop:\n\n• Available from: https://cran.r-project.org/web/packages/albopictus/\n\n• Source code: http://github.com/kerguler/albopictusR\n\n• Archived source code as at time of publication: https://doi.org/10.5281/zenodo.132509514\n\n• License: GPLv3\n\nC and Python implementation of sPop:\n\n• Available from: https://pypi.org/project/albopictus/\n\n• Source code: https://github.com/kerguler/albopictus\n\n• Archived source code as at time of publication: https://doi.org/10.5281/zenodo.132511115\n\n• License: GPLv3\n\nAll data and source code for running the examples and plotting the figures in this manuscript are provided in the Supplementary material.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author would like to thank Murat Can Demirok for testing the packages and providing feedback, and to Johannes (Jos) Lelieveld for valuable feedback on the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: test_spop.R. R script file for Section Implementation:R.\n\nClick here to access the data.\n\nSupplementary File 2: test_spop.py. Python script file for Section Implementation:Python.\n\nClick here to access the data.\n\nSupplementary File 3: test_spop.c. C code file for Section Implementation:C.\n\nClick here to access the data.\n\nSupplementary File 4: case_studies.py. Python script file for Section Use Cases.\n\nClick here to access the data.\n\nSupplementary File 5: plot_test_spop.R. R script file for plotting Figure 1.\n\nClick here to access the data.\n\n\nReferences\n\nRosen G: Time delays produced by essential nonlinearity in population growth models. Bull Math Biol. 1987; 49(2): 253–255. PubMed Abstract \| Publisher Full Text\n\nDelatte H, Gimonneau G, Triboire A, et al.: Influence of temperature on immature development, survival, longevity, fecundity, and gonotrophic cycles of Aedes albopictus, vector of chikungunya and dengue in the Indian Ocean. J Med Entomol. 2009; 46(1): 33–41. PubMed Abstract \| Publisher Full Text\n\nSmith DL, Battle KE, Hay SI, et al.: Ross, macdonald, and a theory for the dynamics and control of mosquito-transmitted pathogens. PLoS Pathog. 2012; 8(4): e1002588. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrouse DT, Crowder LB, Caswell H: A stage-based population model for loggerhead sea turtles and implications for conservation. Ecology. 1987; 68(5): 1412–1423. Publisher Full Text\n\nParham PE, Pople D, Christiansen-Jucht C, et al.: Modeling the role of environmental variables on the population dynamics of the malaria vector Anopheles gambiae sensu stricto. Malar J. 2012; 11: 271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristiansen-Jucht C, Erguler K, Shek CY, et al.: Modelling Anopheles gambiae s.s. Population Dynamics with Temperature- and Age-Dependent Survival. Int J Environ Res Public Health. 2015; 12(6): 5975–6005. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer S, Held L: Incorporating social contact data in spatio-temporal models for infectious disease spread. Biostatistics. 2017; 18(2): 338–351. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStubben C, Milligan B: Estimating and Analyzing Demographic Models Using the popbio Package in R. J Stat Softw. 2007; 22(11): 1–23. Publisher Full Text\n\nJones JH: demogR: A Package for the Construction and Analysis of Age-structured Demographic Models in R. J Stat Softw. 2007; 22(10): 1–28. Publisher Full Text\n\nŠevčíková H, Raftery AE: bayesPop: Probabilistic Population Projections. J Stat Softw. 2016; 75: pii: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKettler H, Nutter D: StagePop: Modelling stage-structured populations in R. Methods Ecol Evol. 2015; 6(12): 1484–1490. Publisher Full Text\n\nErguler K, Smith-Unna SE, Waldock J, et al.: Large-Scale Modelling of the Environmentally-Driven Population Dynamics of Temperate Aedes albopictus (Skuse). PLoS One. 2016; 11(2): e0149282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErguler K, Chandra NL, Proestos Y, et al.: A large-scale stochastic spatiotemporal model for Aedes albopictus-borne chikungunya epidemiology. PLoS One. 2017; 12(3): e0174293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nkerguler: kerguler/albopictusR: The sPop age-structured population dynamics model (Version v1.0.0). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1325095\n\nkerguler, , apolydorides: kerguler/albopictus: Large-scale environment-driven population dynamics and disease spread models for vector-borne diseases (Version v1.0.0). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1325111\n\nGurney WSC, Nisbet RM, Lawton JH: The Systematic Formulation of Tractable Single-Species Population Models Incorporating Age Structure. J Anim Ecol. 1983; 52(2): 479–495. Publisher Full Text\n\nNicholson AJ: An Outline of the Dynamics of Animal Populations. Aust J Zool. 1954; 2(1): 9–65. Publisher Full Text\n\nHastings A: Simple models for age dependent predation. In Simon A. Levin and Thomas G. Hallam, editors, Mathematical Ecology. Berlin, Heidelberg, Springer Berlin Heidelberg. 1984; 114–119. Publisher Full Text\n\nNicholson AJ, Bailey VA: The Balance of Animal Populations.—Part I. Proceedings of the Zoological Society of London. 1935; 105(3): 551–598. Publisher Full Text\n\nRaggett GF: A stochastic model of the Eyam plague. J Appl Stat. 1982; 9(2): 212–225. Publisher Full Text\n\nBrauer F, Castillo-Chavez C: Mathematical models in population biology and epidemiology. Springer, 2 edition, 2010. Publisher Full Text" }	[ { "id": "36953", "date": "28 Aug 2018", "name": "Juliane Liepe", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “sPop: Age-structured discrete-time population dynamics model in C, Python, and R” by Kamil Erguler provides a comprehensive yet well summarised description of a novel software tool to model age-structured discrete-time population dynamics.\n\nThe software tool is implemented in three different programming languages: R, Python and C. All three languages are most commonly used in scientific programming and therefore the presented tools are easily applicable and adaptable for a wide range of users. The manuscript highlights well the differences in implementation for the three programming languages. While R and Python implementations are easy to use and provide an excellent introduction to modellers new to age-structured population dynamics models, the C implementation provides computational speed, which is particularly relevant for parameter optimisation problems in this field.\n\nThe manuscript guides the reader through three examples of the Python implementation. For each example, all necessary scripts are provided to reproduce the figures and the results described. While I have not had much experience with age-structured population dynamic models, I found it straight forward to follow the concept of the underlying model described in the manuscript.\n\nOverall this manuscript is well written and provides a hands-on guide to scientists to implement the discussed methods into their existing workflow.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "39332", "date": "22 Oct 2018", "name": "Matthew Silk", "expertise": [ "Reviewer Expertise Disease ecology", "Wildlife demography", "Animal social structure and behaviour" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Erguler introduces the software tool that can be applied to modelling discrete time, age-structured population dynamics in R, Python and C. The basics of using the software in all three programming languages are well covered and the examples of its use and compelling and clearly explained. The code and equations provided seemed accurate and relevant (albeit with the caveat that I am an R user only, and an ecologist rather than mathematical biologist).\nI feel that a bit more background provided in the introduction would be beneficial, in particular an overview of the research areas where these models are used (as per the examples) and the reasons that age-structured models can be important to use.\nIt would also be good to see some of the caveats/constraints of this sort of modelling framework discussed at some point so that a reader was better able to decide if it is a useful tool for them.\nFinally, there were a couple of places where I felt a bit more detail would be helpful:\nAs someone who doesn't work on insects, a clarification of what constitutes a development cycle would be helpful.\n\nIt would be helpful to more clearly explain the perturbate() function and perhaps provide an illustrative example of when it would be used.\n\nIn the Nicholson's blowflies example it is not clear to me from the figure that relative heights of the minor cycles are \"changing drastically\". I feel this needs to be better depicted or explained.\n\nWhen introducing the plague model it would be helpful to explain how it is age-structured and clarify that this applies to the pathogen.\n\nOtherwise I have a set of very minor changes/suggestions.\nIntroduction: Paragraph 1 - Suggest editing the first sentence to say that populations are heterogeneous, and one of the ways that they are heterogeneous is because age can cause differences among individuals. This feels like a clearer description to me. Paragraph 1 - Add \"For example,\" to the start of the second sentence. Paragraph 1 - Suggest the end of the first paragraph needs some references to support this point. Paragraph 2 - Sentence starting \"This approach\" is difficult to read; perhaps split up to cover the deterministic/stochastic and discrete/continuous points separately?\n\nImplementation: In the R section in paragraph starting \"By defining...\" - \"THE same principles apply....\" In the C section in paragraph starting \"The macro...\" - \"unoptimised implementations of the NEGATIVE binomial\"\nUse Cases: Paragraph 1 - Change guideline to guidelines in final sentence. Age-structured host-parasite interactions - clarifying what Hastings was modelling originally would be helpful - title of reference says predators while in the text it says parasites? Paragraph starting \"Hastings (1984)...\" - change resembles to resemble. Eyam example - It was in 1665-1666 not in 1965-1966 as currently stated in the text.\nSummary: First sentence of the summary reads awkwardly and could do with rephrasing. Figure 4: One of the points is only partially on each graph - it would be good to change this. Figure 4 legend: Misspelling of Eyam Overall this manuscript is clearly written and explained and introduces a potentially useful piece of software available to a wide range of programmers.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "4264", "date": "13 Dec 2018", "name": "Kamil Erguler", "role": "Author Response", "response": "I would like to thank Dr Silk for reviewing the manuscript and for his valuable comments and suggestions. I have submitted a revised version with an aim to address the issues raised in his review. My reply to the specific points raised can be seen below. The rest of his suggestions concerning minor corrections have been addressed in the manuscript accordingly. Addressing specific issues raised in the review: I feel that a bit more background provided in the introduction would be beneficial, in particular an overview of the research areas where these models are used (as per the examples) and the reasons that age-structured models can be important to use. Paragraph 1 - Suggest editing the first sentence to say that populations are heterogeneous, and one of the ways that they are heterogeneous is because age can cause differences among individuals. This feels like a clearer description to me. Paragraph 1 - Add \"For example,\" to the start of the second sentence. Paragraph 1 - Suggest the end of the first paragraph needs some references to support this point. The revised version of the manuscript presents an extended introduction with additional links to the relevant literature and use cases. In addition, the definition of the model is revised and elaborated in the Methods section to convey in a more comprehensive manner the potential areas of use of the proposed modelling strategy. I believe the revised manuscript better describes the approach and places it in the right context. It would also be good to see some of the caveats/constraints of this sort of modelling framework discussed at some point so that a reader was better able to decide if it is a useful tool for them. I added a section named Temporal resolution and accuracy to discuss the effect of time step size on the accuracy of numerical simulations. The section concludes with the assessment that the numerical error increases linearly with increasing step size. I believe that this will help readers to determine an appropriate step size for optimum accuracy given the availability of computational resources. As someone who doesn't work on insects, a clarification of what constitutes a development cycle would be helpful. Development cycle is a concept introduced to the model to accommodate certain biological processes not necessarily related to insect development. In this revision, I used the analogy of human pregnancy to elaborate on the concepts of age (age of the mother), degree of development (duration of her pregnancy), and development cycle (the number of completed development processes which corresponds to the number of births the mother has given in this analogy). I believe that the analogy helps readers better conceptualise these processes and apply them to their work. It would be helpful to more clearly explain the perturbate() function and perhaps provide an illustrative example of when it would be used. In this version of the manuscript, I renamed the method as \"perturb\", elaborated the discussion on its intended use, and provided a simple example to demonstrate how it can be used to model migration. In the Nicholson's blowflies example it is not clear to me from the figure that relative heights of the minor cycles are \"changing drastically\". I feel this needs to be better depicted or explained. I thank Dr Silk for pointing out this issue. In this version, I elaborated on the description of the variability in stochastic simulations and its likely correspondence to the observations of laboratory populations. I believe that readers will find this an improvement. When introducing the plague model it would be helpful to explain how it is age-structured and clarify that this applies to the pathogen. In this version of the manuscript, I explained that the canonical SIR model is not age-structured; however, the underlying transmission dynamics might be better represented with an age-structured model. Especially in a case where disease duration or incubation period is of a certain length, the sPop packages are able to track each individual and trigger a state change only for the right ones and only when it is due. I believe that this revised version offers a clearer description of this capacity. Introduction: Paragraph 2 - Sentence starting \"This approach\" is difficult to read; perhaps split up to cover the deterministic/stochastic and discrete/continuous points separately? Instead of various modelling frameworks, this version of the manuscripts presents examples of various contexts where intermittent stages have been used. Use Cases: Age-structured host-parasite interactions - clarifying what Hastings was modelling originally would be helpful - title of reference says predators while in the text it says parasites? In this version of the manuscript, I stated that Hastings was modelling prey-predator interactions based on the host-parasite model, and presented the analogy between host-parasite and prey-predator interactions as considered by Hastings in his derivations. Summary: First sentence of the summary reads awkwardly and could do with rephrasing. The sentence is rephrased. Figure 4: One of the points is only partially on each graph - it would be good to change this. All Python-generated figures are re-plotted and case_studies.py is updated to improve the visibility of the data points." } ] } ]	1	https://f1000research.com/articles/7-1220
https://f1000research.com/articles/9-1138/v2	25 Sep 20	{ "type": "Research Article", "title": "Mental health status and quality of life among Cambodian migrant workers in Thailand", "authors": [ "Wongsa Laohasiriwong", "Pall Chamroen", "Sim Samphors", "Thiwakorn Rachutorn", "Rebecca S. Dewey", "Vong Pisey", "Wongsa Laohasiriwong", "Sim Samphors", "Thiwakorn Rachutorn", "Rebecca S. Dewey", "Vong Pisey" ], "abstract": "Background: Migrant workers have become a major issue for Thailand. Most of the migrants are from Myanmar, Cambodia, and Laos. Most are employed in jobs referred to as the “3 Ds”; difficult, dangerous and dirty. However, little is known concerning the living and working conditions, or health-related quality of life of these migrant workers. This study aims to determine factors influencing the quality of life of Cambodian migrant workers in Thailand. Methods: A cross-sectional study was conducted among 1,211 Cambodian migrant workers in Thailand, using multistage random sampling from eight districts of the two provinces (Sa Kaeo and Surin) with a structured questionnaire interview. The WHOQOL-BREF was used to measure Quality of Life (QOL) with Cronbach’s alpha of 0.77. Mental health status was assessed using the Perceived Stress Scale (PSS) and Center for Epidemiological Studies-Depression (CES-D) scale with Cronbach’s alpha of 0.83. Descriptive statistics provide participant characteristics. Multilevel logistic regression (MLR) were used to determine which factors significantly impacted the outcome measures in terms of the adjusted odds ratio (AOR). P<0.05 was considered statistically significant. Results: About one third of these migrant workers had a poor quality of life (34.52%; 95%CI: 31.84-37.20), and had moderate-to-high levels of stress (67.96%; 95%CI: 65.33-70.59), and symptoms of depression (69.69%; 95%CI: 67.10-72.29). After controlling other covariate factors, the factors associated with poor QOL were a high level perceived of stress (AOR=3.64; 95%CI: 2.41-5.49; p<0.001); living with family and relatives (AOR=3.63; 95%CI: CI 2.42-5.45; p<0.001); and housing being provided by their employer (AOR=2.66; 95%CI: 1.74-4.08; p<0.001). Conclusion: Stress was strongly associated with QOL. The living environment was found to be the next most influential factor on QOL. Mental health programs aimed at helping migrant workers to cope with stress and to improve their living conditions will help improve QOL in the target group.", "keywords": [ "Quality of Life", "Mental Health", "Adaptation", "Transients and Migrants", "Cambodian migrant workers" ], "content": "Introduction\n\nMigration has become a critical global issue. Reports state the arrival into Thailand of more than 3.5 million migrant workers from Myanmar, Cambodia and Laos, most of whom are employed in jobs described as the “3 Ds” (difficulty, dangerous and dirty), with low pay that would not attract most native Thai employees1,2. According to the Thailand Migration Report, 2011, 41.0% of migrant workers are employed in industry, 27.6% in agriculture, and 31.4% in other services. Because of their working status and only having access to certain sectors of employment, most migrant workers are frequently exposed to hazardous and dangerous conditions: chemical use in agriculture, poor working conditions in industry, forced long hours and work overload. This is coupled with the potential for deportation, arrest, workplace accidents, violence or abuse, and living and working in dirty, dangerous, unhealthy, unclean, uncomfortable or unfavorable conditions, often resulting in illness, disability and death. 90% of low-skilled migrant workers have low levels of educational attainment3,4.\n\nAt present, over one-third of the Cambodian population are migrants to Thailand. Almost half the Cambodian population remains in poverty, with 80% of them are living in rural areas where their quality of life is likely to be lower than those living in urban areas. Migration to Thailand is reported to provide a better quality of life than other migrant worker destinations5–7. However, migration abroad is associated with a worse financial status than working in Phnom Penh due to high levels of stress incurred in relation to money, their future, and consequently the impact on mental health causing a decrease in quality of life8.\n\nThe term quality of life (QOL) refers to the level of an individual’s standard of health, comfort, and happiness. It is a multidimensional construct, involving physical health, psychological health, social relationships, and environmental domains, as defined by the World Health Organization9. Studies on the psychological impact of the working and living environments report low-to-medium QOL levels within migrant workers working in various occupations in Thailand10–12, and adult garment manufacture in Bangladesh13. This has been associated with health difficulties of migrant workers in France14, low-to-medium QOL in rural-to-urban female migrant factory workers in China15, and with an impact on social relationships among Burmese domestic female workers in Singapore16. Agricultural workers have frequently reported work-related injuries and occupational-health issues related to stress and depression17,18. Nearly one third of Cambodian farm workers in eastern Thailand reported occupational injury (back pain/joint pain), and most had limited access to healthcare services19, likely compounding the impact on QOL.\n\nHowever, while most studies have focused on Burmese migrant workers or on workers of unspecified nationality, there has been little attention paid to Cambodian migrant workers, especially on their mental health status and QOL20. Little is known concerning their living and working conditions and health-related QOL. This study aims to determine the factors influencing QOL within Cambodian migrant workers in Thailand.\n\n\nMethods\n\nA cross-sectional study was conducted by using a multistage random sampling method to choose the study sample. Data were collected between March 2018 and May 2018. Cambodian migrant workers are required to register with the Department of Employment Office in each province in order to benefit from Universal Health Coverage (UHC), and to be bestowed with legal working status in Thailand. We applied for and gained permission to use this list from the Department of Employment Office in Thailand. For Sa Kaeo and Surin provinces, a total of 24,256 Cambodian migrant workers were listed during time of study. Finally 1,211 sample size was calculated following the formula from Hsieh FY et al.. (1998) as follows:\n\n\n\nThe approximate sample size was 435 which were further adjusted to control the over-fitting using the rho (ρ) of 0.65 and variance inflation factor (VIF) equal to 2.85. Therefore, the total number of the sample was 1,211. The sample size was calculated to be 1,211. The sampling process was achieved as follows: (I) Sa Kaeo and Surin provinces were randomly selected from the total number of 7 eastern and northeastern Thai provinces that share a boarder with Cambodia, representing a total migrant population in the two provinces of 24,256 (Sa Kaeo represents 89% of this with 21,619 migrant workers, and Surin the remaining 11%, with 2,637 migrant workers). (II) The sample size was split by proportion to the relative populations of the two provinces, with 1,053 samples allocated to Sa Kaeo province and 158 to Surin. (III) Simple random sampling was used to select 8 out of 26 districts. (IV) Systematic random selections were made from the name lists of the total migrant worker population in each of these districts until the completed sample size was achieved.\n\nInclusion criteria: All participants in the study were 18 years of age or above, and had been working in Thailand for at least 1 month before the interview period. Having the required legal documents including a passport, work-permit, border pass required for legal migrant workers, and good physical health and mental health and were willing to participate in the study.\n\nExclusion criteria: Any person with a disability was excluded. Participants with unofficial documents and who were not willing to participate in the study were also excluded.\n\nThe data was collected through in-person interviews conducted by trained interviewers. Interviews took place between March 2018 and May 2018. Prior to data collection five research assistants were trained one-day training on the study objectives and how to administer the questionnaires. Once they understood the data collection process research assistants were paired to test the questionnaire on each other to further ensure they were familiar with all parts of the questionnaire.\n\nAll participants were informed about the purpose, benefits and assured of confidentiality before signing the consent for the study. For convenience, researcher also asked permission from the employer of the migrant workers to interview participants at time that would minimize disruption to their working hours. If participants were illiterate researchers asked a literate volunteer to witness the accurate reading of the consent form, allow the participant to ask any questions and then subsequently signed on their behalf. This study was approved by the Khon Kaen University (KKU) committee for research ethics in human research (Reference no. HE602361), Khon Kaen, Thailand.\n\nA structured questionnaire was developed from reviewed literatures based on research questions, first in English and was then translated into Khmer using forward and backward translation procedures. The questionnaire consisted of 4 sections which were a) individual characteristics and socio-demographic factors, namely gender, age, marital status, educational attainment, occupation, financial status, work environment, incidence of work injury, residential arrangement, house tenure, distance from house to community center (km), daily travel to work, the incidence of work-related diseases during past 12 months, and smoking status. A copy of the questions asked are provided as extended data, b) the Perceived of Stress Scale (PSS) of Cohen et al. (1983), d) depression Scale (CES-D) of Radloff et al. (1977) with Cronbach’s alpha of 0.83, and c) WHOQOL-BREF Khmer version with Cronbach’s alpha of 0.77. The questionnaire was undergone content validation by five experts and was revised to improve validity.\n\nDemographic characteristics of the participants were described using frequency and percentage for categorical data and using mean and standard deviation for continuous data. Inferential statistics comprising simple logistic regression bivariate and multivariate models were used in a multilevel mixed-effects model to reduce clustering effects. Confidence intervals (CI) were taken at 95% and statistical significance was considered at p<0.05. All analyses were performed using Stata version 10.0 (Stata Corp, College Station, TX).\n\n\nResults\n\nOf the 1,211 Cambodian migrant workers, 50.37% were male and 50.29% were working in the agricultural sector. The mean age was 32.54 (±11.13) years (range: 18–67) (Table 1). The majority of respondents were married (62.59%). Most had no formal education (42.69%). The median monthly personal income was 7,500 (range: 7,500-20,000) Baht (equivalent to 237 USD at time of publication; Table 1). Mental health problems were common, with 57.72% of participants reporting moderate levels of perceived stress (32.04% low level and 10.24% high level). Many respondents reported symptoms of depression (69.69% compared to 30.31% not reporting these symptoms) (Table 2). Most of the participants (55.33%) had a moderate QOL level, 34.52% had low QOL and 10.16% had high QOL (Table 3).\n\n(≥16)=Cutoff score of 16 or higher on CES-D is indicative of depression symptoms.\n\nn: number of participants.\n\nThe bivariate analysis using simple logistic regression (SLR) indicated that having a high perceived stress score and having depression symptoms were significantly associated with poor QOL (p<0.001 and p=0.001 respectively). However, sex, age, marital status, educational attainment, job category, financial status, working environment, suffering a work injury in the past 12 months, residential arrangement, house tenure, distance between house and community center, daily travel to work, suffering work-related diseases in the past 12 months, and smoking were also all significantly associated with poor QOL (Table 4).\n\nThe final model was constructed using multivariate analysis, controlling for clustering effects using multilevel logistic regression (MLR) and control covariates. This final model indicated that factors significantly associated with poor QOL were a high perceived stress level (AOR=3.64; 95%CI: 2.41-5.49; p<0.001); living with family/relatives (AOR=3.63; 95%CI: 2.42-5.45; p<0.001); living in housing provided by their employer (AOR=2.66; 95%CI: 1.74-4.08; p<0.001); commuting to work using their employer’s vehicle or by foot (AOR=1.64; 95%CI: 1.11-2.42; p=0.012); and living more than 1 km from the community center (AOR=1.41; 95%CI: 1.07-1.87; p=0.016). However, having symptoms of depression was not associated with poor QOL [Table 1–Table 5].\n\n Simple logistic regression (SLR) shows crude OR\n\n** Multilevel logistic regression (MLR) shows Adjusted OR: 95%CI: CI and p-value after adjusted for other covariates factors.\n\n\nDiscussion\n\nThe prevalence of low QOL in Cambodian migrant workers was 34.52% (95%CI: 31.84-37.20) with moderate QOL at 55.33% (95%CI: 52.52-58.13) and high QOL at 10.16% (95%CI: 8.45-11.86). This result is in contradiction to a previous study on the QOL of Burmese migrant workers in the Chiang Rai province of Thailand10, which reported 0.20% as having low QOL, 56% moderate QOL and 43.8% high levels of QOL. These differences in the prevalence of low QOL may be due to the target population of the previous study being mainly women between 18 and 29 years old, mostly working in industry. The working and living environments of those in industry are less objectionable when compared to that of agricultural workers. In contrast, the population surveyed in the present study had a gender balance nearer parity, mostly within the age range 20 to 49 years, and half of the sample worked at agriculture. Moreover, the socio-cultural settings of Cambodian migrant workers and Burmese migrant workers are different. The findings of the present study are in agreement with a study conducted in Phang-Nga province of Thailand that also reported most migrant workers as having low to moderate QOL12. A study in Dhaka city, Bangladesh showed that 94% of adult migrant workers in garment manufacture had low QOL, with only 3.25% and 2.75% having moderate and high QOL respectively. This may be due to the study in Bangladesh recruiting more female than male workers; within Indianite cultures, the female gender is devalued and females are perceived as the inferior gender, both physically and psychologically, resulting in very low QOL compared to males13. The above results demonstrate the inconsistency of QOL findings across the literature. This inconsistency likely results from the multicultural differences in context and setting of the living and working environments of migrant workers. Another example is the report of female domestic migrant workers in Singapore exhibiting a high QOL within three domains out of four, with the social relationship domain exhibiting a low score and also being associated with stress16 [Figure 1].\n\nMoreover, mental health problems were reported among migrant workers. The prevalence of moderate to high perceived stress was 67.96% (95%CI: 65.33-70.59) and symptoms of depression was 69.69% (95%CI: 67.10-72.29). A comparable study in Europe21 reported 63% of low-skilled workers to exhibit symptoms of distress. Moreover, stress and depression are often cited as the most predominant factors associated with reduced QOL scores, with some studies finding depression to be the most influential on QOL, and vice-versa22. Additionally, stress is commonly reported to impact QOL for migrant workers; such as 62.2% of white-collar migrant workers in China reporting work related-stress23.\n\nAfter controlling for other covariate factors, the factors that were indicated as being significantly associated with poor QOL were high perceived stress (AOR=3.64; 95%CI: 2.41-5.49; p<0.001); living with family/relatives (AOR=3.63; 95%CI: 2.42-5.45; p<0.001); living in employer-provided housing (AOR=2.66; 95%CI: 1.74-4.08; p<0.001); commuting to work using their employer’s vehicle or by foot (AOR=1.64; 95%CI: 1.11-2.42; p=0.012); and living more than 1 km from the community center (AOR=1.41; 95%CI: 1.07-1.87; p=0.016). However, having symptoms of depression was not significantly associated with poor QOL in the final model.\n\nFactors associated with perceived stress have a strong association with poor QOL (AOR=3.64; 95%CI: 2.41-5.49; p<0.001). In stressful situations, migrant workers may turn to alcohol and smoking. This in turn will increase the economic burden, impact on their capacity to work, may cause conflicts in the family, and can sometimes be associated with gambling. Moreover, stressful situations themselves may cause workers to lose focus on their job and worry about earning money to feed their family or reduce the debt incurred in the process of migration. Many workers worry about the expiration of work permits and health insurance documents. A study in China has shown that work related stress was associated with poor QOL15,23. A previous study among migrant workers in India found 14.6% workers to have poor QOL and 25.5% to be in psychological distress, with QOL being significantly (p≤0.05) associated with the factors of age, marital status, low education status24. Stress was associated with both depression and anxiety study among Burmese migrant workers on the borders25. Lifestyle factors were also found to impact migrant workers in the study, for example smoking was associated with low QOL. Smoking was common among the male migrant workers surveyed in the present study in terms of the habitual lifestyle associated with a stressful working environment. Smoking severely increases the economic and health burden on migrant workers. In the study rural to urban Chinese migrant workers, it was found that unhealthy lifestyles caused by many factors, such as the kind of job that they were doing, working in too small a space, low income and long working hours, were significantly related to the poor mental health of migrant workers26.\n\nLiving with family and relatives (AOR=3.63; 95%CI: 2.42-5.45; p<0.001) means that migrant workers have greater levels of responsibility in their daily life. In a Bangladeshi study, living with at least one family member was highly associated with low QOL (p=0.01)13. In the context of Cambodia, migrant workers often live in a small cottage where they work together with many family members, relatives or friends. If this living space becomes too crowded, workers will have difficulty sleeping and further decreased QOL.\n\nHaving their living accommodation provided by their employer (AOR=2.66; 95%CI: 1.74-4.08; p<0.001) was more likely to be associated with low QOL than living in rented housing. In the present study, many migrant workers who lived inside farmworker camps or construction site camps were reported to live in employer-owned accommodation, and also were more likely to be exposed to hazardous living environments27. There are many potential factors associated with this. For example, in worker camps, even though most employer provide employees with a living place free of charge, migrant workers have to build the cottage themselves, often resulting in poor build-quality in terms of dirt, dust and a lack of mains electricity supply. Living together with strangers is not good for psychological health, and often migrant workers from Cambodia end up cohabiting with many colleagues from different places. However, renting a house may cause a low QOL depending on the study context13. Migrant workers in China showed that residential satisfaction was at a moderate level28.\n\nCommuting daily to the workplace using their employer’s vehicle or on foot (AOR=1.64; 95%CI: 1.11-2.42; p=0.012) was more highly associated with low QOL compared to travelling to work using their own vehicle. Daily basic transportation was important among migrant workers where most were living in isolated rural areas. A study among Burmese migrant workers in Thailand found transportation difficulties to be barriers to accessing health services29.\n\nLiving further than 1 km away from the community center was associated (AOR=1.41; 95%CI: 1.07-1.87; p=0.016) with low QOL. It might be that living far from a community center makes it difficult to buy food or to access essential services. Migrant workers who did not own vehicles in Thailand, like bicycles or motorbikes, were more likely to have a low quality of life. This may be further confounded by an unfamiliarity with the area where they live and work, making it difficult to locate the community center if they live far from it. This will make it more difficult when they want to buy food at the market or access to other services in Thailand. Similarly, a study of workers in Russia showed that distance from house to workplace had a significant impact on job satisfaction30.\n\n\nStudy limitations\n\nThis cross-sectional study design was conducted only on a subset of the border provinces in Thailand and therefore may not be generalizable to other settings of migrants in Thailand, or more widely. Moreover, a cross-sectional study design only shows associations, where longitudinal studies will make evident the cause and effect relationships between risk factors and health in this population. Furthermore, study of work related-injuries and mental health problems of migrant workers is required to ascertain the healthcare needs of this population.\n\n\nConclusions\n\nIn summary, workers who participated in the present study had poor health-related QOL, particularly in the environment domain. It could be concluded that perceived stress and living condition are predicators of health-related QOL. Consequently, to improve worker’s health-related QOL, interventional programs should focus on mental health by providing coping strategies for stress and strategies for improving the living environment, obtaining suitable accommodation, providing transportation facilities enabling migrant workers to access essential services, with additional focus on those migrants who do not live directly within the community.\n\n\nRecommendations\n\nThe findings from this study should be used to inform the development of interventional programs to improve QOL in migrant workers, and provide a crucial direction for future research studies to build on the knowledge in this field.\n\n\nData availability\n\nFigshare: 1. Dataset 1 Raw data from a survey mental health status and quality of life among Cambodian migrant workers in Thailand.xlsx. https://doi.org/10.6084/m9.figshare.12769856.v2\n\n- Dataset 1: Raw data from a survey mental health status and quality of life among Cambodian migrant workers in Thailand.xlsx (Questionnaire responses)\n\n- Code book for interpreting the data.docx (Codebook for dataset)\n\nFigshare: 1. Dataset 1 Raw data from a survey mental health status and quality of life among Cambodian migrant workers in Thailand.xlsx. https://doi.org/10.6084/m9.figshare.12769856.v2\n\nThis project contains the following extended data:\n\n- 2. Q_En.docx (Study questionnaire, English translation)\n\n- 2.1 Q_KH.docx (Study questionnaire)\n\n- 3. Information sheet(EN).docx (Study information sheet)\n\n- 3.1 Informed consent(EN).docx (Study consent form)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nMigration IOf: World Migration Report 2018. 2017. Reference Source\n\nHuguet JW, Chamratrithirong A: Thailand migration report 2014. Reference Source\n\nHuguet JW, Chamratrithirong A: THAILAND MIGRATION REPORT 2011. Reference Source\n\nOffice of Foreign Workers Administration, Department of Employment, Ministry of Labor. 2017.\n\nUnited Nations Development Programme. About Cambodia. 2018. Reference Source\n\nChaisuparakul S: Life and Community of Cambodian Migrant Workers in Thai Society. Journal of Population and Social Studies. 2015; 23(1): 2–16. Publisher Full Text\n\nYoungran YB, Bekemeier B, Choi J: Health-related Quality of Life and Related Factors among Rural Residents in Cambodia. Iran J Public Health. 2017; 46(3): 422–424. PubMed Abstract \| Free Full Text\n\nPolicy Briefs on Internal Migration in Southeast Asia. About Cambodia. 2018; Reference Source\n\nWHOQOL-BREF W: Introduction, Administration, Scoring and Generic Version of the Assessment—Field Trial Version. Geneva, Switzerland. 1996. Reference Source\n\nTongprasert M: Factors related to health-related quality of life among adult Myanmar migrant workers at Chiang Rai Regional Hospital and Pirom Clinic in Muang district, Chiang Rai province, Thailand: a cross-sectional study. Chulalongkorn University. 2009. Reference Source\n\nAung T, Pongpanich S, Robson MG: Health seeking behaviors among Myanmar migrant workers in Ranong Province, Thailand. J Health Res. 2009; 23(suppl): 5–9. Reference Source\n\nTi S, Somrongthong R: HEALTH RELATED QUALITY OF LIFE OF MYANMAR MIGRANTS IN TAKUAPA AND KURABURI DISTRICTS, PHANG-NGA PROVINCE, THAILAND. J Health Res. 2008; 22: 79–83. Reference Source\n\nIslam M, Ahmed S, Sarker RN, et al.: Health-related quality of life among adult migrant garment workers in Dhaka City. Bangladesh Medical Journal. 2011; 40(3): 14–17. Reference Source\n\nBaumann M, Chau K, Kabuth B, et al.: Association between health-related quality of life and being an immigrant among adolescents, and the role of socioeconomic and health-related difficulties. Int J Environ Res Public Health. 2014; 11(2): 1694–1714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu C, Geng Q, Yang H, et al.: Quality of life in China rural-to-urban female migrant factory workers: a before-and-after study. Health Qual Life Outcomes. 2013; 11(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnjara SG, Nellums LB, Bonetto C, et al.: Stress, health and quality of life of female migrant domestic workers in Singapore: a cross-sectional study. BMC Womens Health. 2017; 17(1): 98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamos AK, Carlo G, Grant K, et al.: Stress, Depression, and Occupational Injury among Migrant Farmworkers in Nebraska. Safety (Basel). 2016; 2(4): 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiott AE, Grzywacz JG, Davis SW, et al.: Migrant farmworker stress: mental health implications. J Rural Health. 2008; 24(1): 32–39. PubMed Abstract \| Publisher Full Text\n\nThetkathuek A, Jaidee W, Jaidee P: Access to Health Care by Migrant Farm Workers on Fruit Plantations in Eastern Thailand. J Agromedicine. 2017; 22(3): 189–199. PubMed Abstract \| Publisher Full Text\n\nSatawedin P: Health Communication Issues among Migrant Workers in Thailand: A Systematic Review for Health Communication Practices. BU Academic Review. 2017; 16(1): 87–100. Reference Source\n\nEspinoza-Castro B, Vásquez Rueda LE, Mendoza Lopez RV, et al.: Working Below Skill Level as Risk Factor for Distress Among Latin American Migrants Living in Germany: A Cross-Sectional Study. J Immigr Minor Health. 2018; 21(5): 1012–1018. PubMed Abstract \| Publisher Full Text\n\nWong WK, Chou KL, Chow NWS: Correlates of Quality of Life in New Migrants to Hong Kong from Mainland China. Soc Indic Res. 2012; 107(2): 373–391. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai SY: A study of the health-related quality of life and work-related stress of white-collar migrant workers. Int J Environ Res Public Health. 2012; 9(10): 3740–3754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGeethu Mathew NR, Ramesh N, Shanbhag D, et al.: Quality of life and probable psychological distress among male workers at a construction site, Kolar district, Karnataka, India. Indian J Occup Environ Med. 2016; 20(1): 54–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer SR, Decker MR, Tol WA, et al.: Workplace and security stressors and mental health among migrant workers on the Thailand-Myanmar border. Soc Psychiatry Psychiatr Epidemiol. 2016; 51(5): 713–723. PubMed Abstract \| Publisher Full Text\n\nYang H, He F, Wang T, et al.: Health-related lifestyle behaviors among male and female rural-to-urban migrant workers in Shanghai, China. PLoS One. 2015; 10(2): e0117946. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArcury TA, Quandt SA: Living and working safely: challenges for migrant and seasonal farmworkers. N C Med J. 2011; 72(6): 466–70. PubMed Abstract \| Free Full Text\n\nGan X, Zuo J, Ye K, et al.: Are migrant workers satisfied with public rental housing? A study in Chongqing, China. Habitat Int. 2016; 56: 96–102. Publisher Full Text\n\nTschirhart N, Nosten F, Foster AM: Migrant tuberculosis patient needs and health system response along the Thailand-Myanmar border. Health Policy Plan. 2017; 32(8): 1212–1219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpies M: Distance between home and workplace as a factor for job satisfaction in the North-West Russian oil industry. Fennia-International Journal of Geography. 2006; 184(2): 133–149. Reference Source" }	[ { "id": "74162", "date": "08 Dec 2020", "name": "Patchana Hengboriboonpong Jaidee", "expertise": [ "Reviewer Expertise migrant health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary of the article:\nThis paper is the original research, and clearly important to public health workers and policymakers. The aim of the study and the measuring outcomes are defined with appropriate references to the literature.\n\nStudy design:\nThe study design and sample stated that “Cambodian migrant workers are required to register with the Department of Employment Office in each province in order to benefit from Universal Health Coverage (UHC)”. This sentence should be reconsidered about the compulsory migrant health insurance and the Social Security Scheme in Thailand for the migrant workers as the work of McMichael C. & Healy J. (2017)1, Pudpong N. et al. (2019)2, and the United Nations Thematic Working Group on Migration in Thailand (2019).3\n\nMethod:\nA cross-sectional study was appropriated to define the multistage random sampling but from Hsieh FY et al. (1998)’s formula, the authors should explain more about the parameter referenced for calculation of the sample size by simple logistic regression formula and the reference of rho and VIF for adjusting the sample size.\n\nResults:\nThe results showed the final model in table 5 as well as in figure 1, therefore it should be considered that the only illustration should be presented. In my opinion, I will present figure 1 to show the final model.\n\nDetails of the control covariates in the result section should be addressed below the related tables or figures.\n\nDiscussion:\nConsider referring to the work of Wong WKF, Chou KL. & Chow NWS. (2012)4 and Leiler A. et al (2019)5, who found the association between depression and poor QOL. Therefore, please be more specific in detail about how the depression was not related to the quality of life in the discussion topic of the manuscript.\n\nThe minor issues that should be addressed:\n\nIn the details of the research instruments, it showed the section of the research questionnaire. Please check the sequential writing of sections from a,b,c, and d.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "72254", "date": "08 Dec 2020", "name": "Sari Andajani", "expertise": [ "Reviewer Expertise Public health", "health promotion", "global health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is well written. It includes relevant literatures, appropriate methodology and the results are well linked to the conclusion section.\nAs an international -non-Thai readers, I would appreciate more review on current migrant policies in Thailand and possible some historical connection of migrant groups from Cambodia coming to Thailand.\nThis then can be linked to the conclusion, for to influence some policy changes or bilateral collaboration between countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	2	https://f1000research.com/articles/9-1138
https://f1000research.com/articles/9-1170/v1	24 Sep 20	{ "type": "Research Article", "title": "Characterization of flow cytometric immuno-phenotyping of acute myeloid leukemia with minimal differentiation and acute T-cell lymphoblastic leukemia: A retrospective cross-sectional study", "authors": [ "Enass Abdul Kareem Dagher Al-Saadi", "Marwa Ali Abdulnabi", "Faris Hanoon Jaafar", "Marwa Ali Abdulnabi", "Faris Hanoon Jaafar" ], "abstract": "Background: Acute leukemias (ALs) are a heterogeneous group of malignancies with various clinical, morphological, immunophenotypic, and molecular characteristics. Distinguishing between lymphoid and myeloid leukemia is often performed by flow cytometry. This study aimed to evaluate the immunophenotypic characterization and expression of immuno-markers in both acute myeloid leukemia (AML-M0) and acute T-cell lymphoblastic leukemia (T-ALL). Methods: A retrospective cross-sectional study was conducted in the Pathology Department/Teaching Laboratories/Medical City/Iraq and included all patients newly diagnosed with AL from 5 January to 10 December 2018. Immunophenotypic analysis was performed on bone marrow samples, freshly collected in EDTA tubes. Flow cytometry (Canto-2 BD) was used, with laser excitation of blue and red wavelengths. A panel of monoclonal antibodies (MoAbs) was used for diagnosis, using a SSC/CD45 gating strategy. Results: The study showed 41.6% of AML-M0 patients had no aberrant antigen expression, while 33.3%, 16.6%, 8.3%, and 8.3% had aberrant CD7, CD56, CD2, and CD19, respectively. In 16.6% of AML-M0 cases more than one aberrant antigen was expressed. With regard to T-ALL, 7.0% were pro-T type, 58.0% were pre-T, 13.0% were cortical, and 22.0% were mature-T type. In 55.5% of patients with T-ALL there was no aberrant antigen expression. Conclusion: We concluded that most patients with AML-M0 have no aberrant antigen expression. In patients with T-ALL, the pre-T type is most common, according to the European Group for the Immunological Classification of Leukemias (EGIL) classification. Patients with T-ALL also generally lack aberrant antigen expression.", "keywords": [ "Acute leukemia", "Immuno-pheno-typing", "Acute lymphoblastic leukemia", "Multi-parameter flowcytometry" ], "content": "Introduction\n\nThere are many characteristic categories of leukemia, which have well known prognoses and specific treatments. Differentiation between lymphoid and myeloid leukemia is performed using flow cytometry1. The analysis of acute leukemia immunophenotypic done by flow cytometry, this method recently become a tool for distinguishing between myeloid and lymphoid lineages. Several types of acute leukemias identified morphologically with or without enzyme cytochemical analysis, but immunophenotyping stay a vital for the right identification of myeloid lineages in minimally differentiated acute myeloid leukemia (AML-M0) and the differentiation of B or T-cell lineages in acute lymphoblastic leukemia (ALL)2. Flow cytometry (FC) is a newer technology that can be used to detect many CD markers in or on a single malignant cell, and also possesses multi-parametric capabilities for combining the study of physical characteristics, such as cell size and granularity, alongside multiple CD markers. The advantages of FC include simultaneous analysis of multiple antigens on the same cell, assessing small samples, and providing results to make an objective diagnosis within a few hours3. Immunophenotyping permits reproducible lineage detection and assignment of leukemia types. On the other hand, intra-permeable cytoplasmic markers, such as myeloperoxidase, cCD22, cCD3, and cCD79a, are more accurate and specific indicators4. Distinguishing AML-M0 from ALL remains a challenge in a minority of cases. The French–American–British (FAB) Cooperative Group recognized that many cases morphologically classified as types of ALL appeared to be of myeloid origin, as determined by monoclonal antibodies and ultra-structural cytochemistry5.\n\nThis study aimed to evaluate the immunophenotypic characterization and the expression of commonly used immuno-markers in both acute myeloid leukemia with minimal differentiation (AML-M0) and T-cell acute lymphoblastic leukemia (T-ALL), and to define the best use and the role of multi-parameter flowcytometry in the diagnosis and proper classification of these types of acute leukemia. Also, to evaluate the aberrant antigen expression in both AML-M0 and T-ALL and its correlation with clinical and prognostic criteria.\n\n\nMethods\n\nA retrospective cross-sectional study was conducted among 69 participants with newly diagnosed AL (M0 or T-ALL); there were 24 cases of AML-M0, 12 females and 12 males; and 45 cases of T-ALL, 40 males and five females. This study was conducted in the Pathology Department/Teaching Laboratories/Medical City/Iraq, and included all patients newly diagnosed with AL during the period from 5 January to 10 December 2018. Demographic characters of patients like age, and gender were collected from medical records of the participants. General clinical feature of each patients collected either when taking the patients history or during clinical examination included hepatosplenomegaly; lymphadenopathy (LAP); pallor; history of bleeding from gum, gingivae, and skin; fever; effusion of pleura; and presenting with a mediastinal mass.\n\n1. .T-ALL patients aged between (0.5–60) years old.\n\n2. AML-M0 patients aged between (7–76) years old.\n\n3. de novo cases, those not receiving steroid or chemo - therapy before sample collection\n\n4. Those with a positive diagnosis of T-ALL or AML-M0 confirmation.\n\n1. Death of patient prior to treatment\n\n2. Those who received chemotherapy in other hospitals either before referral or after finishing induction.\n\n3. Those whom refused treatment.\n\n4. Those with history of steroid intake of than one week.\n\n1. Those presented have fever, abdominal fullness, and skin rash.\n\n2. Those have pallor, petechiae (bleeding under the skin), sternal tenderness to palpation, lymphadenopathy, and hepatosplenomegaly on physical examination.\n\n3. Those with history of bleeding from gum, and gingivae.\n\n4. Those with elevated WBC count, normal RBC counts, decrease platelets counts in CBC.\n\n5. Those with chest masses on chest radiograph.\n\n6. Those with LAP and organomegaly on CT scans of the chest and abdomen/pelvis.\n\nA sample size of 69 participants was calculated by the following formula:\n\n\n\nwhere\n\nz is the z score for 95% CI=1.96\n\nε is the margin of error=5%\n\nn is sample size\n\np̂ is the population proportion=50%\n\nThis means 69 or more measurements/surveys are needed to have a confidence level of 95% that the real value is within ±5% of the measured/surveyed value. We the population proportion based on historical data from our center, whereby we found 50% of those attending were eligible for the study or in other words 50% were diagnosed with T-ALL or AML-M0.\n\nParticipants were recruited at their first visit to the center. Each patient attending our center meeting the inclusion criteria were invited to be including in our study after which written informed consent was obtained. Follow-up was performed at every subsequent visit. In EDTA anti-coagulated tubes (ATACO / China, Catalogue No.:443N980100E), the bone marrow aspirate (BMA) samples were collected (2–3 ml). The method of collection as follow: we marked and cleaned the area where the biopsy needle will be inserted. The bone marrow fluid (aspirate) and tissue sample (biopsy) were usually collected from the top ridge of the back of the hipbone (posterior iliac crest). A small incision was made, into which the needle was inserted into the bone and into the bone marrow. A sample of the liquid portion of the bone marrow was then drawn. Following sampling pressure was applied to the area of insertion to stop the bleeding and then a bandage will be placed on the site. Bone marrow was either aspirated and tested the same day or stored, if necessary, in a refrigerator (2–8°C), for no more than 48 hours. We pre-washed all the sample using at least 25 volumes excess 1X PBS (BD biosciences, Catalogue No.: 554781), mixed well. Then, pelleted cells by centrifugation and re-suspend in 1X PBS with 0.1% sodium azide to the original volume.\n\nThe panel used for diagnosis of both ALs included the following monoclonal antibodies (MoAbs): CD45, CD34, cMPO, CD13, CD33, CD14, CD15, CD64, CD117, cTdT, cCD79a, CD19, CD20, CD10, CD1a, CD2, sCD3, cCD3, CD4, CD5, CD7, CD8, and CD56, with an SSC/CD45 gating strategy, Table 1.\n\nImmunophenotypic analysis was performed. Cells were stained using fluorescence-conjugated MoAbs and analyzed using a four-color Canto-2 BD flow cytometry device6. The device software was based on the FACS DIVA-8 operating system for multi-parametric data acquisition, display, analysis, and instrument control. The Canto-2 BD is a high performance, accurate, and cost-effective flow cytometry device that employs two lasers (red and blue), six optical parameters, and offers four colors with forward scatter (FSC) and side scatter (SSC) in combination with four fluorescence channels (FL1–FL4)7.\n\nEach surface antibody (6 μl) was added to 100 μl of well-mixed EDTA (BD biosciences, Catalogue No.: 347689) anti-coagulated blood in labeled tubes, then incubated at room temperature in the dark for 15 minutes. Then, 2.5 ml 1X BD FACS lysis solution (BD biosciences, Catalogue No.: 349202) was added, and the samples were again incubated at room temperature in the dark for 15 minutes. The cells were washed three times by centrifugation at 1500 × g for 5 minutes before decanting the supernatant. FACS permeabilizing solution (BD biosciences, Catalogue No.: 347692) (0.5 ml) was added. Here, we vortex gently and incubated for 15 to 20 minutes in the dark at room temperature (20°C–25°C), and then the solution was re-suspended by vigorous shaking to mix. We then added 2 mL of CellWASH solution (BD biosciences, Catalogue No.: 349524) and centrifuged at 1200g for 5 minutes. Finally, we added 0.5 mL of CellFIX solution (BD biosciences, Catalogue No.: 340181) and mixed thoroughly. Then the samples were analyzed using the flow cytometer.\n\nAfter adding surface markers, 0.5 ml of permeabilizing reagent was added to each tube. The tubes were vortexed then incubated for 15 minutes in the dark. Then, 6 μl of each cytoplasmic marker was added and the samples were incubated at room temperature in the dark for 30 minutes. Then, 0.5 ml of FACS solution was added, and the samples were re-suspended by vigorous mixing before being analyzed using the flow cytometer.\n\nPermeabilizing Solution 2 (BD FACS™) is a premixed concentrate formulated specifically for use in flow cytometry. It is intended to permeabilize cell membranes prior to intracellular immunofluorescence staining with monoclonal antibodies.\n\nCell Lyse (BD biosciences, Catalogue No.: 559759), which is an erythrocyte lysing reagent kit for wash and no-wash procedures was used for cell lysis. Residual debris was removed by centrifugation and cell washing. Fixative reagent was used to fix and stabilize leukocytes. The fixed samples were stored for up to 24 hours at 2°C to 8°C before analysis.\n\n1. BD FACS™ lysing solution (10X) (Catalog No. 349202)\n\n2. BD CellWASH™ (Catalog No.349524)\n\n3. BD CellFIX™ (Catalog No. 340181)\n\n4. BD FACS™ permeabilizing solution 2 (Catalog No. 347692)\n\n5. Wash buffer of PBS with 0.1% sodium azide (Catalog No. 554781)\n\n6. Distilled water (BDH Ltd., Catalogue No.:DW005HH2).\n\n1. Adjustable pipettes and their tips (Gelson/ France, Catalogue No.: 111-898-3).\n\n2. Beakers (BD Biosciences, Catalogue No.: 33350410).\n\n3. Flasks (BD Biosciences, Catalogue No.: 645392).\n\n4. Centrifuge (Elite – Medichem/ (India), Catalogue No.: 153-33-IND).\n\n5. Disposable slides (BD Biosciences, Catalogue No.: 335630).\n\n6. EDTA tubes (ATACO / China, Catalogue No.:443N980100E).\n\n7. Racks (BD Biosciences, Catalogue No.: 333489).\n\n8. Refrigerator (HITACHI, Catalogue No.: HRPK0284A_E118-600L4D-STD-EX).\n\n9. Stop watch (CTIZ / Japan, Catalogue No.:44448FHD).\n\n10. Syringes (ATACO / China, Catalogue No.:CCD35560001).\n\n11. Vortex mixer (Memmert/ Germany, Catalogue No.:CR076662088).\n\n12. Wash bottle (BD Biosciences, Catalogue No.: 33635107).\n\n13. Falcon® disposable 12 x 75-mm polystyrene test tubes (BD Biosciences, Catalogue No.: 343514).\n\nThe dominant population of leukemic blasts was identified using side scatter versus CD45 dot plots to isolate the blast population. A 20% cut-off value was used to indicate the positivity of surface markers, while the cut-off value for cytoplasmic markers was 10%.\n\nData acquisition and analysis was performed using FCS DIVA-8, made by the BD Software Company.\n\nWritten informed consent was obtained from all participants, or the parents of those aged less than 18 years. The Medical Ethical Committee at the Oncology Center, Al-Kindy College of Medicine, Baghdad University, Iraq, approved this study (code: 52).\n\nWe used Microsoft Excel (v. 2010) to calculate frequencies and percentages of variables, and to calculate means and standard deviations (SDs).\n\n\nResults\n\nA total of 69 patients were newly diagnosed with AL; 24 patients with AML-M0, 12 females and 12 males; and 45 patients with T-ALL, 40 males and 5 females, as shown in Figure 1, the age and sex of patients is shown in Table 2 and Table 310.\n\nRegarding clinical findings, we listed in Table 410 as follow: hepatomegaly in 24.6%; splenomagaly in 36.2%; LAP in 44.9%; Pallor in 68.1%; bleeding in 71%; fever in 60.9%; pleural effusion in 1.4%; mediastinal mass in 5.8%.\n\nThis study identified 10 (41.6%) patients with AML-M0 who showed no aberrant antigen expression, while 33.3%, 16.6%, 8.3%, and 8.3% had aberrant CD7, CD56, CD2, and CD19 expression, respectively. In four (16.6%) cases of M0, there was more than one aberrant antigen expressed: two cases (8.3%) had both CD7 and CD56, one case (4.2%) had CD7 plus CD19, and one case expressed CD7 plus CD2. With regard to patients with T-ALL, we found the following: 7.0% of T-ALL patients had pro-T type, 58.0% had pre-T type, 13.0% had cortical type, and 22.0% had mature-T type, according to the European Group for the Immunological Classification of Leukemias (EGIL) criteria11. Our study also showed that 55.5% of all patients with T-ALL lacked aberrant antigen expression, while 13.3%, 13.3%, 11.1%, 6.7%, and 2.2% had aberrant CD13, CD117, CD33, CD19, and CD56 expression, respectively. Six cases (13.3%) showed expression of more than one aberrant antigen, mainly CD13 plus CD10, CD19, or CD56; CD13 plus CD117 in two cases; and one case with aberrant CD33 plus CD19, Table 5, Table 6 and Table 710.\n\nSome cases had more than one aberrant antigen expression.\n\nSome cases had more than one aberrant antigen expression.\n\n\nDiscussion\n\nFlow cytometry of leukemic cells plays an essential role in the identification of leukemia cell lines, maturation stage, and detection of residual disease. Immunophenotyping can improve both the accuracy and reproducibility of leukemia classification and is considered particularly useful for identifying AML with lymphoid marker expression and ALL with myeloid marker expression12. Some cases of AML associated with the expression of CD7 have a poor prognosis13, while other studies have reported CD2 and CD19 are better prognostic indicators in cases of AML14. In this study, 69 participants with de novo AL were enrolled, 45 of whom had T-ALL and 24 of whom had AML-M0. Most patients (88%) with T-ALL were male, which was in accordance with a study by Abid Salih and colleagues15 and also similar to another study that showed a predominance of males among patients with T-ALL16. The male:female ratio was 1:1 in patients with AML-M0 in our study, which was consistent with earlier work17.\n\nIn the present study, the most common features patients with T-ALL presented with were pallor and fever14,17–19, followed by hepato-splenomegaly and lymphadenopathy, which is in accordance with the findings of other studies14,17,18. The least common presenting features were CNS involvement, pleural effusion, and mediastinal mass19,20.\n\nRegarding the clinical features of AML-M0, the current study found that pallor and fever were present in 16 (66.7%) of patients, consistent with the findings of Ghosh et al.17. Anemia is a common feature of all acute leukemias and is due to bone marrow infiltration; again, our results are comparable with other studies16. Pahloosye et al. reported pallor and fever were prominent symptoms20. The current study showed that bleeding occurred in 19 (79.2%) of patients with AML-M0. Such bleeding may be due to a number of causes, such as thrombocytopenia due to bone marrow suppression by infiltrating malignant leukemic cells, and by platelet dysfunction that accompanies leukemic involvement in the bone marrow16. Gaydos et al. were the first to report this in 1962, when they found that there was a linear relationship between bleeding and platelet count21. Qazi et al. correlated this feature with thrombocytopenia alone or disseminated intravascular coagulopathy (DIC)22. Extramedullary infiltration by leukemic cells may result in lymphadenopathy, splenomegaly, and/or hepatomegaly. In our patients, we observed hepatomegaly in 24.6% of them. We concluded that pleural effusion was absent in AML-M0 patients but present in one (2.2%) patient with T-ALL. Regarding mediastinal mass, this was absent in cases of AML-M0, and this agree with that noted by Ghosh et al.17.\n\nThe incidence of lymphoid antigen expression in AML-M0 cases was 58.4%, which was higher than that seen by El‑Sissy et al.12 and Khalidi et al.23, who found aberrant lymphoid antigen expression in AML to be 47% and 48.1%, respectively. The incidence of CD56 expression was 16.6% in this study, which was similar to rates recorded in previous studies24–28. Many studies have concluded that overall survival was significantly decreased in individuals who expressed CD5624–26. Regarding CD7, it was expressed in (33.3%) of patients with AML-M0, which was in agreement with other studies that found that CD7 to be aberrantly expressed in 10%–40% of blasts in AML patients26. Kita et al.13 showed that CD7 expression on cells is indicative of phenotype, is functional when immature, and is regarded as a prognostic risk factor.\n\nHere, the most commonly expressed lymphoid marker was CD7 (33.3%), which is the same as recent reports by Bahia et al.29 and Zheng et al.30, although it is higher than the results reported by Venditti et al.31. CD4 was expressed in 25% of patients with AML-M0, which is comparable to one other study32 but higher than values reported by some other studies, e.g., 17.5%33 and 16%23.\n\nIn the current study, CD19 was expressed in 8.3% of patients, which is in accordance with the range of 8.6%–10% found in other studies like Khalidi et al.23, Bahia et al.29 and Venditti et al.31 but contrasts with other reports of 14%34 and even lower than 2.5%35. The current study showed that CD2 was expressed in 8.3% of cases of AML, similar to the proportion reported by Thalhammer-Scherrr et al.36, who found the expression of CD2 to be 3%, while Bahia et al.29 and Venditti et al.31 found higher values, of 11.4% and 14%, respectively.\n\nThe pattern of myeloid expression is correlated with some genetic features of blast cells. CD15, CD33, and CD65 are expressed in ALL cases with a rearranged MLL gene, and CD13 and CD33 are expressed in cases with the ETV6-RUNX1 gene37. The presence of these antigens lacks prognostic significance in contemporary treatment programs38.\n\nThe CD13 and CD33 were expressed in 6 (13.3%) and 5 (11.1%) of cases, respectively, which is consistent with another study39. Acute T-cell lymphoblastic leukemia with aberrant CD117 presented in 6 (13.3%) patients and is also comparable with the proportion seen in other studies40–43. Three (6.7%) patients with T-cell acute lymphoblastic leukemia expressed aberrant CD19, a lower value than other studies which found the expression of CD19 in cases of T-ALL to be 22%44. T-cell acute lymphoblastic leukemia expressing aberrant CD56 was only seen in one (2.2%) patient, and this is consistent with other studies45.\n\n\nConclusion\n\nWe concluded that aberrant expression of CD markers was present in some cases of acute leukemia. The incidence of aberrant antigen expression was comparable with other studies and represents an indicator of poor prognosis. CD13, CD33, and CD117 were the most frequent aberrant myeloid antigens expressed in T-ALL cases, while CD19 and CD56 were expressed in fewer cases. A large number of AML cases had aberrant lymphoid phenotypes, with T-cell markers more common than B‑cell markers. In addition, CD7 was a common aberrantly expressed lymphoid marker in AML. In the future, correlation studies of cytogenetic and lymphoid phenotypes should be performed to determine if there is any association between specific cytogenetic anomalies and the aberrant expression of certain lymphoid markers in patients with AML, or between specific cytogenetic anomalies and myeloid markers in patients with ALL.\n\n\nData availability\n\nZenodo: Leukemia data. http://doi.org/10.5281/zenodo.402133410\n\nThis project contains the following underlying data:\n\n- Pdf files for each participant including a immunophenotyping report with scatter plots\n\n- Data.xlsx (Dataset containing immunophenotyping results for all participants)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nGreat thanks for Adam Bodley, MAEd from Peerwith for his copyediting services of this work.\n\n\nReferences\n\nSexena R, Anand H: Flow cytometry in acute leukemia. Indian J Hematol Blood Transfus. 2008; 24(4): 146–150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee EJ, Pollak A, Leavitt RD, et al.: Minimally differentiated acute nonlymphocytic leukemia: a distinct entity. Blood. 1987; 70(5): 1400–1406. PubMed Abstract \| Publisher Full Text\n\nAbdulsalam A: Diagnosis of Acute Leukemia in Under-Resourced Laboratories. Acute Leukemia - The Scientist's Perspective and Challenge. Prof. Mariastefania Antica (Ed.). Croatia: In Tech. 2011. Publisher Full Text\n\nHoyer JD, Kurtin PJ, Hanson CA: Comparison of cytochemical and immunologic methods for detecting myeloperoxidase (MPO) in acute leukemias. Mod Pathol. 1996; 9(113 A).\n\nBennett JM, Catovsky D, Daniel MT, et al.: Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976; 33(4): 451–458. PubMed Abstract \| Publisher Full Text\n\nPARTEC: Reagents: monoclonal antibodies, antibody Premix, Partec reagents. Germany: Partec 2012; accessed at December, 10, 2019.\n\nPARTEC: CyFlow® SL Healthcare \| Immunology. The ultracompact and portable CyFlow® SL provides most accurate and uniquely affordable 3-colour analysis for use in research and routine applications. (accessed at 20, February, 2019).\n\nBD Biosciences: BD FACS Permeabilizing Solution 2. Accessed at October, 15, 2014. Reference Source\n\nPARTEC: Accessed at October, 17, 2014.\n\nAl-Saadi EA: Leukemia data. F1000reserch. 2020. http://www.doi.org/10.5281/zenodo.4021334\n\nBene MC, Castoldi G, Knapp W, et al.: Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia. 1995; 9(10): 1783–1786. PubMed Abstract\n\nEl-Sissy AH, El-Mashari MA, Bassuni WY, et al.: Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia. J Egypt Natl Canc Inst. 2006; 18(3): 244–9. PubMed Abstract\n\nKita K, Miwa H, Nakase K, et al.: Clinical importance of CD7 expression in acute myelocytic leukemia. The Japan Cooperative Group of Leukemia/Lymphoma. Blood. 1993; 81(9): 2399–405. PubMed Abstract \| Publisher Full Text\n\nDrexler HG, Thiel E, Ludwig WD: Acute myeloid leukemias expressing lymphoid-associated antigens: diagnostic incidence and prognostic significance. Leukemia. 1993; 7(4): 489–98. PubMed Abstract\n\nAbid-Salih B: Evaluation of Oncogene Fusion Transcripts [t(12;21)/TELAML, t(1;19)/E2A-PBX1, t(4;11)/MLL-AF4, and t(9;22)/BCRABL] in Children with Acute Lymphoblastic Leukemia by Multiplex PCR Analysis (PhD in Hematopathology). Baghdad: Al-Nahrain University, College of Medicine. 2013.\n\nHassan MK, Ibrahim JJ, Abbas MS: The adverse effects of chemotherapeutic agents in IRAQI patients with acute lymphocytic drug, the induction phase. I J A B R. 2013; 3(4): 584–592. Reference Source\n\nGhosh S, Shinde SC, Kumaran GS, et al.: Haematologic and immunophenotypic profile of acute myeloid leukemia: an experience of Tata Memorial Hospital. Indian J Cancer. 2003; 40(2): 71–76. PubMed Abstract\n\nCascavilla N, Musto P, Darena G, et al.: Adult and childhood acute lymphoblastic leukemia: clinico-biological differences based on CD34 antigen expression. Haematologica. 1997; 82(1): 31–37. PubMed Abstract\n\nAl-Barazanchi ZA, Al-Sani AK, Naema NF, et al.: Hematological and cytomorphological study of Acute Lymphoblastic Leukemia (ALL). Bahrain Med Bull. 2005; 27(4): 1–4. Reference Source\n\nPahloosye A, Hashemi A, Mirmohammadi SJ, et al.: Presenting Clinical and Laboratory Data of Childhood Acute Lymphoblastic Leukemia. Iran J Ped Hematol Oncol. 2011; 1(3): 71–77. Reference Source\n\nGaydos LA, Freireich EJ, Mantel N: The quantitative relation between platelet count and hemorrhage in patients with acute leukemia. N Engl J Med. 1962; 266: 905–909. PubMed Abstract \| Publisher Full Text\n\nQazi R, Hameed B: Bleeding diathesis in acute Myeloid Leukemia: Thrombocytopenia alone or in Disseminated Intravascular Coagulopathy. Pak J Med Sci. 2002; 13: 682–685.\n\nKhalidi HS, Medeiros LJ, Chang KL, et al.: The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities. Am J Clin Pathol. 1998; 109(2): 211–20. PubMed Abstract \| Publisher Full Text\n\nChang H, Salma F, Yi QL, et al.: Prognostic relevance of immunophenotyping in 379 patients with acute myeloid leukemia. Leuk Res. 2004; 28(1): 43–8. PubMed Abstract \| Publisher Full Text\n\nChang H, Brandwein J, Yi QL, et al.: Extramedullary infiltrates of AML are associated with CD56 expression, 11q23 abnormalities and inferior clinical outcome. Leuk Res. 2004; 28(10): 1007–11. PubMed Abstract \| Publisher Full Text\n\nFerrara F, Morabito F, Martino B, et al.: CD56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated with simultaneous all-trans-retinoic acid and chemotherapy. J Clin Oncol. 2000; 18(6): 1295–300. PubMed Abstract \| Publisher Full Text\n\nRaspadori D, Damiani D, Lenoci M, et al.: CD56 antigenic expression in acute myeloid leukemia identifies patients with poor clinical prognosis. Leukemia. 2001; 15(8): 1161–4. PubMed Abstract \| Publisher Full Text\n\nOgata K, Yokose N, Shioi Y, et al.: Reappraisal of the clinical significance of CD7 expression in association with cytogenetics in de novo acute myeloid leukaemia. Br J Haematol. 2001; 115(3): 612–5. PubMed Abstract \| Publisher Full Text\n\nBahia DM, Yamamoto M, Chauffaille Mde L, et al.: Aberrant phenotypes in acute myeloid leukemia: a high frequency and its clinical significance. Haematologica. 2001; 86(8): 801–6. PubMed Abstract\n\nZheng J, Wang X, Hu Y, et al.: A correlation study of immunophenotypic, cytogenetic, and clinical features of 180 AML patients in China. Cytometry B Clin Cytom. 2008; 74(1): 25–9. PubMed Abstract \| Publisher Full Text\n\nVenditti A, Del Poeta G, Buccisano F, et al.: Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia. Leukemia. 1998; 12(7): 1056–63. PubMed Abstract \| Publisher Full Text\n\nAl-Anizi WM, Al-Mashta MA: The frequency of aberrant lymphoid antigens expression in 202 Iraqi patients with de novo acute myeloid leukemia. Iraqi J Hematol. 2017; 6(2): 49–54. Publisher Full Text\n\nAbdulateef NAB, Ismail MM, Aljedani H: Clinical significance of co-expression of aberrant antigens in acute leukemia: A retrospective cohort study in makah mukaramah, saudi arabia. Asian Pac J Cancer Prev. 2014; 15(1): 221–7. PubMed Abstract \| Publisher Full Text\n\nHaycocks NG, Lawrence L, Cain JW, et al.: Optimizing antibody panels for efficient and cost-effective flow cytometric diagnosis of acute leukemia. Cytometry B Clin Cytom. 2011; 80(4): 221–9. PubMed Abstract \| Publisher Full Text\n\nKaleem Z, Crawford E, Pathan MH, et al.: Flow cytometric analysis of acute leukemias. Diagnostic utility and critical analysis of data. Arch Pathol Lab Med. 2003; 127(1): 42–8. PubMed Abstract\n\nThalhammer-Scherrer R, Mitterbauer G, Simonitsch I, et al.: The immunophenotype of 325 adult acute leukemias: relationship to morphologic and molecular classification and proposal for a minimal screening program highly predictive for lineage discrimination. Am J Clin Pathol. 2002; 117(3): 380–9. PubMed Abstract \| Publisher Full Text\n\nPui CH, Rubnitz JE, Hancock ML, et al.: Reappraisal of the clinical and biologic significance of myeloid-associated antigen expression in childhood acute lymphoblastic leukemia. J Clin Oncol. 1998; 16(12): 3768–3773. PubMed Abstract \| Publisher Full Text\n\nCampana D: Minimal residual disease in acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program. 2010; 2010(1): 7–12. PubMed Abstract \| Publisher Full Text\n\nLarson RA: Acute lymphoblastic leukemia. In: Kaushansky K, Lichtman MA, Prchal JT et al.Wiliams Hematology. 9th ed. China. The McGraw-Hill education. 2016; 91: 1505–1527. Reference Source\n\nBachir F, Bennani S, Lahjouji A, et al.: Characterization of acute lymphoblastic leukemia subtypes in moroccan children. Int J Pediatr. 2009; 2009: 674801. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeegmiller AC, Kroft SH, Karandikar NJ, et al.: Characterization of immunophenotypic aberrancies in 200 cases of B acute lymphoblastic leukemia. Am J Clin Pathol. 2009; 132(6): 940–949. PubMed Abstract \| Publisher Full Text\n\nSupriyadi E, Veerman AJP, Sutaryo, et al.: Myeloid Antigen Expression in Childhood Acute Lymphoblastic Leukemia and Its Relevance for Clinical Outcome in Indonesian ALL-2006 Protocol. J Oncol. 2012; 2012: 135186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRizzo K, Stetler Stevenson M, Wilson W, et al.: Novel CD19 expression in a peripheral T cell lymphoma: A flow cytometry case report with morphologic correlation. Cytometry B Clin Cytom. 2009.; 76(2): 142–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaietta E, Neuberg D, Richards S, et al.: Rare adult acute lymphocytic leukemia with CD56 expression in the ECOG experience shows unexpected phenotypic and genotypic heterogeneity. Am J Hematol. 2001; 66(3): 189–96. PubMed Abstract \| Publisher Full Text\n\nFischer L, Gökbuget N, Schwartz S, et al.: CD56 expression in T-cell acute lymphoblastic leukemia is associated with non-thymic phenotype and resistance to induction therapy but no inferior survival after risk-adapted therapy. Haematologica. 2009; 94(2): 224–9. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77230", "date": "20 Jan 2021", "name": "John K. Choi", "expertise": [ "Reviewer Expertise Flow cytometry based MRD analysis of acute leukemia." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a well written article of a single institution review of their AML-M0 and T-ALL cases in regards to immunophenotype and clinical findings. They conclude that most AML-M0 have no aberrant antigen expression and most T-ALL are in the pre-T development phase, findings compatible with previous publications.\nThere are concerns that question the validity of their conclusions.\nThe authors appear to use EGIL classification scheme for assigning lineage to their leukemia. Unfortunately, the most current classifications use the WHO criteria with the latest being the 2016 publication. Many cases appear misclassified and need to be reclassified using the most current scheme. For example, only 42.2% of the T-ALL cases are cCD3 positive although the current WHO criteria require cCD3 positivity for a diagnosis of T-ALL. It is very likely that the CD3 negative cases would be reclassified as AML-M0 with potentially different conclusions.\n\nThe authors should specify the diagnostic criteria for AML-M0 and T-ALL in their method section.\n\nGiven that the manuscript details the clinical findings and blast immunophenotype at diagnosis, it is unclear why the authors should exclude patients with poor outcome or treatment issue.\n\nIn the introduction, last sentence, the authors wanted to \"...its correlation with ... prognostic criteria.\" The results for this is not presented (see concern 3 above).\n\nThe authors cite the large AML Iraqi study (ref 33) but the authors should also cite the large ALL Iragi study by Jalal et al. ISLH 2017;39:625.\n\nAlthough the results are not novel, they do document the findings in Iraq and the authors are encouraged to include this info in the title for ease of searching.\n\nThe authors conclude that \"most AML-M0 have no aberrant antigen expression\" However, this represent only 41.6% of AML-M0 cases. That would mean that 58.4% or most of AML-M0 cases have aberrant expression.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "76618", "date": "18 Feb 2021", "name": "Buldini Barbara", "expertise": [ "Reviewer Expertise Pediatric leukemias" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors submitted an original paper on immunophenotypic characterization of acute myeloid leukemia with minimal differentiation and T-ALL. The paper must be a 'Not Approved' due to the following points:\nThe Aim of the study is not clear.\n\nCriteria for Antigen expression is not defined.\n\nIntroduction: The cornerstone of leukemia diagnosis, according WHO includes also morphology and cytogenetics. This should be mentioned. The definition of the AML subgroup included in the study is confounding: the authors used indifferently the terms “M0-AML” (FAB classification) and “AML with minimal differentiation” (WHO classification). Whereas the two terms are not interchangeable and refer to different AMLs subtypes. Moreover, authors mentioned to study also “the correlation with clinical and prognostic criteria”: in the paper, survival analysis are missing.\n\nThe study cohort is small and very heterogeneous, including both pediatric and adulthood ALs.\n\nInclusion and exclusion criteria are not clear: e.g., Exclusion criteria 4 contrasts with inclusion criterium 3; Inclusion criteria 4 should mention the diagnostic criteria (according WHO Classification?); Exclusion criteria 1 and 3 are not clear: this is an IF study on AL at diagnosis, why these patients should be excluded?\n\nDiagnostic criteria are not relevant to the study purpose since the authors did not associate them to the study topic (ALs immunophenotype).\n\nThe statistical explanation of sample dimension calculation is not clear. It seems unlikely that T-ALL and M0-AML represent half of the patients with a newly diagnosed AL in the study period.\n\nSamples preparation and acquisition are not accurately described:\n\nto obtain accurate staining, the total amount of MoAbs should be calculated on samples WBC counts rather than blood volume.\n\nNo data on the total number of stained and acquired cells per sample are available.\n\nThe results are not accurately presented and include data that went beyond the aim of the study:\n\nThe authors did not report the total number of newly diagnosed ALs (BCP-, T-, myeloid) in the study period. 69 patients with newly diagnosed AL were included while Figure 1 includes only AML M0 and T-ALL.\n\nThe authors included clinical data without correlating them with immunophenotypic results.\n\nReferences are obsolete (29/45 date more than 10 years) and incomplete. WHO classification in not included, and this is the basis for leukemia diagnosis.\n\nThe manuscript needs copy editing for English language.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1170
https://f1000research.com/articles/8-756/v1	29 May 19	{ "type": "Clinical Practice Article", "title": "Orofacial manifestations of mucocutaneous leishmaniasis: a case series from Brazil", "authors": [ "Gleicy Gabriela Vitória Spinola Carneiro Falcão", "Liliane Lins-Kusterer", "Patricia Miranda Leite-Ribeiro", "Viviane Almeida Sarmento", "Gleicy Gabriela Vitória Spinola Carneiro Falcão", "Liliane Lins-Kusterer", "Patricia Miranda Leite-Ribeiro" ], "abstract": "The dental surgeon plays a fundamental role in the early diagnosis of oral leishmaniasis, since oral mucosa may be the primary site of the disease manifestation. This study reports seven clinical cases of orofacial mucocutaneous leishmaniasis. All had mucocutaneous leishmaniasis with oropharyngeal involvement confirmed by laboratory tests. Five out of the seven cases were males, and in four cases, patients had associated comorbidities. Late diagnosis was observed, resulting in treatment delay and increased hospitalization stay. One patient had severe psychological consequences due to facial deformity. The lack of differential diagnosis due the great variability of clinical presentation of the lesions and frequent unspecific histopathology represent a challenge for the dental surgeon. In two reported cases, there were unspecific biopsy results. The multidisciplinary approach plays an important role in orofacial leishmaniasis diagnosis and treatment. Leishmaniasis should be investigated in case of atypical and persistent lesions in patients from endemic regions. This recommendation may avoid diagnosis delays and decrease dissemination of the disease.", "keywords": [ "Leishmaniasis", "Mucocutaneous", "Diagnosis", "Oral", "Dental Care" ], "content": "Introduction\n\nLeishmaniasis is a parasitic disease caused by several species of the protozoan genus Leishmania1. It is a widely dispersed disease, being endemic in 98 countries, including Brazil. Leishmaniasis classification encompasses different clinical forms2; mucocutaneous leishmaniasis is a chronic form of infection3 that may manifest in the mucosa after months or years of latency4.\n\nThe mucosal involvement of leishmaniasis is uncommon, mainly in immunocompromised individuals5. The lymphatic or hematogenous dissemination of amastigotes may occur from the skin to the nasal, oropharyngeal, laryngeal and/or tracheal mucosa. Delayed diagnosis3,6 and development of primary lesion in the oral mucosa and in the head and neck region can cause dysphagia, dysphonia and dyspnoea3.\n\nThe diagnosis of mucocutaneous leishmaniasis can be difficult7. In older lesions, few parasites are usually detected by microscopy or culture and the clinical aspect may resemble neoplasia1,8. Orofacial symptoms depend on the localization of the lesions and may include nasal obstruction, difficulties in swallowing, mucosal bleeding and/or hoarseness8. Destructive lesions of the mucosa contain few parasites, with high levels of tumor necrosis factor (TNF) suggesting an unmodulated immune response with increased production of proinflammatory cytokines responsible for tissue damage9.\n\nIn this study we report seven clinical cases of orofacial mucocutaneous leishmaniasis from Brazil.\n\n\nCase reports\n\nThis study included seven patients admitted to Edgard Santos University Hospital, Federal University of Bahia, Brazil. All patients had a confirmed diagnosis of mucocutaneous leishmaniasis with oropharyngeal involvement and no visceral involvement, confirmed by laboratory tests. This study was approved by the Ethics and Research Committee of Edgard Santos University Hospital, CAAE 93381518.7.000.0049. All patients (or parents/guardians) provided written informed consent for the publication of their medical data and images.\n\nMale, 24-years-old, Caucasian, unemployed, from Tancredo Neves, State of Bahia, Brazil, was admitted to the University Hospital, in January 2012, presenting diffuse bullous lesions on the body, osteoarthritis of the distal interphalangeal joints and proteinuria 399 mg/day (reference value >150mg/day). He was diagnosed with systemic lupus erythematosus (SLE) and treated with mycophenolate mofetil (MMF). The starting dose for MMF was 0.5 g per day and it was increased up to 1 g per day intravenously. In 2014, two years after SLE diagnosis, he was hospitalized, presenting with ulcerated-painless-skin lesions on the face, upper lip, scalp, neck, upper and lower limbs. Oral examination evidenced crusty upper lip lesions, poor oral health status and amelogenesis imperfecta (Figure 1a and 1b). He developed secondary infection associated with fever, and antibiotic therapy with cephalexin was initiated (1g/day) and a maintenance dose of prednisone (5 mg/day intravenously). On the third day, biopsies were performed on the left nasal mucosa and on the right lower limb lesions. The diagnosis of disseminated leishmaniasis was confirmed (positive PCR and Montenegro intradermal test). Liposomal amphotericin B was introduced on the fourth day of hospitalization at a dose of 150 mg/day up to a maximum dose of 2,400 mg. The patient treatment was followed-up for six months, and lesions were observed to have healed. One month later, during the follow-up for SLE, we observed new development of ulcerated skin lesions on the face and on the upper and lower right limbs. Blisters and fever were absent and the recurrence of disseminated leishmaniasis was confirmed. Few weeks later, the patient was admitted for treatment of new lesions, presenting with erythema, diffuse facial edema, lymphadenopathy and ulcerated and pustular lesions. Patient was treated with liposomal amphotericin B at a cumulative dose of 3,050 mg and followed-up until complete remission of the lesions. Currently, patient is under maintenance treatment for SLE.\n\nOral examination evincing poor oral health status and amelogenesis imperfecta reported at Case-1 (a and b); Infiltrative lesion on hard, soft palate and uvula reported at Case-2 (c); Ulcerated lesions in the lower lip frenulum, reported at Case-3 (d).\n\nIn July 2013, 53-year-old male, Caucasian, unemployed, from Mundo Novo, State of Bahia, Brazil, attended to the Stomatology Clinic at University Hospital, presenting with pain, nasal obstruction, and complaints of odynophagia and dysphagia. Physical examination showed painful, hyperemic and friable lesion in the right nasal cavity, associated with infiltrative lesion on the hard and soft palate, and uvula (Figure 1c). We observed ulcerated lesion on the left eyebrow and right eye with seropurulent secretion, a small ulcer on the lower eyelid, on the lobe of the right ear and a lesion in the malar region. The patient was admitted for diagnosis and treatment of disseminated skin lesions. A biopsy of the palate lesions revealed a non-specific erosive chronic inflammatory process. The patient was HIV negative and positive for Montenegro reaction. Treatment with amphotericin B was initiated at a dose of 150 mg/d up to a maximum dose of 2,410 mg. Lesions regressed after drug treatment and oral treatment was initiated during hospitalization. We removed dental foci without any intercurrence. One month later, the patient was discharged. However, in August 2013, in outpatient medical consultation, the lesions were observed in nasal mucosa and palate. He was followed up in the outpatient clinic and treatment with glucantime 20 mg/kg/day was prescribed for one month. The follow-up period was eight months, and the result was negative.\n\nFemale, 31 years old, Caucasian, unemployed, from Salvador, State of Bahia, was diagnosed (Montenegro positive test) with American Tegumentary Leishmaniasis in October, 2011. The patient was treated with Glucantime, 20 mg/kg/day for 30 days. A lesion in her back region was partially healed. In 2012, two episodes of recurrence occurred and restarted treatment with Glucantime in January and May. In a third recurrence episode (August, 2012), due to the maintenance of the lesion, a lesion biopsy was performed and Leishmania braziliensis was diagnosed. Treatment with amphotericin B was initiated at a dose of 250 mg/d up to a maximum dose of 2,400 mg, resulting in wound healing. In 2013, the patient was admitted with submandibular lymphadenopathy and ulcerated lesions in the lower lip frenulum (Figure 1d), gingiva, nasal septum and in the back region. She was hospitalized for diagnosis and treatment of lesions with liposomal amphotericin B. Due to persistence of the lesions, HIV serology was performed. The patient was HIV positive and antiretroviral therapy was started (efavirenz 600mg, tenofovir 300mg, lamivudine 300mg, per day, one tablet containing the three drugs). Excisional biopsies of oral lesions were performed with unspecific result. Microbiological analysis for fungi was negative. Two months later, the patient was discharged and a maintenance dose of liposomal amphotericin B (150 mg/day) was prescribed.\n\nIn 2017, an eight year-old Caucasian male from Salvador, Bahia, Brazil, presented with a hyperemic and pruritic lesion on the upper lip which had persisted for six months. Patient was treated with acyclovir cream, 5%, 5 times/day and cefadroxil (50 mg/kg/day) for seven days, with no response. He presented worsening of the lesion and Montenegro intradermal examination was performed (Figure 2a). The patient was positive for American Tegumentary Leishmaniasis. Treatment with glucantime (10 mg/day) for 20 days was initiated. After three days of treatment, the patient developed vomiting episodes, intermittent fever, diarrhea, hypoglycemia, dark urine, and began developing a reaction of cardiotoxicity and hepatoxicity. Treatment with liposomal amphotericin B was initiated (3 mg/kg/day for 5 days, followed by 3 mg/kg). One month later, patient was discharged with remission of the lesion (Figure 2b). Two months later, the patient was admitted at University Hospital with a new, erythematous and ulcerated lesion on the upper lip lesion, lymphadenopathy, and facial edema. Therapy with amoxicillin 250 mg (1g/day) and amphotericin B (100 mg/day) for 10 days was started. Patient is currently in psychological follow-up due to trauma caused by facial disfiguration and difficulty in returning to social life. Patient maintained outpatient follow-up and did not present with recurrence of the lesion.\n\nHyperemic and pruritic lesion on the upper lip, reported at Case-4 (a); Aspect of the upper lip one month later, evidenced remission of lesion (b).\n\nIn 2008, a male, 30 years old, Caucasian, unemployed, presented with an isolated nodulation in the right leg and he was diagnosed with Tegumentary leishmaniasis. The patient was treated with Glucantime (10 mg/kg/day for 20 days), achieving complete healing of the lesions. In 2014 the patient presented a papule in the inferior eyelid of the right eye. Patient was PCR positive for Leishmania brasiliensis. Lesions progressively appeared in different body surfaces such as the chest, abdomen, back, feet, and mouth. Ulcerated oral lesions were present in the hard palate, as well as the left and right jugal mucosa (Figure 3a).\n\nUlcerated oral lesions in hard palate reported at Case-5 (a). Leishmaniasis lesion in the left malar region reported at Case-6 (b).\n\nProgression of disease was associate with fever, headache and weight loss. Treatment with glucantime (20 mg/day) for 30 days followed by treatment with amphotericin B at a cumulative dose of 1.5 to 2 g, 50 mg/day. Patient developed acute renal failure secondary to the use of amphotericin B. Treatment was replaced by the liposomal form at a dose of 100 mg/day and patient was discharge one month later with complete remission of lesions.\n\nFemale, 59 years old, Caucasian, unemployed, with diabetes, hypertension, congestive heart failure, chronic renal disease and paraparesis secondary to Human T-cell leukemia virus type 1 (HTLV-1) infection. In June 2012, patient presented with a papule lesion in the left malar region with late ulceration and increasing in size (Figure 3b). After 15 days, another lesion developed in the right knee. Patient was positive for Montenegro intradermal test and diagnosed with mucocutaneous leishmaniasis and was admitted in the University Hospital in September 2012. Patient developed hyperkalemia and, after stabilization of renal function, treatment with liposomal amphotericin B (100 mg/day) was introduced. One day after, the patient developed another episode of renal dysfunction and therapy was discontinued. Five days later, therapy was reintroduced, alternating with dialysis. The culture examination of the malleolar lesion was performed, being positive for Proteus vulgaris and hemoculture was positive for Staphylococcus aureus. In October 2012, patient was transferred to intensive care unit and developed multiple organ failure, dying two weeks later.\n\nMale, 59 years old, mixed ethnicity, unemployed, previously healthy, reported the appearance of an erythematous-crusty lesions in the mental protuberance region, evolving in two months to other parts of the body such as frontal and occipital regions, nasal septum, ears, hands, and lower limbs. Oral cavity clinic-examination showed scattered ulcers on the face, lower labial mucosa, and on the left lip commissure, pseudomembrane on the marginal gingiva, and an exophytic nodule in the left labial mucosa (Figure 4). Patient was Montenegro intradermal test positive and was admitted at the University Hospital in December 2018. After admission, we observed enlarged lymph nodes of hard consistency in the left inguinal region, and an extensive melanocytic lesion in the left plantar region. The lesion was irregular, presenting an area of hyperkeratosis with a grey-bluish center. The patient was biopsied and the diagnostic hypothesis of melanoma was confirmed. We requested laboratory and imaging tests for melanoma staging. After seven days, we accessed the PCR laboratory test and initiated therapy with intravenous liposomal amphotericin B 50 mg at the dose of 200 mg/kg/day for 15 days (Figure 4). Diagnostic confirmation of melanoma resulted in the excision of the melanocytic lesion with left inguinal lymphadenectomy. Patient was referred to an oncology center. The patient has not yet returned for evaluation as they are receiving antineoplastic treatment outside our hospital.\n\nUlcerated lesions in face, marginal gingiva, palate, and labial mucosa, before (a) and after treatment with liposomal amphotericin B (b) reported at Case-7.\n\n\nDiscussion\n\nFive out of the seven cases were males from Brazil’s endemic regions. Four cases had associated comorbidities (SLE, HIV, HTLV infection and melanoma). A multicenter case series study1 with seven patients presenting oral leishmaniasis reported higher frequency of oral lesions in males (86%), tongue (57%) with predominance of exophytic lesions (85%). In our case series, patients were predominantly males with ulcerated lesions in the lips.\n\nThe Montenegro reaction is a diagnosis test of high sensitivity, low cost and minimally invasive. Serological tests, such as immunoenzymatic assays and indirect immunofluorescence, show variation in their results depending on the applied technique and disease classification10. In our series of cases, late diagnosis was observed resulting in treatment delay and extension of hospitalization stay.\n\nFacial involvement of leishmaniasis is a serious complication, since it can lead to disfiguration and be potentially fatal11,12. In one case reported, the patient had severe psychological consequences due to facial deformity, reinforcing the importance of early diagnosis and appropriate therapy.\n\nMucosal leishmaniasis is generally associated with visceral leishmaniasis or immunocompromised individuals13–16. In immunocompetent patients, primary and exclusive mucosal involvement in the head and neck region is uncommon; lesions affecting the buccal mucosa exclusively are even rarer1,15–19. In our series of cases, four cases (57.1%) presented some level of immunological deficiency.\n\nLeishmaniasis is difficult to treat, and may present with spontaneous reactivation20 or be transmitted by a transplanted organ21. Control of cutaneous leishmaniasis depends on case management, early detection and appropriate treatment22. We observed cases of adverse drug reactions during treatment and protocol changes were necessary during the course of treatment. We also observed frequent recurrence of lesions due to inadequate treatment suspension or suboptimal doses.\n\nThe dental surgeon plays a fundamental role in the diagnosis of oral leishmaniasis3. The great variability of clinical presentation of the lesions and frequent unspecific histopathology represent a challenge in regard to differential diagnoses. The dental surgeon can contribute to early diagnosis of mucosal lesions, since oral mucosa may be the primary site of the disease manifestation.\n\nAlthough histopathological techniques describe the inflammatory infiltrate associated to leishmaniasis, they present low diagnostic specificity. The granulomatous aspect of lesions in later stages of cutaneous infection of leishmaniasis hampers histopathological analysis, since few parasites can be found in these lesions1,7,23. In our reported cases, we had two unspecific biopsy results.\n\nThere is no specific standardization for mucocutaneous leishmaniasis therapy17,22. The cases we reported were submitted to different therapeutic plans, adjusted to each patient. In our case series, all patients received systemic treatment for mucocutaneous leishmaniasis, because of this disease well-known resistance. Alternative topical treatment includes use of ointment, cryotherapy, and intralesional injection with antimonials. Multiple and large lesions compromising the face are less suitable for local therapy. The treatment of mucosal leishmaniasis are still based on case reports9.\n\nThe treatment of lesions involving the lip depends on clinical presentation, type of the leishmaniasis. Treatment comprises of intralesional injections, systemic treatment, or a combination of the two. Treatment for large intraoral lesions involves incisional biopsy for diagnosis and use of systemic medication. In the case of small intraoral lesions (5 to 10 mm), excision is recommended, and the patient should be monitored to confirm whether systemic therapy is necessary20. In our cases, all patients were treated with systemic medication. We presented a case with primary and exclusive lesion on the lip. Local treatment was not administered, and the patient is under follow-up.\n\nOur findings present some limitation. First, the few cases reported are not a representative population sample, limiting any possible inference. Due to socioeconomic reasons, patients living far from Salvador are not accessible for a close follow-up and dental care. Diagnosis based on oral biopsies are very limited and the dental surgeon must be aware of the diverse clinical forms of leishmaniasis. Cases of orofacial mucosa leishmaniasis are rare, but we should be aware of them during oral examination. We agree that our report may contribute to a better dental evaluation and early diagnosis of cases of oral leishmaniasis.\n\n\nConclusions\n\nThe multidisciplinary approach in the diagnosis and treatment of orofacial leishmaniasis is highlighted in this case series. We recommend leishmaniasis investigation in the case of patients living in endemic regions and presenting atypical and persistent lesions. Following this recommendation may avoid delays in leishmaniasis diagnosis and decrease the risk of its dissemination.\n\nDifferential diagnosis of mucosal lesions should include mucosal leishmaniasis. The variation in the clinical presentation of leishmaniasis and its ability to mimic different diseases represent a challenge for disease diagnosis. The dentist may play an important role in the early diagnosis of orofacial leishmaniasis.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nMignogna MD, Celentano A, Leuci S, et al.: Mucosal leishmaniasis with primary oral involvement: a case series and a review of the literature. Oral Dis. 2015; 21(1): e70–8. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Control of the leishmaniases. World Health Organ Tech Rep Ser. 2010; (949): xii–xiii, 1–186, back cover. PubMed Abstract\n\nPellicioli AC, Martins MA, Sant'ana Filho M, et al.: Leishmaniasis with oral mucosa involvement. Gerodontology. 2012; 29(2): e1168–71. PubMed Abstract \| Publisher Full Text\n\nCrovetto-Martínez R, Aguirre-Urizar JM, Orte-Aldea C, et al.: Mucocutaneous leishmaniasis must be included in the differential diagnosis of midline destructive disease: two case reports. Oral Surg Oral Med Oral Pathol Oral Radiol. Elsevier Inc.; 2015; 119(1): e20–6. PubMed Abstract \| Publisher Full Text\n\nCelentano A, Ruoppo E, Mansueto G, et al.: Primary oral leishmaniasis mimicking oral cancer: a case report. Br J Oral Maxillofac Surg. British Association of Oral and Maxillofacial Surgeons; 2015; 53(4): 396–8. PubMed Abstract \| Publisher Full Text\n\nCruz AF, Resende RG, Albuquerque DR, et al.: Mucosal leishmaniasis in Brazilian patients: two case reports with similar clinical presentation and different approaches. Oral Surg Oral Med Oral Pathol Oral Radiol. Elsevier Ltd; 2016; 122(6): e199–203. PubMed Abstract \| Publisher Full Text\n\nOkumura Y, Yamauchi A, Nagano I, et al.: A case of mucocutaneous leishmaniasis diagnosed by serology. J Dermatol. 2014; 41(8): 739–42. PubMed Abstract \| Publisher Full Text\n\nCobo F, Rodríguez-Granger J, Gómez-Camarasa C, et al.: Localized mucosal leishmaniasis caused by Leishmania infantum mimicking cancer in the rhinolaryngeal region. Int J Infect Dis. 2016; 50: 54–6. PubMed Abstract \| Publisher Full Text\n\nBlum J, Lockwood DNJ, Visser L, et al.: Local or systemic treatment for New World cutaneous leishmaniasis? Re-evaluating the evidence for the risk of mucosal leishmaniasis. Int Health. Royal Society of Tropical Medicine and Hygiene; 2012; 4(3): 153–63. PubMed Abstract \| Publisher Full Text\n\nGomes CM, Paula NA, Morais OO, et al.: Complementary exams in the diagnosis of American tegumentary leishmaniasis. An Bras Dermatol. 2014; 89(5): 701–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlmeida TF, da Silveira EM, Dos Santos CR, et al.: Exclusive Primary Lesion of Oral Leishmaniasis with Immunohistochemical Diagnosis. Head Neck Pathol. 2016; 10(4): 533–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPassi D, Sharma S, Dutta S, et al.: Localised leishmaniasis of oral mucosa: report of an unusual clinicopathological entity. Case Rep Dent. Hindawi Publishing Corporation; 2014; 2014: 753149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCobo F, Aliaga L, Talavera P, et al.: The histological spectrum of non-granulomatous localized mucosal leishmaniasis caused by Leishmania infantum. Ann Trop Med Parasitol. 2007; 101(8): 689–94. PubMed Abstract \| Publisher Full Text\n\nRamos A, Múñez E, García-Domínguez J, et al.: Mucosal leishmaniasis mimicking squamous cell carcinoma in a liver transplant recipient. Transpl Infect Dis. 2015; 17(3): 488–92. PubMed Abstract \| Publisher Full Text\n\nRathnayake D, Ranawake RR, Sirimanna G, et al.: Co-infection of mucosal leishmaniasis and extra pulmonary tuberculosis in a patient with inherent immune deficiency. Int J Dermatol. 2010; 49(5): 549–51. PubMed Abstract \| Publisher Full Text\n\nTorrico F, Parrado R, Castro R, et al.: Co-Infection of Leishmania (Viannia) braziliensis and HIV: report of a case of mucosal leishmaniasis in Cochabamba, Bolivia. Am J Trop Med Hyg. 2009; 81(4): 555–8. PubMed Abstract \| Publisher Full Text\n\nLee GL, Woods KL, Clark L, et al.: Short communication: mucocutaneous leishmaniasis in HIV-related immune reconstitution syndrome. AIDS Res Hum Retroviruses. 2015; 31(9): 889–92. PubMed Abstract \| Publisher Full Text\n\nGois L, Badaró R, Schooley R, et al.: Immune response to Leishmania antigens in an AIDS patient with mucocutaneous leishmaniasis as a manifestation of immune reconstitution inflammatory syndrome (IRIS): a case report. BMC Infect Dis. 2015; 15(1): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadeddu G, Fiori ML, Ena P, et al.: Mucocutaneous leishmaniasis as presentation of HIV infection in Sardinia, insular Italy. Parasitol Int. Elsevier Ireland Ltd; 2014; 63(1): 35–6. PubMed Abstract \| Publisher Full Text\n\nNadler C, Enk CD, Leon GT, et al.: Diagnosis and management of oral leishmaniasis--case series and literature review. J Oral Maxillofac Surg. American Association of Oral and Maxillofacial Surgeons; 2014; 72(5): 927–34. PubMed Abstract \| Publisher Full Text\n\nBaglieri F, Scuderi G: A case of mucosal leishmaniasis of the tongue in a kidney transplant recipient. Int J Dermatol. 2012; 51(5): 597–600. PubMed Abstract \| Publisher Full Text\n\nGonzález U, Pinart M, Rengifo-Pardo M, et al.: Interventions for American cutaneous and mucocutaneous leishmaniasis. Cochrane Database Syst Rev. 2009; (2): CD004834. PubMed Abstract \| Publisher Full Text\n\nPalmeiro MR, Rosalino CM, Quintella LP, et al.: Gingival leishmaniasis in an HIV-negative patient. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007; 104(6): e12–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "55589", "date": "04 Nov 2019", "name": "Braulio Valencia", "expertise": [ "Reviewer Expertise Infectious and Tropical Diseases", "Neglected Tropical Diseases", "New-World Leishmaniases." ], "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 scope of this article is to provide essential clues about orofacial manifestations of mucocutaneous leishmaniasis. Considering the target readers are Dental Surgeons, this manuscript has several imprecision's and inaccurate messages which need to be corrected.\nAbstract section:\nThe affirmation that \"oral mucosa may be the primary site of the disease manifestation\" is contradictory with the discussion and findings of this report as well as the existent and well-characterised clinical description of MCL. Almost all cases (except case 4) were manifested as disseminated Leishmaniasis with a significant and predominant cutaneous involvement. In the discussion section, paragraph four, line 5, authors state that primary and exclusive oral mucosa involvement is exceptionally uncommon. Then, what is described in the abstract is contradictory.\n\nRegarding that \"All had mucocutaneous leishmaniasis with oropharyngeal involvement\" is also incorrect. Due to the lack of detail in cases 5-7, it is not possible to accept these cases are definitively MCL. Case 6 is not a case of MCL; then, it needs to be removed from this article.\nIntroduction section:\nParagraph 2, line 1-2: It's not clear if authors suggest that MCL is particularly frequent in immunosuppressed individuals. This affirmation is highly dependent on the prevalence of immunosuppressive comorbidities (as HIV). Considering that immunosuppressive conditions are not highly prevalent in endemic areas (except some African or Asian regions), this affirmation is inaccurate. Mucosal involvement in new-world Leishmaniases ranges from 5-20%1. Then, in any case, this affirmation is not correct or requires clarification.\n\nParagraph 2, line 5-6: development of primary lesions in oral mucosa is very infrequent and mainly described in old-world Leishmaniasis2 OR in individuals with immunosuppressive conditions (4/7 cases in this report). For this reason, authors must consider changing the scope (title as well?) of this review from orofacial manifestations of MCL to atypical manifestations of leishmaniasis among immunosuppressed individuals.\nCase reports:\nCase 1: more than MCL, this is a case of disseminated leishmaniasis (DisL) in an immunosuppressed individual. Here the predominant mucosal involvement appears to be nasal (no details are provided regarding the degree of nasal involvement), and the oral involvement is confined to the upper lip involvement. This is unusual even in immunosuppressed individuals, considering that lips, gums, tongue, and hard palate are extremely infrequent in new-world MCL3.\n\nCase 2: again, this is a case of DisL in an immunocompetent individual. Here the mucosal involvement is more typical, but an HIV seronegative status is not enough to classify the patient as immunocompetent. A better characterisation of this individual is required.\n\nCase 3: again, another case of DisL in an immunocompromised patient. It's recommended to improve the quality of picture 1d. As mentioned before gums are unusual in new-world MCL. MCL is not commonly associated with lymphadenopathy. Both findings, and in the absence of parasitologic or histologic characterisation of the oral lesion makes it essential to consider other infectious diseases, importantly in an HIV-seropositive individual.\n\nCase 4: this case is MCL, but the pathophysiology is different from the prior cases. Here, more than a lymphatic/hematologic dissemination, what generated the MCL was a direct inoculation on the lip or a skin inoculation close to a mucosal structure. This case is clearly, utterly different from the rest and hard to classify as an unusual manifestation or among immunosuppressed individuals.\n\nCase 5: again, another DisL in an apparently immunocompetent patient. No details were provided here regarding the HIV serologic status or other potential sources of immunosuppression.\n\nCase 6: again, another DisL in an immunosuppressed individual. In this case, there is not any oral or mucosal involvement (only cutaneous lesions are described). Then, this case must be removed from the report.\n\nCase 7: there is a lack of evidence to catalogue this case as MCL. Besides the facial cutaneous lesions (only localised cutaneous leishmaniasis?), lesions described in the oral mucosa are unusually located, and an alternative explanation must be considered (metastatic melanoma?). It's not clear where the sample for PCR was obtained. With this unclear clinical and parasitological description, it's inaccurate to define the case as MCL.\nDiscussion:\nParagraph 3, lines 1-2: MCL is potentially fatal, mainly when larynx or trachea are affected. From that scenario, it's incorrect to describe them as a facial involvement.\n\nParagraph 4, lines 1-2: as suggested in the first observation of the introduction section, authors are suggesting MCL is generally associated with VL or immunosuppression. That is probably acceptable in the context of Sudanese MCL; however, this is not the epidemiological context of the study. Regarding immunosuppression, as discussed in the same observation, due to the lack of coexistence between Leishmaniasis and immunosuppressive conditions, there is not any evidence supporting this affirmation. The references used to support this statement are case reports which were not designed to measure the prevalence of MCL among immunosuppressed individuals.\n\nParagraph 5, lines 6-7: the high rate of recurrences observed in these cases are not necessarily related to therapeutic issues. Immunosuppression is probably the primary determinant of therapeutic failure.\n\nParagraph 6, lines 6-8: again, this affirmation is contradictory with the findings of this report. Only one case was purely a \"primary\" MCL, in the rest, the predominant manifestation was the development of disseminated cutaneous lesions.\n\nParagraph 8, lines 5-6: the reason why systemic therapy was administered in all these cases is that systemic treatment is the only standard therapeutic regimen for MCL (not due to resistance issues). For a better understanding of current therapeutic recommendations, authors must review citation four4 and update reference 9.\n\nParagraph 9: no local therapy is currently recommended for MCL. This information must be improved or removed.\nConclusions:\nParagraph 2: what other infections must be considered as a differential diagnosis among oral lesions?\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-756
https://f1000research.com/articles/9-488/v1	01 Jun 20	{ "type": "Case Report", "title": "Case Report: IgG multiple myeloma and chronic myeloid leukemia in a single patient", "authors": [ "Neeraja Swaminathan", "Sorab Gupta", "Claudia Dourado", "Sorab Gupta", "Claudia Dourado" ], "abstract": "A 58-year-old man presented with recurrence of chronic myeloid leukemia (CML) after complete molecular remission in the setting of non-compliance with imatinib. He was restarted on imatinib and was also noted to have IgG kappa monoclonal gammopathy of undetermined significance (MGUS). The patient re-achieved molecular remission after resumption of imatinib, but his MGUS progressed to smoldering myeloma and he was eventually diagnosed with multiple myeloma (MM) and initiated on treatment for MM with thalidomide, bortezomib and dexamethasone. He has responded well to treatment of the myeloma and continues concurrent maintenance imatinib treatment for CML and is being evaluated for bone marrow transplant. The association of two concurrent hematological malignancies, CML and MM, is very rare and has been infrequently reported in literature. The pathophysiology of this has not yet been fully understood. This case report reviews the various theories to explain this and discusses the potential challenges of simultaneous treatment of MM and CML.", "keywords": [ "multiple myeloma", "MGUS", "CML", "chronic myeloid leukemia" ], "content": "Introduction\n\nChronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of pluripotent hematopoietic stem cells. It results from a translocation t (9;22) (q34q11) known as the Philadelphia chromosome creating a BCR-ABL fusion gene, which is transcribed into proteins with abnormal tyrosine kinase activity that drives abnormal white blood cell (WBC) proliferation1. Multiple myeloma (MM) is a monoclonal disorder of plasma cells which have differentiated from lymphoid B cells. Therefore, the abnormal cell types in CML and MM are distinctly different. The instances of both MM and CML in the same patient occurring in a synchronous or metachronous manner are extremely rare. There are several factors that have been postulated to be related to this occurrence. These include age, gender, race, exposure to environmental carcinogens or radiation, epigenetic upregulation/downregulation, as a result of progression or treatment of one malignancy potentiating the development of malignant cells (which acquire antiapoptotic ability) and mechanisms to evade immune surveillance, chronic antigenic stimulation, genetic polymorphisms. At present there are multiple theories but insufficient data to make any definite conclusions about the mechanism of co-existence of CML and MM. With the advent of novel therapies and improving survival in patients with CML and MM, there is value in further investigation regarding the pathophysiology and clinical characteristics of such cases2,3. Additionally, the occurrence of more than one hematological malignancy in the same patient presents treatment challenges because it can lead to the possibilities of drug-drug interactions and medication toxicities. Analysis of the treatment protocols of these patients and their follow-up will be required to assess the risks and benefits of different treatment options. At this time, due to paucity of data, the management is tailored according to the patient’s individual risk factors and clinician’s judgement.\n\nThis is a rare case of the occurrence of IgG MM and CML in a single patient and reviews the management of these diseases.\n\n\nCase report\n\nA 58-year-old man with history of CML presented in December 2015 for re-establishment of care after being lost to follow-up. He was first diagnosed in December 2007 with CML and was treated with imatinib (400mg daily). He achieved complete molecular remission but was unfortunately lost to follow-up after 2012. The patient stated that he had stopped taking imatinib in September 2015 due to family issues and stressors. When he presented in December 2015, he had no specific complaints. He was noted to have mild pallor on exam, but no lymphadenopathy or hepatosplenomegaly. Labs were significant for leukocytosis and blasts noted on peripheral smear. BCR-ABL FISH/PCR was also positive indicating relapse of CML.\n\nThe patient was re-initiated on treatment with imatinib 400mg daily. Within 2 weeks of resuming treatment he was noted to have an improvement in WBC count. However, he was incidentally detected to have an elevated total protein level. In view of this serum protein, electrophoresis was ordered and the patient was found to have an IgG kappa monoclonal gammopathy of undetermined significance (MGUS). Skeletal survey was done which showed no lytic lesions. Imatinib was continued for CML and he was monitored closely for progression of plasma cell dyscrasia.\n\nWith regard to the patient’s CML, he achieved molecular remission in November 2016 with 3 log reduction in the 3 tested transcripts b2a2, b3a2, e1a2 and without detectable Philadelphia chromosome. However, he had a steady increase in the paraprotein level from December 2015 to April 2019 without any symptoms. He did not develop myeloma defining events such as hypercalcemia (>1mg/dL over the upper limit of normal OR >11mg/dL), renal insufficiency (creatinine clearance <40mL/min or serum creatinine >2mg/dL), anemia (Hemoglobin > 2g/dL below lower limit of normal or <10g/dL) or bony pain/lytic lesions (on skeletal radiography/CT/PET). These are together referred to as the CRAB phenomenon. His serum free light chain ratio remained at <100mg/L.\n\nIn May 2019, the patient had further rise in creatinine and paraprotein level and had an increase in serum free light chain ratio. Therefore, he underwent a PET CT in June 2019. This showed increased uptake in the left 4th rib, left and right ischium, and a lesion in the second lumbar (L2) vertebra. He also underwent a bone marrow biopsy in July 2019, which showed 50% plasma cells expressing CD 38, CD 138, dim/partial CD 117, CD 56 and kappa light chain restriction. No BCR ABL gene rearrangement was noted. Thus, it confirmed the diagnosis of MM.\n\nMM interphase fluorescence in situ hybridization (FISH) panel analysis of CD138+ enriched plasma cells was positive for three CCND1 signals consistent with trisomy 11 and for extra signals for chromosomes 7, 9 and 15. There were no cells with FGFR3-IGH, CCND1-IGH, or IGH-MAF fusions. Results for 1p/1q, 13q and TP53 were normal. Extra signals for probes targeting the chromosomes reported above suggest the presence of a hyperdiploid clone. Thus, the patient’s cytogenetic testing was negative for high risk factors.\n\nThe trends of serum protein electrophoresis are seen in Table 1, and Figure 1 and Figure 2. Trends of WBC count, hemoglobin, platelet count, and creatinine are seen in Figure 3–Figure 6, respectively.\n\nAbbreviations used: A/G ratio- albumin/globulin ratio, M spike- monoclonal spike, Ig- immunoglobulin, LDH-lactate dehydrogenase.\n\nThe patient was started on treatment for ISS stage II standard risk myeloma with thalidomide (50mg/day daily for 21 days followed by 7 days off), weekly bortezomib (1.3mg/m2) and dexamethasone (40mg/day once a week) in September 2019. This regimen was preferred over the VRd (lenalidomide, bortezomib and dexamethasone) due to the concern for increased risk of pancytopenia with concurrent use of imatinib and lenalidomide. He was also referred to a bone marrow transplant center to assess if he is eligible. Thus far, he has received four cycles of treatment for myeloma along with the concurrent imatinib and has tolerated it well with no dose limiting toxicities.\n\n\nDiscussion\n\nThe occurrence of CML and MM together is very rare. There are multiple explanations that have been suggested for co-existence of more than one hematological malignancy. One of the theories is that there is a common progenitor stem cell. The Philadelphia chromosome is observed not only in granulocytes but also in cells of the monocytic, erythroid, megakaryocytic and lymphoid series. This finding supports the concept of a common pluripotent progenitor cell. The transformation to lymphoid cells in the blast phase of the CML also suggests a relationship between the myeloid and lymphoid lineage4.\n\nThe role of imatinib in promoting development of MM is debatable. There is some evidence from Pandiella et al.5 that imatinib inhibits MM cell proliferation in vitro. On the contrary, it has been reported that imatinib has a stimulatory effect on MM cells through activation of Erk1 and Erk2 mitogen activated protein kinases (MAP kinases). There have also been reports of MM developing in non CML patients such as those with gastrointestinal stromal tumors treated with imatinib6 Carulli et al.7 looked at the possible interference of imatinib with plasma cell phenotype. Their study looked at 30 patients and found that 70% of these patients had an abnormal plasma cell phenotype, which they defined as plasma cells which lack CD 19. Some of these cells showed additional aberrations such as expression of CD 56. Although this cannot establish a causal relationship between imatinib and MM, it merits further investigation.\n\nImatinib mesylate is a tyrosine kinase inhibitor, which is the standard of care for CML as a first line agent. It has activity against different genes involved in cellular transformation such as BCR-ABL1, c-KIT, PGFR-α and β and Jak 2. Imatinib in CML acts by competing with ATP to bind to the BCR ABL1 tyrosine kinase and thereby inhibiting the WBC proliferation that it effects7.\n\nTreatment of MM is based on whether the patient has standard or high-risk disease, which in turn is determined by cytogenetic analysis. High risk features constitute t (14;16), t (4;20), del17p13, t (4;14) and 1q gain. Additionally, patients should be assessed for eligibility to receive an autologous stem cell transplant. In standard risk patients such as ours the standard first line treatment is RVd regimen, which comprises lenalidomide, bortezomib and dexamethasone. Post induction therapy, if patients are eligible for hematopoietic cell transplant (HCT), they may choose either an early HCT or delayed HCT strategy. Autologous HCT is the mainstay, although allogenic HCT is still largely investigational. Post-transplant patients need maintenance therapy as well. Transplant in eligible patients receive either a two/three drug induction regimen followed by maintenance therapy. Melphalan, cyclophosphamide and thalidomide are also part of first line treatment options for myeloma. In patients with relapse, several of the newer agents are being used, which include the new proteasome inhibitor (carfilzomib), immunomodulatory drugs (like pomalidomide), inhibitors of NF-κB, MAPK and AKT, histone deacetylase inhibitors (like vorinostat and panobinostat), and monoclonal antibodies (such as daratumumab, elotuzumab and siltuximab)8.\n\nDue to the rarity of coexistence of more than one hematological malignancy in the same patient, we do not have robust data on treatment regimens. Current treatment is based on factoring in the patient’s individual risk factors and the physician’s judgement and experience. Even though there is concern for imatinib being associated with MGUS and myeloma, co-administration of imatinib with myeloma treatment seems to be reasonable. Myeloma treatment both for high risk and standard risk group patients involves a proteasome inhibitor, such as bortezomib. Both bortezomib and imatinib are metabolized by microsomal enzyme CYP3A4. However, imatinib is a potent inhibitor of CYP3A4, while bortezomib is only a weaker inhibitor. Reduction of bortezomib dosing to once weekly instead of twice seems to be associated with less adverse effects when used in conjunction with imatinib9.\n\nAdditionally, bisphosphonates, which are used as supportive treatment in myeloma, have also been shown to inhibit CML cell lines and induce apoptosis synergistically with imatinib through the inhibition of prenylation of Ras and Ras-related proteins10\n\nThis case report presents an experience with a CML patient who over time progressed to develop the entire spectrum of myeloma, starting with MGUS followed by smoldering myeloma and ultimately MM requiring treatment. At this point it is difficult to say if this association is coincidental or related to some other mechanism and this is a subject that requires further research.\n\n\nConclusion\n\nThe most essential take-home points from this case are as follows:\n\n1. MM can be diagnosed in patients with one of the following: >10% plasma cells in bone marrow OR biopsy proven bony/extramedullary plasmacytoma and any one of the CRAB phenomenon OR any of the following- >/= 60% plasma cells in bone marrow OR serum free light chain ratio >/= 100 OR >1 focal lesion on MRI studies.\n\n2. Both MGUS and smoldering myeloma are characterized by an absence of myeloma defining events. In MGUS: paraprotein level <3g/dL, bone marrow plasma cells <10%; while in smoldering myeloma: paraprotein level >3g/dL or urinary monoclonal protein >/= 500mg in 24 hours and/or clonal bone marrow plasma cells 10 to 60%\n\n3. Co-existence of CML and MM is very rare. Etiology is probably multifactorial but there is a possibility that this is because CML and MM share a common pluripotent progenitor stem cell which can differentiate into both lymphoid and myeloid lines.\n\n4. There is concern that the imatinib may lead to alteration of the plasma cell phenotype and it may be worthwhile to monitor serum electrophoresis and protein levels in patients who have received imatinib treatment.\n\n5. Co-administration of bortezomib and imatinib is feasible. Since CYP3A4 is important for metabolism of both drugs, administration of bortezomib as weekly instead of twice weekly may help to reduce adverse effects of bortezomib.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for the publication of the case report and any associated images was obtained from the patient.", "appendix": "References\n\nHehlmann R, Hochhaus A, Baccarani M, et al.: “Chronic myeloid leukaemia” Lancet. 2007; 370(9584): 342–350. PubMed Abstract \| Publisher Full Text\n\nAli N, Pickens PV, Auerbach HE: Immunoglobulin D multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia in a single patient treated simultaneously with lenalidomide, bortezomib, dexamethasone and imatinib. Hematol Rep. 2016; 8(1): 6295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaerki J, Katava G, Siegel D, et al.: Unusual Case of Simultaneous Presentation of Plasma Cell Myeloma, Chronic Myelogenous Leukemia, and a Jak2 Positive Myeloproliferative Disorder. Case Rep Hematol. 2014; 2014: 738428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlenn PJ, Hyun BH, Lee YH, et al.: Multiple myeloma and chronic myelogenous leukemia: a case report with literature review. Yonsei Med J. 1993; 34(3): 293–300. PubMed Abstract \| Publisher Full Text\n\nPandiella A, Carvajal-Vergara X, Tabera S, et al.: Imatinib mesylate (STI571) inhibits multiple myeloma cell proliferation and potentiates the effect of common antimyeloma agents. Br J Haematol. 2003; 123(5): 858–868. PubMed Abstract \| Publisher Full Text\n\nIde M, Kuwahara N, Matsuishi E, et al.: Uncommon case of chronic myeloid leukemia with multiple myeloma. Int J Hematol. 2010; 91(4): 699–704. PubMed Abstract \| Publisher Full Text\n\nCarulli G, Cannizzo E, Ottaviano V, et al.: Abnormal phenotype of bone marrow plasma cells in patients with chronic myeloid leukemia undergoing therapy with Imatinib. Leuk Res. 2010; 34(10): 1336–1339. PubMed Abstract \| Publisher Full Text\n\nRajkumar SV: Multiple myeloma: Every year a new standard? Hematol Oncol. 2019; 37(Suppl 1): 62–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaral S, Bakanay SM, Yikilmaz AS, et al.: Development of plasma cell leukemia in a patient with chronic myeloid leukemia while on treatment with imatinib mesylate. J Cancer Res Ther. 2018; 14(6): 1431–33. PubMed Abstract \| Publisher Full Text\n\nChuah C, Barnes DJ, Kwok M, et al.: Zolendronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells. Leukemia. 2005; 19(11): 1896–1904. PubMed Abstract \| Publisher Full Text" }	[ { "id": "64202", "date": "08 Jun 2020", "name": "Mahesh Swaminathan", "expertise": [ "Reviewer Expertise Myeloid leukemias", "clinical trials", "Genetic syndromes in leukemia." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 case report, the authors have discussed a patient with chronic myeloid leukemia and metachronous IgG multiple myeloma. The clinical presentation and disease course are well described and discussed.\n\nAdding the following details would add more clarity to the case and help readers:\nPlease mention the transcripts at diagnosis (as knowing typical versus atypical transcripts is important for follow up, the latter might not be detectable in routine PCR and warrants RT-PCR).\n\nAt CML disease relapse, please indicate if the patient was still in the chronic phase. Was there a bone marrow biopsy done at that time, if so what was the percentage of plasma cells.\n\nAs the diagnosis of MGUS requires BMBx? Please indicate if the clonal plasma cells were <10% facilitating the diagnosis of MGUS.\n\nPlease indicate if the x-axis in Figure 1-2 denotes months?\n\nIt will be interesting to know the most common Grade >3 AEs experienced by the patient (to know if there were any specific side effects that were more common with concomitant imatinib and myeloma therapy.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5613", "date": "15 Jun 2020", "name": "Neeraja Swaminathan", "role": "Author Response", "response": "Thank you for the feedback and comments provided. Yes, the transcripts at the time of diagnosis were also typical. At the time of relapse, the patient was in the chronic phase. Only rare blasts were seen on peripheral blood smear. There were fewer than 10% blast cells in the blood and a bone marrow biopsy was not performed. When patient was noted to have an abnormal M-protein spike on serum electrophoresis, a bone marrow biopsy was repeated which showed < 10% plasma cells. The total M protein spike was <3g/dL and the patient had no evidence of end organ damage. All of this helped establish the diagnosis of MGUS and he was monitored clinically and with labs until May-June 2019 when he developed an increase in creatinine and eventually had a bone marrow biopsy which confirmed the diagnosis of multiple myeloma. Yes, the x-axis in figures 1 and 2 denotes time in months. This patient developed a morbilliform pruritic rash over trunk and bilateral upper extremities which has been described as a side effect of thalidomide. This occurred after cycle 2 and was managed with short course of steroids. After cycle 5, he had recurrence of above described rash and also developed conjunctival injection bilaterally. He was seen by ophthalmology and diagnosed to have meibomian gland dysfunction. In view of these symptoms, there was a delay in his myeloma treatment but his symptoms improved with supportive care (oral steroids and steroid eye drops) and resolved completely. He has now completed 6 cycles of treatment for myeloma and is being evaluated for bone marrow transplant. He has had no other side effects." }, { "c_id": "5723", "date": "14 Jul 2020", "name": "Mahesh Swaminathan", "role": "Reviewer Response", "response": "Thanks for updating. Please make necessary inclusions of those comments in the manuscript." } ] }, { "id": "65599", "date": "09 Jul 2020", "name": "Pritish K. Bhattacharyya", "expertise": [ "Reviewer Expertise Leukemia", "Lymphoma", "Myeloma with molecular genetic applications" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 uncommon case presentation of two apparently unrelated hematopoietic malignancies.\nAssociation of Multiple myeloma following CLL or other low grade B cell lymphoma may be related to terminal changes of B cell to Plasma cells (Jaffe et al., NIH)\nWe had published a similar case in 2014 in a patient of CML with t9:22) along with JAK2 mutation and Myeloma (Maerki et al., 20141). We suggested another potential theory of multiple simultaneous malignancies includes the sharing of a common malignant pluripotent progenitor stem cell which may allow further transformation of both lymphoid and myeloid differentiations.\nIt has been proposed that plasma cell myeloma and CML may evolve from the same hematopoietic stem cell [1, 8, 12]. There have been multiple accounts in which plasma cell neoplasms, including plasma cell leukemia and multiple myeloma, have been seen in coexistence with or arising in the background of CML [37–39]. It is suggested that the simultaneous occurrence of CML and multiple myeloma is analogous to acute lymphoblastic leukemia arising in the blastic phase of CML [5, 6, 8, 12, 15]. This reveals the capability of CML in differentiating into either myeloid lineage or lymphoid lineage. Furthermore, the Philadelphia chromosome (Ph) has been linked to increased cell survival, proliferation, and malignant transformation [1, 40] and has been found in all hematopoietic cell lineages including erythropoietic cells, megakaryocytes, macrophages, and B-lymphocytes [1,12, 41].\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-488
https://f1000research.com/articles/9-1167/v1	23 Sep 20	{ "type": "Case Report", "title": "Case Report: A case of immune checkpoint inhibitor therapy in a patient with multiple sclerosis", "authors": [ "Raju Vaddepally", "Soujanya Sodavarapu", "Anupama Kutadi", "Wesley Taylor", "Navneeth Kumar", "Soujanya Sodavarapu", "Anupama Kutadi", "Wesley Taylor", "Navneeth Kumar" ], "abstract": "Immune checkpoint inhibitors (ICIs) have rapidly shifted the landscape of treatments in malignancy with significant improvements in survival paradigm. They have been an attractive armamentarium to the oncologists given the limited immune adverse effects with potential for deeper and durable benefits that haven't been previously noticed with chemotherapy. However, they result in unique toxicities by limiting immune self-tolerance and cause immune-mediated endocrinopathies, such as hypothyroidism, pneumonitis, colitis, hepatitis, myocarditis, meningitis, hypophysitis, etc. As such, they are contraindicated in patients with autoimmune disorders or recipients of organ transplants given the risk for reactivation or flare of the underlying autoimmune disease and rejection of the donor organ in transplants, although sporadic cases have been reported with the use of immunotherapy in such patients. Malignant melanoma is a highly aggressive cancer, with only 15-20% five-year survival rate once it has spread to the lymph nodes or has distant metastasis. ICIs have changed the landscape of advanced melanoma with exponential improvements in survival, the 5-year survival rates are about 50%. Multiple sclerosis (MS) is recognized as T cell-mediated immune response causing inflammation, which causes local inflammatory plaques and demyelination. ICIs are likely to generate an immune response that causes molecular mimicry and cross-react with CNS autoantigens, in turn exacerbating pre-existing immune response and subsequent flare-ups in MS. There is little knowledge about treating such patients with immunotherapy, short of a few case reports and series; in this report, we describe another such case. We present a case of checkpoint inhibitor therapy in a patient with multiple sclerosis who underwent immune checkpoint inhibitor therapy with pembrolizumab for metastatic malignant melanoma who had a complete response to treatment at the cost of MS relapse, which was managed with high-dose steroids.", "keywords": [ "Immunotherapy", "checkpoint inhibitors", "pembrolizumab", "malignant melanoma", "multiple sclerosis", "malignancy", "antigens", "toxicites", "adverse effects", "steroids" ], "content": "Introduction\n\nImmune checkpoint inhibitor therapy has transformed the cancer care landscape1. It is being used to treat various malignancies with significant improvement in survival noted in solid tumors such as melanoma and non-small cell lung carcinoma, etc2. This has been an attractive alternative for many oncologists given its efficacy and minimal toxicity across the spectrum of program death ligand-1 (PDL-1) and program death-1 (PD-1) inhibitors3. However, they result in unique toxicities related to immune modulation and activation such as immune-mediated endocrinopathies such as hypothyroidism, pneumonitis, colitis, hepatitis which are seemingly more common compared to rare complications such as myocarditis, meningitis, hypophysitis, etc4. As such, checkpoint inhibitor therapy is contraindicated in patients with autoimmune disorders or recipients of organ transplants given the risk for reactivation or flare of the underlying autoimmune disease and rejection of the donor organ in transplants, although sporadic cases have been reported with the use of immunotherapy in such patients5. In this report, we present a case of checkpoint inhibitor therapy in a patient with multiple sclerosis (MS) who underwent immune checkpoint inhibitor therapy with pembrolizumab for metastatic malignant melanoma.\n\n\nCase Report\n\nA 73-year-old female with a past medical history of MS with chronic right hemi-somal deficits on baclofen (10 mg twice daily), paroxetine (40 mg daily), and gabapentin (300 mg three times daily). She had prior history of cutaneous malignant melanoma (MM) in 2015 post excision and negative sentinel lymph nodes noticed left inguinal swelling before November in 2018 and a PET scan revealed prominent and bulky metabolic adenopathy involving the left inguinal, external iliac, proximal internal iliac regions, and common iliac region lymphadenopathy (Figure 1 and Figure 2). Left inguinal mass biopsy revealed metastatic malignant melanoma (Figure 3 and Figure 4). Tissue from the biopsy tested positive for HMB-45 and Melan-A antibodies (Figure 5 and Figure 6). Biomarker testing revealed no evidence of BRAF or KIT mutations; TERT, NRAS mutations and MDM2 copy gain was present, MSI-stable, and the TMB high at 8.8 mutation/MB (87th percentile). Magnetic resonance imaging (MRI) of the brain was negative for metastatic malignancy however showed non-specific white matter changes consistent with demyelinating plaques linked to her history of multiple sclerosis without finding of active disease (Figure 7 and Figure 8).\n\nMultiple prominently metabolic nodes are visualized throughout the left external iliac, proximal internal iliac and common iliac regions. 1 of the more prominent nodes situated in the distal external iliac chain spanning 2.8 × 4.2 cm with an SUV max of 18.4 on image 250. Small metabolic nodes are also visualized in the lower retroperitoneum up to the L4 level.\n\nFor example, a left inguinal node is 1.3 × 1.8 cm with an SUV max of 1.0; previously a nodal conglomerate in this region was 2.8 × 5.6 cm and SUV max 21.1. A small residual nodal prominence in the left external iliac region is 0.9 × 2.7 cm, SUV max 2.0; previously 2 adjacent nodes in this region or 3.3 × 7.6 cm and SUV max 19.1. No new metabolic nodal lesions are visualized in the inguinal regions, pelvis or throughout the abdomen\n\nIn the background you can see some normal lymphocytes (smaller dark blue staining cells). Magnification, ×20.\n\nThere are few abnormal mitotic figures and some plasma cells in the background.\n\nNonspecific focal white matter lesions could reflect multiple sclerosis, without finding of active disease.\n\nAfter discussing the pros and cons related to immune checkpoint inhibitors (ICI) versus salvage chemotherapy; she expressed understanding of the potential but significant risk of MS flare-up. She was initiated on pembrolizumab 200 mg every 3 weeks at 4 weeks after diagnosis. Unfortunately, two weeks after the first cycle of pembrolizumab therapy she encountered significant lethargy, slurred speech, word-finding difficulty, stiffness and pain in her bilateral groins, gait imbalance with recurrent falls, generalized weakness requiring assistance for bed transfer. Pertinent physical exam findings included pupils that were reactive to light both directly and consensually; the fundoscopic exam was normal with the cup to disc ration, no edema/hemorrhages; apraxia, tremors in bilateral hands; decreased motor strength (4/5 using the Oxford scale) in the bilateral upper and lower extremity muscle groups; deep tendon reflexes 2/4 in the left and 3/4 in the right biceps, triceps, patellar and achilles. Hoffman, Tromner's, and Babinski signs were present on the right; sustained clonus was noted in right ankle and ataxia was present. Complete blood picture, electrolytes, kidney, and liver function were essentially unremarkable. Her signs and symptoms were concerning for an MS flare-up, and an MRI of the brain with and without contrast revealed enhancing white matter lesion on the right external capsule, larger in size and hyperintense per the gadolinium contrast; EEG was unremarkable. In light of clinical and radiological findings consistent with an MS flare-up, she was started on prednisone 1 mg/kg with a prolonged taper over 8 weeks by 10 mg every week with subsequent significant improvement in treatment-related neurological symptoms in the coming weeks with active participation in physical therapy (PT).\n\nShe had an interval improvement in the tumor size with just one round of treatment, which has been correlated in studies with patients encountering immune-related adverse effects (iAE’s) often having better responses than their counterparts who do not have iAE’s6. CT imaging of the abdomen/pelvis with contrast imaging post-cycle showed significant interval response with the adenopathy decreased by >50%, for example, left inguinal lymph node decreased from 4.6 to 1.3 cm, left external iliac adenopathy decreased from 4.7 to 2.1 cm, and the para-aortic lymph node is essentially resolved, measuring 4 mm where it previously measured 11 mm. Two months into her complete convalescence from neurological adverse effects related to the MS flare-up, she was rechallenged with a second round of pembrolizumab therapy with a baseline of continued prednisone 20 mg, which she tolerated well without MS relapse. She subsequently encountered immune-mediated acute hepatitis and recurrent MS flare-up after cycle 4 of therapy. Labs revealed liver transaminases elevated to 20 times upper limit of normal (ULN)- AST 711 U/L, ALT 978 U/L, and ALP 392 U/L (3 x ULN) with normal total bilirubin 1.1 mg/dl. She was then subjected to another round of high-dose prednisone 1.5 mg/kg with a prolonged taper, requiring a stay at a skilled nursing facility and PT for around 1 month. Restaging PET scan after four cycles of immunotherapy in July 2019 revealed significant interval improvement: previous prominent metabolic adenopathy in the left pelvis/inguinal region and lower retroperitoneum had completely resolved. Small residual nodal lesions remained in the left inguinal region and left external iliac chain, with these demonstrating only low level/background metabolic activity. For example, a left inguinal node was 1.3 × 1.8 cm with an SUV max of 1.0 on axial image 271 - previously a nodal conglomerate in this region was 2.8 × 5.6 cm and SUV max 21.1; a small residual nodal prominence in the left external iliac region was 0.9 × 2.7 cm, SUV max 2.0 on image 256, whereas previously two adjacent nodes in this region were 3.3 × 7.6 cm and SUV max 19.1. At this point, given recurrent and severe CPI-mediated immune-related adverse effects with MS flare-ups and hepatitis, further immunotherapy was discontinued. The patient continued to exhibit an incomplete response thereafter, but with decreased quality of life given the persisting debility from MS relapse.\n\n\nDiscussion\n\nMalignant melanoma is a highly aggressive cancer, with only a 15-20% five-year survival rate once it has spread to the lymph nodes or has distant metastasis7. The development of ICIs has changed the face of melanoma treatment and has improved the survival rates of these patients. The drugs in this class have been directed against CTLA-4, PD-1 and PD-L1, namely ipilimumab, nivolumab and pembrolizumab, respectively3. ICIs have changed the landscape of advanced melanoma with exponential improvements in survival, the 5-year survival rates were about 50%8,9.\n\nICIs can present with a wide range of irAEs due to lack of selectivity and by their generalized immune reaction10. These effects range from systemic autoimmune complications to severe neurological complications11. The neurological complications have been estimated to have an incidence of 2–4% with severe neurological manifestations, including MS, being reported in 0.2–0.4% of patients treated with pembrolizumab and nivolumab11.\n\nMS is recognized as T cell-mediated immune response causing inflammation, which causes local inflammatory plaques and demyelination8. ICIs are likely to generate an immune response that causes molecular mimicry and cross-react with CNS autoantigens, in turn exacerbating pre-existing immune response and subsequent flare-ups in MS11,12.\n\nOn an extensive literature review, we could come across very few reports of ICIs use in patients with MS. Isitan et al. described a 46-year-old with stage IV metastatic non-small cell lung cancer who was treated with one dose of atezolizumab, who subsequently developed relapse of MS with worsening features and refractory to treatment13. Another case, described by Gettings et al., was of a 56-year-old male with MS with recurrent melanoma after multiple resections; he was treated with ipilimumab who developed two MS flare-ups, leaving him with disability from MS relapse but had remission of melanoma on PET scan14. Garcia et al. conducted a retrospective analysis capturing 42,529 adverse events with the use of ICI with 13 cases identified with MS. Of the 13, eight had a previous diagnosis of MS. All the patients in their analysis experienced progression of their MS, and two patients died following their MS relapse15.\n\nOur patient had her first MS relapse after the first dose of pembrolizumab, which resolved with high-dose steroids. She did not develop any flare-ups with the second and third rounds of therapy, but again encountered a relapse during the fourth round, which was again successfully treated with steroids but with delayed convalescence. At the end of the fourth round patient achieved complete remission of metastatic malignant melanoma. Our case adds to the pool of existing clinical data that immune checkpoint inhibitors are universally detrimental to patients with MS, given definitive relapse identified in almost all the patients identified during the literature review and our personal experience.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nHavel JJ, Chowell D, Chan TA: The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nat Rev Cancer. 2019; 19(3): 133–150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoun B, Trikalinos NA, Mor V, et al.: Real-world use and survival outcomes of immune checkpoint inhibitors in older adults with non–small cell lung cancer. Cancer. 2020; 126(5): 978–985. PubMed Abstract \| Publisher Full Text\n\nVaddepally RK, Kharel P, Pandey R, et al.: Review of Indications of FDA-Approved Immune Checkpoint Inhibitors per NCCN Guidelines with the Level of Evidence. Cancers (Basel). 2020; 12(3): 738. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar V, Chaudhary N, Garg M, et al.: Current Diagnosis and Management of Immune Related Adverse Events (irAEs) Induced by Immune Checkpoint Inhibitor Therapy. Front Pharmacol. 2017; 8: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson DB, Sullivan RJ, Menzies AM: Immune checkpoint inhibitors in challenging populations. Cancer. 2017; 123(11): 1904–1911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrior LM, Harrold E, O'Leary CG, et al.: Toxicities in immunotherapy: Can they predict response? J Clin Oncol. 2016; 34(15 suppl): e14534. Publisher Full Text\n\nWeiss SA, Hanniford D, Hernando E, et al.: Revisiting determinants of prognosis in cutaneous melanoma. Cancer. 2015; 121(23): 4108–4123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobert C, Ribas A, Schachter J, et al.: Pembrolizumab versus ipilimumab in advanced melanoma (KEYNOTE-006): post-hoc 5-year results from an open-label, multicentre, randomised, controlled, phase 3 study. Lancet Oncol. 2019; 20(9): 1239–1251. PubMed Abstract \| Publisher Full Text\n\nLarkin J, Chiarion-Sileni V, Gonzalez R, et al.: Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015; 373(1): 23–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYshii LM, Hohlfeld R, Liblau RS: Inflammatory CNS disease caused by immune checkpoint inhibitors: status and perspectives. Nat Rev Neurol. 2017; 13(12): 755–763. PubMed Abstract \| Publisher Full Text\n\nDalakas MC: Neurological complications of immune checkpoint inhibitors: what happens when you 'take the brakes off' the immune system. Ther Adv Neurol Disord. 2018; 11: 1756286418799864. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang WJ, Chen WW, Zhang X: Multiple sclerosis: Pathology, diagnosis and treatments. Exp Ther Med. 2017; 13(6): 3163–3166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIsitan C, Wesley S: Safety of Checkpoint Inhibitors for Cancer Treatment in Patients with Multiple Sclerosis: A Case Report (P1.7-006). Neurology. 2019; 92(15 Supplement): P1.7-006. Reference Source\n\nGettings EJ, Hackett CT, Scott TF: Severe relapse in a multiple sclerosis patient associated with ipilimumab treatment of melanoma. Mult Scler. 2015; 21(5): 670. PubMed Abstract \| Publisher Full Text\n\nGarcia CR, Jayswal R, Adams V, et al.: Multiple sclerosis outcomes after cancer immunotherapy. Clin Transl Oncol. 2019; 21(10): 1336–1342. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "76659", "date": "02 Feb 2021", "name": "Angela Vidal-Jordana", "expertise": [ "Reviewer Expertise Multiple sclerosis", "neuroimmunology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors use different terms regarding Multiple Sclerosis disease that are not correct and/or not commonly used in the literature. Some of them are listed below:\nIn the abstract and the discussion section, they state that MS is recognized as a T cell-mediated immune disease. With the advent of the new therapies targeting B cells (such as ocrelizumab, rituximab, ofatumumab, among others) this statement has been questioned and does not hold true anymore.\n\nThey use the term “flare-ups” where they should use the more correct term “relapses” when referring to neurological worsening due to MS.\n\nIn the case report section, page 3, paragraph 2 the authors state: “MRI (….) showed non-specific white matter changes consistent with demyelinating plaques linked to her history of MS without finding of active disease”. White matter lesions suggestive of MS are not considered non-specific, in fact, they should meet some specific criteria in order to attribute the lesions to MS, not only in shape but also in localization. Also, I recommend the authors to better specify the meaning of “active disease”. Do they refer to new T2 lesions compared to a previous MRI or to Gd-enhancing lesions?\n\nApart from these terminology issues, my main concern is that the neurological symptoms that the patient presented (lethargy, slurred speech, word-finding difficulty, stiffness, pain, gait imbalance, and generalized weakness requiring assistance for transfers) are most probably not related to her MS. Neither the symptoms are typical for an MS relapse (that are also very infrequent in older patients), nor they can be justified by an increase in the size of a previous lesion in the right external capsule, even in the presence of Gd-enhancement, as lesions in this location do not produce the aforementioned symptomatology. It is most likely that the patient might have suffered from ICANS (or other neurotoxicity related to immune checkpoint inhibitors) that usually also improves with corticosteroid treatment.\nTherefore, in my opinion, the case report's interest was to report the evolution of an MS patient receiving immunotherapy and how it impacted her disease. However, having in mind all the stated above, I am afraid I should recommend not to index the case report in its current form.", "responses": [] } ]	1	https://f1000research.com/articles/9-1167
https://f1000research.com/articles/9-332/v1	05 May 20	{ "type": "Research Article", "title": "Determinants of safe delivery utilization among Indonesian women in eastern part of Indonesia", "authors": [ "Ferry Efendi", "Susy Katikana Sebayang", "Erni Astutik", "Setho Hadisuyatmana", "Eka Mishbahatul Mar'ah Has", "Heri Kuswanto", "Susy Katikana Sebayang", "Erni Astutik", "Setho Hadisuyatmana", "Eka Mishbahatul Mar'ah Has", "Heri Kuswanto" ], "abstract": "Background: Improving maternal health and reducing maternal mortality are part of the United Nations global Sustainable Development Goals for 2030. Ensuring every woman’s right to safe delivery is critical for reducing the maternal mortality rate, especially in Indonesia. Our study aimed to identify determinants of safe delivery utilization among women in the eastern Indonesia. Methods: This study was cross-sectional and used data from the 2017 Indonesian Demographic and Health Survey (IDHS). A total of 2,162 women who had their last child in the five years preceding the survey and lived in the eastern part of Indonesia were selected as the respondents. Chi-squared test and binary logistic regression were used to understand the determinants of safe delivery. Results: Higher child rank and interval ≤2 years (OR: 0.30, 95% CI: 0.19-0.47), unwanted pregnancy at time of becoming pregnant (OR: 1.48, 95% CI: 1.05-2.08), richest wealth quintile (OR: 5.59, 95% CI: 3.37-9.30), more than four antenatal care visits (OR: 3.62, 95% CI: 2.73-4.79), rural residence (OR: 0.49, 95% CI: 0.36-0.66), good composite labor force participation (OR: 1.47, 95% CI: 1.15-1.89), and a good attitude towards domestic violence (OR: 1.33, 95% CI: 1.04-1.69) were found to be significantly associated with facility-based delivery. Higher child rank and interval ≤2 years (OR: 0.49, 95% CI: 0.29-0.83), husband/partner having completed secondary or higher education (OR: 2.18, 95% CI: 1.48-3.22), husband/partner having a non-agricultural occupation (OR: 1.35, 95% CI: 1.00-1.81), being in the richest wealth quintile (OR: 15.69, 95% CI: 5.53-44.50), and three other factors were found to be significantly associated with skilled assistance delivery. Conclusions: Safe delivery and facility-based delivery among women in the eastern part of Indonesia were determined by several individual and household factors. An open innovation and partnership process that engages the full range of stakeholders should be developed based on local needs.", "keywords": [ "facility-based delivery", "safe delivery", "skilled birth delivery." ], "content": "Introduction\n\nMaternal morbidity and mortality is a global health concern (World Health Organization, 2017). Every day in 2017, around 830 mothers died due to pregnancy and childbirth (World Health Organization, 2018). The United Nations Sustainable Development Goals set a target to reduce maternal deaths to 70 per 100,000 live births by 2030 (Nations, 2015). In Indonesia, the maternal mortality rate is still high, at 305 per 100,000 live births (BPS, 2015b). A higher rate was found in the eastern part of Indonesia, namely Nusa Tenggara, Maluku, and Papua Island, than in the other islands (BPS, 2015a). One of the major causes of maternal mortality is bleeding, which is followed by eclampsia (Tejayanti et al., 2012). Safe delivery as the critical policy of making motherhood safer requires skilled health professionals and facility-based delivery across the provinces of Indonesia (Efendi et al., 2019; Kementrian Kesehatan RI, 2014). As an archipelago country, institutional delivery and skilled assistant delivery are still a challenge because of the geographical situation (Belton et al., 2014; Ministry of Health [MoH], 2012). To increase safe delivery for Indonesian mothers, the government has set a goal to reach 85% of institutional deliveries in 2019 (Kementrian Kesehatan RI, 2017). Even though the government has not yet set a goal for skilled attendant delivery specifically in this document, it should be assumed that the government demands the highest standard of health attainment.\n\nThe 2017 IDHS found that there is a gap in coverage of institutional delivery and skilled assistant delivery between western provinces and eastern provinces of Indonesia. Eastern provinces of Indonesia, including Bali, Nusa Tenggara Island, Sulawesi Island, Maluku, and Papua Island, have not reached 70% coverage of safe delivery in either institutional delivery or skilled assistant delivery, as depicted in Figure 1 (BPS et al., 2018).\n\nStudies that examine safe delivery have been conducted in some countries. A study conducted in Ethiopia found that residence, religion, educational level, age at first pregnancy, parity, and antenatal care (ANC) attendance have a significant association with safe delivery service utilization (Abera et al., 2011). Another study conducted in Tanzania reported that in addition to socio-demographic factors, women’s empowerment status contributed to the decision to give birth with a health professional (Shimamoto & Gipson, 2015). In a similar vein, studies in 13 sub-Saharan African countries found that living conditions and women’s autonomy are key factors of maternal healthcare utilization (Iacoella & Tirivayi, 2019). In Indonesia, a study about facility-based childbirth found that educational level, place of residence, working status, involvement in decision-making, economic status, and ANC visits are significantly associated with health facility delivery among women (Efendi et al., 2019). Furthermore, the gap in age and education between a woman and her husband/partner, women’s self-esteem, age at first marriage, and age at pregnancy were found have a high association with institutional delivery among Indonesian women (Kurniati et al., 2018).\n\nThe gap in coverage of safe delivery, including institutional delivery and skilled assistant delivery, in western and eastern provinces of Indonesia should be resolved to attain the Sustainable Development Goals by 2030. Therefore, this study aimed to determine safe delivery utilization among Indonesian women in the eastern part of Indonesia.\n\n\nMethods\n\nIDHS ethical clearance was obtained from the Indonesian Ministry of Health (MoH). For this study, permission to use the data was obtained from Inner City Fund (ICF) International. This study used existing IDHS data and re-analysis was done under the original consent provided by the participants. Thus, no further consent was obtained from the participants.\n\nThis was an analytical cross-sectional study that used data from the 2017 IDHS. The 2017 IDHS was conducted in 34 provinces in Indonesia from July to December 2017 by the Central Statistics Agency (BPS), National Population and Family Planning Board (BKKBN), and the Ministry of Health with technical help from ICF. The Individual Recode (IR) dataset was downloaded from www.dhsprogram.com after completing registration.\n\nIn the 2017 IDHS, a total of 49,627 women were finished the survey. Two-stage cluster sampling was used to select the respondents. Interviews were performed as privately as possible with a detailed manual as reported by ICF (ICF Macro, 2020).\n\nThe inclusion criteria for this study were women aged 15–49 years who had their last child in the five years preceding the survey and lived in the eastern provinces of Indonesia. For the purpose of analysis, we divided Indonesia into two greater parts, western and eastern, based on the geographical location. The eastern provinces included Bali, West Nusa Tenggara, East Nusa Tenggara, North Sulawesi, Central Sulawesi, South Sulawesi, Southeast Sulawesi, Gorontalo, West Sulawesi, Maluku, North Maluku, West Papua and Papua. In total, survey data from 2,162 women meeting the criteria were accessed for this study’s analysis.\n\nThe dependent variables in this study were facility-based delivery and assisted delivery. Facility-based delivery was divided into two categories: health facility and non-health facility. Health facility delivery or institutional delivery is delivery that is carried out at a heath facility, including public health centers, clinics or maternity homes, and hospitals. The assisted delivery variable was also divided into two categories: health professional and non-health professional. Health professional or skilled assistant delivery is defined as birth delivered with the assistance of skilled providers such as general practitioners, obstetricians, midwives, and skilled nurses.\n\nThere were several independent variables in this study. Age difference between man and woman was divided into four categories: woman older than husband, 0–4 years younger, 5–7 years younger, and >7 years younger. Birth rank and interval was divided into five categories: second or third child with interval >2 years; first birth, second or third child with interval ≤2 years; fourth or higher child with interval >2 years; and fourth or higher child with interval ≤2 years. Planning status of births, women who had a birth or several births in the five years prior to their interview were asked whether the pregnancy had been wanted at the time it occurred (wanted then) or whether it had been wanted but had occurred sooner than wanted (wanted later), or whether the woman had wanted no further children at the time (unwanted). Husband/partner’s education attainment was divided into three categories: incomplete primary education/none, complete primary or some secondary, and completed secondary or higher. Husband/partner’s occupation was divided into two categories: agricultural and non-agricultural. Wealth quintile was categorized as poorest, poorer, middle, richer, and richest (Rutstein & Johnson, 2004). Number of household members was divided into two categories: households that have less than four members and households with four or more members. Number of ANC visits was categorized as less than four times and four times or more. Covered by health insurance was divided into two categories: yes and no. Residence was categorized as urban and rural. Women’s empowerment variables, including composite labor force participation, attitude towards domestic violence (wife-beating), decision-making power, and women’s knowledge level, were divided into three categories: poor, moderate, and good. Further details on how these variables were assessed can be found in study as conducted by Sebayang et al. (2019).\n\nThe determinants of safe delivery were analyzed using a Chi-square test and binary logistic regression. Both analyses were performed in Stata version 14. The variables were significant at a p-value of 0.05, and the strength of the association was assessed using odds ratio (OR) with a 95% confidence interval (CI).\n\n\nResults\n\nAmong the women who were included in this study, 71.6% used a health facility and 86.2% were assisted by a health professional at their last birth. The majority of the respondents are 0–4 years younger than their husband (41.2%), from the poorest wealth quintile (41.8%), have four or more members in the household (87.6%), are covered by health insurance (64.3%), and live in a rural residence (66.7%). Concerning the husband/partner’s education and occupation, 47.3% have completed secondary or higher education and more than half work in an agricultural occupation (54.1%). For almost half the respondents, their last child was a second or third child with an interval more than two years (44.9%). The majority of respondents had more than four ANC visits (88.4) and their pregnancy was wanted when they became pregnant (82.1%). In terms of women’s empowerment, most respondents have good composite labor force participation (35.7%), a moderate attitude towards domestic violence (34.8%), poor decision-making power (35.3%), and a poor level of knowledge (34.6%). Details about the descriptive characteristics of the respondents are shown in Table 1.\n\nIn the bivariate analysis, most of the variables showed a significant association with a p-value of 0.05 with both outcomes: facility-based delivery and assisted delivery. For the facility-based delivery outcome, three variables have a p-value of more than 0.05 (pregnancy was wanted at time of becoming pregnant, number of household members, decision-making power), while for the assisted delivery outcome, four variables were not significant (age difference between man and woman, pregnancy was wanted at time of becoming pregnant, number of household members, decision-making power). Details about the bivariate analysis are shown in Table 2 and Table 3.\n\np<0.05; p<0.01; *p<0.001\n\np<0.05; p<0.01; p<0.001\n\nIn the binary logistic regression analysis, delivery at a health facility was associated with several variables. Women who lived in a rural residence [AOR=0.49; 95% CI=0.36-0.66] were less likely to deliver in a health facility. A similar result was found for women whose last child was a fourth or higher child with an interval of two years or under [AOR=0.30; 95% CI=0.19-0.47]. Women from the richest wealth quintile family and those who had four or more ANC visits were five [AOR=5.59; 95% CI=3.37-9.30] and three times more likely to deliver in a health facility [AOR=3.62; 95% CI=2.73-4.79], respectively.\n\nWomen who have good composite labor force participation [AOR=1.47; 95% CI=1.15-1.89] and a moderate attitude towards domestic violence [AOR=1.38; 95% CI=1.10-1.73] were more likely to deliver in a health facility. Women whose pregnancy was unwanted when they became pregnant [AOR=1.48 95% CI=1.05-2.08] were also more likely to deliver in a health facility. Details about the binary logistic regression analysis with a facility-based delivery outcome are shown in Table 4.\n\np<0.05; p<0.01; p<0.001\n\nAOR, adjusted odds ratio; CI, confidence interval.\n\nAccording to the assisted delivery outcome, women whose last child was a fourth or higher child with an interval of two years or under [AOR=0.49; 95% CI=0.29-0.83] were less likely to deliver with a health professional. Women whose husband completed secondary or higher education were two times [AOR=2.18; 95% CI=1.48-3.22] more likely to deliver with a health professional. Likewise, women who had four or more ANC visits and were from the richest wealth quintile were three [AOR=3.83; 95% CI=2.77-5.30] and 15 times [AOR=15.69; 95% CI= 5.53-44.50] more likely to be helped by a health professional, respectively.\n\nWomen whose husband worked in a non-agricultural occupation [AOR=1.35; 95% CI=1.00-1.81] were more likely to deliver with a health professional. A similar result was found for women with good composite labor force participation [AOR=1.58; 95% CI=1.11-2.26] and a good level of knowledge [AOR=1.76; 95% CI=1.25-2.46] (Table 5).\n\np<0.05; p<0.01; *p<0.001\n\nAOR, adjusted odds ratio; CI, confidence interval.\n\n\nDiscussion\n\nDelivery was regarded as safe when it was attended by a skilled birth attendant and took place in a health facility. This study found that several variables have a significant association with facility-based delivery and assisted delivery. Women from the richest wealth quintile were more likely to have a delivery in a health facility than those from the poorest wealth quintile. This finding is consistent with that of a previous study in Indonesia. The wealth index of the household would contribute to the access to health care services, including institutional delivery (Caulfield et al., 2016; Do et al., 2015; Efendi et al., 2019; Roro et al., 2014). Women from the richest wealth quintile were more likely to have a delivery with a skilled assistant than those from the poorest wealth quintile. This finding is consistent with that of a previous study conducted in Bangladesh (Muhammed et al., 2017). Women from low-income families may find it difficult to pay for a skilled assistant delivery, so they prefer to give birth without professional assistance (Muhammed et al., 2017). Therefore, the coverage of health insurance must be enhanced so that women in all the wealth quintiles can have equal access to health care services.\n\nA higher child rank and interval of ≤2 years was associated with a lower chance of women having a delivery in a health facility and being assisted by a health professional. This result is similar to those of studies conducted in Ethiopia and Nigeria (Abera et al., 2011; Ononokpono & Odimegwu, 2014). Women with a higher child rank will have more experience with pregnancy and delivery, so they feel that they have the confidence to have a delivery outside a health facility (Abera et al., 2011). Another argument is that women have limited access to health services due to the burden of their economic situation (Ononokpono & Odimegwu, 2014). The results for delivery with a skilled birth attendant are similar to those of studies conducted in Sudan and Ethiopia (Mustafa & Mukhtar, 2015; Wilunda et al., 2015). Women with a higher birth rank tend to rely on their experience from previous pregnancies, believing they already know about childbirth. Consequently, they choose to give birth without professional assistance (Mustafa & Mukhtar, 2015). Review studies conducted in African countries also highlighted the link between higher parity and lower likelihood of facility-based delivery (Moyer & Mustafa, 2013). Therefore, health education about safe delivery should prioritize mothers with a high child rank by giving them greater access to free health care services.\n\nWomen who had more than four ANC visits during their pregnancy were found to be three times more likely to have a safe delivery. This is consistent with the results of studies conducted in Uganda and Ethiopia (Abera et al., 2011; Atusiimire et al., 2019). Furthermore, a population-based study conducted in Bangladesh had a similar result, which emphasized the positive effect of the ANC on utilization of health facility-based delivery (Pervin et al., 2012). The ANC can prevent unsafe delivery because it will provide health education for the mother, giving information and recommending the place of delivery according to the mother’s and fetus’s condition (Atusiimire et al., 2019). Women who had more than four ANC visits during their pregnancy were found to be more likely to have a skilled assistant delivery. This is consistent with the result of a study that was conducted in Kenya (Gitimu et al., 2015). ANC attendance will influence the decision of the mother to have an assisted delivery because the ANC emphasizes the importance of safe delivery (Gitimu et al., 2015). ANC visits must be optimized for pregnant women so that mothers are more exposed to information about safe delivery. The information that the mother receives will influence the decision on where to deliver the baby. Therefore, a minimum number of ANC visits should be given to all pregnant women so they can monitor the condition of the baby and have more knowledge about safe pregnancy and delivery.\n\nAnother finding was that women who wanted no more pregnancies when they became pregnant were more likely to give birth in a health facility. This finding is consistent with that of a study conducted in Egypt (Marston & Cleland, 2003). However, it is inconsistent with the results of a study conducted in Bangladesh, which showed that women who have an unintended pregnancy were less likely to visit an ANC service and more likely to have a home delivery (Kamal, 2013). There was no study that explained this issue, as it may be related to the social norms and health system of the country itself. Therefore, this topic should be analyzed further by considering other variables.\n\nWomen from a rural residence were found to be less likely to have a delivery in a health facility. This finding is similar to those of previous studies conducted in Bangladesh and Indonesia (Efendi et al., 2019; Kamal, 2013; Kenea & Jisha, 2017). Living in an urban residence allows easier access to health facilities than living in rural areas. In addition, access to information is easier in urban areas so information about safe delivery can be spread more easily (Kamal, 2013). Therefore, the gap between rural and urban areas should be taken into consideration by the government regarding the issue of maternal and child health.\n\nWomen who had good composite labor force participation and a good attitude towards domestic violence were more likely to have a delivery in a health facility and be assisted by a health professional. This is consistent with the result of studies conducted in Ethiopia and Bangladesh (Kamal, 2013; Tiruneh et al., 2017). In Bangladesh, women who were against domestic violence and more independent economically were more likely to have four or more ANC service visits, which may lead the women to have a delivery in a health facility (Kamal, 2013). Women who had good composite labor force participation and a good knowledge level were more likely to have a delivery with a skilled assistant. This is consistent with the result of studies conducted in Senegal and Tanzania (Shimamoto & Gipson, 2015). If women have greater empowerment, in terms of knowledge and economic power, this will lead to improvement in their health. They can choose the best for their health, including choosing to have a safe delivery (Prata et al., 2017). Therefore, gender equality needs to be improved so women can make decisions about their health.\n\nHusband/partner’s education attainment was found to be significantly associated with skilled assistant delivery. Women whose husband/partner had completed secondary or higher education were more likely to have a skilled assistant delivery. This is consistent with the results of studies conducted in Kenya and Somalia (Gitimu et al., 2015; Yusuf et al., 2017). Husbands with a higher level of education will have more knowledge about health, including safe delivery (Kifle et al., 2018). As the head of the household, the husband’s knowledge will affect the reproductive health decisions (Yusuf et al., 2017). Engagement of the husband in the issue of maternal health should be expanded in all levels of the community.\n\nWomen whose husband/partner’s occupation was non-agricultural were found to be significantly more likely to have a delivery with professional assistance. This finding is similar to those of studies conducted in Nigeria and Ethiopia (Adewemimo et al., 2014; Fekadu & Regassa, 2015). The husband’s occupation will affect the family’s income. If the family income increases, the decision to have a skilled assistant delivery will also be affected. In addition, another study conducted in Ethiopia found that women whose husbands work in a non-agricultural occupation tend to use an ANC service, which encourages the decision to give birth with professional assistance (Tsegay et al., 2013). Therefore, health promotion about safe delivery is important for the husband/partner, especially for husbands/partners whose occupation is agricultural.\n\n\nLimitations and strengths\n\nThis study used secondary data from the 2017 IDHS, so the selection of the variables was determined by the availability of the data. Another limitation is that some questions in the survey needed respondents to recall what happened five years preceding the survey, so the information may not be precisely stated. In addition to the limitations, however, this study has strengths. The sample of this study was selected using two-stage cluster sampling, so the data were nationally representative. Therefore, the results can provide recommendations for policymakers to develop effective regulation so the coverage gap of safe delivery between western and eastern provinces in Indonesia can be reduced.\n\n\nConclusions\n\nSafe delivery was found to be determined by several factors, which reflected the need for multi-stakeholder intervention in increasing the practice of safe delivery across the country. Programmatic and structured policies that target poor women and those with a low education level and encourage husbands/partners’ participation in this issue may help increase the prevalence of safe delivery in the eastern part of Indonesia. This study gives some recommendations to the policymakers, such as health promotion about safe delivery should be prioritized for women who have a high birth rank. Moreover, not only the women, but also their husband/partner should be involved in health education. The coverage of health insurance and health facilities should be enhanced so that everyone can have equal access to health services. Furthermore, the women’s empowerment program should be maximized so that all the women can choose the best for their health.\n\n\nData availability\n\nData used in this study is available online from the Indonesian 2017 Demographic and Health Survey (DHS) website under the ‘Individual Recode’ section. Access to the dataset requires registration and is granted only for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm.", "appendix": "References\n\nAbera M, Gebremariam A, Belachew T: Predictors of safe delivery service utilization in arsi zone, South-East ethiopia. Ethiop J Health Sci. 2011; 21(Suppl 1): 95–106. PubMed Abstract \| Free Full Text\n\nAdewemimo AW, Msuya SE, Olaniyan CT, et al.: Utilisation of skilled birth attendance in Northern Nigeria: a cross-sectional survey. Midwifery. 2014; 30(1): e7–e13. PubMed Abstract \| Publisher Full Text\n\nAtusiimire LB, Waiswa P, Atuyambe L, et al.: Determinants of facility based–deliveries among urban slum dwellers of Kampala, Uganda. PLoS One. 2019; 14(4): e0214995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelton S, Myers B, Ngana FR: Maternal deaths in eastern Indonesia: 20 years and still walking: an ethnographic study. BMC Pregnancy Childbirth. 2014; 14: 39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBPS: Angka Kematian Ibu Menurut Pulau (per 100.000 kelahiran hidup). 2015a. Reference Source\n\nBPS: Hasil Survei Penduduk Antas Sensus 2015 [Result of the 2015 Intercensal Population Census]. Jakarta: Badan Pusat Statistik. 2015b.\n\nBPS, BKKBN, RI KK, ICF: Indonesia Demographic and Health Survey 2002. 2018.\n\nCaulfield T, Onyo P, Byrne A, et al.: Factors influencing place of delivery for pastoralist women in Kenya: a qualitative study. BMC Womens Health. 2016; 16(1): 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDo M, Soelaeman R, Hotchkiss DR: Explaining inequity in the use of institutional delivery services in selected countries. Matern Child Health J. 2015; 19(4): 755–763. PubMed Abstract \| Publisher Full Text\n\nEfendi F, Ni’mah AR, Hadisuyatmana S, et al.: Determinants of Facility-Based Childbirth in Indonesia. ScientificWorldJournal. 2019; 2019: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFekadu M, Regassa N: Skilled delivery care service utilization in Ethiopia: analysis of rural-urban differentials based on national demographic and health survey (DHS) data. Afr Health Sci. 2014; 14(4): 974–984. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGitimu A, Herr C, Oruko H, et al.: Determinants of use of skilled birth attendant at delivery in Makueni, Kenya: a cross sectional study. BMC Pregnancy Childbirth. 2015; 15: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIacoella F, Tirivayi N: Determinants of maternal healthcare utilization among married adolescents: Evidence from 13 Sub-Saharan African countries. Public Health. 2019; 177: 1–9. PubMed Abstract \| Publisher Full Text\n\nICF Macro: Demographic and Health Survey Interviewer’s Manual. Rockville: ICF International. 2020. Reference Source\n\nKamal SM: Preference for institutional delivery and caesarean sections in Bangladesh. J Health Popul Nutr 2013; 31(1): 96–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenea D, Jisha H: Urban-rural disparity and determinants of delivery care utilization in Oromia region, Ethiopia: Community-based cross-sectional study. Int J Nurs Pract. 2017; 23(1). PubMed Abstract \| Publisher Full Text\n\nKifle MM, Kesete HF, Gaim HT, et al.: Health facility or home delivery? Factors influencing the choice of delivery place among mothers living in rural communities of Eritrea. J Health Popul Nutr. 2018; 37(1): 22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurniati A, Chen CM, Efendi F, et al.: Factors influencing Indonesian women’s use of maternal health care services. Health Care Women Int. 2018; 39(9): 1–16. PubMed Abstract \| Publisher Full Text\n\nMarston C, Cleland J: Do unintended pregnancies carried to term lead to adverse outcomes for mother and child? An assessment in five developing countries. Popul Stud (Camb). 2003; 57(1): 77–93. PubMed Abstract \| Publisher Full Text\n\nMoH: Luas Wilayah dan Kondisi Geografis Menjadi Tantangan Besar Pembangunan Kesehatan Provinsi Papua - Sehat Negeriku. 2012. Reference Source\n\nMoyer CA, Mustafa A: Drivers and deterrents of facility delivery in sub-Saharan Africa: a systematic review. Reprod Health. 2013; 10: 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhammed G, Kibria A, Ghosh S, et al.: Factors affecting deliveries attended by skilled birth attendants in Bangladesh. Matern Health Neonatol Perinatol. 2017; 3: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMustafa MH, Mukhtar AM: Factors associated with antenatal and delivery care in Sudan: analysis of the 2010 Sudan household survey. BMC Health Serv Res. 2015; 15: 452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNations U: About the Sustainable Development Goals. 2015. Reference Source\n\nOnonokpono DN, Odimegwu CO: Determinants of Maternal Health Care Utilization in Nigeria: a multilevel approach. Pan Afr Med J. 2014; 17(Supp 1): 2. PubMed Abstract \| Free Full Text\n\nWH Organizations: WHO Safe Childbirth Checklist. In WHO. Geneva: World Health Organization. 2017. Reference Source\n\nWH Organizations: Maternal mortality. 2018. Reference Source\n\nPervin J, Moran A, Rahman M, et al.: Association of antenatal care with facility delivery and perinatal survival - a population-based study in Bangladesh. BMC Pregnancy Childbirth. 2012; 12(1): 111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrata N, Tavrow P, Upadhyay U: Women's empowerment related to pregnancy and childbirth: introduction to special issue. BMC Pregnancy Childbirth. 2017; 17(Suppl 2): 352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKementrian Kesehatan RI: Rencana Strategis Kementerian Kesehatan 2015-2019. 2017. Reference Source\n\nKementrian Kesehatan RI: Situasi Kesehatan Ibu. In Buletin Jendela Data & Informasi Kesehatan. Jakarta: Pusat Data Kemenkes RI. 2014. Reference Source\n\nRoro MA, Hassen EM, Lemma AM, et al.: Why do women not deliver in health facilities: a qualitative study of the community perspectives in south central Ethiopia? BMC Res Notes. 2014; 7(1): 556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRutstein SO, Johnson K: The DHS wealth index. In DHS Comparative Reports No. 6.2004. Reference Source\n\nSebayang SK, Efendi F, Astutik E: Women's empowerment and the use of antenatal care services: analysis of demographic health surveys in five Southeast Asian countries. Women Health. 2019; 59(10): 1155–1171. PubMed Abstract \| Publisher Full Text\n\nShimamoto K, Gipson JD: The relationship of women's status and empowerment with skilled birth attendant use in Senegal and Tanzania. BMC Pregnancy Childbirth. 2015; 15(1): 154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTejayanti T, Harimat, Sabatini K, et al.: Disparitas Akses dan Kualitas: Kajian Determinan Kematian Maternal di Lima Region Indonesia. Jakarta: MoH-UNFPA. 2012.\n\nTiruneh FN, Chuang KY, Chuang YC: Women's autonomy and maternal healthcare service utilization in Ethiopia. BMC Health Serv Res. 2017; 17(1): 718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsegay Y, Gebrehiwot T, Goicolea I, et al.: Determinants of antenatal and delivery care utilization in Tigray region, Ethiopia: a cross-sectional study. Int J Equity Health. 2013; 12(1): 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilunda C, Quaglio G, Putoto G, et al.: Determinants of utilisation of antenatal care and skilled birth attendant at delivery in South West Shoa Zone, Ethiopia: a cross sectional study. Reprod Health. 2015; 12: 74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYusuf MS, Kodhiambo M, Muendo F: Determinants of Access to Skilled Birth Attendants by Women in Galkacyo District, Somalia. Asian Journal of Medicine and Health. 2017; 4(4): 1–7. Publisher Full Text" }	[ { "id": "63312", "date": "26 May 2020", "name": "Ryan Michael F Oducado", "expertise": [ "Reviewer Expertise Public Health and Community Health Nursing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 looks into the determinants of safe delivery in the eastern part of Indonesia. The report addresses an important topic and contributes to the advancement of knowledge on maternal health and in reducing maternal mortality.\nI only have a few comments regarding the research article:\nThe authors mentioned that the dependent variables were facility-based delivery and assisted delivery. Will it be more appropriate to consider a) place of delivery and b) proportion of women assisted by skilled birth attendants as dependent variables?\n\nIt was reported in the article that 71.6% used a health facility and 86.2% were assisted by a health professional at their last birth. I wonder, isn’t that health professionals typically assist health facility delivery? What is the reason for the discrepancy?\n\nPlease clarify women’s knowledge level. This variable pertains to women’s level of knowledge about what?\n\nAn interesting finding in this study was that women who wanted no more pregnancies when they became pregnant were more likely to give birth in a health facility. The authors mentioned that it might be related to the social norms and health system of the country itself. Can the authors expound on this? Can the researchers offer their plausible explanation of this result?\n\nCan the authors restate the conclusions in the abstract? Can household factors be changed to a more encompassing term reflective of the result? Can the facility-based delivery term be deleted and focus on safe delivery as directed in the title of the article?\n\nCan the use of secondary data be mentioned in the abstract?\nThank you very much for the opportunity to review this article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5895", "date": "23 Sep 2020", "name": "Ferry Efendi", "role": "Author Response", "response": "We thank all reviewers for their generous and positive comments on our manuscript. With regard to their reviews, we have edited the manuscript to respond to their comments. In particular, we have made updates in track changes to indicate changes as suggested. We thank you for the opportunity to submit our revised manuscript. Reviewer 1 This article looks into the determinants of safe delivery in the eastern part of Indonesia. The report addresses an important topic and contributes to the advancement of knowledge on maternal health and in reducing maternal mortality. I only have a few comments regarding the research article: 1. The authors mentioned that the dependent variables were facility-based delivery and assisted delivery. Will it be more appropriate to consider a) place of delivery and b) proportion of women assisted by skilled birth attendants as dependent variables? Thank you for these constructive comments, we have changed the terminology into place of delivery (health facility Vs non-health facility) and type of assistance at delivery (skilled birth attendants Vs unskilled birth attendants) as guided by guide to DHS statistics DHS-7 and your comments. 2. It was reported in the article that 71.6% used a health facility and 86.2% were assisted by a health professional at their last birth. I wonder, isn’t that health professionals typically assist health facility delivery? What is the reason for the discrepancy? Thank you for raising this issue, all delivery in health facility must be assisted by health professionals. However, not all delivery assisted by health professionals occurred in health facility. 3. Please clarify women’s knowledge level. This variable pertains to women’s level of knowledge about what? Thank you very much, we have added the explanation in variables section. 4. An interesting finding in this study was that women who wanted no more pregnancies when they became pregnant were more likely to give birth in a health facility. The authors mentioned that it might be related to the social norms and health system of the country itself. Can the authors expound on this? Can the researchers offer their plausible explanation of this result? Thank you very much, we have added the explanation in discussion section. 5. Can the authors restate the conclusions in the abstract? Can household factors be changed to a more encompassing term reflective of the result? Can the facility-based delivery term be deleted and focus on safe delivery as directed in the title of the article? Thank you very much, we have revised the abstract. 6. Can the use of secondary data be mentioned in the abstract? Thank you very much, we have added in the abstract section." }, { "c_id": "5896", "date": "23 Sep 2020", "name": "Ferry Efendi", "role": "Author Response", "response": "We thank all reviewers for their generous and positive comments on our manuscript. With regard to their reviews, we have edited the manuscript to respond to their comments. In particular, we have made updates in track changes to indicate changes as suggested. We thank you for the opportunity to submit our revised manuscript. Reviewer 2 1. This is a good written article, but there are little things I think to make it perfect if the author follows it. First: in Introduction: the author does not mention study hypotheses. Explain why the current research is important. What value does the paper add? What practical benefits will it provide? Thank you very much, we have added on introduction section for the hypothesis and the value. While, we have added the practical benefits on the limitation and strength section. 2. Second: Sample and sampling, it is important to add and mention the procedure of taking sampling for this huge sample size. Thank you very much, we have added on sample size and sampling section." } ] }, { "id": "66055", "date": "21 Aug 2020", "name": "Asmaa Salah Eldin Mohamed Saleh", "expertise": [ "Reviewer Expertise community health nursing", "geriatric health", "MCH", "health promotion", "school health", "pediatrics", "maternity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good written article, but there are little things I think to make it perfect if the author follows it. First: in Introduction: the author does not mention study hypotheses. Explain why the current research is important. What value does the paper add? What practical benefits will it provide?\n\nSecond: Sample and sampling, it is important to add and mention the procedure of taking sampling for this huge sample size.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-332
https://f1000research.com/articles/9-1166/v1	23 Sep 20	{ "type": "Research Article", "title": "Targeting SARS-CoV-2 RNA-dependent RNA polymerase: An in silico drug repurposing for COVID-19", "authors": [ "Krishnaprasad Baby", "Swastika Maity", "Chetan H. Mehta", "Akhil Suresh", "Usha Y. Nayak", "Yogendra Nayak", "Krishnaprasad Baby", "Swastika Maity", "Chetan H. Mehta", "Akhil Suresh", "Usha Y. Nayak" ], "abstract": "Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), took more lives than combined epidemics of SARS, MERS, H1N1, and Ebola. Currently, the prevention and control of spread are the goals in COVID-19 management as there are no specific drugs to cure or vaccines available for prevention. Hence, the drug repurposing was explored by many research groups, and many target proteins have been examined. The major protease (Mpro), and RNA-dependent RNA polymerase (RdRp) are two target proteins in SARS-CoV-2 that have been validated and extensively studied for drug development in COVID-19. The RdRp shares a high degree of homology between those of two previously known coronaviruses, SARS-CoV and MERS-CoV. Methods: In this study, the FDA approved library of drugs were docked against the active site of RdRp using Schrodinger's computer-aided drug discovery tools for in silico drug-repurposing. Results: We have shortlisted 14 drugs from the Standard Precision docking and interaction-wise study of drug-binding with the active site on the enzyme. These drugs are antibiotics, NSAIDs, hypolipidemic, coagulant, thrombolytic, and anti-allergics. In molecular dynamics simulations, pitavastatin, ridogrel and rosoxacin displayed superior binding with the active site through ARG555 and divalent magnesium. Conclusion: Pitavastatin, ridogrel and rosoxacin can be further optimized in preclinical and clinical studies to determine their possible role in COVID-19 treatment.", "keywords": [ "COVID-19", "SARS-CoV-2", "Docking", "In silico", "RNA-dependent RNA polymerase", "Molecular dynamics simulation", "Drug repurposing" ], "content": "Introduction\n\nA novel disease was first discovered in late 2019 in a 41-year-old patient in China who was admitted to a health facility for severe respiratory syndrome and fever, dizziness, and cough. A new strain of RNA virus of the Coronaviridae family was isolated from the patient's bronchoalveolar lavage fluid and was originally unique initially referred to as 'WH-Human 1' coronavirus (and later known as '2019-nCoV') was. The novel virus was with 60–140 nm diameter with 9–12 nm long spikes, with the virion comparable to the corona of the sun. Viral genome analysis revealed that it is made of ~30k nucleotides, and has 89.1% nucleotide similarity to SARS-like CoV (genus Betacoronavirus, subgenus Sarbecovirus)1. The human CoV B814-strain was isolated in 1965 from the nasal discharge of a patient with a common cold. Since then, 30 specific strains were identified. Seven hCoVs have been reported previously causes disease, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and 2019-nCoV (now established as SARS-CoV-2)2. SARS-CoV emerged during the period 2000–2004, and it was discovered that the infection was descended from animals and bats as intermediate hosts3. MERS-CoV was diagnosed for the first time in Saudi Arabia4. The World Health Organization (WHO) named the disease coronavirus disease 2019 (COVID-19) on 12 January 2020. The same outbreak was declared a public health emergency of international concern by the WHO on 30-January-2020 due to rapid transmission mortality rate5. The International Committee on the Taxonomy of Viruses Coronavirus Study Group (CSG) proposed the name of nCoV as SARS-CoV-26.\n\nCurrently, clinical trials have approved the use of remdesivir, favipiravir, and dexamethasone, but the infection could not be cured or prevented. The remdesivir and favipiravir are comparatively expensive than dexamethasone and not affordable for people at developing countries. Vaccine development, convalescent plasma, interferon-based therapies, small molecule medications, cell-based therapy, and monoclonal antibodies (mAbs) are the various likely future medications and pathways being investigated7. The development of a drug is expensive and time-consuming with a high attrition rate, which is unacceptable in the current global emergency. Hence, there is a great interest in repurposing the existing drugs and speed-up to develop antiviral therapies8. The national health commission (NHC) of the People's Republic of China recommends the use of α-interferon (IFN-α), lopinavir/ritonavir, ribavirin, chloroquine phosphate, and arbidol as antiviral therapy against SARS-CoV-2. Other agents such as inhibition of fusion/entry (camostat mesylate, baricitinib, arbidol and chloroquine phosphate), the agents that disrupt SARS-CoV-2 replication (remdesivir, favipiravir, lopinavir/ritonavir), the agents that suppress excessive inflammatory response (corticosteroid), convalescent plasma, vaccines (inactivated vaccine, recombinant subunits vaccine, nucleic acid-based vaccine, adenoviral vector vaccine, recombinant influenza viral vector vaccine), a combination of traditional Chinese and Western medicine (ShuFeng JieDu capsules and Lianhua Qingwen capsules) are also gaining therapeutic interest9.\n\nThe SARS-CoV-2 genetic material is translated inside the host cell to two distinct groups of proteins; structural proteins, such as Spike (S), Nucleocapsid (N), Matrix (M) and Envelope (E), and non-structural proteins (nsp) such as RNA dependent RNA polymerase (nsp12), helicase (nsp13), papain-like protease (PLpro or nsp3) and main protease Mpro (also known nsp5 or 3CLpro)10–12. SARS-CoV-2 replication is facilitated through a multi-subunit replication/transcription complex of non-structural viral proteins. The key aspect of the nsp complex is an RNA-dependent RNA polymerase (RdRp; nsp12). The central RdRp domain is divided into three subdomains, the thumb, palm, and right-handed cup-like fingers, which is less than 500 long amino acids length11. A high level of homology has been found between SARS-CoV-2, MERS-CoV, and SARS-CoV RdRp13. The RdRp requires extra factors such as nsp7 and nsp8, for its activity14. Remdesivir, which was recently approved by US-FDA for COVID-19, inhibits RdRp15. Furthermore, researchers have explored RdRp-target for in silico repurposing of drugs. Hence, this proves that Nsp12 or RdRp is an attractive target for inhibiting viral replication. In the current study, we assessed USFDA approved drugs to impede the RdRp activity of SARS-CoV-2 through an in silico drug-repurposing strategy.\n\n\nMethods\n\nAll computational simulation experiments were conducted on the Schrödinger Suite's (version 2020-2) Maestro graphical user interface (www.schrodinger.com; v12.3) on an Ubuntu software desktop workstation, Intel ® Xenon ® Gold 6130 CPU @ 2.10 GHz × 64 processors, Quadro P620 / PCle / SSE2 graphics card and 134.8 GB RAM. Alternatively, free software including Autodock 4.2.6, Gromacs 5.1, ArgusLab 4.0 and PDB2PQR 3.0 could be used for such study.\n\nMolecules licensed by US-FDA were downloaded from https://www.drugbank.ca (date of access: 15 Feb 2020). The molecules were configured using LigPrep, wherein the molecules generated the 3D coordinates16. Using the Epik module, the suitable ionization state was predicted at pH 8.0, types of tautomers were produced, and proper chirality was estimated. Finally, the molecules' structure was minimized with energy using the OPLS3e force field17. Alternatively, the Zinc database can be used for FDA approved drug and ArgusLab 4.0, PDB2PQR can be used for ligand configuration and stabilising the ligand state at certain pH respectively.\n\nThe electron microscopical RdRp coordinate structure of SARS-CoV-2 linked to template-primer RNA which was 50- base pair long and remdesivir triphosphate was downloaded from Protein Data Bank (PDB), accession code 7BV2, at a resolution of 2.5 Å. The RdRp-NS7-NS8 complex was linked to three Mg2+ ions (coordinated by Asp760, Asp761, Asp618, and pyrophosphate), double-stranded RNA (14-base template strand and 11-base primary strand) and monophosphate remdesivir (RMP). The Protein Prep Wizard was used for the optimization of protein structure18. ArgusLAb 4.0 software, which is free tool for protein preparation for docking studies can be used for the same. In this process, the missing hydrogens, side chains, and other organic solvent molecules and water residues were eliminated. The proper ionization state was then created at pH 8.0 using PROPKA3, regenerating the hydrogen bond network, and finally minimizing the protein structure using a restrained minimization technique19,20.\n\nLigand docking of molecules licensed by US-FDA (~2800 Nos) was performed using the Schrodinger Glide v8.7 module21. Alternatively, AutoDock 4.2.6, an open access package for ligand docking, can be used for the same function. The binding site of the ligands on the protein was assessed using the Receptor Grid Generation tool using the centroid formed around the bound ligand. The docking was initially performed using high-throughput virtual screening (HTVS). Later, top molecules with strong binding to the active site of RdRp and docking scores were moved ahead for standard precision (SP)-docking. The single best pose of each molecule was saved during ligand docking22.\n\nThe MD-simulation was run on Schrodinger's Desmond module23. Alternatively, the free software NAMD with VMD molecular simulation could be used. The solvated water-soaked system was generated using the Desmond System Builder tool. The solvating system used in the experiment was the TIP3P model. An orthorhombic simulation was a box that generated at least 10 Å from the outer surface of the protein with periodic boundary conditions. The system was neutralized by adding a reasonable amount of counterions. By adding 0.15 M NaCl into the simulation panel, the isosmotic condition was conserved. A pre-defined equilibration procedure was performed before the simulation. The MD simulation was performed at an ambient pressure of 1.013 bar, and a temperature of 300°K, with 1000 frames saved to the trajectory, for 50 nsec period. Later the simulation was analyzed using a simulation interaction diagram, wherein protein-ligand root mean square deviation (RMSD), protein root mean square fluctuations (RMSF), ligand RMSF, protein-ligand contacts, ligand-protein contacts, ligand torsion profile. were analyzed.\n\n\nResults and discussion\n\nSARS-CoV-2 RdRp is a central polymerase involved in the replication of RNA, is a big enzyme consisting of 932 amino acids. The amino acid framework of SARS-CoV RdRp linked to the Nsp7 and Nsp8 cofactors24. Structurally, the RdRp protein is divided into the N-terminal and polymerase domains. The N-terminal domain extends from amino acid residues 1 to 397. The polymerase domain extends from amino acids 398 to 919 and is similar to the previously reported SARS-CoV nsp12 (PDB ID: 6NUR). The RdRp polymerase domain is subdivided into three structurally different subunits, namely the finger subunit (398–581 and 628–687 amino acids), palm subunit (582–627 and 688–815 amino acids), and thumb subunit (816–919 amino acids), which embraces a \"right-handed\" shaped conformation15. The active site is located at the hinge interface between the finger and thumb subunits of the SARS-CoV-2 RdRp, which is an evolutionary conserved in both the coronaviruses reported earlier, ie. MERS and SARS-nsp12. The active site of nsp12 is situated in the middle of the substrate domain where the synthesis of RNA takes place as an RNA template is accessed from the template input channel and nucleoside triphosphate (NTP) from NTP input channel25. Structural assessment of SARS-CoV-2 defined in RdRp displayed the presence of seven retained motifs (A-G motifs), which play a key role in the association of substrate and nucleotides. They also play a key role in catalysis of the attachment of the NTP. Antiviral drugs function by binding to first replicated base pairs and thereby stopping the chain elongation cycle. The sensitivity of the RdRp binding to the RNA prototype has been enhanced by the interaction of the nsp7 and nsp8 co-factors with RdRp. The RNA polymerization activity appeared to be impaired in the presence of remdesivir active triphosphate (RTP), which is being investigated in clinical trials. Remdesivir blocked the polymerization process of RdRp at 1 mM and 10 mM ATP completely. However, remdesivir alone on its own or in its monophosphate form did not demonstrate activity at 5 mM concentration. Remdesivir monophosphate was found to be covalently linked to the primary strand in protein structure to the pyrophosphate moiety and three magnesium as catalytic ions. The ligand-protein interaction of remdesivir also has two hydrogen bonds, with the uridine base, which leads to a stable complex formation. Predominant interactions include those with K545 and R55 side chains, the interaction of magnesium ions with the backbone of the phosphate diester, as well as with the primary strand RNA. Seven conserved motifs from A to G represent the nsp12 RdRp catalytic active site, among which the palm subdomain with an SDD sequence (residues 759-761) in motif C, including amino acid residues D760 and D761 in the coordination of the two magnesium ions. The template-primer RNA, which oriented in the same fashion as template-primer RNA in the poliovirus RdRp elongation complex and residues involved in binding as well as interactions to 2′-OH groups, was found to be highly conserved15.\n\nThe docking provides details about binding affinity and orientation of the interactions between ligand and protein. HTVS was initially performed using ~2800 drug molecules. The HTVS docking is used to screen large numbers of ligands rapidly. However, HTVS seems to have more limited conformational sampling than docking with SP, and hence HTVS can not be used with ranking in place. Hence, molecules were advanced to the higher precision of docking known as SP. The SP docking was performed on 500 molecules, which exhibited good interactions with the RdRp and had topped in the docking score. Among the 500 molecules, 14 displaying top SP-docking scores and the conserved interactions with the active site as that of remdesivir were shortlisted based on visual inspection (Table 1). These molecules were able to interact with RdRp amino acid residues via a predominant metal coordination bond and hydrogen bonding with the active site. The interactions exhibited by rosoxacin, kappadione, tedizolid phosphate, levomefolic acid, etodolac, pitavastatin, montelukast, norfloxacin, cinoxacin, mefenamic acid, diflunisal, fospropofol, fluvastatin, and ridogrel was found to be prominent. Figure 1 depicts the binding mode of Pitavastatin, Ridogrel and Rosoxacin, which emerged as the best molecule by MD simulations.\n\nShown are (A) pitavastatin, (B) ridogrel, and (C) rosoxacin with SARS-CoV-2 RdRp.\n\nThe mode of interaction of selected molecules with the RdRp active site in the SP-docking is depicted in Table 1. The shortlisted molecules interact with conserved residue ARG555 and divalent magnesium in the manner Remdesivir interacted with RdRp. Drugs including rosoxacin, levomefolic acid, etodolac, kappadione, pitavastatin, montelukast, norfloxacin, cinoxacin, diflunisal, fospropofol and ridogrel have demonstrated π-cation interactions with ARG555. Norfloxacin and cinoxacin have demonstrated π-π stacking interactions with 20 unspecified residues: (U P: 20). In SP-docking, the drugs shortlisted made metal coordination and salt bridges with MG A:1004 and MG A:1005 (divalent magnesium ions) in the same way remdesivir was bound (Table 1). Rosoxacin, pitavastatin and ridogrel had strong interactions across metal coordination bonds among the molecules shortlisted by SP-docking, and π-cation with RdRp can be further tested for their in vitro function.\n\nThe RdRp-protein and ligand binding stability were checked based on the RMSD fluctuations during MD-simulations. In the MD simulation, the RMSD fluctuation was measured individually for the protein and ligand structures, and it falls within 3 Å. Hence the complex was considered to be stable. Besides, the persistence of the intermolecular interactions between ligand and RdRp was also monitored during the simulation. The analysis RMSD plot for the structures saved in the trajectory generated during the MD-simulation exhibited stable bindings for 14 drug molecules. For rosaxocin, pitavastatin and ridogrel in complex with the RdRp-protein, the RMSD fluctuations for the ligand and protein remained within 2.0 Å. Figure 2 depicts the ligand and protein RMSD plot for the shortlisted 14 drugs.\n\nDuring the initial phase of MD simulations, the RdRp-bound pitavastatin complex exhibited higher fluctuations due to the equilibration. For the initial 10 nsec of the simulation, the protein backbone exhibited higher RMSD fluctuation. The latter 40 nsec the protein backbone fluctuations remained within the range of 0.6 Å, indicating stabilization of the protein structure. Similarly, the ligand showed consistently stable interactions throughout the simulation. The ligand RMSD fluctuations remained within the range of 0.5 Å for the remaining 40 nsec of the simulation indicated a stable ligand in the complex (Figure 2). The interaction with residues ASP618, ASP760, ASP761, and GLU811 exhibited maximum contacts with the ligand during MD simulation. The bridged hydrogen bonding was exhibited with the ligand was amino acid residue TYR619 over more than 30.0% of the simulation time in the 50 nsec trajectory. A significant metal coordination bond was formed with the divalent magnesium and residues ASP618, ASP760, ASP761, and GLU811. The majority of the interactions observed between the protein and ligand during the docking were consistently retained during the MD simulation (Figure 3). Pitavastatin is used as a primary hyperlipidemic agent and in mixed dyslipidemia. It lowers the elevated total cholesterol, low-density lipoprotein, apolipoprotein-B, triglycerides, and increases high-density lipoprotein. Statins are commonly prescribed in cardiovascular patients. Statins offer protection to vasculatures and are known to have pleiotropic effects in the body, especially in modulating the inflammatory process at the cellular level26. One of the reports on in silico studies analysed the statins interaction with Mpro, and they have also reported the pitavastatin as a hit-drug27. These combined Mpro and RdRp inhibitory properties of statins might help to tackle COVID-19.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\nThe RdRp bound ridogrel complex exhibited a mixture of hydrophilic and ionic interactions during the MD simulation. For the initial ten nsec of the simulation, the protein backbone exhibited higher RMSD fluctuation. The latter 40 nsec the protein backbone fluctuations remained within the range of 0.6 Å, indicating stabilization of the protein structure (Figure 2). Similarly, the ligand showed a higher fluctuation for the initial 15 nsec of the simulation. The ligand RMSD fluctuations remained within the range of 1.5 Å for the remaining 35 nsec of the simulation, indicating a relatively stable ligand-protein complex. After the initial fluctuation for a period of 10 nsec due to the equilibration, the RMSD for the protein structures remained between 2.6-4.4 Å until the end of the simulation. Amino acid residues ASP760 followed by CYS618, ASP622 and ASP623 interacted to the greatest extent with the ligand. The residues CYS622 and ASP623 made bridged hydrogen bonding interaction with the ligand which accounted to have 75 and 74% interaction with the protein residues, respectively. The amino acid residues ASP618 and ASP760 made metal coordination bonds with divalent magnesium. The majority of the interactions observed between the protein and ligand during the docking were consistently retained during the MD simulation (Figure 4). Based on the RdRp interaction, we propose that ridogrel can be repurposed for COVID-19 as an antiviral agent. Ridogrel is a thromboxane synthase inhibitor and thromboxane or prostaglandin/endoperoxide receptor blocker. It is used for the prevention of systemic thromboembolism in acute myocardial infarction28. Redogrel is been replaced with aspirin in the therapy as aspirin was found to be clinical superior. The RdRp inhibitory properties of ridogrel might make it a useful drug in COVID-19. Hence, it can be recommended for further screening and optimize it by in vitro assays.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\nRdRp bound with rosaxocin exhibited a mixture of hydrophobic interactions and hydrophilic interactions during the MD-simulation. For the initial 20 nsec of the simulation, the protein backbone exhibited higher RMSD fluctuation. The latter 30 nsec the protein backbone fluctuations remained within the range of 0.8 Å, indicating stabilization of the protein structure (Figure 2). Similarly, the ligand showed a higher fluctuation for the initial 40 nsec of the simulation. The ligand RMSD fluctuations remained within the range of 1.4 Å for the remaining 30 nsec of the simulation indicated a stable ligand-protein complex. The residues ASP760, ARG555 and ASP623 made maximum contact with the ligand. A predominant π-cation interaction was exhibited with ARG555 residue (54%). A direct salt bridge interaction was observed with ARG555 with the carbonyl carbon, and a bridged hydrogen bonding interactions, which accounted for 92% was observed. Similarly, bridged hydrogen bonding interactions were observed with ARG553 and carboxyl electronegative oxygen (accounted for 64%). The amino acid residues LYS621, CYS622 made direct hydrogen-bonding interactions with carboxyl electronegative oxygen (accounted for 53%, 60%) and ASP623 made direct hydrogen bonding with unsaturated oxygen located in the ligand (58%)respectively over more than 30.0% of the simulation time in the 50 nsec simulation trajectory. Additionally, metal coordination interaction was observed with ASP760 with the divalent magnesium. The majority of the interactions observed between the protein and ligand during the docking were consistently retained during the MD simulation (Figure 5). Rosoxacin is a broad-spectrum quinolone antibiotic for the treatment of urinary tract infections (UTIs) and sexually transmitted diseases29. The popular anti-COVID-19 drugs chloroquine and hydroxychloroquine have the quinoline moiety30. Considering the PK/PD and the safety profiles of rosoxacin, it can be taken forward in clinical trials as a repurposed candidate for COVID-19. This study is the first report of a broad-spectrum antibiotic with a high affinity for RdRp identified by in silico tools.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker colour shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\nThe other molecules short listed are from the diverse pharmacological group can be repurposed by studying in vitro. These molecules include etodolac, montelukast, fluvastatin, kappadione, levomefolic acid, norfloxacin, cinoxacin, tedizolid phosphate, mefenamic acid, diflunisal and fospropofol. The details of these MD-simulation data and molecular interaction are represented in Figure 6–Fiugre 16.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are show.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\n(A) The protein-ligand interaction diagram. (B) The residues that interact with the ligand in each trajectory frame. The residues making more than one contact are shown in darker color shade. (C) Schematic diagram of ligand interaction with the amino acid residues of protein during MD simulation. Interactions that occur more than 30% of the simulation time are shown.\n\nEtodolac is an NSAID with anti-inflammatory, analgesic and antipyretic properties. It inhibits the cyclooxygenase (COX) and prevents the formation of peripheral prostaglandins which causes inflammation. It is officially prescribed for rheumatoid arthritis and osteoarthritis. Etodolac is 50-times more selective for COX-2 than COX-1, and the antipyresis is attributed to hypothalamic actions, cutaneous blood flow, and subsequent heat losses31. Hence, if this drug is repurposed for COVID-19, it will have a multidirectional approach because the drugs will be useful in bringing down the elevated body temperature and anti-inflammatory properties. Montelukast, a leukotriene receptor antagonist, used to treat asthma, exercise-induced bronchoconstriction, and allergic rhinitis32. The anti-inflammatory properties, as well as binding of montelukast to RdRp, might help in tackling COVID-19. Hence, montelukast can be tried for drug repurposing with detailed studies. Fluvastatin is a hypolipidemic agent belongs to the class statins33. Because of their pleiotropic role, statins can be explored further to repurpose to COVID-19. Kappadione or menadiol sodium diphosphate is a highly water-soluble vitamin K analogue approved by the FDA and marketed by Lilly. It is indicated in anticoagulant-induced prothrombin deficiency, and therapy of hypoprothrombinemia due to antibacterial therapy, hypoprothrombinemia secondary to factors limiting absorption or synthesis of vitamin K. Recently, a computational study reported that kappadione would tightly bind to PAX2 transcription factor which regulates the ABC-transporter. PAX2 binding by kappadione inhibits the PAX2-DNA interaction, and this would be beneficial for combating chemoresistance in pancreatic ductal adenocarcinoma34.\n\nLevomefolic acid, also known as 5-methyltetrahydrofolate (5-MTHF) is an active form of folate found in food, which, when administered, increases folate (vitamin B9) concentration in blood. Levomefolic acid is preferred over folic acid supplement due to lower potential for the induction of vitamin B12 deficiency symptoms35. Hence, this drug is safest can be easily repurposed in COVID-19 patients. Norfloxacin is a broad-spectrum fluoroquinolone antibiotic. Norfloxacin acts by blocking DNA gyrase and used to treat UTI36. Similar to quinolones, fluoroquinolones also found to modulate the cytokines, and this property is an added advantage in repurposing norfloxacin for COVID-19. Cinoxacin is currently used to treat UTIs and as an alternative antimicrobial to oxolinic acid and nalidixic acid37. Tedizolid is an oxazolidinone class of antibiotics, a congener of linezolid, which is effective against multidrug-resistant Gram-positive bacteria. Tedizolid is useful in acute bacterial skin infections and was approved by the FDA in 2014. Currently available as both an oral tablet and as a powder for intravenous injection38. Mefenamic acid belongs to the class of aminobenzoic acids effective for the treatment of dysmenorrhea, rheumatoid arthritis, osteoarthritis, mild to moderate pain, inflammation, and fever39. Using this drug will have advantages in COVID-19, as it also tackles the inflammation and has the antipyretic effect. Diflunisal, a salicylate derivative has anti-inflammatory, analgesic, and antipyretic function as NSAID40. It modulates HMGB1 through Toll-like receptor 4 (TLR4). Diflunisal does not inhibit responses that rely on TLR4. The TLR modulation is one among the targets for COVID-19, diflunisal has advantages. Fospropopol is a hypnotic/sedative/anaesthetic short-acting drug prodrug of propofol, which is an anaesthetic drug. It has a high lipid solubility can be tried in COVID-19 based on its interactions with RdRp41.\n\nMolecular docking of FDA-approved drugs for RdRp led us to shortlist 14 molecules. There were many proposals of RdRp inhibitors for repurposing, especially by in silico computational simulations. One previous report used the ZINC drug database to screen SARS-CoV-2 RdRp inhibitors13. They studied 78 commonly used antiviral drugs and drugs which are currently in clinical trials for COVID-19 for molecular simulations and reported the possible in silico repurposing of drugs. They have listed the top 20 drugs from the ZINC-drug database that included the currently used antiviral drug valganciclovir, anti-bacterial drugs such as chlorhexidine, ceftibuten, cefuroxime, novobiocin, the anti-asthmatic drugs fenoterol and cromolyn, the antitumour drugs such as fludarabine, idarubicin, the antimalarial drug atovaquone, the muscle relaxant pancuronium,the anti-allergic cortisone, the contraceptive tibolone, the hepatoprotective silybin, the anti-diarrheal drug diphenoxylate, the anti-amenorrheal bromocriptine, and chenodeoxycholic acid, which is used to dissolve gallstones. In the same study, the investigators shortlisted the top-20 natural compounds as RdRp inhibitors. Another group of researchers studied the feasibility of known RNA-polymerase inhibitors to repurpose as anti-SARS-CoV-2 drugs42. The drugs they studied are broad-spectrum antiviral agents such as remdesivir, 5-fluorouracil, ribavirin and favipiravir. However, they noted that the SARS-CoV-2 might evolve to acquire drug resistance mutations against these nucleoside inhibitors. The virtually combined deep learning and molecular docking simulations approach identified potentially useful 49 most promising FDA-approved drugs for repurposing in COVID-1943. The selected drugs in this study were anidulafungin, velpatasvir, glecaprevir, rifabutin, procaine penicillin G, tadalafil, riboflavin 5'-monophosphate, flavin adenine dinucleotide, terlipressin, desmopressin, elbasvir, oxatomide, enasidenib, edoxaban and selinexor. Another group of computational chemists studied the FDA-approved drugs by virtual screening for the three proteases of SARS-CoV-2, Mpro, PLpro and RdRp44. They have shortlisted promising antiviral drugs such as simeprevir, ledipasvir, idarubicin, saquinavir, ledipasivir, partitaprevir, glecaprevir, and velpatasvir. They also shortlisted several novel drugs such as antiviral raltegravir; antidiabetic amaryl, antibiotics retapamulin, rifimixin, and rifabutin; antiemetic fosaprepitant and netupitant. Another group of researchers used a multi-targetted approach and shortlisted molecules such as δ-viniferin, myricitrin, taiwanhomoflavone A, lactucopicrin 15-oxalate, nympholide A, afzelin, biorobin, hesperidin and phyllaemblicin B. These molecules have the affinity for SARS-CoV-2 Mpro, RdRp and hACE245. Furthermore, a group of researcher have proposed well-tolerated, cost-effective drugs to combat COVID-19 which has the affinity for both Mpro and RdRp. The compounds they have shortlisted are ergotamine, dihydroergotamine, conivaptan, paliperidone, and tipranavir46.\n\nIn the pathogenesis and pathophysiology of COVID-19, both innate and adaptive immunity are activated, and many inflammatory mediators are released47. Mitigating inflammation will reduce the lethality because death due to COVID-19 is partly due to cytokine storm and end-organ failure48–50. The immuno-inflammatory modulators have their role in the treatment of COVID-19, and the drug shortlisted in this study have those potentials have potentials to mitigate inflammation. The shortlisted 14 novel molecules for SARS-CoV-2 RdRp inhibitors from different pharmacological classes. These drugs include antimicrobials such as rosoxacin, norfloxacin, cinoxacin, tedizolid phosphate, vitamin analogues such as kappadione, levomefolic acid, anti-inflammatory agents such as etodolac, mefenamic acid, diflunisal, montelukast, statins such as pitavastatin, fluvastatin, a sedative fospropofol, and a thromboxane inhibitor ridogrel. These drugs are commonly used, well-tolerated and economical. Among the 14-drugs, pitavastatin, is a lipid lowering agent and has pleotropic effects. Ridogrel rosoxacin, a quinolone antibiotic indicated for the treatment of urinary tract infections and certain sexually transmitted diseases, Testing in vitro for RdRp inhibitory activity, and antiviral activity could confirm these molecules for the repurposing or repositioning for COVID-19 treatment.\n\n\nConclusion\n\nIn the absence of approved therapies for treatment or prevention, drug repurposing has provided valuable insight into the treatment of COVID-19. The antiviral drugs such as lopinavir, ritonavir, favipiravir and remdesivir, immunosuppressants such as sirolimus, anthelmintics like ivermectin, corticosteroids such as methylprednisolone, dexamethasone are currently under clinical trials for COVID-19. In this molecular docking study using structure-based virtual screening, we identified favourable drugs, namely rosoxacin, levomefolic acid, etodolac, kappadione, pitavastatin, montelukast, fluvastatin, norfloxacin, cinoxacin, tedizolid phosphate, mefenamic acid, diflunisal, fospropofol, and ridogrel. The MD simulation studies revealed pitavastatin, ridogrel and rosoxacin to be superior compared to other shortlisted drugs. Further, the therapeutic properties of these shortlisted molecules make it suitable for testing RdRp-inhibitory activity and antiviral activity in vitro and in vivo could confirm these molecules for repurposing in COVID-19.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nAuthors are thankful to the Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences for facilities of Schrodinger’s Suit computer simulations.\n\n\nReferences\n\nWu F, Zhao S, Yu B, et al.: A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798): 265–269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu DX, Liang JQ, Fung TS: Human Coronavirus-229E, -OC43, -NL63, and -HKU1. Ref Modul Life Sci. 2020. Publisher Full Text\n\nPeiris JSM, Lai ST, Poon LLM, et al.: Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet. 2003; 361(9366): 1319–1325. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaki AM, Van Boheemen S, Bestebroer TM, et al.: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367(19): 1814–1820. PubMed Abstract \| Publisher Full Text\n\nSohrabi C, Alsafi Z, O’Neill N, et al.: World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg. 2020; 76: 71–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu R, Zhao X, Li J, et al.: Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020; 395(10224): 565–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi G, De Clercq E: Therapeutic options for the 2019 novel coronavirus (2019-nCoV). Nat Rev Drug Discov. 2020; 19(3): 149–150. PubMed Abstract \| Publisher Full Text\n\nLythgoe MP, Middleton P: Ongoing Clinical Trials for the Management of the COVID-19 Pandemic. Trends Pharmacol Sci. 2020; 41(6): 363–382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Zhou Y, Zhang M, et al.: Updated Approaches against SARS-CoV-2. Antimicrob Agents Chemother. 2020; 64(6): e00483–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSatarker S, Nampoothiri M: Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2. Arch Med Res. 2020; 51(6): 482–491. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao Y, Yan L, Huang Y, et al.: Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science. 2020; 368(6492): 779–782. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMirza MU, Froeyen M: Structural elucidation of SARS-CoV-2 vital proteins: Computational methods reveal potential drug candidates against main protease, Nsp12 polymerase and Nsp13 helicase. J Pharm Anal. 2020; 10(4): 320–328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu C, Liu Y, Yang Y, et al.: Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods. Acta Pharm Sin B. 2020; 10(5): 766–788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng Q, Peng R, Yuan B, et al.: Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2. Cell Rep. 2020; 31(11): 107774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYin W, Mao C, Luan X, et al.: Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir. Science. 2020; 368(6498): 1499–1504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen IJ, Foloppe N: Drug-like bioactive structures and conformational coverage with the ligprep/confgen suite: Comparison to programs MOE and catalyst. J Chem Inf Model. 2010; 50(5): 822–839. PubMed Abstract \| Publisher Full Text\n\nRoos K, Wu C, Damm W, et al.: OPLS3e: Extending Force Field Coverage for Drug-Like Small Molecules. J Chem Theory Comput. 2019; 15(3): 1863–1874. PubMed Abstract \| Publisher Full Text\n\nMadhavi Sastry G, Adzhigirey M, Day T, et al.: Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des. 2013; 27(3): 221–234. PubMed Abstract \| Publisher Full Text\n\nRostkowski M, Olsson MH, Søndergaard CR, et al.: Graphical analysis of pH-dependent properties of proteins predicted using PROPKA. BMC Struct Biol. 2011; 11: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlsson MHM, SØndergaard CR, Rostkowski M, et al.: PROPKA3: Consistent treatment of internal and surface residues in empirical pKa predictions. J Chem Theory Comput. 2011; 7(2): 525–537. PubMed Abstract \| Publisher Full Text\n\nHalgren TA, Murphy RB, Friesner RA, et al.: Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. J Med Chem. 2004; 47(7): 1750–1759. PubMed Abstract \| Publisher Full Text\n\nFriesner RA, Banks JL, Murphy RB, et al.: Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J Med Chem. 2004; 47(7): 1739–1749. PubMed Abstract \| Publisher Full Text\n\nBowers KJ, Chow E, Xu H, et al.: Scalable algorithms for molecular dynamics simulations on commodity clusters. Proc 2006 ACM/IEEE Conf Supercomput SC’ 06. 2006. Publisher Full Text\n\nKirchdoerfer RN, Ward AB: Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat Commun. 2019; 10(1): 2342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeersen OB: Picornaviral polymerase structure, function, and fidelity modulation. Virus Res. 2017; 234: 4–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaku K, Zhang B, Noda K: Randomized head-to-head comparison of pitavastatin, atorvastatin, and rosuvastatin for safety and efficacy (quantity and quality of LDL): the PATROL trial. Circ J. 2011; 75(6): 1493–1505. PubMed Abstract \| Publisher Full Text\n\nReiner Ž, Hatamipour M, Banach M, et al.: Statins and the COVID-19 main protease: in silico evidence on direct interaction. Arch Med Sci. 2020; 16(3): 490–496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarty E, Macey M, Mccartney SA, et al.: Ridogrel, a dual thromboxane synthase inhibitor and receptor antagonist: anti-inflammatory profile in inflammatory bowel disease. Aliment Pharmacol Ther. 2000; 14(6): 807–817. PubMed Abstract \| Publisher Full Text\n\nDobson RA, O’connor JR, Poulin SA, et al.: In vitro antimicrobial activity of rosoxacin against Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Antimicrob Agents Chemother. 1980; 18(5): 738–740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandey A, Nikam AN, Shreya AB, et al.: Potential therapeutic targets for combating SARS-CoV-2: Drug repurposing, clinical trials and recent advancements. Life Sci. 2020; 256: 117883. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSvendsen KB, Bech JN, Sùrensen TB, et al.: A comparison of the effects of etodolac and ibuprofen on renal haemodynamics, tubular function, renin, vasopressin and urinary excretion of albumin and alpha-glutathione-S-transferase in healthy subjects: a placebo-controlled cross-over study. Eur J Clin Pharmacol. 2000; 56(5): 383–388. PubMed Abstract \| Publisher Full Text\n\nStorms W: Update on montelukast and its role in the treatment of asthma, allergic rhinitis and exercise-induced bronchoconstriction. Expert Opin Pharmacother. 2007; 8(13): 2173–2187. PubMed Abstract \| Publisher Full Text\n\nAdams SP, Sekhon SS, Tsang M, et al.: Fluvastatin for lowering lipids. Cochrane Database Syst Rev. 2018; 3(3): CD012282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAier I, Semwal R, Raj U, et al.: Comparative modeling and structure based drug repurposing of PAX2 transcription factor for targeting acquired chemoresistance in pancreatic ductal adenocarcinoma. J Biomol Struct Dyn. 2020; 1–8. PubMed Abstract \| Publisher Full Text\n\nPietrzik K, Bailey L, Shane B: Folic acid and L-5-methyltetrahydrofolate: comparison of clinical pharmacokinetics and pharmacodynamics. Clin Pharmacokinet. 2010; 49(8): 538–548. PubMed Abstract \| Publisher Full Text\n\nMajalekar PP, Shirote PJ: Fluoroquinolones: Blessings Or Curses. Curr Drug Targets. 2020; 21(3). PubMed Abstract \| Publisher Full Text\n\nScavone JM, Gleckman RA, Fraser DG: Cinoxacin: mechanism of action, spectrum of activity, pharmacokinetics, adverse reactions, and therapeutic indications. Pharmacotherapy. 1982; 2(5): 266–271. PubMed Abstract \| Publisher Full Text\n\nOng V, Flanagan S, Fang E, et al.: Absorption, distribution, metabolism, and excretion of the novel antibacterial prodrug tedizolid phosphate. Drug Metab Dispos. 2014; 42(8): 1275–1284. PubMed Abstract \| Publisher Full Text\n\nCimolai N: The potential and promise of mefenamic acid. Expert Rev Clin Pharmacol. 2013; 6(3): 289–305. PubMed Abstract \| Publisher Full Text\n\nDe Leo F, Quilici G, Tirone M, et al.: Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment. EMBO Rep. 2019; 20(10): e47788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdelmalak B, Khanna A, Tetzlaff J: Fospropofol, a new sedative anesthetic, and its utility in the perioperative period. Curr Pharm Des. 2012; 18(38): 6241–6252. PubMed Abstract \| Publisher Full Text\n\nNeogi U, Hill KJ, Ambikan AT, et al.: Feasibility of Known RNA Polymerase Inhibitors as Anti-SARS-CoV-2 Drugs. Pathogens. 2020; 9(5): 320. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhammad Umer A, Farjad A, Asma A, et al.: Combined Deep Learning and Molecular Docking Simulations Approach Identifies Potentially Effective FDA Approved Drugs for Repurposing Against SARS-CoV-2. chemRxiv. 2020. Publisher Full Text\n\nHosseini M, Chen W, Wang C: Computational Molecular Docking and Virtual Screening Revealed Promising SARS-CoV-2 Drugs. ChemRxiv Prepr. 2020. Publisher Full Text\n\nJoshi RS, Jagdale SS, Bansode SB, et al.: Discovery of potential multi-target-directed ligands by targeting host-specific SARS-CoV-2 structurally conserved main protease. J Biomol Struct Dyn. 2020; 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeref G, Onur O, Sinan A, et al.: In Silico Identification of Widely Used and Well Tolerated Drugs That May Inhibit SARSCov- 2 3C-like Protease and Viral RNA-Dependent RNA Polymerase Activities, and May Have Potential to Be Directly Used in Clinical Trials. ChemRxiv Prepr. 2020. Publisher Full Text\n\nRao V, Thakur S, Rao J, et al.: Mesenchymal stem cells-bridge catalyst between innate and adaptive immunity in COVID 19. Med Hypotheses. 2020; 143: 109845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore JB, June CH: Cytokine release syndrome in severe COVID-19. Science. 2020; 368(6490): 473–474. PubMed Abstract \| Publisher Full Text\n\nYe Q, Wang B, Mao J: The pathogenesis and treatment of the `Cytokine Storm' in COVID-19. J Infect. 2020; 80(6): 607–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoperchini F, Chiovato L, Croce L, et al.: The cytokine storm in COVID-19: An overview of the involvement of the chemokine/chemokine-receptor system. Cytokine Growth Factor Rev. 2020; 53: 25–32. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71827", "date": "23 Oct 2020", "name": "Harish Holla", "expertise": [ "Reviewer Expertise Synthetic Organic Chemistry", "Medicinal chemistry", "Natural Product Chemistry." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn in silico drug repurposing work is being reported by authors targeting against COVID-19. Authors have attempted to find the possibility of finding drug in the repository of available drug bank which can be repurposed.\nMany of the articles are appearing where computational tools have been utilized targeting the major protease (Mpro), and RNA-dependent RNA polymerase (RdRp) which are two validated target proteins till now.\nAuthors have docked the FDA approved library of drugs against the active site of RdRp using Schrodinger's computer-aided drug discovery tools for in silico drug-repurposing. They have shortlisted 14 drugs from the Standard Precision docking and interaction-wise study of drug-binding with the active site on the enzyme. These drugs are antibiotics, NSAIDs, hypolipidemic, coagulant, thrombolytic, and anti-allergics. Also, they have carried out MD simulation studies on the best among those. The study will be useful for further explorations for in vitro and in vivo studies. The authors have focused only on computational studies and in vitro studies on selected few would have been handy.\n\nOverall, the work is well drafted with minor errors and is suitable for the researchers who are looking for molecules to be studied in vitro on COVID-19.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6088", "date": "04 Nov 2020", "name": "Yogendra Nayak", "role": "Author Response", "response": "Authors thank the reviewer for positive comments" } ] }, { "id": "71828", "date": "03 Nov 2020", "name": "P Hemachandra Reddy", "expertise": [ "Reviewer Expertise Inflammation", "computational biology", "neurodegeneration", "aging and Alzheimer's disease." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a nicely done work, used methods are acceptable - used computational biology is acceptable - Authors shortlisted 14 drugs from the Standard Precision docking and interaction-wise study of drug-binding with the active site on the enzyme. These drugs are antibiotics, NSAIDs, hypolipidemic, coagulant, thrombolytic, and anti-allergics. In molecular dynamics simulations, pitavastatin, ridogrel and rosoxacin displayed superior binding with the active site through ARG555 and divalent magnesium. It is timely and current version is ready to be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6087", "date": "04 Nov 2020", "name": "Yogendra Nayak", "role": "Author Response", "response": "The authors thank the reviewer for positive comments" } ] } ]	1	https://f1000research.com/articles/9-1166
https://f1000research.com/articles/9-681/v1	07 Jul 20	{ "type": "Case Report", "title": "Case Report: Non-microscopic surgical management of incomplete penile amputation", "authors": [ "Donny Eka Putra", "Theddyon Bhenlie Apry Kusbin", "Paksi Satyagraha", "Stephanie Taneysa Widodo", "Theddyon Bhenlie Apry Kusbin", "Paksi Satyagraha", "Stephanie Taneysa Widodo" ], "abstract": "Background: Penile amputation is an emergency urologic condition requiring immediate attention in order to maximize functional outcomes. Unfortunately, there is limited experience and publication of case reports describing the successful replantation of penis after incomplete amputation, especially in facilities without adequate microsurgical tools and means. We hereby present a case of penile amputation caused by a mechanical grass cutter and a discussion of its surgical management. Case description: A 33-year-old Indonesian male presented to the emergency department with incomplete penile amputation six hours post injury. The patient has no prior medical history and presented with penile amputation due to a mechanical grass cutter trauma. He underwent immediate non-microsurgery reconstructive replantation of the penis, reattaching all visible vascular, corporal, and fascia layers. After replantation, the patient recovered well and showed preserved normal appearance and sensitivity of the penis. Subsequent Doppler ultrasound investigation revealed adequate arterial flow at the distal end of the anastomosis. The patient was discharged five days after surgery. Conclusion: In the absence of microsurgical tools and means, the use of non-microsurgical replantation should be the choice of treatment in the case of incomplete penile amputation. The technique showed good outcomes involving adequate functional and cosmetic restoration.", "keywords": [ "traumatic", "penile amputation", "replantation", "case report" ], "content": "Introduction\n\nPenile amputation is an infrequent emergency in the field of urology that needs to be addressed immediately in order to maximize functional outcomes. Frequently, the injury is caused by self-mutilation during an acute psychotic episode. Other etiologies include secondary circumcision, violence, criminal assault, and accidental trauma1. The management of such injury has shifted from the previous inevitable penectomy to a simple reattachment of the organ with re-implantation by microvascular techniques2. In 1929, Ehrich et al. reported the first penile replantation using a macrosurgical technique. In 1977, Cohen et al. and Tamai et al. reported the first successful penile replantation by microsurgical techniques, which includes the re-anastomoses of blood vessels and nerves3,4. However, there is currently no universally accepted regiment to the repair of penile amputation5. There is limited experience and publication of case reports describing the favorable outcomes of penile replantation, especially after incomplete amputation. This case report evaluates therapeutic approach as well as outcome of non-microsurgical replantation of incomplete penile amputation and reviews related literature to summarize the relevance of the current clinical experience6.\n\n\nCase report\n\nA 33-year-old married Indonesian male handyman with no significant past medical and psychiatric history presented to the emergency department of a type-C class rural hospital with partially amputated penis after sustaining a mechanical grass cutter injury six hours prior to the hospital visit. Although compression was immediately performed on the wound, bleeding still persisted (Figure 1A).\n\nA: Penile amputation upon arrival to the emergency department. B: Wound exploration revealed cavernosal and spongiosal body rupture and allowed identification of deep penile arteries and superficial deep dorsal vein. C: Post-surgical evaluation at five days.\n\nAt presentation, his vital signs showed blood pressure of 100/60 mmHg and heart rate of 100 bpm. Physical examination showed a distinctive cut through his spongiosal and cavernosal bodies with diffuse bleeding from the dorsal vessel. The urethra could not be evaluated properly. His scrotum and testicles were found to be intact. Laboratory tests revealed a hemoglobin value of 14.5 g/dl.\n\nFluid infusion and antitetanic injection were administered and the patient was given emergency surgical management by the attending urologist. The patient was placed in the supine position and underwent general anesthesia with 200mg propofol, 50mg atracurium, 10mcg fentanyl, oxygen gas 1 liter/minute, and N2O 1 liter/minute. Subsequent exploration revealed complete detachment of bilateral cavernosal bodies, along with spongiosal body rupture. Povidone-iodine 10% saline irrigation bathing was performed on the wound to allow visualization of the deep dorsal arteries, vein, nerves, and urethra. The urethra was then found to be intact, allowing insertion of a 16Fr foley catheter. Under loupe magnification (2.5x), the spongiosal and cavernosal bodies were sutured circumferentially at both ends with a 5-0 synthetic absorbable suture, followed by suturing the buck’s fascia with the skin with a 4-0 synthetic absorbable suture, and application of a pressure bandage. The deep penile arteries and the superficial deep dorsal vein were not anastomosed. The surgery was completed within two hours and 15 minutes with total ischemia time of nine hours (Figures 1B and 1C).\n\nThe patient was put on total bed rest until the third day post-surgery, with administration of intravenous 3rd generation cephalosporine antibiotics (ceftriaxone 2gr/day), an analgesic (Ketorolac 25mg twice a day), and penile phototherapy (six hours a day) for five days. Five days after reconstruction, the penis showed no significant edema and swelling of the distal penile shaft, and sensation started to return gradually. Evaluation with Doppler ultrasound showed adequate deep and superficial arterial flow at the distal end of the penis. On the fifth day post-surgery, the patient was discharged without urethral catheter following spontaneous micturition (Figure 2).\n\nOne month after reconstruction, the patient underwent further evaluation at a urologic clinic with good skin preservation and adequate wound healing. Erection endured and showed a grade II–III rigidity and also fine penile sensation. There was no urinary fistula formation or difficulties in voiding.\n\n\nDiscussion\n\nGenitourinary injury accounts for as much as 33–66% of hospitalization of patients with external genital injuries7. Males are more prone to genital injury than females due to anatomical differences. Male genitals are more exposed to violence, accidents, and extreme exercise. Of all genital trauma, 80% are caused by blunt injuries. Etiologies and classifications of genital trauma vary based on age (adult and pediatric), anatomical location, and the nature or mechanism of injury. Self-mutilation of the penis is one common etiology of adult genital injury and the majority of cases are associated with mental health problems7,8.\n\nPenile amputation is an uncommon urologic emergency1. It occurs due to a variety of etiologies. The majority of penile amputations are due to self-mutilation due to psychiatric disorders, which accounts for about 87% of all cases. Klingsor syndrome is a psychiatric disease involving self-mutilation characterized by paranoid schizophrenia along with command hallucination as well as disorders of eating such as anorexia and bulimia. The extent of penile injury ranges from minimal skin laceration to total amputation8. A minority of reported cases arise from accidental industrial trauma, masturbatory trauma, and assault by spouses2,9.\n\nThe first documented case of penile replantation was reported by Ehrich in 1929. At that time, penile replantation was conducted on a traumatic injury using a non-microsurgical technique, which involves removal of all necrotic tissue, approximation of related structure, and introduction of a slip graft to cover the penis. A few days after replantation, there was reported hematoma at the glans. Two years later, the patient had urethral stricture and penile shortening with a normal-appearing penis10. However, the organ was functional and apparently in normal shape with few scars. In 1977, Cohen successfully reported the first microsurgical penile replantation5. A systematic review of literature between 1966 to 2007 revealed at least 30 successful penile replantations11.\n\nIn 2017, Morrison et al. conducted a systematic review of 106 patients who underwent penile replantation. They proposed that penile replantation appeared to be safe and effective5. Liu et al. (2019) reviewed 13 published case reports regarding penile amputation in the last five years. It showed that gross contamination or prolonged ischemia time are not factors in successful penile replantation, unless the injury sustained was severe6. However, penile amputation still possesses a great challenge to surgeons due to the current lack of cases, standardized surgical techniques and post-surgical protocols6.\n\nAssessment of the final outcomes of penile replantation have varied widely and is often limited to subjective assessment of both surgeon and patient alike11. This involves survival of the organ, good urinary stream, satisfactory cosmetic appearance and return of sensation as well as erection2,5. Many reports have defined the factors that contribute to favorable outcomes. To name a few, the duration of ischemia time, type and mechanism of injury, severity of injury, as well as microscope use at time of surgery5,11.\n\nMany studies revealed that the ‘golden period’ within six hours post amputation is needed for satisfactory surgical outcomes, but Liu et al. (2019) reported adequate recovery of structure and functional capacity after microsurgical replantation with ischemia time exceeding 10 hours6. In addition, microsurgical repair after 16 hours cold ischemia or injuries of greater than 24 hours has shown promising results12,13. The ischemia time of the patient treated in this report exceeded six hours (about nine hours), but the final outcome of the patient also showed adequate functionality and cosmetic restoration.\n\nA review by Phonsombat et al. of 110 cases of penile amputation showed that gunshot injury (49%) was the most common cause, followed by stab injury or laceration (44%), and bite injury (7%). Surgical reconstruction after penetrating trauma of the penis might be technically easier due to better identification of related structures with intact margins. Blunt penile trauma, however, is more challenging due to the deformed anatomy and unmarked margins6,14. In our case, the injury sustained was due to a mechanical grass cutter at a factory.\n\nBased on the severity of injury, penile amputation can be classified into complete and incomplete15. There is no clear definition regarding incomplete amputation. Liu et al. showed that incomplete amputation with survival of vessels and nerves has a better prognosis compared to those with neurovascular damage6. A retrospective analysis by Morrison et al. concluded that total amputation, increased amount of nerves conglutinated, and anastomosis of the superficial dorsal artery all bear significant association to positive outcomes. In their opinion, complete amputation of the penis tends to have better results because it enables the surgeon to access the neurovascular structurer more clearly. However, numeral illustration and clarification of vessels requiring anastomosis were not available from their data5.\n\nThe preferred surgical techniques, either via microscopic or non-microscopic techniques, are still conflicting. Evaluation of two cases by Liu et al. showed that microsurgical repair was associated with better physical and psychosocial outcomes. Early anastomosis of penile neurovasculature is a critical factor that favors a successful outcome6. Microsurgical techniques enable appropriate anastomosis or coaptation of structure, which allows better sensation and control of sexual function and leads to greater patient satisfaction. Jezior et al. reported that meticulous anastomosis of cavernosal arteries and dorsal structure was associated with erectile function16. A contemporary report recognized the role of microsurgical revascularization in maintaining early and adequate penile blood flow in order to achieve the best appearance and erectile and voiding function outcomes11.\n\nBased on the characteristics of penile blood supply, it is possible to have a good outcome without the need for blood vessels to be re-anastomosed15. Riyach et al. reported a case of incomplete penile replantation using non-microsurgical techniques. The deep penile arteries and superficial deep dorsal vein were not repaired. The outcome was good with a normal-appearing penis, good sensation, ability of penile erection, and ejaculation9. They suggested that the spongiosal bodies may play a role in the arterial supply, venous drainage, and penile erection. Another successful non-microscopic penile replantation was reported by Mensah et al. The aforementioned case reported good voiding flow, cosmetics, and ability of penile erection. They stated that the corporal bodies might play a role in channeling penile blood flow. A review by Kochakarn (2000) concluded that both microsurgical and macrosurgical techniques constituted a good outcome after penile replantation. He reviewed 100 cases with ischemia time of up to 24 hours. The result was satisfying with adequate cosmetics and restoration of erectile ability. The most common complication was skin loss and urethrocutaneous fistula. He underlined that if there was not any microsurgical skills or facilities, penile replantation should be done macrosurgically, because it is proven to show good outcomes17.\n\nMoreover, Li et al. ‘s study involving 109 cases of penile replantation, 51% of which involved non-microsurgical repair, concluded that erectile dysfunction and urethral stricture were more common among patients who underwent non-microsurgical repair. Another report by Mendez et al. showed that a non-microsurgical approach led to necrotic skin, penile sensation loss, urethral stircture, and urethrocutaneous fistulas18.\n\nIn our case, we carried out a non-microsurgical penile replantation in a patient with dorsal structure and cavernosal bodies rupture. As with the case report by Riyach et al., the spongiosal bodies of our patient was partially spared. We used 2.5x loupe magnification to approximate the penile structure. On the fifth day post reconstruction, the penis showed no significant swelling of the distal penile shaft, and sensation started to return gradually. Our case demonstrated a good post-operative result with venous drainage restoration without a microsurgical technique. This raises questions about the role of the spongiosal body in penile blood flow and erection.\n\nThe ischemic time presented in our case was within six hours post injury, and we finished reconstruction in about two hours. Although we did not use a microscope to anastomose a large number of vessels, this led to a shorter ischemic time and therefore better overall outcomes. Moreover, distal penile injuries bear a greater challenge for vascular anastomosis in regards to the involvement of smaller vessels19.\n\nThe limitations of this study include the unavailability of long-term outcomes reported in this study. After coming in for the one month follow-up, the patient ceased to show up for further monitoring. Other limitations include the lack of standardized and validated methods used to report study outcomes. There are currently limited clinical data depicting long term outcomes and functionality of heterogenous surgical repair methods. The strength of this study includes the successful management of a rare case of penile amputation with a partial sparred spongiosal body. Moreover, most cases of penile amputation comprise of complete amputation.\n\nPenile amputation is an infrequent urologic emergency resulting from a variety of factors. This case report outlines the treatment of distal penile amputation with partial spongiosal injury. Although the gold standard treatment for said injury is a microscopic neurovascular reconstruction technique, non-microsurgical penile replantation in a resource-deficient setting seems to yield adequate results.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.", "appendix": "Acknowledgments\n\nThe authors would like to thank all faculty and staff of Urology, Anesthesiology, and Radiology Departments of dr. Dradjat Prawiranegara General Hospital Serang, Banten.\n\n\nReferences\n\nPatel MS, Jensen D, Culp SH: Traumatic Penile Amputation: A Case Report and Acute Management. J Trauma Treat. 2014; 3(4): 1000210. Publisher Full Text\n\nKrishnakumar KS, Petkar KS, Lateef S, et al.: Penile replantation. Indian J Plast Surg. 2013; 46(1): 143–146. PubMed Abstract \| Free Full Text\n\nCohen BE, May JW Jr, Daly JSF, et al.: Successful clinical replantation of an amputated penis by microneurovascular repair. Plast Reconstr Surg. 1977; 59(2): 276–80. PubMed Abstract\n\nTamai S, Nakamura Y, Motomiya Y: Microsurgical replantation of a completely amputated penis and scrotum: Case report. Plast Reconstr Surg. 1977; 60(2): 287–91. PubMed Abstract \| Publisher Full Text\n\nMorrison SD, Shakir A, Vyas KS, et al.: Penile Replantation: A Retrospective Analysis of Outcomes and Complications. J Reconstr Microsurg. 2017; 33(4): 227–232. PubMed Abstract \| Publisher Full Text\n\nLiu X, Liu Z, Pokhrel G, et al.: Two cases of successful microsurgical penile replantation with ischemia time exceeding 10 hours and literature review. Transl Androl Urol. 2019; 8(Suppl 1): S78–S84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKitrey ND, Djakovic N, Hallscheidt P, et al.: EAU Guidelines on Urological Trauma 2020. European Association of Urology Guidelines 2020 Edition [Internet]. Arnhem, The Netherlands: European Association Urology Guidelines Office 2020. Reference Source\n\nKim JH, Park JY, Song YS: Traumatic Penile Injury: From Circumcision Injury to Penile Amputation. Biomed Res Int. 2014; 2014: 375285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiyach O, El Majdoub A, Tazi MF, et al.: Successful replantation of an amputated penis: A case report and review of the literature. J Med Case Rep. 2014; 8: 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhrich WS: Two Unusual Penile Injuries. J Urol. 1929; 2019: 1582047. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaheem OA, Mirheydar HS, Patel ND, et al.: Surgical Management of Traumatic Penile Amputation: A Case Report and Review of the World Literature. Sex Med. 2015; 3(1): 49–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei FC, Hunt McKee N, Javier Huerta F, et al.: Microsurgical replantation of a completely amputated penis. Ann Plast Surg. 1983; 10(4): 317–21. PubMed Abstract \| Publisher Full Text\n\nFaydaci G, Uğur K, Osman C, et al.: Amputation of glans penis: A rare circumcision complication and successful management with primary anastomosis and hyperbaric oxygen therapy. Korean J Urol. 2011; 52(2): 147–149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhonsombat S, Master VA, McAninch JW: Penetrating External Genital Trauma: A 30-Year Single Institution Experience. J Urol. 2008; 180(1): 192–5. PubMed Abstract \| Publisher Full Text\n\nMineo M, Jolley T, Rodriguez G: Leech therapy in penile replantation: A case of recurrent penile self-amputation. Urology. 2004; 63(5): 981–3. PubMed Abstract \| Publisher Full Text\n\nJezior JR, Brady JD, Schlossberg SM: Management of penile amputation injuries. World J Surg. 2001; 25(12): 1602–9. PubMed Abstract \| Publisher Full Text\n\nKochakarn W, Viseshsindh V, Muangman V: Penile fracture: Long-term outcome of treatment. J Med Assoc Thail. 2002; 85(2): 179–82. PubMed Abstract\n\nMendez R, Kiely WF, Morrow JW: Self-emasculation. J Urol. 1972; 107(6): 981–5. PubMed Abstract \| Publisher Full Text\n\nMensah JE, Bray LD, Akpakli E, et al.: Successful penile reimplantation and systematic review of world literature. African J Urol. 2017; 23(13): 253–257. Publisher Full Text" }	[ { "id": "66594", "date": "16 Jul 2020", "name": "Omer A. Raheem", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper and adds to the current literature in penile injuries. I would recommend for publication; however, major revisions need to be added first.\n\nNeed to delineate and describe anatomical injury and orientation: ventral and dorsal? What is preop trauma/injury assessment and how it was managed at the emergency room level?\n\nI understand microscopy was not available but it remains gold standard in penile injury repair and reconstruction especially when anatomizing penile vasculature and corporal tissues and urethral. However, surgical loops were used? Correct?\n\nHow was urethral managed? Did you do preop UA, intra op cysto? Passing catheter? Any resistance? Did you repair urethra?\n\nDescribe in detail penile vascular injury? Was dorsal penile arteries, veins, and nerves all injures? Did you use intrap doppler to assess for arterial signals?\n\nWhy it was not repaired? You also mentioned deep penile artery was not repaired?\n\nWhat is post op follow up? How many days? Months since the injury? Do you have SHIM or AUA SS assessed? Did he retain any erectile or ejaculatory function? Make clear in the paper?\n\nWere other specialities like plastics present to assist or only urology?\n\nWhat is the management of foley cath post op and wound care?\n\nDescribe reconstruction sutures used and what fashion interrupted vs continuous?\n\nIn your conclusion, you mentioned that microscopic repair remains gold standard; however, if there are limited resources, then non micro repair is possible. That’s ok. However you used surgical loops? Correct?\n\nHappy to write a short editorial to accompany this paper after revision is made.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5776", "date": "30 Jul 2020", "name": "Donny Eka Putra", "role": "Author Response", "response": "1. Thank you prof Raheem, we highly appreciate your feedback. We are deeply sorry for the delay in our respond. 2. Need to delineate and describe anatomical injury and orientation: ventral and dorsal? What is preop trauma/injury assessment and how it was managed at the emergency room level? The injury sustained resulted from a grass-cutter blade in the direction from the dorsal side of the penis. The injury consisted of dorsal artery nerves and veins, bilateral corpus cavernosum, and corpus spongiosum. The urethra was intact.Pre-operation assessment in the emergency department was limited to penile amputation only, as we were unable to clearly define structural damages due to diffuse bleeding and excessive pain. At the time of patient arrival, trauma resuscitation protocol including airway, breathing, and circulation assessment was running. The patient was also given antibiotic anti-tetanus, blood specimen testing and on a 23G IV line to handle the earliest signs of shock if necessary.The patient was then treated with compression bandage on the penis to minimize bleeding.3. I understand microscopy was not available but it remains gold standard in penile injury repair and reconstruction especially when anatomizing penile vasculature and corporal tissues and urethral. However, surgical loops were used? Correct? Yes, we used surgical loupe with 2.5x magnification.4. How was urethral managed? Did you do preop UA, intra op cysto? Passing catheter? Any resistance? Did you repair urethra? At the time of surgical exploration, urethra was found to be intact and the catheter was able to pass through without any resistance. I did not do prep UA and i did not have the need to repair the urethra. 5. Describe in detail penile vascular injury? Was dorsal penile arteries, veins, and nerves all injures? Did you use intrap doppler to assess for arterial signals? Surgical exploration revealed dorsal penile veins and nerves injury along with corporal bodies. I did not use doppler US because it was not available during surgery.6. Why it was not repaired? You also mentioned deep penile artery was not repaired? What I meant by ‘not repaired’ is that I did not repair it by micro-surgical means to create a proper end-to-end anastomosis. I however did an anastomoses by approximating the deep dorsal artery end with the available 2.5x loupe magnification.7. What is post op follow up? How many days? Months since the injury? Do you have SHIM or AUA SS assessed? Did he retain any erectile or ejaculatory function? Make clear in the paper?The patient was discharged from the hospital on the 5th day after surgery. At day 5, the patient underwent doppler ultrasound to assess vascularization of penile distal to the injury. The patient was discharged without catheter following no difficulty in voiding. At one month after surgery, the patient was evaluated with IPSS, IIEF, and EHS score. Yes, the patient retained erection and ejaculation function at the level of grade 3 EHS, IIEF score 19, IPSS score 2, QOL 1.8. Were other specialities like plastics present to assist or only urology?No, Only Urology.9. What is the management of foley cath post op and wound care? On the 3rd day after surgery we applied a new sterile dressing on the wound and observed no significant edema or pus or any leakage. On the fifth day after surgery, we adopted a post operative open wound treatment care plan. The catheter was also removed at the 5th day post surgery with close observation for hematuria or urinary retention. 10. Describe reconstruction sutures used and what fashion interrupted vs continuous? I used vicryl 5/0 interrupted suture on the corporal bodies and 4/0 vicryl interrupted suture on fascia and skin.11. In your conclusion, you mentioned that microscopic repair remains gold standard; however, if there are limited resources, then non micro repair is possible. That’s ok. However you used surgical loops? Correct? Yes. My apologies, i will revise my conclusion with the recommendation of doing repair with at least a 2,5x surgical loupe like the way i treated the patient in the paper. 12. Happy to write a short editorial to accompany this paper after revision is made.Thank you, we highly appreciate the time you spent to review our paper. We would love to have your short editorial to accompany our paper." } ] }, { "id": "69356", "date": "18 Aug 2020", "name": "David J. Ralph", "expertise": [ "Reviewer Expertise Andrology and penile reconstruction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNeed more anatomical description.\n\nWas this a division through the glans and distal corpora - urethral vessels were, therefore, supplying the glans?\n\nWhat about the dorsal neuromuscular bundle - please comment?\n\nUse the correct anatomy for each vessel, nerve, vein etc.\n\nNeed a more subjective assessment of sensation and erectile function.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/9-681
https://f1000research.com/articles/7-628/v1	22 May 18	{ "type": "Software Tool Article", "title": "SeqAcademy: an educational pipeline for RNA-Seq and ChIP-Seq analysis", "authors": [ "Syed Hussain Ather", "Olaitan Igbagbo Awe", "Thomas J. Butler", "Tamiru Denka", "Stephen Andrew Semick", "Wanhu Tang", "Ben Busby", "Olaitan Igbagbo Awe", "Thomas J. Butler", "Tamiru Denka", "Stephen Andrew Semick", "Wanhu Tang", "Ben Busby" ], "abstract": "Quantification of gene expression and characterization of gene transcript structures are central problems in molecular biology. RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) are important methods, but can be cumbersome and difficult for beginners to learn. To teach interested students and scientists how to analyze RNA-Seq and ChIP-Seq data, we present a start-to-finish tutorial for analyzing RNA-Seq and ChIP-Seq data: SeqAcademy (source code: https://github.com/NCBI-Hackathons/seqacademy, webpage: http://www.seqacademy.org/). This user-friendly pipeline, fully written in Jupyter Notebook, emphasizes the use of publicly available RNA-Seq and ChIP-Seq data and strings together popular tools that bridge that gap between raw sequencing reads and biological insight. We demonstrate practical and conceptual considerations for various RNA-Seq and ChIP-Seq analysis steps with a biological use case - a previously published yeast experiment. This work complements existing sophisticated RNA-Seq and ChIP-Seq pipelines designed for advanced users by gently introducing the critical components of RNA-Seq and ChIP-Seq analysis to the novice bioinformatician. In conclusion, this well-documented pipeline will introduce state-of-the-art RNA-Seq and ChIP-Seq analysis tools to beginning bioinformaticians and help facilitate the analysis of the burgeoning amounts of public RNA-Seq and ChIP-Seq data.", "keywords": [ "RNA-Seq", "ChIP-Seq", "alignment", "differential gene expression", "peak-calling", "education", "tutorial", "pipeline" ], "content": "Introduction\n\nRNA sequencing (RNA-Seq) is a rapidly expanding technique used to answer broad questions in the life sciences, ranging from mitochondrial function (Mercer et al., 2011) to the pathogenesis of breast cancer (Li et al., 2017). Chromatin immunoprecipitation sequencing (ChIP-Seq) is a genome-wide technique for profiling histone modifications, protein interactions, and transcription factor binding sites (Barski et al., 2007). Using this technique to analyze protein interactions involves very large data sets for computational analysis. The computational steps can identify the locations of features such as DNA-binding enzymes, modified histones, chaperones, nucleosomes, and transcription factors (TFs) (Bailey et al., 2013).\n\nThe expanding importance of RNA-Seq and ChIP-Seq data is reflected by its explosive growth in terabytes in the primary public repository for storing this data - the Sequence Read Archive (SRA) (Wheeler et al., 2008). This incredible increase in the amount of public data has not been met with an equal increase in the number of scientists who can skillfully and thoughtfully analyze this important resource. Given the fundamental role that RNA-Seq and ChIP-Seq data, among other next-generation sequencing data types, are likely to play in the coming decades, there is a critical need to teach RNA-Seq and ChIP-Seq analysis to life scientists with diverse interests and backgrounds.\n\nThe goal of analyzing RNA-Seq data is often to identify and characterize quantitative differences in gene expression between biological samples from two or more groups. For ChIP-Seq, the goal is to characterize DNA-protein interactions. Biological samples may originate from several different study designs including: different tissue types from the same individual (e.g. cancerous tissue vs. non-cancerous tissue), the same strain of cells under different environmental conditions, or the same tissue under a time-course experiment.\n\nThere are major barriers to the novice bioinformatician who is interested in learning how to analyze RNA-Seq and ChIP-Seq data. RNA-Seq and ChIP-Seq data are costly to generate (>$1,000/sample) and cumbersome to store; with data from a single sample often occupying several gigabytes of storage space. However, recent advances, including a greater impetus to deposit sequencing data in SRA (“Principles and Guidelines for Reporting Preclinical Research,” 2015) and the innovative alignment of streamed sequencing data (Kim et al., 2015), offer new opportunities to overcome these long-standing problems. The second barrier to entry is inherent to RNA-Seq and ChIP-Seq data. These datasets are large and complex: there are over 20,000 known genes in the human genome (Naidoo et al., 2011) and the transcriptional diversity of the human genome is not yet fully characterized (Yamashita et al., 2011).\n\nFurthermore, RNA-Seq data is susceptible to “batch effects” and other confounders that can occlude real biological effects or, worse, mislead the un-skeptical researcher. Thus, appropriate analysis of these data requires advanced algorithms and sophisticated statistical methods, coupled with traditional scientific skepticism, to uncover biological insight buried in the data.\n\nThese difficulties dissuade many from attempting RNA-Seq and ChIP-Seq analysis, particularly those lacking previous data analysis experience, but the genomics community needs more scientists who can adeptly analyze RNA-Seq and ChIP-Seq data. Moreover, shared understanding of RNA-Seq and ChIP-Seq analysis will produce higher quality discourse between the biologists who are responsible for conducting RNA-Seq and ChIP-Seq experiments and the bioinformaticians who are experts at analyzing the resulting data produced from these experiments. Several well-developed pipelines currently exist for processing RNA-Seq and ChIP-seq data from start to finish (Djebali et al., 2017; Park et al., 2017; Torres-García et al., 2014; Yalamanchili et al., 2017); however, these pipelines are generally designed for advanced bioinformaticians who often have existing practical experience in analyzing high-throughput data. A pipeline designed to teach those with little experience how to analyze high-throughput sequencing data is therefore needed. Thus, we developed a proof-of-concept, well-documented “tutorial pipeline” over the course of a three-day NCBI-sponsored hackathon intended to teach RNA-seq and ChIP-seq analysis to beginners. This tutorial pipeline, “SeqAcademy,” incorporates state-of-the-art RNA-Seq and ChIP-seq analysis tools into a simple, easy to use workflow tutorial and we demonstrate its use with publicly available data.\n\n\nMethods\n\nSeqAcademy uses a self-contained Jupyter Notebook, which runs Python, R, and Bash scripts among others, all from the document itself (Kluyver et al., 2016). It requires about 16 GB of memory storage. Jupyter Notebook facilitates open science and reproducible code by mixing code chunks with notes and markup. This format, known as “literate programming,” is particularly amenable to teaching bioinformatics because it allows learners to follow along in the document while running each code step directly within the notebook.\n\nThe tutorial begins with an explanation of how to install necessary dependencies and select interesting data from the BioProjects browser. Alignment while streaming the data is done with HISAT2 version 0.1.6 and subsequent quality control with MultiQC version 1.5. The tutorial then splits into two separate protocols: one for RNA-seq, the other for ChIP-seq analysis.\n\nThe workflow involved setup, alignment, quality control, analysis, and visualization steps for publicly available RNA-Seq and ChIP-seq data sets. There are many appropriate tools available for each step of RNA-seq and ChIP-seq analysis. Our goal is to present an easy to use and understandable pipeline rather than an exhaustive list of analysis tools. For each step below, we will explain the role of the bioinformatic tool, as well as our rationale for including it in this tutorial pipeline (Figure 1). Here, we present an overview of the steps; further details for each subsection can be found on the project’s Github page.\n\nThe setup step uses the Bioconda channel (Grüning et al., 2017) for the conda package manager to install all of the programmatic dependencies for the entire pipeline. The data sets were selected by searching NCBI BioProject web browser (Barrett et al., 2011). For our use case, we searched for publically available RNA-Seq and ChIP-Seq datasets that were relatively small and thus could be easily downloaded and processed, and would be relatively straightforward to interpret biologically. We therefore selected RNA-Seq and ChIP-Seq data from yeast (Saccharomyces cerevisiae) samples (Mulla et al., 2017; Rawal et al., 2018).\n\nThe RNA-Seq data demonstrates the differences in genetic expression between aneuploid and euploid yeast (Mulla et al., 2017). The ChIP-Seq data demonstrates the effects of 3-Amino-1,2,4-triazole (3-AT) on chromatin accessibility (Rawal et al., 2018). We downloaded the reference sequence for Saccharomyces cerevisiae from Ensembl version 84 (RNA-Seq SRA study number: SRP106028 ChIP-Seq SRA study number: SRP132584). We note that the SraRunTables file can be adjusted to specific user data, different from the RNA-Seq or ChIP-seq data sets used in this project. Thus, this lightweight, portable educational pipeline can be adapted to meet the usage needs and interests of a broad base of bioinformatics beginners and teachers.\n\nHISAT2 is a software program used for the alignment of raw sequence data, consisting of FASTQ files (Kim et al., 2015. We chose to use HISAT2 because it allows users to stream raw sequence data rather than downloading it to the local machine, reducing disk space and time requirements for users of the SeqAcademy educational tool - an exemplary use of “edge-computing” in bioinformatics. One disadvantage of this approach is that it requires a stable internet connection, as the aligned raw sequence files are downloaded as SAM (sequence alignment mapping) files along with the log files. Nevertheless, by choosing to use HISAT2 for alignment, we reduced required disk space and broadened the potential user base of this pipeline.\n\nTo generate a quality control report about the success of the alignment, we used MultiQC (Ewels et al., 2016). MultiQC reports the number of reads mapped to one unique location, reads mapped to multiple unique locations, and reads not mapped to any location in the reference genome. MultiQC can provide reports for both RNA-Seq and ChIP-seq data. Reads mapped to one unique location have a higher confidence level of being correctly mapped, as reads mapped to multiple unique locations cannot be localized to the reference with a high degree of probability. While MultiQC is not strictly necessary for this pipeline--the plots and statistics it produces are based off of the HISAT2 alignment summary files - we chose to include it to introduce users to a useful tool that is built for quality control.\n\nAfter alignment and quality control, users convert the SAM files to BAM files with the samtools package version 1.8 (Li et al., 2009). Then, gene expression is quantified with HTSeq version 0.9.1 (Anders et al., 2015). Afterwards, we demonstrate how to extract biological significance from these various analyses, by showing students how to visualize gene expression patterns and undertake exploratory data analysis with principal component analysis (PCA). Finally, we show how to undertake differential expression analysis using DESeq2 version 1.21.0 (Love et al., 2014) and how to visualize these differences with volcano plots and experiment-specific visualizations in the R package ggplot2 version 2.2.1 (Wickham, 2009). Thus, students can learn how to quantify gene expression, answer biologically relevant questions through differential gene expression analysis, and visualize gene expression patterns.\n\nAfter alignment, we perform peak-calling to determine protein-binding locations in the ChIP-seq data. The peak-calling step of ChIP-Seq involves finding differentially binding sites between the two ChIP-Seq signals (input and immunoprecipitate). Numerous peak callers exist to distinguish biologically relevant signal peak from technical noise for the Chip-Seq experiments. Here, we used the peak-calling algorithm MACS (Model-based Analysis for ChIP-Seq) version 1.4.2 (Zhang et al., 2008). MACS is a commonly used peak-caller and has been shown to have more accurate results than competing peak-callers (Hocking et al., 2017). After calling peaks, the results are sorted and analyzed for intersections using bedtools version 2.27.0, a set of tools for analyzing genomic data (Quinlan & Hall, 2010). Lastly, bedtools output is visualized with Integrative Genomics Viewer (IGV) version 2.4, a genomic data set viewer that allows for visualization of genomic features (Robinson et al., 2011).\n\n\nUse cases\n\nThis educational pipeline is designed for students without previous programming experience who are looking for an introduction to the acquisition, processing, analysis, and visualization of either RNA-Seq or ChIP-seq data. Students of next-generation sequencing analysis may range the academic spectrum, from undergraduates to professors, all of whom share an interest in learning to analyze sequencing data. SeqAcademy also offers a useful introduction to the core steps of RNA/ChIP-Seq analysis for use by bioinformatics educators who are teaching a class or mentoring students. Motivated individual learners, for instance a graduate student who is attempting RNA-Seq analysis, may also benefit by working through SeqAcademy. The Jupyter Notebook file is completely self-contained, so users do not need to manage additional input files or tools beyond what is provided directly in the notebook document—every line of code to be run has already been written and tested. Thus, this flexible tutorial may be a suitable introduction to RNA-seq and ChIP-seq analysis for workshops, graduate school classes, or motivated individual learners. We also hope that fellow bioinformatics educators will build off of SeqAcademy to teach intermediate and advanced bioinformatics concepts and skills. The pipeline is simple and modular, so it can easily be adapted to analyze different datasets and customized to meet different user needs.\n\nThe learning objectives of SeqAcademy are two-fold. The first and most immediate or practical objective is for a student to learn how to conduct the core steps of an RNA/ChIP-seq analysis, beginning with a search for publicly available sequencing data and ending with biologically meaningful results. The second objective is to foster a greater understanding of the concepts behind each step. This includes biological reasons behind why certain experiments such as ChIP-Seq and RNA-Seq are run, and the logic behind alignment, differential gene expression, and peak-calling. The tutorial pipeline is purposefully simple, as this will introduce important component of next generation sequencing more gently, and will encourage students to build off of it to create more advanced pipelines that will meet the unique goals of the student.\n\nTable 1 and Table 2 illustrate the sample input yeast data for RNA-Seq and ChIP-Seq, respectively. The RNA-Seq data examines aneuploidy while the ChIP-Seq data shows induction by 3-Amino-1,2,4-triazole (3-AT). Results of the principal component analysis, an unsupervised data reduction technique, of the RNA-Seq data are shown in Figure 2a. The slight clustering of the data into two different groups, euploid and aneuploid can be observed. A volcano plot is used to visualize significant differentially expressed genes between two groups, in this case euploid and aneuploid (Figure 2b). Figure 2c displays the enrichment of chromosome X for differentially expressed genes, consistent with the aneuploid sample having an extra X chromosome. Figure 3 shows an IGV screenshot of how peaks of protein-enrichment are distributed across the yeast genome. The corresponding genes can be examined to determine proteins involved in 3-AT induction.\n\nThis data presents the RNA-Seq data used in this tutorial. This tutorial observes RNA-Seq data of aneuploidy in yeast.\n\nThis data presents the ChIP-Seq data used in this tutorial. This tutorial observes ChIP-Seq data of induction by 3-AT in yeast.\n\nPCA suggests gene expression for euploid yeast samples (haploid) clusters distinctly from that of the aneuploid yeast samples (diploid chromosome X). The first two Principal Components account for ~70% of the variance in expressed genes). Data provided by Mulla et al., 2017.\n\nThe x-axis represents the difference in gene expression between the conditions. False discovery rate (FDR), a method for controlling for multiple testing, is along the y-axis. Each point represents a tested gene (N=3,926). Red points are those reaching genome-wide significance (at FDR<0.05, N=663), whereas grey points are genes not reaching statistical significance (FDR>0.05, N=3,263). Data provided by Mulla et al., 2017.\n\nThe relative enrichment of chrX for differentially expressed genes suggests the downstream results of this processing pipeline are consistent with biological expectations. The RNA-seq experiment was performed on yeast colonies with an extra chromosome X. Data provided by Mulla et al., 2017.\n\nThis IGV screenshot shows in the bottom row the intersected peaks between the two treatment conditions of the yeast samples. The matching genes with each intersected peak can be analyzed. Data provided by Rawal et al., 2018.\n\n\nConclusion and next steps\n\nThere are several limitations to take into account with this tutorial and future directions for further work. In this tutorial, we focused on using RNA-seq on “bulk” or homogenate tissue samples, as opposed to single-cell RNA-seq, which has distinct analytical considerations. Our pipeline is currently limited to only two of the various next generation sequencing analyses, and we would like to broaden the scope to also include DNA sequencing and other epigenetic sequencing protocols, such as whole-genome bisulfite sequencing. Our platform can also be developed further to incorporate more advanced features, such user interfaces for performing bioinformatics analyses from the web browser, login systems for users to keep track of their own progress, and forums and messaging systems for community feedback. We would also like to translate the pipeline into other languages to broaden its scope. In subsequent improvements, we plan to make the pipeline easily individualized for a user’s own data sources by adjusting SraRunTables. Future hackathons may offer a useful setting to further improve this developing resource. Despite these limitations, SeqAcademy provides a solid starting foundation for beginners to learn the fundamentals.\n\nWe have presented a novel, standalone educational tool for two types of next generation sequencing data: RNA-Seq and ChIP-Seq data. This project offers a simple guidebook to an introductory analysis pipeline used in RNA-Seq and ChIP-Seq data. We introduced a cutting-edge bioinformatics tools frequently used for the acquisition, alignment, processing, analysis, and visualization of large-scale sequencing data and referenced further resources for continued learning. SeqAcademy meets the need for an educational analysis pipeline which can be used to teach undergraduate and graduate students with limited bioinformatics experience how to analyze publically available sequencing data.\n\n\nData availability\n\nUse case data is available for the NCBI Sequence Read Archive Run Selector under accession numbers – SRP132584 and SRP106028.\n\n\nSoftware availability\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.1233661 (Ather et al., 2018)\n\nThe code for this project is deposited under an MIT License on GitHub: https://github.com/NCBI-Hackathons/seqacademy", "appendix": "Competing interests\n\n\n\nNo competing interests are disclosed.\n\n\nGrant information\n\nThis research was supported by the Intramural Research Program of the NIH, National Library of Medicine.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Lisa Federer for help organizing the manuscript and some revision suggestions. We would also like to thank the Intramural Research Program of the National Library of Medicine for supporting this work.\n\n\nReferences\n\nAnders S, Pyl PT, Huber W: HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2): 166–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAther SH, Awe OI, Butler TJ, et al.: SeqAcademy: an educational pipeline for RNA-Seq and ChIP-Seq analysis. Zenodo. 2018. Data Source\n\nBailey T, Krajewski P, Ladunga I, et al.: Practical guidelines for the comprehensive analysis of ChIP-seq data. PLoS Comput Biol. 2013; 9(11): e1003326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarrett T, Clark K, Gevorgyan R, et al.: BioProject and BioSample databases at NCBI: facilitating capture and organization of metadata. Nucleic Acids Res. 2011; 40(Database issue): D57–D63. PubMed Abstract \| Publisher Full Text \| Free 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): 823–37. PubMed Abstract \| Publisher Full Text\n\nDjebali S, Wucher V, Foissac S, et al.: Bioinformatics Pipeline for Transcriptome Sequencing Analysis. Methods Mol Biol. 2017; 1468: 201–219. PubMed Abstract \| Publisher Full Text\n\nEwels P, Magnusson M, Lundin S, et al.: MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016; 32(19): 3047–3048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: A sustainable and comprehensive software distribution for the life sciences. bioRxiv. 2017. Publisher Full Text\n\nHocking TD, Goerner-Potvin P, Morin A, et al.: Optimizing ChIP-seq peak detectors using visual labels and supervised machine learning. Bioinformatics. 2017; 33(4): 491–499. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim D, Langmead B, Salzberg SL: HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12(4): 357–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKluyver T, Ragan-Kelley B, Pérez F, et al.: Jupyter Notebooks – a publishing format for reproducible computational workflows. Loizides, Fernando and Schmidt, Birgit (eds.) In Positioning and Power in Academic Publishing: Players, Agents and Agendas. IOS Press. 2016; 87–90. Publisher Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Liu X, Tang H, et al.: RNA Sequencing Uncovers Molecular Mechanisms Underlying Pathological Complete Response to Chemotherapy in Patients with Operable Breast Cancer. Med Sci Monit. 2017; 23: 4321–4327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMercer TR, Neph S, Dinger ME, et al.: The human mitochondrial transcriptome. Cell. 2011; 146(4): 645–658. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMulla WA, Seidel CW, Zhu J, et al.: Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast. eLife. 2017; 6: pii: e27991. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaidoo N, Pawitan Y, Soong R, et al.: Human genetics and genomics a decade after the release of the draft sequence of the human genome. Hum Genomics. 2011; 5(6): 577–622. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark SJ, Kim JH, Yoon BH, et al.: A ChIP-Seq Data Analysis Pipeline Based on Bioconductor Packages. Genomics Inform. 2017; 15(1): 11–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrinciples and Guidelines for Reporting Preclinical Research. Retrieved April 18, 2018, 2015. Reference Source\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRawal Y, Chereji RV, Valabhoju V, et al.: Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast. Mol Cell. 2018; 70(2): 297–311.e4. PubMed Abstract \| Publisher Full Text\n\nRobinson JT, Thorvaldsdóttir H, Winckler W, et al.: Integrative genomics viewer. Nat Biotechnol. 2011; 29(1): 24–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorres-García W, Zheng S, Sivachenko A, et al.: PRADA: pipeline for RNA sequencing data analysis. Bioinformatics. 2014; 30(15): 2224–2226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWheeler DL, Barrett T, Benson DA, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2008; 36(Database issue): D13–D21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag. 2009. Publisher Full Text\n\nYalamanchili HK, Wan YW, Liu Z: Data Analysis Pipeline for RNA-seq Experiments: From Differential Expression to Cryptic Splicing. Curr Protoc Bioinformatics. 2017; 59: 11.15.1–11.15.21. PubMed Abstract \| Publisher Full Text\n\nYamashita R, Sathira NP, Kanai A, et al.: Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res. 2011; 21(5): 775–789. 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" }	[ { "id": "34335", "date": "05 Jun 2018", "name": "Philip Ewels", "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\nBioinformatics training is of critical importance for the field of genomics, especially given the current shortage of experienced bioinformaticians and the utility of cross-training lab scientists in data analysis. In this manuscript, the authors describe a training tool developed to help teach people new to bioinformatics how to handle data from RNA and ChIP high throughput sequencing experiments.\nSuch a tool is valuable to the genomics community - it is a resource for interested parties to use in their own time and could also be very useful for those designing bioinformatics courses.\n### Manuscript\nThe manuscript does a good job of describing the resource, as well as the reason it is helpful. In my opinion, it is longer than needed - the Introduction could be more concise and the Use cases section would feel more natural in the introduction. The figures could also easily be condensed into one and the table be a supplementary figure. Making the manuscript shorter would make it more accessible.\n### Tutorial\nThe authors take an excellent approach to the design of their tutorial, using a Jupyter notebook and software installations from bioconda. These are appropriate, user friendly and provide an excellent starting point for well documented and reproducible analyses. Some dependencies are required, but these are well described on the tool's homepage and commonly used in bioinformatics. I attempted to run the tutorial on my laptop, but I ran into a few issues that required some debugging.\nBroken steps I came across a couple of problems in the notebook: I couldn't get the MultiQC commands to work - the mix of Python variables in the bash commands failed in my Jupyter notebook. I changed the command to use the following command:\n!multiqc test/{\"* test/\".join(RNASeqoutrun)}* --quiet --outdir test/multiqc_rnaseq --force\nNext, the Bedtools steps didn't work - the intersect command refers to a sample SRR6703663, which does not seem to be included anywhere in the tutorial before this point. I gave up at this point, so I'm not sure about the final steps. If the authors think that I did something wrong, I'd be happy to have another go.\nI recommend trying to get some colleagues to run this tutorial from scratch to get more feedback.\nExternal scripts My main concern with the tutorial is that much of the downstream processing is done within R scripts outside of the Jupyter notebook. For me, this sort of misses the point - it would be preferable to have this code mixed in with the tutorial without any external scripts. It should be possible to have both Python (bash) and R in the same notebook, and this would make those steps part of the exercise rather than an abstract \"runDeseq.R\" script that requires the user to go and find and read through.\nData subsampling Although the authors have endeavoured to use a small dataset (Yeast, not Human), the data used is still large. The authors note in the tutorial that the alignment \"will most likely take several hours.\", which limits the utility of the tutorial in any teaching scenario.\nIt should be quite easy to use the -u/--qupto flag in HiSAT2 to limit the number of alignments performed. I imagine that most of the downstream analysis will still work with subsampled data, and will run much faster. I tried doing this with 1,000,000 reads and the alignment step completed in a couple of minutes.\n### Tutorial - Minor points\nSoftware requirements I had trouble getting the bioconda installation command to first install and then activate inside the the notebook. I would instead recommend creating the conda environment on the terminal first (including jupyter as a package to install), activating it and then running jupyter. This is what I ended up doing to get the tutorial to work.\nStreaming Input Data The approach of streaming data from the SRA for the alignment step is a little non-standard. The reasoning is described in the paper as helping to reduce the disk space footprint required for the analysis. Whilst its cool that this step works, I would argue that it lessens the value of the tutorial, as most bioinformatics projects can not use such a method. Starting with regular static FastQ files would be more typical and useful for future analyses. This would also allow the additional step of FastQC for raw data analysis, which is pretty typical for such pipelines. The downside of this is that it invalidates my above subsampling idea! But SRA data could still be streamed to FastQ files with a similar subsampling approach.\nTwo-in-one analysis The analysis presented uses common alignment and quality control steps for both sets of data, before splitting into bespoke RNA and ChIP-seq analysis. I see the reasoning for this but I think this complicates issues a little for users. Personally, I would prefer to see two separate Jupyter notebooks and separate analyses for the two different data types to avoid confusing the two. This would simplify future expansion of the resource across additional data types, which may not be able to share such analysis steps.\nMultiQC reporting The Quality Control step using MultiQC is placed after the HiSAT2 alignment. It would be better placed at the end of the analysis, as MultiQC also supports outputs from MACS, HTSeq and deepTools. A more complete report would be generated if it were run with all of these outputs.\n### Conclusions I think that the aim of this paper is worthwhile and the approach is very nice. The tutorial itself feels a little unpolished and could do with a few trial runs on willing volunteers to iron out the issues I encountered. With a few tweaks and the changes above, I think that this tutorial could be the beginning of an excellent resource for budding bioinformaticians and educators.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "34336", "date": "12 Jun 2018", "name": "Norann A. Zaghloul", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a streamlined tool aimed toward novice bioinformaticians who wish to analyze RNA-Seq or ChIP-Seq data in one simple tool. The premise for this tool is excellent. Investigators who are new to these approaches often find the analysis of data daunting and are unsure of the tools to use for each step. SeqAcademy provides a single tool for all the steps in an easy to download package. Given that the target for this tool is investigators who do not have extensive expertise in the tools and analyses available, the article would benefit from more detailed descriptions of some individual components. Specifically, the following: 1. Alignment: The rationale for use of HISAT2 is clearly described. The time needed for this step should be discussed and, if possible, it would be helpful to provide an alternate local tool that could be used if users are unable to maintain consistent internet connectivity over the time needed for alignment completion. 2. RNA-Seq: A critical piece of RNA-Seq analyses are the statistical tools used to define differential expression. Some brief discussion of important cut-offs would be helpful. 3. Most tools incorporated into the package are described in 1-2 sentences. This should be consistent throughout, including for HTSeq, DESeq2.\nThe tool is really quite useful and I believe will be very valuable to novice investigators and students learning the pipeline of RNA-Seq or ChIP-Seq. Given that, it would be very helpful to provide more, not less, explanation of some of the basic tools such that the educational goal of this package will be served.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-628
https://f1000research.com/articles/9-571/v1	08 Jun 20	{ "type": "Brief Report", "title": "Workplace exposure to carbon dioxide during routine laparoscopy – is it safe?", "authors": [ "Malin af Petersens", "Fritiof Andersson Fenger-Krog", "Jan G. Jakobsson", "Malin af Petersens", "Fritiof Andersson Fenger-Krog" ], "abstract": "Background: Minimally invasive surgeries have increased dramatically during the last decades. Carbon dioxide (CO2) is the gas used for insufflation during laparoscopies, creating space and visibility. The CO2 leaks into ambient air through ports where instruments are inserted. If the CO2 reaches a certain concentration it affects personnel health. There are national occupational exposure limits (OEL) for CO2, including a level limit value (LLV) of 5000 ppm. We are not aware of any previous studies addressing occupational exposure to CO2 during laparoscopies. The aim of this study was to assess the compliance to national OELs for CO2 during laparoscopies. Methods: A gas detector was placed in the breathing zone of personnel in the operating theatre. The detector measured CO2 concentrations every tenth minute during laparoscopies in three locations. Results: During 27 laparoscopies, the measured CO2 reached a maximum concentration of 1100 ppm, less than one fourth of the LLV. Median CO2 concentration was 700 ppm. Conclusion: Results show that the occupational exposure to CO2 during laparoscopies is well below set OELs. Our findings support personnel safety associated with routine use of CO2 during laparoscopies.", "keywords": [ "Ambient air", "Carbon dioxide", "Laparoscopy", "Minimally invasive surgery", "Occupational exposure", "Work place exposure" ], "content": "Introduction\n\nMinimally invasive surgical techniques aim to achieve surgical therapeutic goals with minimal trauma1. Minimal invasive surgery (MIS) has increased dramatically and is today well-established for huge numbers of procedures. During all forms of MISs, e.g. classic laparoscopy, gas insufflation is the most commonly used technique to create enough space to allow surgery2. The preferred, most commonly used, gas for insufflation is carbon dioxide (CO2)3. Characteristics of a perfect gas for insufflation include being colorless, incombustible, easily soluble in blood, non-toxic, inexpensive and easily removed from the body. CO2 is the gas that best matches these characteristics. To establish a gaseous cushion, an insufflator is used to pump CO2 into the abdominal cavity or other surgical field. CO2 will leak into the ambient air from the cavity where the instruments later are inserted, hence the CO2 concentration in ambient air in the operating theatre may be elevated and thus potentially cause personnel health concern.\n\nSymptoms of acute hypercapnia include flushed skin, headaches and sweating. Higher CO2 concentrations in ambient air may also cause anxiety and dizziness. High levels may further cause confusion and shortness of breath and eventually dimmed sight, tremor, unconsciousness or even death4,5. The individual response to elevated CO2 concentrations in ambient air varies depending on the time of exposure and CO2 concentration4.\n\nRecent work suggests that chronic exposure to higher concentration of CO2 may cause negative health effects, potentially having effects on fertility6,7.\n\nTo prevent ill health, many countries have provisions regarding the highest acceptable concentrations of air pollutants at workplaces. The highest acceptable average concentration of an air pollutant in workplace air, calculated as time weighted average is known as the occupational exposure limit (OEL). There are two often used OEL values, the level limit value (LLV) and the short-term exposure limit (STEL). LLV is the OEL value for exposure during a working day, normally eight hours. STEL is the OEL value for a reference period of 15 minutes exposure8. The Swedish OELs are based on the EU’s binding OELs, which includes an LLV of 5000 ppm for CO29. The US National Institute for Occupational Safety and Health (NIOSH) has a similar level10. The LLV is binding, unlike the STEL for CO2 at 10000 ppm which is the recommended highest value8.\n\nPersonnel workplace safety is of huge importance and OELs has been set to secure good working condition, securing personnel health. The workplace CO2 concentrations may constitute a safety risk. We are not aware of previous studies explicitly addressing the adherence to OELs in operating theatres (OT) during routine use of CO2 for insufflation during laparoscopies.\n\nThe aim of this study was to assess the occupational exposure to CO2 in OTs during laparoscopies to verify the compliance to set national (Swedish) OELs.\n\n\nMethods\n\nThis was an explorative, non-interventional study of CO2 concentrations in ambient air during laparoscopies conducted at Danderyd Hospital during October 2019. The CO2 concentration was measured at three locations: old general surgery ward (OGSW; n=2), new general surgery ward (NGSW; n=1) and day surgery unit (DSU; n=1). The ventilation differed between the locations. In the two older OTs, the air volume flow was 710 l/s (liter/second) and 650 l/s. The air volume flow in the new OT was 2160 l/s during surgeries and 100 l/s during basic ventilation. In the DSU, the air volume flow was between 720 and 2160 l/s.\n\nThe laparoscopies included in this study were aggregated into three groups based on the type of surgery: cholecystectomies, hernia repairs and intestinal surgeries. Five groups (A-E) were created depending on the type of surgery and the location: cholecystectomy DSU (A), cholecystectomy NGSW (B), hernia repair NGSW (C), intestinal surgery OGSW (D), intestinal surgery NGSW (E) (Table 1).\n\nSurgery duration is presented as mean and standard deviation. Intra-abdominal pressure, number of people in the operating theatre and carbon dioxide concentration are presented as median and range. Number of people in operating theater excludes the patient.\n\nIAP intra-abdominal pressure, CO2 carbon dioxide, OT operating theatre, OGSW old general surgery ward, NGSW new general surgery ward, DSU day surgery unit, ppm parts per million\n\nA gas detector (TM Dräger X-am 5600, Germany) was used to record point measurements of CO2 concentrations during the surgeries. This detector has a measurement range of 0–5%, hence the full-scale value is 5% (50000 ppm). The manufacturer of the sensor states an accuracy of ±800 ppm if the CO2 concentration is 25000 ppm or less. The resolution of the sensor is 100 ppm, thus the scale is divided into 500 equal divisions (400 ppm, 500 ppm, 600 ppm etc.). The exact value displayed depends on the span value set during calibration.\n\nThe primary outcome of the study was the concentration of CO2 in ambient air during MIS. The gas detector was positioned at a height of 153 cm in the OT at the IV pole on the right side of the patient. The CO2 concentration was noted manually every tenth minute starting on the hour. Observations were collected from the point measurement before the start of surgery until the point measurement after the end of surgery. Start and end of surgery were determined by start- and endpoint as noted by the nurse anesthetist in the medical record.\n\nOne of the secondary outcomes was the CO2 concentration at different heights in the OT. During a laparoscopic hernia repair in the NGSW (group C) the gas detector was placed as previously described. During the first two observations (20 minutes) the detector was placed at a height of 153 cm and was then moved to 105 cm for the next two observations. The following two observations were collected at a height of 15 cm and the detector was then moved back to a height of 153 cm. Observations were collected by changing the height as described every 20 minutes until the end of surgery.\n\nThe other secondary outcome of the study was the maximum concentration of CO2 when gas is allowed to freely enter the OT by disconnecting the insufflation tube from the insufflator. This was conducted in an OT in the NGSW. The gas detector was placed as described previously. The insufflator was set at high flow and the intra-abdominal pressure (IAP) was set to 14 mmHg. The central gas was turned on for five minutes and the highest observed CO2 concentration, the CO2 concentrations at the beginning and end of the attempt were noted manually. The attempt was conducted three times, the first time with basic ventilation and the third time with operation ventilation.\n\nData is presented as mean, standard deviation, or median and range as applicable. For descriptive analysis and Kruskal-Wallis test, Microsoft Excel (version 16.32, 2019) was used. Descriptive analysis was performed to show measured CO2 concentrations, characteristics and possible confounding factors among the five groups. Kruskal-Wallis test was used to analyze if there was a significant difference among the three different heights where the CO2 concentration was measured. A P value <0.05 was considered statistically significant. To illustrate CO2 concentrations in different groups, box plots were created using R programming language (version 3.6.1, 2019-07-05).\n\nThis was an explorative non-interventional air quality study and the CO2 concentrations in OTs were monitored only to ensure personnel health. Personnel safety and health is of great importance and this study was significant to assess the personnel safety related to CO2 in OTs. No patient or personnel data were collected. There was no need for ethical approval. The Head of the Department of Anesthesia and Intensive Care as well as the Head of the Department of Surgery approved the study.\n\n\nResults\n\nThe CO2 concentration was measured during 20 surgeries where a total of 210 observations were collected with the gas detector. The number of observations in each group ranged from 30 to 57. Possible confounding factors such as the number of people in the OT and IAP showed little variation between groups (Table 1).\n\nDuring the surgeries, the measured CO2 concentration showed minor variation. Point measurements from one of the surgeries were selected to show an example of intraoperative variation of CO2 concentration measured with the gas detector (Figure 1). During this surgery, 65% of the measured CO2 concentrations were at 600 ppm and observations ranged from 600 to 1000 ppm. Variation of intraoperative CO2 concentration was occasionally coherent with emptying and insufflations of gas in the peritoneal cavity. All measured CO2 concentrations during this surgery were less than or equal to 20% of the LLV (Figure 1).\n\n(minutes on x-axis and CO2 concentration ppm on Y-axis.\n\nOut of all observations collected in groups A-E, none exceeded 1100 ppm (Figure 2). The CO2 concentration was 600 ppm or 700 ppm in 81% of the observations (Figure 2). Concentration of CO2 exceeding 700 ppm was seen in 12% of observations.\n\nCO2 concentration measured on x-axis, number of observations on y-axis.\n\nThe CO2 concentration during the surgeries was measured at 400–1100 ppm and never exceeded 22% of the LLV at 5000 ppm (Figure 3). Because of the scarce variation in all the groups, sporadic variations are frequently shown as outliers in the box plot.\n\nProcedures on x-axis, CO2 concentration on y-axis. A and B cholecystectomy; A Days surgical unit, B in News Surgical unit, C Hernia repair in new surgical unit, D and E Intestinal surgery; D in old surgical unit, E in new surgical unit.\n\nWhen the CO2 concentration was measured at different heights in the OT, results of Kruskal-Wallis test showed no significant difference between heights (χ2 = 1.371, p = 0.504).\n\nDuring the three attempts when CO2 was allowed to freely enter the OT for five minutes, the highest value measured was 2000 ppm. This value is a fifth of the STEL and less than half of the LLV (Figure 4).\n\nAttempt on x-axis and peak CO2 concentration ppm on y-axis.\n\n\nDiscussion\n\nThe primary aim of this study was to measure the concentration of CO2 in ambient air during laparoscopies to verify compliance to the set national OELs. During 20 laparoscopies in three different locations, the measured CO2 concentration did not exceed 1100 ppm, which is less than one fourth of the LLV and one ninth of the STEL. Furthermore, when gas was allowed to freely enter the OT for five minutes, mimicking an accidental user error, the measured CO2 reached a maximum concentration of two fifths of the LLV. Thus, all measured CO2 concentrations were well below set OELs, hence the findings are reassuring.\n\nPersonnel health is of great importance and it is the obligation of all healthcare organizations to secure proper workplace safety including ambient air quality. However, we are not aware of previous studies reporting CO2 concentrations during laparoscopies. Air quality indices including CO2 concentrations have nonetheless recently been studied during other types of MIS in a gastrointestinal endoscopy unit11. Similar to our findings, the CO2 concentration in the procedural area was well below set OELs with a median concentration of 593.1 ppm (range 400–1645.9 ppm).\n\nIn our study we measured CO2 as a direct pollutant. Conversely, like the study in the gastrointestinal endoscopy unit, the CO2 concentration in other hospital environments has previously been studied as an indicator of air quality rather than as a direct pollutant12–14. CO2 as an indicator of air quality has also been studied in other environments such as classrooms15,16. Overall, results in these studies of hospital environments and classrooms show concentrations considerably lower than the set OELs.\n\nThe results must be put in perspective of some limitations. The gas detector can only assume certain fixed values, thus small changes in concentration were not detected. Nevertheless, it was important in this study to distinguish between measured CO2 concentrations and national OELs and detection of smaller changes in CO2 concentration were not needed for this purpose.\n\nPossible confounding factors include the number of trocars, the ventilation systems, conversion to open surgery, the number of people in the OT and the IAP. Nevertheless, the CO2 concentration varied little among groups and thus these factors did not seem to have a considerable impact on the concentrations in this study.\n\nData was handled manually due to the inability to store data and the inability to transfer data to a computer in the gas detector. Concentrations at different heights were only measured during one surgery and differences between heights can therefore not be established. Moreover, concentrations were only measured at one hospital and during rather few surgeries and results may not be generalizable to other hospitals, although all concentrations in the different locations were very low. CO2 concentrations were only studied at adult surgery departments and because of differences in abdominal cavity volume, amount of CO2 used and other unrecognized factors, results may be less transferable to children.\n\nIn addition, we looked solely at laparoscopies in this study. Other MIS techniques, such as colonoscopies and robotic surgeries, can be performed in other environments or have a longer duration which might affect CO2 concentrations.\n\n\nConclusion\n\nThis study shows that the occupational exposure to CO2 in OTs during laparoscopies is well below set OELs. Our findings also suggest that CO2 concentrations are distributed the same way at different heights in the OT. Even when gas is freely entering the OT for five minutes, mimicking an accidental user error, the CO2 concentrations are well below OELs, hence the results are reassuring. Our findings support personnel safety associated with routine use of CO2 for insufflation during laparoscopy.\n\n\nData availability\n\nOpen Science Framework: CO2 measurement, https://doi.org/10.17605/OSF.IO/6S5UQ17.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWilliams NS, O’Connell PR, McCaskie AW: Chapter 8, Principles of laparoscopic and robotic surgeries. Bailey & Love’s short practice of surgery. 27th edition. Boca Raton, FL: CRC Press; 2017; 104–18.\n\nGurusamy KS, Koti R, Davidson BR: Abdominal lift for laparoscopic cholecystectomy. Cochrane Database Syst Rev. 2013; (8): CD006574. PubMed Abstract \| Publisher Full Text\n\nYu T, Cheng Y, Wang X, et al.: Gases for establishing pneumoperitoneum during laparoscopic abdominal surgery. Cochrane Database Syst Rev. 2017; 6(6): CD009569. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUSDA FSIS Environmental Safety and Health Group: Carbon Dioxide Health Hazard Information Sheet. Maryland: USDA FSIS. [cited 2019 Dec 9]. Reference Source\n\nCDC - The National Institute for Occupational Safety and Health (NIOSH): Pocket Guide to Chemical Hazards - Carbon dioxide. Washington D.C.: CDC - NIOSH. [cited 2019 Dec 9]. Reference Source\n\nSharabi K, Hurwitz A, Simon AJ, et al.: Elevated CO2 levels affect development, motility, and fertility and extend life span in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2009; 106(10): 4024–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHelenius IT, Krupinski T, Turnbull DW, et al.: Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection. Proc Natl Acad Sci USA. 2009; 106(44): 18710–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwedish Work Environment Authority; Arbetsmiljöverket: AFS 2018: 1 - Hygieniska gränsvärden. Stockholm: Arbetsmiljöverket; 2018. Reference Source\n\nSwedish Work Environment Authority: Arbetsmiljöverket. EUs indikativa och bindande gränsvärden. Stockholm: Arbetsmiljöverket; 2018. Reference Source\n\nhttps://www.cdc.gov/niosh/npg/npgd0103.html\n\nBang CS, Lee K, Yang YJ, et al.: Ambient air pollution in gastrointestinal endoscopy unit. Surg Endosc. 2019. PubMed Abstract \| Publisher Full Text\n\nTang CS, Wan GH: Air quality monitoring of the post-operative recovery room and locations surrounding operating theaters in a medical center in Taiwan. PLoS One. 2013; 8(4): e61093. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChung FF, Lin HL, Liu HE, et al.: Aerosol distribution during open suctioning and long-term surveillance of air quality in a respiratory care center within a medical center. Respir Care. 2015; 60(1): 30–7. PubMed Abstract \| Publisher Full Text\n\nLee ST, Liang CC, Chien TY, et al.: Effect of ventilation rate on air cleanliness and energy consumption in operation rooms at rest. Environ Monit Assess. 2018; 190(3): 178. PubMed Abstract \| Publisher Full Text\n\nMuscatiello N, McCarthy A, Kielb C, et al.: Classroom conditions and CO2 concentrations and teacher health symptom reporting in 10 New York State Schools. Indoor Air. 2015; 25(2): 157–67. PubMed Abstract \| Publisher Full Text\n\nGaihre S, Semple S, Miller J, et al.: Classroom carbon dioxide concentration, school attendance, and educational attainment. J Sch Health. 2014; 84(9): 569–74. PubMed Abstract \| Publisher Full Text\n\njakobsson J: CO2 measurement. 2020. http://www.doi.org/10.17605/OSF.IO/6S5UQ" }	[ { "id": "65631", "date": "13 Jul 2020", "name": "Colin F. Royse", "expertise": [ "Reviewer Expertise Anesthesiology", "Echocardiography", "cardiac surgery outcomes", "Quality of recovery after surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a safety study to measure the Carbon Dioxide concentration that could be inhaled by staff in the operating room during laparoscopic surgery.\n\nThey chose three different locations, each of which have different ventilation and air volume turnover methodologies. They conducted serial assessments over time for multiple surgeries. They also conducted a brief experiment where they ran CO2 freely into the operating room.\n\nThey found that there is little variance between each of the operating locations or types of surgery. Importantly, all will well below the acceptable limits (level limit value of 5000 PPM). It was interesting, that even the range of CO2 was relatively small, with the maximum amount being around 1100 PPM. When CO2 is freely introduced into the air, the values were higher-approximating 2000 PPM, which was still well below the level limit value that is mandated in Europe.\n\nThis paper adds to our literature of occupational safety. The use of different theatre locations (old and new) with different air volume turnover rates allows this knowledge to be transferred to other locations and hospitals. It is highly unlikely, even with old theatres, that laparoscopic surgery will produce high levels of carbon dioxide, sufficient to affect health care workers.\n\nThe conclusion is consistent with the data, and supports that even an accidental user error, where carbon dioxide is freely running into the theatre, is still unlikely to cause a health issue for workers-this is very encouraging data.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "70136", "date": "11 Sep 2020", "name": "Jakob Walldén", "expertise": [ "Reviewer Expertise Anesthesiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study evaluating carbon dioxide levels during laparoscopies in operating theaters in relation to occupational exposure limits. The study design and results are clear and easy to understand and the conclusions are sound in relation to the results- carbon dioxide levels are not a health related issue.\nHowever there are a few minor issues I would like to address to improve the manuscript:\nThe levels of carbon dioxide detected are low. What are normal background levels? Outdoor, indoor, operating rooms with high ventilation? Please relate your findings and include in the discussion.\n\nHow is CO2 levels affected by the the high ventilation? Is it even possible to detect high levels, are CO2 washed out quickly? Consider including the issue in discussion.\n\nThe accuracy of the method is stated as +/- 800 ppm. This means that there is a possibility that the levels detected at 800 ppm is in the range 0-1600 ppm. The rise in CO2 during flushing shows that sensor work. However, if related to the primary aim, even if the real CO2 levels are in the higher interval, it is far below OEL. Please include briefly in discussion.\n\nPlease use L/min instead of l/min.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5932", "date": "21 Sep 2020", "name": "Jan Jakobsson", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Referee. Thank you for important comments. The average ambient air CO2 has passed 400 ppm and is today higher than ever before. Operating rooms are indeed well ventilated. Merely a rather limited increase was seen in the operating room CO2 concentration when a 5 minute free release of CO2 accident, user error, was mimicked The instrument used was calibrated but the accuracy must of course be acknowledged. We have now revised and incorporated these important aspects into the discussion." } ] } ]	1	https://f1000research.com/articles/9-571
https://f1000research.com/articles/9-645/v1	25 Jun 20	{ "type": "Case Report", "title": "Case Report: Multifocal non-invasive follicular thyroid neoplasm with papillary-like nuclear features presenting in a female child", "authors": [ "Asmaa Gaber Abdou", "Hayam Aiad", "Nancy Asaad", "Hayam Aiad", "Nancy Asaad" ], "abstract": "Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) was introduced as a separate entity by the World Health Organization in 2017 with strict inclusion and exclusion criteria. Most NIFTP cases have been reported in adults and few cases have been diagnosed in children. Here, we present a classic case of NIFTP affecting a 10-year old female child. We also review previous reports of NIFTP in children regarding size, focality, nodal metastasis, recurrence, type of operation and follow-up data. The present report adds a new case of NIFTP in the paediatric age group characterized by multifocality, absence of nodal invasion and indolent course until last follow-up, recommending less aggressive management.", "keywords": [ "NIFTP", "children", "multifocality" ], "content": "Introduction\n\nGenerally, the diagnosis of papillary thyroid carcinoma (PTC) has increased over the past several decades1, partly due to increased recognition of the follicular variant of PTC2. The subjectivity in diagnosis of this variant and the indolent behaviour of encapsulated or non-invasive forms, led to revision and follow-up of a large number of these cases by international multidisciplinary collaborative group3,4. Consequently, the encapsulated variant of PTC was reclassified as non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), which had strict inclusion and exclusion criteria for this diagnosis. The term NIFTP was then introduced as a separate entity by the World Health Organization in 2017, with a category of follicular tumour of uncertain malignant potential and well-differentiated tumour of uncertain malignant potential5. The majority of NIFTP reports have been in adults. Here, we present a classic case of NIFTP affecting a 10-year old female child.\n\n\nCase report\n\nA female patient of 10 years presented to our department with an enlarged thyroid that had been observed by her mother. No previous relevant family history was recorded.\n\nUltrasound revealed two suspicious nodules on the right side of the thyroid lobe. No pathological lymph node enlargement was reported. Ultrasound guided fine needle aspiration cytology was performed and the results showed sheets of follicular epithelial cells, some were elongated with occasional nuclear grooves and inclusions (Figure 1A). This was diagnosed as atypical thyroid lesion indefinite for malignancy (THY3a).\n\n(A) Cytologic features of fine needle aspiration cytology showing cohesive sheet of follicular epithelial cells, including some which were rounded and others that were elongated with occasional grooved nuclei (hematoxylin and eosin, mag. ×600). (B) Gross picture of affected right lobe after total thyroidectomy showing two well circumscribed whitish nodules. (C) Histopathological examination of nodule of resected thyroid revealing a capsulated nodule formed of microfollicles lined by follicular epithelial cells, which had enlarged pale crowded nuclei together with nuclear grooves and inclusions (nuclear features of papillary thyroid carcinoma)(hematoxylin and eosin, mag. ×400).\n\nThe patient was submitted for total thyroidectomy within one month from her first presentation. On resection, the right thyroid lobe measured 5.5 × 3.5 × 3 cm with two well-defined, firm, grayish white nodules. One nodule measured 2 × 1.5 cm and the other measured 1.5 × 1.5 cm (Figure 1B). The left lobe and isthmus measured 4.5 × 3 cm and 1 × 0.5 cm, respectively.\n\nHistological examination of the two nodules resected from the right thyroid lobe revealed well-circumscribed capsulated nodules formed of microfollicles, lined by follicular epithelial cells with wide-spread nuclear features of papillary thyroid carcinoma (Figure 1C). There was no evidence of capsular or vascular invasion, true papillae, trabeculae or solid arrangement. The patient did not receive any specific medications before surgery and she was followed up for 12 months with no evidence of recurrence or nodal involvement.\n\n\nDiscussion\n\nMost NIFTP cases have been previously reported in adults and data concerning this diagnosis in children is scarce; only 21 cases in children have been reported in the English literature within the last two years (Table 1)6–10. Preoperative diagnosis of our case was based on ultrasound data and the cytology was not obviously malignant. NIFTP cytology is commonly interpreted as indeterminate (categories III and IV according to Bethesda system), followed by suspicion for malignancy11. Although the above findings would suggest lobectomy, our patient was submitted for total thyroidectomy and as has been done in previously reported cases6,7,9,10.\n\nF:M, female to male ratio, NA: not available\n\nThe current report demonstrated a classic case of NIFTP affecting a young female child, agreeing with previous reports that there are more cases in women than men (Table 1). Although not common, multifocality has been reported previously for NIFTP in adults12 and in children9. The size of NIFTP lesion is usually small, rarely exceeding 2 cm in diameter (Table 1).\n\nMore aggressive therapy is recommended for PTC in childhood and adolescence13 but the indolent behaviour reported for NIFTP necessitates less aggressive management in children, as well as adults. Therefore, completion lobectomy is not recommended for postoperative cases diagnosed as NIFTP8. NIFTP in children has a similar outcome as cases reported in adults, suggesting that paediatric NIFTP behaves indolently, as evidenced by the absence of local recurrence and nodal metastasis6.\n\nThe present report adds a new case of NIFTP in the paediatric age group characterized by multifocality, absence of nodal invasion and indolent course - until last follow-up, recommending less aggressive management of this disease.\n\n\nConsent\n\nWritten informed consent was obtained from the patient's father for the publication of this case report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nDavies L, Welch HG: Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA. 2006; 295(18): 2164–2167. PubMed Abstract \| Publisher Full Text\n\nJung CK, Little MP, Lubin JH, et al.: The increase in thyroid cancer incidence during the last four decades is accompanied by a high frequency of BRAF mutations and a sharp increase in RAS mutations. J Clin Endocrinol Metab. 2014; 99(2): E276–E285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNikiforov YE, Seethala RR, Tallini G, et al.: Nomenclature revision for encapsulated follicular variant of papillary thyroid carcinoma: a paradigm shift to reduce overtreatment of indolent tumors. JAMA Oncol. 2016; 2(8): 1023–1029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson LD: Ninety-four cases of encapsulated follicular variant of papillary thyroid carcinoma: a name change to noninvasive follicular thyroid neoplasm with papillary-like nuclear features would help prevent overtreatment. Mod Pathol. 2016; 29(7): 698–707. PubMed Abstract \| Publisher Full Text\n\nLloyd RV, Osamura RY, Klöppel J, et al.: WHO Classification of Tumours of Endocrine Organs. (4th edition), IARC: Lyon, France. 2017. Reference Source\n\nWang H, Correa H, Sanders M, et al.: Noninvasive Follicular Thyroid Neoplasm With Papillary-Like Nuclear Features in Children: An Institutional Experience and Literature Review. Pediatr Dev Pathol. 2020; 23(2): 121–126. PubMed Abstract \| Publisher Full Text\n\nRosario PW, Mourão GF: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) inchildren and adolescents. Endocrine. 2018; 61(3): 542–544. Publisher Full Text\n\nRossi ED, Mehrotra S, Kilic AI, et al.: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features in the pediatric age group. Cancer Cytopathol. 2018; 126(1): 27–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMariani RA, Kadakia R, Arva NC: Noninvasive encapsulated follicular variant of papillary thyroid carcinoma: Should it also be reclassified in children? Pediatr Blood Cancer. 2018; 65(6): e26966. PubMed Abstract \| Publisher Full Text\n\nSamuels SL, Surrey LF, Hawkes CP, et al.: Characteristics of Follicular Variant Papillary Thyroid Carcinoma in a Pediatric Cohort. J Clin Endocrinol Metab. 2018; 103(4): 1639–1648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosario PW, Mourão GF, Nunes MB, et al.: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Endocr Relat Cancer. 2016; 23(12): 893–897. PubMed Abstract \| Publisher Full Text\n\nFonseca D, Bhuyan S, Murthy SS, et al.: Noninvasive follicular thyroid neoplasm with papillary-like nuclear features: A distinct clinicopathologic entity. Indian J Pathol Microbiol. 2018; 61(3): 380–382. PubMed Abstract \| Publisher Full Text\n\nFrancis GL, Waguespack SG, Bauer AJ, et al.: Management Guidelines for Children with Thyroid nodules and Differentiated Thyroid Cancer. Thyroid. 2015; 25(7): 716–59. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70314", "date": "08 Sep 2020", "name": "Hanan Alshenawy", "expertise": [ "Reviewer Expertise histopathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a case report about a case of NIFTP in thyroid.\nThe case is well clinically presented with full clinical data.\n\nThe procedure as FNAC is also presented well with clear figure.\n\nHistopathology is shortly presented with clear good figure.\n\nThe discussion should be in more details.\n\nIs there any role for immunohistochemistry?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-645
https://f1000research.com/articles/9-174/v1	10 Mar 20	{ "type": "Data Note", "title": "Ribosome profiling of HEK293T cells overexpressing codon optimized coagulation factor IX", "authors": [ "Aikaterini Alexaki", "Jacob Kames", "Gaya K. Hettiarachchi", "John C. Athey", "Upendra K. Katneni", "Ryan C. Hunt", "Nobuko Hamasaki-Katagiri", "David D. Holcomb", "Michael DiCuccio", "Haim Bar", "Anton A. Komar", "Chava Kimchi-Sarfaty", "Aikaterini Alexaki", "Jacob Kames", "Gaya K. Hettiarachchi", "John C. Athey", "Upendra K. Katneni", "Ryan C. Hunt", "Nobuko Hamasaki-Katagiri", "David D. Holcomb", "Michael DiCuccio", "Haim Bar", "Anton A. Komar" ], "abstract": "Ribosome profiling provides the opportunity to evaluate translation kinetics at codon level resolution. Here, we describe ribosome profiling data, generated from two HEK293T cell lines. The ribosome profiling data are composed of Ribo-seq (mRNA sequencing data from ribosome protected fragments) and RNA-seq data (total RNA sequencing). The two HEK293T cell lines each express a version of the F9 gene, both of which are translated into identical proteins in terms of their amino acid sequences. However, these F9 genes vary drastically in their codon usage and predicted mRNA structure. We also provide the pipeline that we used to analyze the data. Further analyzing this dataset holds great potential as it can be used i) to unveil insights into the composition and regulation of the transcriptome, ii) for comparison with other ribosome profiling datasets, iii) to measure the rate of protein synthesis across the proteome and identify differences in elongation rates, iv) to discover previously unidentified translation of peptides, v) to explore the effects of codon usage or codon context in translational kinetics and vi) to investigate cotranslational folding. Importantly, a unique feature of this dataset, compared to other available ribosome profiling data, is the presence of the F9 gene in two very distinct coding sequences.", "keywords": [ "Ribosome profiling", "codon optimization", "Ribo-seq", "RNA-seq", "translation kinetics", "codon usage", "codon pair usage", "protein therapeutics" ], "content": "Introduction\n\nThe ribosome profiling (footprinting) technique has only been around for a decade1 but has already contributed tremendously to our understanding of translation efficiency and kinetics. Initially developed to systematically monitor protein translation in yeast1, it has since been adapted to work in a range of organisms2,3 and to tackle a variety of questions. Ribosome profiling data typically consist of a set of sequences of ribosome protected fragments (RPF), designated as Ribo-seq data, which is accompanied by sequences from total RNA (RNA-seq). The availability of Ribo-seq and RNA-seq data from the same sample provides a treasure trove of information, enabling to quantitatively study the translation efficiency, rate and kinetics of every mRNA sequence in the pool4. Given that these sequences cover the entire transcriptome, and also include tRNA and rRNA, typically only a fraction of the data is presented and constructively used, within its initial publication. Further analyses, and comparisons of different ribosome profiling datasets can yield significant new information.\n\nWe recently conducted a ribosome profiling study to examine the translation kinetics of blood coagulation factor IX5, a protein with great pharmaceutical interest. Two human embryonic kidney 293T (HEK293T) cell lines were lentivirally transduced, one with the wild type (WT) version of the gene and one with a codon optimized (CO) F95. Codon optimization is a widely used technique that aims at increasing the protein expression levels by replacing multiple codons within a coding sequence with synonymous ones. In doing so, the amino acid sequence of the protein remains unaltered, therefore these changes were assumed to be inconsequential for the structure and function of the protein. However, this is not always true; through our ribosome profiling study, we described that these synonymous changes drastically altered translational kinetics and led to protein conformational changes5.\n\nThe translational kinetics of the F9 variants, along with the control genes, GAPDH and ACTB, were analyzed in detail in the original publication5. Similarly, any other gene of interest can be investigated in this dataset in terms of their rate of synthesis and translational kinetics; genes in the entire transcriptome can be compared to each other. Since there are several other HEK293T ribosomal profiling datasets available, these could be used to examine the reproducibility of the results6. Furthermore, by looking into ribosome profiling datasets from other cell types, such as other human cells7 and/or across species, it would be valuable to examine whether a given gene maintains the same translation kinetics or if there are significant differences that could reflect on the conformation of the protein. Clearly, since a rather large inter-experiment variation is expected, the accumulation of several ribosome profiling databases would be very useful for this type of analysis.\n\nInnovative computational approaches of analyzing ribosome profiling data have led to the identification of novel CDSs that lead to the production of previously unidentified peptides and variants of known proteins8. Such coding sequences may be found in what is typically designated as untranslated regions (UTRs) of the mRNA, particularly the 5’UTRs, and may originate from non-AUG start sites9–11. However, such approaches have not been applied yet to this dataset and it would be intriguing to see if they could lead to new discoveries12. Importantly, since the genome of the HEK293T used to generate this dataset contains part of lentiviral vector and the cytomegalovirus (CMV) promoter to drive expression of F9, it would be interesting to examine whether any part of this sequence is actively translated. These analyses may be particularly insightful in studies of immunogenicity.\n\nFurther analysis of this dataset will help elucidate the effect of codon usage, codon context and possible other factors in translational kinetics. By looking at the global rate in which each codon is translated, and examining adjacent sequences on a transcriptome level, it may be possible to predict translational kinetics of recombinant genes and to make inferences on whether cotranslational folding may be affected. This may be particularly important in gene therapy applications where the cell type expressing the gene of interest may be different from the naturally expressing cells, e.g. expression of coagulation factor VIII from hepatocytes in gene therapy. A recent study in yeast13 showed promising results in this direction; however, increasing availability of ribosome profiling datasets from other cell types will allow further comparisons. A unique feature of this dataset that may be pivotal in these types of studies is the presence of F9 in two genes with very different codon usage.\n\n\nMaterials and methods\n\nWT (RefSeq NM_000133.3) and CO (accessible at https://github.com/FDA/Ribosome-Profiling F9_opt1_construct_100bpUTRs.fasta)14 F9 ORFs were sub-cloned into pcDNA3.1/V5-His-TOPO (Invitrogen/Life Technologies) according to manufacturer’s instructions to generate pcDNA3.1-F9-V5-His plasmids. Each fusion construct (WTF9-V5-His and COF9-V5-His) was sub-cloned into a lentiviral vector pTK642 (gift from Dr. Kafri, University of North Carolina at Chapel Hill) at the Pacl/Sfil site.\n\nHuman embryonic kidney cells (HEK293T; ATCC) were grown in Dulbecco’s Modified Eagle Medium (Quality Biological, Inc) with 1% L-glutamine (Quality Biological), 1% penicillin- streptomycin (Hyclone) and 10% fetal bovine serum (Quality Biological) at 37°C in 5% CO2. HEK293T cells stably expressing WT or CO FIX were established following transduction with lentiviral vectors, as previously described15.\n\nAn equivalent number of cells were plated in T-flasks and supplemented with 10 ng/ml of Vitamin K3, one day prior to all experiments. The culture medium was replaced with Opti-MEM Reduced Serum Medium (Life Technologies) at approximately 80–90% cell confluency and cells were harvested after an additional 24 hours of incubation.\n\nRibosome profiling was conducted as described previously7 using the Illumina TruSeq Ribo Profile (Mammalian) Kit according to manufacturer’s instructions with modifications in harvest, RNA isolation/purification (isopropanol isolation used to improve the yield) and ribosome protected fragments size selection (~20–32 nt). During harvest, media was carefully removed, and cells were immediately flash-frozen. All equipment used from hence forth was pre-chilled. Cells were quickly scraped into 1 ml of ice-cold lysis buffer (5X Mammalian Polysome Buffer, 10% Triton-X100, 100 mM DTT, DNase I, Nuclease-free water) and homogenized on ice by passing through a 26G needle 10 times. Lysate was then spun at 4°C for 10 minutes at 20,000 × g. Supernatant was aliquoted into cryovials and immediately frozen in liquid nitrogen for future use. Samples were sequenced using Illumina HiSeq 2500.\n\nThe complete ribosome profiling pipeline analysis is described in Figure 1: Sequencing data were pre-processed and aligned as described by Alexaki et al.5 as well as the step by step guide found in the README.txt accessible on GitHub.\n\nColored arrows indicate steps that first require execution of utility script (blue and yellow) or require manual input by the user (red). Pipeline steps are represented as ovals (main step) or pentagons (validation / analysis step). Rectangles represent input / output data. UTR: untranslated region, CDS: coding sequence, RPF: ribosome protected fragments, RPKM: reads per kilobase of transcript per million mapped reads.\n\nRPF sequences were analyzed based on fragment length (Figure 2a), alignment distribution between coding sequences (CDS) and 5’- and 3’-UTRs (Figure 2b), triplet periodicity (Figure 3a) and reading frame (Figure 3b). RPF fragments 20–22 nt and 27–29 nt in length were used for further analysis with an A-site offset of 15 nucleotides from the 5’ end of the fragment. Pearson’s correlation was used to evaluate the reproducibility between samples using a common subset of moderately to highly expressed genes (reads per kilobase of transcript per million mapped reads, RPKMCDS ≥10) and considering reads with the ribosome A site annotated at least 20 nt downstream of the coding sequence start codon (Table 1).\n\n(a) Fragment size distribution of Ribo-seq and RNA-seq reads. The average of 6 experiments (3 WT and 3 CO F9) was plotted, s.e.m. are shown. (b) Distribution of Ribo-seq (left) and RNA-seq (right) reads in mRNA coding regions (CDS) and untranslated (5’UTR and 3’UTR) regions. The average of 6 experiments (3 WT and 3 CO F9) was plotted, s.e.m. are shown.\n\n(a) Profiles of the 5′ end positions of all 20–22 nt (top) and 27–29 nt (bottom) fragments relative to the start codon of their genes. The average of 6 experiments (3 WT and 3 CO F9) was plotted. (b) Positions of 20–22 nt and 27–29 nt fragments relative to the reading frame of the Ribo-seq (left) and RNA-seq (right) reads. The average of 6 experiments (3 WT and 3 CO F9) was plotted, s.e.m. are shown.\n\nRPKM of each gene in the Ribo-seq and RNA-seq datasets were calculated, considering reads with the ribosome A site annotated at least 20 nt downstream of the start codon. A comparison between each pair of experiment within the 3 replicates was performed\n\n\nDataset validation\n\nThe quality of the sequencing files is presented in Table 2. A pipeline was created to process the data (Figure 1). A number of steps allow for validation of the data and confirmation of their quality. The fragment length distributions for the whole genome were plotted, indicating that the vast majority of the fragments from the Ribo-seq data are either 20–21 or 27–28 nucleotides in length (Figure 2a), and as expected the RNA-seq data have a more flat distribution. The distribution of the Ribo-seq data in the UTRs and CDSs of the mRNA was also plotted. As expected, most of the sequences aligned within the CDSs (Figure 2b), while a smaller fraction of the RNA-seq data aligned with the CDSs. It should be noted that as the 3’ UTR, and 5’ UTR are typically shorter in length than the CDSs, it is not surprising that about 60% of the RNA-seq data align with the CDSs (Figure 2b). In addition, Ribo-seq data exhibit periodicity, characteristic of the RPFs (Figure 3a and Figure 3b), which is not observed in the RNA-seq data (Figure 3b). In accordance with previously published data16, we can infer that the 5′-most peaks in (Figure 3a) represent ribosomes with the start codon in the P site and the second codon in the A site, for both large and short fragments. Very tight correlation between the experiments, both for Ribo-seq and RNA-seq data, supports the reproducibility of the results (Table 1).\n\nSample ID, index, yield, number of clusters, percent Q30 and above and mean Q score for all sequencing experiments.\n\nSequencing for 3 replicates of RNA-seq and Ribo-seq of HEK293T cells stably expressing WT and CO FIX was performed by Eurofins Genomics (Louisville, KY, USA), resulting in 12 raw data files (3 WT and 3 CO F9 for both Ribo-seq and RNA-seq) in FASTQ format. Raw data are accessible at the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA591214. File names, SRA accession numbers (experiment and sample) and descriptions of data are summarized below in Table 3.\n\nFilenames, SRA experiment accession, SRA sample accession and brief description of the 12 Ribo-seq and RNA-seq FASTQ files. All data files are accessible from SRA BioProject accession PRJNA591214. Data files represent three replicates of each condition (WT F9 Ribo-seq, WT F9 RNA-seq, CO F9 Ribo-seq and CO F9 RNA-seq).\n\nThe custom ribosome profiling analysis pipeline has been deposited in GitHub in the FDA/Ribosome-Profiling directory14. Raw data files may be accessed from SRA and downloaded to the ‘./Ribosome_profiling/Raw_data/X/’ folder. In our descriptions and instructions, ‘X’ is replaced with ‘S12’, but the user may choose any designation they prefer. Detailed instructions for running the data analysis pipeline are included in the ‘README.txt’ file. Execution of the pipeline requires the following tools be installed on the user’s system: Python 2.7 (Python Software Foundation, Wilmington, DE, USA), GFF Utilities (Johns Hopkins University, Baltimore, MD, USA), Bowtie (Johns Hopkins University, Baltimore, MD, USA), TopHat (Johns Hopkins University, Baltimore, MD, USA), FASTX-Toolkit (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) and Samtools (Genome Research Limited, Hinxton, Cambridgeshire, UK).\n\n\nData availability\n\nNCBI BioProject: Ribosome profiling of HEK-293T cells stably expressing wild-type and codon-optimized coagulation factor IX. Accession number PRJNA591214; https://identifiers.org/NCBI/bioproject:PRJNA591214.\n\nThis project collates the raw data, held at the NCBI Sequence Read Archive (SRA).\n\n\nSoftware availability\n\nThe pipeline, including the code used to process the presented dataset and instructions for use, is available: https://github.com/FDA/Ribosome-Profiling\n\nArchived pipeline at time of publication: https://doi.org/10.5281/zenodo.367870914.\n\nLicense: MIT License.", "appendix": "Acknowledgements\n\nThe authors would like to thank Dr. Nicholas T. Ingolia and Dr. Estelle Russek-Cohen, CDER, FDA for very useful discussions.\n\n\nReferences\n\nIngolia NT, Ghaemmaghami S, Newman JR, et al.: Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; 324(5924): 218–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoolstenhulme CJ, Guydosh NR, Green R, et al.: High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep. 2015; 11(1): 13–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGobet C, Naef F: Ribosome profiling and dynamic regulation of translation in mammals. Curr Opin Genet Dev. 2017; 43: 120–127. PubMed Abstract \| Publisher Full Text\n\nIngolia NT: Ribosome Footprint Profiling of Translation throughout the Genome. Cell. 2016; 165(1): 22–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlexaki A, Hettiarachchi GK, Athey JC, et al.: Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies. Sci Rep. 2019; 9(1): 15449. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunt, R, Hettiarachchi G, Katneni U, et al.: A Single Synonymous Variant (c.354G>A [p.P118P]) in ADAMTS13 Confers Enhanced Specific Activity. Int J Mol Sci. 2019; 20(22): pii: E5734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHettiarachchi GK, Katneni UK, Hunt RC, et al.: Translational and transcriptional responses in human primary hepatocytes under hypoxia. Am J Physiol Gastrointest Liver Physiol. 2019; 316(6): G720–G734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFields AP, Rodriguez EH, Jovanovic M, et al.: A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation. Mol Cell. 2015; 60(5): 816–827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIngolia NT, Lareau LF, Weissman JS: Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes. Cell. 2011; 147(4): 789–802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung DJ, Guydosh NR, Zhang F, et al.: Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3'UTRs In Vivo. Cell. 2015; 162(4): 872–884. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpealman P, Naik AW, May GE, et al.: Conserved non-AUG uORFs Revealed by a Novel Regression Analysis of Ribosome Profiling Data. Genome Res. 2018; 28(2): 214–222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichel AM, Ahern AM, Donohue CA, et al.: GWIPS-viz as a Tool for Exploring Ribosome Profiling Evidence Supporting the Synthesis of Alternative Proteoforms. Proteomics. 2015; 15(14): 2410–2416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTunney R, McGlincy N, Graham ME, et al.: Accurate Design of Translational Output by a Neural Network Model of Ribosome Distribution. Nat Struct Mol Biol. 2018; 25(7): 577–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCBER-Source: FDA/Ribosome-Profiling: Ribosome profiling (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3678709\n\nSuwanmanee T, Hu G, Gui T, et al.: Integration-deficient lentiviral vectors expressing codon-optimized R338L human FIX restore normal hemostasis in Hemophilia B mice. Mol Ther. 2014; 22(3): 567–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLareau LF, Hite DH, Hogan GJ, et al.: Distinct Stages of the Translation Elongation Cycle Revealed by Sequencing Ribosome-Protected mRNA Fragments. eLife. 2014; 3: e01257. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "64172", "date": "05 Jun 2020", "name": "Stefano Biffo", "expertise": [ "Reviewer Expertise Translational control of gene expression" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report is certainly interesting and the possibility to access data relatively nice. However, in the present form there is not much advantage compared to a paper supplementary section and accession number. In the current pipeline one should install - as per explicit requirements - Python 2.7, Bowtie, Tophat, Samtools, which still requires bioinformatician work.\nSome programs are not used anymore. Python 2 has been disconnected since January. Tophat as mapping resource is not suggested even by the developers, since now there are better ones. In the present form the pipeline can clearly work. In order to facilitate the use of the pipeline the authors could have wrapped everything in a folder that could have been run by the readers more easily. The biological data seem very nice to me (Stefano Biffo with the help of a bioinformatician).\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "5640", "date": "26 Jun 2020", "name": "Chava Kimchi-Sarfaty", "role": "Author Response", "response": "Dr. Biffo,Thank you very much for the time you put into reviewing the article and for your suggestions. We would like to inform you that we are currently working on upgrading the pipeline as per your comments. We will be updating the code to Python 3, bundling individual Python scripts into a more easily executable shell script and replacing the current Tophat alignment with a more modern tool. We very much appreciate your comments and we will notify you when we have completed the upgrades.Chava Kimchi-Sarfaty and Jacob Kames" }, { "c_id": "5901", "date": "10 Sep 2020", "name": "Chava Kimchi-Sarfaty", "role": "Author Response", "response": "We appreciate the suggestion and have made changes accordingly. The Python scripts used in the code have been updated to Python 3.7. Furthermore, the alignment software used in the pipeline has been changed from TopHat to Hisat2. Finally, two bash scripts have been created to run the pipeline. The first, build_hisat_index.sh, carries out the commands to set up the reference index with the two F9 constructs used in this study. The second, RP_analysis_pipeline.sh, runs the remaining steps of the pipeline. Both are run from within the Ribosome_profiling directory and have the dataset defined within each script.We hope the reviewer agrees that the current changes make the software more user friendly and thus usable and accessible." } ] }, { "id": "65801", "date": "13 Jul 2020", "name": "Jordan Berg", "expertise": [ "Reviewer Expertise Ribosome profiling library creation and data analysis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe provided dataset provides the opportunity for more in-depth study of two versions of the F9 gene with identical protein amino acid sequence but different nucleotide coding sequences, thus allowing for an exploration of the consequences of differential codon usage. The dataset itself is of biological and pharmacological interest and offers a valuable data contribution. More specific comments and questions regarding key points of the data note are provided below.\nIntroduction:\nAt face value, this seems like a re-publishing of a dataset that has already been described in the literature (albeit with some additional detail and the pipeline). Were any data added or modified between the original publication and this data note? It is not clear why this dataset was not published with the original study describing these data (https://doi.org/10.1038/s41598-019-51984-2)1.\n\nIf part of the intended contribution of this data note is the pipeline, it would be helpful to at least provide a bash script that ties together the different scripts and flexibly accepts input FASTQ files. Currently, with all the hard-coding present, it makes it difficult to reuse these scripts, especially when input references or sequence files vary from those used when analyzing this particular dataset.\n\nMethods:\nA Pearson's correlation assumes the data are normally distributed. As sequencing data follows a negative binomial distribution, the use of a Spearman rank-order coefficient would be more appropriate. It might be helpful to clarify that this is for comparing biological replicates.\n\nIs there a reason the A-site offset is set at a strict 15 nt? Recent methods, such as those found in the RiboWaltz (https://doi.org/10.1371/journal.pcbi.1006169)2 package allow for a more optimized P-site offset determination that could probably be applied to A-site offset determination. On that note, is there a downstream reason you are interested in calculating the offset of the A-site? It looks like P-sites were used when calculating periodicity in the figures and scripts and thus it is not clear why the A-site offset is mentioned.\n\nThe pipeline uses deprecated software (TopHat has been deprecated for nearly 5 years now) and should be updated to use a more accurate splice-aware option (such as STAR, HISAT2). Figures might need to be updated as appropriate. Is the installation of dependencies included in a script, or does the user need to handle that? For example, BioPython and pysam are included as a dependency in the python scripts, but I don't see them listed in the manuscript.\n\nGitHub Documentation:\nTo aid in readability, some formatting updating (new lines in example code) would be helpful. Currently, there are several commands on the same line as a comment, making the commands and comments difficult to read. Using markdown syntax to display the code sections would aid in readability as well.\n\nExplicitly state the gene annotation used for the preparation of this dataset. Comprehensive, basic gene, etc? Same for the GFF3 file. It is not clear from the documentation itself what versions were used and would be nice from an archiving/reproducibility stand-point. The same goes for listing software versions used for processing the dataset as presented in the paper.\n\nCode:\nI could not get the trim-adapters.py script to work using the deposited data. I had empty trimmed files output and no error information was displayed to aid in debugging.\n\nAs far as functionality, all of the indexing scripts appeared to function, but I was unable to test past the trim-adapters.py script due to the issue mentioned above.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "5902", "date": "10 Sep 2020", "name": "Chava Kimchi-Sarfaty", "role": "Author Response", "response": "Thank you very much for your thorough review. Below we provide a point by point response. At face value, this seems like a re-publishing of a dataset that has already been described in the literature (albeit with some additional detail and the pipeline). Were any data added or modified between the original publication and this data note? It is not clear why this dataset was not published with the original study describing these data (https://doi.org/10.1038/s41598-019-51984-2)1. Regrettably, due to space restrictions we did not have the opportunity to publish the pipeline and the raw data with a detailed description in the original publication, which focused on the comparison of the wild type and codon optimized coagulation factor 9. We strongly believe that the raw ribosome profiling data can be of value to the scientific community and should be public with sufficient description to make it accessible. Similarly, we hope that the pipeline could be of use to researchers, and we note that despite the multitude of ribosome profiling papers that have been published so far, the software used in the analysis is not always readily accessible. If part of the intended contribution of this data note is the pipeline, it would be helpful to at least provide a bash script that ties together the different scripts and flexibly accepts input FASTQ files. Currently, with all the hard-coding present, it makes it difficult to reuse these scripts, especially when input references or sequence files vary from those used when analyzing this particular dataset. We appreciate the suggestion and have made changes accordingly. The pipeline has been modified to be more user friendly, and updates have been made as described in the response to Reviewer 1. Methods: A Pearson's correlation assumes the data are normally distributed. As sequencing data follows a negative binomial distribution, the use of a Spearman rank-order coefficient would be more appropriate. It might be helpful to clarify that this is for comparing biological replicates. Thank you for pointing this out. We have added a Spearman rank-order coefficient to Table 1 for each of the dataset comparisons. The text describing Table 1 has been updated. Is there a reason the A-site offset is set at a strict 15 nt? Recent methods, such as those found in the RiboWaltz (https://doi.org/10.1371/journal.pcbi.1006169)2 package allow for a more optimized P-site offset determination that could probably be applied to A-site offset determination. On that note, is there a downstream reason you are interested in calculating the offset of the A-site? It looks like P-sites were used when calculating periodicity in the figures and scripts and thus it is not clear why the A-site offset is mentioned. Thank you for pointing out the lack of clarity regarding the A- and P-site offset. The A-site offset at 15 nt is equivalent to a P-site offset at 12 nt. We made changes to consistently refer to the P-site offset. We agree that when the translation frame is undetermined a hard 12 nt P-site offset can lead to inaccuracies, and an optimized method for P-site determination is paramount. However, our analysis did not incorporate unannotated open reading frames (ORFs), non-conventional translation initiation sites and we did not attempt to reveal novel translated regions. As a result, the triplet periodicity in our dataset is unambiguous and the P-site offset can be determined at 12 nt. For ribosome-protected fragments that aligned in reading frame one or two, a +/- 1 offset was additionally added to properly annotate the codons within each site of the ribosome. The pipeline uses deprecated software (TopHat has been deprecated for nearly 5 years now) and should be updated to use a more accurate splice-aware option (such as STAR, HISAT2). Figures might need to be updated as appropriate. Is the installation of dependencies included in a script, or does the user need to handle that? For example, BioPython and pysam are included as a dependency in the python scripts, but I don't see them listed in the manuscript. We agree with your concern. When we started developing our pipeline, TopHat was the software of choice and we retained it to allow duplication of the data in our published paper [1]. We have now updated the pipeline to use HISAT2; all the figures have been updated accordingly. The readme file and manuscript have been updated to reflect the complete list of software dependencies and their versions. GitHub Documentation: To aid in readability, some formatting updating (new lines in example code) would be helpful. Currently, there are several commands on the same line as a comment, making the commands and comments difficult to read. Using markdown syntax to display the code sections would aid in readability as well. We thank you very much for pointing this out. We have removed all unnecessary commands and lines of code that had been commented out. We believe this will aid in readability of the code. Explicitly state the gene annotation used for the preparation of this dataset. Comprehensive, basic gene, etc? Same for the GFF3 file. It is not clear from the documentation itself what versions were used and would be nice from an archiving/reproducibility stand-point. The same goes for listing software versions used for processing the dataset as presented in the paper. We agree with your concern. We have updated the GitHub documentation to explicitly state which annotation and assembly versions were used in the in the analysis of this dataset. I could not get the trim-adapters.py script to work using the deposited data. I had empty trimmed files output and no error information was displayed to aid in debugging.As far as functionality, all of the indexing scripts appeared to function, but I was unable to test past the trim-adapters.py script due to the issue mentioned above. Thank you very much for pointing this out. This is an interesting result. We have re-run the analysis pipeline using the new bash scripts and code updated to Python 3.7, and the adapter trimming step ran correctly. However, we have added an error message in the Trim_adapters.py script to notify the user if an error occurs to aid in the debugging process. Are the protocols appropriate and is the work technically sound? Partly We have updated the alignment tool used in the pipeline to HISAT2 and the code used in the individual scripts to Python 3.7. We hope that these updates to the pipeline have improved the appropriateness of the protocol. Are sufficient details of methods and materials provided to allow replication by others? Partly We have bundled all scripts in the pipeline into two shell scripts and there is now a warning message in the Trim_adapters.py script that notifies the user when it does not execute properly. We have also updated the dependency list to accurately reflect all additional software required as well as versions that were tested. We hope that these updates have made replication of the work easier for the user. Are the datasets clearly presented in a useable and accessible format? Partly We hope that the updates to the pipeline described above have made the datasets more useable and accessible. 1 Alexaki, A. et al. Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies. Sci Rep 9, 15449, (2019)." } ] }, { "id": "65798", "date": "14 Jul 2020", "name": "Rafal Bartoszewski", "expertise": [ "Reviewer Expertise Protein trafficking", "protein expression and folding (Collawn)", "mRNA structure", "translational expression", "and silent polymorphisms (Bartoszewski)." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 by Dr. Kimchi-Sarfaty examines ribosome profiling to follow the translational kinetics of factor IX (F9) and is a follow up of a previous study of theirs (DOI: 10.1038/s41598-019-51984-2)1. They compare two HEK293T cell lines with one expressing a WT F9 gene and another with a codon-optimized F9 gene. In their previous study, they demonstrated the importance of this type of analysis by illustrating that these two cell lines had different translational kinetics and furthermore, different effects on protein conformation. In this study, they compared 3 replicates of the RNA-seq and Ribo-seq of the two cell lines. The correlations between the pairs of experiments seemed quite good and we believe this is an important type of study, however, we do have two concerns. First of all, the housekeeping control genes they utilize are GAPDH and ACTB, both of which are translated on free ribosomes in the cytosol. F9, however, is a secreted protein that is translated on ER ribosomes. Therefore, a rationale for the appropriateness of the two controls needs to be discussed. Secondly, they used a Box-Cox variance-stabilizing transformation for raw data followed by a Kolmogorov-Smirnov test which tests for probability distribution functions but is a less selective normality test in our view. Why not utilize Saphiro-Wilk (https://doi.org/10.1093/biomet/52.3-4.591)[ref-2] or Arednson-Darling tests (doi:10.2307/2281537)3? Some more detail here about the rationale for the statistical analyses is therefore warranted and whether additional controls and samples would have any effect on the outcomes of the studies should be discussed.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5903", "date": "10 Sep 2020", "name": "Chava Kimchi-Sarfaty", "role": "Author Response", "response": "Thank you very much for raising this interesting question. Although they are not secreted proteins as F9, ACTB and GAPDH were chosen as they have a relatively high level of expression and therefore generate sufficient sequencing reads to lead to a well resolved ribosome profile. Moreover, these two genes’ expression is stable and not changing due to the transfection or cell culture growth. It is not our intention to make any conclusions regarding the translation kinetics of secreted versus non-secreted proteins. Rather, the control genes are compared to themselves between experimental groups (cells expressing WT F9 and cells expressing CO F9). In this context, we believe that ACTB and GAPDH are appropriate to use as control genes. Regarding the statistical method, we want to point out that the Kolmogorov-Smirnov method is considered more conservative than the methods mentioned by the reviewer. Therefore, we feel that by using it we are able to detect a difference between two cumulative curves, then we should be more confident that the difference is real. Second, our aim is not to test for normality, but rather to compare two distributions. The main aims of the normalization step via the Box-Cox transformation is to stabilize the variance (of the originally skewed distribution), and to put the distributions on a comparable scale, so that we can compare the cumulative plots." } ] } ]	1	https://f1000research.com/articles/9-174
https://f1000research.com/articles/9-1158/v1	21 Sep 20	{ "type": "Research Article", "title": "The prevalence of secondhand smoke exposure and related factors among schoolchildren in Northeast Thailand", "authors": [ "Nirun Intarut", "Piyalak Pukdeesamai", "Piyalak Pukdeesamai" ], "abstract": "Background: The prevalence of tobacco consumption in Thailand has gradually declined; however, the prevalence of exposure to secondhand smoke (SHS) is still high. The objective of this study is to estimate the prevalence of SHS exposure and examine the association between exposure to SHS and depressive symptoms among schoolchildren, and test for moderation by the number of smokers in household. Methods: We conducted a cross-sectional study of 1105 schoolchildren. Socioeconomics factors, depressive symptoms and exposure to SHS variables were collected. We used the chi-square test for testing the factors associated to SHS exposure. In addition, we used the Mantel Haenszel test for testing interaction effect of depression to SHS exposure by the number of smokers in home. Multiple logistic regression was used to test the factors related to SHS exposure adjusted for confounders. Results: The prevalence of exposure to SHS was 58.2% (95%CI: 55.2, 61.1). The schoolchildren with abnormal depression status were 1.8 times more likely to have been exposed to SHS (95%CI: 1.3, 2.5). In addition, the number of smokers in the home did not modify the association between exposure to SHS and depressive symptoms (P: 0.964). Conclusions: An association between exposure to SHS and depressive in schoolchildren was observed, but this relationship was not affected by the number of smokers in children’s homes.", "keywords": [ "schoolchildren", "adolescent", "secondhand smoke exposure", "depression", "number of smokers" ], "content": "Introduction\n\nIn Thailand, the prevalence of tobacco consumption among people aged 15 years old or above has decreased from 32.0% in 1991 to 19.1% in 2017; however, the prevalence of exposure to secondhand smoke (SHS) in the home remains quite high, 32.7% in 20171. Exposure to SHS has been linked to several diseases including cancer and heart disease, especially in children and non-smokers2. Many strategies have been implemented to reduce non-smokers’ exposure to SHS, including legislation to prevent smoking in public places and health promotion3. Laws have been passed to ban smoking on public transport, in airports, in restaurants, and in schools; however, this legislation does not cover private places such as the home.\n\nHome is a place where family members in a range of activities, and is a common location for exposure to SHS4–6. Most studies to date show that exposure to SHS is linked to depression, including SHS exposure occurring in the home7–11. Just one study found no such connection12. Limited data is presently available on the association between SHS exposure and depression in schoolchildren. We therefore conducted this study to investigate whether or not there is an association between SHS exposure and depressive symptoms in this population, and whether the number of smokers in children’s households affects this association.\n\n\nMethods\n\nThis cross-sectional survey was conducted in nine schools located in northeast Thailand from August in 2018 to March in 2019.\n\nWe calculated the sample size for this survey as the following formula: n=(zα/22×p×q)/d2 in which α is 0.05; p is 0.232 that was reported from Leung L et al.13, q=1-0.232=0.768; d=0.035. In addition, we multiplied the total sample size with design effect 1.5. So, in total at least 900 participants were needed.\n\nSchools were selected by simple random sampling from the list of schools provided by the Office of the Basic Education Commission (OBEC). We contacted nine schools to describe our study and invite them to participate in the study. In each school, students in grades 6 to 8 (13 to 15 years old), were asked to participate in the study. The rationale for using these grades was that students of this age group still mostly stay within their homes outside of school and therefore would be more likely to be exposed to SHS if a smoker was present in the household. The total number of students in grade 7 (Mattayomsuksa 1), grade 8 (Mattayomsuksa 2), and grade 9 (Mattayomsuksa 3) from the selected school was 2278. All were asked to complete the questionnaire by themselves following instructions from a research assistant. Students that were part of the same household as an existing participant or had any psychiatric disorders as identified by their teacher were excluded. After filling in the self-report questionnaire, students were asked to deliver questionnaires to their guardians to collect information on household variables. A guardian was defined as mother, father, or relation. They were asked to return these questionnaires to a teacher within 7 days. The questionnaire for student and parents are available as extended data14. A total of 1103 families completed the questionnaires, equating to a response rate of 48.4% (1103/2278). We excluded 4 cases because there was not reporting of SHS exposure, so a total of 1099 participants were analyzed. Participants provided written informed consent form to participate in this study. A consent form was provided to the children who then took them home for their parents/guardians to review. This included a consent form for the parent/guardian and assent form for the child to complete. The child then returned these to school.\n\nDepressive symptoms were measured using the Center for Epidemiological Studies-Depression Scale (CESD) questionnaire. This questionnaire comprises of 20 questions and has a Cronbach's alpha of 0.86, and was translated into the Thai language15. For each question, schoolchildren could respond never (0-score), rarely (1-score), often (2-score), or all the time (3-score). The scores for each question were summed, with totals ranging from 0 to 60. If the total scores of the CESD were more than 22, the schoolchildren were identified as having depressive symptoms16.\n\nFor exposure to SHS, the schoolchildren were asked “In the past 7 days, have you inhaled any tobacco smoke?” The available answers were “inhaled on 1–3 days”, “inhaled on 4–6 days”, “inhaled every day”, or “No”. Schoolchildren who reported “no” were classified as not having been exposed to SHS, and the others were classified as having been exposed to SHS. We also collected information on variables such as the number of years of school attended, household’s income per month, number of smokers in the home, school grade (grade 7, grade 8, and grade 9), perceived danger of exposure to SHS and third hand smoke, self-confidence in avoidance of SHS (scores ranged 0-20), SHS attitude (scores ranged 0-10), SHS knowledge (scores ranged 0-10), and place of exposure to SHS.\n\nThe association between variables and exposure to SHS were examined using Chi-square tests or Fisher’s exact tests as appropriate. We also tested the effect of the number of smokers per household on the association between depressive symptom and exposure to SHS by using the Mantel-Haenszel Test. For multivariable analysis, we used multiple logistic regression for adjusting potential confounders. All data analysis was performed using R version 3.6.017.\n\n\nEthics\n\nThe Mahasarakham University Research Ethics Board provided ethical approval for this study (Approval number:115/2018).\n\n\nResults\n\nA total of 1099 subjects were included in the analysis. The prevalence of exposure to SHS was 58.2% (95%CI: 55.2, 61.1). In this study, 92.9% of guardians had attended 1–6 years of school, 85.2% of participants had a household income less than 10000 Thai Baht (THB; equivalent to 218.37 USD at time of publication), and 20.8% of households had more than one person who was a smoker. Most students were in 7th grade (39.8%), and 63.4% were female. Awareness of the harm caused by SHS exposure and third hand smoke exposure was 35.1% and 57.2%, respectively. The mean scores of self-confidence in avoidance of SHS was 12.7 (SD: 5.5). The mean scores of attitude and knowledge of secondhand smoke exposure were 27.5 (SD: 3.1) and 4.5 (SD: 1.3), respectively. The characteristics of the participants are shown in Table 1 and full details for each participant available as underlying data14.\n\neSHS = Exposure to secondhand smoke\n\nIn Table 1, information is presented on the association between participant risk factors and SHS exposure. The results show there was a statistically significant association between SHS exposure and the number of smokers per household (P value: < 0.001), gender (P value: 0.004), awareness of the harm caused by third hand smoke exposure (P value: 0.046), and self-confidence in avoidance of SHS exposure (P value < 0.001). A statistically significant association was not detected between SHS exposure and relation of the parent/guardian to the school child (P value: 0.957), the number of years attended in school (P value: 0.540), household income per month (P value: 0.342), school grade placement (P value: 0.565), perception about the harm of eSHS (P value:0.778), SHS attitude (P value:0.636), and SHS knowledge (P value:0.513). In Table 2, we present results from the multiple logistic regression model to control for confounder factors. Our analysis shows that schoolchildren with an abnormal CESD state are 1.8 times more likely to have been exposed to SHS (95%CI: 1.3, 2.5; P value <0.001) than those with a normal CESD state, with adjustment made for the number of years of school attended, household income per month, perception about the harm caused by SHS exposure, perception about the harm caused by third hand smoke exposure, SHS attitude, SHS knowledge, and self-confidence in avoidance of SHS exposure.\n\nHomogeneity test (Mantel-Haenszel Testing), P value = 0.964, eSHS = Exposure to secondhand smoke\n\nWe also investigated the effect of the number of smokers per household. In households with no smokers, schoolchildren who reported depressive symptoms were 1.8 times (95%CI: 1.0, 3.1) more likely to exposed to SHS than those who were not. In households with one smoker, schoolchildren who had depressive symptoms were 1.9 times (95%CI: 1.1, 3.3) more likely to exposed to SHS. Lastly, in households with two or more smokers, schoolchildren who reported depressive symptoms were 1.7 times (95%CI: 0.8, 3.6) more likely to be exposed to SHS than those who reported no depressive symptoms (Table 3). The results of the Mantel-Haenszel test show there was no statistically significant difference between the number of smokers per household and the association between SHS exposure and depression symptoms.\n\nAdjusted for the number of years attended in schools, household income per month, perception about the harm of SHS exposure, perception about the harm of third hand smoke exposure, SHS attitude, SHS knowledge, and self-confidence in avoiding SHS exposure scores.\n\n\nDiscussion\n\nThis study shows there is a high prevalence of SHS exposure in schoolchildren in northeast Thailand, and a statistically significant association between SHS exposure and depressive symptoms in these children. The number of smokers per household did not affect this association.\n\nThe prevalence of SHS exposure in this study was 58.2%, a higher rate than a comparable study in the US where exposure was 48%18, and a study in lower-middle-income countries where exposure was 55.9%19. The prevalence of tobacco use in Thailand has declined gradually, but the prevalence of SHS exposure is still quite high1. This may be due to non-smokers not being aware of the harms of SHS exposure or to smokers continuing to smoke in locations near non-smokers.\n\nWhen a univariate analysis was performed to test the association between different factors and SHS exposure, and SHS exposure and depressive symptoms, several statistically significant associations were identified. After adjusting for potential confounding factors, we also showed that schoolchildren exposed to SHS tend to have more depressive symptoms than unexposed children. This finding is similar to several previous studies7,20–22, except one conducted in the Netherlands12. Possible reasons for the disparity include differences in sample population and depressive symptom measurement. Previous studies that detected an association between SHS exposure and depressive symptoms used a predominantly adolescent population7,20–22, while the study that did not used an adult population. It may be that children and adolescents who are exposed to SHS are not in a position to move away from the smoker, and depressive symptoms are stimulated. Adults, by comparison, have greater autonomy and can more readily avoid SHS exposure.\n\nOur study also investigated whether the number of smokers in a household affects the association between SHS exposure and depression. No interactive effect was detected. The association between SHS exposure and depression might be of equal magnitude regardless of the number of household smokers. Very few studies consider the interaction between known risk factors and the association between SHS exposure and depression. Our assumption was that, if there are more smokers in a household, there would be a greater chance of SHS exposure and depression.\n\n\nStrengths and limitations\n\nThis study has two major strengths. It is the first study to examine how the number of smokers per household affects the association between SHS exposure and depressive symptoms. Also, the sample size was large, so the findings should be more accurate than smaller-sized studies. Set against these two strengths are the following three limitations. Firstly, the schoolchildren were selected from northeast Thailand, and may not be representative of schoolchildren nationally. Secondly, a cross-sectional study design was used, so we cannot conclude there is a causal relationship between SHS exposure and depressive symptoms. Finally, the survey relied on self-reports, so there may be recall bias with regard to history of exposure.\n\n\nConclusion\n\nIn this study, we observed the association between SHS exposure and depressive symptoms. However, the number of smokers per household did not affect this association.\n\n\nData availability\n\nHarvard Dataverse: Secondhand smoke: https://doi.org/10.7910/DVN/I7KCVH14\n\nThis project contains the following underlying data:\n\n- datashsfandcesd1000research.tab (data secondhand smoke and cesd – not that Grade 1, 2 and 3 correspond with grade 7 (Mattayomsuksa 1), grade 8 (Mattayomsuksa 2), and grade 9 (Mattayomsuksa 3))\n\nHarvard Dataverse: Secondhand smoke: https://doi.org/10.7910/DVN/I7KCVH14\n\nThis project contains the following extended data:\n\n- parent_questionaire_eng.pdf (parent questionnaire English)\n\n- parent_questionaire_thai.pdf (parent questionnaire Thai)\n\n- student_questionaire_eng.pdf (student questionnaire English)\n\n- student_questionaire_thai.pdf (student questionnaire Thai)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank school staff for assistance with data collection, and Dr. Tim Cushnie for assistance with manuscript presentation.\n\n\nReferences\n\nPitayarangsarit S, Punkrajang P: The current situation of tobacco control in Thailand 2018. Ed. 2., Bangkok: Jaruenmunkong Publisher, 2017.\n\nOberg M, Jaakkola MS, Woodward A, et al.: Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries. Lancet. 2011; 377(9760): 139–46. PubMed Abstract \| Publisher Full Text\n\nPitayarangsarit S, Punkrajang P, Sitabutr D: 25 years: Tobacco Control in Thailand from 1992 to 2017. Bangkok: Jaruenmunkong Publisher, 2017.\n\nTsai J, Homa DM, Gentzke AS, et al.: Exposure to Secondhand Smoke Among Nonsmokers - United States, 1988-2014. MMWR Morb Mortal Wkly Rep. 2018; 67(48): 1342–1346. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJallow IK, Britton J, Langley T: Prevalence and factors associated with exposure to secondhand smoke (SHS) among young people: a cross-sectional study from the Gambia. BMJ Open. 2018; 8(3): e019524. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoku DT: Prevalence and socioeconomic inequalities in indoor exposure to secondhand smoke at home among children 0-5years in Ghana. Addict Behav. 2018; 79: 68–73. PubMed Abstract \| Publisher Full Text\n\nPatten SB, Williams JVA, Lavorato DH, et al.: Major depression and secondhand smoke exposure. J Affect Disord. 2018; 225: 260–264. PubMed Abstract \| Publisher Full Text\n\nKim SY: Secondhand Smoke Exposure, Depression Symptoms, and Suicidal Ideation in Adults. Korean J Fam Med. 2016; 37(2): 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaha F, Goodwin RD: Secondhand smoke exposure across the life course and the risk of adult-onset depression and anxiety disorder. J Affect Disord. 2014; 168: 367–372. PubMed Abstract \| Publisher Full Text\n\nLee KJ: Current smoking and secondhand smoke exposure and depression among Korean adolescents: analysis of a national cross-sectional survey. BMJ Open. 2014; 4(2): e003734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandiera FC, Caban-Martinez AJ, Arheart KL, et al.: Secondhand Smoke Policy and the Risk of Depression. Ann Behav Med. 2010; 39(2): 198–203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBot M, Vink JM, Willemsen G, et al.: Exposure to secondhand smoke and depression and anxiety: A report from two studies in the Netherlands. J Psychosom Res. 2013; 75(5): 431–436. PubMed Abstract \| Publisher Full Text\n\nLeung LT, Ho SY, Wang MP, et al.: Secondhand Smoke From Multiple Sources, Thirdhand Smoke and Respiratory Symptoms in Hong Kong Adolescents. Nicotine Tob Res. 2018; 20(2): 192–198. PubMed Abstract \| Publisher Full Text\n\nIntarut N: \"Secondhand smoke\". Harvard Dataverse, V3, 2020. http://www.doi.org/10.7910/DVN/I7KCVH\n\nNilmanut S, Kuptniratsaikul V, Pekuman P, et al.: The Study of The Center for Epidemiologic Studies-Depression Scale (CES-D) in Thai People Siriraj Hospital. ASEAN J Rehabil Med. 2000; 6(3): 6. Reference Source\n\nJiamjaroenkul J, Limsuwan N: Depression among Junior High School Students in Muang District, Chiang Mai Province. J Psychiatr Assoc Thailand. 2015; 60(4): 10. Reference Source\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna, Austria, 2013. Reference Source\n\nAgaku IT, Singh T, Rolle I, et al.: Prevalence and Determinants of Secondhand Smoke Exposure Among Middle and High School Students. Pediatrics. 2016; 137(2): e20151985. PubMed Abstract \| Publisher Full Text\n\nXi B, Liang Y, Liu Y, et al.: Tobacco use and second-hand smoke exposure in young adolescents aged 12-15 years: data from 68 low-income and middle-income countries. Lancet Glob Health. 2016; 4(11): e795–e805. PubMed Abstract \| Publisher Full Text\n\nHuang JY, Xu B, Guo D, et al.: Dose-Response Relationships between Second-Hand Smoke Exposure and Depressive Symptoms among Adolescents in Guangzhou, China. Int J Environ Res Public Health. 2018; 15(5): 985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim NH, Park JH, Choi DP, et al.: Secondhand Smoke Exposure and Depressive Symptoms among Korean Adolescents: JS High School Study. PLoS One. 2016; 11(12): e0168754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe XH, Li L, Gao Y, et al.: Dose-response relations between second-hand smoke exposure and depressive symptoms among middle-aged women. Psychiatry Res. 2015; 229(1–2): 533–538. PubMed Abstract \| Publisher Full Text" }	[ { "id": "71748", "date": "06 Oct 2020", "name": "Pongdech Sarakarn", "expertise": [ "Reviewer Expertise Biostatistics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have some comments and issues for adding and revising as follows;\nIn introduction, I don't find the literature regarding depression in schoolchildren, could you please add for completion?\n\nIn data collection, \"in grades 6 to 8 \" or \"in grade 7 to 9\", please clarify for this words.\n\nPlease clarify regarding the number of smokers in house (persons) and place of exposure in home (Table 1). I think both of these variables should be related (e.g. the number of place of exposure is 409, but the number of smokers in house (persons) (including 1,>=2) is 460).\n\nIn the results (page 5), please check the position of table; may mistake between \"table2\" and \"table3\".\n\nIn practice, I think not only the number of smokers in house (persons) can affect the exposure of smoking, but also the quantity of smoking (per persons) can relate as well. Therefore, this point should be added in the discussion or limitation parts of this study.\n\nFinally, this study uses depression of schoolchildren for analyzing with the prevalence of secondhand smoke exposure, in my opinion \"the depression of schoolchildren\" should be added in the title name, if possible.\nThese preliminary results can be important for explanation of the effects of smoking. I think this paper can be useful, especially the schoolchildren group.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "71749", "date": "06 Oct 2020", "name": "Kyaw Ko Ko Htet", "expertise": [ "Reviewer Expertise Public health", "NCD", "TB" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 some comments:\n1) Introduction:\nWhy do the investigators want to investigate the association between the secondhand smoke exposure and depression among the children? It would be better to explain the magnitude of depression among children in the introduction part.\n\n2) Methods:\nWhat is the meaning of p is 0.232?\n\nHow can response rate 48.4% affect the study result? Is it a study limitation?\n\n3) Measurement:\nFor information about number of smokers in household, who completed this information? It is not clear which information was completed by children and household members.\n\nIn some countries, grade 8 & 9 students have experience on smoking, no information was found in this study. Can it affect the study result?\n\n4) Results:\nTable 2: It may need to explain how schoolchildren were exposed to secondhand smoke although there was no smoker in the house.\n\nThe authors may need to explain the definition of second-hand smoke and third-hand smoke. In the result on perception about the harm of exposure to thirdhand smoke, is it third- or second-hand smoke?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71747", "date": "08 Oct 2020", "name": "Hendra Kurniawan", "expertise": [ "Reviewer Expertise public health", "pulmonology", "health social study" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 background: please clarify clearly which province in Thailand that covers the northeast Thailand area?\n\nIf you used the CESD questionnaire, did you translate it to Thai language and validate it before or did you already have the valid Thai version?\n\nThe category of income; household income less than 10000 Thai Baht, is mean enough or which category? Please state in your discussion part.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71751", "date": "26 Oct 2020", "name": "Roongnapa Khampang", "expertise": [ "Reviewer Expertise Epidemiology", "health policy or program evaluation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nCould you please elaborate more on the rationale of the study and the significance of the study to public health? Why did you choose to study the association between SHS and depression instead of other diseases? Is there a lack of evidence in Thailand? Could you also provide a bit more detail on the psychological or biological mechanisms of SHS and depression so that the readers understand why depression is important here and how could SHS associate with depressive symptoms?\n\nIn the method, you stated that you selected nine schools from northeast Thailand, please also provide detail in the introduction why you are interested in this area and how your findings would benefit the responsible organizations?\n\nMethods:\nYou stated that 9 schools were randomly selected from a list of schools but did not show the distribution of the schools among provinces in northeast Thailand. Were there any schools located in the same province? What were the sizes of the school included in the study? How many schools were located in urban areas and how many were in rural areas? This detail may help readers to understand and justify the representativeness of the study samples and the generalizability of the findings especially the prevalence of SHS.\n\nThere is no detail on how the students (unit of analysis) were selected. Were all eligible students recruited?\n\nResults:\nThere are some typos in the manuscript such as in Table 1, mother, strongly disagree.\n\nThere is no information on current smoking status of the students who were interviewed. Many studies found a positive association between smoking and depression and anxiety. Would this missing information influence the main findings?\n\nDiscussion:\nYou may compare the characteristics of your study samples (both schools and students) with the figures of northeast Thailand to clarify about similarity of your samples and the population of northeast Thailand.\n\nHow would the smoking status of the students influence your findings?\n\nIt would be nice if you could develop policy recommendations drawing from your findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1158
https://f1000research.com/articles/9-1157/v1	21 Sep 20	{ "type": "Research Article", "title": "Molecular docking analysis of selected phytochemicals on two SARS-CoV-2 targets", "authors": [ "Amaka Ubani", "Francis Agwom", "Oluwatoyin Ruth Morenikeji", "Nathan Yakubu Shehu", "Emmanuel Arinze Umera", "Uzal Umar", "Simeon Omale", "John Chinyere Aguiyi", "Nnaemeka Emmanuel Nnadi", "Pam Dachung Luka", "Amaka Ubani", "Francis Agwom", "Oluwatoyin Ruth Morenikeji", "Nathan Yakubu Shehu", "Emmanuel Arinze Umera", "Uzal Umar", "Simeon Omale", "John Chinyere Aguiyi", "Pam Dachung Luka" ], "abstract": "Background: The coronavirus spike (S) glycoprotein and M protease are two key targets that have been identified for vaccines and drug development against COVID-19. Methods: Virtual screening of some compounds of plant origin that have shown antiviral activities were carried out on the two targets, the M protease (PDB ID 6LU7) and S glycoprotein (PDB ID 6VSB), by docking with PyRx software. The binding affinities were compared with other compounds and drugs already identified as potential ligands for the M protease and S glycoprotein, as well as chloroquine and hydroxychloroquine. The docked compounds with best binding affinities were also filtered for drug likeness using the SwissADME and PROTOX platforms on the basis of physicochemical properties and toxicity, respectively. Results: The docking results revealed that scopadulcic acid and dammarenolic acid had the best binding affinity for the S glycoprotein and Mpro protein targets, respectively. Silybinin, through molecular docking, also demonstrated good binding affinity for both protein targets making it a potential candidate for further evaluation as repurposed candidate for SARS-CoV-2, with likelihood of having multitarget activity as it showed activities for both targets. Conclusions: The study proposes that scopadulcic acid and dammarenolic acid be further evaluated in vivo for drug formulation against SARS-COV-2 and possible repurposing of Silybinin for the management of COVIV-19.", "keywords": [ "SARS-CoV-2", "Molecular docking", "COVID-19" ], "content": "Introduction\n\nSince 2003, three coronaviruses have been associated with pneumonia, the first was severe acute respiratory syndrome coronavirus (SARS-CoV-1)1 which affected 8,098 people causing 774 deaths between 2002 and 20032, the second was Middle-East respiratory syndrome coronavirus (MERS-CoV)3 and the third is SARS-CoV-2 which, as at 8th July, 2020, has affected 11,669,259 globally and is responsible for 539,906 deaths. SARS-CoV-2 is a human pathogen which has been declared a global pandemic by the World Health Organization4 and is responsible for the Coronavirus disease 2019 (COVID-19) (1). . The bats have been identified as the possible reservoir and origin for both the SARS-CoV-2 and SARS-COV-13. Unfortunately, there has been no known cure for COVID-19 to date.3\n\nThe entry into the host cell by the coronaviruses is usually mediated by the spike (S) glycoprotein3. This glycoprotein interacts with the angiotensin-converting enzyme 2 (ACE2), enabling the virus penetration into the host. The main protease (M protease, also known as 3CL protease) has also been found to be essential for processing of translated polyproteins for the SARS_CoV-2 virus. The two targets, the S glycoprotein, Sgp and MPro proteins have therefore been considered as important drug targets against the SARS-CoV-2 virus.5. For this study, these two drug targets were selected and used to virtually screen some phytochemicals for possible activity against the SARS-CoV-2 virus.\n\nPossibly, potent inhibitors of these two targets will be able to interfere with the SARS-CoV-2 replication process and thus serves as potential drugs for the management of the COVID-19. Hence, this work is aimed at identifying some potential lead compounds of plant origin that can serve as candidates for testing against the SARS-CoV-2 virus.\n\n\nMethods\n\nPlant compounds reported in the literatures that have been demonstrated to have antiviral activities (list of all the compounds analyzed is available as Extended data6) were selected, alongside also hydroxychloroquine, remdesivir and favipiravir, which have been used in the treatment and management of COVID-19 and mined from the PubChem database.\n\nTwo proteins including crystal structure of the SARS-CoV-2 main protease (PDB ID 6LU7)7 in complex with an inhibitor N3 and the S glycoprotein in complex with N-acetyl-D-glucosamine (NAG) (PDB ID 6VSB) were downloaded from the Protein Data Bank (www.rcsb.org). The proteins were prepared by removing water molecules and co-crystalized ligands (highlighting the water molecules and co-crystalized ligands and deleting) on the proteins using Discovery Studio version 2017R2 (19). The UCSF Chimera molecular modeling package is an open access equivalent that could be sued to perform the same function8.\n\nThe already prepared protein and ligands were loaded on to the PyRx docking software (PyRx-Python Prescription 0.8), where molecular docking was done in the AutodockVina mode. Visualization to see how the ligands fitted and bound into the binding pockets on the protein and also the interactions between the protein and the ligands was done using Discovery Studio version 2017R2 (19) by first loading the saved PDBqt output file of the target protein from PyRx and then inserting the output of the binding modes of the different ligands and then viewed under the Receptor-Ligand interaction platform of Discovery Studio software.\n\nThe Physicochemical properties and drugability of selected compounds were predicted using the free online versions of SwissADME9 and Molinspiration10 platforms and their predicted toxicity profile also compared using the PROTOX toxicity prediction platform11. In each case the ligands were loaded onto the platforms as SMILES structures obtained from PubChem.\n\n\nResults\n\nA library of 22 compounds of plant origin known to have antiviral activity was obtained from the PubChem database (see Extended data6). Though the compounds are chemically diverse, they consist of largely flavonoids and terpenes. Some compounds from the citrus family were found among the library, though they could not make it among the top six selected compounds that demonstrated good binding affinities for the two targets. Most of the compounds has showed similar binding affinities to the selected protein targets (6LU7 (M protease) and 6VSB (S glycoprotein)) compared to the training sets of known ligands to the selected targets (see Table 1).\n\nHowever, the top six compounds with most favorable binding affinity were selected for each of the targets. The outcomes of the binding affinities of the selected compounds on the 6LU7 and 6VBS targets are presented in Table 2 and Table 3, respectively.\n\nThe binding affinities of the top six compounds (Table 1) on the 6vsb target are comparable to each other, i.e. they all lie within a close range of 9 to 9.6 kcal/mol indicating that they might likely have equal or comparable potential as lead compounds for the 6vsb S glycoprotein.\n\nOne of the compounds sylibinin12 is an FDA approved drug, which showed up as active on both M protease and S glycoprotein will make a good candidate of repurposing. Finding Quercetin as a potential inhibitor of the M protease Protein (6LU7) of SARS-CoV-2 corresponds with an earlier report13.\n\nLooking at the octanol–water coefficient (cLogP) of the compounds, there was no correlation observed between the lipophilicity and the interaction with the receptors. However, for the compounds acting on 6LU7 (Serial numbers 3, 4, 7, 8, 9 and 10 in Table 4), interaction with the receptor is correlated with low lipophilicity, with the exception of solanidine and dammarenolic acid, which have high cLogP values, although both compounds also use their polar functional groups in interacting with the receptor. Bacailin (Figure 1) and naringenin showed good hydrogen bond interaction with the 6VSB receptor due to their polarity.\n\ncLogP: octanol-water coffecient.\n\nBest binding pose and interaction of (A) scopodulcic acid and (B) baicalin on the spike glycoprotein.\n\nFiltering the compounds for drug likeness on the basis of Linpinski’s and/or Veber’s rule showed that all the compounds have drug-like properties except baicalin, which failed the two filtering scales applied (Table 5). This implies that baicalin is not worth considering further without any structural modification. The predicted toxicity profile of the selected compounds shows (Table 6) that all the compounds are likely to be relatively safe, which makes them good potential candidates for anti-infectives because the chances of achieving selective toxicity is high. Baicalin is thus most likely the safest.\n\nMol. Wt.: molecular weight; TPSA: total polar surface area; HBA: number of hydrogen bond acceptors; HBD: number of hydrogen bond donors; RB: number of rotatable bonds; cLogP: mean of calculated logP values.\n\n--: inactive; -: less inactive; +: active; ++: more active; AR: androgen receptor; AO: amine oxidase A; PGS: prostaglandin G/H synthase 1.\n\n\nDiscussion\n\nTwo compounds among the top six selected for each target, solanidine and sylibinin, were observed to have good binding affinity on both the 6VSB and the 6FLU7 proteins. This makes them potential multitarget acting inhibitors on the SARS-CoV-2. Solanidine is a steroidal glycoalkaloid found in potatoes14. Although toxic to humans and animals, solanidine has been reported to be effective against herpes viruses (HSV), herpes genitalis and herpes zoster15 Its activity against HSV is attributed to the presence of a sugar moiety16. In silico drug screening using PROTOX II showed that solanidine is very likely to be cytotoxic and immunotoxic. PROTOTOX II is a cost- and time-saving approach for testing and determining the toxicity of a compound to be considered a drug of choice17. PROTOTOX II predicts the toxicity outcome of a potential drug of choice, it incorporates machine-learning models which use a combination of fragment propensities, molecular similarity, pharmacophores, to predict toxicity endpoints, such as acute toxicity, cytotoxicity, , carcinogenicity , hepatotoxicity, immunotoxicity, mutagenicity and toxicity targets17\n\nA safe drug must not be toxic to its host target. Based on the PROTOX II evaluation of toxicity, dammarenolic acid emerges as the compound of choice with the least toxicity. Dammarenolic acid has been reported as effective antiviral agents dammarenolic acid potently inhibited the in vitro replication of other retroviruses, including simian immunodeficiency virus and murine leukemic virus in vector-based antiviral screening studies and has been proposed as a potential lead compound in the development of anti-retrovirals18. The compound is cytotoxic and demonstrate potential against respiratory syncytial virus19. We therefore propose that the evaluation of dammarenolic acid might hold the key to a safe and effective anti-SARS-CoV-2 drug considering its drugability and low toxicity.\n\nThis study proposes a potential re-purposing of silybinin for the management of COVID19 diseases. Silybinin (silymarin) possesses antiviral ability against hepatitis C virus (HCV)20,21 It has been reported to have activities against a wide range of viral groups including flaviviruses (HCV and dengue virus), togaviruses (Chikungunya virus and Mayaro virus), influenza virus, human immunodeficiency virus, and hepatitis B virus20. In an in vivo and in vitro study, Silymarin has been proposed to inhibit HCV entry, RNA synthesis, viral protein expression and prevent infectious virus production; it can also block cell-to-cell spread of the virus22. In silico analysis of silybinin in this present study has shown that it can likely inhibit SARS-CoV-2 S glycoprotein and Mpro targets, making it a drug to be considered with a possible multi-target activity against the SARS-CoV-2 virus.\n\n\nConclusions\n\nFrom the 22 phytocompounds that were virtually screened, scopodulcic acid and dammarenolic acid showed the best binding energies with the S glycoprotein and Mpro, respectively. This makes them potential lead compounds for development into candidates against the SARS-CoV-2. Furthermore, the FDA-approved drug silybinin (Legalon) had good binding affinity for the two targets, so could be evaluated further for possible repurposing against the SARS-CoV-2 virus.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: Replication Data for:Molecular docking analysis of some phytochemicals on two SARS-CoV-2 targets. https://doi.org/10.7910/DVN/BB0QQK6.\n\nThe file within this project contains the compounds obtained from the PubChem database that were analyzed in this study.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nDrosten C, Günther S, Preiser W, et al.: Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003; 348(20): 1967–76. PubMed Abstract \| Publisher Full Text\n\nWalls AC, Park YJ, Tortorici MA, et al.: Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020; 181(2): 281–292.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaki AM, Van Boheemen S, Bestebroer TM, et al.: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367(19): 1814–20. PubMed Abstract \| Publisher Full Text\n\nNeher RA, Dyrdak R, Druelle V, et al.: Potential impact of seasonal forcing on a SARS-CoV-2 pandemic. Swiss Med Wkly. 2020; 150: w20224. PubMed Abstract \| Publisher Full Text\n\nHilgenfeld R: From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 2014; 281(18): 4085–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNnadi N: Replication Data for:Molecular docking analysis of some phytochemicals on two SARS-CoV-2 targets. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/BB0QQK\n\n10.2210/pdb6LU7/pdb.\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–12. PubMed Abstract \| Publisher Full Text\n\nDaina A, Michielin O, Zoete V: SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017; 7: 42717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJarrahpour A, Motamedifar M, Zarei M, et al.: Petra, osiris, and molinspiration together as a guide in drug design: predictions and correlation structure/antibacterial activity relationships of new N-Sulfonyl monocyclic β-lactams. Phosphorus, Sulfur, and Silicon and the Related Elements. 2010; 185(2): 491–7. Publisher Full Text\n\nDrwal MN, Banerjee P, Dunkel M, et al.: ProTox: a web server for the in silico prediction of rodent oral toxicity. Nucleic Acids Res. 2014; 42(Web Server issue): W53–W58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerenci P, Scherzer TM, Kerschner H, et al.: Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. Gastroenterology. 2008; 135(5): 1561–7. PubMed Abstract \| Publisher Full Text\n\nChen L, Li J, Luo C, et al.: Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CLpro: Structure–activity relationship studies reveal salient pharmacophore features. Bioorg Med Chem. 2006; 14(24): 8295–306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGinzberg I, Tokuhisa JG, Veilleux RE: Potato steroidal glycoalkaloids: biosynthesis and genetic manipulation. Potato Research. 2009; 52(1): 1–15. Publisher Full Text\n\nFriedman M, McDonald GM, Filadelfi-Keszi M: Potato glycoalkaloids: chemistry, analysis, safety, and plant physiology. Anal Bioanal Chem. 1997; 16(1): 55–132. Publisher Full Text\n\nThorne HV, Clarke GF, Skuce R: The inactivation of herpes simplex virus by some Solanaceae glycoalkaloids. Antiviral Res. 1985; 5(6): 335–43. PubMed Abstract \| Publisher Full Text\n\nBanerjee P, Eckert AO, Schrey AK, et al.: ProTox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018; 46(W1): W257–W263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEsimone CO, Eck G, Nworu CS, et al.: Dammarenolic acid, a secodammarane triterpenoid from Aglaia sp. shows potent anti-retroviral activity in vitro. Phytomedicine. 2010; 17(7): 540–7. PubMed Abstract \| Publisher Full Text\n\nEsimone CO, Eck G, Duong TN, et al.: Potential anti-respiratory syncytial virus lead compounds from Aglaia species. Pharmazie. 2008; 63(10): 768–73. PubMed Abstract \| Publisher Full Text\n\nLiu CH, Jassey A, Hsu HY, et al.: Antiviral Activities of Silymarin and Derivatives. Molecules. 2019; 24(8): 1552. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolyak SJ, Ferenci P, Pawlotsky JM: Hepatoprotective and antiviral functions of silymarin components in hepatitis C virus infection. Hepatology. 2013; 57(3): 1262–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagoner J, Negash A, Kane OJ, et al.: Multiple effects of silymarin on the hepatitis C virus lifecycle. Hepatology. 2010; 51(6): 1912–21. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "84269", "date": "04 Jun 2021", "name": "Yogendra Nayak", "expertise": [ "Reviewer Expertise Pharmacy and Pharmacology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Ubani et al., on molecular docking analysis of phytochemicals on SARS-CoV-2 is well presented. I recommend and endorse the study. However, few points can be improved, they are as follows:\nIn the abstract the abbreviation for M protease can be mentioned, it is mentioned but not corresponding to M protease.\n\nGenerally, the molecular dynamic simulation is said to be the best indicator/predictor of biological activity. Why this was not performed? or is it not the limitation of the study?\n\nWhy only 22 compounds from plant origin are selected? How it is shortlisted? What was the rationale? Because there are more than 22 natural plant product having antiviral properties. Why only silybin, why not curcumin, which is also FDA approved. (Mohan et al. (20201))\n\nThe ligand protein interaction can be further discussed. In the discussion, at least top five compounds can be discussed.\n\nWhy not just try the silybin in clinical trial?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "94590", "date": "06 Oct 2021", "name": "Adel S Girgis", "expertise": [ "Reviewer Expertise Organic", "medicinal chemistry with special interest in computational chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current study titled “Molecular docking analysis of selected phytochemicals on two SARS-CoV-2 targets”, deals with an important subject. Investigation of promising anti-SARS-CoV-2 agents is a recent universal need. Computational technique can accelerate the drug discovery. Major revisions are needed for this study.\n\nUpdate what mention in the first paragraph of introduction section “the third is SARS-CoV-2 which, as at 8th July, 2020, has affected 11,669,259 globally and is responsible for 539,906 deaths”.\n\nFigures exhibiting the best fit docking of all the tested compounds should be inserted in the supplementary file with short comment(s) explaining the interactions (hydrogen bonding, π-interactions, etc.)\n\nExperimental data for the most successful agents discovered are the perfect support for the hypothesized/computational studies. This point is considered the major revision requested.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1157
https://f1000research.com/articles/9-333/v1	06 May 20	{ "type": "Research Article", "title": "Tracking and forecasting milepost moments of the epidemic in the early-outbreak: framework and applications to the COVID-19", "authors": [ "Huiwen Wang", "Yanwen Zhang", "Shan Lu", "Shanshan Wang", "Huiwen Wang", "Yanwen Zhang", "Shan Lu" ], "abstract": "Background: The outbreak of the 2019 novel coronavirus (COVID-19) has attracted global attention. In the early stage of the outbreak, the most important question concerns some meaningful milepost moments, including the time when the number of daily confirmed cases decreases, the time when the number of daily confirmed cases becomes smaller than that of the daily removed (recovered and death), and the time when the number of daily confirmed cases and patients treated in hospital becomes zero. Unfortunately, it is extremely difficult to make right and precise prediction due to the limited amount of available data at the early stage of the outbreak. To address it, in this paper, we propose a flexible framework incorporating the effectiveness of the government control to forecast the whole process of a new unknown infectious disease in its early-outbreak. Methods: We first establish the iconic indicators to characterize the extent of epidemic spread. Then we develop the tracking and forecasting procedure with mild and reasonable assumption. Finally we apply it to analyze and evaluate the COVID-19 using the public available data for mainland China beyond Hubei Province from the China Centers for Disease Control (CDC) during the period of Jan 29th, 2020, to Feb 29th, 2020, which shows the effectiveness of the proposed procedure. Results: Forecasting results indicate that the number of newly confirmed cases will become zero in the mid-early March, and the number of patients treated in the hospital will become zero between mid-March and mid-April in mainland China beyond Hubei Province. Conclusions: The framework proposed in this paper can help people get a general understanding of the epidemic trends in counties where COVID-19 are raging as well as any other outbreaks of new and unknown infectious diseases in the future.", "keywords": [ "COVID-19", "Prediction Method", "Epidemic Development Index System" ], "content": "1 Introduction\n\nThe atypical pneumonia caused by the 2019 novel coronavirus (COVID-19), which is a highly infectious human disease, was first reported in Dec 31st, 2019 in Wuhan, the capital of Hubei Province in China (WHO et al., 2020). To mitigate the effect of epidemics spreading across China and other countries, Wuhan was temporarily shut-down from Jan 23th, 2020, which has proved to be efficient in the timely stopping the spread of the coronavirus (Chinazzi et al., 2020). However, due to the “Spring Festival travel rush”, there was still a rising number of confirmed cases in China in the following two months, which has caused great strain on medical resources (Li et al., 2020).\n\nThe questions that draw the most concerns are how COVID-19 will spread, and when it will end. People were always asking when the number of the daily confirmed cases will become smaller than the previous days, and when the daily confirmed cases will become smaller than that of the removed (recovered and death). These are not only of highly important for the general public, but also for government, who plays an important role in controlling the disease within a short period as much as possible. Since the decline of the number of newly confirmed cases and the number of patients in hospital imply the alleviation of epidemic, the emergence of these turning points convey useful information for decision making on medical resources allocation and isolation policies in the post-stage of the epidemic.\n\nMeanwhile, it is also important to predict when will the number of daily confirmed cases become “zero”, as well as when the number of infectious cases in hospital will be “zero”. The latter indicates the end of the epidemic. These two “zero points” can also help the government to consider loosening population migration restriction in cities. Additionally, authorities in economic departments can use the forecasting results to assess the impact of the epidemic on the economy in advance, and plan for the restoration of normal production and living order.\n\nThere have been various publications on COVID-19 from different perspectives, i.e., the origin of COVID-19, the clinical features as well as epidemic transmission characteristics. Specifically, for the origin of the virus, Fan et al. (2019) and Luk et al. (2019) pointed out that COVID-19 is an infectious disease caused by a virus closely related to SARS-CoV, while others believed that the COVID-19 virus was originally derived from wild animals (Benvenuto et al., 2020; Huang et al., 2020). For the epidemic transmission characteristics, Holshue et al. (2020) and Hui et al. (2020) found that the virus can be transmitted from person to person and that it has a high interpersonal transmission rate. Zhao et al. (2020) investigated the preliminary estimation of the basic reproduction number R0, which ranged from 2.24(95%CI : 1.96 − 2.55) to 3.58(95%CI : 2.89 − 4.39) in the early outbreak, while Prasse et al. (2020) estimated it around 2.2, Tang et al. (2020) applied likelihood-based and model-based methods to the analysis of early reported cases, and the results showed that R0 could be is as high as 6.47. Zhou et al. (2020) used the SEIR model and stated that the range of R0 of COVID-19 is 2.8–3.3, indicating that the early pathogenic transmission capacity of COVID-19 is close to or slightly higher than SARS. Other studies related to R0 are Anastassopoulou et al. (2020); Zhang et al. (2020) and referenced therein. Unfortunately, each of these models may results in different estimations of R0, which may cause any predictions based on R0 to be unstable.\n\nRecently, a number of publications have been related to trend prediction of the COVID-19 outbreak in China. Zeng et al. (2020) proposed a multi-model ordinary differential equation set neural network and model-free methods to predict the interprovincial transmission in mainland China, especially those from Hubei Province, and predicted that COVID-19 in China is likely to decelerate before Feb 18th and to end before April 2020. Chen et al. (2020) made prediction based on epidemiological surveys and analyses, which showed that the total number of diagnoses would be 2–3 times that of SARS, and the peak is predicted to be in early or middle February. Yu et al. (2020) revised the SIR model based on the characteristics of the COVID-19 epidemic development, and proposed a time-varying parameter-SIR model to study the trend of the number of infected people. Peng et al. (2020) used the SEIR method to predict the end of the epidemic in most cities in mainland China. Wu et al. (2020) used the Markov chain Monte Carlo method to estimate R0, and inferred from the SEIR model that the peak COVID in Wuhan would be reached in April, and other cities in China would be delayed by 1 to 2 weeks.\n\nHowever, there are some obvious shortcomings of forecasting methods based on epidemic models in terms of outbreak prediction. For example, the SEIR model is a mathematical method relying on an assumption of epidemiological parameters for disease progression, which are absent for a novel pathogen. For instance, the basic infection number R0, the daily recovery rate, the characteristics of the disease itself (such as the infection rate and the conversion rate of the latent to the infected), the daily exposure rate of the latent and infected, and their initial population infection status (total population, infected, the initial value of the latent, the susceptible, the healer, etc.) and many other key parameters need to be set. For infectious diseases that have already appeared in the past, or those who have a large amount of data, it is not difficult to obtain these parameters. However, for unknown, sudden and early infectious diseases, obtaining these parameters is full of difficulties, which leads to a great uncertainty and limitations in the prediction of the epidemic situation using the SEIR model.\n\nMoreover, there exist many challenges for the prediction of a new epidemic situation similar to COVID-19. First, little prior knowledge that can be refered to or analogized for a brand new epidemic; secondly, the existence of government management will make the development of the epidemic completely different from that under free development, thus how to incorporate the influence of government measures into the fitting process of parameters and build a statistical model from this needs to be considered; thirdly, in the early-outbreak the initial data often fluctuates violently and the data quality is low, thus many commonly used parameter estimation methods are not applicable anymore; furthermore, the amount of data in the early stage is too small, making it difficult to directly rely on the inertia of the data to make forward prediction. In summary, in the early stages of a brand new epidemic, how to use some low-quality and small data sets to make basic and relatively accurate forecast judgements for the entire process of the epidemic, is a long-term pain point.\n\nTo cope with these challenges, we propose a simple and effective framework incorporating the effectiveness of the government control to forecast the whole process of a new unknown infectious disease in its early-outbreak, from which we emphasis the prediction of meaningful milepost moments. Specifically, we first propose a series of iconic indicators to characterize the extent of epidemic spread, and describe four periods of the whole process corresponding to the four meaningful milepost moments: two turning points and two “zero” points; then we develop the proposed procedure with mild and reasonable assumptions, specfically without relying on an assumption of epidemiological parameters for disease progression. Finally we apply it to analyze and evaluate COVID-19 using publicly available data from mainland China beyond Hubei Province from the China CDC during the period of Jan 29th, 2020, to Feb 29th, 2020, which shows the effectiveness of the proposed procedure.\n\nFrom the empirical study, we can suggest that the proposed method may cast a flexible framework and perspective for early prediction of a sudden and unknown new infectious disease with effective government control. Specifically, in the early stage of the epidemic when some regular information is initially displayed, the proposed method can be used to predict the process of epidemic development and to judge which stage of development the situation is at, when the peak will be reached, and when the turning point will appear. Moreover, by continuously accumulating data and updating the model during the development of the epidemic, we can also predict when the epidemic will basically end. Finally, the proposed method enjoys great generalizability, which can be generalized to understand the epidemiological trend of COVID-19 spread in other counties, which will provide useful guidance for fighting against it.\n\nThe reminder of this paper is organized as follows. In Section 2, we proposed the main methodology, where we defined the iconic indicators to characterize the extent of epidemic spread in Section 2.1, yielding four periods of the whole process corresponding to the four meaningful milepost moments: two turning points and two “zero” points in Section 2.2, then Section 2.3 presents the proposed procedure with mild and reasonable assumption. Then we applied the proposed method to the COVID-19 using the public available data in mainland China beyond Hubei Province from the China CDC during the period of Jan 29th, 2020, to Feb 29th, 2020, and describe the trend of the COVID-19 spread in detail in Section 3. Some conclusions and discussions are finally given in Section 4.\n\n\n2 Methods\n\nThe data we used are provided by China CDC via public data sources, in which the cumulative confirmed cases up to the given day t, the daily confirmed cases at day t, the daily recovered ones and the daily deaths at day t are included. All the data analysis results are done with R software, version 3.6.0 and higher is recommended. The main code for the implementation of the proposed procedure as well as the data and its full description are available from Github (See data availability for more detail (Zhang (2020))).\n\nIn order to assess and predict the epidemic, we first define a set of necessary indicators that can reflect the status of disease contagion. We then divide the cycle of the epidemic into four stages, which are divided by the turning points of the proposed indicators. Finally, we propose a computational framework to predict the turning points.\n\nIt is obvious that the contagion process of an unknown virus in different regions would be diverse with respect to the number of patients and the growth pattern of the epidemic, because of population density, population mobility, public health conditions, as well as disease prevention and control measures. Therefore, we first constructed a set of indicators to monitor the essential laws of the development of the disease.\n\nThere are several requirements for the monitoring indicators. Firstly, as the number of patients can vary greatly across regions, the scale of the data should be eliminated so that the analysis methods and results are comparable. Secondly, they should well reflect the general laws and characteristics of the epidemic process as well as accurately and coherently describe the entire process of the epidemic from the beginning to the end. Particularly, they should be able to answer the question of when the turning point of the epidemic would appear. Thirdly, they should be as simple and convenient as possible so that it can be applied with publicly available data. Last but not least, the indicators should have clear meaning and be easily interpreted.\n\nFollowing the above, we first adopt three basic indicators that are published daily by the provincial and municipal governments of China. That is, for time t, the daily confirmed cases Et, the daily recovered cases Ot, and the daily deaths Dt. Then we define a few monitoring indicators to characterize the epidemic stages, that is the number of infectious cases in hospital Nt, the daily infection rate Kt and the daily removed (the sum of recovered and deaths) rate It, which are defined as follows.\n\nThe number of infectious cases in hospital Nt is defined as the cumulative confirmed cases with recovered ones and deaths removed up to t, that is\n\nThe daily infection rate Kt is defined as the ratio of the daily confirmed cases at time t and the number of infectious cases in hospital at time t − 1, i.e.\n\nSimilarly, the daily removed rate It is defined as the ratio of the daily removed cases at time t and the number of infectious cases in hospital at time t − 1, i.e.\n\nUsing the above indicators, we further define Rt as the outbreak status on day t as follow:\n\nObviously, it holds that\n\nIn this section, we will describe the whole process of a epidemic under the assumption that the government has implemented effective control measures, which can be divided into four stages, i.e. “outbreak period”, “controlled period”, “mitigation period” and “convergence period” successively. And we will quantify the iconic features for each stage, which corresponds to the two turning points and two “zero” points, respectively.\n\nStage 1: Outbreak Period\n\nIn the initial stage of an epidemic outbreak, there is delay of social response due to the limited knowledge of the epidemic, and the power of contagion prevention and control is inevitably not enough. Thus the daily infection rate Kt would be high. At the same time, the recovery process in the initial stage is relatively long, and the number of severe patients is small, leading the daily removed rate It to be close to “zero”. Therefore, the outbreak status indicator Rt during this period is usually much larger than 1, that is:\n\nAs the epidemic exacerbates, if the government begins to intervene through a series of emergency measures, where a disease prevention and control system is quickly established, the daily infection rate Kt will significantly decrease. Usually, the new daily confirmed cases will begin to decline as well. During the epidemic prevention and control process, once the situation improves, we will see the emergence of the first turning point denoted as T1. Then after the data T1, the newly diagnosed patients Et changes from a rapid rise in the outbreak period to a descending channel (Et < Et−1). In summary, the emergence of the first turning point T1 indicates that the disease control measures have begun to work, which implies the end of the “Outbreak Period”.\n\nStage 2: Controlled Period\n\nThe emergence of the first turning point is a very positive signal, indicating that the public health management measures have obviously taken effect and the epidemic has entered the “controlled period”. However, due to the fact that the completion rate It at this stage is still relatively low, the number of patients treated in hospital will continue to increase. The controlled period will continue until the second turning point T2 appears, that is, patients in hospital Nt reaches the peak and starts to decline. This is because the completion rate increase so significantly that Kt = It is fulfilled after a long period of treatment in the previous stage. When the completion rate It surpasses infection rate Kt, the number of patients treated in the hospital begins to decline from peak.\n\nStage 3: Mitigation Period\n\nThe sign of the end of the controlled period is Kt = It. Thereafter, Kt will continue to fall with the rise of It, which gives\n\nStage 4: Convergence Period\n\nThe “convergence period” will end at the second “zero” point Z2, which means that the number of people treated in the hospital is equal to or close to “zero”. After reaching the second “zero” point, the epidemic is completely over.\n\nFor clarity, we summarize the iconic features and the corresponding milepost moments of each stage in the whole process of the epidemic in Table 1.\n\nAccording to Section 2.2, the modeling and predicting of the epidemic need to be divided into two parts. The first part corresponds to the outbreak period, where the intervention and disease curing is not effective enough. The infection rate Kt increases rapidly and the completion rate It is small. Thus, the number of newly diagnosed patients Et increases rapidly, and the number of patients treated in hospital Nt increases. The pressure on medical resources will soon be overwhelmed. According to equation (2), Nt will be in an exponential growth trend without forming a convex curve, nor will the so-called two turning points or two “zero” points appear.\n\nThe second part, which is the focus of this article, is when the Kt starts to decrease and It starts to increase due to effective intervention and improved recovery level for individual patients. Only in this situation will the turning points and “zero” points T1, T2, Z1, Z2 successively appear, and then the epidemic could end. Therefore, we will model the development of the epidemic under the assumption of effective intervention, then we can obtain the early prediction of two turning points and two “zero” points based on the predicting modeling of Et and Nt.\n\nSuppose that the infection rate Kt and the removed rate It change gently within a time window m before time t0 with exponential growth, then given m and t0, denote VK\|(t0,m) and VI\|(t0,m) as the average change rate of Kt and It respectively, that is,\n\nAccording to the prediction process, it can be seen that the prediction results mainly depend on VK\|(t0,m) and VI\|(t0,m), whose value is up to the selection of time window m and starting point t0. However, it is worth noting that the selection of m and t0 is not arbitrary, which is suggested as in the follow assumption.\n\nAssumption 1. The time window m and the starting point t0 should be chosen satisfying VK\|(t0,m) < 1 and VI\|(t0,m) > 1. Meanwhile, keeping It < 1 due to interpretability constraints, and the starting point t0 should be close to the date of the latest published data as much as possible.\n\nIn summary, here we describe details of the proposed procedure in Algorithm 1.\n\n1: Initial setting m and t0, which satisfying Assumption 1;\n\n2: Compute VK and VI according to (3); Set t = t0 + 1.\n\n3: Prediction: updating the predicted results at time t via the forecasting value ahead of l = t−t0-step as follows:\n\n4: Prediction of the milepost moments: If Êt−1\|t0 < Êt\|tc, then T1 = t − 1; If N^t−1\|t0<N^t\|t0, then T2 = t − 1; If Êt−1\|t0 < E0 = 1, then Z1 = t − 1; If N^t−1\|t0<N0=1, then Z2 = t − 1; If none of the above is satisfied, turn to the next step.\n\n5: Set t = t + 1, return to Step 2 until T1, T2, Z1, Z2 are obtained.\n\nIt is also worth noting that in practice, the more data we accumulate, the clearer the underlying law of the epidemic. Therefore, we can also continuously modify the iterative prediction model according to the actual data, so that the prediction of the next stage and the prediction of the long-term situation can be more accurate.\n\n\n3 Application: Analysis of the COVID-19 in mainland China beyond Hubei Province\n\nWe apply our model to analyze and evaluate the COVID-19 using publicly available data from mainland China beyond Hubei Province from the China CDC during the period of Jan 29th, 2020, to Feb 29th, 2020. Here we first show the actual trend of the COVID-19, and then compared with the predicted ones via the proposed method. Finally, we will show the effect of m on the predicted results. All these results are implemented via R software.\n\nAfter the shutdown of most parts of Hubei province in Jan 23rd, other parts of China also immediately launched prevention and control strategies, including regional isolation, admission of all confirmed patients, isolating all suspected patients and so on. The effective implementation of these intervention policies quickly controlled the rapid spread of the epidemic in these areas. As can be seen in Figure 1, the parameter infectious rate Kt, which reflects the intensity of the spread of the epidemic, has shown a significant downward trend since Jan 27th after severe fluctuations from Jan 22nd to 26th. As can be seen in Figure 1, we find out that the daily confirmed cases peaked on Jan 30th, 2020, with 761 confirmed cases and then continued to decline for two consecutive days.\n\nHowever, the migration raised from people returning to work after Chinese New Year on Feb 3rd undermines the continuous decline of Et. Since Feb 2nd, the number of daily confirmed patients in mainland China beyond Hubei Province has increased for two consecutive days, where the Et on Feb 3rd has increased by 23% compared to that on Feb 2nd. It can be concluded that these fluctuations are caused by the resuming of social activities, which leads Et to continue to decline since Feb 4th. In many literature and media reports, Feb 3rd is used as the time point when the number of newly confirmed patients starts to decline. But considering the fact that the epidemic was already under control, here we still view Jan 30th as the first turning point.\n\nAfter that, the second turning point T2, which is the time point when the number of infectious cases in hospital Nt starts to decline, is also observed. Figure 2 shows the true curves of the daily infection rate Kt, daily removed rate It, and Nt calculated based on the actual data from mainland China beyond Hubei from Jan 22th, 2020 to Mar 13th, 2020. It can be seen that the second turning point T2 appeared on Feb 11th, with the emergence of Kt < It on that day, and the number of patients in the hospital continued to decreases since then.\n\nAs for the first “zero” point Z1, the definition is the time when the number of daily confirmed cases is equal to “zero”, which is too strict for the real situation. Thus, in this article, we take the criteria for cancelling travel warnings developed by the WTO during SARS as a reference, and make some adjustments to the definition of the first “zero” point: the time when the daily confirmed cases Et continues to be less than 5 for 3 days is revised to be Z1. Then, if we exclude confirmed cases that originated from abroad, daily confirmed cases has already become less than 5 since Mar 3rd in mainland China beyond Hubei Province, thus according to our revised definition, Mar 5th is Z1. However, there were still 1,089 patients in hospital on that day. Therefore, it would still take some extra time to reach the second “zero” point Z2.\n\nStarting from Jan 29th, we use the proposed forecasting method to make real-time predictions on the two turning points T1 and T2 and two ”zero” points Z1 and Z2 with window size m = 5. The specific and predicted results are as follows.\n\nWe first conducted the proposed prediction model on Jan 29th, which indicated that the first turning point T1 would arrive on Jan 31st, i.e., Et < Et − 1. In reality, the first turning point did arrive on Jan 30th, which is only one day away from our predicted result.\n\nAs for the second turning point, since the true T2 occurred on Feb 11th, we summarize the frequency of the prediction results obtained with t0 varying from Jan 29th to Feb 10th, 2020 and m = 5 in Figure 3(a). From it we can see that the prediction of the second turning point mainly concentrated in the range from Feb 9th to Feb 11th, which is consistent with the observed second turning point in reality. It is worth mentioning that we got the general information of T2 at a very early stage: we predicted on Feb 2nd that the second turning point T2 would arrive on Feb 11th, which is exactly the same as the second turning point that observed in reality. Since then, we have continuously tracked the rolling predictions, which have not yet changed much.\n\nSimilarly, Figure 3(b) and Figure 3(c) show the frequency of the prediction results for two “zero” points obtained with t0 varying from Jan 29th to Feb 29th, 2020 and m = 5, respectively. Specifically, for the predicted first “zero” point Z1 in Figure 3(b), we divide the prediction results from these days into 5 intervals, which can be seen that the prediction results of the first “zero” point Z1 are mainly concentrated on Mar 1st to 5th, which is consistent with the actual result. There is also a “pessimistic” prediction as a result of the sudden fluctuation of data on Feb 3rd, which predicted that the first “zero” point would arrive on Mar 17th. For the predicted second “zero” point Z2 in Figure 3(c), it can be seen that the second “zero” point will be reached from early-March to late-March. However, there is a prediction result that Z2 will appear on May 11th, which is far away from other results. The reason for this uncommon result is that the starting point of this forecast is Jan 29th, when the epidemic situation in mainland China beyond Hubei was still in the outbreak period with Et still rising, It very small, so the prediction result about the finish of the epidemic may not be accurate.\n\nFurthermore, we also present the forecast results of the four milepost moments together with the trend of the cumulative number of infectious cases in hospital N^t and the cumulative number of infectious ∑l=1tE^l in Figure 4 when the prediction starting point t0 fixed at Jan 29th, Jan 31st, Feb 12th and Feb 26th, 2020, respectively. As can be seen from Figure 4(a), in Jan 29th, which is the very early stage of the epidemic, we predicted that the first turning point would appear on Jan 31st, which is only one day behind the actual observation. Additionally, the time of the second turning point result predicted on that day was Feb 14th, which is only 3 days away from the reality. The first ”zero” and second ”zero” forecast results are Mar 7th and May 11th, respectively.\n\nForecasting results of the four milepost moments together with the trend of the cumulative number of infectious cases in hospital N^t and the cumulative number of infectious ∑l=1tEl compared with their observed cases Nt and ∑l=1tEl when the prediction starting point t0 fixed at Jan 29th (a), Jan 31st (b), Feb 12th (c) and Feb 26 (d), 2020, respectively.\n\nFigure 4(b) shows the prediction results when the first turning point have already appeared, from which we can see that the prediction for T2 on Jan 31st is accurately with the second turning point possible occurring on Feb 11th. Meanwhile the first “zero” point and the second “zero” point are predicted to appear around Mar 4th and Mar 23rd, respectively.\n\nSimilarly, after the arrival of the second “zero” point, Figure 4(c) shows the forecast results of the first and second “zero” points predicted on Feb 12th, which show the forecast results for Z1 and Z2 are on Mar 9th and Mar 25th, respectively. From the fitting results, we know that our prediction of the cumulative number of patients in hospital Nt and the total number of confirmed patients is very similar to the actual situation, so our prediction results are likely reliable. Finally, we also give a very recent (Feb 26th) forecast in Figure 4(d), which is similar to the results mentioned above.\n\nNote that the number of m plays an important role in the proposed procedure, and all the results we discussed in the section 3.2 are obtained with fixed m = 5. In this section, we will illustrate the impact of different choice of m on the results, and give the empirical choice in real data analysis. Parallel to Section 3.2, here we obtain the results for the second turning point and both “zero” points via implementation of the proposed procedure with m =3, 4, and 6, respectively. We summarize all these results for the second turning point and both “zero” points in Figure 5, respectively.\n\nFrom Figure 5, we can see that the highest frequency of prediction results for the second turning point occur around the period from Feb 9th to 11th for all choice of m, which means that the second turning point is most likely to occur during this period; similar results hold for the forecast of the first “zero” with the most likelihood of appearance around the early March. Both results show the limited influence of m on the results. From Figure 5(c), although the results of forecast frequency distributions for the second “zero” point with different m seem not as concentrated as those for the second turning point and the first “zero”, it varies slightly, with its occurrence from mid-March to mid-April. Overall, the choice of m seems not to be a critical value for the forecasting results, and we recommend its empirical choice from 3 to 6.\n\n\n4 Discussion and conclusion\n\nFocusing on the four meaningful mileposts, we put forward a simple and effective framework incorporating the effectiveness of the government control to forecast the whole process of a new unknown infectious disease in its early-outbreak. Specifically, we first propose a series of iconic indicators to characterize the extent of epidemic spread, and describe four periods of the whole process corresponding to the four meaningful milepost moments: two turning points and two “zero” points; then we develop the proposed procedure with mild and reasonable assumption, especially without relying on an assumption of epidemiological parameters for disease progression.\n\nWe examine our model with COVID-19 data in mainland China beyond Hubei province, which can detect the gross process of the epidemic at its early-outbreak. Specifically, in the first predicting task that conducted on Jan 29, the predicted date when the number of newly confirmed patients Et would fall for the first time is only one day behind the observation in reality. On Feb 2nd, our model predicted that the date when the number of patients in the hospital Nt reaches its peak is Feb 11th, which is consistent with the real world situation. Later, the forecasting results fluctuated but were overall stable and close to the true observation. Meanwhile, we predict that the first “zero” point Z1 will arrive between the end of Feb and the beginning of March. And the second “zero” point Z2 will arrive at mid-March to mid-April. We also checked the robustness of our model under different time windows and found that the selection of the time window has little effect on the prediction of turning points. As a prediction model for the task of early warning of a new epidemic, our prediction model is proved to be quite efficient.\n\nAt present, many countries around the world are overwhelmed by the COVID-19 epidemic, which calls for global efforts. While our method is able to depict and predict the trend of an epidemic at a very early stage, it can be used to predict the current COVID-19 epidemic internationally, or any other new, unknown, explosive epidemic in the future. We believe that the prediction results of this method can provide decision support for epidemic control and intervention. It is worth noting that, due to the short-term dependence of our method, our model may show poor performance for wildly fluctuating data. Thus, more data preprocessing methods like data smoothing need to be developed within our framework, in order to allow for wider use of our method.\n\n\nData availability\n\nThe underlying data and code required to replicate the studies finding are available from GitHub (https://github.com/Vicky-Zh/Tracking_and_forecasting_milepost_moments_of_COVID-19/tree/v1.0.0) and archived with Zenodo (Yanwen Zhang (2020)).\n\nZenodo: Vicky-Zh/Tracking and forecasting milepost moments of COVID-19: First release. http://doi.org/10.5281/zenodo.3755197 (Zhang (2020)).\n\nThis project contains the following underlying data:\n\nData of China Mainland Beyond Hubei.csv (A csv file with data collected from China CDC and four variables: the cumulative confirmed cases up to the given day t, the daily confirmed cases at day t, the daily recovered ones and the daily deaths at day t, with t from Jan 29th to Feb 29th, 2020)\n\nZenodo: Vicky-Zh/Tracking and forecasting milepost moments of COVID-19: First release. http://doi.org/10.5281/zenodo.3755197 (Zhang (2020)).\n\nThis project contains the following extended data:\n\ncode for prediction.R (R code for replication)\n\nData are available under the terms of the Creative Commons Zero ”No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAnastassopoulou C, Russo L, Tsakris A, et al.: Data-based analysis, modelling and forecasting of the novel coronavirus (2019-ncov) outbreak. medRxiv. 2020. Publisher Full Text\n\nBenvenuto D, Giovanetti M, Salemi M, et al.: The global spread of 2019-ncov: a molecular evolutionary analysis. Pathog Glob Health. 2020; 114(2): 64–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Z, Zhang W, Lu Y, et al.: From sars-cov to wuhan 2019-ncov outbreak: Similarity of early epidemic and prediction of future trends. bioRxiv. 2020. Publisher Full Text\n\nChinazzi M, Davis JT, Ajelli M, et al.: The effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak. Science. 2020; 368(6489): 395–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan Y, Zhao K, Shi ZL, et al.: Bat coronaviruses in China. Viruses. 2019; 11(3): pii: E210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolshue ML, DeBolt C, Lindquist S, et al.: First case of 2019 novel coronavirus in the united states. N Engl J Med. 2020; 382(10): 929–936. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHui DS, EI Azhar E, Madani TA, et al.: The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health - The latest 2019 novel coronavirus outbreak in Wuhan, China. Int J Infect Dis. 2020; 91: 264–266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q, Guan X, Wu P, et al.: Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med. 2020; 382(13): 1199–1207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuk HKH, Li X, Fung J, et al.: Molecular epidemiology, evolution and phylogeny of sars coronavirus. Infect Genet Evol. 2019; 71: 21–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng L, Yang W, Zhang D, et al.: Epidemic analysis of covid-19 in China by dynamical modeling. medRxiv. 2020. Publisher Full Text\n\nPrasse B, Achterberg MA, Ma L, et al.: Network-based prediction of the 2019-ncov epidemic outbreak in the chinese province Hubei. arXiv preprint arXiv: 2002.04482. 2020. Reference Source\n\nTang B, Wang X, Li Q, et al.: Estimation of the transmission risk of the 2019-ncov and its implication for public health interventions. J Clin Med. 2020; 9(2): pii: E462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Novel coronavirus (2019-ncov) situation reports. 2020. Reference Source\n\nWu JT, Leung K, Leung GM: Nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in Wuhan, China: a modelling study. Lancet. 2020; 395(10225): 689–697. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu WB, Tang GD, Zhang L, et al.: Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2 / HCoV-19) using whole genomic data. Zool Res. 2020; 29: 1–11. PubMed Abstract \| Publisher Full Text\n\nZeng T, Zhang Y, Li Z, et al.: Predictions of 2019-ncov transmission ending via comprehensive methods. arXiv preprint arXiv: 2002.04945. 2020. Reference Source\n\nZhang Y: Vicky-zh/tracking and forecasting milepost moments of covid-19: First release. 2020. https://www.zenodo.org/record/3755197\n\nZhang H, Kang Z, Gong H, et al.: The digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes. BioRxiv. 2020. Publisher Full Text\n\nZhao S, Lin Q, Ran J, et al.: Preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in China, from 2019 to 2020: A data-driven analysis in the early phase of the outbreak. Int J Infect Dis. 2020; 92: 214-217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou T, Liu Q, Yang Z, et al.: Preliminary prediction of the basic reproduction number of the wuhan novel coronavirus 2019-ncov. J Evid Based Med. 2020; 13(1): 3–7. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63126", "date": "17 Jun 2020", "name": "Rosanna Verde", "expertise": [ "Reviewer Expertise Statistical Data Analysis", "Data Stream Analysis", "Functional Data Analysis", "Symbolic Data Analysis", "Clustering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 deals with a forecasting procedure to analyse and evaluate the early stage of the COVID-19 outbreak in China. Inspite the classical SEIR model, the authors define a set of iconic indicators to study the status of disease contagion and the extent of epidemic spread. Then, they divide the cycle of the epidemic in four stages corresponding to the four meaningful milepost moments: two turning points and two “zero” points, related to the tuning of the proposed indicators.\nThe authors characterize the first stage: the Outbreak period, by its end, which is represented by the arrival of the first tuning point (T_1), when the newly diagnosed patients (denoted E_t) after a rapid rise, begin to decrease (E_t < E_{t-1}). The second stage is the Controlled period, that sees an increasing of hospitalised patients (denoted N_t) until a second turning point (T_2), when N_t, after to have reached a peak, starts to decline.\nThe Mitigation stage corresponds to the third stage and it continues until the daily confirmed cases reach the zero (E_t=0). That represents the first “zero” point (Z_1). This condition is also reformulated and referred to the time when the daily confirmed cases are less then “5” for 3 consecutive days.\n\nThe Convergence Period, the four stage, is characterized to reach a second “zero” point (Z_2), when the number of hospitalised patients is closed to zero (N_t=0). That signs the end of the epidemic.\nThe procedure is corroborated on public available data in mainland China beyond Hubei Province from the China CDC during the period of Jan 29th, 2020, to Feb 29th, 2020. The results show the proposed procedure has provided to a prediction of the tuning and “zero” points, very near to the exact dates also in a very large stage of the epidemic.\nAn analysis of the robustness of the method has also been performed on the time windows and on the four milepost moments. The results have revealed the influence of the parameter “m”, related to the width of the time windows used to estimate the average change rate of the daily diagnosed cases and of the daily removed cases.\n\nThe paper is clear and smoothly written. I agree for its indexing after to have kept into consideration the following remarks:\n\nSection 2. Methods Equation (1)\n\n1) N_l in brackets (1+K_l - N_l) should to be replaced with I_l\n\nSection 2.3\n\n2) I suggest of referring explicitly the exponential model of the infection rate K_t and of the removed rate I_t (in the window m);\n\n3) the term “average” for V_{K\|(t0,m)} (and V_{I\|(t0,m)}) is confused, it would be better to refer to it as “unitary rate of change” (and “unitary rate of removed”). I think, that is more properly the “rate of change” (and the “rate of removed”) associated with a unitary time step.\n\nAlgorithm 1 Main Prediction Procedure. 3 Prediction: updating the predicted results …..\n\n4) The first equation R_t\|to = \\hat{R_t\|to (l)} should to be replaced with K_t\|to = \\hat{K_t\|to (l)}\n\nSection 3: Application\n\n5) All the Figures must be improved, especially the Figure 2. And the Figure 4.\n\n6) The proposed strategy has been verified on data referred to a very early stage of the epidemic and it performs short-term dependency. Given to the spread of the epidemic in world wide, maybe it can be proved on data related to the outbreak of the pandemic in another Countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "63123", "date": "14 Jul 2020", "name": "Paula Brito", "expertise": [ "Reviewer Expertise Multivariate Data Analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors propose a methodology aimed at forecasting milepost moments of an epidemic in the early outbreak. This is then applied to the COVID-19 outbreak in China. The method lays on the definition of the relevant involved variables :\nEt : number of new confirmed cases at date t Ot : number of recovered cases at date t Dt : number of deaths at date t Nt : number of infectious cases in hospital at date t Kt : infection rate at date t It : removal rate at date t Rt : outbreak status at date t\nThe milepost moments to be predicted are\nthe first turning point, T1 – when the number of newly diagnosed patients Et starts decreasing; the second turning point, T2 – when number of active cases Nt starts decreasing; the first zero point, Z1– when the number of newly diagnosed patients Et becomes null ; the second zero point, Z2– when the number of active cases Nt becomes null.\nThe procedure depends on the starting point t0, and on the size of the time window used, m. It is assumed that Kt and It change gently within time window m before t0, so that the average change rate of both Kt and It within that period are reliable values. Under those assumptions, recurrence formulas allow predicting the required time points.\n\nThe results reported from the application of the proposed methodology to the China Covid-19 data are quite good, predicting well the observed time points, and hence show that the method is well founded and useful.\n\nThe manuscript is quite well written, and is easy to follow.\nThe pertinence and actuality of the topic go without saying. The method appears sound to me, and is not extremely complicated, neither relying on extraordinary assumptions.\nI consider this is an interesting and nice piece of work, and I am therefore in favour of acceptance.\n\nHowever, a few issues need consideration/correction, as described here below.\nThe authors should revise the manuscript taking these aspects into account.\nMajor points\nThe success of the proposed methodology lays on the fact that the values of the average change rate of both Kt and It, within the considered time window period, are reliable and stable - so that they may be used for prediction. It is not clear to me how this condition may be assessed, or what are the criteria involved. This should be clarified. The fact that the reported predictions for China are indeed good, result, in my opinion, from the fact that the local situation has been stable, measures taken and local conditions not changing before the general end of the outbreak (or so it is perceived from the outside). If this were not the case, I wonder whether the method would still apply – which then questions its applicability in other countries /regions where such stability is not guaranteed/observed. This emphasises the need for a clear establishment of applicability conditions. This is my major concern.\n\nI am curious about the prediction results if the starting point is a bit earlier, and not as close to T1 as January 29th.\n\nNt is defined as “number of infectious cases in hospital at date t”. I believe this is what is generally referred to as “active cases” – also because in many countries not all diseased people are actually in hospital. I suggest using this terminology, so that it becomes clear to a general reader. In that case, the reference to “in hospital” should be updated along the manuscript.\n\nAlgorithm 1 : there are some typos here, that require attentive correction: in point 3, - formula 1: it should be K and not R , in both left and middle terms - formula 3, right term: R should be replaced by K\n\nMinor issues\n\nPage 1, line 15 of the Abstract : assumption -> assumptions Page 1, line 16 of the Abstract : COVID-19 -> COVID-19 outbreak Page 1, line 25 of the Abstract : counties -> countries\n\nPage 3, line 35 : results -> result\n\nPage 4, line 30 : generalized -> used Page 4, line 36 : assumption -> assumptions\n\nPage 8, Algorithm 1, line 2 : satisfying -> satisfy Page 8, Section 3, line 7 : in Jan 23rd -> on Jan 23rd\n\nPage 9, line 12 : decreases -> decrease\n\nPage 10, line 15 : in Jan 29th -> on Jan 29th Page 10, line (-13) : zero -> turning\n\nPage 11 – Figure 5 : the labels along the horizontal axis are not centered, therefore the figure is not clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-333
https://f1000research.com/articles/9-602/v1	12 Jun 20	{ "type": "Opinion Article", "title": "Poikilosis – pervasive biological variation", "authors": [ "Mauno Vihinen" ], "abstract": "Biological systems are dynamic and display heterogeneity at all levels. Ubiquitous heterogeneity, here called for poikilosis, is an integral and important property of organisms and in molecules, systems and processes within them. Traditionally, heterogeneity in biology and experiments has been considered as unwanted noise, here poikilosis is shown to be the normal state. Acceptable variation ranges are called as lagom. Non-lagom, variations that are too extensive, have negative effects, which influence interconnected levels and once the variation is large enough cause a disease and can lead even to death. Poikilosis has numerous applications and consequences e.g. for how to design, analyze and report experiments, how to develop and apply prediction and modelling methods, and in diagnosis and treatment of diseases. Poikilosis-aware new and practical definitions are provided for life, death, senescence, disease, and lagom. Poikilosis is the first new unifying theory in biology since evolution and should be considered in every scientific study.", "keywords": [ "biological heterogeneity", "poikilosis", "noise", "unifying theory", "lagom", "effective variation" ], "content": "Poikilosis\n\nBiological systems are dynamic and display ubiquitous heterogeneity and variation at all levels and processes. To investigate, describe and understand the entirety of variation and its significance, a new concept – poikilosis (poikilos, ποικιλός in Ancient Greek for “variable” or “variegated”, and -osis, -ωσις for a suffix of “state, condition or action” – is defined. Variation is considered as an integral and important property that has numerous consequences.\n\nPoikilosis is inherent pervasive variation, heterogeneity and fluctuation in living organisms, populations, ecosystems, biosphere and in their components and in processes within them.\n\nAlthough PubMed lists 353,467 articles about heterogeneity (May 2020), until now there has been no general theory or framework to combine and explain the effects and properties of heterogeneity and variation. Poikilosis is discussed here in life sciences, however, it appears everywhere in nature and is relevant for chemistry and physics, as well for social sciences, humanities, economics and other disciplines. Poikilosis is an intrinsic property of all living organisms and a driver and cause of several phenomena.\n\nHeterogeneity, and more generally poikilosis, has been largely regarded in science as noise and negative nuisance to be get rid of and to be avoided. Noise and poikilosis together affect what can be measured and perceived. Noise relates to measurements, according to the Wikipedia article for signal processing it is “unwanted (and in general, unknown) modifications that a signal may suffer during capture, storage, transmission, processing or conversion”. Poikilosis is inherent variation within biological systems, not in the measurements.\n\nSome examples of biological heterogeneity include uncertainty of positions of electrons1, stochastic gene expression2, DNA sequence differences that lead to >10,000 amino acid substitutions in each individual in comparison to human reference sequence3, variants in one gene may be related to several diseases4 and one variant can lead to different phenotypes5, differences between individual genomes and in comparison to pangenome6, heterogeneity of isogenic bacteria7 and human cells8, protein structural flexibility9 and dynamics10, fluctuating enzyme catalytic rates11, heterogeneity in cellular machineries like ribosomes12, differences in protein post translational modifications13, asymmetric inheritance of degradative machineries and cell fates14, protein abundance differences between individuals including twins15, phenotypic plasticity16, continuum of sex17, incomplete penetrance of diseases18, differential cellular19 and individual20 drug responses, diversity of gut microbiota21, and predator-prey dynamics22. Phenotypic and genetic variation23 and ecological heterogeneity24,25 have been extensively reviewed.\n\nEvery system and process can be thought to represent its own level. In cells there are levels e.g. for genetic information, DNA, RNA and protein activity and expression, metabolic and signalling pathways. All biological processes, molecules and systems display heterogeneity and many levels are interconnected and affect each other (Figure 1). Poikilosis has a huge number of origins of intrinsic and extrinsic type. These include stochastic processes and reactions, promiscuity and non-specificity of reactions and interactions, germline and somatic genetic variations, epigenetic alterations, erroneous repair mechanism, unspecific post translational modifications and other regulatory mechanisms, environmental effects etc. (for a review of variation generating cellular mechanisms see 26 and for protein variations27). A factor can be both intrinsic and extrinsic depending on the level, e.g. what is extrinsic at the cellular level may be intrinsic for a tissue and organism.\n\nInterconnected tori in red, magenta and green indicate three of the multiple levels that interact and overlap and thereby can affect each other. Processes in living organisms are cyclic, therefore the torus shapes. Matter, energy and information flow in cyclic processes. In disease there is a deviation and by curative treatment it is still possible to return back to normal, lagom level. A large and severe deviation, which is not treated with curative care, can permanently reduce the function and adaptation capacity of the organism. Death in individual level and extinction in population level are irreversible escapes from the system. Reproduction generates new individuals that have their own interconnected levels.\n\nPoikilosis emerges both actively and passively and due to intrinsic and extrinsic factors and effects. It penetrates all levels in biological systems and time wise ranges from less than a femtosecond for atom bond length and angle vibrations, to hundreds or thousands of years for individual organisms and millions of years for evolution. Poikilosis facilitates biodiversity of species, populations and ecosystems within biosphere, differences between cells, individuals and in populations, differences at genetic, molecular, structural, physiological, interindividual and other levels, and thereby a large pool of possible responses to changes in conditions.\n\nAlthough biological variations have had largely negative connotations, there are some accounts of positive effects e.g. in increased cell-cell variability to cope with acute environmental stress28, in mutation rate heterogeneity to increase odds of survival29, in gene expression and signal transduction30, in robustness of populations31, and in ecological resilience32. Large body of literature deals with biological noise and how to avoid and treat it, reviewed in 33. It is more fruitful to consider variation as a neutral or positive property, which is intrinsic to every system.\n\nPoikilosis provides a new unifying theory for biology. It is compatible with many current concepts such as evolution, inheritance and selection, process regulation, continuum of pathogenicity, as wells as modern and post-modern synthesis, but it has much wider application area ranging from subatomic level to populations and ecosystems. On the other hand, poikilosis replaces some established concepts such as homeostasis and other fixed standard state conceptions.\n\n\nNew definition for life\n\nTo further discuss the properties, characteristics and consequences of poikilosis we have to start by defining life. Although there is no lack of definitions for life, see e.g. 34,35, none of them takes poikilosis fully into account. The closest has come Rollin D. Hotchkiss, who defined “Life is repetitive reproduction of ordered heterogeneity.”36 However, this definition is too general for our purpose and for the treatise in here it is sufficient to make the following definition:\n\nLife is cyclic flow of compartmentalized information, matter and energy in processes that form a self-reproductive system. Poikilosis emerges in organisms at all levels and can be selected at population level.\n\nCompartmentalization is essential to prevent molecules, energy and matter from being diluted to the environment. Living organisms contain highly increased or decreased concentrations of many molecules and atoms. The known life forms are compartmentalized to cells, which act as the basic units of life both for single- and multi-cell organisms.\n\nLife forms produce, degrade and convert matter and consume and convert energy based on information that guides processes such as metabolism, catabolism, and signalling networks and development of new individuals. One type of poikilosis, genetic variation, can alter the inherited information and forms the basis for evolution. Variations have to be fixed to have a wider impact in a population. This happens via natural selection. The definition of life contains thus individual and population level components and includes enrichment of variations from individuals to population.\n\nInformation, energy and matter flow in a cyclic manner. For example, biomolecules are synthetized and degraded in cycles, and similarly genetic information in the form of polynucleotides is replicated and expressed in cycles. Information types include genetic information coded into DNA or RNA, epigenetic information, and information in signalling pathways, regulatory networks, immune system and others.\n\nReproduction is essential for the continuation and renewal of life forms. Life is self-reproducing and does not require outside forces for continuation. Life is penetrated by poikilosis, every living thing is unique and somewhat different from others, in its constitution, function and responses and even in its dysfunction.\n\nLife can be visualized as concentric overlapping tori that indicate the cyclic renewing nature at different levels and the interactions of these levels (Figure 1). Each toroid represents one level. Living organisms are in constant interaction with external and internal factors and conditions. When variations exceed acceptable levels, disease appears as a consequence. Escape from the system leads to death at individual level and to extinction at population level.\n\nLife appears in numerous forms all of which follow the same general principles as exemplified by shared metabolic pathways and almost universal genetic code. The purpose of life is survival and continuation by adjusting to the prevailing conditions and by reproduction. An organism can adapt by adjusting itself and its responses to internal and external challenges. In populations, selection and survival facilitate evolution and adaptation.\n\nIn death, an organism loses control of variation effects, which leads to irreversible collapse of vital processes. In a multicellular organism, systems and cells die at different pace depending on their vulnerability ref. 37 and references therein.\n\n\nEffective variation\n\nVariations and heterogeneity have a spectrum of effects. The total magnitude of a variation V can be presented as\n\nRecently functional effects of protein variants were reviewed and TARAR countermeasures were defined as biological processes that reduce variation effects (Vihinen, submitted38). The model was introduced in relation to protein functional effects, but it is generic and applies to all types of effects. The T stands for tolerance, A for avoidance, R for repair, the second A for attenuation, and the last R for resistance. Similar processes apparently reduce and limit effects at all levels. Behind these five features there are a plethora of mechanisms, different at different levels.\n\nAvoidance relates to threat management39 and tolerance to ability to survive and thrive with a perturbation such as an infectious agent or genetic variation40. Disease tolerance was introduced in relation to immunology to describe processes that reduce the negative impact of infections with no or even positive effect on the fitness of the infectious organism41. Mounting immune system may cause more collateral damage than the tolerance of the agent. The concept of tolerance has subsequently been expanded to other effects and disease areas.\n\nNumerous repair and rescue mechanisms actively reduce the consequences of variants at several levels. For example, genomic and dosage suppression42 can restore or limit effects of genetic variants. Chaperones as general rescue molecules assist proteins to fold correctly43, even if they contain a variant(s) causing somewhat defective structure and/or function. Certain activity effects can be overcome completely or partly also by promiscuity of related proteins44.\n\nAttenuation mechanisms are active or passive and include in-built resilience and robustness45 as well as canalization46 that returns the system back to the original path after perturbation. Robustness means resistance to intrinsic variation or environmental change. Redundancy is one of the simplest forms of attenuation47. Rewiring of metabolic and signalling pathways crosslinks and reduces effects on pathways48. Metabolic rewiring is in fact a hallmark for many cancers49. Resistance reactions and processes actively and passively resist and reduce effects of variations and perturbations. In many diseases, genetic variants show variable expressivity or incomplete penetrance50. The combined contribution of TARAR mechanisms reduces the extent of E. Consequences on a level may be limited to that level if the effect in other level(s) does not have a major contribution.\n\n\nLagom: poikilosis under control\n\nAlthough poikilosis is pervasive, all variations and their extents are not compatible with biological processes and systems and thereby are not acceptable. Acceptable variation ranges are here called as lagom.\n\nLagom means suitable, sufficient, allowed and tolerated extent of variation at any level in an organism, population, biological system or process.\n\nLagom is a central concept in Sweden and in Swedish, where it means sufficient, not too much not too little, in other words balanced and just right. Lagom carries the connotation of appropriateness, but not perfection.\n\nVariation zone is an artificial reconstruction of the lagom extent of variation for a poikilosis component. Variation zones are dynamic, both the positioning of the zone and extent of variation within it are variable and dependent on situation, environmental condition etc. see Figure 2A. The large pipe indicates the universe of possible variation within one level, while the smaller shape indicates dynamic lagom variation. TARAR mechanisms limit and reduce consequences of variations to lagom extent in normal situations.\n\nA. Part of one torus indicating the possible range of variation (outer tube) and variation zone (inner shape) within a level. B. Cost of feedback control the efficiency of which is in quadric power. The smaller the allowed variation, the larger the cost. Variation within lagom extent is costless, whereas set point-based homeostasis would mean extensive cost. The circles indicate the reduction of heterogeneity from original situation to half, one fifth and to one tenth. The increasing control costs are shown to the right.\n\nBiological systems and organisms contain many regulated processes. Well known examples are human blood glucose level and body temperature. Even these processes display heterogeneity. Recently, single-cell studies have revealed wide variations in many systems that previously were anticipated to be homogeneous51,52.\n\nHomeostatis53,54, and its updated versions homeorhesis55 and allostasis56, is based on the concept of a static ideal state, a “set point”, to which the system is actively returned by negative feedback loops after any change or perturbation. Homeostasis and other set point-based conceptions are not compatible with poikilosis. Poikilosis restricted by lagom performs similar regulation but is conceptually exactly opposite idea of variation and its restriction.\n\nLife does not strive towards perfection, instead at lagom i.e. sufficient and relevant reactions and responses. As an example, enzymes are essential catalysts that facilitate reactions spontaneous rates of which are far too slow for living systems. They can increase reaction rates up to 1026 fold57, although most enzymes are much less efficient, since there is no need for the highest reaction rate and nature does not optimize reactions and systems beyond sufficient performance. Similarly, enzymes and reactions are not entirely specific. The rigid lock and key model58 does not describe reality of biological interactions, since enzymes are promiscuous and process a range of substrates and may have several activities. The goal of life is survival, not perfection, which means that it is relevant and sufficient to have lagom activities and processes, not more efficient. Thus, there is no selection pressure to increase activity or functionality beyond relevant and sufficient extent or to regulate a system beyond what is pertinent for it.\n\nExcessive variation is harmful and e.g. fetuses with too large variations are not viable and cause miscarriage in higher organisms59. On the other hand, too restricted or limited variation has negative consequences. Endangered species degenerate because their pool of genetic variation is too restricted60. Similarly, consanguinity leads to limited variation and enrichment of genetic defects and diseases in a population61.\n\n\nRedefining disease and death\n\nNon-lagom variations have negative effects which affect interconnected levels. The consequences do not remain at one level, unless the effects on the other level(s) are within lagom extent for that level. Once the effects are large enough a disease emerges. Too large a variation has multilevel effects first locally but can evolve and spread to become systemic. A new definition for disease is warranted.\n\nDisease is a systemic deviation, defect or failure due to non-lagom variation leading to cumulative consequences in several levels.\n\nThis is related to but different from naturalist definitions including biostatistical theory62.\n\nPhenotypic heterogeneity within a disease can hamper accurate diagnosis as the phenotype, signs, symptoms, and laboratory values could match with several diseases. Since many levels are connected to others, similar effects and signs can originate from different primary variations in different levels. Disease consequences and symptoms vary according to non-lagom variation extent and cause, as well as due to progression, duration and severity of the condition. A pandemic is a disease at (sub)population level occurring when a large number of individuals has the same vulnerability for an infectious agent.\n\nThe extent of multilevel effects has wide individual variation range. In the case of smaller variations, the system returns back to lagom level relatively quickly and without major consequences. Larger deviations may lead to damage of some kind and possibly impair or reduce the functionality and adaptability of the system or organism. In extreme cases of most severe conditions, there is a domino-like effect spreading effects to new levels and eventually leading to death. The systemic extent varies markedly between diseases and between individuals suffering of the same disease. Low grade inflammation is an example of mainly tolerated condition, which however is a risk factor for a number of diseases.\n\nDeath is caused by excessive multilevel variations that irreversibly collapse vital processes and functions, and spread to become systemwide.\n\nCurative treatments aim to reconstitute the system back to lagom extent of variation on all the affected levels. Such treatments are available just for a small fraction of known diseases, therefore, many conditions are treated with palliative care to reduce the extent of variation effects. Multimorbidities are challenging to diagnose and treat since many connected levels, systems and processes are simultaneously affected.\n\nOrganisms change gradually during time. Senescence per se is not a disease but can contribute to many diseases. It can be defined as follows:\n\nSenescence originates from lifetime accumulation of variations in an organism. Although many of these variations are corrected, attenuated, resisted or tolerated, the increasing burden of variations eventually leads to permanent non-lagom effects and costs for the individual.\n\nDepending on the combination of variations, senescence-related effects and their severity vary between individuals. Persistent variations cause chronic effects and become a burden.\n\nAs shown above, poikilosis is an integral component in medicine, therefore diagnosis and treatment will require a new line of thinking how to define diseases. The changes may not have to be extensive since some aspects of variation and heterogeneity are already taken into account in certain specialities. Many-valued logic with more than two or even with infinite number of truth values has been applied to diagnosis and other applications in some diseases63,64. One way to take heterogeneity in diseases into account is to apply pathogenicity model that describes the continuum of a disease as a joint outcome of three factors: extent, modulation and severity65. Implementation of the pathogenicity model will facilitate the comprehension of variation as well as its consequences for making diagnosis, decision on treatment and e.g. pharmacogenetics and patient stratification66.\n\n\nCost for poikilosis is moderate\n\nThe costs of poikilosis are estimated here from three perspectives. First, effective variation restricts and reduces costs of many variants and lagom means that a range of variations is accepted without extra costs. Second, poikilosis reduces the generated Gibbs free energy in living organisms to nominal level. Third, maintenance of a system at lagom level bears only modest costs.\n\nCost C of a variation at level i can be formulated as\n\nLagom variation extent reduces the net costs of biological systems as variations within the variation zone, where variation lies for most of the time in normal situations, do not bear any extra cost. Perfect performance or phenotype would require extensive costs in surveillance, repair and other expenses. Life and nature do not gain any benefit from perfection. For example, fluctuating asymmetry has often been considered as a developmental instability and deviation67, however there is no benefit for perfect symmetry. Similarly, protein activity has just to be sufficient, not at highest possible speed, specificity etc.\n\nOrganisms are open systems, thus increased order caused by life is not against the second law of thermodynamics. According to this law the total entropy in a closed system remains the same or increases, but does not decrease, over time. Organisms are not closed systems, they are part of their surroundings. They input free energy and export entropy in the form of waste and heat. As life requires sufficient and not perfect i.e. lagom organization, the effect on entropy, more precisely on Gibbs free energy, is not excessive. In comparison, homeostasis would require substantial contribution to free energy to keep the system at a set point.\n\nThe magnitude of the Gibbs energy difference due to life could be analogous to stabilizing effects in globular proteins, where there is only a small 3-15 kcal/mol difference between the folded and unfolded states68, an amount that equals the sum of just a few bonds and interactions.\n\nHomeostasis means a stable constant state that has to be actively maintained with negative feedback control. To implement such a system, extensive and costly monitoring and regulation is needed. Even the most effective biological feedback circuits reduce the variation with the fourth root of the number of signalling events (number of control molecules)69. To reduce the standard deviation of variation to half requires 16-fold (24) excess of control molecules (Figure 2B). More stringent regulation by 10-fold would demand at least 10,000 i.e. 104 times excess of the monitoring molecules. Thus, set point-based control mechanisms (homeostasis, homeorhesis, allostasis, proteostasis etc.) are not feasible due to the excessive cost for the production and maintenance of the control machinery. Maintenance of poikilosis at lagom level within variation zone introduces only a low or modest cost (Figure 2B) and is energetically and cost-wise feasible but it still facilitates the required control.\n\n\nCorrelation to evolution and survival\n\nPoikilosis is fully compatible with the evolutionary theory and in fact it facilitates evolution as it provides states from which to choose fitted combinations by natural selection. It provides material for selection, but not only genetic variations.\n\nThe concept of the survival of the fittest actually means the survival of the individuals which have a relevant combination of tolerance, resistance and attenuation and suitable lagom variation. It does not mean that the strongest or fastest or any other property described with a superlative would be the fittest. Large enough poikilosis in a population guarantees the survival of at least some individuals in all but the most drastic changes in environmental conditions. However, poikilosis has to be kept at lagom level since in a stable situation excessive variation would have negative effects. Which genetic and possibly epigenetic variants are essential for adaptation depends on the situation. Founder variants may not have been the most optimal alterations for survival but were enriched due to being present in the population when needed.\n\nWhich variants are fixed in a population depends on many factors, population size being an important one. Natural selection, genetic drift and genetic variations are weak evolutionary forces at generation level, their strength comes over extended time frames70. Protein structural epistasis, where a compensatory variant rescues and saves from harmful effects of a variant71, has a strong impact on evolutionary trajectories.\n\nMathematical formulation of evolution as a differential equation of motion revealed that evolution can be described with the second law of thermodynamics as an energy transfer process. Based on this model, natural selection favours variants that lead to faster entropy increase in the system72. Thus, the most probable path of evolution follows the steepest energy descent.\n\n\nPoikilosis-aware study design, experimentation and data analysis\n\nBy considering poikilosis more realistic analysis, prediction and modelling of biological systems can be achieved. Changes will be required to concepts, experiments, analyses, predictors, models and simulations to fully include poikilosis as an intrinsic feature of systems instead of trying to get rid of unwanted “noise”. Full consideration of poikilosis requires five steps: understanding the investigated phenomenon and variation and its lagom extent, knowing and testing effects of noise, detailed description and annotation of experiments, experimental design including poikilosis, and data analysis and interpretation that are aware of poikilosis.\n\nThe first step for including poikilosis is understanding the investigated phenomenon or process and variation within it. Variation Ontology (VariO) is an example of systematics for describing variation within a knowledge domain73. It was designed to describe effects, consequences, mechanisms and types of variations at DNA, RNA and protein levels. Detailed explanations and examples have been published for protein and DNA variants74,75 and for RNA variations (Vihinen, submitted76). This kind of framework lends power for describing the type, extent and context of observed poikilosis.\n\nMeasurements and experiments contain components of both poikilosis and noise. Second, it is essential to discern the effects of noise that confound the true signal from experiments. How that should be done varies for the investigated systems and used measurement instruments as the signal to noise ratio may not be linear over the investigated measurement range and therefore may command for use of advanced approaches.\n\nTo facilitate true comparisons of experiments, the third step demands very detailed description of the used methods, instruments, reagents, samples, experimental conditions and other details. These annotations have to be much more thorough than currently customary in many scientific journals. Several best and good practice guidelines and minimum information requirements have been published to describe various aspects of experiments, many of which are available at FAIRsharing77. Systematics and harmonization of these annotations are of utmost importance to facilitate reproducibility and analyses and comparisons of data sets from different laboratories and consortia.\n\nFourth, poikilosis has to be included already into experimental design and conduct. Noise and poikilosis jointly affect studies and have to be divided into components. It is likely that in many studies the number of replicates has to be increased compared to the current approaches to chart the extent and characteristics of poikilosis and noise. To confirm the observations, it is recommended to repeat the experiments in another independent but related system, like cell line, population or habitat, whatever is relevant for the study.\n\nIn the fifth stage, data analysis has to be geared towards poikilosis. Some steps have already been taken to consider poikilosis as an intrinsic component of systems. Examples include probabilistic trait loci in genetics78, cell population modelling in biology79 and pathogenicity model in diseases65. However, it is obvious that new physical and mathematical models are needed in many fields to fully capture the extent and significance of variation80.\n\nTraditionally, many studies have been based on metrics for the point estimates of population average for investigated items and thereby completely neglecting poikilosis. Gough et al.81 discuss metrics of heterogeneity in regard to the shape (modality) of the distribution, extent or diversity, and the tails of the distribution. They list approaches that have been used to address these aspects including univariate, Gaussian statistics, Gaussian models and nonparametric statistics, entropy, spatial, temporary and combined metrices. Squared coefficient of variation and Fano factor have been applied in some areas, however have assumptions that do not hold with real data26. Noise filtering methods, like Kalman filter, and information measures including Shannon entropy and Gini index, quantify heterogeneity and with suitable data could be used for studies of poikilosis. The shape of the distribution and its visualisation inform about the type of modality. Current methods for the analysis of the overall distribution and tails each have their pros and cons but do not fully cover poikilosis.\n\nThe majority of available prediction methods in many fields are binary in design without consideration of the continuum of variation. This is the case also in tools for genetic variation interpretation. Most variation tolerance/pathogenicity predictors consider two states, benign and pathogenic. More realistic approaches are needed. For example, PON-P282 predicts variants in three categories, benign, pathogenic and of unknown or variable effect. The first generic variant severity predictor PON-PS83 has also three categories for benign, mild and severe phenotypes. Whether more detailed grading is required, depends on the application, however, the number of predicted classes is often limited by the amount of known experimentally validated cases.\n\n\nConnotations and implications of poikilosis\n\nAlthough literature on heterogeneity is voluminous, there are not many studies that have tried to organize or provide theory for it. Heraclitus of Ephesus had as a cornerstone of his philosophy panta rhei (πάντα ῥεῖ), meaning everything flows and changes i.e. pervasive flux, change or becoming (quoted in Simplicius' Commentary on Aristotle's Physics). The idea of pervasive cellular variation was presented by Elsasser84, and more recently variation was divided into three categories: population, spatial and temporary heterogeneity81, but there are many more levels as shown above.\n\nEvolution has been the only unifying theory in biology. It was subsequently combined with genetics to form modern synthesis. More recently, efforts have been made for post-modern or extended synthesis by including e.g. epigenetics and evo-devo aspects. Poikilosis is compatible with these central theories and goes much wider and beyond inherited traits. Poikilosis is a generic concept describing and based on variation at all possible levels in organisms and systems.\n\nEvolution is facilitated by poikilosis. The inherent heterogeneity of all processes and levels including genetic variation means that populations contain wide spectra of variations and states from which to choose the fitted ones, if needed. The old statement of the survival of the fittest could be rephrased as survival of the variable meaning that traits that facilitate adaptation to new situations are selected. Poikilosis provides variations for organisms to adapt, for phenotypic heterogeneity, and it facilitates strategies as bet-hedging85 and eventually it provides material for natural selection and evolution.\n\nDuring recent years, reproducibility of scientific studies and their results have been brought up since many investigations published even in the most prominent journals could not have been repeated86,87. There are numerous reasons for the irreproducibility, the lack of consideration of inherent poikilosis being one of them. Poikilosis should be taken into account already in the design of experiments, in conduction of studies and analysis and interpretation of results. Recent suggestion to (again) retire the concept of statistical significance88 has emerged due to erroneous description of differences and their meaning. Knowing the intrinsic poikilosis of a system is a prerequisite for understanding differences. As example, very large genetic, transcriptional, translational and turnover rate differences were noted in widely used cell lines, in 27 strains of the breast cancer cell line MCF7, and 14 stocks of HeLa cells from different laboratories, respectively89,90. Cell lines show marked differences also for cancer drug responses89. Single cell study of human fibroblasts, which are considered as a very homogenous cell population, revealed large variations in three dimensional global genome organization91. Thus, studies even on standard systems without considering poikilosis at many levels are likely to fail or at least provide somewhat misleading outcomes.\n\nRelated to reproducibility and overall reporting of poikilosis, scientific literature has to start to demand detailed descriptions of conducted studies as well as of the investigated samples and systems. Reporting guidelines are available e.g. for systematic reviews and meta analyses92, prediction method description and performance assessment93,94, multiple sequence alignments95, and more than 200 guidelines for health reporting have been reviewed96. Existing systematics should be used, or developed if not available. However, even when guidelines and standards are available, they are often not followed or applied only selectively. By following FAIR principles97 the sharing of data will facilitate confirmatory and repeated experiments. Combined with open access to data principle soon to be implemented in several countries, reproducibility will be increased and the extent of poikilosis can be revealed.\n\nIncreasing evidence in scientific literature supports individual parts and areas of the theory of poikilosis. Some of these are mentioned above, further examples will be given in here, but there are far too many to cover but a small fraction of the existing literature. However, none of these studies has taken poikilosis fully into account.\n\nNature takes benefit of variations in many ways and certain processes have evolved to generate huge variability. Recognition molecules of adaptive immune system, B- and T-cell receptors and antibodies, are produced by a specific variation generation machinery. The combination of V(D)J recombination, somatic hypermutation and class switch recombination generates a vast array of molecules with different binding sites to detect foreign compounds and organisms. There are in the order of 1010 different possible recognition molecules. Somewhat similar outcome, but in smaller scale, is produced by errors in transcription, translation and expression machineries, especially under stress situations. Alternative splicing, initiation and termination are among processes that can generate numerous mRNA and protein forms. Drosophila melanogaster Dscam gene for Down syndrome cell adhesion molecule has totally 38,016 possible splicing isoforms by different combinations of its 24 exons98. The protein has two alternative transmembrane fragments, the other variants appear in three immunoglobulin domains.\n\nVariation in cell populations can enable information coding and transfer as well as rapid responses based on crowd control, as only some cells in a population may detect a signal but they can launch a coordinated response99. Diversity of cell states in a population makes it possible to adapt to environmental changes. This is called bet hedging and means that the fitness of the population is somewhat decreased in stable conditions, but significantly increased in stressful conditions85. Synchronization of cell populations to act as on-off switches may not be the optimal strategy to respond, since dose-dependent responses are smoother99. Still another cell level process where heterogeneity is beneficial is fate plasticity of stem cells100.\n\nDiseases are systemic deviations due to non-lagom variation affecting several levels. By considering poikilosis in medicine, the continuum of the conditions becomes apparent. As pathogenicity model65 indicates, similar disease states in patients can be sums of different disease components. Curative medicine aims at returning the system back to lagom variation extent while palliative care reduces the effects of variation but does not lead to full recovery. Poikilosis-aware strategy has been presented for diagnosis, prognosis, patient stratification and drug development for COVID-1966.\n\nCancer is an example of a disease where variations exceed lagom at many levels. Cancers indicate also how robust and resilient organisms are even for excessive variations. Despite even more than 1 million genetic variants e. g. in lung cancer101 affecting multiple levels, even the most aggressive forms of cancer require extended time to finally collapse the systems.\n\nPoikilosis explains differences also in drug treatments. Individuals respond differently due to their heterogeneity indicating the need for personalized medicine19,20. Similarly, adverse drug reactions have a wide spectrum as individuals react differently.\n\nMany biological and bodily functions and processes are tightly regulated, but instead of a fixed set point each system display some variation. Currently, many disease diagnoses and treatments are based on the idea of homeostasis despite of its flaws. Poikilosis is conceptually totally different although many outcomes are similar from the surface. In homeostasis the system strives to keep some ideal stable state. Homeostasis and related concepts are not feasible since they would require extensive energy and other resources for monitoring and controlling, especially since the efficiency of feedback control systems is very low69. Synthesis of monitoring molecules in vast excess to controlled compounds would be extremely costly and require substantial portion of energy available for an organism. Dynamically adjusted poikilosis kept at lagom level facilitates sufficient control at reasonable and acceptable cost (Figure 2B).\n\n\nConclusions and prospects\n\nPoikilosis means constant changes that in biology are controlled at lagom level. Lagom fluctuates on the variation zone, which changes dynamically depending on the situation and even the extent of lagom variation varies in time, space and due to perturbations. Lagom is relevant for biological systems and processes instead of perfection, which does not provide any benefit, but which would require extensive control machinery and costs in many ways.\n\nPoikilosis as a concept is in certain extent analogous to junk DNA, as the non-coding part of genomes was still recently called due to ignorance of its meaning, function and purpose. To reveal the full importance of poikilosis, something similar to The Encyclopedia of DNA Elements (ENCODE) project102 for the noncoding genome would be needed. The project could start by analysing large volumes of existing information and learn about poikilosis, its extent and consequences in systems of interest. Reanalysis of obtained results and observations could be the solution at some instances or could indicate how more comprehensive studies should be performed.\n\nPoikilosis is not restricted to scientific endeavours. It penetrates also human culture. Many forms of art are based on heterogeneity and take benefit of it. For example, symphonies are based on variation and development of a theme. Many artists generate variations of the same central ideas and motifs throughout their careers, cf. self-portraits of Vincent van Gogh or female figures in Pablo Picasso’s paintings.\n\nAwareness of poikilosis could hopefully contribute towards acceptance of differences between people. Wide adaptation of the concept of poikilosis could increase understanding and acceptance of e. g. social, sexual and ethnic variation. Evolution has in addition to its biological significance had wide effects including social and cultural aspects and poikilosis has a potential to make similar contribution.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nCarola Tilgmann is thanked for valuable discussions.\n\n\nReferences\n\nHeisenberg W: Über den anschaulichen Inhalt der quantentheoretischen Kinematik und Mechanik. Zeitschrift für Physik. 1927; 43: 172–198. Publisher Full Text\n\nBlake WJ, KAErn, M, Cantor CR, et al.: Noise in eukaryotic gene expression. Nature. 2003; 422(6932): 633–637. PubMed Abstract \| Publisher Full Text\n\nAbecasis GR, Auton A, Brooks LD, 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\nZhu X, Need AC, Petrovski S, et al.: One gene, many neuropsychiatric disorders: lessons from Mendelian diseases. Nat Neurosci. 2014; 17(6): 773–781. PubMed Abstract \| Publisher Full Text\n\nKammenga JE: The background puzzle: how identical mutations in the same gene lead to different disease symptoms. Febs j. 2017; 284(20): 3362–3373. PubMed Abstract \| Publisher Full Text\n\nSherman RM, Forman J, Antonescu V, et al.: Assembly of a pan-genome from deep sequencing of 910 humans of African descent. Nat Genet. 2019; 51(1): 30–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpudich JL, Koshland DE: Non-genetic individuality: chance in the single cell. Nature. 1976; 262(5568): 467–471. PubMed Abstract \| Publisher Full Text\n\nKamitani M, Miyatsuka T, Miura M, et al.: Heterogeneity of autophagic status in pancreatic beta cells under metabolic stress. Biochem Biophys Res Commun. 2018; 496(2): 328–334. PubMed Abstract \| Publisher Full Text\n\nVihinen M: Relationship of protein flexibility to thermostability. Protein Eng. 1987; 1(6): 477–480. PubMed Abstract \| Publisher Full Text\n\nHvidt A, Linderstrom-Lang K: Exchange of hydrogen atoms in insulin with deuterium atoms in aqueous solutions. Biochim Biophys Acta. 1954; 14(4): 574–575. PubMed Abstract \| Publisher Full Text\n\nEnglish BP, Min W, van Oijen AM, et al.: Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat Chem Biol. 2006; 2(2): 87–94. PubMed Abstract \| Publisher Full Text\n\nGenuth NR, Barna M: Heterogeneity and specialized functions of translation machinery: from genes to organisms. Nat Rev Genet. 2018; 19(7): 431–452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarino K, Bones J, Kattla JJ, et al.: A systematic approach to protein glycosylation analysis: a path through the maze. Nat Chem Biol. 2010; 6(10): 713–723. PubMed Abstract \| Publisher Full Text\n\nLoeffler D, Wehling A, Schneiter F, et al.: Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells. Nature. 2019; 573(7774): 426–429. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Buil A, Collins BC, et al.: Quantitative variability of 342 plasma proteins in a human twin population. Mol Syst Biol. 2015; 11(1): 786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBradshaw AD: Evolutionary significance of phenotypic plasticity in plants. Adv Genet. 1965; 13: 115–155. Publisher Full Text\n\nAinsworth C: Sex redefined. Nature. 2015; 518(7539): 288–291. PubMed Abstract \| Publisher Full Text\n\nCaporali L, Maresca A, Capristo M, et al.: Incomplete penetrance in mitochondrial optic neuropathies. Mitochondrion. 2017; 36: 130–137. PubMed Abstract \| Publisher Full Text\n\nSingh DK, Ku CJ, Wichaidit C, et al.: Patterns of basal signaling heterogeneity can distinguish cellular populations with different drug sensitivities. Mol Syst Biol. 2010; 6: 369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSweeney GD: Variability in the human drug response. Thromb Res Suppl. 1983; 4: 3–15. PubMed Abstract \| Publisher Full Text\n\nDavid LA, Maurice CF, Carmody RN, et al.: Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014; 505(7484): 559–563. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaatz M, van Velzen E, Gaedke U: Co-adaptation impacts the robustness of predator-prey dynamics against perturbations. Ecol Evol. 2019; 9(7): 3823–3836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHallgrímsson B, Hall BK: Variation: A central concept in biology. Elsevier, Amsterdam. 2005. Reference Source\n\nKolasa J, Pickett STA: Ecological Heterogeneity. Springer. 1991. Reference Source\n\nHutchings ML, John EA, Stewart AJA: The ecological consequences of environmental heterogeneity. Blackwell, University of Sussex. 2000. Reference Source\n\nEling N, Morgan MD, Marioni JC: Challenges in measuring and understanding biological noise. Nat Rev Genet. 2019; 20(9): 536–548. PubMed Abstract \| Publisher Full Text\n\nHarper JW, Bennett EJ: Proteome complexity and the forces that drive proteome imbalance. Nature. 2016; 537(7620): 328–338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlake WJ, Balazsi G, Kohanski MA, et al.: Phenotypic consequences of promoter-mediated transcriptional noise. Mol Cell. 2006; 24(6): 853–865. PubMed Abstract \| Publisher Full Text\n\nMatic I: Mutation rate heterogeneity increases odds of survival in unpredictable environments. Mol Cell. 2019; 75(3): 421–425. PubMed Abstract \| Publisher Full Text\n\nHarton MD, Batchelor E: Determining the limitations and benefits of noise in gene regulation and signal transduction through single cell, microscopy-based analysis. J Mol Biol. 2017; 429(8): 1143–1154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaszek P, Ryan S, Ashall L, et al.: Population robustness arising from cellular heterogeneity. Proc Natl Acad Sci U S A. 2010; 107(25): 11644–11649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevine NM, Zhang K, Longo M, et al.: Ecosystem heterogeneity determines the ecological resilience of the Amazon to climate change. Proc Natl Acad Sci U S A. 2016; 113(3): 793–797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsimring LS: Noise in biology. Rep Prog Phys. 2014; 77(2): 026601. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrifonov EN: Vocabulary of definitions of life suggests a definition. J Biomol Struct Dyn. 2011; 29(2): 259–266. PubMed Abstract \| Publisher Full Text\n\nPopa R: Between necessity and probability: Searching for the definition and origin of life (Advances in Astrobiology and Biogeophysics). Springer, Berlin. 2004. Reference Source\n\nGerard RW, Stevens RB: Concepts of Biology. National Academy of Sciences- National Research Council, Washington, D.C. 1958. Reference Source\n\nVrselja Z, Daniele SG, Silbereis J, et al.: Restoration of brain circulation and cellular functions hours post-mortem. Nature. 2019; 568(7752): 336–343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihinen M: Functional effects of protein variants. (Submitted) 2020.\n\nNeuberg SL, Kenrick DT, Schaller M: Human threat management systems: self-protection and disease avoidance. Neurosci Biobehav Rev. 2011; 35(4): 1042–1051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedzhitov R, Schneider DS, Soares MP: Disease tolerance as a defense strategy. Science. 2012; 335(6071): 936–941. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarville JL, Ayres JS: Disease tolerance: concept and mechanisms. Curr Opin Immunol. 2018; 50: 88–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHodgkin J: Genetic suppression. WormBook. 2005; 1–13. Reference Source\n\nEllis J: Proteins as molecular chaperones. Nature. 1987; 328(6129): 378–379. PubMed Abstract \| Publisher Full Text\n\nMartinez-Martinez M, Coscolin C, Santiago G, et al.: Determinants and prediction of esterase substrate promiscuity patterns. ACS Chem Biol. 2018; 13(1): 225–234. PubMed Abstract \| Publisher Full Text\n\nBarkai N, Leibler S: Robustness in simple biochemical networks. Nature. 1997; 387(6636): 913–917. PubMed Abstract \| Publisher Full Text\n\nWaddington CH: Canalization of development and the inheritance of acquired characters. Nature. 1942; 150: 563–565. Publisher Full Text\n\nGu Z, Steinmetz LM, Gu X, et al.: Role of duplicate genes in genetic robustness against null mutations. Nature. 2003; 421(6918): 63–66. PubMed Abstract \| Publisher Full Text\n\nWard PS, Thompson CB: Metabolic reprogramming: a cancer hallmark even warburg did not anticipate. Cancer Cell. 2012; 21(3): 297–308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanahan D, Weinberg RA: The hallmarks of cancer. Cell. 2000; 100(1): 57–70. PubMed Abstract \| Publisher Full Text\n\nZlotogora J: Penetrance and expressivity in the molecular age. Genet Med. 2003; 5(5): 347–352. PubMed Abstract \| Publisher Full Text\n\nQian M, Wang DC, Chen H, et al.: Detection of single cell heterogeneity in cancer. Semin Cell Dev Biol. 2017; 64: 143–149. PubMed Abstract \| Publisher Full Text\n\nEvans CR, Fan Y, Weiss K, et al.: Errors during Gene Expression: Single-Cell Heterogeneity, Stress Resistance, and Microbe-Host Interactions. mBio. 2018; 9(4): e01018–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCannon WB: Organization for physiological homeostasis. Psychological Review. 1929; 9(3): 399–431. Publisher Full Text\n\nBernard C: Introduction à l’étude de la médicine expérimentale. Baillière, Paris. 1865. Reference Source\n\nWaddington CH: The strategy of the genes. A discussion of some aspects of theoretical biology. George Allen & Unwin, London. 1957. Reference Source\n\nSterling P, Eyer J: Allostasis: A new paradigm to explain arousal pathology. In Handbook of life stress, cognition, and health. (Fisher, S., and Reason, J. eds.), Wiley, New York. 1988; 629–649. Reference Source\n\nEdwards DR, Lohman DC, Wolfenden R: Catalytic proficiency: the extreme case of S-O cleaving sulfatases. J Am Chem Soc. 2012; 134(1): 525–531. Publisher Full Text\n\nFischer E: Einfluss der Configuration auf die Wirkung der Enzyme. Ber Dtsch Chem Ges. 1894; 27(3): 2985–2993. Publisher Full Text\n\nVaiman D: Genetic regulation of recurrent spontaneous abortion in humans. Biomed J. 2015; 38(1): 11–24. PubMed Abstract \| Publisher Full Text\n\nHedrick PW, Miller PS: Conservation Genetics: Techniques and Fundamentals. Ecol Appl. 1992; 2(1): 30–46. PubMed Abstract \| Publisher Full Text\n\nRomdhane L, Mezzi N, Hamdi Y, et al.: Consanguinity and Inbreeding in Health and Disease in North African Populations. Annu Rev Genomics Hum Genet. 2019; 20: 155–179. PubMed Abstract \| Publisher Full Text\n\nBoorse C: On the distinction between disease and ilness. Philos Public Aff. 1975; 5(1): 49–68. Reference Source\n\nAhmadi H, Gholamzadeh M, Shahmoradi L, et al.: Diseases diagnosis using fuzzy logic methods: A systematic and meta-analysis review. Comput Methods Programs Biomed. 2018; 161: 145–172. PubMed Abstract \| Publisher Full Text\n\nSakhanenko NA, Galas DJ: Probabilistic logic methods and some applications to biology and medicine. J Comput Biol. 2012; 19(3): 316–336. PubMed Abstract \| Publisher Full Text\n\nVihinen M: How to define pathogenicity, health, and disease? Hum Mutat. 2017; 38(2): 129–136. PubMed Abstract \| Publisher Full Text\n\nVihinen M: Strategy for disease diagnosis, progression prediction, risk group stratification and treatment – Case of COVID-19. Front Med. 2020. Reference Source\n\nJuarez-Carreño S, Morante J, Dominguez M: Systemic signalling and local effectors in developmental stability, body symmetry, and size. Cell Stress. 2018; 2(12): 340–361. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPakula AA, Sauer RT: Genetic analysis of protein stability and function. Annu Rev Genet. 1989; 23: 289–310. PubMed Abstract \| Publisher Full Text\n\nLestas I, Vinnicombe G, Paulsson J: Fundamental limits on the suppression of molecular fluctuations. Nature. 2010; 467(7312): 174–178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkashi H, Osada N, Ohta T: Weak selection and protein evolution. Genetics. 2012; 192(1): 15–31. PubMed Abstract \| Publisher Full Text \| \|Free Full Text\n\nGasser R, Hamoudi M, Pellicciotta M, et al.: Buffering deleterious polymorphisms in highly constrained parts of HIV-1 envelope by flexible regions. Retrovirology. 2016; 13(1): 50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaila V, Annila A: Natural selection for least action. Proc R Soc A. 2008; 464(2099): 3055–3070. Publisher Full Text\n\nVihinen M: Variation Ontology for annotation of variation effects and mechanisms. Genome Res. 2014; 24(2): 356–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihinen M: Types and effects of protein variations. Hum Genet. 2015; 134(4): 405–421. PubMed Abstract \| Publisher Full Text\n\nVihinen M: Systematics for types and effects of DNA variations. BMC Genomics. 2018; 19(1): 974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihinen M: Systematics for types and effects of RNA variations. (Submitted) 2020.\n\nSansone SA, McQuilton P, Rocca-Serra P, et al.: FAIRsharing as a community approach to standards, repositories and policies. Nat Biotechnol. 2019; 37(4): 358–367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYvert G: 'Particle genetics': treating every cell as unique. Trends Genet. 2014; 30(2): 49–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Q, Herring CA, Sheng Q, et al.: Quantitative assessment of cell population diversity in single-cell landscapes. PLoS Biol. 2018; 16(10): e2006687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMontévil M, Mossio M, Pocheville A, et al.: Theoretical principles for biology: Variation. Prog Biophys Mol Biol. 2016; 122(1): 36–50. PubMed Abstract \| Publisher Full Text\n\nGough A, Stern AM, Maier J, et al.: Biologically relevant heterogeneity: Metrics and practical insights. SLAS Discov. 2017; 22(3): 213–237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiroula A, Urolagin S, Vihinen M: PON-P2: Prediction method for fast and reliable identification of harmful variants. PLoS One. 2015; 10(2): e0117380. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiroula A, Vihinen M: Predicting severity of disease-causing variants. Hum Mutat. 2017; 38(4): 357–364. PubMed Abstract \| Publisher Full Text\n\nElsasser WM: Outline of a theory of cellular heterogeneity. Proc Natl Acad Sci U S A. 1984; 81(16): 5126–5129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen D: Optimizing reproduction in a randomly varying environment. J Theor Biol. 1966; 12: 119–129. PubMed Abstract \| Publisher Full Text\n\nBegley CG, Ellis LM: Drug development: Raise standards for preclinical cancer research. Nature. 2012; 483(7391): 531–533. PubMed Abstract \| Publisher Full Text\n\nHaibe-Kains B, El-Hachem N, Birkbak NJ, et al.: Inconsistency in large pharmacogenomic studies. Nature. 2013; 504(7480): 389–393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmrhein V, Greenland S, McShane B: Scientists rise up against statistical significance. Nature. 2019; 567(7748): 305–307. PubMed Abstract \| Publisher Full Text\n\nBen-David U, Siranosian B, Ha G, et al.: Genetic and transcriptional evolution alters cancer cell line drug response. Nature. 2018; 560(7718): 325–330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y, Mi Y, Mueller T, et al.: Multi-omic measurements of heterogeneity in HeLa cells across laboratories. Nat Biotechnol. 2019; 37(3): 314–322 . PubMed Abstract \| Publisher Full Text\n\nFinn EH, Pegoraro G, Brandao HB, et al.: Extensive heterogeneity and intrinsic variation in spatial genome organization. Cell. 2019; 176(6): 1502–1515.e1510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097 . PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihinen M: How to evaluate performance of prediction methods? Measures and their interpretation in variation effect analysis. BMC Genomics. 2012; 13(Suppl 4): S2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihinen M: Guidelines for reporting and using prediction tools for genetic variation analysis. Hum Mutat. 2013; 34(2): 275–282. PubMed Abstract \| Publisher Full Text\n\nVihinen M: Guidelines for systematic reporting of sequence alignments. Biol Meth Protoc. 2020; 5(1): bpaa001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanno M, Tsujimoto Y, Kataoka Y: The majority of reporting guidelines are not developed with the Delphi method: a systematic review of reporting guidelines. J Clin Epidemiol. 2020; 124: 50–57. PubMed Abstract \| Publisher Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR guiding principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeves G, Zucker J, Daly M, et al.: Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Nat Genet. 2004; 36(3): 240–246. PubMed Abstract \| Publisher Full Text\n\nDueck H, Eberwine J, Kim J: Variation is function: Are single cell differences functionally important?: Testing the hypothesis that single cell variation is required for aggregate function. Bioessays. 2016; 38(2): 172–180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChacon-Martinez CA, Koester J, Wickstrom SA: Signaling in the stem cell niche: regulating cell fate, function and plasticity. Development. 2018; 145(15): dev165399. PubMed Abstract \| Publisher Full Text\n\nAlexandrov LB, Nik-Zainal S, Wedge DC, et al.: Signatures of mutational processes in human cancer. Nature. 2013; 500(7463): 415–421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAn integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414): 57–74. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "67620", "date": "11 Aug 2020", "name": "Emil Alexov", "expertise": [ "Reviewer Expertise Personalized Medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript reports a novel approach to deal with dynamics and heterogeneity in biological systems. This new approach is called “Poikilosis” and it is defined by the author as “inherent pervasive variation, heterogeneity and fluctuation in living organisms, populations, ecosystems, biosphere and in their components and in processes within them”.\n\nMajor comment: Throughout the manuscript “molecules” and “matter” are considered to be different entities. The same is about “atoms” and “matter”. However, “atoms” and “molecules” are the building block of the matter.\nMinor comments:\n\nFigure 1 is not informative. Perhaps it can be reconsidered and the effect of disease/curative treatment shown in some cartoonish presentation. Another note, the reproduction (presumably an offspring) is shown with smaller tori, which can be wrongly interpret as lower complexity than in the parent.\n\np. 2: “Every system and process can be thought to represent its own level.” What level?\n\nP.3. The paragraph starting with “Information, energy and matter flow…”. Epigenetic information, and information in signalling pathways, regulatory networks, immune system are encoded in DNA. Why they are considered to be independent source of information?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "68772", "date": "01 Sep 2020", "name": "Xavier de la Cruz", "expertise": [ "Reviewer Expertise Clinical and Translational Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting contribution in which the author presents and develops the concept of poikilosis which unifies the different types of heterogeneity observed in the different complexity levels of biological organisms. A variety of examples of poikilosis are discussed in the text and, importantly, life, disease and death are redefined in terms of this central concept. This work, presented as an Opinion Article, is of interest to those interested in understanding how effects repeating through the biological hierarchy can be unified. I have a few comments on some specific issues that, from my point of view, require clarification or minor action.\nIn p2, the author mentions that 'Some examples of biological heterogeneity include uncertainty of positions of electrons...' I believe that this example, considering the reference given, is closer to physics rather than biology. I feel that its extreme nature dilutes the value of the examples listed afterwards, more related to biological studies.\n\nIn p5, the author states ‘Life does not strive towards perfection, instead at lagom i.e. sufficient and relevant reactions and responses.’ Finalism slipped into the sentence, clashing with the idea of life as a physico-chemical process.\n\nIn p6, the sentence 'Second, poikilosis reduces the generated Gibbs free energy in living organisms' expresses the existence of a relationship between poikilosis and a thermodynamics variable, the Gibbs free energy. I believe that, even if short, an explanation should be provided justifying relationship, because it is not easy to derive from the definition of poikilosis.\n\nIn the same line, in the section 'Correlation to evolution and survival', it would be valuable to briefly mention the relationship between poikilosis and fitness, a key parameter in evolutionary studies that is related to free energy change upon mutation in proteins (DePristo et al., Nature Rev. Genet, 20051).\n\nIn p6, the sentence 'Perfect performance or phenotype would require...' suggests the existence of perfect phenotypes. This concept is not very clear and may have undesired implications in a work where all levels of biological complexity are considered, included the population level. I suggest that the idea of perfect phenotype is either clarified or discarded.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-602
https://f1000research.com/articles/9-1053/v1	27 Aug 20	{ "type": "Brief Report", "title": "Characterization of buccal cell DNA after exposure to azo compounds: a cross-sectional study", "authors": [ "Juni Handajani", "Urfa Tabtila", "Nadia Rully Auliawati", "Abdul Rohman", "Urfa Tabtila", "Nadia Rully Auliawati", "Abdul Rohman" ], "abstract": "Background: Azo compounds, containing naphthol and diazonium salts, are synthetic dyes widely used in the batik industry. Azo compounds are considered toxic when they are exposed to human tissue. The purpose of this study was to analyze buccal cell DNA exposed to azo compounds in batik workers. Methods: A cross-sectional study involving 20 male subjects divided into two groups (n=10 group), namely azo-exposed and non-exposed (control group). Inclusion criteria were batik workers of the colouring division who have been exposed to azo for at least 5 years. Buccal cells were taken using cytobrush then DNA were isolated from buccal cell. DNA isolation was done by buccal DNA kit, while the purity and concentration of the DNA was determined using spectrophotometer and electrophoresis. Results: The azo-exposed group revealed higher purity DNA than those in the control group. The purity of the DNA in the azo-exposed group and control group was 0.61±0.93 and 0.21±0.09, respectively, while the concentration of DNA was of 59.02 and 19.35 ng/UL, respectively. The ratio at 260/280 nm was 1.84-1.94 (azo-exposed) and 1.85-1.92 (control). Principal component analysis using the first principle component (PC1) and second principle component (PC2) could successfully classify subjects in the control and azo-exposed groups. Conclusion: Characteristics of DNA could be used as an indication of exposure to azo compounds in workers of batik industries.", "keywords": [ "DNA", "buccal cell", "azo-exposed" ], "content": "Introduction\n\nThe oral mucosa is the first defence against particles entering the body. The oral epithelial mucosa functions to protect the body from chemical, microbial, and physical challenges1,2. The buccal epithelium is the thickest region in the squamous stratification epithelium. Keratinization is influenced by endogenous or exogenous factors. Exogenous factors include the use of drugs, nutritional factors, and irritant factors, such as plaque and calculus, artificial teeth, and smoking or exposure to other substances3,4.\n\nThe use of azo synthetic dyes and their derivatives, especially those with benzene groups, are increasing in the batik industry5,6. Azo dyes are compounds characterized with one or more azo functional groups (-N=N-), linked to benzene. They are readily reduced to hydrazines and primary amines. The benzene group in azo compounds is difficult to degrade because it takes a long time7,8. Chemicals in the batik industry are known to cause irritation to the skin and eyes, and cause interference with the respiratory system8. Azo compounds are also known to be carcinogenic and mutagenic if they are in the environment for a long time, and they are suspected to be a source of disease9,10.\n\nExposure to synthetic azo dyes, which are continuously inhaled by batik workers, may cause changes in the oral mucosa. Daily exposure to azo dyes needs to be analysed to assess the possibility of the risk of oral cavity abnormalities, although there have been no reports of batik workers that mention oral cavity abnormalities due to azo exposure. Exposure to azo dyes for more than 5 years in batik artisans has been known to significantly increase the frequency of micronuclei, karyolysis, and pyknosis in buccal mucosal epithelial cells11–13. In addition, exposure to azo dyes significantly increases the expression of cytokeratin 5 and 19 in the buccal mucosa14,15. The results of these studies have not yet explained the changes in buccal cell DNA exposed to azo compounds; therefore, the objective of this study was to evaluate the profile of buccal cell DNA exposed to synthetic azo dyes to determine the possibility of cellular damage.\n\n\nMethods\n\nThe method was cross-sectional to compare subjects exposed and not exposed at the same time. We conducted the study in batik industries (for exposed group) and non- batik industries (for control group) in Yogyakarta-Indonesia from May to August 2019. The procedure of this study was approved by Research Ethics Committee of the Faculty Dentistry, Universitas Gadjah Mada (Ethical Clearance No.00107/KKEP/ FKG-UGM/EC/2019).\n\nParticipants of exposed group were from batik industries in Yogyakarta Indonesia whereas participants of the control group were students and staff at the Faculty of Dentistry, Universitas Gadjah Mada, Indonesia. For the exposed group, batik factories were identified from a list online and information about the study was sent to the manager of the factories (letter No. 5189/UN1/FKG/Set.KG1/PT/2019 from Universitas Gadjah Mada to the factories), who allowed the researchers to interview their workers. For the control group, information about this study was sent to students at our university that asked them to participate in the study. All participants agreed to participate by providing written informed consent.\n\nInformation collected from the participants were age, past medical and dental history, occupational history, lifestyle (smoking and alcohol consumption) and if they wore a dental apparatus. Oral Hygiene Index-Simplified (OHI-S) were calculated from calculus index (CI(S)) and debris index (DI(S)): OHI-S = CI(S) + DI(S). Interpretation: 0 - 1.2 is good; 1.3 - 3.0 is fair; and 3.0 - 6.0 is poor16.\n\nInclusion criteria were aged between 18 and 45 years old (age group most likely to be working), male (to provide continuity among participants), OHI-S status of ‘good’, worked in colouring batik for a minimum of 5 years (for exposed group), and did not work in coloring batik (for control group).\n\nStudy size was calculated according to Notoatmodjo17\n\n\n\nn = number of samples\n\nZ1-α/2 = the Z value at 95% degree of significance is 1.96\n\nP = proportion of subject azo-exposed around 50% (0.5)\n\nd = degree of deviation to population, by 5% (0.05)\n\nBased on the formula, n = 9.8 ≈ 10. In this study, the number of subjects for each group is 10 participants.\n\nParticipants were asked to rinse out their mouths first to remove debris in the oral cavity. Buccal epithelial cell harvesting was carried out using the smear method using sterile foam Tipped Swab (Product Code: PW1174, Himedia, India). Swab was done by turning in the direction of at least 360° in the buccal mucosa then put in a microtube. Samples were transported in the microtube with 1x PBS to the lab. Sample collection was carried out at the batik factories for the exposed participants and at the university for the non-exposed participants.\n\nDNA isolation was done following the protocol from HiPurATM Buccal DNA Purification Kit (Product Code: MB531; Himedia, India). Briefly, the buccal swab sample was placed into a 2.0 ml microcentrifuge tube, 400 µl of resuspension solution was added, and the tube was centrifuged at 14,000 rpm for 5 minutes. The pellet was discarded and the supernatant was transferred to a new collection tube. 20 µl of Proteinase K solution (20 mg/ml) was added to the tube containing the supernatant, and this was vortexed for 10–15 seconds. 20 µl of RNase A solution (20 mg/ml) was added, and the tube was again vortexed for 10–15 seconds. The sample tubes were incubated for 2 minutes at room temperature (15–25°C).\n\nThe lysis reaction was done by added 400 µl of lysis solution to the tube, which was vortexed thoroughly for a few seconds to obtain a homogenous mixture. Samples were incubated at 55°C for 10 minutes. For the binding step, 400 µl of ethanol (96–100%) was added to the lysate, which was then mixed thoroughly by vortexing for 5–10 seconds. The lysate was added to the HiElute Miniprep Spin Column (Capped) and samples was centrifuged at 6,500 x g (10,000 rpm) for 1 minute. The flow-through liquid was discarded, and the procedure was repeated with any remaining lysate. A prewash was performed by adding 500 µl of diluted prewash solution to the column and centrifuged at 6,500 x g (10,000 rpm) for 1 minute. The flow-through liquid was discarded and the same collection tube was re-used with the column.\n\nSubsequently, samples were washed by adding 500 µl of diluted Wash Solution to the column and centrifuged at 12,000–16,000 x g (13,000–16,000 rpm) for 3 minutes to dry the column then the flow-through was discarded and a new uncapped 2.0 ml collection tube was placed in the column. DNA Elution was done by pipetting 150 µl of the Elution Buffer directly onto the column without spilling on the sides. The samples were incubated for 1 minute at room temperature and centrifuged at >6500 x g (10,000rpm) for 1 minute to elute the DNA. Storage of the eluted purified DNA was done at 2–8°C for short-term (24–48 hours) or -20°C for long-term storage.\n\nPurity and concentration of DNA buccal cells were characterized using electrophoresis and spectrophotometer. Agarose gel was prepared in concentration of 2%. Agarose (Biotechnology Grade, 1st Base, Singapore; 1 gr) was added to 50 ml Tris/Borate/EDTA (TBE) buffer, then put in microwave for around 2 minutes until completely dissolved. After agarose solution was cooled to about 50°C (around 5 minutes), this was poured into a gel tray with the well comb in place and then let sit at room temperature for around 20 minutes until agarose gel had completely solidified. Loading buffer was added to each DNA sample. Agarose gel was placed into the gel box (electrophoresis unit), then filled gel box with 1x TBE until the gel was covered. Electrophoresis was run at 100 mA and then visualized with Florosafe DNA Stain (Genetika Science, PT. Genetika Science Indonesia) using a UV transilluminator. Concentration of DNA buccal cell were measured using a spectrophotometer. Purity of DNA was analysed using spectrophotometer at 280 and 260/280 nm.\n\nData analysis was performed using stat Shapiro-Wilk and Levene’s test, to see if the data were normal and homogenous. Mann-Whitney U test was used to describe the comparison between azo-exposed group and the control group. Statistical measurement was performed using IBM SPSS Statistics v22. The classification of the azo-exposed group and control group was performed using chemometrics of principal component analysis (PCA) using Minitab version 17. P<0.05 was taken as significant.\n\n\nResults\n\nCharacteristics of study participants are described in Table 1.\n\nThe presence of high-molecular weight DNA was evaluated by gel electrophoresis and visualized using a UV illuminator. Figure 1 shows that bands for high-molecular weight DNA only appeared for the azo-exposed group, but it did not appear in control group. There was no significant difference between groups for the purity of the DNA at A280 nm (p=0.076), ratio A260/280 (p=0.718), or the concentration of the DNA (p=0.076) (Table 2).\n\nPCA could successfully classify participants in azo-exposed and control groups. The score plot for the ﬁrst principal components (PC1) and second principle component (PC2) is shown in Figure 2. In addition, the loading plot for the evaluation of variables contributing to the separation is shown in Figure 3. The concentration of DNA contributed the most in PCA, as it was the fartherest variable from the initial points (0.0).\n\n\nDiscussion\n\nThe method of this study was done using exfoliative buccal epithelial cells by swab tip or cytobrush then purity and concentration of the DNA was analysed. The exfoliative method was non-invasive. One of the important procedures in the study for DNA extraction was the collection method of the sample. According to Mulot et al.18, cytology brushes (cytobrush) are the most appropriate method and provide good quality cell collection compared to mouthwashes, swabs, or collected from saliva.\n\nIn the present study, DNA electrophoresis revealed a band for high-molecular weight DNA in azo-exposed group only (Figure 1). This result indicated that the concentration of the DNA from buccal epithelial cells in azo-exposed group was higher than controls. However, we noticed that not all samples in the azo-exposed group revealed a band. This may be because of the low concentration of DNA in the collected buccal epithelial cells. This result was supported by our spectrophotometer measurements (Table 2), showing that DNA concentration in the control group was lower than in the azo-exposed group. DNA quality may have been affected by collection and isolation methods. This result showed the mean OD 260/280 ratio was 1.89 both in the azo-exposed and control groups, which indicates that the bulk of the proteins were removed successfully.\n\nThe standard deviation for the purity of the DNA at A280 nm and the concentration of the DNA (Table 2) in azo-exposed groups was higher than the mean. This indicates that the purity of the DNA from azo-exposed participants varied, which may be due to exposure of azo that has induced DNA damage. According to Ferraz et al.19, the azo dye, Disperse Orange 1, which is used in textiles, induces a frameshift mutation and cytotoxic effect in the human hepatoma cell line HepG2. Mutagenicity was shown by enhanced nitroreductase and o-acetyltransferase, which are important enzymes in mutagenicity. This result was also supported by a previous study that showed that azo dye exposure increases the number of micronuclei, karyolysis, pyknosis, and expression of cytokeratin 5 and 19 in oral epithelial cells11–15. However, these results have not yet revealed how the mechanism of DNA damage occurs in oral epithelial cells due to azo exposure.\n\nIn order to classify participants into azo-exposed and control groups, principal component analysis (PCA) was used. PCA is capable of projecting the initial variable data in reduced dimensions deﬁned by principal components (PCs). The value corresponding to the PC is known as score plot20,21. PCA was done in this study using three variables, namely the purity of DNA at 280 nm, the concentration of DNA, and the ratio of absorbance values at 280 and 260 nm (A280/260 nm). Our results showed that the azo-exposed group could be separated successfully and easily differentiated from control group using PC1 and PC2 score plots (Figure 2). The loading plot of PCA was performed to evaluate the variables having the most significant contribution to the separation and classification of participants as azo-exposed and controls. The loading plot can explain the projection of variables used during PCA in the same plane as the score plot22. The absolute value of loading in the variables explains the importance of the contribution of each region. Therefore, the further the variables are from the origin of the variable point, the larger the contribution of that variable to the PCA model23,24. The results of the loading plot indicated that all three variables made a significant contribution to the PCA model.\n\n\nConclusion\n\nBuccal cell DNA of batik workers exposed to azo compounds had higher purity of DNA, concentration of DNA and absorbance ratio at 260/280 than buccal cell DNA of controls (not exposed to azo compounds). Principal component analysis, based on score plot, could successfully classify participants as controls and azo-exposed individuals. The characteristics of DNA could be used as an indication of exposure to azo compounds in workers in batik industries.\n\n\nData availability\n\nFigshare: Raw data-Dana Masyarakat 2019, https://doi.org/10.6084/m9.figshare.12733055.v525.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would also like to show our gratitude to Dr. Niken Satuti Nur Handayani, M.Sc; Indra Lesmana, S.Si., M.Sc; and Nailil Husna, S.Si from Faculty of Biology, Universitas Gadjah Mada for sharing their knowledge with us during the course of this research.\n\n\nReferences\n\nMorton W, Richard HC: Toxic Metal Syndrome. New York: Avery Pub Group, New York. 1995; 26–31. Reference Source\n\nKrejei CB, Bissada NF: Women's health issues and their relationship to periodontitis. J Am Dent Assoc. 2002; 133(3): 323–9. PubMed Abstract \| Publisher Full Text\n\nGarant PR: Oral Cells and Tissues. Chicago: Quintessence. 2003. Reference Source\n\nGroeger S, Meyle J: Oral Mucosal Epithelial Cells. Front Immunol. 2019; 10: 208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWidodo S, Sinarya IK, Iswahyudi: Coloring of Natural Materials in Lurik Batik \"Batik Natural Sarwidi\" Bayat Klaten, Central Java (Pewarnaan Bahan Alam pada Batik Lurik Karya \"Batik Natural Sarwidi\" Bayat Klaten Jawa Tengah). UNY Journal. 2012; 1(2): 7–13.\n\nDani I: Beautiful in style with Batik and Tenun (Cantik Bergaya dengan Batik dan Tenun). Jakarta: Niaga Swadaya, 2012; 4. Reference Source\n\nGratha B: Easy Guide to Learning Batik (Panduan Mudah Belajar Membatik). Jakarta: Demedia Pustaka, 2012; 4–6. Reference Source\n\nLestari F: Chemical: Sampling and measurement of contaminants in the air (Bahan Kimia: Sampling dan Pengukuran Kontaminan di Udara). Jakarta: Penerbit Buku Kedokteran EGC, 2007; 205–21. Reference Source\n\nCamargo-Ventura B de C, Maltempi PPP, Marin-Morales MA: The Use of the Cytogenetic to Identify Mechanisms of Action of an Azo Dye in Allium Cepa Meristematic Cells. J Toxicology. 2011; 1(3): 5–12. Publisher Full Text\n\nWidjajanti E, Regina TP, Prajonto UM: Zeolite adsorption pattern against azo red methyl dyes and orange (Pola Adsorpsi Zeolit Terhadap Pewarna Azo Metil Merah dan Jingga, Seminar Nasional Penelitian). National Research Seminar, Faculty of Math and Science, Universitas Negeri Yogyakarta, 2011; 1–14. Reference Source\n\nNarissi DH, Handajani J, Tandelilin RTC: Analyze frequency of micronucleus on buccal mucosa of batik workers caused by azo dyes exposure. 2014.\n\nAziz M, Handajani J, Tandelilin RTC: the exposure effect of azo dyes in the increase of karyolysis nucleus in buccal mucosal epithelial cells of batik workers in Yogyakarta. 2014.\n\nRahmawati AS, Handajani J, Jonarta AL: The frequency of pyknosis cell in buccal mucosa epithelium caused by azo dyes. 2014.\n\nHandajani J, Narissi DH: Analyze the expression of cytokeratin 5 on the epithelial cells of the buccal mucosa in batik workers. IJSR. 2016; 5(2): 510–4. Publisher Full Text\n\nHandajani J, Hanindriyo L: Expression of Cytokeratin 19 in the epithelial cell of Azo-exposed buccal mucosa. Med J Islam Repub Iran. 2018; 32(2): 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreene JC, Vermillion JR: The Simplified Oral Hygiene Index. J Am Dent Assoc. 1964; 68: 7–13. PubMed Abstract \| Publisher Full Text\n\nNotoatmodjo S: Health Research Methodology (Metodologi Penelitian Kesehatan). Jakarta: Rineka Cipta, Jakarta, 2010; 127–128.\n\nMulot C, Stücker I, Clave; J, et al.: Collection of human genomic DNA from buccal cells for genetics studies: comparison between cytobrush, mouthwash, and treated card. J Biomed Biotechnol. 2005; 2005(3): 291–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerraz ERA, Grando MD, Oliveira DP: The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri. J Hazard Mater. 2011; 192(2): 628–33. PubMed Abstract \| Publisher Full Text\n\nCheng XL, Wei F, Xiao XY, et al.: Identification of five gelatins by ultra performance liquid chromatography/time-of-flight mass spectrometry (UPLC/Q-TOF-MS) using principal component analysis. J Pharm Biomed Anal. 2012; 62: 191–5. PubMed Abstract \| Publisher Full Text\n\nChe Man YB, Rohman A, Mansor TST: Differentiation of Lard from Other Edible Fats and Oils by Means of Fourier Transform Infrared Spectroscopy and Chemometrics. J Am Oil Chem Soc. 2011; 88: 187–92. Publisher Full Text\n\nWidodo H, Asmara W, Sismindari RA: Antioxidant activity, total phenolic and flavonoid contents of selected medicinal plants used for liver diseases and its classification with chemometrics. J Appl Pharm Sci. 2019; 9(6): 99–105. Publisher Full Text\n\nMarina AM, Che Man YB, Ismail A: Use of the SAW sensor electronic nose for detecting the adulteration of virgin coconut oil with RBD palm kernel olein. J Am Oil Chem Soc. 2010; 87(3): 263–70. Publisher Full Text\n\nRohman A: Application of FTIR spectroscopy for quality control in pharmaceutical products: a review. Indonesian J Pharm. 2012; 23: 1–8. Reference Source\n\nHandajani J: Raw data-Dana Masyarakat 2019. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12733055.v5" }	[ { "id": "70437", "date": "04 Sep 2020", "name": "Futoshi Nakazawa", "expertise": [ "Reviewer Expertise Oral Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, authors analysed buccal cell DNA exposed to azo compound in batik workers. And, they showed that the characteristics of DNA could be used as an indication of exposure to azo compound in workers of batik industries.\n\nAlthough the number of subjects, 10 for each group, was small, the authors performed statistical comparisons. And they provided sufficient Tables and Fig.s for the conclusion. Also, descriptions of Introduction, Methods, Results and Discussion were reasonable. Therefore, I think that this article is acceptable as Brief Report.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "70435", "date": "04 Sep 2020", "name": "Boy M. Bachtiar", "expertise": [ "Reviewer Expertise Oral microbiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: I suggest “Risk assessment of DNA damage exposed to azo compound”\n\nIntroduction\nI suggest, in the introduction section, the author needs to write a strong background in this study. Please add the fundamental reason behind it. For example: whether the author has observed/found that the population (batik worker) were consisting of those with and without a “clinical symptom” of cellular damage.\n\nTherefore, the objective of this study was to compare the profile of buccal cell DNA exposed to synthetic azo dyes between the batik worker with and without clinical symptoms of cellular damage.\n\nMethods\nI think this study did not need to use control of those who come from not exposed subjects.\n\nThere is no explanation of the concentration of DNA that was put into the agarose well?\n\nResults\nIt would be better if the characteristics of study participants incorporated with pictures showing the buccal mucosa of participants with and without clinical symptoms.\n\nDiscussion\nIn general, this section needs to be revised. Please do not repeat the result but discuss it to answer the research question (this needs to clearly explain in the introduction section).\nSpecific questions:\n\"This result was supported by our spectrophotometer measurements (Table 2), showing that DNA concentration in the control group was lower than in the azo-exposed group. DNA quality may have been affected by collection and isolation methods. This result showed the mean OD 260/280 ratio was 1.89 both in the azo-exposed and control groups, which indicates that the bulk of the proteins were removed successfully.\"\nIn the second paragraph, “DNA electrophoresis revealed a band for high-molecular weight DNA in azo-exposed group”, indicating the DNA concentration was higher in BEC in azo exposed people. What does it mean? I only show the same band derived from either group. How much of the DNA concentration was added into the well of agarose?\n\nSimilarly, “This result was also supported by a previous study that showed that azo dye exposure increases the number of micronuclei…………, …………,…However, the author needs to explain what is the obtained data they have to support this statement.\n\nAlso, ……… ”not all samples in the azo-exposed group revealed a band. The author argues that the unrevealed band could be due to low concentration of DNA in the collected buccal epithelial cells”. I suggest, that they need to explain this result with the possibility of the relationship between the unrevealed DNA and azo exposure.\n\nConclusion\nPlease revise the conclusion. T should be related to this study’s aim. Particularly, what does the author mean with the “characteristics of DNA”? It could be used as indication (indicator?) of clinical symptom of azo exposure? The meaning is not clear.\n\nReferences\nSome references (no 11, 12, and 13) can be searched. Please use references that have been indexed in database.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5933", "date": "18 Sep 2020", "name": "Juni Handajani", "role": "Author Response", "response": "Title: I suggest “Risk assessment of DNA damage exposed to azo compound” Answer:Thank you for your suggestion.In this study, we did not know yet the occurrence of DNA damage, but it was to examine the change of DNA characteristics. We need further study to identify possible DNA damage. IntroductionI suggest, in the introduction section, the author needs to write a strong background in this study. Please add the fundamental reason behind it. For example: whether the author has observed/found that the population (batik worker) were consisting of those with and without a “clinical symptom” of cellular damage.Therefore, the objective of this study was to compare the profile of buccal cell DNA exposed to synthetic azo dyes between the batik worker with and without clinical symptoms of cellular damage.Answer:We added in introduction:Although previous studies stated that exposure to azo dyes significantly increased the expression of cytokeratin 5 and 19, but clinically it has not shown changes in the buccal mucosa. Until now, there is limited study on the effects of azo exposure on the buccal mucosa. MethodsI think this study did not need to use control of those who come from not exposed subjects.Answer:In this study we need control to compare between azo-exposed group and without exposed-azo. There is no explanation of the concentration of DNA that was put into the agarose well? Answer:We added explanation:Loading dye 1μl was prepared on parafilm then added 5 µl DNA, aspirated in the micropipette, put into the agarose well ResultsIt would be better if the characteristics of study participants incorporated with pictures showing the buccal mucosa of participants with and without clinical symptomsAnswer:We apologize, we did not take pictures of the buccal mucosa each subject. All subject did not show any clinical symptoms.DiscussionIn general, this section needs to be revised. Please do not repeat the result but discuss it to answer the research question (this needs to clearly explain in the introduction section).Specific questions:\"This result was supported by our spectrophotometer measurements (Table 2), showing that DNA concentration in the control group was lower than in the azo-exposed group. DNA quality may have been affected by collection and isolation methods. This result showed the mean OD 260/280 ratio was 1.89 both in the azo-exposed and control groups, which indicates that the bulk of the proteins were removed successfully.\"Answer:We added explanation:According Desjardins and Conklin that pure nucleic acids typically was in yield a 260/280 ratio of ~1.8 for DNA. In the second paragraph, “DNA electrophoresis revealed a band for high-molecular weight DNA in azo-exposed group”, indicating the DNA concentration was higher in BEC in azo exposed people. What does it mean? I only show the same band derived from either group. How much of the DNA concentration was added into the well of agarose.Answer:The results from DNA electrophoresis showed band in azo-exposed group while band was not seen in control group. When prepared the electrophoresis, we used loading dye to visualize the DNA. We added 5 µl DNA into loading dye. If we compared to DNA ladder, a band indicated that concentration in azo-exposed group is higher than in control group. Similarly, “This result was also supported by a previous study that showed that azo dye exposure increases the number of micronuclei…………, …………,…However, the author needs to explain what is the obtained data they have to support this statement.Answer:The results of this study have not yet revealed how the mechanism of DNA damage occurs in oral epithelial cells due to azo exposure even previous study showed there was some alteration in the buccal cell e.g. increases the number of micronuclei, karyolysis, pyknosis, and expression of cytokeratin 5 and 19 in the epithelial cell may be due to dye exposure. Also, ……… ”not all samples in the azo-exposed group revealed a band. The author argues that the unrevealed band could be due to low concentration of DNA in the collected buccal epithelial cells”. I suggest, that they need to explain this result with the possibility of the relationship between the unrevealed DNA and azo exposure.Answer:We added explanation:Another possibility, azo may cause DNA breakage in the human cell. In accordance with previous study, the component azo dye Disperse Orange 1 may cause apoptosis and DNA breakage in HepG2 cell derived from human hepatoma ConclusionPlease revise the conclusion. T should be related to this study’s aim. Particularly, what does the author mean with the “characteristics of DNA”? It could be used as indication (indicator?) of clinical symptom of azo exposure? The meaning is not clear.Answer:The result of this study was a preliminary study, and we have successfully to classify participants as controls and azo-exposed individuals based on score plot. We also characterized DNA of buccal cell according purity and concentration DNA, and absorbance ratio at 260/280. ReferencesSome references (no 11, 12, and 13) can be searched. Please use references that have been indexed in database.Answer:We revised some references by adding link." } ] } ]	1	https://f1000research.com/articles/9-1053
https://f1000research.com/articles/8-115/v1	29 Jan 19	{ "type": "Research Article", "title": "The effect of an electronic module about drug abuse prevention on teachers’ beliefs in Indonesia", "authors": [ "Ghozali Ghozali", "Ahmad Azuhairi A", "Nor Afiah Mohd Zulkefli", "Faisal Ibrahim", "Ahmad Azuhairi A", "Nor Afiah Mohd Zulkefli", "Faisal Ibrahim" ], "abstract": "Background: Drug abuse is a serious global health problem. Globally, 210 million people use illicit drugs each year, of which 200,000 die from drugs. Evidence shows that most drug addicts start using drugs in adolescence (<15-years-old). Adolescents need role models who are able to guide them; teachers have important roles as they are primary role models for students. Therefore, teachers should have positive beliefs to guide students effectively, i.e. they should have good awareness about the threat of drug abuse and high confidence to implement required prevention. This research developed an alternative electronic delivery method of learning material to empower teachers in preventing drug abuse. This study aimed to compare the effect of the electronic and a printed teaching module on teachers’ beliefs about drug abuse prevention. Methods: 260 junior high school teachers were selected randomly. These teachers were split into intervention and control groups. Before intervention, a questionnaire was completed by both groups. The teachers then completed the learning material: electronic module in intervention group and printed module in control group. One month later, data was collected from both groups using the same questionnaire to assess the beliefs of the teachers Results: There was significant positive effect on teachers’ beliefs, both in the intervention and control groups. All categories of beliefs at one month after intervention were significantly higher than those at baseline (P<0.001). Based on between group comparison analysis of mean changes, perceived susceptibility in the intervention group was significantly higher than the control group (P<0.001), while perceived severity, benefits, barriers and efficacy were not significantly different (P>0.05). Conclusions: Electronic and printed module intervention significantly increased teachers’ beliefs in drug abuse prevention. The printed module was still effective to be used as learning media, while the electronic module was an alternative with some advantages.", "keywords": [ "electronic module", "teachers’ beliefs", "drug abuse", "prevention" ], "content": "Introduction\n\nDrug abuse is a global health problem. Globally, 210 million people in the world use illicit drugs each year, and almost 200,000 of them die as a result of drug use1. The United Nations Office on Drugs and Crime (UNODC) also reported that in 2013, around 246 million people in the world or 1 in 20 people aged 15 to 64 years abused drugs. This was increased from ~3 million individuals from 2012. Approximately 12.19 million people out of 246 million were injecting drug users; 1.65 million individuals with HIV/AIDS1. In a study of HIV testing experience of drug users in Bali, there is evidence that most of the subjects in this study (60%) started using drugs in junior high school, aged 15-years-old or less2.\n\nAdolescence is a critical period because in this period there will be many changes in individual development, physically, psychologically and socially. In this period, adolescents will undergo many conflicts, including between the need for self-control and the need to be independent. In these conditions, adolescents require other persons to guide them. There seems to exist a general consent that education, and teachers in particular, have an important role to play by imparting knowledge, values and skills, as well as by acting as role models for students. Teachers should have adequate preparation to play their role sufficiently and effectively, which involves a combination of cognitive and practical knowledge and skills, values, motivation and attitudes. The lack of this set of preparation may impart a bad influence on the students’ learning outcomes and also students’ behaviors.\n\nTeaching is one enabling and reinforcing factor of student’s behavior, including student’s health behavior3. Positive beliefs of teachers are needed in their role to reinforce factors and be good models for students, including in drug abuse prevention. Hanley et al. studied the influence of teacher training on the fidelity of substance use prevention programs implementation in the United States. This study concluded that teacher training on this subject significantly increased the fidelity of implementation of the prevention program4.\n\nNowadays in Indonesia and many other developing countries, in the field of media and health promotion methods on drugs and the prevention of drug abuse, it is still very rare to find media or methods of delivering messages about the danger of drug abuse and the importance of its prevention that utilizes technological advances, especially for specific individuals like teachers. The electronic messaging media are mostly just short messages via television or radio for universal targets. Media with more complete message content in the form of books or printed brochures generally only target teenagers and general society. Macedo-Rouet et al. concluded that there was a strong motivation for using electronic books, and the level of users’ satisfaction was generally very high with this type of medai5. Many studies have also showed that the positive valuation towards electronic books was due to their accessibility and availability6,7. Shelburne8 stated that the increased availability of electronic books has influenced students’ perception and students value electronic books more than printed ones.\n\nThis research developed an alternative electronic delivery method of learning media to empower teachers in preventing drug abuse in the school setting. This study aimed to compare the effect of the electronic module with a printed module on teachers’ beliefs in drug abuse prevention among students.\n\n\nMethods\n\nThis study used comparative design to determine the effects of educational intervention using drug abuse prevention module towards changing and improving teachers’ beliefs in drug abuse prevention. The study was conducted from 10th October to 5th December 2016. In this study, the intervention group was given an educational intervention using an electronic module, and the control group received the usual printed module with the same content. Before the intervention, a pre-test was conducted to measure teachers’ beliefs. A post-test to measure teachers’ beliefs was conducted at one month after intervention. Pretest and post test used the same instrument.\n\nThe final participants of this study were 260 junior high school teachers in Balikpapan, Indonesia. The study sample size was calculated using Jekel’s formula9. The minimum sample size resulted from the calculation was 46 subjects per-group. In accordance with design effect, this number was multiplied by 2; therefore, the minimum number was 92 subjects per-group. With the consideration of 20% attrition rate, the number of expected sample was 115 persons per-group or 230 persons for both groups.\n\nA cluster random sampling was used in this study to select 6 schools from the total of 22 junior high schools. The inclusion criteria of participants were teachers with permanent and full-timer status, and all teachers who signed a written consent form to participate in the study. A total of 278 teachers met the inclusion criteria of this study, from 6 schools selected. Then, the random number assignment was used to allocate each selected school to intervention or control groups.\n\nTo prevent selection bias, a researcher assistant performed the allocation process with the instruction that they had to allocate participants to group 1 and group 2, without identifying which group is the intervention or control groups. The result of random assignment were three schools in the intervention group and the other three schools in control group, with the total number teacher who agreed to partake in the study being 133 teachers in the intervention group and 145 teachers in the control group. Therefore, 278 teachers in both groups was set as participants in this study, but 18 teachers could not complete the study or dropped-out from the study with various reasons. Therefore, a total of 260 teachers in both groups completed the study: 128 teachers in the intervention group and 132 teachers in the control group.\n\nIn the purpose to prevent bias from the participants, this study was set as a single-blinded study, where all of participants was unaware of whether they are in the intervention or control group. All of investigators made an agreement not to inform to participants about which group they were in, throughout the period of the study.\n\nThis study involved three experts in training module development and two practitioners in drug and drug abuse prevention to determine the validity of the module and questionnaire used in this study. Before being used in this study, the module and questionnaire were pretested in teachers who did not participate in this study to examine the reliability. The values of Cronbach alpha of the questionnaire was 0.701.\n\nThe content of the educational module included definition and types of drugs, factors affecting drug abuse, early detection of drug abuse, usual characteristics of drug abuser, effects of drug abuse, strategies of drug abuse prevention, the role of school and teacher in drug abuse prevention, and how to build a free drugs school.\n\nThe questionnaire was used to measure teachers’ beliefs about drug abuse and drug abuse prevention. This questionnaire consisted of four statements about perceived susceptibility, four statements about perceived severity, three statements about perceived benefits, three statements about perceived barriers, and seven statements about perceived self-efficacy.\n\nQuestionnaire and modules are available: http://doi.org/10.5281/zenodo.254653210\n\nDescriptive statistics was used to analyze sociodemographic variables and component of beliefs from the questionnaire at baseline. Chi-squared and Mann Whitney U test were used to compare these variables between groups at baseline. Paired-t and Wilcoxon signed-rank test were used to determine the effect of intervention in each group, and independent-t and Mann Whitney U test were used to compare effect of intervention between groups. All statistical analysis was performed using SPSS Version 24, with significance level of 95%.\n\n\nEthical approval\n\nThis study obtained approval from Balikpapan District Office of Ministry of Education (420/2180/SKT-VIII/2016) and The University Research Ethics Committee of the Universiti Putra Malaysia (UPM/TNCPI/RMC/1.4.18.1 (JKEUPM)/F1). Written informed consent was obtained from the teachers before they were recruited into the study.\n\n\nResults\n\nTable 1 describes the sociodemographic characteristics of study participants in each group. Most of the participants in both groups were female and Javanese. The mean of age was 41.53 ± 9.031 years in the intervention group and 43.19 ± 9.167 years in the control group. The mean of duration of work was 15.79 ± 8.890 years in the intervention group and 17.00 ± 9.388 years in the control group. There were no significant differences between intervention and control groups on the mean of age and duration of work, field of teaching, proportion of gender, and ethnicity.\n\nTable 2 describes each category of beliefs mean score from the questionnaire of participants in the intervention and control groups at baseline. There were no significant differences between intervention and control groups on participants’ categories of beliefs at baseline (P value > 0.05). Table 3 describes within group comparison of participants’ beliefs between baseline and one month after intervention. Overall the results indicated that the teachers’ beliefs at one month after intervention were significantly higher compared with baseline, in both groups. Table 4 describes between group comparison of mean changes in each component of beliefs from baseline to one month after intervention. There were significant differences between the intervention and control groups in mean changes of perceived susceptibility from baseline to one month after intervention (P<0.05). There were no significant differences between intervention and control groups in mean changes of perceived severity, benefits, barriers, efficacy, and total beliefs from baseline to one month after intervention (P value: 0.086, 0.283, 0.261, 0.834 and 0.129, respectively).\n\nSignificant level at P<0.05\n\nSignificant at level p<0.05\n\nSignificant difference at P<0.05\n\nSignificant difference at P<0.05\n\n\nDiscussion\n\nThe objective of this study was to evaluate the effects of educational intervention for teachers using a printed and electronic module on drug abuse prevention among junior high school students. The intervention group in this study received the educational intervention using an electronic module which has developed by the researchers. The control group received the usual printed module, which contained the same materials as the electronic module.\n\nThe findings of this study showed that there was a significant positive effect of the intervention on teachers’s beliefs about drugs and drug abuse prevention, both in the intervention group and the control group. All categories of beliefs at one month after intervention were significantly higher than those at baseline condition (P<0.001), both in intervention and control groups. Based on the between groups comparison analysis of mean changes from baseline to one month after intervention, there was found that mean changes of perceived susceptibility in the intervention group was significantly higher than control group (P<0.001). The mean changes of perceived severity, benefits, barriers, efficacy and total beliefs were not significantly different between intervention and control group (P>0.05). Based on these findings, we conclude that there was almost the same effects of the electronic module and the printed module on all categories of teachers’ beliefs in drug abuse prevention.\n\nSimilarly to these findings, Dusenbury et al.11 carried out a study to examine the influence of training on teacher beliefs and perceptions about norm setting and student drug use. They found that there was a significant pretest-to-posttest improvement on teacher beliefs and perceptions for several items. There was a significant improvement in teachers expectations that students to not go on to use substances (t=3.391, p=0.001); all teachers better understood how to develop lesson plans for drug education (t=5.886, p<0.001); and finally all teachers had marked improvement in their confidence to use norm setting in teaching (t=9.018, t<0.001). Our findings were also in line with a previous study which found that there was a significant correlation between knowledge on risk factors and the general score of attitude (r Pearson= 0.373, p < 0.001)12. This means that by giving specific knowledge, a person’s attitude toward specific issues will be improved. Belief is a form of attitude.\n\nAzwar13 stated that media is one of the factors influencing the formation of a closed response. Media has a fundamental task in the delivery of information. Media provides information and messages that contain suggestions which will direct one's opinion. When strong enough, the messages brought by the information will provide an effective basis for judging things, so that certain beliefs are formed. In this study, the module which was used in the intervention and control groups is described as learning media. This finding was also similar with that found by Mahmoodabad et al.14, who concluded that there was significant improvement on health belief model components average scores among male students two months after receiving an educational intervention about preventive drug dependency in Iran.\n\n\nConclusions\n\nEducational intervention using an electronic module in the intervention group and printed module in the control group significantly increased teachers’ beliefs in drug abuse prevention among students. The findings also indicated that in general both methods equally gave a positive effect on teachers' beliefs in preventing drug abuse. Therefore, both forms of delivery method of learning materials can be used as methods for teacher empowerment efforts in the prevention of drug abuse. The usual printed module was still effective and relevant to be used as learning media in drug abuse prevention, while the electronic module was an alternative that had some advantages in one category, and additionally is easy to carry everywhere, cheaper in production costs, durable, and environmentally friendly. To the best of our knowledge, there are not many studies that have evaluated the effectiveness of this module in the prevention of drug abuse especially on the target of teachers.\n\n\nData availability\n\nZenodo: Dataset, Module and Questionnaire of Teachers’ Beliefs About Drug Abuse, http://doi.org/10.5281/zenodo.254653210. Demographic data of participants is contained in ‘DEMOGRAPHIC_DATA.xlsx’. Data of comparison of sociodemographic characteristics and participants’ beliefs between study groups, within and between group comparison of beliefs changes is contained ‘RAWDATA_VAR.xlsx’.\n\nQuestionnaire used to assess teacher’s beliefs about drug abuse prevention: http://doi.org/10.5281/zenodo.254653210.\n\nElectronic/printed module used for the intervention and control groups: http://doi.org/10.5281/zenodo.254653210.\n\nUnderlying and extended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to express the gratitude to the head of Balikpapan District Office of Ministry of Education who made it possible for the authors to conduct this study.\n\n\nReferences\n\nUnited Nations Office on Drugs and Crime (UNODC): World Drug Report 2011. New York: UNODC. 2011. Reference Source\n\nSawitri AA, Sumantera GM, Wirawan DN, et al.: HIV testing experience of drug users in Bali, Indonesia. AIDS Care. 2006; 18(6): 577–88. PubMed Abstract \| Publisher Full Text\n\nGreen LW, Kreuter MW: Health Promotion And Planning: An Educational And Environmental Approach. 4th edition. Mountain View, CA: Mayfield Publishing Co. 1999.\n\nHanley S, Ringwalt C, Vincus AA, et al.: Implementing evidence-based substance use prevention curricula with fidelity: the role of teacher training. J Drug Educ. 2009; 39(1): 39–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacedo-Rouet M, Rouet JF, Epstein I, et al.: Effects of online reading on popular science comprehension. Sci Commun. 2003; 25(2): 99–128. Publisher Full Text\n\nAnuradha KT, Usha HS: Use of e-books in an academic and research environment: A case study from the Indian Institute of Science. Program: electronic library and information systems. 2006; 40(1): 48–62. Publisher Full Text\n\nCarlock D, Perry A: Exploring faculty experiences with ebooks: a focus group. Library Hi Tech. 2008; 26(2): 244–54. Publisher Full Text\n\nShelburne WA: E-book usage in an academic library: user attitudes and behaviors. Libr Collect Acquis. 2009; 33(2/3): 59–72. Publisher Full Text\n\nKatz DL, Elmore JG, Wild DMG, et al.: Jekel’s Epidemiology, Biostatistics, Preventive Medicine, and Public Health. Fourth Edition. Philadelphia: Elsevier Saunders. 2014.\n\nGhozali G, Azuhairi AA, Nor Afiah MZ, et al.: Dataset, Module and Questionnaire of Teachers’ Beliefs About Drug Abuse [Data set]. Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2546532\n\nDusenbury LA, Hansen WB, Giles SM: Teacher training in norm setting approaches to drug education: a pilot study comparing standard and video-enhanced methods. J Drug Educ. 2003; 33(3): 325–36. PubMed Abstract \| Publisher Full Text\n\nMoreira FG, Da Silveira DX, Andreoli SB: Knowledge and attitudes related to drug abuse and prevention displayed by public school educators. Braz J Psychiatry. 2009; 31(2): 95–100. PubMed Abstract \| Publisher Full Text\n\nAzwar S: Sikap Manusia Teori dan Pengukurannya, edisi 2. Yogyakarta: Pustaka Pelajar. 2007.\n\nMahmoodabad SSM, Khosab S, Vafa FS, et al.: The Effect of Health Education Based on Health Belief Model on Preventive Actions of Synthetic Drugs Dependence in Male Students of Kerman, Iran. Soc Behav Res Health. 2017; 1(2): 100–107. Publisher Full Text" }	[ { "id": "45218", "date": "15 Mar 2019", "name": "William B. Hansen", "expertise": [ "Reviewer Expertise drug prevention" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCorrect this grammar: with the total number teacher who agreed to partake in the study being 133 teachers in the intervention group and 145 teachers in the control group. Should be: with the total number of teachers who agreed to partake in the study being 133 teachers in the intervention group and 145 teachers in the control group. With a nested design, teachers within schools and schools being assigned to condition, the required number of teachers needed would be greater than the number apparently calculated. I suggest software such as Optimal Design. https://www.theanalysisfactor.com/sample-size-randomized-trials/ Only one alpha coefficient is reported but it is apparent that there are five subsclasses. Coefficients should be calculated for each separately, unless there is statistical evidence that they all load on one factor. No such evidence is provided. I appreciate that the questionnaire was made available. It is not clear if a pre-test and post-test were given, or if just a post-test was given. It sounds like there might have been a pre-test (baseline is mentioned), but it is not clear. Significance should be p<.05, not 95% (this was correctly noted in the tables, but not in the narrative). The data presented in Table 3 is very promising; however, a one-way ANOVA with repeated measures or MANOVA would have been a more appropriate analytic method. Also, it is a bit bothersome to have t values presented some of the time and Z values the rest of the time. If consistency cannot be maintained, it should be explained. Tables 3 and 4 present results that could be combined. The second paragraph of the discussion includes a wordy rehash of the results paragraph about the same topic. I think the discussion should focus more on interpretation of results. There really is not a treatment and a control group. Both sets of teachers are exposed to an intervention; therefore, both groups are treatment groups. They are just different in the format with which they received training. There really is no control group and it is misleading to call the group that received paper instruction as such. I would change the title of the paper to fit this reality.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4536", "date": "17 Sep 2020", "name": "Ghozali Ghozali", "role": "Author Response", "response": "Thank you so much for your review and your suggestions. We will make some corrections and explanations based on your suggestions. The corrections will be in our revised article. Some explanations are below: The number of teachers recruited in each group has considered the design effect and attrition rate. Based on the minimum sample calculation, the minimum number of samples was 46 per-group, considered the design effect then the number multiplied by 2 to 92 people per-group. Taking into account the attrition rate of 20%, the number of samples was 115 per-group. The actual number of samples recruited at the initial stage was 133 people in group 1 and 145 people in group 2 (greater than the optimum sample size, 115). Questionnaire was available at https://zenodo.org/record/2542702#.XKOtEuszYTE A pre-test and post-test were given. We mean baseline measurement of variables as a pre-test. Anyway, we will make correction in the narrative. We did not use ANOVA or MANOVA since there was only two groups and two times measurement of dependent variable. The t and Z values were retained in table 3 because of the different statistical tests used. The t value was obtained from the paired-t test (parametric) , while the Z value of the Wilcoxon signed rank test (non-parametric). However, we will accommodate suggestions for combining tables 3 and 4 in the revised article." }, { "c_id": "5927", "date": "18 Sep 2023", "name": "Ghozali Ghozali", "role": "Author Response", "response": "Dear Dr William B. Hansen, Thank you so much for reviewing our article and giving us some great suggestions. We have carefully considered all your comments and suggestions and we have accommodated them in the revised version of the manuscript we have uploaded. The points of explanation and updates are as follows: The grammar of the third paragraph in the section of study participants has been corrected according to the suggestion. This study has used a sample size larger than the number apparently calculated. The results of the minimum sample calculation in this study according to the formula and added with the attrition rate were 115 teachers per group or 230 teachers for two groups. The actual number of respondents in this study was 128 for group 1 and 132 for group 2. The alpha coefficients have been added for each of the five subclasses of beliefs, as suggested In the study design section, there has been made clear that the “pretest” is referred to as “baseline” conditions. Significance level in the narrative of data analysis section has been corrected to P < 0.05 Tables 3 and 4 have been combined into table 3, following the suggestion. Paired t and Wilcoxon tests have been changed with repeated measure ANOVA, so that the statistical values have also been adjusted and consistent. The description of table 3 in the results section has been updated The second paragraph in the discussion has been improved, focusing more on the interpretation of the results. We agreed with the comment that both groups in our study are treatment groups. The title of manuscript was updated by adding “and printed”. The words of “intervention group” and “control group” have been changed to “group 1” and “group 2” in the whole of the manuscript. We sincerely hope that the responses and revisions that we have made can fulfill the suggestions that you have provided to make our article better. Best regards." } ] }, { "id": "66158", "date": "14 Jul 2020", "name": "Manop Kanato", "expertise": [ "Reviewer Expertise Drug abuse", "Health behavior", "Epidemiology", "Policy evaluation", "Preventive Medicine & Public Health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough this article is interesting, minor revision is needed. UNODC world drug report is now 2020, reference WDR 2011 may be too old. The article needs more details on the intervention and implementation process. Characteristics of the teacher should be taken into account in the analysis. Statistics regarding covariate need to be considered rather than paired and independent t-test.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5928", "date": "17 Sep 2020", "name": "Ghozali Ghozali", "role": "Author Response", "response": "Dear Dr Manop Kanato,Thank you very much for reviewing our article and giving some great suggestions to make our article better. We have made corrections according to your suggestions and the revised article has also been uploaded. The first paragraph of the introduction was partly changed according to the newer reference of World Drug Report as suggested. The reference of World Drug Report has been changed to the 2020 version. The more detailed explanation in the study design especially to clarify the intervention and implementation process has been added according to the suggestion. Characteristics of the teachers analysis were not fully presented in this article because there were too many descriptions, so that the material was being prepared to be made in another separate publication article. This article is focused on the comparison of the effects between the electronic module and the printed module, table 1 on the comparison of the characteristics of teachers between group 1 and group 2 is intended to provide an illustration that in terms of the characteristics of teachers the two groups were comparable. The statistical data analysis has been improved according to suggestion. T test, Wilcoxon and Mann Whitney U tests have been replaced with repeated measure ANOVA and Kruskal Wallis Test. Tables 3 and 4 have been combined into table 3 which contains the results of statistical test comparisons within and between groups using repeated measure ANOVA and Kruskal Wallis Tests. We sincerely hope that the improvements we have made can fulfill the suggestions you have given to make our article better.Best regards" } ] } ]	1	https://f1000research.com/articles/8-115
https://f1000research.com/articles/9-1144/v1	16 Sep 20	{ "type": "Software Tool Article", "title": "BCO App: tools for generating BioCompute Objects from next-generation sequencing workflows and computations", "authors": [ "Nan Xiao", "Soner Koc", "David Roberson", "Phillip Brooks", "Manisha Ray", "Dennis Dean", "Nan Xiao", "Soner Koc", "David Roberson", "Phillip Brooks", "Manisha Ray" ], "abstract": "The BioCompute Object (BCO) standard is an IEEE standard (IEEE 2791-2020) designed to facilitate the communication of next-generation sequencing data analysis with applications across academia, government agencies, and industry. For example, the Food and Drug Administration (FDA) supports the standard for regulatory submissions and includes the standard in their Data Standards Catalog for the submission of HTS data. We created the BCO App to facilitate BCO generation in a range of computational environments and, in part, to participate in the Advanced Track of the precisionFDA BioCompute Object App-a-thon. The application facilitates the generation of BCOs from both workflow metadata provided as plaintext and from workflow contents written in the Common Workflow Language. The application can also access and ingest task execution results from the Cancer Genomics Cloud (CGC), an NCI funded computational platform. Creating a BCO from a CGC task significantly reduces the time required to generate a BCO on the CGC by auto-populating workflow information fields from CGC workflow and task execution results. The BCO App supports exporting BCOs as JSON or PDF files and publishing BCOs to both the CGC platform and to GitHub repositories.", "keywords": [ "BCO App", "BioCompute Object", "Common Workflow Language", "precisionFDA", "Cancer Genomics Cloud", "IEEE 2791-2020" ], "content": "Introduction\n\nThe BioCompute Object (BCO) is an IEEE standard (IEEE 2791-2020) titled Bioinformatics Analyses Generated by High-Throughput Sequencing (HTS) to Facilitate Communication1. BCOs provide a systematic approach for documenting next-generation sequencing (NGS) data analysis workflows in order to facilitate communication of these complex computations between stakeholders2. The need for the BCO standard emerged from the realization that documenting NGS data analysis tool choices and parameter settings is equally as crucial for ensuring reproducibility as documenting experimental methods3. Whereas there are elaborate methodologies for documenting experiments, there is no gold standard for documenting NGS computation. Consequently, the goal of developing BCO software tools is to facilitate the generation and adoption of BCOs from a range of computational architectures in support of government, academic, and industrial applications.\n\nThe BCO, in its simplest form, supports the documentation of workflows through nine domains (provenance, usability, extension, description, execution, parametric, input/output, error, and top-level fields), each with two to twelve fields that specify domain characteristics (i.e., domain fields). The BCO supports documenting execution components (such as computational implementations and computational platforms) through the execution domain and the description domain. The specification aims to further clarify the workflow execution via the input/output domain and the error domain that defines expected errors. It also allows additional information describing the appropriate use of a workflow through the usability and parametric domains. A primary design principle of the standard is to reduce the effort required to create BCOs that conforms to the specification, by only requiring plaintext entries for each field. The simplest BCO instantiation, by definition, is a JSON file with text entries corresponding to the domain fields.\n\nWe present the BCO App, a web application that assists in the rapid generation of BCOs from bioinformatics workflows and their execution results. The application accepts plaintext user inputs, workflow contents written in the Common Workflow Language (CWL), and task execution results from the Cancer Genomics Cloud (CGC), an NCI funded computational platform4 and other similar informatics platforms. By connecting to the CGC, the application enables the users to automatically populate the workflow metadata, the fields in the execution domain, the fields in the input/output domain, and the fields in the parameter domain, which already exist within workflow written in CWL and task information on the CGC. Reusing workflow and task information reduces the time required to construct a BCO and allows users to focus on authoring content for description domains and usability domains. The BCO App can be deployed and accessed on local machines, dedicated hosting servers, and the CGC. Additional details on the supported running environments and cloud platform integrations can be found in the “Deployment” section. The application’s implementation and operation details are described below. An example bioinformatics pipeline for RNA-seq differential expression analysis is used to demonstrate the BCO generation flow.\n\n\nMethods\n\nFigure 1 shows an overall schematic of the BCO App’s architecture. The web interface is the central component of the application (Figure 1C). The web interface provides an optional authentication module, accepts user inputs, supports interactive updates to the BCO field entries, displays generated outputs, and can optionally connect users to informatics platforms via an API. The backend of the web application (Figure 1B, Figure 1D) receives user inputs, including workflow information (Figure 1A), and composes the BCO output as either a JSON file or a PDF formatted file (Figure 1F). The BCO App supports multiple deployment options, including local workstation support through a Docker container, persistently running instances on a remote hosting server, and the CGC (Figure 1E). The modularized application also allows a user-contributed extension component to add support for additional cloud-based informatics platforms (Figure 1G).\n\nA) The BCO App allows for text, workflows written in Common Workflow Language (CWL), and CGC task information as inputs. The BCO app parses complex inputs, as in the case for CWL workflow files and CGC task information. For the CWL input case, the BCO App extracts workflow metadata required to generate a BCO from the CWL file. B) The R package tidycwl handles CWL workflow processing, including reading, parsing, and visualizing CWL workflows. C) The BCO App offers BCO generation and validation capabilities powered by the tidycwl and biocompute R packages. D) The biocompute package implements the BioCompute standard. The package can compose, validate, and export BCOs. E) The BCO App can be deployed locally or remotely, with a fully-managed or self-managed approach. F) BCOs generated by the BCO App can be exported as JSON and PDF, or persistently saved to informatics platforms or GitHub repositories. G) The BCO App offers informatics platform integration that can be easily extended to include additional integration options.\n\nWe use the R web framework Shiny5 to implement the user interface and interaction logic of the BCO App. The functional components behind the application are two R packages: biocompute and tidycwl. The biocompute package is an implementation of the BioCompute standard in R. The package offers the capabilities to compose, validate, convert, and export BioCompute Objects. The tidycwl package can read, parse, and visualize CWL workflows from their JSON or YAML representations. These packages ensure that the application’s core components are separate from the interface code and interaction logic, while still being standardized and reusable for other applications developed for working with BCO and CWL. The architecture of both R packages employs the tidyverse design guide to ensure their consistency and interoperability within the existing R package ecosystem.\n\nIn this section, we provide a summary of the BCO App’s features and deployment options. See the “BCO App User Manual” for more installation and operational details.\n\nThe BCO App architecture supports the generation of a BCO through the web application or by using the R packages biocompute and tidycwl directly. For advanced users or developers who prefer creating BCOs programmatically, please see the vignette “A Grammar for Tidying CWL Workflows” for processing CWL workflows, and the vignette “Create and Manipulate BioCompute Objects with R” for generating BCOs.\n\nFeatures. The primary features of the BCO App include 1) the BCO Composers, 2) the BCO Validator, and 3) the BCO Browser, with each feature arranged as an individual page accessible from the navigation bar. The application includes an optional authentication module, which allows the application administrators to control user access and manage permissions in scenarios such as collaborative BCO editing for a team of contributors and reviewers. Users can quickly search and browse definitions of specific BCO domains or fields from an interactive, tabular version of the BioCompute standard by visiting the “Utilities - Standard” page without losing the BCO content editing progress or focus. We describe the primary features below.\n\nBCO Composers. The BCO App includes three types of composers that facilitate each of the three use cases driven by the source and type of inputs, detailed as follows:\n\nThe Text Composer features a form wizard user interface for creating BCOs. This interface allows users to fill out the standard BCO fields as forms with plaintext input. After paging through the forms that facilitate user editing of fields by the BCO domain, the user can generate and review the BCO presented in JSON format. There is an option in the final step to download the BCO as a JSON file.\n\nThe CWL Composer generates BCOs with the computational workflow information from uploaded CWL files. It offers semi-automated generators for creating BCOs from local workflows written in CWL. Generation of the BCO proceeds similarly to the Text Composer after the workflow is uploaded and parsed, with options to download the BCO as a JSON or PDF file.\n\nThe Platform Composer can generate BCOs with the workflow and its execution information from computational platforms. It takes a user-specified workflow or task (a completed workflow execution archive) as input. It then uses this input to pre-populated workflow execution-related fields defined in the standard. It also includes additional options to publish the generated BCO to a CGC project or to GitHub repositories.\n\nBCO Validator. The BCO Validator supports the two types of validation recommended by the BioCompute standard (IEEE 2791-2020). After uploading a BCO file, the validator computes and validates the SHA-256 checksum of all non-top-level domains, to ensure its content integrity. Next, the validator verifies each BCO domain against the BCO JSON schema and advises users about potential structural issues, such as a type mismatch or required fields being left blank.\n\nBCO Browser. The BCO Browser includes an interactive BCO viewer that supports domain-specific BCO inspection, data type highlighting, collapsed/expanded view for nested BCO components, and copying the components selectively to the clipboard for further inspection.\n\nDeployment. The BCO App supports multiple testing and production scenarios by offering flexible, off-the-shelf installation or deployment options. Currently, there are three options to deploy and access the application.\n\nSelf-managed local installation. We offer a containerized version of the application, with all software dependencies packaged as a Docker image. Users can pull the pre-built Docker image from Docker Hub, or build the image locally, then run the Docker container to start the application.\n\nFully-managed cloud deployment. A pre-configured application is packaged with required dependencies, and it can execute inside the “Data Cruncher” environment on the CGC. This method enables CGC users to access and run the application inside a CGC-hosted RStudio Server instance, directly facilitating access to over 500 public CWL tools and workflows on the CGC.\n\nSelf-managed cloud deployment. Users can choose to host the BCO App with a dedicated hosting server using their existing cloud infrastructure. This approach provides a self-managed solution with secure, browser-based access to the application, suitable for large-scale distribution within organizations.\n\nThese deployment options aim to maximize the deployment flexibility while lowering the deployment barriers due to possible constraints in software access and security policies. The BCO App user manual provides detailed steps and additional information regarding the deployment.\n\n\nUse cases\n\nWe demonstrate the process of generating a BioCompute Object using the BCO App with an NGS data analysis workflow and its execution results available from the CGC. We specifically use an RNA-seq workflow with publicly available NGS data from a study of bi-ventricular heart failure (accession number GSE120852)6. The workflow demonstrates a complete RNA-seq data analysis procedure, beginning from raw FASTQ files and ending with differential expression and pathway enrichment analysis results.\n\nWe used the Platform Composer in the BCO App to generate a BCO from a completed RNA-seq workflow execution. The Platform Composer guides the user from workflow selection through six steps resulting in a generated BCO. The first step involves selecting a specific workflow on the CGC. The application then populated multiple BCO fields across multiple domains automatically. More specifically, the application successfully captured the 102 input files, 187 output files, and four workflow steps with their associated input and output parameter lists. The application then populated the appropriate fields in the description and input/output domain with the captured information.\n\nWe then added additional workflow design details and a description of appropriate use to the usability domain. For the provenance domain, we provided detailed review and contributor information to ensure the traceability of changes made to the BCO. Finally, we exported the generated BCO as a JSON file. Figure 2 shows the first and the last form inputs (steps 1 and 6) of the BCO generation. See the Data availability section for additional screenshots taken during the BCO generation.\n\nThe figure shows the first step (step 1) and the last step (step 6) for generating a BCO from a workflow stored on the Cancer Genomics Cloud (CGC). A) Step 1 shows the workflow import panel that includes the CGC project selector, workflow selector, task selector, and the authentication field. B) Step 6 shows the review and export panel that displays the BCO preview generated from the RNA-seq data analysis. The panel also shows buttons (features) to export the generated BCO as JSON or PDF and save it to the platform or GitHub repositories.\n\nA major advantage of using CWL-based input is that the BCO App can access all the information within the CWL file, including the structured data that describes the workflow inputs, outputs, and steps. With the workflow graph data, the application can automatically generate a workflow wiring diagram which allows the user to review the workflow visually. Figure 3 presents the automatically constructed RNA-seq workflow visualization with the provenance, usability, and extension domain’s forms (step 2).\n\nThe figure highlights the automatically generated visualization of the RNA-seq workflow in the provenance, usability, and extension domain’s forms (step 2 of 6). The figure also shows some of the automatically populated fields in the provenance domain.\n\nNotably, we submitted the generated BCO to the beginner track of the precisionFDA BioCompute Object App-a-thon in October 2019. The generated BCO received full scores on basic qualifications. The BCO App received high scores in terms of functionality, documentation, usability, and aesthetics as an advanced track submission.\n\nThe CWL workflow, the example RNA-seq data, and the generated BCO can be downloaded from the repositories mentioned under the Data availability section.\n\n\nDiscussion\n\nWe developed the BCO App to facilitate the adoption of the BioCompute standard. Multiple practical use cases and deployment options are supported, from working on a local machine to working in cloud computing environments. Providing strong support for CWL processing makes documenting workflows more detailed, less error-prone, and reduces the time required to generate BCOs. Moreover, enabling the BCO App to access workflow and task information from the CGC exemplifies integrating the application with other informatics platforms. Designed with extensibility and modularity in mind, the application can be used as-is on platforms like the CGC. It can be easily extended to access workflow and task information from several other research platforms, including the NHLBI BioData Catalyst and Cavatica. Thus, BCOs and the BCO App could play a role in enhancing the computational reproducibility of NGS data analysis.\n\n\nData availability\n\nThe example BVHF RNA-seq dataset was obtained from NCBI GEO: WIPI1 is a Genetic Hub that Mediates Right Ventricular Failure, accession number GSE120852, and Sequence Read Archive (SRA), accession number SRP163468.\n\nFigshare: BioCompute Object - RNA-Seq Differential Expression & Pathway Analysis - Generated by BCO App, https://doi.org/10.6084/m9.figshare.10257659.v47.\n\nThis project contains the following underlying data:\n\nrnaseq-de-pathway.cwl.json (CWL workflow for RNA-seq differential expression and pathway analysis)\n\nrnaseq-de-pathway.bco.json (A BioCompute Object generated by the BCO App from the RNA-seq differential expression and pathway analysis workflow and execution results on CGC.)\n\nFigshare: BCO App User Interface, https://doi.org/10.6084/m9.figshare.12793457.v28.\n\nThis project contains the following extended data:\n\n1-landing-page.png (Landing page)\n\n2-composer-text-step-1...-5.png (Text Composer)\n\n3-composer-cwl-step-1...-6.png (CWL Composer)\n\n4-composer-platform-step-1...-6.png (Platform Composer)\n\n5-browser.png (BCO Browser)\n\n6-validator.png (BCO Validator)\n\n7-standard.png (BCO Standard Viewer)\n\n8-help.png (Application help page)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSource code available from:\n\nhttps://github.com/sbg/bco-app (BCO App)\n\nhttps://github.com/sbg/biocompute (R package biocompute)\n\nhttps://github.com/sbg/tidycwl (R package tidycwl)\n\nArchived source code at the time of publication:\n\nhttp://doi.org/10.5281/zenodo.3967760 (BCO App)9\n\nhttp://doi.org/10.5281/zenodo.3967769 (R package biocompute)10\n\nhttp://doi.org/10.5281/zenodo.3967767 (R package tidycwl)11\n\nLicense: GNU Affero GPL v3.\n\nAccess to the Cancer Genomics Cloud is free for all academic and nonprofit researchers, but it requires the creation of a login before use. Users can log in with either an email and password, or they can log in with their eRA Commons credentials to access controlled data. Data access restrictions according to each dataset apply. See here for more information: https://www.cancergenomicscloud.org/controlled-access-data.", "appendix": "Acknowledgments\n\nWe appreciate the support from the Mazumder Lab at George Washington University in deploying the BCO App as part of an extra credit assignment for a first-year graduate bioinformatics course (BIOC-6223). We especially thank Dr. Raja Mazumder, Dr. Jonathon Keeney, Charles Hadley King, Janisha Patel, and all the students who used the tool as part of their assignments.\n\n\nReferences\n\nIEEE Standard for Bioinformatics Analyses Generated by High-Throughput Sequencing (HTS) to Facilitate Communication. IEEE Std 2791-2020. 2020; 1–16. Reference Source\n\nAlterovitz G, Dean D, Goble C, et al.: Enabling precision medicine via standard communication of HTS provenance, analysis, and results. PLoS Biol. 2018; 16(12): e3000099. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimonyan V, Goecks J, Mazumder R: Biocompute objects - A step towards evaluation and validation of biomedical scientific computations. PDA J Pharm Sci Technol. 2017; 71(2): 136–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLau JW, Lehnert E, Sethi A, et al.: The cancer genomics cloud: Collaborative, reproducible, and democratized - A new paradigm in large-scale computational research. Cancer Res. 2017; 77(21): e3–e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWojciechowski J, Hopkins AM, Upton RN: Interactive pharmacometric applications using R and the Shiny package. CPT Pharmacometrics Syst Pharmacol. 2015; 4(3): 146–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTzimas C, Rau CD, Buergisser PE, et al.: WIPI1 is a conserved mediator of right ventricular failure. JCI Insight. 2019; 5(11): e122929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiao N: BioCompute Object - RNA-Seq Differential Expression & Pathway Analysis - Generated by BCO App. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10257659.v4\n\nXiao N: BCO App User Interface. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12793457.v2\n\nXiao N, Koc S: sbg/bco-app: BCO App 1.0.0 (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3967760\n\nXiao N: sbg/biocompute: biocompute 1.0.3.9000 (Version v1.0.3.9000). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3967769\n\nXiao N: sbg/tidycwl: tidycwl 1.0.4.9000 (Version v1.0.4.9000). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3967767" }	[ { "id": "71511", "date": "23 Sep 2020", "name": "Ezekiel Maier", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis software tool article describes the BCO App, which enables users to easily generate of BioCompute Objects. BioCompute Objects are instances of the IEEE approved BioCompute specification, and provide a standardized reporting framework to enhance the reproducibility of computational workflows. The BCO App can be deployed locally or in the cloud (including the Cancer Genomics Cloud), and provides text, CWL, and platform based mechanisms for composing BCOs. In additions the BCO App provides a BCO validator to ensure compliance of the BCOs to the BioCompute specification, and a browser for viewing BCOs. The BCO App was designed and developed with modularity and interoperability in mind. The BCO App substantially reduces the burden of generating, validating, and viewing BCOs.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "71509", "date": "26 Nov 2020", "name": "Yuriy Gusev", "expertise": [ "Reviewer Expertise Bioinformatics", "genomics data science", "cancer research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA concept of biocompute objects (BCOs) was developed about 3 years ago to satisfy regulatory research needs for evaluation, validation, and verification of bioinformatics pipelines. Since then BioCompute Object (BCO) standard was established as an IEEE standard for communicating NGS data analysis pipelines primarilhy across regulatory agencies such as FDA.\n\nThis article describes a new web application that allows to generate BCOs on a range of computational platforms with major focus on cloud computational environments. Importantly, the BCO App accepts plaintext user inputs, workflow contents written in the Common Workflow Language (CWL), and task execution results from the Cancer Genomics Cloud (CGC), that has became one of the major platforms of choice for bioinformaticians conducting cancer genomics research. The BCO App provides fast and efficient way to generate BCO from existing CGC pipelines or workflows and task execution results. The BCO App can be extended to access workflow and task information from several similar cloud based computational platforms.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1144
https://f1000research.com/articles/9-1142/v1	16 Sep 20	{ "type": "Research Article", "title": "Population structure of Salmonella enterica serotype Mbandaka reveals similar virulence potential irrespective of source and phylogenomic stratification", "authors": [ "Linto Antony", "Gavin Fenske", "Radhey S Kaushik", "Tiruvoor G Nagaraja", "Milton Thomas", "Joy Scaria", "Linto Antony", "Gavin Fenske", "Radhey S Kaushik", "Tiruvoor G Nagaraja", "Milton Thomas" ], "abstract": "Background: Salmonella enterica serotype Mbandaka (Salmonella ser. Mbandaka) is a multi-host adapted Non-typhoidal Salmonella (NTS) that can cause foodborne illnesses in human. Outbreaks of Salmonella ser. Mbandaka contributed to the economic stress caused by NTS due to hospitalizations. Whole genome sequencing (WGS)-based phylogenomic analysis facilitates better understanding of the genomic features that may expedite the foodborne spread of Salmonella ser. Mbandaka. Methods: In the present study, we define the population structure, antimicrobial resistance (AMR), and virulence profile of Salmonella ser. Mbandaka using WGS data of more than 400 isolates collected from different parts of the world. We validated the genotypic prediction of AMR and virulence phenotypically using an available set of representative isolates. Results: Phylogenetic analysis of Salmonella ser. Mbandaka using Bayesian approaches revealed clustering of the population into two major groups; however, clustering of these groups and their subgroups showed no pattern based on the host or geographical origin. Instead, we found a uniform virulence gene repertoire in all isolates. Phenotypic analysis on a representative set of isolates showed a similar trend in cell invasion behavior and adaptation to a low pH environment. Both genotypic and phenotypic analysis revealed the carriage of multidrug resistance (MDR) genes in Salmonella ser. Mbandaka. Conclusions: Overall, our results show that the presence of multidrug resistance along with adaptation to broad range of hosts and uniformity in the virulence potential, isolates of Salmonella ser. Mbandaka from any source could have the potential to cause foodborne outbreaks as well as AMR dissemination.", "keywords": [ "Salmonella", "Mbandaka", "pathogenesis", "foodborne pathogen" ], "content": "Introduction\n\nFor the last two decades, Salmonella has remained the major foodborne pathogen in the U.S. according to the Centers for Disease Control and Prevention (CDC)1. Non-typhoidal Salmonella (NTS) is estimated to cause 1 million foodborne illnesses annually2. Salmonella enterica subspecies enterica serotype Mbandaka (Salmonella ser. Mbandaka) has been identified by the CDC as an important outbreak-causing serotype of Salmonella3. Classified as one of the top ten Salmonella serotypes that cause human foodborne illnesses in Europe, a clonal population of Salmonella ser. Mbandaka (sequence type ST413) has been shown to be capable of surviving for many years and associated with animal feed, poultry, and human food4. Following its initial isolation from the Belgian Congo (Central Africa) in 1948, Salmonella ser. Mbandaka has been reported as a cause of human salmonellosis in several countries, making this serotype globally important for human and animal health4–6.\n\nHuman foodborne illnesses caused by Salmonella ser. Mbandaka have rarely been reported in the U.S.; nevertheless, three multistate outbreaks were reported by the CDC between 2013 and 20187–9. Based on annually compiled data from several sources, Hayward et al. reported that cattle, poultry, and pigs are the major hosts of Salmonella ser. Mbandaka10. However, the sources of two of the abovementioned outbreaks were from food preparations7,9, indicating the spread of this multi-host adapted serotype by other means.\n\nAlthough Salmonella ser. Mbandaka has been involved in several multi-serotype comparative studies, its epidemiological and evolutionary characteristics are not well understood. Previous studies on Salmonella ser. Mbandaka have focused on either a very small number of isolates11 or isolates from a specific geographical location4. Comparative genomic analysis has been widely used as a powerful tool for elucidating genomic diversity across Salmonella serotypes as well as epidemiological investigation of outbreaks12–14. In the present study, we defined the population structure and associated genotypic features of Salmonella ser. Mbandaka in a global context using whole genome sequence (WGS)-based analysis of 403 Salmonella ser. Mbandaka genomes. We assessed the antimicrobial resistance (AMR) and virulence gene repertoire of this Salmonella serotype to understand the potential capability of this serovar to act as an important zoonotic pathogen and public health hazard. To verify the genotypic prediction of the AMR and virulence, we examined these characteristics phenotypically using an available set of isolates that represented the study population of 403 Salmonella ser. Mbandaka isolates.\n\n\nResults\n\nTo define the phylogenomic characteristics of Salmonella ser. Mbandaka, we used genome sequence data previously deposited in the National Center for Biotechnology Information (NCBI) sequence read archive (SRA) in conjunction with our newly sequenced genomes. To ascertain their serotype as Salmonella ser. Mbandaka with an antigenic formula of z10 e,n,z15, we performed in silico genoserotyping of all genomes using the web-accessible tool Salmonella In Silico Typing Resource (SISTR)15. Samples that showed a discrepancy in their serotype between the available metadata and our serotyping results were removed from further study. This resulted in a final set of 403 Salmonella ser. Mbandaka genomes, of which 66 were newly sequenced and the remaining 337 were accessed from the NCBI-SRA. Based on their isolation sources, we grouped them into 12 different categories (Table 1; Supplementary Table 1, Underlying data16).\n\nThe distribution of selected isolates based on their geographical origin and isolation source.\n\n*Median\n\nWe performed core genome multilocus sequence typing (cgMLST) analysis of the genomes using the web tool ‘SISTR’ and identified five different sequence types (STs) in Salmonella ser. Mbandaka. ST413 was the most common type (93% of the isolates) followed by ST1602 (5% of the isolates) (Supplementary Table 2, Underlying data16). ST413 showed a global prevalence, while others were limited to certain geographical areas. For example, ST1602, ST2238, and ST2444, were found only in European or Asian isolates, while ST2404 was found only in North American isolates (Supplementary Figure 1, Extended data17). A similar trend was observed in the case of isolation sources of these STs. ST143 showed a wide distribution of isolation sources, while a narrower source distribution was found in other STs of this serotype (Supplementary Figure 1, Extended data17).\n\nWe hypothesized that the population structure of Salmonella ser. Mbandaka may have host-specific clades. To test our hypothesis, we constructed a core genome phylogeny and elucidated the pan-genome of the serotype. Figure 1 illustrates the phylogeny of the serotype as predicted by Bayesian evolutionary analysis sampling trees (BEAST). The results show that the isolates bifurcate into two major groups (Figure 1A). Contrary to our hypothesis, no major host-specific clades were identified in this analysis. The constituent genomes of groups 1 and 2 did not show any clear pattern with respect to their isolation source, geographical origin, or date of isolation (Supplementary Figure 1, Extended data17). We generated a pairwise single nucleotide polymorphism (SNP) distance matrix from the core gene alignment of 403 genomes. Interestingly, hierarchal clustering of these genomes based on the Pearson correlation showed the same biphasic clustering of the genomes (Figure 2), further supporting the core genome phylogeny. The pairwise SNP difference between the members within a group (either group 1 or group 2) differed by a median of 46 SNPs in both groups. However, the difference was large between group 1 and group 2 members, with a median of 293 SNPs (Figure 2).\n\nMonomorphic single nucleotide polymorphism (SNP) sites extracted from the multi-FASTA alignment of all the core genes were analyzed for the reconstruction of Salmonella ser. Mbandaka phylogeny using the Bayesian approach (BEAST. v.2.5.1). Maximum clade credibility (MCC) tree rooted to outgroup isolates (KY1 and ALT1) showed a separation of the total population into two major groups (colored red and green). Figure was generated using FigTree v.1.4.4. B.Pan-genome analysis of Salmonella ser. Mbandaka isolates using Roary identified almost 4100 genes (29%) as core genes (present in ≥ 99% of the strains).\n\nA) Heatmap showing the hierarchical clustering of 403 Salmonella ser. Mbandaka genomes based on the Pearson correlation of their pairwise SNP distance. B) Box plot representation of the number of pairwise SNP differences between members of group 1 (i), group 2 (ii), and group 1 and group 2 (iii).\n\nBased on the clustering pattern in the Markov chain Monte Carlo (MCMC) tree, we divided each group into different subgroups and determined the distribution of isolates according to their origin and isolation source. We found that all subgroups contained isolates from multiple sources (Figure 1A); however, there was a closer association of food isolates with Asian countries and human isolates with Europe. These associations were found in both groups 1 and 2. Pan-genome analysis of Salmonella ser. Mbandaka revealed a core genome size of 4,044 genes and a pan-genome size of ~ 13,000 genes. The core genes that represented 30% of the pan-genome were found in ≥ 99% of the genomes analyzed; however, the cloud genes i.e., those present in only < 15% of the total genomes analyzed, represented the major share (65%) of the Salmonella ser. Mbandaka pan-genome (Figure 1B).\n\nThe presence of antimicrobial-resistant pathogenic bacteria in food has been addressed as a direct hazard to public health18. We determined the AMR profile of Salmonella ser. Mbandaka at both the phenotypic and genomic levels.\n\nOur analysis revealed 40 AMR genes in 403 Salmonella ser. Mbandaka genomes (Figure 3; Supplementary Figure 2, Extended data17; Supplementary Table 3, Underlying data16). These genes were related to resistance against 12 classes of antibiotics. Most resistance was found against tetracycline (16.87% genomes), followed by aminoglycosides (13.89%), sulfonamide (8.4%), QAC (6.9%), trimethoprim (5.7%), and quinolone (3.47%). The gene tet(B) (Supplementary Figure 3, Extended data17), which confers resistance to the tetracycline group of antibiotics, was most abundant and found in 10.66% of the genomes. This was followed by two aminoglycoside resistance genes aph(6)-Id and aph(3'')-Ib, which had a percentage occurrence of 8.93% and 8.68%, respectively. We identified isolates carrying resistance genes against quinolones, lincosamide, bleomycin, and rifampin. Thirty-six genomes (8.9%) were found to carry genes conferring resistance against ≥ 3 classes of antimicrobial agents. A total of five quinolone resistance genes (qnrB1, qnrB19, qnrB6, qnrB9, and qnrS1) were predicted in 14 isolates. Of the 403 isolates, only six (1.5%) carried genes conferring resistance to β-lactam antimicrobials. The blaCMY-2, blaTEM-1, and blaLAP-2 genes were found in three, two, and one genome(s), respectively. These isolates were distributed in the bovine, porcine, food, and human categories of sources and were from North America, Europe and Asia.\n\nWe used a representative set of 66 isolates to perform a phenotypic level antibiotic sensitivity assay using a panel of 12 antibiotics [Sensititre™ Gram negative plate (CMV3AGNF, ThermoFisher)]. More than 60% of the isolates showed resistance to at least one antibiotic (Figure 3). Most resistance was observed against streptomycin (41 isolates, 62%), followed by tetracycline (six isolates, 9%). There were isolates that showed resistance (intermediate) against cefoxitin, chloramphenicol, and sulfonamide; however, no resistance was found against quinolones in these 66 tested isolates (Supplementary Table 4, Underlying data16).\n\nAMR genes were predicted from the genomic assemblies and categorized into different groups based on the antibiotic class, as shown in the legend. A cladogram of the MCC tree rooted to outgroup (KY1 and ALT1) is shown at the center. Tree branches are colored to visualize the two major groups. The first circular layer immediately around the tree shows the presence (colored triangle) or absence (no color) of genes resistant to the corresponding antibiotic class. The next two circular layers represent the types of point mutations (pmrB T147B – blue, gyrA D87Y/S83Y – red) identified in Salmonella ser. Mbandaka genomes. Newly sequenced isolates (n = 66) formed a representative dataset in the context of their phylogenetic location. The phenotypic resistance profiles of these representative isolates against 12 different antibiotics are depicted in the next circular layer. This is followed by an outermost circular layer that shows the presence (dark) and absence (light) matrix of 26 plasmid replicons in the analyzed genome assemblies. The isolates that showed a match to both phenotypic and genotypic prediction of AMR are marked with a dark color for the tree leaf node. Figure was generated using iTOL v.4.3.219.\n\nComparison of genotypic predictions with phenotypic susceptibility results found 100% sensitivity for genotypic prediction of phenotypic resistance to nine of 12 antimicrobials, with a specificity of ≥ 95% for all antimicrobials tested (Table 2). Disagreement was found in 49 (6.2%) of a possible 792 resistance/susceptibility combinations of 12 antimicrobials. The overall specificity was 99%, with that for streptomycin being 100%.\n\nIsolates that showed intermediate resistance to any antimicrobials were also considered resistant.\n\nWe subjected all 403 genomes of Salmonella ser. Mbandaka to screening of plasmid replicons and point mutations that may confer drug resistance. A total of 26 different plasmids were predicted in our analysis (Figure 3). With the highest abundance, the IncHI2 and IncHI2A plasmids were predicted in 11.16% of the genomes (Supplementary Figure 4, Extended data17; Supplementary Table 5, Underlying data16). ColpVC (3.9%), IncFIB(K) (3.47%), and IncI1 (2.23%) were the other predominant plasmids found in Salmonella ser. Mbandaka. Using the CLC Genomics Workbench v.12 (Qiagen) and the PointFinder database (accessed on December 2018), we checked chromosomal point mutations associated with AMR in the studied isolates. Of the three types of mutations found, pmrB T147P was the major one, being predicted in 4.7% of genomes. The remaining two were gyrA mutations (gyrA D87Y and S83Y), of which one was detected in a food isolate from Taiwan (FDA187) and the other in a poultry isolate from Nigeria (EUR004) (Supplementary Table 6, Underlying data16).\n\nTo determine the potential virulence capability of Salmonella ser. Mbandaka isolates, we screened the genomes for the presence of virulence genes and Salmonella pathogenicity islands (SPIs). The virulence behavior was further evaluated by an in vitro invasion assay in Caco2 cells using a representative set of available isolates from different isolation sources. On average, 92 of the 97 predicted virulence factors were present in all 403 Salmonella ser. Mbandaka genomes (Figure 4; Supplementary Figure 5, Extended data17; Supplementary Table 7, Underlying data16). With 95% homology, the number of virulence determinants ranged from 89 to 93, with a median value of 92. This indicates that the virulence gene repertoire of the studied genomes was homogenous, irrespective of isolation host or geographical region. A screening of different SPIs was performed using SPIFinder v.1.0 (Center for Genomic Epidemiology). Of the 23 SPIs previously reported in Salmonella10,20, we identified seven (C63PI, SPI-1, SPI-2, SPI-4, SPI-5, SPI-13, and SPI-14) in these genomes (Figure 4; Supplementary Table 8, Underlying data16). Centisome 63 pathogenicity island (C63PI) was found in all 403 genomes. SPI-5 was found only in one poultry isolate (FDA166) collected from the U.S. SPI-13 and SPI-14 were predicted in only a few genomes (each were found in three different isolates) from different sources. We could not identify CS54, SPI-3, SPI-6, SPI-9, SPI-11, SPI-12, or SPI-18 reported in a previous comparative genomic analysis of two Salmonella ser. Mbandaka strains10. However, prediction of SPI-13, SPI-14, and C63PI islands in our analysis is in agreement with results obtained from WGS mapping of SPIs in the Salmonella ser. Mbandaka ATCC 51958 strain performed in another study21. In accordance with these two previous analyses, we could not predict the presence of SPI-7, 8, 10, 15, 16, 17, 19, 20, 21, or 22 in Salmonella ser. Mbandaka genomes.\n\nScreening of genome assemblies was performed to predict the Salmonella pathogenicity islands (SPIs) and virulence factors. A cladogram of the MCC tree rooted to KY1 is shown at the center. Tree branches are colored to visualize the two major groups. The presence (dark color) of different SPIs is shown as different circles around the tree. The number of virulence factors predicted per genome (red) and the number of colony forming units (CFUs) that invaded Caco2 cells (blue) by the representative set of newly sequenced isolates are shown in different circles of simple bar charts. Figure was generated using iTOL v.4.3.219.\n\nHost cell invasion and survival in the acidic environment of phagosomes are two critical steps in Salmonella pathogenicity; therefore, we used these as surrogate measurements of their phenotypic behavior inside the host. Consistent with our genomic prediction showing uniformity of virulence factors, all tested isolates displayed a similar ability to invade Caco2 cells (Figure 4; Supplementary Figure 6, Extended data17; Supplementary Table 9, Underlying data16) and to survive under low pH conditions (Supplementary Figure 7, Extended data17; Supplementary Table 10, Underlying data16). All 66 tested isolates showed an increase in their growth after three and six hours of exposure to an acidic environment without any prior adaptation. Taken together, the genomic and phenotypic results show the potential virulence capability of Salmonella ser. Mbandaka isolates, irrespective of their isolation source, geographical location, and population structure.\n\n\nDiscussion\n\nIn Salmonella enterica species, host specificity and the ability to cause disease in different hosts are serotype-dependent22. Some serotypes are “host-restricted,” that is, they are only able to infect one specific host23; others such as Salmonella ser. Mbandaka have a broad host range. In addition to humans and farm animals, the main sources of Salmonella ser. Mbandaka are dogs, wild birds, and fish. According to outbreak investigation reports, Salmonella ser. Mbandaka can originate from both live animals8,24 and processed food7,9. Geographically, as well as host distribution wise, ST413 was the most prevalent sequence type in Salmonella ser. Mbandaka. Association of ST413 with sources such as animal feed, livestock, food, and humans aids its survival in the food chain and renders Salmonella ser. Mbandaka a serious threat for foodborne outbreaks. Unlike other serotypes25, we could not find any specific pattern in the Salmonella ser. Mbandaka population structure in relation to either geographical origin or isolation source, disproving our hypothesis that host-specific clades may be emerging in this serotype (Figure 1A). We presume that this was not due to sampling bias, since a similar clustering pattern was observed in a previous study4 based on pulsed-field gel electrophoresis (PFGE) profiles of a smaller number (n = 70) of Salmonella ser. Mbandaka isolates from a geographically restricted area.\n\nAntibiotic use in agricultural settings and animal-based food production provides major contributions to the overall problem of antibiotic resistance26. Due to the widespread use of antimicrobial agents in livestock farming, resistant Salmonella strains are more frequently found in animals used for food22,27. WGS is an excellent technique for the prediction of AMR due to its fast turnaround and affordability. It has been proven to be a successful method for genotypic AMR prediction in several gastrointestinal pathogens including Salmonella28–31. Our study also shows a high sensitivity and specificity for the comparison of genotypic prediction using WGS with phenotypic resistance to nine antimicrobials out of the 12 tested. There was a high discrepancy in the case of streptomycin resistance, since we found 38 isolates that were phenotypically resistant but genotypically susceptible. There could be two reasons for this. Firstly, we used a low minimum inhibitory concentration (MIC) breakpoint of ≥ 16, since there is not a precise clinical breakpoint for streptomycin susceptibility in Salmonella32. Secondly, there may exist unknown resistance mechanisms or resistance determinants, that may be absent in the reference database used for prediction28.\n\nPlasmids are one of the main vehicles for dissemination of AMR genes. Resistance genes are assembled on plasmids as arrays by transposition and site-specific recombination mechanisms33. For example, the AMR genes blaTEM, tetA, tetB, and tetC have been found to be associated with plasmids in Salmonella ser. Typhimurium. Acquisition of plasmids is not a universal phenomenon in all Salmonella enterica subspecies enterica serotypes. There are many serotypes in this subspecies that do not possess any plasmids34. Above all, animals used for food and food products have been reported as major sources of AMR plasmids35. We predicted 26 different plasmids in 403 draft genomes, of which incompatibility group HI2 (IncHI2) plasmids were the most predominantly identified in our analysis (Figure 3; Supplementary Figure 4, Extended data17; Supplementary Table 5, Underlying data16). The presence of IncHI2 plasmids in antibiotic-resistant Salmonella, as well as their association with MDR in Salmonella, has been reported previously36,37. In addition, we found the presence of chromosomal mutations associated with quinolone resistance38,39 (gyrA D87Y and S83Y) and resistance to colistin40 (pmrB), an antibiotic that can be used against carbapenemase-producing Enterobacteriaceae as a last-resort treatment option41. This indicates the potential capability of these mobile genetic elements to spread AMR among Salmonella.\n\nPrediction of virulence determinants in Salmonella ser. Mbandaka in the present analysis revealed similar virulence gene distribution in all 403 genomes (Figure 4; Supplementary Figure 5, Extended data17; Supplementary Table 7, Underlying data16). In addition to virulence factors, we also made use of WGS-based genotypic prediction to elucidate the distribution of pathogenicity islands, large distinct regions on chromosomes that contain virulence genes42. Of the 23 previously reported SPIs in Salmonella10,20, seven were detected in our study isolates, including C63PI (Figure 4; Supplementary Table 8, Underlying data16). Salmonella virulence factors are necessary for Salmonella pathogenicity, which involves survival in the extreme acidic environment of the host’s stomach, host cell invasion, and survival inside the acidic vacuoles of host immune cells (macrophages)42,43. Since the results of the cell invasion assay using a representative dataset of available isolates showed similar invasiveness, the virulence at the genomic and phenotypic levels shows high correlation. This may indicate that any Salmonella ser. Mbandaka strains may have the potential to cause human infection if ingested by a susceptible individual.\n\n\nMethods\n\nTo study the phylogeny of Salmonella ser. Mbandaka, we sequenced 66 isolates (sample name ADRDL-01 to ADRDL-76 in Supplementary Table 1, Underlying data16) of this serotype collected from various centers. Genomic DNA was extracted from the overnight cultures of these isolates using a Qiagen DNeasy blood and tissue kit (Qiagen, Inc., Valencia, CA; Cat no. 69506) according to the manufacturer’s protocol and stored at -20°C until use. For WGS, all 66 DNA samples were processed using a Nextera XT DNA sample preparation kit (Illumina inc. San Diego, CA; Cat no. FC-131-1096). Following bead-based normalization, DNA libraries were pooled at an equal volume and sequenced with Miseq reagent (version 2.0) (Illumina Inc., CA) on an Illumina Miseq platform using 2× 250 bp paired-end V2 chemistry. Genome sequences of the remaining isolates (n = 337) were downloaded from the NCBI-SRA (National Center for Biotechnology Information – Sequence Read Archive) database using the sra tool kit v.2.8.1-2. Prior to further analysis, we verified the serotype (Supplementary Table 2, Underlying data16) and assembly statistics (Table 1) of the 403 selected Salmonella ser. Mbandaka genomic data using in silico methods- Salmonella In Silico Typing Resource (SISTR)15 and assembly-stats, respectively. Metadata for all 403 isolates used in the present study are shown in Supplementary Table 1 (see Underlying data16).\n\nSequencing reads from the Salmonella isolates used in the present study (Salmonella ser. Mbandaka n = 403, Salmonella ser. Kentucky n = 1, and Salmonella ser. Altona n = 1) were assembled into contigs using SPAdes v.3.044. To ensure that the assemblies were not greatly fragmented, those containing more than 500 contigs (minimum contig length: 200 bp) were removed from the analysis. In silico serotyping and multilocus sequence typing (MLST) of all genomes were performed using the SISTR webserver15. Annotation of genomes was performed using Prokka v1.1245. A genus-specific database for Salmonella was created using a manually annotated reference genome assembly of Salmonella enterica ser. Typhimurium str. LT2 (RefSeq assembly accession: GCF_000006945.2) and formatted to a Prokka database as described elsewhere (https://github.com/tseemann/prokka). Prokaryote pan-genome analysis pipeline Roary v.3.12.046 was used for pan-genome analysis and the generation of a multi-FASTA alignment of core genes from the isolates using the aligner PRANK47. The software SNP-sites v2.4.048 was used to format the core gene alignment output from Roary to remove gaps and N characters (suitable format for BEAST2 phylogeny). A pairwise SNP distance matrix was created using snp-dists v0.6.3.\n\nTo infer the phylogenetic relationship of Salmonella ser. Mbandaka isolates, we used the Bayesian maximum clade credibility approach. For this purpose, we used the BEAST2 (v.2.5.1) platform, which employs the MCMC49,50 method for phylogenetic tree inference. We performed a model testing of the alignment of all SNPs using ‘ModelTest-NG.v.0.1.5’ and generated a maximum likelihood tree using a generalized time-reversible (GTR+I+G4) substitution model. This tree was constructed to infer the relationship between genetic divergence and time using Tempest51. The resulting phylogeny provided a weak temporal signal (R < 0.10); therefore, tip dates were not included in the final BEAST2 phylogeny. Multiple analyses using both relaxed clock (Log normal) and strict clock models were carried out in combination with coalescent constant population for priors. A MCMC chain length of 100 million generations with 10% preburnin and sampling at every 1000 generations were used for each analysis. The traces from each analysis were examined using Tracer v.1.7.152 and the strict clock coalescent constant population model, which showed a better convergence and > 100 effective sample sizes (ESSs) for many of the traces, was selected as a best-fit model for our dataset. Information from 100,000 sample trees produced by BEAST2, after removing 10% burnin, was summarized to a final target MCC tree using TreeAnnotator v2.5.1. We used two other Salmonella serotypes [Salmonella ser. Kentucky (KY1) and Salmonella ser. Altona (ALT1)] as outgroups, since said serotypes were identified as the nearest neighbors to Salmonella ser. Mbandaka by SISTR15.\n\nAntimicrobial resistance (AMR) and virulence gene homologs were identified in assemblies using ABRicate. A minimum sequence identity of 95% and a coverage of 60% were used against the NCBI Bacterial Antimicrobial Resistance Reference Gene Database and Virulence Factor Database (VFDB) for AMR gene prediction and virulence gene profiling, respectively. PlasmidFinder v.2.0 (Center for Genomic Epidemiology) and SPIFinder v.1.0 (Center for Genomic Epidemiology) were used for screening plasmid replicons and Salmonella pathogenicity islands (SPIs) in the genome assemblies53. Point mutations were identified using the CLC Genomics Workbench v.12 (Qiagen) after downloading the PointFinder database for Salmonella (accessed on December 2018). An open source alternative for finding point mutation is ResFinder 4.0 offered by the Center for Genomic Epidemiology.\n\nSusceptibility to 12 antimicrobial agents was determined for 66 Salmonella ser. Mbandaka isolates using Sensititre™ Gram negative plates (CMV3AGNF, ThermoFisher). Resistance to antimicrobial agents was determined in accordance with the Clinical and Laboratory Standards Institute (CLSI) and National Antimicrobial Resistance Monitoring System (NARMS) guidelines. Five beta lactams (amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, and ceftriaxone), two quinolones (ciprofloxacin and nalidixic acid), two aminoglycosides (gentamicin and streptomycin), trimethoprim/sulfamethoxazole, and chloramphenicol were the antibiotics included in the screening panel. Isolates reported as displaying intermediate resistance to any antimicrobials were also considered as resistant.\n\nHuman colorectal adenocarcinoma (Caco2) cells obtained from ATCC were used for the cell invasion assay. Cells were seeded onto a 24-well plate at a density of 0.3 × 105/well and were grown in DMEM medium containing glutamine (DMEM (1×) + glutamine; Gibco) supplemented with 10% (v/v) FBS and 1% antibiotic and antimycotic solution at 37°C with 5% CO2 (v/v) for 48 hours. Overnight cultures of bacteria were used to infect Caco2 cell monolayers at a multiplicity of infection (MOI) of 1:100. The invasion assay described by Lee et al.54 was used with some modifications. Briefly, bacteria were resuspended in cell culture medium (DMEM (1×) + glutamine; Gibco, with no supplementation) after washing with sterile PBS. Cells were subsequently incubated with this bacterial suspension for 2 hours at 37°C with 5% CO2. After infection, the media was removed, and the cells were washed with sterile PBS. Cells were then treated with 400 µL DMEM supplemented with the antibiotic gentamicin (100 µg/mL) to kill extracellular non-invading bacteria. Plates were incubated for 1 hour at 37°C with 5% CO2 followed by washing with sterile PBS. A colony forming unit (CFU) count of intracellular bacteria was taken using serial dilution and plating. Cells were lysed with 1% Triton X-100 (Sigma) for 10 minutes to release intracellular bacteria, following which the cell lysate was serially diluted, plated on LB plates, and incubated overnight. Two independent assays, each in triplicate, were performed for each Salmonella ser. Mbandaka isolate (total n = 66).\n\nAn overnight bacterial culture with an OD600 adjusted to 0.4 was used to inoculate (20% v/v) low pH LB broth (pH = 4.0 ± 0.1, adjusted using 1M HCl). The experiment was performed in flat- bottomed, non-treated 96-well plates, in which triplicate wells were used for each bacterial sample. The OD600 was measured using an ELISA plate reader (BioTek, ELx808) to assess the growth of bacteria over time. The initial OD600 was taken immediately after inoculation (T0) and then after three hours (T1) and six hours (T2) of incubation at 37°C in an aerobic environment. The assay was performed as two independent experiments for all 66 isolates.\n\n\nData availability\n\nThe raw sequence data for Salmonella strains sequenced in this study have been deposited in NCBI sequence read archive (NCBI SRA) for public access. The full list of NCBI SRA accession numbers is given Supplementary Table 1 (see below).\n\nZenodo: Underlying Data: Population structure of Salmonella serotype Mbandaka. https://doi.org/10.5281/zenodo.400497016.\n\nThis project contains the following underlying data:\n\n- Supplementary Table 1.xlsx. Metadata for the 403 Salmonella ser. Mbandaka isolates used in the present study. Major information on the isolates, including both the biosample and SRA run number. With the exception of newly sequenced isolates (n = 66), sequence data for all other isolates in the FASTQ format was acquired with the help of NCBI SRA toolkit.v.2.8.1-2. using the SRA run number. More detailed metadata for individual isolates can be obtained from the NCBI database using the biosample number. (XLSX format)\n\n- Supplementary Table 2.xlsx. In silico serotyping and sequence typing of the 403 Salmonella ser. Mbandaka genomes using SISTR. Detailed results of WGS-based serotype and sequence type prediction of the 403 Salmonella ser. Mbandaka genomes using SISTR. (XLSX format)\n\n- Supplementary Table 3.xlsx. Details of antimicrobial resistance genes predicted in Salmonella ser. Mbandaka genomes. Details of genes identified in the genomes following a search of the NCBI Bacterial Antimicrobial Resistance Reference Gene Database using ABRicate. A gene was reported as present if there were 60% coverage and 95% homology. (XLSX format)\n\n- Supplementary Table 4.xlsx. Phenotypic assay results of antimicrobial susceptibility using Sensititre™ Gram negative MIC plates. (XLSX format)\n\n- Supplementary Table 5.xlsx. Details of plasmids identified in the Salmonella ser. Mbandaka genomes following a search using the PlasmidFinder-2.0 web tool. A sequence identity of 95% and a minimum coverage of 60% were the criteria used for a positive hit. (XLSX format)\n\n- Supplementary Table 6.xlsx. Details of the point mutations identified in the Salmonella ser. Mbandaka genomes using CLC genomics workbench v.12 (Qiagen) after downloading the PointFinder database for Salmonella. (XLSX format)\n\n- Supplementary Table 7.xlsx. Details of virulence determinants identified in the genomes following a search of the virulence factor database using ABRicate. A gene was reported as present if there were 60% coverage and 95% homology. (XLSX format)\n\n- Supplementary Table 8.xlsx. Details of Salmonella pathogenicity island (SPI) genes identified in the genomes following a search using SPIFinder.v.1.0. A gene was reported as present if there were 60% coverage and 95% homology. (XLSX format)\n\n- Supplementary Table 9.xlsx. Bacterial count (CFU/mL) of Salmonella ser. Mbandaka isolates that invaded Caco2 cells in in-vitro cell invasion assay. (XLSX format)\n\n- Supplementary Table 10.xlsx. Growth of Salmonella ser. Mbandaka in low pH condition was assessed by OD600 readings taken at three different time points after inoculation. (XLSX format)\n\nZenodo: Extended Data: Population structure of Salmonella serotype Mbandaka. https://doi.org/10.5281/zenodo.400500817.\n\nThis project contains the following extended data:\n\n- Supplementary Figure 1. Defining the population structure of Salmonella ser. Mbandaka isolates.\n\nAn MCC tree of 403 Salmonella ser. Mbandaka isolates along with two outgroup strains of Salmonella ser. Kentucky (KY1) and Salmonella ser. Altona (ALT1). The tree was rerooted to outgroup strains as shown in the circular cladogram. The tree is colored based on the two major groups identified in the phylogeny (Figure 1). Different circles around the tree represent cgMLST sequence type as well as different metadata attributes of the genomes. Figure was generated using iTOL v.4.3.219. (PNG format)\n\n- Supplementary Figure 2. Antimicrobial resistance gene prediction in Salmonella ser. Mbandaka isolates.\n\nHeat map showing the predicted AMR genes (n = 40) in the Salmonella ser. Mbandaka isolates. The dark color represents the presence of a predicted gene at 90% sequence identity with a minimum coverage of 60%. The tree represents the MCC tree created using BEAST v.2.5.1. Figure was generated using iTOL v.4.3.219. (PNG format)\n\n- Supplementary Figure 3. Abundance distribution of AMR genes in the 403 Salmonella ser. Mbandaka isolates.\n\nSimple bar graph showing the percentage of Salmonella ser. Mbandaka genomes harboring each predicted AMR gene. More than 10% of genomes carried the tet(B) gene that confers resistance to tetracycline antibiotics, followed by the aminoglycoside resistance genes aph(6)-ld and aph(3”)-lb (8.9% and 8.7%, respectively). (PNG format)\n\n- Supplementary Figure 4. Distribution of plasmid replicons in Salmonella ser. Mbandaka.\n\nIncHI2 and IncHI2A plasmids were found to be the most abundant replicons in Salmonella ser. Mbandaka genomes. Simple bar graph with plasmids on the ‘X’ axis and the percentage of genomes on the ‘Y’ axis. (PNG format)\n\n- Supplementary Figure 5. WGS-based profiling of virulence genes in Salmonella ser. Mbandaka.\n\nHeat map showing the predicted virulence factors in Salmonella ser. Mbandaka genomes at a minimum sequence identity of 95% and a minimum coverage of 60%. Virulence factors were categorized into six groups as shown in the color legend. Approximately 87% of predicted genes were found in all 403 Salmonella ser. Mbandaka genomes. Figure was generated using iTOL v.4.3.219. TTSS (SPI-1) - Type three secretion system encoded by Salmonella pathogenicity island-1; TTSS (SPI-2) - Type three secretion system encoded by Salmonella pathogenicity island-2. (PNG format)\n\n- Supplementary Figure 6. Invasiveness of Salmonella ser. Mbandaka isolates in Caco2 cells.\n\nThe 66 newly sequenced Salmonella ser. Mbandaka isolates were used for the invasion assay in Caco2 cells. Bar plot showing the count of intracellular bacteria (log CFU/mL) retrieved after an incubation time of two hours under aerobic conditions followed by treatment with gentamicin to kill all extracellular bacteria. Cell lysates were serially diluted and plated on LB plates in duplicate. Figure was generated using Prism 7 (GraphPad software, Inc.). (TIF format)\n\n- Supplementary Figure 7. Adaptation of Salmonella ser. Mbandaka isolates to low pH.\n\nThe ability of Salmonella ser. Mbandaka isolates to tolerate an acidic environment was tested using the 66 newly sequenced isolates as representatives. All tested isolates were able to withstand the immediate exposure to a low pH environment and showed an increase in growth after three hours (A) and six hours (B) of incubation at 37°C under aerobic conditions. Bar graph showing the OD600 immediately after exposure to LB broth at pH 4.0 (T0) and after three hours (T1) and six hours (T2) of incubation. Figure was generated using Prism 7 (GraphPad software, Inc.). (PNG format)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the Section of Bacteriology, Animal Disease Research and Diagnostic Laboratory, SD for their assistance with the antibiotic sensitivity assay. Computations supporting this project were performed on high-performance computing systems managed by the Research Computing Group, part of the Division of Technology and Security at South Dakota State University.\n\n\nReferences\n\nBasler C, Forshey TM, Machesky K, et al.: Multistate outbreak of human Salmonella infections linked to live poultry from a mail-order hatchery in Ohio--March-September 2013. MMWR Morb Mortal Wkly Rep. 2014; 63(10): 222. PubMed Abstract \| Free Full Text\n\nScallan E, Hoekstra RM, Angulo FJ, et al.: Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis. 2011; 17(1): 7–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGragg SE, Loneragan GH, Brashears MM, et al.: Cross-sectional study examining Salmonella enterica carriage in subiliac lymph nodes of cull and feedlot cattle at harvest. Foodborne Pathog Dis. 2013; 10(4): 368–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoszowski A, Zając M, Lalak A, et al.: Fifteen years of successful spread of Salmonella enterica serovar Mbandaka clone ST413 in Poland and its public health consequences. Ann Agric Environ Med. 2016; 23(2): 237–41. PubMed Abstract \| Publisher Full Text\n\nHoszowski A, Wasyl D: Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates. Vet Microbiol. 2001; 80(2): 139–48. PubMed Abstract \| Publisher Full Text\n\nScheil W, Cameron S, Dalton C, et al.: A South Australian Salmonella Mbandaka outbreak investigation using a database to select controls. Aust N Z J Public Health. 1998; 22(5): 536–9. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention (CDC): Multistate Outbreak of Salmonella Montevideo and Salmonella Mbandaka Infections Linked to Tahini Sesame Paste (Final Update). 2013. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Eight Multistate Outbreaks of Human Salmonella Infections Linked to Live Poultry in Backyard Flocks (Final Update). 2016. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Multistate Outbreak of Salmonella Mbandaka Infections Linked to Kellogg’s Honey Smacks Cereal (Final Update). 2018. Reference Source\n\nHayward MR, Jansen VAA, Woodward MJ: Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK. BMC Genomics. 2013; 14: 365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHayward MR, Petrovska L, Jansen VAA, et al.: Population structure and associated phenotypes of Salmonella enterica serovars Derby and Mbandaka overlap with host range. BMC Microbiol. 2016; 16(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nden Bakker HC, Switt AIM, Cummings CA, et al.: A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type. Appl Environ Microbiol. 2011; 77(24): 8648–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson MR, Brown E, Keys C, et al.: Whole Genome DNA Sequence Analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks. PLoS One. 2016; 11(6): e0146929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeekitcharoenphon P, Nielsen EM, Kaas RS, et al.: Evaluation of whole genome sequencing for outbreak detection of Salmonella enterica. PLoS One. 2014; 9(2): e87991. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshida CE, Kruczkiewicz P, Laing CR, et al.: The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies. PLoS One. 2016; 11(1): e0147101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntony L, Fenske G, Thomas M, et al.: Underlying Data: Population structure of Salmonella serotype Mbandaka [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4004970\n\nAntony L, Fenske G, Thomas M, et al.: Extended Data: Population structure of Salmonella serotype Mbandaka. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4005008\n\nCollineau L, Boerlin P, Carson CA, et al.: Integrating Whole-Genome Sequencing Data Into Quantitative Risk Assessment of Foodborne Antimicrobial Resistance: A Review of Opportunities and Challenges. Front Microbiol. 2019; 10: 1107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLetunic I, Bork P: Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016; 44(W1): W242–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFookes M, Schroeder GN, Langridge GC, et al.: Salmonella bongori provides insights into the evolution of the Salmonellae. PLoS Pathog. 2011; 7(8): e1002191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoer L, Hendriksen RS, Leekitcharoenphon P, et al.: Is the Evolution of Salmonella enterica subsp. enterica Linked to Restriction-Modification Systems? mSystems. 2016; 1(3): e00009–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndino A, Hanning I: Salmonella enterica: survival, colonization, and virulence differences among serovars. ScientificWorldJournal. 2015; 2015: 520179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJajere SM: A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and antimicrobial resistance including multidrug resistance. Vet World. 2019; 12(4): 504–521. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenters for Disease Control and Prevention (CDC): Multistate Outbreak of Human Salmonella Infections Linked to Live Poultry (Final Update). 2013. Reference Source\n\nMakendi C, Page AJ, Wren BW, et al.: A Phylogenetic and Phenotypic Analysis of Salmonella enterica Serovar Weltevreden, an Emerging Agent of Diarrheal Disease in Tropical Regions. PLoS Negl Trop Dis. 2016; 10(2): e0004446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanders TF, Cohen B, Wittum TE, et al.: A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep. 2012; 127(1): 4–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu LH, Chiu CH, Chu C, et al.: Antimicrobial resistance in nontyphoid Salmonella serotypes: a global challenge. Clin Infect Dis. 2004; 39(4): 546–51. PubMed Abstract \| Publisher Full Text\n\nNeuert S, Nair S, Day MR, et al.: Prediction of Phenotypic Antimicrobial Resistance Profiles From Whole Genome Sequences of Non-typhoidal Salmonella enterica. Front Microbiol. 2018; 9: 592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoesser N, Batty EM, Eyre DW, et al.: Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data. J Antimicrob Chemother. 2013; 68(10): 2234–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcDermott PF, Tyson GH, Kabera C, et al.: Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella. Antimicrob Agents Chemother. 2016; 60(9): 5515–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadouki Z, Day MR, Doumith M, et al.: Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Shigella sonnei isolated from cases of diarrhoeal disease in England and Wales, 2015. J Antimicrob Chemother. 2017; 72(9): 2496–2502. PubMed Abstract \| Publisher Full Text\n\nPornsukarom S, van Vliet AHM, Thakur S: Whole genome sequencing analysis of multiple Salmonella serovars provides insights into phylogenetic relatedness, antimicrobial resistance, and virulence markers across humans, food animals and agriculture environmental sources. BMC Genomics. 2018; 19(1): 801. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBennett PM: Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. 2008; 153 Suppl 1(Suppl 1): S347–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRychlik I, Gregorova D, Hradecka H: Distribution and function of plasmids in Salmonella enterica. Vet Microbiol. 2006; 112(1): 1–10. PubMed Abstract \| Publisher Full Text\n\nMadec JY, Haenni M: Antimicrobial resistance plasmid reservoir in food and food-producing animals. Plasmid. 2018; 99: 72–81. PubMed Abstract \| Publisher Full Text\n\nGlenn LM, Lindsey RL, Folster JP, et al.: Antimicrobial resistance genes in multidrug-resistant Salmonella enterica isolated from animals, retail meats, and humans in the United States and Canada. Microb Drug Resist. 2013; 19(3): 175–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen W, Fang T, Zhou X, et al.: IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates. Front Microbiol. 2016; 7: 1566. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins KL, Davies RH, Threlfall EJ: Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents. 2005; 25(5): 358–73. PubMed Abstract \| Publisher Full Text\n\nZankari E, Allesøe R, Joensen KG, et al.: PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens. J Antimicrob Chemother. 2017; 72(10): 2764–2768. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun S, Negrea A, Rhen M, et al.: Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother. 2009; 53(6): 2298–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu YY, Wang Y, Walsh TR, et al.: Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016; 16(2): 161–8. PubMed Abstract \| Publisher Full Text\n\nHensel M: Evolution of pathogenicity islands of Salmonella enterica. Int J Med Microbiol. 2004; 294(2–3): 95–102. PubMed Abstract \| Publisher Full Text\n\nKenney LJ: The role of acid stress in Salmonella pathogenesis. Curr Opin Microbiol. 2019; 47: 45–51. PubMed Abstract \| Publisher Full Text\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19(5): 455–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeemann T: Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014; 30(14): 2068–9. PubMed Abstract \| Publisher Full Text\n\nPage AJ, Cummins CA, Hunt M, et al.: Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015; 31(22): 3691–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoytynoja A: Phylogeny-aware alignment with PRANK. Methods Mol Biol. 2014; 1079: 155–70. PubMed Abstract \| Publisher Full Text\n\nPage AJ, Taylor B, Delaney AJ, et al.: SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom. 2016; 2(4): e000056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrummond AJ, Nicholls GK, Rodrigo AG, et al.: Estimating mutation parameters, population history and genealogy simultaneously from temporally spaced sequence data. Genetics. 2002; 161(3): 1307–20. PubMed Abstract \| Free Full Text\n\nBouckaert R, Heled J, Kühnert D, et al.: BEAST 2: a software platform for Bayesian evolutionary analysis. PLoS Comput Biol. 2014; 10(4): e1003537. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRambaut, A, Lam TT, Carvalho LM, et al.: Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). Virus Evol. 2016; 2(1): vew007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRambaut A, Drummond AJ, Xie D, et al.: Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Syst Biol. 2018; 67(5): 901–904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarattoli A, García-Fernández A, Zankari E, et al.: In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother. 2014; 58(7): 3895–903. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee CA, Falkow S: The ability of Salmonella to enter mammalian cells is affected by bacterial growth state. Proc Natl Acad Sci U S A. 1990; 87(11): 4304–8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71450", "date": "25 Sep 2020", "name": "Mohamed N. Seleem", "expertise": [ "Reviewer Expertise Microbiology and antimicrobial resistance." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the whole genome sequencing-based analysis of population structure, antibiotic resistance, and virulence profile of Salmonella Mbandaka, a serotype that primarily colonizes animals but also causes human infections. Authors have sequenced 66 isolates and compared the data against 337 publicly available genomes of S. Mbandaka. A subset of strains were also tested phenotypically to determine virulence and antibiotic resistance profile. The analysis did not show any host specific clustering. The virulence and antibiotic resistance profile was not host or region specific. CaCo2 cell based virulence assay showed that most strains are invasive, indicating that any strain could possibly cause infection in a susceptible host. Overall, this manuscript is written well. However, a few modifications could improve the clarity of the results. Minor points to consider are:\nFigure 2 is confusing, and the row and column headers are not readable. I assume that row and column headers are isolates. However, that is not mentioned in the legend. Please correct.\n\nWhat is the significance of groups in Figure 2B? Are these groups related to group 1 and 2 in Figure 1? Are you calculating the SNPs in these groups based on Beast phylogeny? Please include the analysis details in the figure legend.\n\nThe inner labels in Figure 3 are not readable. The figure overall is faint and hard to understand. The font size of the ring labels needs to be increased to make it readable.\n\nThe same problem is in Figure 4. This figure is better than Figure 3. However, the inner labels are still hard to read.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71448", "date": "09 Nov 2020", "name": "Shaankumar Mooyottu", "expertise": [ "Reviewer Expertise Pathology and microbiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current study defines the population structure and associated genotypic features of Salmonella ser. Mbandaka in a global context using whole-genome sequence (WGS)-based analysis of 403 Salmonella ser. Mbandaka genomes. The authors assessed the antimicrobial resistance and virulence gene repertoire of this Salmonella serotype to understand the potential capability of this serovar to act as an important zoonotic pathogen and public health hazard. The research method, results, and presentation look sound, and no significant shortcomings are identified.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1142
https://f1000research.com/articles/9-356/v1	13 May 20	{ "type": "Brief Report", "title": "High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum", "authors": [ "Yiran Wang", "Hoda Yaghmaiean", "Yuelin Zhang", "Yiran Wang", "Hoda Yaghmaiean" ], "abstract": "Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD600≈2.0) in large cultures and resuspended in inoculation medium to OD600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees relatively high transformation rate in Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.", "keywords": [ "Arabidopsis", "floral dip", "transformation" ], "content": "Introduction\n\nPlant transformation integrates foreign genes into the plant nuclear genome. The development of different transformation protocols in various plants has enabled advances in plant molecular biology and crop improvements. Agrobacterium is routinely used as a plant gene transformation vehicle as it naturally possesses the ability to transfer a segment of its plasmid DNA (T-DNA) into its host nucleus, which ultimately leads to integration of the T-DNA into the nuclear genome (Tzfira et al., 2004). During 1980s and early 1990s, generating transgenic plants by leaf disc-based Agrobacterium-mediated transformation requires laborious plant tissue culture and regeneration steps. In 1993, a simple floral vacuum infiltration method was developed in Arabidopsis for stable transformation, overcoming the tedious tissue culture requirements (Bechtold et al., 1993). Later, the vacuum infiltration step was replaced by floral dipping where the developing floral tissues are dipped into a solution containing Agrobacterium, sucrose and the surfactant Silwet L-77 (Clough & Bent, 1998). Because of the simplicity and reliability of this floral dip method, it is now the commonly used transformation method in Arabidopsis. This protocol has also been shown to work in certain Brassicaceae plants (Bent, 2006). Floral dip transformation may be feasible in plants such as wheat and Setaria viridis (Agarwal et al., 2009).\n\nIn Agrobacterium-mediated transformation protocols, the concentration of bacterial inoculum has been considered crucial to the success of plant transformation. In the commonly used floral dip protocol, bacterial cells are grown to stationary phase (OD600=2.0), pelleted and resuspended in inoculation medium to OD600≥0.8 (Clough & Bent, 1998; Zhang et al., 2006). Here, we tested whether low concentration of Agrobacterium inoculum affects the plant transformation rate. Our data showed that, in contrary to our expectation, using extremely low density of Agrobacterium inoculum (OD600=0.002) in floral dip method still warrants relatively high transformation rate in Arabidopsis.\n\n\nMethod\n\nArabidopsis Col-0 wild type plants were grown in a growth room under long day (16 h light/8 h dark cycle) at 23°C. Seedlings were grown at a density of 30–40 per 64 cm2 (8 cm × 8 cm) pot in moistened potting soil initially and transplanted to 64 cm2 pots with eight plants per pot when they were two weeks old. After plants bolted and floral buds are formed (~30-day-old), they were used in floral dip transformation.\n\nThe plasmid pCambia1305-3flag-NOS was transformed into Agrobacterium tumefaciens strain GV3101 (Van Larebeke et al., 1974) by mixing the plasmid DNA with the bacterial cells in a 1mm gap cuvette (BTX, #45-0124) followed by electroporation for 5 millisecond at 1,500 volts using the ECM 399 Electroporation System (BTX, #45-0000) (Gao et al., 2009). The resulting strain was used in the plant transformation experiments. Bacteria were grown overnight in sterilized 4 ml LB media (Bio Basic Inc., #SD7002) with kanamycin, gentamicin and rifampicin antibiotics (50 μg/ml each, Bio Basic Inc. #KB0286, #GB0217, #RB0808) in a 28°C shaker (New Brunswick Scientific Co G25 Controlled Environment Incubator Shaker). Then the overnight culture was diluted into 100 ml LB media with kanamycin (50 μg/ml) and allowed to grow further for 8 h in the same shaker. The bacteria were collected by centrifugation (Thermo Scientific, Sorvall Legend X1R) at 6000 g for 10 min at room temperature and then resuspended in 100 ml floral dip medium to final OD600 of 1, 0.1, 0.01, and 0.002 (measured by BioSpec-1601 UV-visible spectrophotometer from SHIMADZU) prior to use. The floral dip medium contained 5.0% (w/v) sucrose (Bio Basic Inc. #SB0498) and 0.01% (v/v) Silwet L-77 (PhytoTechnology Laboratories #S7777) in distilled water.\n\nFor floral dip, pots were tilted and floral buds were submerged in bacterial suspension with 30 sec of gentle agitation. The dipped plants were then covered with a tall clear-plastic dome to maintain humidity. Plants were placed in a dark room overnight before being moved back to the growth room. The domes were removed approximately 48 h after the floral dip treatment. Plants were grown for another 30–32 days until siliques became brown and dry. Each pot with 8 plants were transformed separately. For each concentration of Agrobacterium inocula, 4–6 pots of plants were transformed depending on the number of plants available for transformation in each experiment, this varied mainly due to the uneven germination of the seeds in each experiment. About 6000 seeds were bulk harvested from the plants grown in a pot. Seeds were harvested by gentle stripping of dried inflorescences by fingers above a piece of clean paper. The debris from the stem and pods was removed from the seeds by gentle blowing. Seeds were kept in a 37°C incubator for two days for desiccation.\n\nPrior to selection, seeds were surface sterilized with 20% (v/v) bleach (Clorox Regular Bleach) containing 0.1% (v/v) Tween20 (Sigma-Aldrich #P1379) for 1min, followed by three times rinse with sterile water. The sterilized seeds were suspended in 0.1% (w/v) sterile agar (Bio Basic Inc. #FB0010) and plated on hygromycin selection plates (1/2 MS medium, Murashige & Skoog Basal Medium with Vitamins from PhytoTechnology Laboratories #M519 and 50 μg/ml hygromycin, Bio Basic Inc. #HD0230) at a density of approximately 3000 seeds (0.06 gram by weight) per 92×12mm (diameter×height) petri plate (Sarstedt #82.1473.001). Seeds collected from each pot (4–6 pots for each concentration of Agrobacterium inocula) were plated on a separate selection plate. Plates were placed in 4°C refrigerator for two days before moved to a plant growth chamber (16 h light/8 h dark cycle, Conviron Model A1000). The plants were grown at 23°C for 10 days before transformants were identified as hygromycin-resistant seedlings that produced green leaves and well-established roots grown on the selective medium. The experiment was repeated three times by transforming independently grown plants with different concentrations of Agrobacterium inocula.\n\nAnalysis of statistical differences between transformation rates from different concentrations of Agrobacterium inocula was performed by one-way ANOVA using Microsoft® Office Excel version 16.35 (20030802).\n\n\nResults and discussion\n\nFour Agrobacterium inocula from high to low concentrations (OD600=1, 0.1, 0.01, 0.002) were used in floral dip transformation to test the effect of bacterial concentration on the transformation rate. As shown in Figure 1 (Underlying data (Wang, 2020)), similar transformation rate (approximately 0.60%) was observed under all tested bacterial concentration. Notably, the transformation efficiency remains unchanged even though the Agrobacterium inoculum was diluted 500 times form OD600 = 1 to OD600 = 0.002. Therefore, it is feasible to dramatically reduce the Agrobacterium inoculum concentration in the floral dip method. Regardless of the inoculum concentration, transforming eight Arabidopsis plants grown in a single pot produced about 36 T1 transgenic lines on average, which is sufficient for most studies.\n\nTransformation rates were calculated as [(# of hygromycin-resistant seedlings) / (total # seedlings tested)] × 100%. The data are shown as mean ± SE from six independent repeats. The same letters denote no statistically significant difference according to one-way ANOVA (p<0.05).\n\nStandard floral dip protocols use high concentrations of Agrobacterium inoculum, which requires growing large bacterial cultures (Clough & Bent, 1998; Zhang et al., 2006). Our study showed that Agrobacterium inoculum can be diluted to as low as OD600 = 0.002 without sacrificing the transformation efficiency. Thus, the volume of bacterial culture used in each transformation experiment could be greatly reduced. For example, diluting 0.1 ml of overnight culture (OD600 = 2) to OD600 = 0.002 gives ~100 ml bacterial inoculum, which is sufficient in most transformation experiments. Such improvement allows researchers to culture small volume of a large number of Agrobacterium strains in parallel and use the diluted cultures to carry out high-throughput transformation of a large number different constructs into Arabidopsis plants.\n\n\nData availability\n\nOpen Science Framework: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum project.\n\nhttps://doi.org/10.17605/OSF.IO/YF6AE (Wang, 2020)\n\nThis project contains the following underlying data:\n\nTransformation efficiency.xlsx (raw data of results from transformation using different Agrobacterium concentrations)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAgarwal S, Loar S, Steber C, et al.: Floral transformation of wheat. Methods Mol Biol. Humana Press. 2009; 478: 105–113. PubMed Abstract \| Publisher Full Text\n\nBechtold N, Ellis J, Pelletier G: In-planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis-thaliana plants. Comptes Rendus De l Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences. 1993; 316(10): 1194–1199. Reference Source\n\nBent A: Arabidopsis thaliana floral dip transformation method. Methods Mol Biol. Humana Press. 2006; 343: 87–103. PubMed Abstract \| Publisher Full Text\n\nClough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16(6): 735–743. PubMed Abstract \| Publisher Full Text\n\nGao M, Wang X, Wang D, et al.: Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe. 2009; 6(1): 34–44. PubMed Abstract \| Publisher Full Text\n\nTzfira T, Li J, Lacroix B, et al.: Agrobacterium T-DNA integration: molecules and models. Trends Genet. 2004; 20(8): 375–383. PubMed Abstract \| Publisher Full Text\n\nVan Larebeke N, Engler G, Holsters M, et al.: Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 1974; 252(5479): 169–170. PubMed Abstract \| Publisher Full Text\n\nWang Y: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum. 2020. Publisher Full Text\n\nZhang X, Henriques R, Lin SS, et al.: Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006; 1(2): 641–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "63373", "date": "26 May 2020", "name": "Ying Wang", "expertise": [ "Reviewer Expertise Plant development", "Arabidopsis thaliana", "molecular genetics", "flowering", "meristems", "transcription factors", "gene expression", "transgenic plants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe floral dip method for the transformation of Arabidopsis plants has been a revolutionary tool in plant biology. Several refinements to the popular protocol of Clough and Bent (1998) have been published.\n\nThis report shows that Agrobacterium cultures diluted to as low as OD600=0.002 yielded a transformation rate similar to infection with regular-density suspensions (OD600=1). Down-scaling in this way can save time, space, and expense -- especially important for high-throughput transformation experiments.\nThe article is well-written and the data convincing. Other steps of the transformation process such as plant preparation and downstream selection remain labor intensive, so follow-up experiments are important. The Arabidopsis Col-0 ecotype used in this study is relatively easy to transform. It will be interesting to see if low Agro concentrations are equally suitable for other ecotypes / mutant genetic backgrounds or if the method can be combined with other efficiencies like making the Agrobacterium solution directly from plates to further avoid culturing and sub-culturing steps.\n\nMinor comments:\nPlease see attached PDF for small corrections in grammar.\n\nFloral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein).\n\nClarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods.\n\nUnable to access the underlying raw dataset - check the file is attached to the link.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5904", "date": "10 Sep 2020", "name": "Yuelin Zhang", "role": "Author Response", "response": "We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments:1. Please see attached PDF for small corrections in grammar.Thanks a lot for your suggestion. We have made the suggested corrections.2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein).References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested.3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods.Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link.We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data:Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\"" } ] }, { "id": "63860", "date": "27 May 2020", "name": "Zhanyuan J Zhang", "expertise": [ "Reviewer Expertise Plant tissue culture and transformation and plant molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work showed an interesting result that is contrary to the common practice in Arabidopsis floral dip transformation. The authors demonstrated that the use of an unusually low concentration of Agrobacterium inoculum (OD600=0.002) could achieve the same transformation rate as a much higher concentration (OD600=1) does. They also discover a practical implication by using a small amount of Agrobacterium culture. The manuscript is well-written and results are convincing with a sound conclusion. The statistical method was appropriate as well.\nMinor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important.\n\"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper.\nThe other reviewer has pointed out all the written and grammatical issues that I agree with, so no need for me to raise them again.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5905", "date": "10 Sep 2020", "name": "Yuelin Zhang", "role": "Author Response", "response": "We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested." } ] } ]	1	https://f1000research.com/articles/9-356
https://f1000research.com/articles/9-1141/v1	16 Sep 20	{ "type": "Software Tool Article", "title": "Methods developed during the first National Center for Biotechnology Information Structural Variation Codeathon at Baylor College of Medicine", "authors": [ "Medhat Mahmoud", "Alejandro Rafael Gener", "Michael M. Khayat", "Adam C. English", "Advait Balaji", "Anbo Zhou", "Andreas Hehn", "Arkarachai Fungtammasan", "Brianna Sierra Chrisman", "Chen-Shan Chin", "Chiao-Feng Lin", "Chun-Hsuan Lo", "Chunxiao Liao", "Claudia M. B. Carvalho", "Colin Diesh", "David E. Symer", "Divya Kalra", "Dreycey Albin", "Elbay Aliyev", "Eric T. Dawson", "Eric Venner", "Fernanda Foertter", "Gigon Bae", "Haowei Du", "Joyjit Daw", "Junzhou Wang", "Keiko Akagi", "Lon Phan", "Michael Jochum", "Mohammadamin Edrisi", "Nirav N. Shah", "Qi Wang", "Robert Fullem", "Rong Zheng", "Sara E Kalla", "Shakuntala Mitra", "Todd J. Treangen", "Vaidhyanathan Mahaganapathy", "Venkat Sai Malladi", "Vipin K Menon", "Yilei Fu", "Yongze Yin", "Yuanqing Feng", "Tim Hefferon", "Fritz J. Sedlazeck", "Ben Busby", "Medhat Mahmoud", "Michael M. Khayat", "Adam C. English", "Advait Balaji", "Anbo Zhou", "Andreas Hehn", "Arkarachai Fungtammasan", "Brianna Sierra Chrisman", "Chen-Shan Chin", "Chiao-Feng Lin", "Chun-Hsuan Lo", "Chunxiao Liao", "Claudia M. B. Carvalho", "Colin Diesh", "David E. Symer", "Divya Kalra", "Dreycey Albin", "Elbay Aliyev", "Eric T. Dawson", "Eric Venner", "Fernanda Foertter", "Gigon Bae", "Haowei Du", "Joyjit Daw", "Junzhou Wang", "Keiko Akagi", "Lon Phan", "Michael Jochum", "Mohammadamin Edrisi", "Nirav N. Shah", "Qi Wang", "Robert Fullem", "Rong Zheng", "Sara E Kalla", "Shakuntala Mitra", "Todd J. Treangen", "Vaidhyanathan Mahaganapathy", "Venkat Sai Malladi", "Vipin K Menon", "Yilei Fu", "Yongze Yin", "Yuanqing Feng" ], "abstract": "In October 2019, 46 scientists from around the world participated in the first National Center for Biotechnology Information (NCBI) Structural Variation (SV) Codeathon at Baylor College of Medicine. The charge of this first annual working session was to identify ongoing challenges around the topics of SV and graph genomes, and in response to design reliable methods to facilitate their study. Over three days, seven working groups each designed and developed new open-sourced methods to improve the bioinformatic analysis of genomic SVs represented in next-generation sequencing (NGS) data. The groups’ approaches addressed a wide range of problems in SV detection and analysis, including quality control (QC) assessments of metagenome assemblies and population-scale VCF files, de novo copy number variation (CNV) detection based on continuous long sequence reads, the representation of sequence variation using graph genomes, and the development of an SV annotation pipeline. A summary of the questions and developments that arose during the daily discussions between groups is outlined. The new methods are publicly available at https://github.com/NCBI-Codeathons/, and demonstrate that a codeathon devoted to SV analysis can produce valuable new insights both for participants and for the broader research community.", "keywords": [ "Structural Variant", "Graph Genome", "Human Genomics", "Clinical Annotation", "Quality Control", "Codeathon" ], "content": "Introduction\n\nStructural variants (SVs) are large-scale genomic alterations, frequently defined as greater than 50 bases (bp) in length, involving deletions, duplications, insertions, inversions and/or translocations, and can occur in combinations. SVs have been linked to multiple phenotypic differences between organisms, as well as within populations of the same species1,2. SVs are known to play roles in myriad diseases, including neurological (e.g. Parkinson, Huntington), Mendelian3, and other genomic alterations such as those seen in many cancers and constitutional diseases4–7. In contrast to single nucleotide variants (SNVs), involving substitution of a single nucleotide, SVs remain understudied due to their more complex nature1. Our understanding of these larger forms of genomic alterations is limited by the sequencing technology and computational methods available to analyze ever-increasing amounts of sequence or similar data. A special type of SV is copy number variant (CNV). These are unbalanced SVs that could either increase or decrease total DNA content through duplications and deletions, respectively. CNVs of high importance are associated with different physical diseases like obesity8,9, type 1 diabetes10, rheumatoid arthritis10, and neurological disorders such as autism11, schizophrenia12, Mendelian diseases and other genomic disorders. More general studies examine the relationship between copy number variation and a range of diseases13,14.\n\nRecent studies report between 20,000 to 25,000 SVs per human genome1. Although the number of SVs per individual is smaller than that for SNVs, SVs account for more altered nucleotides per diploid genome15. Recently, there has been a prominent shift towards studying SVs at the population level. The most prominent and early example of this was the 1000 Genomes Project16, representing different ethnic groups. Other projects include National Institutes of Health (NIH)-sponsored programs such as the National Human Genome Research Institute (NHGRI)’s Centers for Common Disease Genomics (CCDG), and National Heart, Lung, and Blood Institute (NHLBI)’s Trans-Omics for Precision Medicine (TOPMed) program that includes ~155,000 participants sequenced. With data from newer, high-throughput sequencing platforms, investigators are able to capture the genetic variability at a genomic scale of SVs across more geographically and genetically distinct populations. The Icelandic project17 studied 1,817 Icelandic individuals, including 369 trios (mother+father+child). Accordingly, many tools exist to study SVs in cohorts (e.g. svtools18 and the StructURal Variant majorIty VOte (SURVIVOR)19).\n\nOur understanding of genomic variability in humans is tied to the technologies used to study those genomes, typically involving DNA sequencing. Short-read (SR) DNA sequencing (resulting in reads usually less than 150 bp in length) has been the most common way to evaluate DNA samples directly, and RNA samples indirectly after cDNA conversion20,21. When short reads are mapped to reference genomes, they either map entirely or partially22. Partial mapping can be accomplished by locally aligning part of the read and dropping the rest. This is known as soft-clipping. However, short reads do not align well to large variants when there is a significant gap between the last anchored reference position of the read and position of the soft-clipped portion (e.g. a variant with repetitive sequences longer than the read itself)23. Therefore, the aligner may not produce a global alignment between the read and reference, either choosing not to map the entire read or leaving the soft-clipped portion unmapped1.\n\nThe linear reference genome (LRG), which is based on a linear coordinate system, is a common way to represent variability within a genome24,25. While this may be efficient for applications using one or few genomes, more complex applications using multiple LRGs do not scale well. For example, when mapping reads from RNA sequencing (RNA-seq) to a single genome, the use of an LRG requires a baseline amount of compute resources. However, if mapping to multiple linear reference genomes, the task scales roughly linearly, (increasing by a factor n, where n is the number of LRGs to which data can be mapped).\n\nBy contrast, graph genomes can explicitly encode many alternate paths. Variants can be represented in this data structure, and reads can then be mapped exactly to both a reference path and variant sequences. Graphs have been shown to reduce reference bias and improve read mappings to variants26–28. Variants can also be quickly genotyped from such structures. However, adding more variations can rapidly increase the complexity of the graph. In order to make the problem computationally tractable, one must carefully choose which genomic regions to include. Tools for working with graphs are still nascent, and there are few open-source graph implementations that enable mapping directly to SVs (e.g. vg27 and Paragraph28). Graphs are a non-linear alternative to represent genomes; different paths are presented in the graph data structure. Consequently, 1.) they can decrease reference bias and improve read mappings to variants26–28; and 2.) variants can be efficiently genotyped. Adding more genomes to the graph enhances it, but increases its complexity. To decrease complexity and make this problem computationally tractable, the added regions must be chosen carefully.\n\nRegardless of how drastically human individuals may differ in features such as their physical characteristics and behaviors, the human genomes between any two people are actually relatively well-conserved across individuals. The completion of de novo individual genomes is costly, and population-scale comparisons impossible with current technology infrastructure. There are a small number of individuals represented in the reference human genome, while in comparison any given human individual has on average ~4 million single-nucleotide polymorphisms (SNPs) and ~2,500 CNVs16,29. Consequently, there is a burgeoning awareness that the current standard reference genome assemblies do not include available human variation data. Moreover, there is an urgent need for improved tools, which more precisely represent rare genotype(s) of individuals bearing haplotype(s) absent from the human genome30,31. This is particularly relevant for genomic variants leading to clinical phenotypes such as in Mendelian diseases and genomic disorders where rare and ultra-rare variants have a prominent role. A reliable and scalable solution to this comprises developing a more comprehensive reference genome data structure that represents variations that exist in a given population, such as a graph genome8. The variants in a given cohort’s genomic sequences are represented as independent walks along the graph, allowing it to represent the reference cohort.\n\nOverall, the quality assessment, representation, and annotation of SVs across multiple disciplines (e.g. whole-genome sequencing (WGS) and metagenomic sequencing) remain challenging. Thus, our SV codeathon groups focused on seven topics, which led to development of seven new computational methods. These topics included: 1.) quality assessment of population-scale VCF files; 2.) metagenomic assembly quality assessment; 3.) CNV detection and identification of de novo SVs using long-reads; 4.) fast genome graph generation; 5.) SV annotation; 6.) graph making and graph-based read-mapping with GPUs; and 7.) CNV detection quality control. Here we describe our progress, giving details about our tools’ implementations and applications to foster continuous development beyond the current scope of the tools as they were at the end of this codeathon. All methods are open source licensed, and have been made available on GitHub (https://github.com/NCBI-Codeathons/).\n\n\nMethods\n\nClouseau: rapid quality assessment of population-scale VCF files. As we progress from comparing SVs between a sample and its corresponding reference (capturing differences between a given sample and its reference genome) to large cohort genomic datasets (variants across multiple hundreds to thousands of samples), large variant call format (VCF) files are being generated. Generating these large VCF files often involves customized scripts or analysis methods leaving the possibility of human or other runtime errors. For example, undetected errors such as incomplete files or artificially missing data might be mistaken for real biological phenomena (deletions/truncations). Leaving these unchecked may lead to erroneous conclusions. We developed Clouseau to address these challenges. Clouseau is a command line tool allowing users to rapidly validate VCF file formatting, generate multiple QC statistics, providing rapid QC insights from the input VCF file.\n\nMasQ: Metagenomic assemblies-focused Quality assessment. While metagenomic assemblies have significantly improved since the early days of the Human Microbiome Project (HMP), intragenomic and intergenomic repetitive sequences remain as confounders. Individual reads spanning microbial strains (either via long-read technology or by generating synthetic long-reads) may be pieced together to resolve variation within a given microbial community. However, this process is imperfect. A major concern is that detected structural variants could actually result from misassembled genomic data instead of actual strain-specific variation. The goal of this project is to identify errors in any metagenomic assembly based on both short and long read mapping, in the hope of eliminating some of the uncertainty and error in metagenomic studies. Examples of errors to detect include falsely called (false positive) inversions, chimeras (translocation), INDELs (less than 50 bases long), and replacements (large substitutions). We created a containerized quality control pipeline called MasQ to locate, classify, and rectify errors in metagenomic assemblies. Out of concern for the integrity of current and future metagenomic studies from short-read and long-read genome sequencing data, we found that the quality control of metagenomic assemblies could be substantially improved by using a combination of sequence alignment tools and VCF file outputs from SV callers such as Sniffles v1.0.832 and Manta v1.5.033. The resulting SV call sets are subsequently compared and merged using packages like Truvari v0.1.2018.08.1 and SURVIVOR19. The current version of MasQ neatly packages this workflow and integrates it with novel correction and validation steps to fix any erroneous contigs found.\n\nDeNovoSV: CNV detection and identification of de novo SV events using long reads. We developed a pipeline to identify de novo structural variants (SVs) from long-read (LR) sequencing data collected from trios (probands + parents). As described below, in a pilot study, we analyzed data from an individual who carries multiple de novo CNVs, initially identified by array-based comparative genomic hybridization (aCGH). Prior to this work, we lacked an integrated bioinformatics pipeline to identify high-confidence, de novo structural variants called from LR DNA sequencing (e.g., Oxford Nanopore Technology - ONT). Accordingly, to select further SV calls for orthogonal validation, we merged de novo LR SV calls with de novo SV calls independently identified by short-read (SR) DNA sequencing (i.e. PCR-free paired-end Illumina DNA sequencing, 150 paired-end reads, 40x depth of coverage) in this trio. DeNovoSV facilitates identification and prioritization of high-quality de novo SV calls that can be further validated by using targeted orthogonal methodologies such as Sanger sequencing or droplet digital PCR (ddPCR).\n\nSWIGG (SWIft Genomes in a Graph): fast genome graph generation. There is a growing consensus across genomics that linear genome representation is suboptimal for representing variants across populations. While graph genomes have been steadily gaining popularity, many challenges remain (complexity, visualization, etc.). In this project, we developed a heuristic approach that quickly generates graphs and represents genomes in an efficient and succinct way. We created a simple algorithm and tool to build a graphical model that captures variability in the genomes at multiple scales. Moreover, there are regions across the human genome that are conserved among species while bearing modest amounts of variability that are suitable for understanding relationships of genome structure among individuals and/or organisms. We used a k-mer approach to create a sparse representation of such regions (anchors) at a large scale so as to allow visualizing the entire genome easily. These \"anchored\" graphs can then be further iteratively improved to include local sequence differences, and in turn, to help with genotyping existing variants and identifying new variants in new genomes.\n\nASAP (Automated Structural Variation Annotation Pipeline). ASAP is an automated and robust pipeline to annotate structural variations. The pipeline integrates annotations including allele frequency, colocated gene, functional features, domains, regulatory elements, and transcription levels. To facilitate further development, we took a pseudo-multistage build approach. Specifically, during stage one, we pull the main program, AnnotSV v2.234 from its remote source, as we expect this step to change relatively infrequently and the program itself is large. In stage two, based on the previously-built base image, we pull in its dependencies including BEDtools v2.29.035 and build the docker. The workflow is as follows: 1.) User provides a VCF containing SV as input into AnnotSV 2.) AnnotSV annotates the variations and outputs a tab-delimited file 3.) the R script “postprocess.R” is used to process the TSV file generated by AnnotSV and extracts essential annotations like ranking score. AnnotSV has many default annotation sources (https://lbgi.fr/AnnotSV/), and can also accept user-provided annotations as input. The output of this pipeline used the default annotation.\n\nSuper-minityper: graph making and graph-based read-mapping with GPUs in the cloud. We present a set of cloud-based workflows, composed mostly of preexisting and optimized tools, to 1.) construct graphs containing structural variants and 2.) map reads to these graphs. Our workflows allow users to make arbitrary SV calls, construct a graph, and map reads to these graphs. This workflow prioritizes ease-of-use and speed, accepting common input formats and returning results in minutes on commodity cloud virtual machines. Our approach is an example of what can be done now, and is generalizable to newer graph tools.\n\nScanCNV. Many current medical genomic studies still rely on CNV calling to identify de novo events that could have led to a certain disease phenotype. Nevertheless, a common problem for CNV detection is a high rate of false positives36 due to multiple biases in leading short-read sequencing technologies (e.g. GC bias, repetitiveness and general unevenness of the sequence data). To identify false positives and thus improve the reliability of CNV calling pipelines, we designed ScanCNV, which includes multiple QC steps. These QC steps currently include FASTQC v0.11.9, XYAlign v1.1.637, and Plink v2.038 where all the information is currently assembled and vetted within ScanCNV. Future work is still required to automate the process and fully leverage the obtained QC results.\n\nAll methods were tested on DNA-Nexus instances azure:mem1_ssd1_x, 4, cores, 8G ram or azure:mem1_ssd1_x16, 16 cores, 32G ram.\n\nClouseau. Clouseau requires python 3.5 or above. Users supply optional parameters such as the expected maximum distance between variations to identify missing entries. Importantly, the input VCF file needs to be sorted by coordinates. The Clouseau pipeline reads in a VCF file and performs a sample-level basic statistic carried out to ensure that the VCF file is complete and was not truncated during the VCF generation. Next, the VCF is parsed and sample-level QC is carried out. This consists of checking the number of samples in the VCF, the number of chromosomes, the names of all chromosomes/contigs, the distribution and number of variants (single nucleotide variants, insertions, deletions, structural variants) in each chromosome/contig for all samples, and the coordinates for the start and end of each variant for each chromosome and sample. Clouseau further tries to identify missing entries based on long stretches of no variations, which might represent file errors (as occur in incomplete files) or real biological phenomena (as in deletion/truncation). The full workflow is shown in Figure 1.\n\nClouseau starts with a VCF/pVCF/gVCF (variant call format) file that is assessed to ensure the completeness of the previous run. Furthermore, Clouseau assesses the overall statistics to give insights into the per sample quality control (QC). VCF, variant call format. pVCF, project variant call format. gVCF, genome variant call format. QC, quality control.\n\nMasQ. The MasQ pipeline comprises four stages for metagenomic study quality control and validation: assembly, classification, correction, and validation. Our pipeline takes read files in FASTQ format as input, and classifies possible assembly errors as inversions, INDELs, substitutions, chimeras, or N/A, and then compares them with the make-up of the original assembly.\n\nThe purpose of the assembly stage is to do basic quality control on the sequencing data, generate a mock assembly from alignments of the read data, and generate VCF files informing about identified errors. Short-reads are pre-processed and visually assessed for quality, using FastQC. Then, all the reads are aligned to the MegaHIT assembly using BWA-MEM v0.7.439. The alignment results are processed through Manta v1.5.033, a structural variant caller, to produce a VCF file containing the detected errors. Optionally, if long reads are also available for the same sequencing sample, they are passed through a similar pipeline. The long reads are inspected for quality using NanoporeQC. Minimap2 v2.840 is used to align the long reads instead of BWA, then the long-read alignments are provided to Sniffles v1.0.832 to call SVs, condensing this information into a VCF file.\n\nIn the classification stage, VCF files for the short and long reads are compared to each other using Truvari and a merged VCF file is generated with SV data. Short-read and long-read data each present their own sets of challenges while building metagenomic assemblies, so comparison of the Truvari results from short and long sequencing reads of the same samples decreases false positive results.\n\nThe correction stage takes the regions of assembly error ascertained from the classification stage and performs the necessary changes to make a more accurate metagenomic assembly. From here, the validation stage uses sequence aligners such as Minimap2 and BWA to compare the edited assembly to the original inputs, looking to confirm an increase in the percentage of mapped reads in the corrected assembly, which would show the success of the pipeline in locating and correcting assembly errors.\n\nMasQ is implemented using Docker and is freely available on DockerHub. MasQ can also be run online using DNAnexus. All relevant parameters, as well as python scripts for the correction and validation steps, can be found on the GitHub repository. The MasQ pipeline is shown below in Figure 2.\n\nOver multiple steps MasQ utilizes the short and long reads available to assess the quality of the before obtained metagenomic assembly.\n\nDeNovoSV. DeNovoSV takes input VCF files produced by long-read SV callers such as Sniffles v1.0.1132 and short-read SV callers such as Lumpy v0.2.1341, Delly v0.8.242, and Manta v1.6.033. We developed a custom shell script to remove calls that genotyped as a homozygous reference (GT=0/0), with read support less than 5 and aligned to haplotype chromosome, unplaced or unlocalized contigs (GL, KI, etc). Second, the filtered outputs are combined and compared by the pipeline using SURVIVOR v1.0.619 merge allowing a maximum distance of 1000 bp measured pairwise from the start and end breakpoints of each SV, respectively; SVs classified as the same type; and SVs larger than 30 bp. Lastly, CNV calls are performed independently, using ONT data calculated on the resulting alignments using mosdepth v0.2.343 with the following parameter set “-F 3588 -Q 1”. This calculates the bp coverage in 100 kb bins and includes only primary and supplemental alignments. Normalization of the coverage signal is performed based on a custom script, which generates bedgraph data as output. Each individual bedgraph file pertaining to proband and parents is processed by dividing the 100 bp bins scores by the median of the coverage windows. Using this normalization, the majority of the genome shows a score of 1 (ie. similar coverage between the samples) while a CNV on the proband results in a score of 2 for a duplication. We load these files into JBrowse44 with the multibigwig plugin for visualization, and to facilitate downstream analyses of the putative CNV calls. The resulting output is a list of high-confidence de novo SVs. The overall workflow of DeNovoSV is shown in Figure 3.\n\nThe schematic shows the required inputs and final outputs, along with intermediate steps. DeNovoSv starts with already aligned reads (BAM) from long reads and short reads to filter candidate SVs based on a trio to identify de novo SVs in the proband.\n\nSWIGG. The SWIGG (SWIft Graph in a Genome) pipeline can take multiple FASTA sequences as input and outputs a graph that would be a sparse representation for a multiple sequence alignment which can be visualized for larger scale differences. First, the input FASTA sequence is processed to identify appropriate k-mers. Appropriate k-mers are those that are not repetitive within and across all FASTA sequences. The thresholds for appropriate k-mers can be modified using the script arguments. K-mers are then sorted based on their positions and collapsed into a node-edge list. This node-edge list can be visualized using a graph visualizer such as Gephi.\n\nOur motivation to implement SWIGG (Figure 4) is that the human genome essentially contains two types of regions, those that are quite stable, and those that are hypervariable45,46. For the stable regions, short reads can be either hashed as exact matches to these relatively static regions, or mapped with very small bubbles. Nevertheless, stable regions are interspersed by variable and hypervariable regions (e.g. Kir, MSC). We used k-mers to provide a backbone for these complex regions. Likewise, we have also looked at those regions that are very unstable for mapping in the reference genome to extrapolate to graphs. We used 64 of such regions located on human chromosome 6. They are graphically depicted using the NCBI Genome Data Viewer47 in Figure 5. SWIGG is implemented in python 3 and publicly available both on GitHub.\n\nASAP. The user inputs an SV VCF file(s) produced from any SV caller, and the SV file is then ranked and annotated using AnnotSV and produces a tab-delimited file which will be processed using contemporary versions of R. The complete pipeline can be executed automatically as a single WDL script on GitHub. Below is the workflow Figure 6.\n\n\nResults/Use cases\n\nBefore proceeding with SV analysis, the user needs to have insight into the SV file to avoid issues with downstream analyses. Clouseau facilitates this best-practice quality control check by analyzing an input SV file and providing detailed information about its content. Clouseau takes as input an SV VCF file, and returns the number of samples in the file, chromosomes analyzed, distribution of SVs in all samples, and detailed information about each sample in the file. The output is a list of files for all the samples and each sample in the input file. Clouseau was benchmarked using the SV file from the 1000 Genomes Project16. The data set consists of 2,505 samples and consumed 1 minute (wall clock time) to analyze 10,000 variant lines, Figure 7 shows an example of the output results per sample.\n\nMasQ uses short- and long-read sequencing data as input. It outputs VCF files with the locations of structural variants, as well as a classification of assembly errors. Among the two existing methods of assembly validation, reference-based or de novo assembled, we chose to develop a de novo validation pipeline, and benchmarked it using two widely-used datasets, Zymo Microbial Community Standards48 and Shakya49. The Zymo dataset consisted of both Illumina pair-ended short reads and Oxford Nanopore long reads, while the Shakya dataset was made up of pair-ended short reads. The current version of MasQ is publicly available as a Docker container that combines existing tools for assembly, creation and comparison. It includes a Python classifier and correction to identify and fix assembly errors, and is open source on GitHub.\n\nFor the Shakya49 data, metaSPAdes50 assembly, metaQUAST51 reports that there are 328 misassemblies and the total misassembled contigs length is 12,555,565 base pairs. For Shakya, MEGAHIT52 assembly, metaQUAST reports that there are 673 misassemblies and the total misassembled contigs length is 20,244,280 base pairs. For both of the finished assemblies, metaQUAST reports more than hundreds of misassemblies and the total length is more than tens of million base pairs. Also, on the Shakya dataset, according to metaQUAST, the performance of metaSPAdes is slightly better than that of the MEGAHIT. For the MasQ result of both assemblies, both Manta and Sniffles call hundreds of mis-assemblies in two assemblies, which are about the same size of the result outputted by metaQUAST. From the long-read perspective, Sniffles calls less insertions and duplications on the metaSPAdes assembly. Even though metaSPAdes required significantly more time to finish assembling, the extension of edges through repetitive regions and careful decision to remove low coverage edges contributed to a lower insertion and duplication rate.\n\nIn a prototype trial of DeNovoSV, we investigated available genomics data obtained from a trio of samples (i.e. father, mother and proband), with a particular focus on identification of de novo SVs in the proband. Available data included ONT long-read sequences, Illumina short-read WGS from the trio and aCGH. Data from the latter were used as a positive control for known de novo CNVs for the purpose of developing this pipeline. Working with ONT long-read sequence data, we used the NGMLR32 aligner to map the data against the human genome reference assembly (hg38). Sniffles32 used BAM files from the previous step to identify candidate de novo SVs. Read depths of SVs were calculated using mosdepth43. Sniffles outputs were used as source dataset for VCF files which were filtered using our DeNovoSV custom script to remove SVs for which supporting reads were limited in number (RE < 5); homozygous reference SVs; SV branching from autosomes to decoy; and contigs identified with GL numbers, which represent unlocalized contigs. De novo SVs in the child were defined by merging and comparing with parents in the trio using SURVIVOR19. After filtering for de novo SVs in the child using the DeNovoSV pipeline, we compared these de novo SVs and CNV candidates with results extracted from CGH array data.\n\nWe used the DeNovoSV pipeline to prioritize high-confidence, de novo SVs called from long-read (LR) technology. Starting with about 3 million SV calls identified by Sniffles for each individual, the de novo LR pipeline detected 4,509 high-quality SV calls. We further enriched for potentially true-positive calls by merging the long-read calls with SVs calls independently identified with short-read sequencing in this trio (N = 2599 high-quality SV calls). After merging the calls from short-read sequencing to filter for consensus calls, we obtained a list of 67 high-quality SV calls (Figure 8 and Figure 9).\n\nDel: deletion; Dup: duplication; Inv: inversion; INS: insertion; TRA: translocation; UNK: unknown.\n\nEight de novo duplications in the proband were detected using normalized read depth coverage data from mosdepth and visualized in JBrowse (Figure 10).\n\nJBrowse screenshots displaying normalized Oxford Nanopore read coverage for four out of eight de novo duplications spanning 900 kb to 1 Mb genomic segments from Chromosomes 4, 5, 6 and 10. Parent 1 is represented in a blue line, parent 2 in a light blue line and proband in a dark red line. Red rectangles denote duplications.\n\nIn SWIGG we created a simple algorithm and tool to build genome graphs, which is suitable for understanding relationships of genome structure among individuals and/or organisms. Our approach captured variations in a hierarchical way. The idea was to create a sparse representation of large-scale differences (anchors) so as to allow visualizing the entire genome in a succinct way. These \"anchored\" graphs can then be further iteratively improved to include local sequence differences, and in turn, facilitate genotyping existing variants and identifying new variants in new genomes. We tested SWIGG by creating a graph of the human MHC region (4.5Mb in size) Figure 11 using 128-mers in less than three minutes on seven alternative haplotypes. To evaluate our approach at a smaller scale, we tried building a graph for 10 HIV viral genome (each ~10kb) using 10-mers (see SWIGG Github repository).\n\nASAP was benchmarked using 53 non-diseased tissue sites across nearly 1000 individuals. GTEx RNA-seq data (retrieved from RNA-seq gene read counts GCT file https://gtexportal.org/home/datasets) and GTEx Metadata (manually curated from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-5214/E-MTAB-5214.sdrf.txt) from NA12878 Germline Whole Genome were used. V2 Chromium Genome dataset by Long Ranger 2.2.1 downloaded WGS v2 deletions VCF file from https://support.10xgenomics.com/genome-exome/datasets/2.2.1/NA12878_WGS_v2. Messenger RNA features were extracted from NCBI latest RefSeq Annotation53 on GRCh38 using instructions provided here https://www.ncbi.nlm.nih.gov/refseq/functionalelements/. The workflow for running benchmarking and results are shown in Figure 12 below.\n\n\nConclusions and next steps\n\nThe current version of Clouseau sought to validate VCF integrity and to provide informative statistics. Clouseau can read an SV file and return relevant reports. A next step would be to implement modules to accept SNVs, more than one file as an input, visualize the statistics results using publicly available modules, and reimplement the tool to guarantee better performance.\n\nMasQ currently provides a smooth, convenient workflow to locate genome assembly errors and a solid foundation for a machine learning model to classify assembly errors. In the future, MasQ would benefit from further testing with simulated data, larger datasets, and manual annotation of training data for a more sophisticated classifier. We intend to treat this problem as a multiclass text classification problem, which could be successfully handled using an RNN, LSTM, SVM, or HAN framework (Figure 13). Additionally, the outputs from the classification model must be visualized with histograms of the assembly error types, as well as with visual indications of where the errors are located within the assembly. The assembly errors could possibly be organized so that each type of error could be queried within the given assembly and made easily accessible to users.\n\nIn a pilot study to analyze de novo SVs identified in a proband from a genetic trio, we evaluated capabilities of DeNovoSV to confirm those SVs and potentially to discover additional SVs. Using the DenovoSV pipeline with parameters described in Figure 3, all eight genome-wide chromosomal segments with known de novo duplications identified by aCGH in the proband were also identified by short- and long-reads. In addition, 59 additional potential de novo SVs were identified, which need further validation. Independently, the read depth information generated by mosdepth using LR data from ONT also successfully identified all eight genomic duplications, suggesting that analysis of LR data alone can be valuable for de novo CNV detection.\n\nOnce simple and variable regions are solved for graphs, including those with many structural variants and those with complex regions across a number of populations and diseases, then we will be able to phase new genomes automatically. Thus, we will be able to observe traversals of previously unmanageable paths through graphs made from both long and short read data as shown in Figure 14.\n\nNow that we are developing tools for generating genome graphs quickly and efficiently, it is desirable to find optimal parameters (e.g. size of k-mer and occurrence of a k-mer) to determine whether a given graph should be retained or not. This remains challenging because there is no simple way to evaluate genome graphs. Numbers of nodes and edges can be starting points. Comprehensive investigations of potential factors are needed, such as determining to what extent known features are recapitulated in the graphs.\n\n\nData availability\n\nClouseau - SV data from 2,505 samples from the 1000 Genomes Project (http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/).\n\nMasQ - Zymobiomics Microbial Community Standards Assemblies (10 genomes; 5 gram positive, 3 gram negative, 2 yeast). Each has a known reference. Synthetic bacteria included: Bacillus subtilis, Cryptococcus neoformans, Enterococcus faecalis, Escherichia coli, Lactobacillus fermentum, Listeria monocytogenes, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Salmonella enterica, Staphylococcus aureus. Illumina pair-ended short reads from IMMSA dataset (ftp://ftp-private.ncbi.nlm.nih.gov/nist-immsa/IMMSA/). Shakya Assembly (assembled using MegaHIT and MetaSPAdes) - Each has a known reference. Synthetic bacteria included: Acidobacterium capsulatum, Aciduliprofundum boonei, Akkermansia muciniphila, Archaeoglobus fulgidus, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bordetella bronchiseptica, Burkholderia xenovorans LB400, Caldicellulosiruptor bescii, Caldicellulosiruptor saccharolyticus, Chlorobium limicola, Chlorobium phaeobacteroides, Chlorobium phaeovibriodes, Chlorobium tepidum, Chloroflexus aurantiacus, Clostridium thermocellum, Deinococcus radiodurans, Desulfovibrio piger, Desulfovibrio vulgaris, Dictyoglomus turgidum, Enterococcus faecalis, Fusobacterium nucleatum, Gemmatimonas aurantiacus, Geobacter sulfurreducens, Haloferax volcanii, Herpetosiphon aurantiacus, Hydrogenobaculum, Ignicoccus hospitalis, Leptothrix cholodnii, Methanocaldococcus jannaschii, Methanococcus maripaludis C5, Methanococcus maripaludis S2, Methanopyrus kandleri, Methanosarcina acetivorans C2A, Nanoarchaeum equitans, Nitrosomonas europaea, Nostoc sp. PCC 7120, Pelodictyon phaeoclatharatiforme, Persephonella marina EX-H1, Porphyromonas gingivalis, Pyrobaculum aerophilum IM2, Pyrobaculum arsenaticum, Pyrobaculum calidifontis, Pyrococcus furiosus, Pyrococcus horikoshii, Rhodopirellula baltica, Ruegeria pomeroyi, Salinispora arenicola, Salinispora tropica, Shewanella baltica OS185, Shewanella baltica OS223, Sulfitobacter sp.EE-36, Sulfitobacter sp.NAS-14.1, Sulfolobus tokodaii, Sulfurihydrogenibium sp.YO3AOP1, Sulfurihydrogenibium yellowstonense, Thermoanaerobacter pseudethanolicus, Thermotoga neapolitana DSM 4359, Thermotoga petrophila RKU-1, Thermotoga sp.RQ2, Thermus thermophilus HB8, Treponema denticola, Zymomonas mobilis. From Illumina pair-ended short reads. Number of sequences: 54814748. Read length: 101 bp.\n\nDeNovoSV - For the development of the pipeline we used in-house control data from patients.\n\nSWIGG - For data to construct the graph, we used the alternative sequences of the MHC region given in the latest reference Human Genome (HGR38). These were downloaded using NCBI accession numbers GL000250, GL000251, GL000253, GL000254, GL000255 and GL000256. HIV sequences were obtained from Los Alamos National Laboratories54, [with specific accession numbers] Human immunodeficiency virus type 1 (HXB2), complete genome; HIV1/HTLV-III/LAV reference genome [K03455], HIV-1 isolate 2106 from Democratic Republic of the Congo, complete genome [MH705158], HIV-1 isolate 50 from Democratic Republic of the Congo, complete genome [MH705161], HIV-1 isolate 70641 from Democratic Republic of the Congo, complete genome [MH705151], HIV-1 isolate P4039 from Democratic Republic of the Congo, complete genome [MH705157], HIV-1 isolate PBS6126 from Democratic Republic of the Congo, complete genome [MH705153], HIV-1 isolate PBS888 from Democratic Republic of the Congo, complete genome [MH705133], HIV-1 isolate HIV/CH/BID-V3538/2003 from Switzerland, partial genome [JQ403028], Human immunodeficiency virus 1 proviral env gene for envelope protein, isolate 98CM.MP1014 [AM279354], HIV-1 isolate 09NG010499 from Nigeria gag protein (gag) gene, complete cds; pol protein (pol) gene, partial cds; and vif protein (vif), vpr protein (vpr), tat protein (tat), rev protein (rev), vpu protein (vpu), envelope glycoprotein (env), and nef protein (nef) genes, complete cds. [KX389622].\n\nASAP - Data sources and Data Description: 53 non-diseased tissue sites across nearly 1000 individuals: GTEx RNASeq Data: Downloaded RNA-Seq gene read counts GCT file from https://gtexportal.org/home/datasets. GTEx Metadata: manually curated from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-5214/E-MTAB-5214.sdrf.txt from NA12878 Germline Whole Genome v2 Chromium Genome v2 Dataset by Long Ranger 2.2.1: Downloaded WGS v2 dels VCF file from from https://support.10xgenomics.com/genome-exome/datasets/2.2.1/NA12878_WGS_v2. RefSeq Features: mRNA features are extracted from NCBI latest RefSeq Annotation on GRCh38 using instructions provided here: https://www.ncbi.nlm.nih.gov/refseq/functionalelements/.\n\nScanCNV - Simulated CNV 6 samples (males and females) FASTQ data (truth set: “In silico CNVs - Sheet1.csv”), and 1000 Genomes 10 random samples (males and females) mapped data (in file names) in GitHub repository (see below).\n\n\nSoftware availability\n\nClouseau is available from GitHub: https://github.com/NCBI-Codeathons/Clouseau.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395042155\n\nLicense: MIT\n\nMasQ is freely available from GitHub: https://github.com/NCBI-Codeathons/MASQ.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395044156\n\nLicense: MIT\n\nDeNovoSV is freely available from GitHub: https://github.com/NCBI-Codeathons/DeNovoSV.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395043957\n\nLicense: MIT\n\nSWIGG is freely available from GitHub: https://github.com/NCBI-Codeathons/SWIGG.git.\n\nDockerHub: ncbicodeathons/swigg.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395042558\n\nLicense: MIT\n\nASAP is freely available from GitHub: https://github.com/NCBI-Codeathons/ASAP.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395044459\n\nLicense: MIT\n\nSuper-minityper is freely available from GitHub: https://github.com/NCBI-Codeathons/super-minityper.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.395043560\n\nLicense: MIT\n\nScanCNV is freely available from GitHub: https://github.com/NCBI-Codeathons/SCANCNV.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.397728361\n\nLicense: MIT", "appendix": "Acknowledgements\n\nWe would like to thank the administrative staff of the Human Genome Sequencing Center at Baylor College of Medicine (especially Meagan Elizaheth Sam and Chelette Darlene Gaskin) who helped to organize and run the event. We thank DNAnexus Inc. for sponsoring cloud computing resources. We thank Drs. Pengfei Liu and James R. Lupski for generously providing data from their Multiple CNVs project for the DenovoSV pipeline development and Oxford Nanopore Technology team for helping with data generation and data analysis. We thank Dr. Nick Matinyan for helpful suggestions during manuscript review.\n\n\nReferences\n\nMahmoud M, Gobet N, Cruz-Dávalos DI, et al.: Structural variant calling: the long and the short of it. Genome Biol. 2019; 20(1): 246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo SS, Urban AE, Mills RE: Structural variation in the sequencing era. Nat Rev Genet. 2020; 21(3): 171–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanchis-Juan A, Stephens J, French CE, et al.: Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing. Genome Medicine. 2018; 10(1): 95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStransky N, Cerami E, Schalm S, et al.: Abstract 954: The landscape of kinase fusions in cancer. Mol Cell Biol. 2015. Publisher Full Text\n\nFriedman LS, Ostermeyer EA, Szabo CI, et al.: Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet. 1994; 8(4): 399–404. PubMed Abstract \| Publisher Full Text\n\nCarvalho CMB, Lupski JR: Mechanisms underlying structural variant formation in genomic disorders. Nat Rev Genet. 2016; 17(4): 224–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYi K, Ju YS: Patterns and mechanisms of structural variations in human cancer. Exp Mol Med. 2018; 50(8): 98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaten B, Novak AM, Eizenga JM, et al.: Genome graphs and the evolution of genome inference. Genome Res. 2017; 27(5): 665–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJacquemont S, Reymond A, Zufferey F, et al.: Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus. Nature. 2011; 478(7367): 97–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWellcome Trust Case Control ConsortiumCraddock N, Hurles ME, et al.: Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls. Nature. 2010; 464(7289): 713–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinto D, Delaby E, Merico D, et al.: Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. Am J Hum Genet. 2014; 94(5): 677–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalhotra D, Sebat J: CNVs: harbingers of a rare variant revolution in psychiatric genetics. Cell. 2012; 148(6): 1223–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNowakowska B: Clinical interpretation of copy number variants in the human genome. J Appl Genet. 2017; 58(4): 449–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZarrei M, MacDonald JR, Merico D, et al.: A copy number variation map of the human genome. Nat Rev Genet. 2015; 16(3): 172–83. PubMed Abstract \| Publisher Full Text\n\nSedlazeck FJ, Lee H, Darby CA, et al.: Piercing the dark matter: bioinformatics of long-range sequencing and mapping. Nat Rev Genet. 2018: 19(6): 329–46. PubMed Abstract \| Publisher Full Text\n\nSudmant PH, Rausch T, Gardner EJ, et al.: An integrated map of structural variation in 2,504 human genomes. Nature. 2015; 526(7571): 75–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeyter D, Ingimundardottir H, Eggertsson HP, et al.: Long read sequencing of 1,817 Icelanders provides insight into the role of structural variants in human disease. Publisher Full Text\n\nLarson DE, Abel HJ, Chiang C, et al.: svtools: population-scale analysis of structural variation. Bioinformatics. 2019; 35(22): 4782–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeffares DC, Jolly C, Hoti M, et al.: Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nat Commun. 2017; 8: 14061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShendure J, Balasubramanian S, Church GM, et al.: DNA sequencing at 40: past, present and future. Nature. 2017; 550(7676): 345–53. PubMed Abstract \| Publisher Full Text\n\nStark R, Grzelak M, Hadfield J: RNA sequencing: the teenage years. Nat Rev Genet. 2019; 20(11): 631–56. PubMed Abstract \| Publisher Full Text\n\nFonseca NA, Rung J, Brazma A, et al.: Tools for mapping high-throughput sequencing data. Bioinformatics. 2012; 28(24): 3169–77. PubMed Abstract \| Publisher Full Text\n\nSedlazeck FJ, Rescheneder P, Smolka M, et al.: Accurate detection of complex structural variations using single-molecule sequencing. Nat Methods. 2018; 15(6): 461–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChurch DM, Schneider VA, Graves T, et al.: Modernizing reference genome assemblies. PLoS Biol. 2011; 9(7): e1001091. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider VA, Graves-Lindsay T, Howe K, et al.: Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly. Genome Res. 2017; 27(5): 849–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarrison E, Sirén J, Novak AM, et al.: Variation graph toolkit improves read mapping by representing genetic variation in the reference. Nat Biotechnol. 2018; 36(9): 875–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHickey G, Heller D, Monlong J, et al.: Genotyping structural variants in pangenome graphs using the vg toolkit. Genome Biol. 2020; 21(1): 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen S, Krusche P, Dolzhenko E, et al.: Paragraph: a graph-based structural variant genotyper for short-read sequence data. Genome Biol. 2019; 20(1): 291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n1000 Genomes Project ConsortiumAuton A, Brooks LD, et al.: A global reference for human genetic variation. Nature. 2015; 526(7571): 68–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmeur A: Goodbye reference, hello genome graphs. Nat Biotechnol. 2019; 37(8): 866–8. PubMed Abstract \| Publisher Full Text\n\nRakocevic G, Semenyuk V, Lee WP, et al.: Fast and accurate genomic analyses using genome graphs. Nat Genet. 2019; 51(2): 354–62. PubMed Abstract \| Publisher Full Text\n\nSedlazeck FJ, Rescheneder P, Smolka M, et al.: Accurate detection of complex structural variations using single-molecule sequencing. Nat Methods. 2018; 15(6): 461–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen X, Schulz-Trieglaff O, Shaw R, et al.: Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics. 2016; 32(8): 1220–2. PubMed Abstract \| Publisher Full Text\n\nGeoffroy V, Herenger Y, Kress A, et al.: AnnotSV: an integrated tool for structural variations annotation. Bioinformatics. 2018; 34(20): 3572–4. PubMed Abstract \| Publisher Full Text\n\nQuinlan AR, Hall IM: BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarchuk DS, Crooks K, Strande N, et al.: Increasing the diagnostic yield of exome sequencing by copy number variant analysis. PLoS One. 2018; 13(12): e0209185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebster TH, Couse M, Grande BM, et al.: Identifying, understanding, and correcting technical artifacts on the sex chromosomes in next-generation sequencing data. Gigascience. 2019; 8(7): giz074. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPurcell S, Neale B, Todd-Brown K, et al.: PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses. Am J Hum Genet. 2007; 81(3): 559–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34(18): 3094–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLayer RM, Chiang C, Quinlan AR, et al.: LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 2014; 15(6): R84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRausch T, Zichner T, Schlattl A, et al.: DELLY: structural variant discovery by integrated paired-end and split-read analysis. Bioinformatics. 2012; 28(18): i333–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedersen BS, Quinlan AR: Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics. 2018; 34(5): 867–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuels R, Yao E, Diesh CM, et al.: JBrowse: a dynamic web platform for genome visualization and analysis. Genome Biol. 2016; 17: 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZook JM, Chapman B, Wang J, et al.: Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat Biotechnol. 2014; 32(3): 246–51. PubMed Abstract \| Publisher Full Text\n\nDolgalev I, Sedlazeck F, Busby B: DangerTrack: A scoring system to detect difficult-to-assess regions [version 1; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2017; 6: 443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSayers EW, Beck J, Brister JR, et al.: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2020; 48(D1): D9–D16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNicholls SM, Quick JC, Tang S, et al.: Ultra-deep, long-read nanopore sequencing of mock microbial community standards. Gigascience. 2019; 8(5): giz043. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShakya M, Quince C, Campbell JH, et al.: Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities. Environ Microbiol. 2013; 15(6): 1882–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNurk S, Meleshko D, Korobeynikov A, et al.: metaSPAdes: a new versatile metagenomic assembler. Genome Res. 2017; 27(5): 824–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikheenko A, Saveliev V, Gurevich A: MetaQUAST: evaluation of metagenome assemblies. Bioinformatics. 2016; 32(7): 1088–90. PubMed Abstract \| Publisher Full Text\n\nLi D, Liu CM, Luo R, et al.: MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 2015; 31(10): 1674–6. PubMed Abstract \| Publisher Full Text\n\nO’Leary NA, Wright MW, Brister JR, et al.: Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016; 44(D1): D733–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSayers EW, Cavanaugh M, Clark K, et al.: GenBank. Nucleic Acids Res. 2020; 48(D1): D84–D86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedhat, Malladi V, Fungtammasan A, et al.: GenerGener/Clouseau v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950421\n\nMitra S, Albin D, Treangen TJ, et al.: GenerGener/MASQ v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950441\n\nLo CH, Symer D, Idraktt, et al.: GenerGener/DeNovoSV v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950439\n\nShah N, Chrisman B, Chunxiao, et al.: GenerGener/SWIGG v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950425\n\nlonphan, Busby B, Huang S, et al.: GenerGener/ASAP v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950444\n\nDawson ET, Bae G, Daw J, et al.: GenerGener/super-minityper v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3950435\n\nEdrisi M, Vaidhymg A, Busby B: GenerGener/SCANCNV v0.1.0 (Version v0.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3977283" }	[ { "id": "74008", "date": "12 Nov 2020", "name": "Istvan Albert", "expertise": [ "Reviewer Expertise Bioinformatics", "Software development" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 and other similar works fulfill an important need of demonstrating cutting edge methodologies, recommending new practices, and educating the broader community on what may be considered state of the art in a given subfield of bioinformatics.\nThat being said I wish the authors did a better job at organizing and distributing the code. The current practice of creating a separate disconnected repository for every single (sometimes exploratory) project feels verbose, counterproductive and long term misses the whole point of sharing code.\n\nTo make a better point of this criticism I took the time to rework the repository for this paper in what I think is far superior approach. See it here:\nhttps://github.com/ialbert/Structural-Variation-Baylor-College-Codeathon-2019\nThe important difference in the structure I recommend is there is that more information should be shared right there on Github to avoid the constant referral back to the paper, chasing down links (by the way the links in the abstract do not match the text!) and scrolling the paper back and forth.\nThere should be a single entry point for each Codeathon that contains all the code developed during the event. During the events, the speed of development is of critical importance, thus it is very unlikely that the product would be a package that is worth maintaining separately. In addition, a lot of value can be gained from seeing what someone actually did during the Codeathon.\nCopying over the code to a new project, if that is worthy would be a trivial exercise. It helps no one that from now on there will be an abandoned repository called Clouseau on Github that hangs out there with no context.\n\nI would strongly urge NCBI in general, and theses author, in particular, to start sharing the results of Codeathons not as a random list of seemingly unrelated packages but as complete, standalone event snapshots that are worth visiting and stand the test of time (as I show in my repository). The current organization neither scales nor allows Codeathons to reach their full potential.\n\nAs for the code I noted several shortcomings, all summarized as a lack of educational focus. Education starts with not just telling someone what to do but also showing how to do it. Each project developed during the Codeathon should have a simple example (with a simple data published there as well) that shows the full usage.\n\nCloseau: needs a simple example run shown.\n\nASAP: there is a title header saying Execution Example but does not contain the example.\n\nDenovoSNV: please show an example run.\n\nMASQ: has run code but please do not hardcode the full link to it, instead use relative links otherwise cloned repositories will be incorrect.\n\nSCANCVN: please compress your data files! In my repository I compressed the VCF and BED files and that dropped the repository size from 340MB to 40 MB! Makes an enormous difference. Also please do not use an IMAGE to demonstrate numbers or content in a shell. Just copy over the text into a markdown codeblock. There is a usage example, so that is covered.\n\nSWIGG - is done nicely and it is how one imagines a complete report. It is informative, uses images as Mother Nature intended ;-), has usage instructions. Well done.\nOverall I think the paper is a valuable addition and a good fit for the Hackathons series if the authors take time to make their code more usable and meaningful to the larger community.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "99140", "date": "08 Nov 2021", "name": "Yuriy Orlov", "expertise": [ "Reviewer Expertise bioinformatics", "genomics", "e-Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is good and interesting work on actual bioinformatics applications. It should be indexed.\nI have only minor remarks (on authors’ discretion):\n\nIn the abstract: need to give the abbreviations in full, variant call format (VCF), avoid abbreviation QC since it is not used in the Abstract, and fix the phrase: \"including quality control (QC) assessments of metagenome assemblies and population-scale VCF files\"\n\nThe keywords might be updated by the Variation word - 'Structural Variant, Graph Genome, Human Genomics, Clinical Annotation, Quality Control, Codeathon'\n\n'constitutional diseases[4-7].'- it is kind of a bulk citation. Reference 6 could be cited separately.\n6. Carvalho CMB, Lupski JR: Mechanisms underlying structural variant formation in genomic disorders. Nat Rev Genet. 2016;\n\nReference 8 is not in place. It is about graph genome, not a disease (see citation text “different physical diseases like obesity [8,9])\n\n\"CNVs of high importance are associated with\" - It is kind of logical repeat with the previous phrase listing the diseases and reference\n\n\"Partial mapping can be accomplished by locally aligning part of the read and dropping the rest. This is known as soft-clipping.\" - I’d recommend adding a reference here, for the term ‘soft-clipping’ and mention the most used Illumina sequencing platform.\n\nCodeathon - need to give a definition of ‘codeathon’ earlier in the text, mention other hackathons and computer competitions. - currently, it starts from ‘Thus, our SV codeathon groups focused...’\n\nThe hyperlink in the abstract ‘https://github.com/NCBI-Codeathons/’ leads to MASQ ‘https://github.com/NCBI-Codeathons/MASQ’ - need to fix.\n\nOverall, the manuscript fits F1000Research, is well prepared, and is very useful for education.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1141
https://f1000research.com/articles/9-512/v1	04 Jun 20	{ "type": "Software Tool Article", "title": "ExploreModelMatrix: Interactive exploration for improved understanding of design matrices and linear models in R", "authors": [ "Charlotte Soneson", "Federico Marini", "Florian Geier", "Michael I. Love", "Michael B. Stadler", "Federico Marini", "Florian Geier", "Michael I. Love", "Michael B. Stadler" ], "abstract": "Linear and generalized linear models are used extensively in many scientiﬁc ﬁelds, to model observed data and as the basis for hypothesis tests. The use of such models requires speciﬁcation of a design matrix, and subsequent formulation of contrasts representing scientiﬁc hypotheses of interest. Proper execution of these steps requires a thorough understanding of the meaning of the individual coefﬁcients, and is a frequent source of uncertainty for end users. Here, we present an R/Bioconductor package, ExploreModelMatrix, which enables interactive exploration of design matrices and linear model diagnostics. Given a sample annotation table and a desired design formula, the package displays how the model coefﬁcients are combined to give the ﬁtted values for each combination of predictor variables, which allows users to both extract the interpretation of each individual coefﬁcient, and formulate desired linear contrasts. In addition, the interactive interface displays informative characteristics for the regular linear model corresponding to the provided design, such as variance inﬂation factors and the pseudoinverse of the design matrix.", "keywords": [ "Linear Model", "Experimental Design", "Design Matrix", "Shiny", "R", "Interactivity" ], "content": "Introduction\n\nLinear and generalized linear models are ubiquitous tools in a wide variety of scientific disciplines, and encompass well-known special cases such as linear and logistic regression, ANOVA and Student’s t-test. Of particular interest to us, they are also the basis for many of the most widely used tools for analysis of high-throughput biological data. This includes limma1,2 for linear modeling of gene expression microarray and similar data, as well as edgeR3,4 and DESeq25 for differential expression analysis of RNA-seq and other count data, missMethyl6, DMRcate7 and minfi8 for differential methylation analysis, DiffBind9 for differential binding analysis, msmsTests10 for mass spectrometry, and many others. Since the linear model is a special case of the generalized linear model, and particularly as the aspects of defining the design matrix are shared between the two, we will generally refer to generalized linear models in the rest of this manuscript.\n\nFitting a generalized linear model requires observations of a response variable y (e.g., inferred abundance levels of a gene) as well as a set of continuous or categorical predictor variables or sample annotations (e.g. the sample genotype, age, or treatment condition). In addition, in the R statistical programming environment, the user provides a design formula, specifying which, and how, provided predictor variables should be used to model the expected value of the response. The design formula in R is a version of a syntax for model specification originally proposed in 1973 by Wilkinson and Rogers11. This design formula and a specification of a type of contrast coding define a numeric N × J design matrix X, where N is the number of observations and J the number of model coefficients. The expected response values are then modeled by\n\nwhere β = (β1, . . . , βJ) are the regression coefficients for the respective columns of the design matrix, and g is a link function12,13. X β is typically referred to as the linear predictor. After fitting the model, statistical tests can be performed to test the null hypothesis that a given combination of coefficients (referred to as a linear contrast) is zero. In this manuscript, we will focus on reference cell coding, or “treatment” coding for contrasts, though in general other schemes may also be considered. For more details on how R’s design formula functionality is implemented, we refer to the reference for statistical modeling in S14.\n\nThe way that the model is specified, that is, the definition of the design matrix, naturally determines how the model coefficients should be interpreted. As an example, consider a situation with a linear model and a single categorical predictor with two levels. Defining a model including an intercept (a column of the design matrix with the value 1 for all observations) implies that the second regression coefficient represents the difference between the average response values for the two levels of the predictor, while without the intercept, the two regression coefficients directly represent the average response values for the two factor levels. Given the versatility of generalized linear models, determining the proper contrast to use for testing a specific biological hypothesis of interest requires an understanding of the interpretation of the individual regression coefficients, and can be challenging for users of generalized linear model-based tools.\n\nHere, we present ExploreModelMatrix15, an R package for interactive exploration of generalized linear model designs, coefficients, and contrasts. Given a table of predictor variables, the user can specify the desired design formula and explore the value of the linear predictor for each combination of predictor values, expressed in terms of the model coefficients. From this type of visualization, it is often straightforward to determine the contrast corresponding to a given comparison of interest. We envision that ExploreModelMatrix can be useful for both research and teaching purposes. Specifically for the latter, the application contains several built-in example data sets, corresponding to some of the most commonly used experimental design setups. The underlying function in ExploreModelMatrix that processes the input data and generates visualizations can also be directly called by the user, enabling the generation of static plots for inclusion in reports and educational material.\n\n\nMethods\n\nExploreModelMatrix15 is implemented as an R package16, using the Shiny framework17. The package is available via Bioconductor18, with the current development version accessible via GitHub. The package has been tested with R version 3.6 and later.\n\nAn instance of the interactive application is launched by calling the ExploreModelMatrix() function. This function accepts two optional arguments; a data.frame with one row per observation and each column corresponding to a measured predictor variable (below referred to as the sample information table), and a design formula. If the ExploreModelMatrix() function is called without any arguments, the user can either explore one of the built-in designs, or load a sample information table from a tab-separated text file. The design formula can always be specified or modified interactively in the application. If the user wishes to generate the visualizations independently of the interactive interface, this can be achieved via the VisualizeDesign() function, which is also called internally by ExploreModelMatrix().\n\nThe user interface of ExploreModelMatrix consists of a side bar with control widgets and a main window containing a set of fixed, but collapsible, panels, each illustrating a different aspect of the design matrix or the associated standard linear model (Figure 1). A more detailed explanation of each panel is accessible via the guided tour of the interface, implemented via the rintrojs package19 and accessible by clicking on the question mark icon in the top right corner (represented by the letter P in Figure 1).\n\nThis example shows a model with two predictors (genotype and treatment), each with two levels, and with the assumption that their effects are additive. Red circles with letters were added to be able to refer to specific parts of the interface in the text.\n\nGiven a sample information table and a design formula, either provided by the user or obtained via one of the built-in designs, ExploreModelMatrix will first check that the two objects are compatible, i.e., that the terms in the design formula use only variables that are present in the sample information table, and that the design formula is supported by the package. If no problems are detected, ExploreModelMatrix will create a design matrix using the model.matrix() R function. The full sample table, a summary of its columns, and the resulting design matrix are all displayed in the application interface for convenience (see H-J in Figure 1). In addition, the rank of the design matrix is calculated (K). If the design matrix is not full rank, ExploreModelMatrix will display a warning, together with an indication of the coefficients that are not estimable (using the nonEstimable() function from the limma R package1,2). In addition, ExploreModelMatrix will inform the user if the number of rows (observations) in the design matrix is the same as its rank, in which case there are no residual degrees of freedom, and the variance or dispersion cannot be estimated.\n\nExpressed in terms of the model coefficients, the panels in the first row of the application (F-G) illustrate, in graphical and tabular form, the value of the linear predictor in a generalized linear model, for each combination of levels for the predictors used in the design formula. This provides an intuitive understanding of the interpretation of each of the model coefficients, and can be helpful for specifying appropriate contrasts.\n\nThe panels in the lower part of the interface (L, M, O) should largely be interpreted in the context of standard linear models, where coefficient estimates are obtained using least squares fitting. The pseudoinverse (XT X)−1 XT20–22 represents the way each observed response value would contribute to the coefficient estimates. More precisely, in such a linear model represented by\n\nthe estimated regression coefficients are given by\n\nExploreModelMatrix also estimates variance inflation factors and correlations among the coefficient estimates. Finally, the co-occurrence plot in the bottom left panel (N) shows the number of observations in the data set for each combination of levels of the predictor variables.\n\nThe controls in the left-hand sidebar can be used to interactively modify the studied design as well as the display parameters of the panels. The text box in the top (A) allows the user to type in a design formula (starting with the ~, or “tilde” character), and the displayed figures will be updated accordingly. The dropdown menu immediately below (B) contains the built-in example designs. To use the sample information table provided either as an argument to ExploreModelMatrix() or uploaded into the app at run time, select -- here. The next section of controls (C) lets the user control which level should be considered the “baseline” or reference level for each categorical or factor variable in the model. ExploreModelMatrix will convert each character variable to a factor when a sample information table is loaded; by default the baseline level will be the first in alphabetical order.\n\nIn cases where the design matrix is not of full rank, it may be desirable to exclude a subset of the columns in the design matrix (for example, columns with all zero values or columns that are linear combinations of other columns). This can be done in the \"Drop columns\" section (D). As mentioned above, in the case of a non-full rank design matrix, ExploreModelMatrix will indicate which coefficients are not estimable and thus candidates for being dropped. The final group of controls (E) provide the ability to change the way the panels are displayed, e.g. by setting the height of the plot panels and changing the size and display mode of the text.\n\n\nUse case\n\nTo illustrate how ExploreModelMatrix15 can be used to interpret the coefficients in a complex experimental design, we consider the example of differential allele-specific expression analysis with RNA-seq data. Generalized linear models for count data often use the log link function, and we assume this to be the case in some of the interpretations below. This type of experiment contains different groups of subjects (e.g., from different experimental conditions), where each subject contributes two columns in the read count matrix: one representing the read counts for the reference allele, and one representing those for the alternative allele, for each considered gene. Typical scientific questions of interest are whether there are differences between the expression of the two alleles within each condition, and whether there are differences in the allele-specific expression patterns between the conditions. Similar setups can be observed, for example, in differential methylation experiments (where the two columns for each sample would correspond to methylated and unmethylated read counts for a feature), or in situations where individuals from different groups are each given the same set of treatments.\n\nThe sample annotation table considered here is provided in Table 1. In addition to the columns containing the subject identifier, the condition and the count type (reference or alternative allele), we include a column corresponding to a within-condition relabeling, or dummy encoding, of the subject identifier. Note that this dummy subject identifier has only three levels, compared to six for the original subject identifier. This design setup is available among the example designs provided within ExploreModelMatrix, denoted “Two crossed, one nested factor (manuscript example)”. We will illustrate two equivalent ways of setting up the design formula, and show how ExploreModelMatrix can help in the interpretation of the model coefficients.\n\nFirst, we specify the design formula\n\nincluding an overall condition effect, a term to account for sample-specific effects, and an interaction between the condition and count type columns to capture allele-specific expression within each condition. In R’s design formula syntax, a “:” between two variable names indicates the addition of an interaction term between these two variables, which may have a different effect on columns of X depending on whether these are numeric or factor variables, and what other terms are in the design. Given this design formula together with the sample annotation table from Table 1 as the input arguments, the ExploreModelMatrix functions determine the composition of the linear predictor for each combination of predictor variables shown in Figure 2A (corresponds to panel (F) in Figure 1, shown here separately for increased readability). The Rank panel in the application further indicates that the design matrix is of full rank and that the residual degrees of freedom is non-zero, allowing also estimation of variances or dispersions for use in statistical hypothesis tests involving the estimated coefficients. The illustration in Figure 2A can be used to extract appropriate contrasts for statistical testing. For example, comparing the values of the linear predictor for each sample in the control group, we can see that the conditioncontrol:countalt coefficient represents the allele-specific expression effect (alt/ref expression log-ratio) in this group. Similarly, the conditiontreated:countalt coefficient represents the allele-specific expression in the treated group. As a consequence, the condition-dependent allele-specific expression effect is obtained as the difference between the allele-specific effects within the respective conditions, that is, by conditiontreated:countalt - conditioncontrol:countalt.\n\nA. Using the design formula ~ condition + condition:subjectdummy + condition:count. B. Using the design formula ~ conditioncount + subject.\n\nNext, we illustrate an alternative way of setting up the design matrix, by specifying the design formula as\n\nHere, we use the original subject ID (not the dummy encoded), and include main effects for condition and count type as well as an interaction between the condition and the count type. In R’s design formula syntax, a “” between two variable names indicates the addition of both main effects and an interaction term between these two variables. Upon changing the design formula in ExploreModelMatrix, we are notified that the design matrix is no longer full rank, as a consequence of having different subjects in the different conditions. Dropping the subjectS4 column results in a full-rank design matrix, and the composition of the linear predictor is shown in Figure 2B. The rank of the design matrix, as well as the residual degrees of freedom, are the same as with the previous formulation. However, the composition of the linear predictor for each combination of input variables is different. Comparing the alternative and reference allele groups for the control condition shows that with this formulation, the allele specificity in the control group is encoded by the countalt coefficient. Similarly, the allele specificity in the treated group is represented by the sum of the countalt and conditiontreated:countalt coefficients. Consequently, the difference in allele specificity between the treated and control group is now directly encoded in the conditiontreated:countalt coefficient.\n\nThis example stresses that knowing how to interpret a given coefficient in a generalized linear model is critical, that identically labelled coefficients can have different meanings depending on the chosen design formula, and that ExploreModelMatrix can help the user interpret the resulting coefficients for a given choice of design formula and set up an appropriate contrast.\n\n\nSummary\n\nWe have described the ExploreModelMatrix R/Bioconductor package15, which enables interactive exploration for increased understanding of model coefficients in linear and generalized linear models. To the best of our knowledge, this is the first package of its kind, and we envision applications for both research and educational purposes. The application requires minimal input and can be launched from a local R session, as well as be deployed on a Shiny server. An example instance of the latter is available at http://shiny.imbei.uni-mainz.de:3838/ExploreModelMatrix/, and the process for deploying an instance of the application on a Shiny server is documented in one of the vignettes accompanying the software.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nExploreModelMatrix is available at: http://www.bioconductor.org/packages/ExploreModelMatrix/\n\nSource code available at: https://github.com/csoneson/ExploreModelMatrix.\n\nSource code at time of publication: https://doi.org/10.5281/zenodo.383740215.\n\nLicense: MIT License.", "appendix": "References\n\nSmyth GK: Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004; 3(1): Article 3. PubMed Abstract \| Publisher Full Text\n\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\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarthy DJ, Chen Y, Smyth GK: Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012; 40(10): 4288–4297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhipson B, Maksimovic J, Oshlack A: missMethyl: an R package for analyzing data from Illumina’s HumanMethylation450 platform. Bioinformatics. 2016; 32(2): 286–288. PubMed Abstract \| Publisher Full Text\n\nPeters TJ, Buckley MJ, Statham AL, et al.: De novo identification of differentially methylated regions in the human genome. Epigenet Chromatin. 2015; 8: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAryee MJ, Jaffe AE, Corrada-Bravo H, et al.: Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics. 2014; 30(10): 1363–1369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStark R, Brown G: DiffBind: differential binding analysis of ChIP-Seq peak data. R package version 2.15.2. 2011. Publisher Full Text\n\nGregori J, Sanchez A, Villanueva J: msmsTests: LC-MS/MS Differential Expression Tests. R package version 1.25.0 . 2019. Publisher Full Text\n\nWilkinson GN, Rogers CE: Symbolic description of factorial models for analysis of variance. J R Stat Soc Ser C (Applied Statistics). 1973; 22(3): 392–399. Publisher Full Text\n\nNelder JA, Wedderburn RWM: Generalized linear models. J R Stat Soc Ser A. 1972; 135(3): 370–384. Publisher Full Text\n\nMcCullagh P, Nelder JA: Generalized Linear Models. Second Edition. Chapman and Hall/CRC Monographs on Statistics and Applied Probability Series. Chapman & Hall, 1989. ISBN 9780412317606. Reference Source\n\nChambers JM, Hastie T: Statistical Models in S. Wadsworth & Brooks/Cole computer science series. Wadsworth & Brooks/Cole Advanced Books & Software, 1992. ISBN 9780534167646. Reference Source\n\nSoneson C, Marini F, Geier F, et al.: ExploreModelMatrix. May 2020. http://www.doi.org/doi.org/10.5281/zenodo.3837403\n\nR Core Team: R: A language and environment for statistical computing. 2020. Reference Source\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. R package version 1.4.0.2. 2020. Reference Source\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanz C: rintrojs: A wrapper for the intro.js library. J Open Source Softw. 2016; 1. Publisher Full Text\n\nMoore EH: On the reciprocal of the general algebraic matrix. B Am Math Soc. 1920; 26: 394–395.\n\nBjerhammar A: Application of calculus of matrices to method of least squares; with special references to geodetic calculations. Trans Roy Inst Tech Stockholm. 1951; 49.\n\nPenrose R: A generalized inverse for matrices. Math Proc Cambridge. 1955; 51(3): 406–413. Publisher Full Text" }	[ { "id": "64392", "date": "18 Jun 2020", "name": "Jean Fan", "expertise": [ "Reviewer Expertise Bioinformatics", "Applied Statistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExecutive Summary\nChoosing between different model designs is a common first step in hypothesis testing. Here, Soneson et al. created an R/Bioconductor package called ExploreModelMatrix that provides an interactive Shiny interface for exploring such model designs. While the potential utility of such a package in a guided teaching setting is evident, its utility in a research setting is currently limited by its inability to accommodate larger, more complex designs common to real biological research. Most pressingly, it remains unclear how each of the explorer's modules can be used to guide a non-mathematical user to choose the appropriate model design.\n\n---\nMajor comments:\nOverall, it is unclear to me who is intended to be the target user for this package. The authors suggest that ExploreModelMatrix can be use in biological research or teaching, where many users will not be formal mathematicians.\nHowever, if I try to model using redundant variables, I get a warning \"The design matrix is not full rank,\" which is a difficult message to interpret, particularly for non-mathematicians.\n\nLikewise, if the number of observations in the design matrix is the same as its rank, I get a warning \"The residual degrees of freedom is 0. Values such as variances or dispersions can not be estimated from data with this design,\" which again is a difficult message to interpret, particularly for non-mathematicians.\n\nA more actionable jargon-free set of recommendations would be important for users.\n\nIn biological research, it is not uncommon to have dozens of patients and dozens of cell-types for multiple treatments across multiple time points for example. For designs with more than a few options per predictor, the current interface becomes quickly unusable. For example:\n``` celltype <- factor(sapply(1:10, function(x) rep(paste0('celltype', x), 30))) patient <- as.factor(sample(1:10, 300, replace=TRUE)) levels(patient) <- paste0('patient', 1:10) names(celltype) <- names(patient) <- paste0('cell', 1:300)\nsampleData <- data.frame(patient, celltype) head(sampleData) ExploreModelMatrix(sampleData = sampleData,\n\ndesignFormula = ~ patient + celltype) ```\nThe 'Fitted values', 'Pseudoinverse of design matrix', and 'Correlation plot', all become overlapping and illegible, rendering the interface unusable.\n\nTesting two different design formula (one with and one without intercepts):\n``` ExploreModelMatrix(sampleData = sampleData,\n\ndesignFormula = ~ genotype + treatment) ExploreModelMatrix(sampleData = sampleData,\n\ndesignFormula = ~ 0 + genotype + treatment) ```\nI was unable to achieve the author's stated goals of \"extract[ing] the interpretation of each individual coefﬁcient, and formulat[ing] desired linear contrasts\" and ultimately deciding which design was best suited for my data. It is not clear how the 'variance inflation factors', 'Pseudoinverse of design matrix', 'Co-occurrence plot', and 'Correlation plot' should be used to inform my decision.\n\nA video tutorial or walkthrough showing how this could be done would be useful.\n\n---\nMinor comments:\nI was unable to install the package using the instructions provided:\n``` > BiocManager::install(\"ExploreModelMatrix\") Bioconductor version 3.9 (BiocManager 1.30.10), R 3.6.0 (2019-04-26) Installing package(s) 'ExploreModelMatrix' Warning message: package ‘ExploreModelMatrix’ is not available (for R version 3.6.0) ```\nInstead, I had to use:\n``` devtools::install_github('csoneson/ExploreModelMatrix') ```\n\nThe \"Choose reference levels\" dropdown panels order the options alphabetically rather by the given level ordering. The fitted values plot does use the correct order though.\n\nAll the functions and utilities that are available are not clear. For example, the \"Flip coordinates\" toggle was very useful and it took me awhile to find it. Again, video tutorial or walkthrough highlighting these features will be very helpful.\n\nIf the annotation names (\"subjectdummy\", \"condition\", \"count\", etc) is too long, the Fitted values visualization becomes illegible as the text overlaps each other without wrapping.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "5915", "date": "10 Sep 2020", "name": "Charlotte Soneson", "role": "Author Response", "response": "Executive SummaryChoosing between different model designs is a common first step in hypothesis testing. Here, Soneson et al. created an R/Bioconductor package called ExploreModelMatrix that provides an interactive Shiny interface for exploring such model designs. While the potential utility of such a package in a guided teaching setting is evident, its utility in a research setting is currently limited by its inability to accommodate larger, more complex designs common to real biological research. Most pressingly, it remains unclear how each of the explorer's modules can be used to guide a non-mathematical user to choose the appropriate model design. Thank you for your constructive comments. We provide point-by-point responses below. Generally, we would like to emphasize that the main purpose of the application is to help users interpret a given design, and to understand how changing the design influences the way you would perform a given comparison of interest, rather than choosing a design for a specific experiment (the latter needs to be guided by insights and expectations about the system at hand, and possibly in consultation with a statistical collaborator). ---Major comments:Overall, it is unclear to me who is intended to be the target user for this package. The authors suggest that ExploreModelMatrix can be use in biological research or teaching, where many users will not be formal mathematicians.However, if I try to model using redundant variables, I get a warning \"The design matrix is not full rank,\" which is a difficult message to interpret, particularly for non-mathematicians. Likewise, if the number of observations in the design matrix is the same as its rank, I get a warning \"The residual degrees of freedom is 0. Values such as variances or dispersions can not be estimated from data with this design,\" which again is a difficult message to interpret, particularly for non-mathematicians. A more actionable jargon-free set of recommendations would be important for users. In the revised version, we have expanded on these messages and added links to external documentation. We have also expanded the built-in tour, which provides additional interpretation assistance for the different concepts. In biological research, it is not uncommon to have dozens of patients and dozens of cell-types for multiple treatments across multiple time points for example. For designs with more than a few options per predictor, the current interface becomes quickly unusable. For example:```celltype <- factor(sapply(1:10, function(x) rep(paste0('celltype', x), 30)))patient <- as.factor(sample(1:10, 300, replace=TRUE))levels(patient) <- paste0('patient', 1:10)names(celltype) <- names(patient) <- paste0('cell', 1:300)sampleData <- data.frame(patient, celltype)head(sampleData)ExploreModelMatrix(sampleData = sampleData, designFormula = ~ patient + celltype)```The 'Fitted values', 'Pseudoinverse of design matrix', and 'Correlation plot', all become overlapping and illegible, rendering the interface unusable. It is true that with a large number of levels for each factor, the available screen space can be too small. There are several ways around this. First, the size of the displayed text as well as of the panels can be modified. In the revised package we have implemented a reactive panel height for the fitted values plot, which should avoid the need for the user to change it manually. Furthermore, by combining the plot and table representations of the fitted values in one panel, the full width of the application is used for the plot. Second, if the number of levels becomes very large, it may be better to use the non-interactive interface where the output can be written to a file of arbitrary dimensions. Third, in order to understand a given type of model, it is often possible to work with a reduced version, with fewer levels per predictor but the same underlying structure. Testing two different design formula (one with and one without intercepts):```ExploreModelMatrix(sampleData = sampleData, designFormula = ~ genotype + treatment)ExploreModelMatrix(sampleData = sampleData, designFormula = ~ 0 + genotype + treatment)```I was unable to achieve the author's stated goals of \"extract[ing] the interpretation of each individual coefﬁcient, and formulat[ing] desired linear contrasts\" and ultimately deciding which design was best suited for my data. It is not clear how the 'variance inflation factors', 'Pseudoinverse of design matrix', 'Co-occurrence plot', and 'Correlation plot' should be used to inform my decision. A video tutorial or walkthrough showing how this could be done would be useful. The main purpose of the application is to help users interpret a given design, and to understand how changing the design influences the way you would perform a given comparison of interest, rather than choosing a design for a specific experiment (the latter needs to be guided by insights and expectations about the system at hand). In the example above, the fitted values panel will inform the user about, e.g., the contrast required to compare two of the treatment groups. Neither design is more 'suitable' for the data, but depending on which one is selected, the contrast needs to be adapted.---Minor comments:I was unable to install the package using the instructions provided:```> BiocManager::install(\"ExploreModelMatrix\")Bioconductor version 3.9 (BiocManager 1.30.10), R 3.6.0 (2019-04-26)Installing package(s) 'ExploreModelMatrix'Warning message:package ‘ExploreModelMatrix’ is not available (for R version 3.6.0) ```Instead, I had to use:```devtools::install_github('csoneson/ExploreModelMatrix')``` ExploreModelMatrix was added to Bioconductor in release 3.11 (the current release, since April 2020). Thus, in order to install it via BiocManager::install() you need to have the most recent Bioconductor release. We now mention this in the manuscript, and we have expanded a bit on the installation instructions in the README to clarify this point. The \"Choose reference levels\" dropdown panels order the options alphabetically rather by the given level ordering. The fitted values plot does use the correct order though. This was intentionally designed in this way - if the user wants to choose a new reference level, the current level ordering doesn't matter, and we reasoned that it's typically easier to find the desired level in a list if the options are listed alphabetically. All the functions and utilities that are available are not clear. For example, the \"Flip coordinates\" toggle was very useful and it took me awhile to find it. Again, video tutorial or walkthrough highlighting these features will be very helpful.The application contains a walkthrough (accessible via the question mark in the top-right corner), pointing out useful features. Moreover, we have added a question mark button to each panel, which will open up the corresponding step in the tour. We have also moved the \"Flip coordinates\" toggle inside the panel with the fitted values plot for easier access. If the annotation names (\"subjectdummy\", \"condition\", \"count\", etc) is too long, the Fitted values visualization becomes illegible as the text overlaps each other without wrapping. This is difficult to manage automatically, since the actual width of the panel depends on the size of the browser window (as opposed to the height, which is set in pixels). It is best remedied by changing the font size of the displayed text in the side bar. We have intentionally kept the full coefficient name on a single line, to avoid misinterpretations and make it easier to match it back to the column names of the design matrix." } ] }, { "id": "64393", "date": "07 Jul 2020", "name": "Lucy D'Agostino McGowan", "expertise": [ "Reviewer Expertise Biostatistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThis article describes an R package and corresponding shiny application, ExploreModelMatrix, that can be used to explore design matrices for linear models. I can see this having a lot of utility for helping partitioners understand their design for simple research questions or for pedagogical use in the classroom. While I see this being extremely useful, the current design falls somewhere in the middle, a bit basic for complex research questions and a bit too complex for those with little statistical background. My suggestions assume that the primary user will be in the latter group.\nMajor Comments\nThe main take away seems to be from the \"fitted values\" boxes (both the plot and the table at the top of the application display this information). These seem to show the exact same content, so it feels repetitive to have both. The table is much more legible, but I can also see the utility of visualizing this. Perhaps these should be a single box with two tabs, one with the table and one with the plot. I find the content in these depictions of the fitted values confusing, the variable names here are referring to the beta coefficients from the model, not the variable values themselves. To someone familiar with R / model output this may be obvious, but I'm not sure that is the case for the target user. Additionally, the use of : to indicate an interaction is not something I would expect novice users to know. If this is meant to help users interpret/calculate fitted values after they fit their models, perhaps the \"pseudo\" model output could be printed above the \"fitted values\" plot/table. This would explain where the values that are being plugged into each of these variable names come from. Additionally, this fitted values plot becomes quickly illegible, it would be great to have some simple defaults built in to expand the plot space if there are several inputs (see #5).\n\nThere are several terms used in the application that may not be familiar to those without statistical/mathematical backgrounds. It would be great to have definitions provided to explain the plots/terms. For example, you could have a question mark next to each term that will pop up an explanation when the user hovers. Terms that need defining in the application: Design Matrix, Pseudoinverse of the design matrix, Variance inflation factors, Co-occurrence plot.\n\nSeveral of the boxes output `code` type texts (for example \"Design matrix\" and \"Sample table summary\") I am not sure what the utility of this being in this format is. If this was something the user could copy and paste into R, for example, this design choice would make sense, but I don't believe that is the purpose.\n\nThe first argument of the R function is `sampleData`, however everywhere else in the application/documentation this is referred to as a `sample table`. This should be consistent.\n\nThere are several pieces for the user to control that could have some better defaults based on user inputs (for example the height of the plots, the size of the text, etc). Especially for a novice user, it would be great if the defaults for these values were reactive (updated based on the user input) and were mostly correct, allowing for tweaking only if absolutely necessary. In that case, you could hide these options in an \"Advanced plot settings\" tab in the sidebar, rather than having them visible when the user first opens the application. I think this would greatly improve the user experience.\n\nMinor Comments\nThe \"Rank\" box outputs text using the textOuput() function, this means that you end up with a [1] prepending the text (which may be confusing to a novice user). Using renderUI() and uiOutput() instead would remove this [1].\n\nIf you remove the value from some of the numeric inputs, you end up with an error that is hard to parse (\"An error has occurred. Check your logs or contact the app author for clarification.\"). You can check for whether the values needed for each value/plot are input using the validate() function and provide better error messages if not. (It looks like this is done for the formula input, just needs to be done for other inputs, for example, plot height, text size, etc.)\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5916", "date": "10 Sep 2020", "name": "Charlotte Soneson", "role": "Author Response", "response": "Summary This article describes an R package and corresponding shiny application, ExploreModelMatrix, that can be used to explore design matrices for linear models. I can see this having a lot of utility for helping partitioners understand their design for simple research questions or for pedagogical use in the classroom. While I see this being extremely useful, the current design falls somewhere in the middle, a bit basic for complex research questions and a bit too complex for those with little statistical background. My suggestions assume that the primary user will be in the latter group. Thank you for your constructive comments. Please find point-by-point responses to each comment below. Major Comments The main take away seems to be from the \"fitted values\" boxes (both the plot and the table at the top of the application display this information). These seem to show the exact same content, so it feels repetitive to have both. The table is much more legible, but I can also see the utility of visualizing this. Perhaps these should be a single box with two tabs, one with the table and one with the plot. This is a good idea, and also frees up more of the horizontal screen space for the plot. In the revised version of the package (v1.1.5, available on GitHub and in the devel branch of Bioconductor), these two representations are now shown in a single box with two tabs. I find the content in these depictions of the fitted values confusing, the variable names here are referring to the beta coefficients from the model, not the variable values themselves. To someone familiar with R / model output this may be obvious, but I'm not sure that is the case for the target user. Additionally, the use of : to indicate an interaction is not something I would expect novice users to know. If this is meant to help users interpret/calculate fitted values after they fit their models, perhaps the \"pseudo\" model output could be printed above the \"fitted values\" plot/table. This would explain where the values that are being plugged into each of these variable names come from. Additionally, this fitted values plot becomes quickly illegible, it would be great to have some simple defaults built in to expand the plot space if there are several inputs (see #5). The main motivation for this representation is to simplify the interpretation of the beta coefficients and the generation of appropriate contrasts. The actual values of the predictor variables are given as row and column names, and when combined provide a \"label\" for each box in the plot. For example, in the example shown in Figure 1, to find the estimated genotype effect in the control group, one would take the fitted value for the \"genotype B\" control samples ((Intercept) + genotypeB) and subtract the fitted value for the \"genotype A\" control samples ((Intercept)). The result (genotypeB) is the coefficient (column) in the design matrix to test for significance. Thus, it is essential that what is shown in the figure/table are the coefficient names (as generated by R's model.matrix()), since they are directly referred back to the column names of the design matrix. It is, in a way, less important to understand exactly why the coefficients are named as they are - the important thing is to be able to extract the proper combination of them for testing. We have addressed the automatic space allocation for the panel depending on the number of predictors and levels - see comment below. There are several terms used in the application that may not be familiar to those without statistical/mathematical backgrounds. It would be great to have definitions provided to explain the plots/terms. For example, you could have a question mark next to each term that will pop up an explanation when the user hovers. Terms that need defining in the application: Design Matrix, Pseudoinverse of the design matrix, Variance inflation factors, Co-occurrence plot. The tour provided with the application (accessible via the question mark icon in the top-right corner) provides some of these descriptions and additional help for interpretation. In the revised version, we have expanded on these and also added links to external documentation. In addition, we have added a question mark to each panel. Clicking on this will open the corresponding step of the built-in tour. Several of the boxes output `code` type texts (for example \"Design matrix\" and \"Sample table summary\") I am not sure what the utility of this being in this format is. If this was something the user could copy and paste into R, for example, this design choice would make sense, but I don't believe that is the purpose. The original purpose of this formatting was to display the output as it would be shown in R, to make it easier for users to make the connection. For the sample table summary, it is also important to display the class of each column (to see, for example, if a column that displays as numbers is really a factor). In the revised version of the package, the user has the choice of whether to display the design matrix as a data table or as 'regular' R output. The first argument of the R function is `sampleData`, however everywhere else in the application/documentation this is referred to as a `sample table`. This should be consistent. We have updated the application to be more consistent by replacing 'sample table' by 'sample data table'. We have also updated the text in the manuscript for consistency. There are several pieces for the user to control that could have some better defaults based on user inputs (for example the height of the plots, the size of the text, etc). Especially for a novice user, it would be great if the defaults for these values were reactive (updated based on the user input) and were mostly correct, allowing for tweaking only if absolutely necessary. In that case, you could hide these options in an \"Advanced plot settings\" tab in the sidebar, rather than having them visible when the user first opens the application. I think this would greatly improve the user experience. In the revised package, we have implemented a reactive panel size for the fitted values plot, which changes with the total number of displayed 'rows' (number of panels x number of levels of the predictor shown on the y-axis). It is still possible to tweak if desired. We now also collapse all plot settings in the sidebar by default. Minor Comments The \"Rank\" box outputs text using the textOutput() function, this means that you end up with a [1] prepending the text (which may be confusing to a novice user). Using renderUI() and uiOutput() instead would remove this [1]. We have changed the renderPrint() in these statements to renderText(), which removes the [1]. If you remove the value from some of the numeric inputs, you end up with an error that is hard to parse (\"An error has occurred. Check your logs or contact the app author for clarification.\"). You can check for whether the values needed for each value/plot are input using the validate() function and provide better error messages if not. (It looks like this is done for the formula input, just needs to be done for other inputs, for example, plot height, text size, etc.) This has been fixed, and the app now displays more informative messages if numeric inputs are not properly specified." } ] }, { "id": "64395", "date": "13 Jul 2020", "name": "Matthew Ritchie", "expertise": [ "Reviewer Expertise Gene expression analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUnderstanding how to set up a design matrix is a significant challenge in genomic data science, especially for new analysts. The manuscript by Soneson et al. presents the ExploreModelMatrix R package which aims to give the user better intuition on this for any arbitrary design.\nThis is great contribution and would be really useful in a teaching setting (e.g. when running an Intro to RNA-seq analysis course) to help participants understand how a linear model is parameterised and how this can be changed to suit different biological questions. At the other extreme it is also helpful in the interpretation of coefficients in more complicated designs that include interactions.\nThe ExploreModelMatrix output is provided via a shiny app which allows the user to interactively change either their own design based on the data.frame supplied, or choose from a series of standard examples, which was easy to navigate. I particularly liked the interactive tour of the different elements of the interface provided by rintrojs.\nOverall, I really enjoyed using this package and list a few optional suggestions below to help further improve the work.\nIt might be useful to add a sentence about the intended audience of the package to the abstract.\n\nCan a window be given to show what the line of code looks like to make the design matrix? Just thinking about beginners, who could start the app with ExploreModelMatrix(), choose an example design and then immediately see what code they would need to run at the R command prompt to create the design they're interested in (again helpful for teaching). This would provide a concrete output that the user could take forward in their analysis with a simple copy and paste. Likewise, if they provided their own data frame they will know what to do with it in terms of specifying model.matrix().\n\nWould it be possible to create a dummy x vs y plot (perhaps by simulating data) to show what a fit might look like for theoretical y? This might be rather complicated to implement in practice given the wide array of models one could envisage, however, such a display would provide an intuitive view of what the coefficients represent graphically.\n\nIf a model is parameterised without an intercept, it will generally be necessary to define contrasts between coefficients. Is there a way to point this out to the user in the app, or can a module be added to help set-up contrasts to show how this is done and again provide code that the analyst could then use in their analysis?\n\nOn page 3, column 2, paragraph 4, line 2 there appears to be a formatting problem with th'e' in 'the ExploreModelMatrix()'\n\nIn the 'Use cases' section, I wondered whether the simple example that features in Figure 1 could be stepped through (again to appeal to beginners) in addition to the complex ASE analysis example of Table 1 and Figure 2 which is likely to be more niche.\n\nIn the interactive tour of the interface, the rendering of some equations hasn't occurred properly in steps 19 and 22.\n\nWhen you 'Flip coordinates' in the Fitted values / Co-occurrence plot, the y-axis labels aren't preserved in the flip (not sure if this is intentional)\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5917", "date": "10 Sep 2020", "name": "Charlotte Soneson", "role": "Author Response", "response": "Understanding how to set up a design matrix is a significant challenge in genomic data science, especially for new analysts. The manuscript by Soneson et al. presents the ExploreModelMatrix R package which aims to give the user better intuition on this for any arbitrary design. This is great contribution and would be really useful in a teaching setting (e.g. when running an Intro to RNA-seq analysis course) to help participants understand how a linear model is parameterised and how this can be changed to suit different biological questions. At the other extreme it is also helpful in the interpretation of coefficients in more complicated designs that include interactions. The ExploreModelMatrix output is provided via a shiny app which allows the user to interactively change either their own design based on the data.frame supplied, or choose from a series of standard examples, which was easy to navigate. I particularly liked the interactive tour of the different elements of the interface provided by rintrojs. Overall, I really enjoyed using this package and list a few optional suggestions below to help further improve the work. Thank you for your constructive comments and careful evaluation of the software. Point-by-point responses are provided below. It might be useful to add a sentence about the intended audience of the package to the abstract. We have added a sentence about this in the abstract. Can a window be given to show what the line of code looks like to make the design matrix? Just thinking about beginners, who could start the app with ExploreModelMatrix(), choose an example design and then immediately see what code they would need to run at the R command prompt to create the design they're interested in (again helpful for teaching). This would provide a concrete output that the user could take forward in their analysis with a simple copy and paste. Likewise, if they provided their own data frame they will know what to do with it in terms of specifying model.matrix(). This is an interesting point and something that we may consider in a future version. One challenge is that different analysis packages require the design to be specified in different ways (e.g., as a design formula, or as a design matrix), and in some cases will automatically deal with e.g. non-estimable parameters. Thus, it is not obvious how to provide this code to make it as useful as possible. Would it be possible to create a dummy x vs y plot (perhaps by simulating data) to show what a fit might look like for theoretical y? This might be rather complicated to implement in practice given the wide array of models one could envisage, however, such a display would provide an intuitive view of what the coefficients represent graphically. We agree that such plots are often instructive for the user, and some examples are included e.g. in the Data Analysis for the Life Sciences book, which we now also refer to in the application as an additional resource. Implementing them in a very general context, on the other hand, is a non-trivial task, and furthermore there is a risk that a user is misled if the coefficients estimated from the simulated response are different (perhaps with opposite signs) compared to those obtained from their actual data. For these reasons, we currently consider this out of scope for ExploreModelMatrix. If a model is parameterised without an intercept, it will generally be necessary to define contrasts between coefficients. Is there a way to point this out to the user in the app, or can a module be added to help set-up contrasts to show how this is done and again provide code that the analyst could then use in their analysis? We have expanded on the use case section in the manuscript to provide additional guidance on how the ExploreModelMatrix interface can be used to generate contrasts. On page 3, column 2, paragraph 4, line 2 there appears to be a formatting problem with th'e' in 'the ExploreModelMatrix()' Thanks for pointing this out. We hope that this has been resolved in typesetting of the revised version. In the 'Use cases' section, I wondered whether the simple example that features in Figure 1 could be stepped through (again to appeal to beginners) in addition to the complex ASE analysis example of Table 1 and Figure 2 which is likely to be more niche. In the revised manuscript, we have added a paragraph walking through the design in Figure 1 and showing how ExploreModelMatrix can be used to interpret coefficients and generate contrasts of interest. In the interactive tour of the interface, the rendering of some equations hasn't occurred properly in steps 19 and 22. This is correct - unfortunately the rintrojs package currently doesn't support proper rendering of equations via e.g. MathJax. We have initiated a discussion with the developer and hope that a solution can be engineered - in the meantime, we hope that the non-rendered equations still provide some help with the interpretation. When you 'Flip coordinates' in the Fitted values / Co-occurrence plot, the y-axis labels aren't preserved in the flip (not sure if this is intentional) We were not able to reproduce this behaviour - when the coordinates are flipped, the labels are flipped as well. If you would be willing to open an issue in the GitHub repository with a reproducible example, that would be super helpful!" } ] } ]	1	https://f1000research.com/articles/9-512
https://f1000research.com/articles/9-1139/v1	16 Sep 20	{ "type": "Opinion Article", "title": "COVID-19 and blood groups – there is an elephant in the room, but who cares? Do we need additional rules for preprints?", "authors": [ "Joern Bullerdiek" ], "abstract": "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) not only can cause very severe disease but, less obviously, the virus can also infect science in unpredicted ways. It seems that during these times some basic rules of science will lose validity and we do not know if they will come back. Though not necessarily always being the case, problems can arise from messages that make their way to public media straight from preprints. An impressive example is a recent study on an association between ABO blood groups and the severity of COVID-19. The study was first published as a preprint which almost immediately gathered an enormous amount of public interest though major drawbacks of the study had been identified by members of the scientific community. One of the major advantages of preprints is to present data, even if still incomplete, to the scientific community for an early discussion. It does not serve the quality of science if possible critical considerations are not addressed adequately until these preliminary studies go public and are submitted for publication in classical journals. Accordingly, clear additional rules for handling data derived from preprints are advocated herein. Speed does not have an advantage on its own.", "keywords": [ "COVID-19", "disease severity", "blood group", "preprints" ], "content": "\n\nIn general, associations between blood groups and infectious diseases are well known. They may be either related to the probability of becoming infected or to a more or less severe course of the disease. Recently, associations of ABO blood groups and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) have attracted a lot of scientific interest, due to a number of studies where such associations have been investigated. One such study has generated enormous public interest.\n\nAs to SARS-COV-2 and blood types, initial interest in the field was stimulated by a preprint by Zhao et al., which was initially published on March 16, 20201. Based on their results, individuals with blood group A had a significantly higher risk for acquiring COVID-19 compared with non-A blood groups, whereas individuals with blood group O were found to be at a significantly lower risk for the infection. Less than one month later, another preprint appeared also addressing a possible link between COVID-19 and blood groups, but did not find any significant associations between blood groups and intubation or death2. Again one month later, the first report on a genome-wide association study (GWAS), carried out by a European consortium, appeared as a preprint3, which was followed by a press release. This was then published as a New England Journal of Medicine (NEJM) paper4, again accompanied by a corresponding press release, and apparently all based on the same group of COVID-19 patients receiving oxygen and a control group. The critical considerations outlined herein mainly relate to the preprint by the European consortium, the paper published in NEJM, and the two press releases. The message of all four was that people with blood group A have a much higher risk for respiratory failure during the course of COVID-19 than those with B or 0. This caused a huge public reaction (Table 1), resulting in a lot of anxiousness due to headlines being published in the media, such as “People with blood type A more likely to suffer severe coronavirus symptoms, research finds\" (Telegraph, UK, 4th June 2020a) and TV reports (e.g. News9 India of June 5, 2020b). Both the high public interest, as well as possible clinical applications of the findings, warrant a critical analysis of the methods and data applied.\n\n\nThe preprint and its weakness\n\nThe European consortium (with K. Ellinghaus and F. Degenhardt from Kiel, Germany, as shared first authors and A. Franke, Kiel, and T. H. Karlsen, Oslo, Norway, as corresponding authors) published the results of a GWAS with COVID-19 patients from Spain and Italy who all received oxygen during the course of the disease3. One of two significant association signals was located at chromosomal band 9q34, corresponding to the ABO blood group locus. It was claimed that the ABO blood groups thus are associated with COVID-19 induced respiratory failure \"with higher risk for A-positive individuals and a protective effect for blood group O\". Shortly after the preprint’s appearance the headlines and news went viral (Table 1).\n\nIn a few media articles, the message was overreached, for example by Medscape, Germany, who stated that an association between COVID-19 mortality and blood groups has been found (12th June 2020c). In particular in German-speaking countries, interest in the results of the study was also stimulated by a tweet from Karl Lauterbachd, professor of Health Economics and Clinical Epidemiology at the University of Cologne and member of the Deutscher Bundestag, which was frequently cited in public media (Figure 1).\n\n[A very remarkable result shown in a robust study. Blood group A about 50% higher risk for severe course of COVID-19. Risk twice as high as with blood group 0. Blood group B in between. Because immune response depends on the blood group it makes sense]. (https://twitter.com/Karl_Lauterbach/status/1268522737573728257, translated from German). Permission to reproduce this tweet was obtained from Karl Lauterbach’s office.\n\nDespite this wide-spread interest, the value of the study suffers from its control group, which comprises a total of 2,381 individuals. According to the preprint, the control group, among others, included “998 randomly selected blood donors at Fondazione IRCCS Cá Granda Ospedale Maggiore Policlinico, Milan with no evidence of Covid-19 who were genotyped for the purpose of the present study.“3. Two further control panels with genotype data derived from previous studies using the same genotyping array were included as well (from Italy n=396 and from Spain n=987). Due to the selection of the control group, the findings do not allow for the conclusion that ABO blood types influence the severity of symptoms, since no comparison has been made between people receiving oxygen versus those mildly affected (e.g. non-hospitalized) as a control group. Accordingly, similar results have to be expected, if blood groups affect one’s chance getting infected at all. This problem was pinpointed early on by some in the public media, for example by The Scientist Magazine (“Ideally, a GWAS analysis would analyze the genomes of people with COVID-19 and compare those who didn’t get very sick to those who experienced severe symptoms, instead of using population-based controls whose exposure to the virus is unknown, says Priya Duggal, a genetic epidemiologist at the Johns Hopkins Bloomberg School of Public Health who did not participate in the study.“; 8th June 2020e) and the German Sueddeutsche Zeitung (15th June 2020f).\n\nAlready at this time, at least one of the authors of this study apparently was well aware of this major shortcoming: “The study was not perfect. Ideally there would be another full group of patients for comparison who were infected but who did not develop the severe disease. This was one reason, said Professor Franke, that he could not be confident of the exact risk increase linked to blood type: “It may be 20 per cent, it may be 30 per cent, it may be 50 per cent.” (as quoted in The Australian, 12th June 2020g). Nevertheless, a press releaseh by the lead author’s own institution (Universitätsklinikum Schleswig-Holstein) on 10th June 2020 revealed that according to their results “people with blood group A have a roughly 50% elevated risk for a severe course of COVID-19 than people with other blood groups. Vice versa, people with blood group O were protected against severe disease by nearly 50%” (translated from German).\n\n\nThe study received accolade, but doubt remains\n\nOn 17th June 2020, the paper was published in the New England Journal of Medicine4, and the study received recognition. Unfortunately, the problem with the control group became even more complicated because the description was changed from the preprint: “We recruited 998 randomly selected blood donors at Fondazione IRCCS Cá Granda Ospedale Maggiore Policlinico, Milan, who underwent genotyping for the purpose of the present study. A total of 40 of these participants had evidence of the development of anti–SARS-CoV-2 antibodies, all of whom had mild or no Covid-19 symptoms.“4\n\nIn contrast, according to the preprint3, all 998 blood donors did not show evidence of COVID-19. Also, it is unclear if all had been tested for antibodies and by which test. What was the control group then? A pre-COVID-19 population? Apparently not, but maybe in part. Patients infected, but with mild or absent symptoms of COVID-19 constituting part of the controls, make interpretation of the data even more difficult if not impossible. Despite this, a second press releasei by the lead author’s institution was published on 18th June 2020, which unequivocally concludes: “The study had shown that people with blood group A have an approximately 50 percent higher risk of severe Covid-19 progression than people with other blood groups. In contrast, people with type O blood groups were almost 50 percent better protected against serious covid-19 disease.”. The appearance of the now peer-reviewed paper caused a second wave of headlines (see Table 1). Nevertheless, most notably a large part of the media coverage was gained in the two-week period when only the preprint was available (Figure 2).\n\nNEJM: New England Journal of Medicine. UKSH: Universitätsklinikum Schleswig-Holstein, Germany.\n\nThe enormous attention is also reflected by the preprint and article metrics, e.g. compared to a paper by Latz et al.5 also addressing a possible association between blood groups and the severity of COVID-19, but which came to an opposite conclusion (Figure 3). Simultaneously, the publication caused an enormous level of public interest and anxiety as witnessed by the coverage gained in public media (Table 1).\n\nMetrics for 5 and 14 days after publication are given.\n\n\nAre additional rules for preprints needed?\n\nHow could it happen that, to say the least, a misleading message spread so rapidly and at a preprint stage? What makes the story so appealing for public recognition? It’s not the association with an anonymous gene. Instead, people know about blood groups and many even know about their own. Besides genetic association there is obviously a direct interest in personal risk. Can we thus blame the hunger of the public for intriguing messages on COVID-19 that might affect people personally? At least this is not the only factor explaining the high public interest in this case. It seems reasonable to speculate that a scientifically more correct interpretation of the limits of the study at the preprint stage and in the press releases might have led to a drastically decreased level of interest by public media, but the message of the study has been made more fascinating by the authors themselves – with their press releases and the preprint. Should thus the preprint system of publishing a piece of work without peer review be blamed?\n\nOne of the main arguments in favour of sharing work in its preliminary preprint form is that articles are improved by feedback from a wider group of readers instead of only formal peer review by few experts, and that science works faster if results are made available sooner after being completed6. Basically, both principles have worked here. As for the speed, the preprint was published some two weeks earlier than the paper in NEJM. However, it could be said that there was no such need for speed, and more important was the opportunity to gather feedback from a larger community, which has worked nearly perfectly. Currently, there are 39 comments for the preprint by Ellinghaus and coworkers3. Of these, some comment on the issues faced with the controlsj, and this problem was also pinpointed very early on by experts in the public media and, curiously enough, even at least by one of the authors (see above). Moreover, there were also contradictory findings, which were neither appropriately mentioned in the preprint nor in the NEJM paper, e.g. the preprint by Zietz and Tatonetti2 that appeared earlier on the same server (medRxiv).\n\nOne of the main advantages of a preprint, i.e. the chance to have it critically considered by a larger group of peers, has evaporated here due to the short time between the preprint and appearance of the paper, giving the authors no chance to consider comments and criticism. Of note, even now the preprint has not been corrected, though the control group is apparently not correctly described given that the description in the NEJM is correct.\n\nThere is a low level of control until preprints go public, which clearly increases the author’s responsibilities. Aimed at the benefit for the scientific community, preprints have to balance a possible need for speed with “suitable safeguards to protect the public”6 as well. The case presented here illustrates the adverse effects associated with a loss of such balance and clearly advocates additional rules for preprints. To put it simply, authors should not leave the room while the elephant is still in it. Whenever submitting a manuscript which corresponds to a preprint, a time of some weeks should be given to the extended group of peers to comment on and vice versa, authors should communicate possible comments along with the manuscript and they should declare that they have read and considered all these comments carefully. During that time, there should not be any press releases from the authors. In a comment on their study5 investigating possible associations between the severity of COVID-19 and ABO blood groups, Latz et al. made the rigorous statement that they are confident their principle finding “will help debunk the kinds of clinically unfounded rumours and misinformation that can readily gain traction in the midst of a pandemic, and in some cases become part of accepted medical practice”k. Certainly, we as scientists have a direct responsibility to avoid this type of misinformation.\n\nAnd what on the actual news on COVID-19 severity and blood groups? “Risk does not depend (much) on blood type” stated The New York Times on 15th July 2020l. The Latz et al.5 study, based on 1,289 COV+ individuals, concluded that blood type was not associated with intubation or mortality in COVID-19 patients. Similarly, the recently published second version of the preprint by Zietz and Tatonetti7 even revealed a significantly lesser risk for intubation for individuals with blood group A.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Acknowledgements\n\nThe many helpful critical comments and suggestions of Dr. B. Rommel and Prof. Dr. R. Stick, University of Bremen, are acknowledged.\n\n\nNotes\n\nahttps://www.telegraph.co.uk/global-health/science-and-disease/people-blood-type-likely-suffer-severe-coronavirus-symptoms/\n\nbhttps://www.youtube.com/watch?v=OfRQ-DYCV6s).\n\nchttps://deutsch.medscape.com/artikelansicht/4908983?faf=1&src=soc_fb_200701_mscpmrk_de_single_covid\n\ndhttps://www.hsph.harvard.edu/voices/events/karl-lauterbach-professor-of-health-economics-and-clinical-epidemiology-university-of-cologne-member-of-the-deutsche-bundestag/\n\nehttps://www.the-scientist.com/news-opinion/two-genetic-regions-linked-with-severe-covid-19-67619\n\nfhttps://www.sueddeutsche.de/wissen/genetik-covid-19-coronavirus-krankheit-verlauf-1.4934939?reduced=true\n\nghttps://www.theaustralian.com.au/world/the-times/coronavirus-blood-type-puts-you-at-worse-risk-of-virus-susceptibility-and-severity/news-story/bb0e6fc15ce75b15a23521e7addf6696\n\nhhttps://www.uksh.de/Service/Presse/Presseinformationen/2020/Weltweit+erste+gro%C3%9Fe+genomweite+Studie+_+Kieler+Forschungsteam+findet+Genvarianten+f%C3%BCr+schweren+Verlauf+von+Covid_19-p-184658.html\n\nihttps://idw-online.de/de/news?print=1&id=749683\n\njhttps://www.medrxiv.org/content/10.1101/2020.05.31.20114991v1\n\nkhttps://hms.harvard.edu/news/covid-19-blood-type\n\nlhttps://www.nytimes.com/2020/07/15/science/coronavirus-blood-type.html\n\n\nReferences\n\nZhao J, Yang Y, Huang HP, et al.: Relationship between the ABO Blood Group and the COVID-19 Susceptibility. medRxiv. preprint, version 1, posted March 16, 2020. Publisher Full Text\n\nZietz M, Tatonetti NP: Testing the association between blood type and COVID-19 infection, intubation, and death. medRxiv. preprint, version 1, posted April 11, 2020. Reference Source\n\nEllinghaus D, Degenhardt F, Bujanda L, et al.: The ABO blood group locus and a chromosome 3 gene cluster associate with SARS-CoV-2 respiratory failure in an Italian-Spanish genome-wide association analysis. medRxiv. preprint, posted June 2, 2020. Publisher Full Text\n\nEllinghaus D, Degenhardt F, Bujanda L, et al.: Genomewide Association Study of Severe Covid-19 with Respiratory Failure. Published on June 17, 2020, at New Engl J Med. 2020; NEJMoa2020283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLatz CA, DeCarlo C, Boitano L, et al.: Blood type and outcomes in patients with COVID-19. Ann Hematol. 2020; 99(9): 2113–2118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRawlinson C, Bloom T: New preprint server for medical research. BMJ. 2019; 365: l2301. PubMed Abstract \| Publisher Full Text\n\nZietz M, Tatonetti NP: Testing the association between blood type and COVID-19 infection, intubation, and death. medRxiv. preprint, version 2, posted July 21, 2020. Reference Source" }	[ { "id": "71681", "date": "25 Sep 2020", "name": "Charles Bangham", "expertise": [ "Reviewer Expertise Virology", "immunology", "genetics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Bullerdiek makes some cogent criticisms of the rapid publication of preprints, with specific reference to a recent publication on the association between ABO blood groups, SARS-CoV-2 infection and the resulting disease, COVID-19. He concludes by proposing that additional rules should be observed by the authors of preprints. Specifically, three rules are suggested:\n\nA time of some weeks should be given to the extended group of peers to comment on and vice versa;\n\nauthors should communicate possible comments along with the manuscript and they should declare that they have read and considered all these comments carefully; and\n\nduring that time, there should not be any press releases from the authors.\nComments\nThe main purposes of publishing a preprint are to make findings widely known without delay; to solicit responses and critical appraisal; and to establish the authors' priority in a competitive field. There is little logic, on any of these grounds, in publishing a preprint a very short time (two weeks, as in the present instance) before the print version. I concur with Bullerdiek on this principle.\n\nThe publication of a preprint increases the normal responsibilities on the authors to be accurate and, in particular, to respond appropriately to criticism both in the final 'print' version and also in revised preprints if serious issues are identified or if there will be a long delay before the print version is published.\n\nIt is impossible to foresee, and still less to prevent, the distortion or misinterpretation that may follow the publication of a scientific paper. However, in circumstances of exceptionally wide and intense public interest, such as in the present coronavirus pandemic, it is proportionally more important to make public announcements such as press releases that are worded cautiously, in such a way as to minimize the chance of exaggeration or misinterpretation by others, especially in the popular press.\n\nGenetic association studies, by definition, can identify associations but cannot identify the mechanism of any observed association. In the present case of the preprint on ABO blood groups and SARS-CoV-2 infection by Ellinghaus, Degenhardt et al., there is a stark difference between two potential causes of the observed association. That is, ABO blood group might influence either the risk of acquiring SARS-CoV-2 infection, or the severity of a resulting illness, COVID-19; these are not mutually exclusive possibilities, and both might be true. The broad conclusion of the Ellinghaus, Degenhardt et al preprint, that ABO blood group influences the risk of COVID-19, is consistent with recent publications from several other groups, but it is not yet certain whether the association is due to an effect on acquisition of infection or on the pathogenesis of the resulting disease. The recently revised preprint by Zietz and Tatonetti, cited by Bullerdiek, favours the notion that the primary effect of ABO blood group in SARS-CoV-2 infection is on the risk of acquisition of the infection, not on disease severity. Further studies are needed to identify potential mechanisms, in particular of the apparent protective effect of blood group O.\n\nWhat, then, of the proposed additional rules for the publication of preprints, listed above? I believe that the first two should be strongly recommended, but it is difficult and inappropriate to mandate strict numerical limit on the time between preprint and final publication of a peer-reviewed paper. However, I believe that press releases should not be forbidden: the authors remain responsible for the decision whether it is appropriate and helpful to make a press release, and for the content and style of any release.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "71682", "date": "08 Oct 2020", "name": "Laura Cooling", "expertise": [ "Reviewer Expertise Transfusion Medicine", "Immunohematology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 highlights the conundrum that preprint servers can play in bypassing the formal publication peer review process. The particular case study focuses on two papers listed on medical preprint servers that asserted an association between ABO blood groups and CoV-SARS-2 incidence and/or morbidity. The conclusions from these two papers (and an online study by 23andme) were widely discussed in the lay press, often with dramatic tabloid headlines (see Table 1). The “Elephant in the Room” is the author’s increased scientific and public responsibility for the information on the preprint server—even after manuscript acceptance. The subsequent formal peer review and later studies subsequently disclosed problems with the control groups (blood donors and not COVID patients).\n\nComments: Bullerdiek raised several issues that arose from institutional press announcements regarding a paper that was listed on preprint server but not formally accepted. The author’s institution also bears responsibility for the public perception the preprint was a peer-reviewed, accepted findings. It sadly demonstrates the need for public institutions to aggressively “market” science discoveries in the lay press.\n\nIt is surprising that scientific journals have not pushed back at the listing of manuscripts on pre-print servers. In these two instances, the preprint server acted to undermine the regular peer review process. All journals require that authors state that the submitted manuscript is NOT under consideration by other journals. In the cited example, the authors effectively had a dual submission to NEJM and the preprint server because of public interest, which was driven by institutional marketing. As Bullerdiek exposed, the time between preprint listing and NEJM acceptance was a mere two weeks. This obviated any insight or useful criticism from a preprint listing.\n\nI have personal insight into these two papers and was contacted by multiple scientific and lay press organizations. In addition, I received emails from scared citizens for weeks, wondering if they needed to have their blood typed. Although I refused to comment on the articles as preprints, I did offer some opinions and skepticism once the European GWAS article was accepted by NEJM. My skepticism was based on the control groups, as well as the US experience, in which COVID19 appeared worse among African Americans, who have a higher incidence of group O than whites. As noted by Bullerdiek, subsequent US studies found no association between ABO and COVID19.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1139
https://f1000research.com/articles/9-1137/v1	16 Sep 20	{ "type": "Data Note", "title": "The genome of the American groundhog, Marmota monax", "authors": [ "Daniela Puiu", "Aleksey Zimin", "Alaina Shumate", "Yuchen Ge", "Jiabin Qiu", "Manoj Bhaskaran", "Steven L. Salzberg", "Daniela Puiu", "Aleksey Zimin", "Alaina Shumate", "Yuchen Ge", "Jiabin Qiu", "Manoj Bhaskaran" ], "abstract": "We sequenced the genome of the North American groundhog, Marmota monax, also known as the woodchuck. Our sequencing strategy included a combination of short, high-quality Illumina reads plus long reads generated by both Pacific Biosciences and Oxford Nanopore instruments. Assembly of the combined data produced a genome of 2.74 Gbp in total length, with an N50 contig size of 1,094,236 bp. To annotate the genome, we mapped the genes from another M. monax genome and from the closely related Alpine marmot, Marmota marmota, onto our assembly, resulting in 20,559 annotated protein-coding genes and 28,135 transcripts. The genome assembly and annotation are available in GenBank under BioProject PRJNA587092.", "keywords": [ "genome assembly", "groundhog", "woodchuck", "genome annotation" ], "content": "Introduction\n\nGroundhogs (Marmota monax), also known as woodchucks, belong to the same family of ground squirrels as the alpine marmot, Marmota marmota. Groundhogs are found throughout the eastern United States and across much of Canada. They are small, ground-dwelling rodents that weigh ~4 kg as adults.\n\nThe woodchuck is of interest to biomedical science as a model for Hepatitis B virus (HBV) infection in humans, due to endemic infections of woodchucks with woodchuck hepatitis virus (WHV), which is genetically similar to human HBV and causes a similar course of infection1. Unlike some animal models of hepatocellular carcinoma (HCC) that require immunocompromised animals, woodchucks can develop HCC spontaneously after WHV infection. This propensity makes the woodchuck a promising model of HBV-induced hepatocellular carcinoma in humans. This in turn motivated our efforts to sequence, assemble, and annotate its genome.\n\n\nDNA isolation\n\nDNA was collected from a healthy, wild-caught adult male woodchuck (WC2) captured in 2016 near Ithaca, New York by Northeastern Wildlife, Inc. The gDNA was isolated from the left medial lobe of the liver from animal WC2. All DNA used for sequencing came from the same animal.\n\n\nResults\n\nWe generated 3.17 billion paired, 150-bp Illumina reads, for a total of 951 Gbp or approximately 390X genome coverage. We generated 32 million reads using Pacific Biosciences sequencing technology, of which 2.59 million were at least 10,000 bp long. The long PacBio reads contained 42.0 Gbp and had an N50 length of 16,554 bp. We also generated 6.4 million Oxford Nanopore (ONT) reads, of which 1.57 million were at least 10,000 bp long. The long ONT reads totaled 22.2 Gbp and had an N50 length of 13,815 bp. We then assembled the Illumina reads, the PacBio 10Kb+ reads, and the ONT 10Kb+ reads using MaSuRCA v3.2.72.\n\nThe resulting assembly, Woodchuck_1.0, consists of 8,860 contigs containing 2,737,034,741 bp, with an N50 contig size of 1,094,236. We compared our assembly to a recently published assembly of another woodchuck from the same species, GenBank accession GCA_901343595.13. That assembly (MONAX5) was generated entirely from Illumina reads, and it has a total length of 2,552,052,516 bp in 48,534 scaffolds, with a scaffold N50 of 892 kb and a contig N50 of 74,495 bp. The earlier assembly is thus ~185 Mbp shorter than Woodchuck_1.0.\n\nWe aligned all contigs and scaffolds between the two assemblies, and found that 3791 scaffolds in MONAX5 were contained within longer contigs in Woodchuck_1.0, with an average identity of 99.24%. In contrast, only 84 contigs from Woodchuck_1.0 were contained in MONAX5 scaffolds, consistent with the much larger contig sizes in our assembly.\n\nWe mapped the annotation from MONAX5 to Woodchuck_1.0 using Liftoff4. To assign functions to the mapped transcripts, we aligned them to transcripts annotated in the Alpine marmot (M. marmota, GenBank accession GCA_001458135.15. This yielded 20,559 protein-coding genes with 28,135 transcripts (including alternative splice variants). 10,664 of the genes were assigned functions based on near-identical matches with the Alpine marmot annotation, and the rest were labeled as hypothetical proteins. The average transcript contains 7.9 exons.\n\n\nData availability\n\nData from Marmota monax is available at NCBI under BioProject PRJNA587092, including the assembly with annotation at GenBank accession WJEC00000000, and the read data in the Sequence Read Archive under the same BioProject. The assembly and annotation are also available at ftp://ftp.ccb.jhu.edu/pub/data/Groundhog.", "appendix": "References\n\nMenne S, Cote PJ: The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World J Gastroenterol. 2007; 13(1): 104–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimin AV, Puiu D, Luo MC, et al.: Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm. Genome Res. 2017; 27(5): 787–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlioto TS, Cruz F, Gomez-Garrido J, et al.: The Genome Sequence of the Eastern Woodchuck (Marmota monax) - A Preclinical Animal Model for Chronic Hepatitis B. G3: Genes, Genomes, Genetics. 2019; 9(12): 3943–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShumate A, Salzberg SL: Liftoff: an accurate gene annotation mapping tool. bioRxiv. 2020. Publisher Full Text\n\nGossmann TI, Shanmugasundram A, Borno S, et al.: Ice-Age Climate Adaptations Trap the Alpine Marmot in a State of Low Genetic Diversity. Curr Biol. 2019; 29(10): 1712–1720.e7.PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71457", "date": "09 Nov 2020", "name": "Markus Ralser", "expertise": [ "Reviewer Expertise Metabolism" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Puiu et al. announces an improved, better annotated, and more complete genome of the American groundhog (M. monax), a ground squirrel species. The authors justify the importance of the genome as a model for Hepatitis infections, which sounds plausible. Obviously, such a genome is also important for other disciplines like evolutionary genetics, and as Marmots are exquisitely niche adapted species, a better understanding of their biology helps to understand problems of global importance, i.e. climate change, which warrants highlighting.\nThe paper is well written and technically of high quality. I have some questions concerning the relationship to the annotated M. monax genome deposited in GenBank (MONAX5), which the authors use as comparison of their genome quality. The authors conclude that their genome is more complete than the deposited genome. As the better quality is a key aspect of this submission, the manuscript would profit to disentangle the relative contribution of technical differences (e.g. due to novel sequencing technologies and assembly strategies) and biological differences (e.g. species variation) or what the relative contribution of each of the parts are.\n\nAlso, the improved genome annotation builds upon comparative mapping of the Alpine marmot and the deposited M. monax genome - meaning that “novel” regions in the new assembly are not covered in a similar quality (potential annotation bias). Also the genome assemblies’ DNA stems from the same individual, which means that the authors may want to include information on heterozygosity to conclude how representative the chosen individuals’ genome is of the species.\nMinor remark: I do not think that a 4 kg rodent would be referred to as “small”; that perception may come from a comparison to some other marmots, but it may feel different in the context of other rodents.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "74309", "date": "20 Nov 2020", "name": "Mihai Pop", "expertise": [ "Reviewer Expertise Genomics", "genome assembly." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the sequencing, assembly, and annotation of the groundhog genome. Overall the manuscript is clearly written and provides a good level of detail about the experimental setup and validation.\nIt would be beneficial for the readers if full details of the analytical pipeline (including specific parameters used and quality control/trimming steps) were made available either as supplementary material or through some other online platform (e.g. the authors' FTP server).\nGiven the substantial investment in the sequencing of the genome I am surprised that the project did not also generate RNA-seq data from the same animal. Since the rationale for this project is to support the use of the groundhog as a model organism for HCC, it's likely that animals are sacrificed during research making available a range of tissues from which a fairly comprehensive picture of the transcriptome can be gleaned.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6144", "date": "26 Nov 2020", "name": "Steven Salzberg", "role": "Author Response", "response": "Response to reviewer 2: we produced the assembly using the default parameters for MaSuRCA 3.2.7, utilizing ~120x coverage from Illumina reads (a subset of the full data set) and all of the PacBio (15x) and Nanopore (8x) reads. Trimming and error correction steps are built into the MaSuRCA pipeline. The long reads were all labeled as the “NANOPORE” type for input to MaSuRCA. Our annotation process used the default settings for the Liftoff program." } ] } ]	1	https://f1000research.com/articles/9-1137
https://f1000research.com/articles/9-1136/v1	16 Sep 20	{ "type": "Research Article", "title": "Green tea influence on iron overload in thalassemia intermedia patients: a randomized controlled trial", "authors": [ "Hayder Al-Momen", "Hussein Khudhair Hussein", "Zaid Al-Attar", "Mohammed Jalal Hussein", "Hussein Khudhair Hussein", "Zaid Al-Attar", "Mohammed Jalal Hussein" ], "abstract": "Background: Although iron chelation therapies have been available for many years for thalassemia intermedia patients, iron accumulation remains the major cause of death. Therefore, the need for additional chelation options is in demand. This randomized controlled study aimed to understand the effects of green tea on iron balance in thalassemia intermedia patients. Methods: Using a random selection method, 141 thalassemia intermedia patients were initially screened for inclusion in this trial; only 68 patients included after applying exclusion criteria. Two equal groups were generated (n=34/group): green tea (three cups/day after meals) + usual treatment (deferasirox iron chelator and on demand blood transfusion); and control (only usual treatment). The study lasted for a period of 12 months. Patients failing to comply to the trial methodology were excluded, leaving a final total of 29 patients in the green tea group and 28 patients in the control group. Liver iron concentration, and serum ferritin were assessed at baseline and 12 months, while hemoglobin levels were assessed monthly. Results: At baseline, both groups were matched regarding general demographics. At 12 months, the net drop of liver iron concentration in the green tea group (7.3 mg Fe/g dry weight) was significantly higher than the control group (4.6 mg Fe/g dry weight) (p<0.05). This was also seen with serum ferritin; net reduction in green tea and control groups were 1289 ng/ml and 871 ng/ml, respectively (p<0.05). Hemoglobin levels were slightly higher in the green tea group compared with the control group, but this was not significant. Conclusions: Regular green tea consumption had a significant capability to improve iron deposition in thalassemia intermedia patients who already undergo deferesirox iron chelation therapy. Trial registration: UMIN-CTR Clinical Trials Registry, UMIN000040841 (retrospectively registered June 21, 2020).", "keywords": [ "Deferasirox", "Liver iron concentration", "Serum ferritin", "Thalassemia." ], "content": "Introduction\n\nThalassemia intermedia (TI) is part of the thalassemia syndromes, which are the commonest genetic disorders worldwide, and is a member of the non-transfusion-dependent thalassemia (NTDT) group. By definition, β-thalassemia intermedia is a hemoglobin disorder characterized by a reduction in the expression of the β-globin gene1,2. Iron overload in TI has more than one cause, the most important is increased gut absorption. In addition, ineffective erythropoiesis and hemolysis may increase the need for blood transfusions with the eventual result of iron overload3,4. High morbidity rates usually accompany iron accumulation in NTDT and TI and has a significant impact on free radicals and oxidative stress tissue damage. Iron chelators are the best solution to alleviate these pathologies5,6.\n\nDeferasirox is the main oral iron chelation therapy according to international guidelines, but it is not free from side effects, leading to issues surrounding compliance, resulting in excessive body iron. Accordingly, recent research aims to assist, improve, or even substitute the use of iron chelating drugs using certain plants7,8. The common tea plant Camellia sinensis has been utilized globally as a tea drink in various subtypes based on the manufacturing process: black (fermented), green (non-fermented), and oolong (semi-fermented). Tea belongs to the group of common beverages worldwide and it is considered as the number one beverage (after water) in Iraq and some other parts of the world9,10.\n\nRegarding green tea (GT), polyphenols are the main ingredient, representing 24–36% of dry weight. Catechins are the mainstay of these polyphenol compounds, which have inhibitory effects on iron (heme and non-heme) absorption across gut cells. This may lead to extraordinary negative iron balance in humans who consume GT as part of their daily diet11,12.\n\nConsequently, this randomized controlled trial aimed to assess the iron accumulation levels in TI patients treated with deferasirox with additional GT intake using serum ferritin (SF) and liver iron concentration (LIC). Also, hemoglobin (Hb) levels were assessed regularly.\n\n\nMethods\n\nThis is a randomized controlled (parallel-group; 1:1) study performed prospectively at the Baghdad Hereditary Anemia Center at Ibn Al-Baladi Hospital in Baghdad, Iraq, which is the largest thalassemia center in the country. The study was conducted over a period from 1st March, 2019 until 29th February, 2020.\n\nAll β- thalassemia intermedia patients aged ≥10 years old who were regularly managed by the Baghdad Hereditary Anemia Center were entered in a baseline screen. No sample size was calculated as all patients were considered.\n\nPatients with chronic illnesses such as diabetes mellitus, heart failure, asthma, renal failure, liver disease, and any hematological problem other than thalassemia, were excluded from the study. Patients that failed to have full compliance with blood transfusion and iron chelation therapy according to physicians’ instructions in the last three months before baseline recruitment were also excluded.\n\nAt the initial screening, 141 patients were eligible. In total, 73 patients were excluded as they did not meet the inclusion criteria, leaving 68 patients who were randomly divided into two groups with matched age, sex, and BMI. Patients were asked to take part in the study during routine check-up appointments.\n\nIron overload status through measurements of SF and LIC at baseline and end of the study period, in addition to regular monthly assessment of Hb levels.\n\nThe patients were randomly divided into two groups (n=34/group): GT group and control group.\n\nGT groups: Patients in the GT group were instructed to drink a cup of GT after meals (within an hour) three times daily. Patients were given GT tea bags from Lipton™ containing 2 grams of GT (Camellia sinensis), which was collected from Kenya and Indonesia and made in January 2019, in Jebel Ali, United Arab Emirates by Unilever Gulf FZE (P.O. Box 17055, Dubai). Patients were instructed to infuse the tea bag in a hot cup of water (150°C) for 2–3 minutes, according to the manufacturer’s instructions. Patients were undergoing deferasirox treatment and blood transfusion (when needed as decided by the attending physician) as their usual treatment.\n\nControl group: Patients in the control group were instructed drink one cup or more of either warm or cold drinking water after each meal, and to avoid any beverage other than water for three hours after each meal daily. Patients were undergoing deferasirox treatment and blood transfusion (when needed as decided by the attending physician) as their usual treatment.\n\nPatients of both groups were asked to report their daily meals through a phone call (twice a week) to ensure low-iron food consumption, which might be considered as a confounding factor. Follow-up visits were performed at least every month with the patients attending physician.\n\nDuring the study interval (12 months), participants failing to follow these instructions for three days a month (a total of nine drinking events during 30 days, which represents 10%) were excluded from analysis.\n\nSimple randomization was performed to allocate participants into GT and control groups in an equal pattern with matched age, sex, and BMI. Researchers have given members of each group a unique sequential number which was applied to all laboratory blood samples and MRI results. The researchers have enrolled and assigned participants to GT and control groups. The attending physicians and staff of the laboratory and MRI units were blinded to the study groups.\n\nFull medical history and examination with the decision for blood transfusion requirement and iron chelation therapy (deferasirox 20–40 mg/kg/day) were made by the attending physician during each follow-up visit (at least once a month) during the study period. Body mass index (BMI (kg/m2)) was calculated at the start of the study for all patients. Also, packed red blood cells (RBC) transfusion volume during the previous year, calculated just before the start of study period, was estimated for all participants.\n\nThe following measurements were taken at baseline and 12 months for all patients:\n\n- Serum ferritin (SF) readings (ng/ml), determined using a MINI VIDAS analyzer (bioMerieux S.A., Lyon, France) through an electrofluorescence assay method;\n\n- Liver iron concentration (LIC) values (mg Fe/dry gram weight), determined using R2 magnetic resonance imaging (MRI; Siemens MRI machine with 3 Tesla and Medis suite software);\n\n- Hemoglobin levels (g/dl) (documented monthly), determined using microprocessor hemoglobin meter (model number LE182, and manufactured by GPC Medical Ltd.)\n\nData are expressed as mean±standard deviation (SD) unless otherwise indicated. Shapiro-Wilk test was used for normal distribution. Pearson's chi-square test (for discrete or nominal variables) or Student t-tests (for continuous variables) were used to compare the characteristics between GT and control groups. When p < 0.05, results were assumed to be significant. SPSS 22.0 for Windows (IBM Corp., NY, USA) was utilized in the analysis.\n\nEthical approval was obtained from Ethical and Scientific Committee of Al-Kindy College of Medicine at University of Baghdad (No.: 367). Baghdad Hereditary Anemia Center is affiliated with Al-Kindy College of Medicine at University of Baghdad. Written informed consent for participation was taken from all participants or their caregivers/legal guardians for those aged <18 years. All work was done according to the most recent version of the Declaration of Helsinki. The trial was registered retrospectively on UMIN-CTR Clinical Trials Registry UMIN000040841 (June 21, 2020) according to the University of Baghdad recommendations.\n\n\nResults\n\nDue to non-compliance, the GT group had 29 patients (five patients excluded) and the control group had 28 patients (six patients excluded) at the end of the study (Figure 1).\n\nThe two groups had comparable (non-significant) general demographic data at baseline (Table 1). Iron overload (measured by SF or LIC) and volume of packed red blood cells (RBC) transfusion during the previous year calculated just before the start of study period showed no significant differences between groups. Deferasirox mean doses for the GT (36.89±7.24 mg/kg/day) and control (37.21±1.13 mg/kg/day) groups were high and near to the maximum approved doses, but were not significantly different between the groups.\n\na: Student t-tests, b: Pearson's chi-square test.\n\nLIC was significantly reduced (P<0.05) after 12 months in both the GT group (baseline, 19.4±2.3 Fe/gram/dry weight; 12 months, 12.1±1.3 Fe/gram/dry weight), and control groups (baseline, 19.8±1.7 Fe/gram/dry weight; 12 months, 15.2±1.6 Fe/gram/dry weight) (Figure 2). Similarly, SF levels were significantly decreased at the end of study period (P<0.05) in the GT (baseline, 2617±549 ng/ml; 12 months, 1328±127 ng/ml) and control (baseline, 2684±164 ng/ml; 12 months, 1813±235 ng/ml) groups.\n\nDeferasirox use was comparable at the start and end of the study period for both the GT (baseline, 36.89±7.24 mg/kg/day; 12 months, 36.24±6.83 mg/kg/day) and control (baseline, 37.21±1.13 mg/kg/day; 12 months, 37.15±3.78 mg/kg/day) groups.\n\nThe mean volume of transfused packed RBC during the study period was estimated after 12 months from the baseline and revealed no statistical significance between GT (70.64±7.84 ml/kg) and control (72.14±8.35 ml/kg) patients. The net reduction of LIC (Fe/gram/dry weight) in GT patients was 7.3 (baseline, 19.4; 12 months, 12.1), while for control patients the net result was 4.6 (baseline, 19.8; 12 months, 15.2). A significant reduction (P<0.05) of LIC was found in the GT group in comparison with the control cases. The drop of serum ferritin in GT patients after 12 months [1289 ng/ml (2617-1328)] was significantly (P<0.05) higher than that of the control group [871 ng/ml (2684-1813)] (Table 2).\n\na: Student t-tests.\n\nHemoglobin readings gradually increased in both the GT and control groups over the study period. GT patients had higher Hb levels after 12 months than the control group (8.4 versus 8.2), but this was not statistically significant (P=0.37) (Figure 3).\n\n\nDiscussion\n\nIron overload is a major consequence of TI. Physicians have several challenges when using medications that eliminate excessive iron, including type, dose, expected adverse effects, and the best time to start an iron chelator. Therefore, the need for a naturally mediated iron chelator based on plants is increasing along with the increase of thalassemia patients around the world13–15. Green tea (GT) seems to be one of the best available options due to its natural properties to counteract iron absorption in addition to other promising advantages16,17.\n\nThere are several ways to estimate the overload of iron in thalassemia patients, such as the widely available test of SF, and the most accurate non-invasive recent method of LIC using the R2 MRI technique18,19.\n\nPatients with TI usually have high iron content loading in their tissues as evidenced by other studies from different areas of the world13,20,21. Our sample of patient had even higher iron content, which is in line with other reports from Middle East3,14. This might be due to compliance deterioration, background cultural beliefs, possible side effects of iron chelating drugs, and the frequent use of mimic medications in this part of the world22.\n\nGT consumption in our sample was associated with decreased ferritin levels mirroring better iron chelation. This was also noticed by other works done on both humans and animals with transfusion dependent thalassemia23–25. SF could be affected by the antioxidant properties of GT26,27. However, a recent study that added curcumin to GT for thalassemia patients discovered a slight increase of SF after two months, with the reasons for this unknown to the authors28.\n\nA more sophisticated method of iron accumulation assessment in TI is LIC, and in this study, we found a significant drop in the GT group compared to the control group. This has only been previously reported in animal studies, and an online search conducted by the present authors failed to reveal human trials15. It should be noted that iron deficiency has been documented in healthy individuals consuming large amounts of GT29.\n\nSurprisingly, in the GT group, TI patients developed slightly higher hemoglobin readings than controls. Again, no previously published data was found to verify this, but it might be related to better iron removal and effects of antioxidant characteristics of GT, which could be associated with improved hematopoiesis and less red blood cells hemolysis. However, GT is historically associated with anemia in a healthy population, especially in high doses30.\n\nAlthough this trial was conducted in the largest thalassemia center within Iraq country borders, its small sample size could be considered as a limitation. We recommend other interested scientists throughout the world to involve more patients with more variables and risk factors to deeply investigate the effects of GT in TI and discover more plant-based iron chelation therapies with better effects.\n\n\nConclusion\n\nThalassemia intermedia patients having usual medical care, including blood transfusion and deferasirox oral iron chelation therapy, have significantly superior levels of iron chelation and slightly better hemoglobin levels when consuming GT regularly compared with a control group.\n\n\nData availability\n\nMendeley Data: Green tea influence on iron overload in thalassemia intermedia, http://dx.doi.org/10.17632/p7558gcrmw.231.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nMendeley Data: CONSORT checklist for ‘Green tea influence on iron overload in thalassemia intermedia patients: a randomized controlled trial’, http://dx.doi.org/10.17632/9c5bx4p65c.132.", "appendix": "Acknowledgements\n\nMany thanks for the enormous encouragements, and great deal of support offered by the medical and management staff of Baghdad Hereditary Anemia Center at Ibn Al-Baladi Hospital.\n\n\nReferences\n\nBen Salah N, Bou-Fakhredin R, Mellouli F, et al.: Revisiting beta thalassemia intermedia: past, present, and future prospects. Hematology. 2017; 22(10): 607–16. PubMed Abstract \| Publisher Full Text\n\nAsadov C, Alimirzoeva Z, Mammadova T, et al.: β-Thalassemia intermedia: a comprehensive overview and novel approaches. Int J Hematol. 2018; 108(1): 5–21. PubMed Abstract \| Publisher Full Text\n\nYassin MA, Soliman AT, De Sanctis V, et al.: Final height and endocrine complications in patients with β-thalassemia intermedia: our experience in non-transfused versus infrequently transfused patients and correlations with liver iron content. Mediterr J Hematol Infect Dis. 2019; 11(1): e2019026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVichinsky E: Non-transfusion-dependent thalassemia and thalassemia intermedia: epidemiology, complications, and management. Curr Med Res Opin. 2016; 32(1): 191–204. PubMed Abstract \| Publisher Full Text\n\nAl-Momen H: Iron chelation therapy in sickle cell/beta thalassemia syndrome, a 2 years’ extension study. Al-Kindy College Medical Journal. 2017; 13(1): 76–81. Reference Source\n\nKontoghiorghe CN, Kontoghiorghes GJ: Efficacy and safety of iron-chelation therapy with deferoxamine, deferiprone, and deferasirox for the treatment of iron-loaded patients with non-transfusion-dependent thalassemia syndromes. Drug Des Devel Ther. 2016; 10: 465–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaher AM, Al-Momen H, Jasim SK: Deferasirox in thalassemia: a comparative study between an innovator drug and its copy among a sample of Iraqi patients. Ther Adv Drug Saf. 2019; 10: 2042098619880123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoayedi B, Gharagozloo M, Esmaeil N, et al.: A randomized double-blind, placebo-controlled study of therapeutic effects of silymarin in β-thalassemia major patients receiving desferrioxamine. Eur J Haematol. 2013; 90(3): 202–9. PubMed Abstract \| Publisher Full Text\n\nJasim SK, Al-Momen H, Al-Asadi F: Maternal Anemia Prevalence and Subsequent Neonatal Complications in Iraq. Open Access Maced J Med Sci. 2020; 8(B): 71–5. Reference Source\n\nShafik SS, Rejah BK: Measurement of the uranium concentration in different types of tea used in Iraqi kitchen. Iraqi Journal of Science. 2014; 55(3A): 1039–43. Reference Source\n\nXing L, Zhang H, Qi R, et al.: Recent advances in the understanding of the health benefits and molecular mechanisms associated with green tea polyphenols. J Agric Food Chem. 2019; 67(4): 1029–43. PubMed Abstract \| Publisher Full Text\n\nNawab A, Farooq N: Review on green tea constituents and its negative effects. J Pharm Innov. 2015; 4(1): 21–4. Reference Source\n\nTaher AT, Porter JB, Viprakasit V, et al.: Defining serum ferritin thresholds to predict clinically relevant liver iron concentrations for guiding deferasirox therapy when MRI is unavailable in patients with non-transfusion-dependent thalassaemia. Br J Haematol. 2015; 168(2): 284–90. PubMed Abstract \| Publisher Full Text\n\nAl-Momen H, Jasim SK, Hassan QA, et al.: Relationship between liver iron concentration determined by R2-MRI, serum ferritin, and liver enzymes in patients with thalassemia intermedia. Blood Res. 2018; 53(4): 314–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaewong T, Ounjaijean S, Mundee Y, et al.: Effects of green tea on iron accumulation and oxidative stress in livers of iron-challenged thalassemic mice. Med Chem. 2010; 6(2): 57–64. PubMed Abstract \| Publisher Full Text\n\nChacko SM, Thambi PT, Kuttan R, et al.: Beneficial effects of green tea: a literature review. Chin Med. 2010; 5: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKe X, Qin N, Zhang T, et al.: Highly Augmented Antioxidant and Anticancer Effect of Biocompatible MIL-100 (Fe)@ SiO 2-Immobilized Green Tea Catechin. J Inorg Organomet Polym Mater. 2020; 30(3): 935–42. Publisher Full Text\n\nAl-Momen HH, Obed AA, Mahdi ZK, et al.: Isdeferasirox as Effective as Desferrioxamine in Treatment of Iron Overload in Patients with Thalassemia Major?\n\nBharadwaj NS, Jain R, Bhatia P, et al.: Assessment of Iron Overload and Role of Genetic Modifiers in Patients with Beta Thalassemia Intermedia. Pediatric Hematology Oncology Journal. 2019; 4(2): S39.\n\nSaliba AN, Musallam KM, Cappellini MD, et al.: Serum ferritin values between 300 and 800 ng/mL in nontransfusion-dependent thalassemia: A probability curve to guide clinical decision making when MRI is unavailable. Am J Hematol. 2017; 92(3): E35–7. PubMed Abstract \| Publisher Full Text\n\nSarhan MM, El Asmy AE, Mohamed GM: Magnetic Resonance Imaging Versus Serum Ferritin Levels in Detection of Liver and Cardiac Iron Overload in Non-Transfusion Dependent Thalassemia. Pediatr Therapeut. 2017; 7(03): 10–4. Publisher Full Text\n\nMattar M, Alwan AF, Boukhelal H, et al.: On the use of substandard medicines in hematology: An emerging concern in the Middle East and North Africa region. Eur J Intern Med. 2018; 48: e40–1. PubMed Abstract \| Publisher Full Text\n\nOunjaijean S, Thephinlap C, Khansuwan U, et al.: Effect of green tea on iron status and oxidative stress in iron-loaded rats. Med Chem. 2008; 4(4): 365–70. PubMed Abstract \| Publisher Full Text\n\nBadiee MS, Nili-Ahmadabadi H, Zeinvand-Lorestani H, et al.: Green tea consumption improves the therapeutic efficacy of deferoxamine on iron overload in patients with [Beta]-thalassemia major: a randomized clinical study. InBiological Forum. 2015; 7(2): 383. Research Trend. Reference Source\n\nSoeizi E, Rafraf M, Asghari-Jafarabadi M, et al.: Effects of Green Tea on Serum Iron Parameters and Antioxidant Status in Patients with β–Thalassemia Major. Pharm Sci. 2017; 23(1): 27–36. Publisher Full Text\n\nBou-Abdallah F, Paliakkara JJ, Melman G, et al.: Reductive mobilization of iron from intact ferritin: mechanisms and physiological implication. Pharmaceuticals (Basel). 2018; 11(4): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeluso I, Serafini M: Antioxidants from black and green tea From dietary modulation of oxidative stress to pharmacological mechanisms. Br J Pharmacol. 2017; 174(11): 1195–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoonyosying P, Tantiworawit A, Hantrakool S, et al.: Consumption of a green tea extract–curcumin drink decreases blood urea nitrogen and redox iron in β-thalassemia patients. Food Funct. 2020; 11(1): 932–943. PubMed Abstract \| Publisher Full Text\n\nFan FS: Iron deficiency anemia due to excessive green tea drinking. Clin case Rep. 2016; 4(11): 1053–1056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRani S, Rasyid R, Desmawati D: High intake of green tea decreased hemoglobin and hematocrit levels in Rattus novergicus strain wistar albino. International Journal of Research in Medical Sciences. 2018; 6(11): 3688. Publisher Full Text\n\nAl-Momen H, Hussein KH, Al-Attar Z, et al.: “Green tea influence on iron overload in thalassemia intermedia ”, Mendeley Data, v2, 2020. http://www.doi.org/10.17632/p7558gcrmw.2\n\nAl-Momen H, Al-Musawi H, Al-Attar Z, et al.: CONSORT Checklist for (Green tea influence on iron overload in thalassemia intermedia patients). Mendeley Data, V1, 2020. http://www.doi.org/10.17632/9c5bx4p65c.1" }	[ { "id": "71684", "date": "24 Sep 2020", "name": "Alaa Alwan", "expertise": [ "Reviewer Expertise benign and malignant hematology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is interested subject which has covered important issue in beta thalassemia.\n\nThe introduction is well covered with recent references.\n\nMaterials and method done appropriately especially with use of statistical methods and randomization.\n\nRegarding the results, they are clear and in my opinion it is obvious.\n\nThe discussion part is well written and it is good to see comparison with other studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71683", "date": "25 Sep 2020", "name": "Hassan Abolghasemi", "expertise": [ "Reviewer Expertise pediatric hematology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article the impact of green tea on reduction of liver iron concentration have been studied in a randomized clinical trials by authors. They showed liver iron was decreased in the case groups compared with control group significantly. The level of hemoglobin was not significantly changed at the end of trial. The trial was done appropriately. The only problem with the study was the level of ferritin at the start of trial which was higher than usual for thalassemia intermedia and the dose of Deferasirox. It seems that some of the patients were transfusion dependent.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1136
https://f1000research.com/articles/9-231/v1	02 Apr 20	{ "type": "Systematic Review", "title": "Supine versus prone position in percutaneous nephrolithotomy: a systematic review and meta-analysis", "authors": [ "Ponco Birowo", "William Tendi", "Indah S. Widyahening", "Nur Rasyid", "Widi Atmoko", "William Tendi", "Indah S. Widyahening", "Nur Rasyid", "Widi Atmoko" ], "abstract": "Background: The decision for using supine or prone position in percutaneous nephrolithotomy (PCNL) is still debatable. The aim of this study is to compare the efficacy and safety profile of the supine and prone position when performing PCNL. Methods: A systematic electronic search was performed using the database from MEDLINE, Cochrane library and Google Scholar from January 2009 to November 2019. The outcomes assessed were stone free rate, major complication rate, length of hospital stay and mean operation time. Results: A total of 11 articles were included in qualitative and quantitative analysis. The efficacy of PCNL in supine position as determined by stone free rate is significantly lower than in prone position (OR: 0.74; 95% CI: 0.66 – 0.83; p<0.00001), However, major complication rate is also lower in the supine group compared with the prone group (OR: 0.70; 95% CI: 0.51 – 0.96; p=0.03). There is no statistically significant difference in the length of hospital stay and mean operation time between both groups. Conclusion: Prone position leads to a higher stone free rate, but also a higher rate of major complication. Thus, the decision of using which position during PCNL should be based on the surgeon’s experience and clinical aspects of the patients.", "keywords": [ "Complication rate", "Percutaneous nephrolithotomy", "Prone", "Stone free rate", "Supine" ], "content": "Introduction\n\nNephrolithiasis is one of the most common urological diseases worldwide. It is defined as a condition where mineral deposits are found in the kidney, either free in the renal calyces and pelvis or attached on the renal papillae1. The prevalence is varied between regions, ranging between 7–13% in North America, 5–9% in Europe, and 1–5% in Asia2. The most common stone composition is calcium, comprising about 80% of all urolithiasis3.\n\nDepending on stone burden, the treatment of nephrolithiasis also has a wide range of options. Active management includes extracorporeal shockwave lithotripsy (ESWL), retrieval by ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL). The current guideline generally recommends ESWL for smaller stones (up to 20 mm) and PCNL for larger stones (>20 mm) regardless of the location inside the kidney4.\n\nWhile PCNL has higher free stone rates with a similar recurrence and complication rate compared with ESWL, this procedure also has its own preparation including a guiding system, anesthesia, and positioning of the patient. The conventional position of PCNL is prone, which allows direct access to the posterior calyx with minimal risk of bowel puncture. However, this positioning method limits the possibility of switching anesthesia from regional to general. The alternative position is supine, which allows general anesthesia switching and combination technique of antegrade and retrograde approaches. Moreover, this position is also more preferred in patients with cardiac comorbidity. However, working space and the possibility of multiple channels are limited5. The aim of this study is to determine whether one position is more superior than the other, by comparing efficacy and safety profiles using a systematic review and meta-analysis approach.\n\n\nMethods\n\nThe target population in this study is patients with renal stone of 20 mm or more in size who underwent PCNL. The intervention to the patients is PCNL in prone position, compared with PCNL in supine position. Prone is a classic position in PCNL procedure, described in 1976 when PCNL was first introduced. The original prone position consists of a two-stage procedure. The first stage is in supine position, where anesthesia is given and retrograde access to the upper urinary tract is established. The patient is then repositioned to a prone position, and supports are placed under the thorax and upper abdomen. All pressure points are also padded6.\n\nIn contrast, a supine prone only needs one stage, in which the patient is placed supine with ipsilateral flank held up with a 3-liter saline bag. This original position was first introduced by Valdivia-Uria et al. and has been modified over time7. One popular modification of Valdivia position is the Galdakao modification. This position is slightly more lateral; the contralateral leg of the patient is flexed and abducted, while the ipsilateral leg is extended. A 3-liter bag is also placed to raise the flank6.\n\nApart from the Valdivia position and its modifications, a complete supine position was also introduced by Falahatkar et al.8 This position does not require an elevation of the flank. The patient is simply put in a supine position at the edge of the table, with legs extended. The patient’s arms are stretched, abducted and supported.\n\nThe outcome of this study is the efficacy of both positions, determined by stone free rate and safety profile, determined by the occurrence of major complications.\n\nA systematic search was carried out with the date last searched in 14 February 2020, using the database from MEDLINE, with keywords of “(((supine[Title/Abstract]) AND prone[Title/Abstract])) AND ((PCNL[Title/Abstract]) OR percutaneous nephrolithotomy[Title/Abstract])”, and Cochrane library, with keywords of “prone in Title Abstract Keyword AND supine in Title Abstract Keyword AND PCNL in Title Abstract Keyword”, and Google Scholar with keywords of “prone AND supine AND percutaneous nephrolithotomy”. After we identified the articles, we removed the duplicates and further screened the articles. The reporting is based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) algorithm.\n\nTwo reviewers (PB and WT) independently appraised the articles, and a discussion was conducted when disagreement occurred. The relevance of the articles is determined by reading through the titles and abstracts. The inclusion criterion is a comparative study between the supine and prone position in PCNL procedure, and the articles were written in English. The exclusion criteria are non-comparative studies, studies that combine PCNL with other techniques of stone extraction such as URS or retrograde intrarenal surgery, not focused on comparing supine and prone position in PCNL, and inclusion of confounding factors such as a difference in guiding method when performing PCNL in each position, since this difference will lead to intervention bias. The quality of each article included were then tested using Jadad scale for randomized controlled trials (RCT) and Newcastle-Ottawa scale for non-RCTs9,10.\n\nData extraction from the articles was performed by two authors (NR and WA), and any disagreement was settled by consensus. The variables extracted from the articles included the first author’s name, year of publication, stone free rate, percentage of major complications, length of hospital stay, and mean operation time. Stone free condition is defined as the absence of residual fragments of ≤ 4 mm after procedure. Major complications are defined as those with a Clavien score of III or more11.\n\nMeta-analysis was performed by Review Manager 5.3. The results were described as odds ratio (OR) with 95% confidence interval (CI) for dichotomous variables, and as a mean difference with 95% CI for continuous variables. Heterogeneity was analyzed using a Chi square and I2 test. The data was analyzed using the random-effect model when I2 >25%, and fixed-effect model when I2 is less than 25%. The analysis is considered statistically significant when p value is less than 0.05. For studies that provided the minimum and maximum value instead of standard deviation (SD) for the mean difference analysis, estimated SD were calculated with the formula derived from a study by Walter and Yao (2007)12. In addition, for studies that provided 95% Confidence Interval (CI) instead of SD, the value of SD was calculated using the formula described in the Cochrane Handbook13.\n\n\nResults\n\nFollowing the result of article screening and the application of exclusion criteria, a total of 156 articles were found from the three databases. After removing duplicates, a total of 131 studies were screened for relevance, of which only 11 articles were included in qualitative and quantitative analysis (Figure 1).\n\nQuality assessment of the articles is shown in Table 1. Study characteristics, including the study design, mean age, and stone burden, is shown in Table 2.\n\nRCT, randomized controlled trial.\n\nAll 11 studies reported the stone free rate of both supine and prone groups. A meta-analysis of these studies showed that there was a statistically significant lower stone free rate in the supine group (OR: 0.74; 95% CI: 0.66 – 0.83; p<0.00001; Figure 2).\n\nMajor complication rate is defined as Clavien score of 3 of more in this study. There were only 5 articles that reported the complication rate using Clavien score. Figure 3 showed that there is a statistically significant lower complication rate in the supine group (OR: 0.70; 95% CI: 0.51 – 0.96; p=0.03).\n\nThere were nine studies that reported mean days of hospital stay in both groups. The forest plot in Figure 4 shows that there is no difference in the length of hospital stay between groups (Mean difference: -0.01; 95% CI: -0.27 – 0.24; p=0.92).\n\nMean operation time was reported in all studies. The meta-analysis in this parameter showed that there is no difference in mean operation time between these two groups (Mean difference: -2.68; 95% CI: -12.36 – 7.00; p=0.59; Figure 5).\n\n\nDiscussion\n\nAccording to our review, supine and prone position during PCNL share a similar mean operation time and duration of hospital stay. This result is important so that the surgeons will be able to confidently decide the position based on their experiences and the patient’s comorbidities. In addition, the study conducted by Melo et al. showed similarities between both positions in terms of the number of puncture tracts, blood transfusion rate, and mean drop in hemoglobin level11.\n\nHowever, despite the similarities, this study also found significant differences between these two groups. The authors choose stone free rate and major complication as the main outcome of this article to help identify which position is safe in PCNL and whether there is a difference in the efficacy. Interestingly, both of these outcomes were statistically different.\n\nOur study found that the supine position had a lower major complication rate than prone position. Literature revealed that the original (Valdivia) position is reportedly safe, and endoscopic instruments can be moved more freely because the puncture site of the abdominal wall is performed more laterally and away from the lumbar muscles. The tract in this position also preserves a low pressure in the renal pelvis, reducing the risk of fluid absorption. Moreover, risk of colonic puncture might be reduced because the bowel is not pressed towards the kidney. Should a rigid ureteroscopy be needed simultaneously with PCNL, a modified Valdivia position can be performed by flexing and supporting the patient’s ipsilateral leg, and the contralateral leg descended. The supine position also has the advantage of easier management of cardiac and respiratory emergencies6.\n\nMoreover, the Galdakao-modified position allows more instrument manipulation than the original supine position. Furthermore, it also enables simultaneous retrograde access to the kidney and there is no need to reposition thus the asepsis and antisepsis procedure needs to be performed only once6.\n\nIn the complete supine position, the lack of flank support allows more feasible access to the upper pole of the kidney because there is no risk of cephalad sliding of the kidney, as observed in the supine position with flank support6. The supine position also has the advantages of easier access to the upper pole after lower pole puncture23.\n\nHowever, it should also be noted that while there are many advantages to the supine position, the flank in this position is not fully exposed, therefore reducing the possibility of multiple access when needed. In addition, the state of low compressed abdomen allows the kidney to move more freely, making the navigation of the instrument towards the kidney more challenging, and the chance of failed access is higher6,7,16.\n\nAdditionally, our study found that stone free rate was significantly higher in prone position. The major advantage in this position is the fully exposed lumbar area. This allows a possibility of several puncture sites, and easier access to the upper pole kidney. Moreover, the working area is greater, providing enough space for instrument manipulation6.\n\nHowever, the two-stage nature of this position usually prolongs the operating time, and a prone position makes it difficult for the anesthetists to attend cardio-respiratory emergency. The risk of ocular complications has also been described because of the increase in intra-ocular pressure6.\n\nThe limitation in our study is that the number of articles providing data of major complication rate in terms of Clavien score was limited and there were too many heterogeneities in the length of hospital stay and mean operation time variables. Therefore, the authors believe that another comprehensive study should be performed in urology centers in which the surgeons excel in both supine and prone position when performing PCNL and have a larger sample size.\n\nThe implication of this study is that it exposed the benefit and disadvantages of both supine and prone position, which in turn can be used as a decision guide for clinicians who want to perform PCNL.\n\n\nConclusion\n\nIn conclusion, the prone position leads to a higher stone free rate than supine position. However, in terms of safety profile, supine position provides a better choice than the prone position. There is no difference in both the length of hospital stay and mean operation time between prone and supine position. Therefore, it can be inferred that there is no position that has absolute superiority and it is important to note that both supine and prone position in PCNL procedure have their respective advantages and disadvantages. Thus, the decision of choosing the position when performing PCNL should be based on clinical status of the patient and the experience of the surgeon.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: PRISMA checklist for article ‘Supine versus prone position in percutaneous nephrolithotomy: a systematic review and meta-analysis’, https://doi.org/10.17605/OSF.IO/23GND24.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors would like to thank you to the Cipto Mangunkusumo National Hospital for the support and permission for the authors to finish the production of this article.\n\n\nReferences\n\nKhan SR, Pearle MS, Robertson WG: Kidney stones. Nat Rev Dis Primers. 2016; 2: 16008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSorokin I, Mamoulakis C, Miyazawa K, et al.: Epidemiology of stone disease across the world. World J Urol. 2017; 35(9): 1301–20. PubMed Abstract \| Publisher Full Text\n\nAlelign T, Petros B: Kidney Stone Disease: An Update on Current Concepts. Adv Urol. 2018; 2018: 3068365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurk C, Skolarikos A, Neisius A, et al.: EAU Guidelines on Urolithiasis. The Netherlands: EAU Guidelines Office; 2019.\n\nGanpule AP, Vijayakumar M, Malpani A, et al.: Percutaneous nephrolithotomy (PCNL) a critical review. Int J Surg. 2016; 36(Pt D): 660–664. PubMed Abstract \| Publisher Full Text\n\nKaraolides T, Moraitis K, Bach C, et al.: Positions for percutaneous nephrolithotomy: Thirty-five years of evolution. Arab J Urol. 2012; 10(3): 307–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValdivia JG, Scarpa RM, Duvdevani M, et al.: Supine versus prone position during percutaneous nephrolithotomy: a report from the clinical research office of the endourological society percutaneous nephrolithotomy global study. J Endourol. 2011; 25(10): 1619–25. PubMed Abstract \| Publisher Full Text\n\nFalahatkar S, Moghaddam AA, Salehi M, et al.: Complete supine percutaneous nephrolithotripsy comparison with the prone standard technique. J Endourol. 2008; 22(11): 2513–7. PubMed Abstract \| Publisher Full Text\n\nCho HJ, Chung JH, Jo JK, et al.: Assessments of the quality of randomized controlled trials published in International Journal of Urology from 1994 to 2011. Int J Urol. 2013; 20(12): 1212–9. PubMed Abstract \| Publisher Full Text\n\nLuchini C, Stubbs B, Solmi M, et al.: Assessing the quality of studies in meta-analyses: advantages and limitations of the newcastle ottawa scale. World J Meta Anal. 2017; 5(4): 80–4. Publisher Full Text\n\nMelo PAS, Vicentini FC, Perrella R, et al.: Comparative study of percutaneous nephrolithotomy performed in the traditional prone position and in three different supine positions. Int Braz J Urol. 2019; 45(1): 108–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalter SD, Yao X: Effect sizes can be calculated for studies reporting ranges for outcome variables in systematic reviews. J Clin Epidemiol. 2007; 60(8): 849–52. PubMed Abstract \| Publisher Full Text\n\nHiggins JPT, Green S: Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0 [updated March 2011]. The Cochrane Collaboration; 2011. Reference Source\n\nGokce MI, Ibis A, Sanci A, et al.: Comparison of supine and prone positions for percutaneous nephrolithotomy in treatment of staghorn stones. Urolithiasis. 2017; 45(6): 603–8. PubMed Abstract \| Publisher Full Text\n\nMahmoud M, Abdel AA, Abdel AM, et al.: Flank suspended supine position versus standard supine and prone position in percutaneous nephrolithotomy. J Endourol. 2017; 31: A95–A98.\n\nWood GJA, Torricelli FCM, Vicentini FC, et al.: Supracostal punctures in supine percutaneous nephrolithotomy are safe. Can J Urol. 2017; 24(2): 8749–53. PubMed Abstract\n\nAstroza G, Lipkin M, Neisius A, et al.: Effect of supine vs prone position on outcomes of percutaneous nephrolithotomy in staghorn calculi: results from the Clinical Research Office of the Endourology Society Study. Urology. 2013; 82(6): 1240–4. PubMed Abstract \| Publisher Full Text\n\nKan RW, Fu KK, Wong BT, et al.: Percutaneous nephrostomy, nephrolithotomy and combined ureteroscopic lithotripsy using the supine approach. Hong Kong Med J. 2013; 19(2): 142–9. PubMed Abstract\n\nKarami H, Mohammadi R, Lotfi B: A study on comparative outcomes of percutaneous nephrolithotomy in prone, supine, and flank positions. World J Urol. 2013; 31(5): 1225–30. PubMed Abstract \| Publisher Full Text\n\nSanguedolce F, Breda A, Millan F, et al.: Lower pole stones: prone PCNL versus supine PCNL in the International Cooperation in Endourology (ICE) group experience. World J Urol. 2013; 31(6): 1575–80. PubMed Abstract \| Publisher Full Text\n\nArrabal-Martin M, Arrabal-Polo MA, Lopez-Leon V, et al.: The oblique supine decubitus position: technical description and comparison of results with the prone decubitus and dorsal supine decubitus positions. Urol Res. 2012; 40(5): 587–92. PubMed Abstract \| Publisher Full Text\n\nWang Y, Hou Y, Jiang F, et al.: Percutaneous nephrolithotomy for staghorn stones in patients with solitary kidney in prone position or in completely supine position: a single-center experience. Int Braz J Urol. 2012; 38(6): 788–94. PubMed Abstract \| Publisher Full Text\n\nSofer M, Giusti G, Proietti S, et al.: Upper Calyx Approachability through a Lower Calyx Access for Prone Versus Supine Percutaneous Nephrolithotomy. J Urol. 2016; 195(2): 377–82. PubMed Abstract \| Publisher Full Text\n\nTendi W: Supine versus Prone Position in Percutaneous Nephrolithotomy: A Systematic Review and Meta-Analysis. 2020. http://doi.org/10.17605/OSF.IO/23GND" }	[ { "id": "61982", "date": "06 Apr 2020", "name": "Noor Buchholz", "expertise": [ "Reviewer Expertise Stone surgery", "PCNL" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 need to be congratulated for this effort. They produced a comprehensive review on supine versus prone position in PCNL. This former method has been around for now almost 15 years, and still surgeons remain unsure whether there is a benefit in adopting the supine position over the established and older prone position. In that sense, this article may be a good decision tool.\n\nThe authors systematically reviewed the relevant literature. The reviewer misses some evidence but the authors used stringent selection criteria which may have led to their exclusion but in turn make the data more robust. An example would be:\nKachrilas S, Papatsoris A, Bach C, Kontos S, Faruquz Z, Goyal A, Masood J,BUCHHOLZ N. Colon perforation during percutaneous renal surgery: a 10-year experience in a single endourology centre. Urol Res. 2012 Jun;40(3):263-81.\nThis article shows on a large number of patients that there is no difference in complications in the right hands.\n\nAlthough in most experienced hands, the differences between the two methods are minimal in all aspects, and the advantages of supine are evident to most surgeons who use it, somewhat surprisingly to me the analysed data show better safety in supine, and better stone-free rate in prone PCNL. Since there seems to be no flaw in their analysis, we will have to believe this data.\n\nThat makes it even more important to index.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "67732", "date": "31 Jul 2020", "name": "Mohammed Said ElSheemy", "expertise": [ "Reviewer Expertise Urolithiasis", "endourology", "PCNL", "Mini-PCNL", "URS", "SWL" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented their meta-analyses comparing prone versus supine PCNL for the management of renal stones > 2 cm. They reported a higher SFR with prone technique but a better safety profile with supine technique. However, there are many points that require more details especially in the results and discussion section. The authors did not comment on studies heterogenousities or quality. Many points were not presented in each study (detailed in the comments on the results and discussion section). The authors did not discuss previous meta-analyses on the same issue nor clarifying the addition or difference in the current study.\nIntroduction:\nThe authors reported that: [While PCNL has higher free stone rates with a similar recurrence and complication rate compared with ESWL,...]. This should be cited. Additionally, the complication rates are different between both procedures. Similarly, the recurrence rate is different.\n\nMethod:\nThe authors should clarify the age of patients and the design of the study (RCT or cohort) in the inclusion criteria.\n\nResults:\nThe authors did not comment on the quality of included studies as well as their heterogenicity.\n\nFigure 1:\nThe authors should clarify why 103 studies were excluded out of 131.\n\nThe authors should perform a separate analysis (or at least a separate comment) for blood transfusion, sepsis, pleural effusion or visceral injury complications.\n\nTable 2: the authors should add many points to describe each study adequately including:\nNumber and shape of stone SFR for each study. The method and timing for evaluation of SFR. If SFR was calculated after first session or after 2nd look PCNL. The complications for each technique (and its rate). If any procedure was Mini-PCNL and the caliber of used renal track (sheath). If there were congenital renal anomalies. Lithotripsy technique. Operative imaging modality.\n\nDiscussion:\nThe authors should give more details of the included studies.\n\nThe authors should discuss previous meta-analyses comparing both procedures. Additionally, the authors should compare the results of the present study to previous meta-analyses clarifying any difference and any addition.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5819", "date": "24 Aug 2020", "name": "Ponco Birowo", "role": "Author Response", "response": "Introduction Reviewers’ comment: The authors reported that: [While PCNL has higher free stone rates with a similar recurrence and complication rate compared with ESWL,...]. This should be cited. Additionally, the complication rates are different between both procedures. Similarly, the recurrence rate is different. Authors’ response: Thank you very much for the comment, we have added the citation of the statement mentioned in the introduction section: [While PCNL has higher free stone rates with a similar recurrence and complication rate compared with ESWL, this procedure also has its own preparation including a guiding system, anesthesia, and positioning of the patient]. The citation was the reference number 5, which is [Ganpule AP, Vijayakumar M, Malpani A, Desai MR. Percutaneous nephrolithotomy (PCNL) a critical review. Int J Surg. 2016; 36(Pt D): 660-4.] (Page 3, Paragraph 3, Introduction section). Methods Reviewers’ comment: The authors should clarify the age of patients and the design of the study (RCT or cohort) in the inclusion criteria. Authors’ response: Thank you very much for the comment, we have revised the inclusion criteria accordingly. (Page 4, Paragraph 6, Methods section, subsection Study Selection). Results Reviewers’ comment: The authors did not comment on the quality of included studies as well as their heterogenicity. Figure 1: The authors should clarify why 103 studies were excluded out of 131. The authors should perform a separate analysis (or at least a separate comment) for blood transfusion, sepsis, pleural effusion or visceral injury complications. Table 2: the authors should add many points to describe each study adequately including: Number and shape of stone SFR for each study. The method and timing for evaluation of SFR. If SFR was calculated after first session or after 2nd look PCNL. The complications for each technique (and its rate). If any procedure was Mini-PCNL and the caliber of used renal track (sheath). If there were congenital renal anomalies. Lithotripsy technique. Operative imaging modality. Authors’ response: Thank you very much for the comment, we have added a summary of the quality assessment of each article (Page 5, Paragraph 5, Results section, subsection Study Characteristics). Furthermore, we also showed the heterogeneity of each variable measured in this article (Page 9-11, Results section, subsection Stone Free Rate, Major Complication Rate, Length of Hospital Stay, and Mean Operation Time). Thank you very much for the kind comment. The reason of which 103 articles were excluded because as we screened through all the titles and abstracts, we only found 28 articles that has the main topic of our interest, which is a study with either trial design or observational study (Page 5, Paragraph 4, Results section, subsection Literature Search). Thank you very much for the suggestion, we have added a further separate analysis regarding the need of blood transfusion, sepsis, and visceral injuries complication presented in a forest plot (Page 10, Results section, Figure 4). Thank you very much for the suggestion, we have added more details on the Table 2 accordingly (Page 7, Results section, Table 2). Discussion Reviewers’ comment: The authors should give more details of the included studies. The authors should discuss previous meta-analyses comparing both procedures. Additionally, the authors should compare the results of the present study to previous meta-analyses clarifying any difference and any addition. Authors’ response: Thank you very much for the kind comment, we have added more detail of our included studies on the discussion section (Page 11, Paragraph 3, Discussion section). Thank you for the suggestion, we have added the comparison of the previous meta-analyses to our study (Page 12, Paragraph 6, Discussion section). Regarding to this addition, we have added the references accordingly (Page 15, References number 24 and 25)." } ] }, { "id": "64021", "date": "13 Dec 2021", "name": "Doddy Moesbadianto Soebadi", "expertise": [ "Reviewer Expertise Stone disease", "male infertility", "andrology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 good review. We do need the evidence based for choosing the method. The authors could also state their opinions concerning the training of the procedures, whether the two methods have to be taught or directly to one method.\nIn conclusion, it can also be stated the positive and negative sides of each procedures, in consideration the needs for training.\nCongratulations to the authors.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-231
https://f1000research.com/articles/9-582/v1	09 Jun 20	{ "type": "Study Protocol", "title": "Effect of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial", "authors": [ "Parisa Rasouli Fard", "Farnoush Jarollahi", "Seyyed Jalal Sameni", "Mohammad Kamali", "Parisa Rasouli Fard", "Seyyed Jalal Sameni", "Mohammad Kamali" ], "abstract": "Background: Age-related hearing loss (presbycusis) is a form of hearing loss in over 60-year-olds and has a negative impact on quality of life. The pathophysiology of presbycusis is multifactorial and is predominately characterised with a loss of speech perception in noise. In the cochlea, auditory filters decompose broadband sound into a series of narrowband output signals, which contains two kinds of temporal information: slow changes in overall amplitude envelope (ENV) and faster variations in temporal fine structure (TFS). TFS is important for recognition of target speech in noise. The main aim of the study is to evaluate the effect of TFS rehabilitation training in participants over the age of 60 years with mild to moderate hearing loss. Methods: A randomised clinical trial will be conducted on 30 participants with mild (loss of 20-39dB) to moderate (40-69dB) hearing loss, aged between 60 and 75 years old. Participants with conductive hearing loss, abnormal middle ear pathology and central nerve system disease will be excluded. Participants will be randomly selected to an intervention and control group with a 1:1 ratio. Rehabilitation for the intervention group will be 30-minute sessions three times a week for a total five weeks of vowel consonant vowel words that are used to eliminate ENV and keep only TFS. Word in noise test, binaural TFS test, and Speech, Spatial and Qualities of Hearing Scale scores will be performed at the beginning and end of study to evaluate the effect of rehabilitation training. Conclusion: Life expectancy in the elderly has improved, leading to an increased prevalence of age-related diseases including presbycusis. A literature review highlighted that TFS damage is permanent; however, in this study we will attempt to prove that TFS training may lead to speech in noise perception restored. Trial registration: Registry of Clinical Trials, IRCT2019625044006N1 (7th August 2019).", "keywords": [ "Age related hearing", "Presbycusis", "Temporal Fine Structure", "Rehabilitation Training" ], "content": "Introduction\n\nPresbycusis (age-related hearing loss) is one the most common disorders worldwide1,2. The cause of presbycusis is multifactorial, including pathophysiological degeneration, extrinsic and intrinsic damage, genetic predisposition and comorbidities (conditions like diabetes, hypertension and stroke)3–5.\n\nIn cochlea high frequency sounds evoke greatest vibration of the basilar membrane at the base while lower frequency sounds evoke greatest vibration at the apex6–9. Sounds are decomposed to narrow band signals (envelope; ENV) and rapid oscillations (temporal fine structure; TFS)9–12. The ENV frequency range is between 2-50 Hz. One of the most important task of ENV is to identify speech in quiet environments11,12. TFS frequency range is between 0.6-10 kHz13, and TFS cues are important in perception of pitch, tone separation14, and identify target speech in interfering sounds15. Presbycusis is associated with loss of speech perception in noisy environments16 and deterioration of the processing of TFS information14,15,17.\n\nPrevious studies indicate that sensorineural hearing loss is associated with a reduction in speech recognition and is dependent on deterioration of TFS18, showing the importance of TFS for listening with background sounds8. Studies by Hopkins et al. suggest that TFS is important to recognise the temporal dips in fluctuating background noise19,20. In an elderly population with high frequency hearing loss, even when absolute thresholds are within the normal range, the TFS can be damaged21. It is speculated that TFS information is useful for separation of the target speech in background speech22.\n\nThe main aim of the study is to evaluate the impact of special rehabilitation training based on TFS on improvement of speech in noise perception in an elderly population with mild to moderate hearing loss.\n\n\nProtocol\n\nThis is version 1 of the protocol. There is no plan for further trial modifications.\n\nWe will conduct a randomised clinical trial of rehabilitation training on speech in noise perception performance on an elderly population with mild to moderate hearing loss at the Audiology Clinic of School of Rehabilitation Sciences, Iran University of Medical Sciences (Tehran, Iran). It is assumed that the inability to use TFS speech cues is the main cause of speech perception problem in noise in elderly individuals, and it is possible by designing appropriate rehabilitation exercises to reduce the difficulty of speech perception in noise.\n\nThe Medical Ethics Committee at the Iran University of Medical Sciences approved the registered study protocol (IR.IUMS>REC.1398.003). The study was registered on the Iranian Registry of Clinical Trials (registration number, IRCT2019625044006N1), a Primary Registry in the World Health Organization Clinical Trials Registry Network.\n\nThe protocol does not involve complications for precipitants in the study. All participants will be informed both orally and writing about the study process. Written consent to participant will be obtained from the participants before the study start (see Extended data: S1).\n\nMild to moderate hearing loss: auditory thresholds ≤25dB within the frequency<2000 Hz and 25-70 dB with frequency 2000-8000 Hz.\n\nTFS-LF test: software designed by Hopkins and Moore in 2010. The test is originally based on measuring the inter-aural phase differences15.\n\nInter aural phase difference (IPL): lowest difference in the phase of the wave in each ear and dependent frequency sound waves and difference in time between ears15.\n\nSignal to noise ratio (SNR): ratio of the power of a signal (meaningful information) to the power of background noise (unwanted signal), expressed in decibels (dB). Larger numbers for signal characteristics mean better and more useful than unwanted noise information23. In this study the signal-to-noise ratio levels were 0, 4, 8, 12, 16, 20, and 24 dB.\n\nSpeech in noise score: measured by PARWIN list, which is expressed as a percentage by performing a single syllable word test. The PARWIN test is a version of the Richard H. Wilson WIN test, in which the background noise in this test is baffled noise24. PARWIN test is used to estimate SNR (50%) using Spearman Karber equation.\n\nSpeech, Spatial and Qualities of Hearing Scale (SSQ) questionnaire: used in previous studies in elderly individuals with communication disorders caused by hearing loss. From the original version of the SSQ questionnaire, its validity and reliability native version were confirmed (validity, 96% reliability) and included 47 items in three subgroups of speech perception, spatial hearing, and auditory quality. Based on the results of the questionnaire, the mean score of each item and item of each index will be measured for the research participants.\n\nRehabilitation training: auditory rehabilitation will be based on TFS. The intervention group will be asked to identify vowel consonant vowel words (VCVs) that have only TFS preserved and their envelope discarded. It is based on that VCVs that processing and converting to TFS speech. In this process the ENV of VCVs will be eliminated and only TFS will be kept.\n\nParticipants will be recruited from elderly people, aged between 60 and 75 years old, referred to the audiology clinics of Iran University of Medical Sciences and will be informed by phone about the study. They will be selected based on previous clinical examination, including otoscopy, tympanometry and pure tone audiometry test (PTA) to identify type and level of a hearing loss. In a preliminary interview, speech perception difficulty will be evaluated with a question if they had difficulty in understanding speech in noise. Those who respond yes will be entered into the study. We will perform Mini Mental State Examination (MMSE) questionnaire in order to rule out prominent cognition difficulty in participants.\n\nParticipants can withdraw from the study at any time. Privacy concerning information and results of participants will be respected.\n\nThe schematic diagram of study procedures is shown below Extended data: Figure S1.\n\nInclusion and exclusion criteria. Inclusion criteria: individuals with mild to moderate hearing loss and aged between 60-75 years, having diploma or higher degree; right-handedness (assessed using Edinburgh handedness inventory); speaking native language and being monolingual; complaint about speech in noise perception difficulties and normal condition of middle ear function.\n\nExclusion criteria: those who do not meet the inclusion criteria, unwillingness for participation in each step of study, conductive hearing loss and abnormal middle ear, central nervous system disease, head trauma, history of seizure attack and epilepsy, and use of psychiatric and nervous system drugs. Individuals with obvious cognitive problems, as diagnosed by Mini Mental State Examination (MMSE), will also be excluded.\n\nSample size. The following formula is used to determine the number of samples in each group with the concern that the two groups are independent and dependent variables in this study are quantitative.\n\nα1: standard deviation of the studied variable in the first group (case, exposed, or intervened)\n\nα2: standard deviation of the studied variable in the second group (control, unexposed, or compared)\n\nμ1: mean of the studied variable in the first group\n\nμ2: mean of the studied variable in the second group\n\nα=0.05\n\nβ=80%\n\nZ= 1.96\n\nBased on previous studies, a power of 85% and level of significance of 95% was determined for this study. We obtained a sample size of 15 individuals for each group (total = 30), which takes into consideration a 20% drop out.\n\nThe study will not involve complications for participants, but if there is extreme difficulty with cooperation for participants the test will be discontinued. All participants will be informed both orally and in writing about the study process. Written consent to participant will be obtained before the study start. There is no criteria for intervention modification in this study protocol. To improve adherence to intervention protocols, every training session the examiner will provide feedback to all participants and will inform them about the training progress. The rehabilitation sessions and duration are flexible for participant.\n\nWe will randomly assign participants in 1:1 ratio, intervention and control group. The intervention group will undergo the rehabilitation training program.\n\nThe two groups will be matched for age and gender. Those in the control group will not receive any rehabilitation programs during the study. The randomization will be applied by random number table (those assigned an odd number, control group; those assigned an even number, intervention group).\n\nStudy procedures. Pre-rehabilitation, the SNR (50%) of all participants will be measured using the WIN test. In addition, a binaural TFS test and the SSQ questionnaire score of all participants will be evaluated (see section Outcomes below).\n\nRehabilitation training. Participants will identify the set of 16 consonants using one-interval forced-choice procedure and feedback with correct answer. On each test the participants will select one of the stimuli from the set of 16 syllables. The participants will be informed while the stimulus is presented that they should identify its middle consonant. Following each stimulus presentation, a 4 × 4 visual display of the response alternatives will appear on a computer monitor and the participant will select the response by using the computer mouse.\n\nThe participant will select a box, if they click the box correctly, the box will turn green and if they chooses the wrong answer, the box will turn red. The participant will be given visual feedback by showing the correct VCV with a yellow box. No time limit will be imposed on the participant's responses. Each experimental run consists of 64 trials derived from a different random-order presentation of the 64 syllables in the stimulus set. Each run will last 16 to 30 min depending on the participant's response time. The total duration of rehabilitation sessions will be five weeks. Experiments are controlled by a desktop PC.\n\nOnly the intervention group will undergo the rehabilitation and control group will not be informed about details of the intervention study procedure.\n\nThe TFS speech consists of single syllable recorded in / a / C / a / with various 16 consonant format which included Aja, Aka, Ara,…. and it will be pronounced by a native-speaking man. The analogue signals will be converted to digital a 16-bit at 44.1 KHz sampling frequency. The stimulus synthesis process will be performed in MATLAB software and the software will be provided in C programming language.\n\nThe original bandpass will be filtered into 16 bands of equal bandwidth on a log frequency scale spanning 80 to 8020 Hz. Each Bandpass signal will be decomposed to ENV and TFS by Hilbert transform. The ENV component will be discarded and TFS component will be normalized and TFS component in each band summed lastly creating TFS speech.\n\nAfter rehabilitation, the SNR (50%) using the WIN test, and a binaural TFS test and SSQ questionnaire will be evaluated again in intervention group. Results will be compared in intervention and control groups before and after the rehabilitation program.\n\nAfter rehabilitation, the SNR (50%) using the WIN test, and a binaural TFS test and SSQ questionnaire will be evaluated again. Results will be compared in intervention and control groups before and after the rehabilitation program.\n\nOutcomes. SNR (50%): single syllable words in the presence of noise at different signal-to-noise ratios (0, + 4, + 8, + 12, + 16, + 20, + 24) as binaural in two study groups and compare the SNR (50%). Differences in scores before and after rehabilitation training between the two groups will be compared.\n\nThe Words-in-Noise (WIN) materials were developed to evaluate the ability of listeners to understand words in multitalker babble. The WIN involves in which the level of the noise is fixed and five words are presented at seven signal-to-noise ratios from 24 to 0 dB in 4 dB decrements. The 35 words are spoken by a native male speaker. The metric of interest is the signal-to-noise ratio (S/N) at which recognition performance is 50%, which is a value determined with the Spearman Karber equation see (Extended data: S2).\n\nBinaural TFS test: determines the binaural change of phase difference at different frequencies in intervention and control groups before and after rehabilitation training.\n\nThe correlation between the results of speech perception test scores in the presence of noise with the results of binaural TFS test in the two groups after rehabilitation program will be assessed.\n\nSSQ questionnaire score: provided to each group before and after rehabilitation training. Scores between intervention and control groups will be compared (Extended data: S3).\n\nIn descriptive analysis of data, central tendency and dispersion indices (mean, median and standard deviation) will be used. Kolmogorov–Smirnov test will be used to test whether two random samples are drawn from the same normal distribution. Otherwise its nonparametric equivalent will be used. Depending on the circumstances, paired t-test and analysis of covariance will be used to compare pre- and post-rehabilitation program. Other analytical tests will be used as required during the data processing phase. SPSS software (V20.0, IBM Corporation, New York, USA) will be used for statistical data analysis and the significance level for all tests will be 0.05.\n\nThe results of our research will be disseminated through presentations at regional and national audiology conferences. The study outcomes will be published through peer-reviewed journals. There is no limit in the publication of the trial results.\n\nEight independent audiologists expert who are the academic members of rehabilitation schools in Shahid Beheshti University of Medical Sciences (SBMU) and Iran University of Medical Sciences (IUMS) will monitor patient safety and treatment efficacy. They approved the relevance, clarity and simplicity material of the study.\n\n\nStudy status\n\nThe enrolment of the patients has been performed and the allocation will be performed in the near future. The study started in November 2019 and will continue until December 2020.\n\n\nDiscussion\n\nElderly populations are growing rapidly worldwide, and this higher number of older individuals is associated with an increase in prevalence and incidence of age-related disorders. Age-related hearing loss (presbycusis) is one the most common disorders with an increase in age. Speech perception in noisy environments is very serious difficulty with presbycusis, which can impact negatively on the quality of life of individuals. Loss of speech perception in noisy environments with presbycusis is mostly caused by damage of processing of TFS information. In this study, we will attempt to prove by special rehabilitation training based on TFS damage that age-related hearing loss can be re-established.\n\n\nData availability\n\nNo data is associated with this article.\n\nOpen Science Framework: Effect of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial, https://doi.org/10.17605/OSF.IO/VU9CH25.\n\nThis project contains the following extended data:\n\nS1: Questionnaire.\n\nS2: Words-in-Noise (WIN) test to measure single syllable words (SNR) 50%\n\nS3: Speech, Spatial and Qualities of Hearing Scale (SSQ) questionnaire\n\nFigure S1: Schematic diagram of study procedures and timeline\n\nOpen Science Framework: Effect of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial, https://doi.org/10.17605/OSF.IO/A4KGM, registered on 1st June 202026.\n\nThis project contains the following extended data:\n\n- Informed consent: Form has been uploaded to OSF https://osf.io/a4kgm\n\nOpen Science Framework: SPIRIT checklist for ‘Effect of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial’, https://doi.org/10.17605/OSF.IO/VU9CH25.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank Dr Tony Lewis and Dr Hanri Afghahi for English editing of the manuscript.\n\n\nReferences\n\nGates GA, Cooper JC Jr, Kannel WB, et al.: Hearing in the elderly: the Framingham cohort, 1983-1985. Part I. Basic audiometric test results. Ear Hear. 1990; 11(4): 247–56. PubMed Abstract\n\nAgrawal Y, Platz EA, Niparko JK: Prevalence of hearing loss and differences by demographic characteristics among US adults: data from the National Health and Nutrition Examination Survey, 1999-2004. Arch Intern Med. 2008; 168(14): 1522–30. PubMed Abstract \| Publisher Full Text\n\nCooper JC Jr: Health and Nutrition Examination Survey of 1971-75: Part I. Ear and race effects in hearing. J Am Acad Audiol. 1994; 5(1): 30–6. PubMed Abstract\n\nCruickshanks KJ, Wiley TL, Tweed TS, et al.: Prevalence of hearing loss in older adults in Beaver Dam, Wisconsin. The Epidemiology of Hearing Loss Study. Am J Epidemiol. 1998; 148(9): 879–86. PubMed Abstract \| Publisher Full Text\n\nHelzner EP, Cauley JA, Pratt SR, et al.: Race and sex differences in age-related hearing loss: the Health, Aging and Body Composition Study. J Am Geriatr Soc. 2005; 53(12): 2119–27. PubMed Abstract \| Publisher Full Text\n\nSmith ZM, Delgutte B, Oxenham AJ: Chimaeric sounds reveal dichotomies in auditory perception. Nature. 2002; 416(6876): 87–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu L, Pfingst BE: Relative importance of temporal envelope and fine structure in lexical-tone perception. J Acoust Soc Am. 2003; 114(6 Pt 1): 3024–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorenzi C, Gilbert G, Carn H, et al.: Speech perception problems of the hearing impaired reflect inability to use temporal fine structure. Proc Natl Acad Sci U S A. 2006; 103(49): 18866–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGilbert G, Lorenzi C: The ability of listeners to use recovered envelope cues from speech fine structure. J Acoust Soc Am. 2006; 119(4): 2438–44. PubMed Abstract \| Publisher Full Text\n\nMoore BCJ: The role of temporal fine structure processing in pitch perception, masking, and speech perception for normal-hearing and hearing-impaired people. J Assoc Res Otolaryngol. 2008; 9(4): 399–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrullman R: Temporal envelope and fine structure cues for speech intelligibility. J Acoust Soc Am. 1995; 97(1): 585–92. PubMed Abstract \| Publisher Full Text\n\nShannon RV, Zeng FG, Kamath V, et al.: Speech recognition with primarily temporal cues. Science. 1995; 270(5234): 303–4. PubMed Abstract \| Publisher Full Text\n\nRosen S: Temporal information in speech: acoustic, auditory and linguistic aspects. Philos Trans R Soc Lond B Biol Sci. 1992; 336(1278): 367–73. PubMed Abstract \| Publisher Full Text\n\nFullgrabe C, Moore BCJ, Stone MA: Age-group differences in speech identification despite matched audiometrically normal hearing: contributions from auditory temporal processing and cognition. Front Aging Neurosci. 2014; 6: 347. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins K, Moore BCJ: The effects of age and cochlear hearing loss on temporal fine structure sensitivity, frequency selectivity, and speech reception in noise. J Acoust Soc Am. 2011; 130(1): 334–49. PubMed Abstract \| Publisher Full Text\n\nHenry KS, Heinz MG: Effects of sensorineural hearing loss on temporal coding of narrowband and broadband signals in the auditory periphery. Hear Res. 2013; 303: 39–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins K, Moore BCJ: Moderate cochlear hearing loss leads to a reduced ability to use temporal fine structure information. J Acoust Soc Am. 2007; 122(2): 1055–68. PubMed Abstract \| Publisher Full Text\n\nBuss E, Hall JW, Grose JH: Temporal fine-structure cues to speech and pure tone modulation in observers with sensorineural hearing loss. Ear Hear. 2004; 25(3) : 242–50. PubMed Abstract \| Publisher Full Text\n\nHopkins K, Moore BCJ, Stone MA: Effects of moderate cochlear hearing loss on the ability to benefit from temporal fine structure information in speech. J Acoust Soc Am. 2008; 123(2): 1140–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopkins K, Moore BC: The contribution of temporal fine structure to the intelligibility of speech in steady and modulated noise. J Acoust Soc Am. 2009; 125(1): 442–6. PubMed Abstract \| Publisher Full Text\n\nLorenzi C, Debruille L, Garnier S, et al.: Abnormal processing of temporal fine structure in speech for frequencies where absolute thresholds are normal. J Acoust Soc Am. 2009; 125(1): 27–30. PubMed Abstract \| Publisher Full Text\n\nLunner T, Hietkamp RK, Andersen MR, et al.: Effect of speech material on the benefit of temporal fine structure information in speech for young normal-hearing and older hearing-impaired participants. Ear Hear. 2012; 33(3): 377–88. PubMed Abstract \| Publisher Full Text\n\nKillion MC, Niquette PA, Gudmundsen GI, et al.: Development of a quick speech-in-noise test for measuring signal-to-noise ratio loss in normal-hearing and hearing-impaired listeners. J Acoust Soc Am. 2004; 116(4 Pt 1): 2395–405. PubMed Abstract \| Publisher Full Text\n\nWilson RH, Farmer NM, Gandhi A, et al.: Normative data for the Words-in-Noise Test for 6- to 12-year-old children. J Speech Lang Hear Res. 2010; 53(5): 1111–21. PubMed Abstract \| Publisher Full Text\n\nFard PR: Effect of rehabilitation training on an eldery population with mild to moderate hearing loss: study protocol for a randomised clinicla trial. 2020. http://www. doi.org/10.17605/OSF.IO/VU9CH\n\nFard PR: Effect of rehabilitation training on eldery population with mild to moderate hearing loss: study protocol for a randomised clinical trial. 2020. http://www. doi.org/10.17605/OSF.IO/A4KGM" }	[ { "id": "64606", "date": "23 Jun 2020", "name": "Mojtaba Tavakoli", "expertise": [ "Reviewer Expertise Hearing aid" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for the valuable article posted.\nThe selection of cases is well done and the research path is well described. Given the problems of people with hearing loss with more falls, please explain if the results are positive, is it possible to do this protocol in more hearing loss or not?\nIn the section on formal validity and reliability, please indicate the number obtained from the information collected from eight academic members of the university.\nIn the future perspective of this protocol, if the results are positive, it is appropriate to have auditory rehabilitation in the control group. Please explain why the degree of education must be at least a diploma?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5662", "date": "25 Jun 2020", "name": "Parisa Rasouli Fard", "role": "Author Response", "response": "Dear Reviewer, Thank you very much for very important comments and suggestions. In our study the primary results is very positive and participants in intervention group have significantly better speech perception in noisy environments. We will perform rehabilitation training program in subjects with other causes of hearing loss like noise related hearing loss (NRHI). The Waltz & Bausell method is used to examine validity of the test. The stimulus was sent to eight independent audiologists expert. They assessed relevance, clarity and simplicity of the test by Likert scale from one (non-relevant, non-simple and non-clarity) to four scale (complete-relevant, complete-simple and complete-clarity) in each item.The analyze of their evaluation showed content validity index (CVI) 87% validity for the test. In this study to increase homogeneity of our study population we decided to eliminate education as nuisance variable (unwanted variable) (1). All participants had to have at least high school diploma which it is more applicable to evaluate the difficulty of test by participants. In future to increase the power of the study and higher external validity the study population with education degree less than high school diploma will be included. It is very interesting suggestion, we will perform the test auditory rehabilitation in the control group. 1. Fregni F et al, Critical Thinking in Clinical Research, Oxford University Press, 2018." } ] }, { "id": "67180", "date": "21 Jul 2020", "name": "Suna Tokgoz‐Yilmaz", "expertise": [ "Reviewer Expertise Audiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is excellent, but How to do TFS rehabilitation training for 5 weeks is not detailed.\n\nIn the study status there was \"The enrollment of the patients has been performed and the allocation will be performed in the near future. The study started in November 2019 and will continue until December 2020.\" But looking at the dates, the work should be finished, but in many sentences, the future tense is used.\n\nIn the inclusion criteria there was \"In a preliminary interview, speech perception difficulty will be evaluated with a question if they had difficulty in understanding speech in noise.\" For this criteria please look at this article \"The role of the medial olivocochlear system in the complaints of understanding speech in noisy environments by individuals with normal hearing\"1.\n\nThe discussion and results sections are insufficient.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "5786", "date": "11 Aug 2020", "name": "Parisa Rasouli Fard", "role": "Author Response", "response": "Dear Dr. Tokgoz-Yilmaz, Thank you very much for your positive response to our paper. We have now carefully evaluated your comments and revised the manuscript. Below we have specified in detail our responses. We hope you will find the new version of the manuscript acceptable for publication, The study is excellent, but How to do TFS rehabilitation training for 5 weeks is not detailed. Respons: The TFS rehabilitation trainings is performed in our clinic. Rehabilitation sessions are three times a week and 30 minutes each session. However later a considerable number of participants need more than 30 minutes in each session. The total duration of rehabilitation sessions will be five weeks. In the study status there was \"The enrollment of the patients has been performed and the allocation will be performed in the near future. The study started in November 2019 and will continue until December 2020.\" But looking at the dates, the work should be finished, but in many sentences, the future tense is used. Respons: Thank you for very important comment. We started to write the protocol before to enroll the participants but it is ongoing study now. We changed several sentences from future to past or present. In the inclusion criteria there was \"In a preliminary interview, speech perception difficulty will be evaluated with a question if they had difficulty in understanding speech in noise.\" For this criteria please look at this article \"The role of the medial olivocochlear system in the complaints of understanding speech in noisy environments by individuals with normal hearing. Respons: It is very interesting article. It is defined very excellent how participants complaint of speech understanding in noise by asking seven questions. The population in this study had normal hearing but our subjects had mild to moderate hearing loss due to presbycusis. However we will use the questionnaire about difficulty to speech understanding in noise in this study in our population too. We mentioned the study by Tokgoz-Yilmaz et al as a reference. The discussion and results sections are insufficient. Response: This is study protocol and results of our study are not completed yet. Primary results are shown rehabilitation training associated with significantly better test results of SNR (50%) by WIN test, binaural TFS test and SSQ questionnaire. The completed results will be published. As reviewer demanded we changed the discussion part to become more sufficient." } ] }, { "id": "65520", "date": "07 Aug 2020", "name": "Ali Danesh", "expertise": [ "Reviewer Expertise Audiology", "Auditory Neuroscience and Auditory Electrophysiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease note that each comment is prefixed by AAD and a number. You can see the comments at the end of this report. The report is also attached as a file here. Thank you.\nEffect[AAD1] of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial [version 1; peer review: 1 approved] Parisa Rasouli Fard[AAD2]\n\nhttps://orcid.org/0000-0003-4985-435X , Farnoush Jarollahi, Seyyed Jalal Sameni, Mohammad Kamali Author details Abstract Background: Age-related hearing loss (presbycusis) is a form of hearing loss in over 60-year-olds and has a negative impact on quality of life. The pathophysiology of presbycusis is multifactorial and is predominately characterised with a loss of speech perception in noise[AAD3] . In the cochlea, auditory filters decompose broadband sound into a series of narrowband output signals, which contains two kinds of temporal information: slow changes in overall amplitude envelope (ENV) and faster variations in temporal fine structure (TFS). TFS is important for recognition of target speech in noise. The main aim of the study is to evaluate the effect of TFS rehabilitation training in participants over the age of 60 years with mild to moderate hearing loss. Methods: A randomised clinical trial will [AAD4] be conducted on 30 participants with mild (loss of 20-39dB[AAD5] ) to moderate (40-69dB[AAD6] ) hearing loss, aged between 60 and 75 years old. Participants with conductive hearing loss, abnormal middle ear pathology and central nerve[AAD7] system disease will[AAD8] be excluded. Participants will [AAD9] be randomly selected to an intervention and control group with a 1:1 ratio. Rehabilitation for the intervention group will be 30-minute sessions three times a week for a total five weeks of vowel consonant vowel words that are used to eliminate ENV and keep only TFS. Word in noise test, binaural TFS test, and Speech, Spatial and Qualities of Hearing Scale scores will be performed at the beginning and end of study to evaluate the effect of rehabilitation training. Conclusion:[AAD10]\n\nLife expectancy in the elderly has improved, leading to an increased prevalence of age-related diseases including presbycusis. A literature review highlighted that TFS damage is permanent; however, in this study we will attempt to prove [AAD11] that TFS training may lead to speech in noise perception restored.[AAD12] Trial registration: Registry of Clinical Trials, IRCT2019625044006N1 (7th August 2019). Keywords Age related hearing, Presbycusis, Temporal Fine Structure, Rehabilitation Training[AAD13] Corresponding author: Farnoush Jarollahi Competing interests: No competing interests were disclosed. Grant information: This study was part of a Ph.D. Dissertation approved by Iran University of Medical Sciences (IUMS), Tehran, Iran and is financially supported by IUMS (Contract No: 98-1-6-14345). The funders [AAD14] had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2020 Rasouli Fard P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Rasouli Fard P, Jarollahi F, Sameni SJ and Kamali M. Effect of rehabilitation training on an elderly population with mild to moderate hearing loss: study protocol for a randomised clinical trial [version 1; peer review: 1 approved]. F1000Research 2020, 9:582 (https://doi.org/10.12688/f1000research.23332.1)First published: 09 Jun 2020, 9:582 (https://doi.org/10.12688/f1000research.23332.1)Latest published: 09 Jun 2020, 9:582 (https://doi.org/10.12688/f1000research.23332.1) Introduction Presbycusis (age-related hearing loss) is one the most common disorders worldwide1,2. The cause of presbycusis is multifactorial, including pathophysiological degeneration, extrinsic and intrinsic damage, genetic predisposition and comorbidities (conditions like diabetes, hypertension and stroke)3–5. In cochlea high frequency sounds evoke greatest vibration of the basilar membrane at the base while lower frequency sounds evoke greatest vibration at the apex6–9. Sounds are decomposed to narrow band signals (envelope; ENV)[AAD15] and rapid oscillations (temporal fine structure; TFS[AAD16] )9–12. The ENV frequency range is between 2-50 Hz. One of the most important task o[AAD17] f ENV is to identify speech in quiet environments11,12. TFS frequency range is between 0.6-10 kHz13, and TFS cues are important in perception of pitch, tone separation14, and identify target speech in interfering sounds15. Presbycusis is associated with loss of speech perception in noisy environments16 and deterioration of the processing of TFS information14,15,17. Previous studies indicate that sensorineural hearing loss is associated with a reduction in speech recognition and is dependent on deterioration of TFS18, showing the importance of TFS for listening with background sounds8. Studies by Hopkins et al. suggest that TFS is important to recognise the temporal dips in fluctuating background noise19,20. In an elderly population with high frequency hearing loss, even when absolute thresholds are within the normal range, the TFS can be damaged21. It is speculated that TFS information is useful for separation of the target speech in background speech22. Objectives The main aim of the study is to evaluate the impact of special rehabilitation training based on TFS on improvement of speech in noise perception in an elderly population with mild to moderate hearing loss. Protocol This is version 1 of the protocol. There is no plan for further trial modifications.[AAD18] Study overview We will conduct a randomised clinical trial of rehabilitation training on speech in noise perception performance on an elderly population with mild to moderate hearing loss at the Audiology Clinic of School of Rehabilitation Sciences, Iran University of Medical Sciences (Tehran, Iran). It is assumed [AAD19] that the inability to use TFS speech cues is the main cause of speech perception problem in noise in elderly individuals, and it is possible by designing appropriate rehabilitation exercises to reduce the difficulty of speech perception in noise. The Medical Ethics Committee at the Iran University of Medical Sciences approved the registered study protocol (IR.IUMS>REC.1398.003). The study was registered on the Iranian Registry of Clinical Trials (registration number, IRCT2019625044006N1), a Primary Registry in the World Health Organization Clinical Trials Registry Network. The protocol does not involve complications for precipitants [AAD20] in the study. All participants will be informed both orally[AAD21] and writing [AAD22] about the study process[AAD23] . Written consent to participant will be [AAD24] obtained from the participants before the study start (see Extended data: S1). Terminology used in this study Mild to moderate hearing loss: auditory thresholds ≤25dB [AAD25] within the frequency<2000 Hz and 25-70 dB with frequency 2000-8000 Hz. TFS-LF test: software designed by Hopkins and Moore in 2010. The test is originally based on measuring the inter-aural phase differences15. Inter aural [AAD26] phase difference (IPL): lowest difference in the phase of the wave in each ear and dependent frequency sound waves and difference in time between ears15. Signal to noise ratio (SNR): ratio of the power of a signal (meaningful information) to the power of background noise (unwanted signal), expressed in decibels (dB). Larger numbers for signal characteristics mean better and more useful than unwanted noise information23. In this study the signal-to-noise ratio levels were 0, 4, 8, 12, 16, 20, and 24 dB. Speech in noise score: measured by PARWIN l[AAD27] ist, which is expressed as a percentage by performing a single syllable word test. The PARWIN test is a version of the Richard H. Wilson WIN test, in which the background noise in this test is baffled noise24. PARWIN test is used to estimate SNR (50%) using Spearman Karber equation. Speech, Spatial and Qualities of Hearing Scale (SSQ) questionnaire: used in previous studies in elderly individuals with communication disorders caused by hearing loss. From the original version of the SSQ questionnaire, its validity and reliability native version were confirmed (validity, 96% reliability) and included 47 items in three subgroups of speech perception, spatial hearing, and auditory quality. Based on the results of the questionnaire, the mean score of each item and item of each index will be measured for the research participants. Rehabilitation training: auditory rehabilitation will be based [AAD28] on TFS. The intervention group will be asked to identify vowel consonant vowel words (VCVs) that have only TFS preserved and their envelope discarded. It is based on that VCVs that processing and converting to TFS speech. In this process the ENV of VCVs will be eliminated and only TFS will be kept. Participants Participants will be recruited [AAD29] from elderly people, aged between 60 and 75 years old, referred to the audiology clinics of Iran University of Medical Sciences and will be informed by phone about the study. They will be selected based on previous clinical examination, including otoscopy, tympanometry and pure tone audiometry test (PTA) to identify type and level of a hearing loss. In a preliminary interview, speech perception difficulty will be evaluated with a question if they had difficulty in understanding speech in noise. Those who respond yes will be entered into the study. We will perform Mini Mental State Examination (MMSE) questionnaire in order to rule out prominent cognition difficulty in participants. Participants can[AAD30] withdraw from the study at any time. Privacy concerning information and results of participants will be respected.[AAD31] The schematic diagram of study procedures is shown below Extended data: Figure S1. Inclusion and exclusion criteria. Inclusion criteria: individuals with mild to moderate hearing loss and aged between 60-75 years, having diploma or higher degree; right-handedness (assessed using Edinburgh handedness inventory); speaking native language and being monolingual; complaint about speech in noise perception difficulties and normal condition of middle ear function. Exclusion criteria: those who do not meet the inclusion criteria, unwillingness for participation in each step of study, conductive hearing loss and abnormal middle ear, central nervous system disease, head trauma, history of seizure attack and epilepsy, and use of psychiatric and nervous system drugs. Individuals with obvious cognitive problems, as diagnosed by Mini Mental State Examination (MMSE), will also be excluded. Sample size. The following formula is used to determine the number of samples in each group with the concern that the two groups are independent and dependent variables in this study are quantitative. n=(Z1−α2+Z1−β)2(σ12+σ22)(μ1−μ2)2n=(Z1−α2+Z1−β)2(σ12+σ22)(μ1−μ2)2 α1: standard deviation of the studied variable in the first group (case, exposed, or intervened) α2: standard deviation of the studied variable in the second group (control, unexposed, or compared) μ1: mean of the studied variable in the first group μ2: mean of the studied variable in the second group α=0.05 β=80% Z= 1.96 Based on previous studies, a power of 85% and level of significance of 95% was determined for this study. We obtained a sample size of 15 individuals for each group (total = 30), which takes into consideration a 20% drop out. Study design The study will not involve complications for participants, but if there is extreme difficulty with cooperation for participants the test will be discontinued. All participants will be informed both orally and in writing about the study process. Written consent to participant will be obtained before the study start. There is no criteria for intervention modification in this study protocol. To improve adherence to intervention protocols, every training session the examiner will provide feedback to all participants and will inform them about the training progress. The rehabilitation sessions and duration are flexible for participant. We will randomly assign participants in 1:1 ratio, intervention and control group. The intervention group will undergo the rehabilitation training program. The two groups will be matched for age and gender. Those in the control group will not receive any rehabilitation programs during the study. The randomization will be applied by random number table (those assigned an odd number, control group; those assigned an even number, intervention group). Study procedures. Pre-rehabilitation, the SNR (50%) of all participants will be measured using the WIN[AAD32] test. In addition, a binaural TFS test and the SSQ questionnaire score of all participants will be evaluated (see section Outcomes below). Rehabilitation training. Participants will identify the set of 16 consonants using one-interval forced-choice procedure and feedback with correct answer. On each test the participants will select one of the stimuli from the set of 16 syllables. The participants will be informed while the stimulus is presented that they should identify its middle consonant. Following each stimulus presentation, a 4 × 4 visual display of the response alternatives will appear on a computer monitor and the participant will select the response by using the computer mouse. The [AAD33] participant will select a box, if they click the box correctly, the box will turn green and if they chooses t[AAD34] he wrong answer, the box will turn red. The participant will be given visual feedback by showing the correct VCV with a yellow box. No time limit will be imposed on the participant's responses. Each experimental run consists of 64 trials derived from a different random-order presentation of the 64 syllables in the stimulus set. Each run will last 16 to 30 min depending on the participant's response time. The total duration of rehabilitation sessions will be five weeks. Experiments are controlled by a desktop PC. Only the intervention group will undergo the rehabilitation and control group will not be informed about details of the intervention study procedure. Speech stimulus process The TFS speech consists of single syllable recorded in / a / C / a / with various 16 consonant format which included Aja, Aka, Ara,…. and it will be pronounced by a native-speaking man. The analogue signals will be converted to digital a 16-bit at 44.1 KHz sampling frequency. The stimulus synthesis process will be performed in MATLAB software and the software will be provided in C programming language. The original bandpass will be filtered into 16 bands of equal bandwidth on a log frequency scale spanning 80 to 8020 Hz. Each Bandpass signal will be decomposed to ENV and TFS by Hilbert transform. The ENV component will be discarded and TFS component will be normalized and TFS component in each band summed lastly creating TFS speech. After rehabilitation, the SNR (50%) using the WIN[AAD35] test, and a binaural TFS test and SSQ questionnaire will be evaluated again in intervention group. Results will be compared in intervention and control groups before and after the rehabilitation program. After rehabilitation, the SNR (50%) using the WIN test, and a binaural TFS test and SSQ questionnaire will be evaluated again. Results will be compared in intervention and control groups before and after the rehabilitation program.[AAD36] Outcomes. SNR (50%): single syllable words in the presence of noise at different signal-to-noise ratios (0, + 4, + 8, + 12, + 16, + 20, + 24) as binaural in two study groups and compare the SNR (50%). Differences in scores before and after rehabilitation training between the two groups will be compared. The Words-in-Noise (WIN) materials were developed to evaluate the ability of listeners to understand words in multitalker babble. The WIN involves in which the level of the noise is fixed and five words are presented at seven signal-to-noise ratios from 24 to 0 dB in 4 dB decrements. The 35 words are spoken by a native male speaker. The metric of interest is the signal-to-noise ratio (S/N) at which recognition performance is 50%, which is a value determined with the Spearman Karber equation see (Extended data: S2). Binaural TFS test: determines the binaural change of phase difference at different frequencies in intervention and control groups before and after rehabilitation training. The correlation between the results of speech perception test scores in the presence of noise with the results of binaural TFS test in the two groups after rehabilitation program will be assessed. SSQ questionnaire score: provided to each group before and after rehabilitation training. Scores between intervention and control groups will be compared (Extended data: S3). Statistical analysis In descriptive analysis of data, central tendency and dispersion indices (mean, median and standard deviation) will be used. Kolmogorov–Smirnov test will be used to test whether two random samples are drawn from the same normal distribution. Otherwise its nonparametric equivalent will be used. Depending on the circumstances, paired t-test and analysis of covariance will be used to compare pre- and post-rehabilitation program. Other analytical tests will be used as required during the data processing phase. SPSS software (V20.0, IBM Corporation, New York, USA) will be used for statistical data analysis and the significance level for all tests will be 0.05. Dissemination The results of our research will be disseminated through presentations at regional and national audiology conferences. The study outcomes will be published through peer-reviewed journals. There is no limit in the publication of the trial results.[AAD37] Monitoring Eight independent audiologists expert [AAD38] who are the academic members of rehabilitation schools in Shahid Beheshti University of Medical Sciences (SBMU) and Iran University of Medical Sciences (IUMS) will monitor patient safety and treatment efficacy. They approved the relevance, clarity and simplicity material of the study. Study status The enrolment of the patients has been performed and the allocation will be performed in the near future. The study started in November 2019 and will continue until December 2020. Discussion[AAD39] Elderly populations are growing rapidly worldwide, and this higher number of older individuals is associated with an increase in prevalence and incidence of age-related disorders. Age-related hearing loss (presbycusis) is one the most common disorders with an increase in age. Speech perception in noisy environments is very serious difficulty with presbycusis, which can impact negatively on the quality of life of individuals. Loss of speech perception in noisy environments with presbycusis is mostly caused by damage of processing of TFS information. In this study, we will attempt to prove by special rehabilitation training based on TFS damage that age-related hearing loss can be re-established.\n\n[AAD1]effects\n\n[AAD2]Good study! This paper has a potential to add to our current knowledge of presbycusis!\n\n[AAD3]loss of speech perception in noise is not considered as a pathophysiologic reason for presbycusis. It is a functional consequence of presbycusis. Pathophysiology can be due to loss of hair cells etc. Please change accordingly.\n\n[AAD4]was\n\n[AAD5]dB HL\n\n[AAD6]dB HL\n\n[AAD7]nervous\n\n[AAD8]were\n\n[AAD9]change all of these from future tense to past tense. The study has been completed so it can be stated in past tense.\n\n[AAD10]You are missing the results section. Please put your results and findings before the conclusions.\n\n[AAD11]Did you prove it? Did your rehabilitation changed or caused outcomes? Did the participants improve their speech recognition in noise. All of theses are your results and should be expanded and explained.\n\n[AAD12]Restoration? Improvement?\n\n[AAD13]Remove comma\n\n[AAD14]The funding agency\n\n[AAD15]Need to spell out the ENV here as it is the first place it shows up in the article.\n\n[AAD16]Same as above\n\n[AAD17]Tasks of\n\n[AAD18]Not necessary, remove please.\n\n[AAD19]Hypothesized\n\n[AAD20]spelling\n\n[AAD21]verbally\n\n[AAD22]in writing\n\n[AAD23]procedures\n\n[AAD24]were\n\n[AAD25]dB HL, please change in the rest of the manuscript\n\n[AAD26]interaural\n\n[AAD27]spell this out please\n\n[AAD28]was based on\n\n[AAD29]This study has been completed, right? Then you should change the whole article to past tense.\n\n[AAD30]Participants were informed that they can withdraw …\n\n[AAD31]What does this mean? I guess you mean they will be confidentially and anonymously saved.\n\n[AAD32]Spell out this please.\n\n[AAD33]Each? Every?\n\n[AAD34]choose\n\n[AAD35]spell this out please.\n\n[AAD36]this paragraph is repeated above.\n\n[AAD37]Remove. Not necessary\n\n[AAD38]Audiology experts\n\n[AAD39]This section is too short and it needs to be expanded.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "5804", "date": "15 Sep 2020", "name": "Parisa Rasouli Fard", "role": "Author Response", "response": "Dear Dr. Danesh Thank you very much for your positive response to our paper. We have now carefully evaluated your comments and revised the manuscript. We hope the new version of the manuscript will be acceptable. In the new version of the study protocol we changed most of your very important comments as you recommended. We started to write the protocol before to enroll the participants but it is ongoing study now. We changed several sentences from future to past or present. The results of our study protocol are not completed yet but primary results indicated speech in noise was improved significantly after rehabilitation training program. In the new version the discussion section was expanded and became more adequate. Best regards" } ] } ]	1	https://f1000research.com/articles/9-582
https://f1000research.com/articles/9-1131/v1	14 Sep 20	{ "type": "Research Article", "title": "A new virtue of phantom MRI data: explaining variance in human participant data", "authors": [ "Christopher P. Cheng", "Yaroslav O. Halchenko" ], "abstract": "Background: Magnetic resonance imaging (MRI) is an important yet complex data acquisition technology for studying the brain. MRI signals can be affected by many factors and many sources of variance are often simply attributed to “noise”. Unexplained variance in MRI data hinders the statistical power of MRI studies and affects their reproducibility. We hypothesized that it would be possible to use phantom data as a proxy of scanner characteristics with a simplistic model of seasonal variation to explain some variance in human MRI data. Methods: We used MRI data from human participants collected in several studies, as well as phantom data collected weekly for scanner quality assurance (QA) purposes. From phantom data we identified the variables most likely to explain variance in acquired data and assessed their statistical significance by using them to model signal-to-noise ratio (SNR), a fundamental MRI QA metric. We then included phantom data SNR in the models of morphometric measures obtained from human anatomical MRI data from the same scanner. Results: Phantom SNR and seasonal variation, after multiple comparisons correction, were statistically significant predictors of the volume of gray brain matter. However, a sweep over 16 other brain matter areas and types revealed no statistically significant predictors among phantom SNR or seasonal variables after multiple comparison correction. Conclusions: Seasonal variation and phantom SNR may be important factors to account for in MRI studies. Our results show weak support that seasonal variations are primarily caused by biological human factors instead of scanner performance variation. The phantom QA metric and scanning parameters are useful for more than just QA. Using QA metrics, scanning parameters, and seasonal variation data can help account for some variance in MRI studies, thus making them more powerful and reproducible.", "keywords": [ "Neuroimaging", "seasonal variation", "MRI QA", "MRI", "reproducibility" ], "content": "Introduction\n\nMagnetic resonance imaging (MRI) is an important data acquisition technology used to unravel the mysteries of the brain, and it is very complex. The exact constitution of MRI signals is not entirely known since it could also potentially be affected by factors such as temperature and humidity variations across seasons1. Notably, a study by Meyer et al. noted that seasonal variations may not even correspond to the four seasons, indicating a complicated relationship between environmental factors and brain functions2; thus, whether or not this variance in MRI is due to scanner effects or biological causes is unclear. This unexplained variance in MRI data hinders the statistical power of MRI studies and affects their reproducibility.\n\nMRI quality assurance (QA) metrics are indicators of the condition of the scanner at the time of a given scan, and are used for quality control in MRI centers3. In cases of significant deviation from the norm, MRI personnel look into resolving underlying hardware or software issues. Otherwise, QA results are not used for anything else, and not shared alongside large shared datasets, such as Human Connectome Project (HCP)4 or the ABCD study where data is acquired across different scanners and potentially affected by scanner idiosyncrasies. It is typically unknown how seasonal and operational factors affect different types of scanning (on phantom and real subjects). We hypothesize that there may be a relationship between the QA metrics of a scanner (obtained on a phantom) and the characteristics of the MRI scans (on human participants), which affect the consecutive data analysis results and drawn conclusions.\n\nThe purpose of this study was to evaluate if phantom data could be used as a useful proxy for overall scanner operational characteristics that can help explain variance in real human subject data acquired using the same MRI scanner on dates nearby phantom QA scans. The first stage of this study analyzed the influence of phantom scanning parameters on signal-to-noise ratio (SNR), which is known to be a fundamental QA metric for MRI. The second stage of this study used SNRs from stage one from phantom data to model morphometric measures obtained using human MRI data.\n\n\nMethods\n\nThis study was approved by the Dartmouth Committee for the Protection of Human Subjects (CPHS 31408). Data collection in the individual studies was approved by the same committee (CPHS 17763, 28486, 29780, 21200 and 30389). All participants gave written informed consent for participation and re-analysis of data.\n\nWe used the data from 206 participants (261 scans) collected from October 30, 2017 to August 28, 2018 who participated in five studies17–20 of three labs at the Dartmouth Brain Imaging Center (DBIC). Participants ranged from 18–64 years of age. There were 78 male and 128 female participants. The MRI scanner used was the 3.0 Tesla Siemens MAGNETOM Prisma whole-body MRI system from Siemens Medical Solutions. Human participant and phantom data were collected using a 32-channel head coil.\n\nThe DBIC collects MRI QA data weekly (typically each Monday) on an agar phantom. For the purposes of this study we did not use DBIC QA estimates but carried out QA using MRIQC BIDS-App7.\n\nQA data, converted to a BIDS dataset (including original DICOM data under sourcedata/), is available as a ///dbic/QA DataLad dataset8. Subject “qa” within that dataset contains data for the agar phantom used in weekly QA scans. QA scans contain a single T1 weighted anatomical image (192×256×256 matrix at 0.90×0.94×0.94mm) and two functional T2* weighted echo planar imaging (EPI) scans (80×80×30 matrix at 3.00×3.00×3.99mm with 200 volumes acquired with time of repetition (TR) of two seconds; not used in this study).\n\nAll data at the DBIC is collected following the ReproIn convention on organizing and naming scanning protocols9. To guarantee that the data would not contain variance caused by different conversion software versions through time, data from all phantom and human subjects was reconverted from raw DICOMs into BIDS datasets using consistent versions of ReproIn/HeuDiConv and dcm2niix10. All phantom QA data was re-converted using ReproIn/HeuDiConv with dcm2niix (v1.0.20171215 (OpenJPEG build) GCC6.3.0), and human data from different studies re-converted using ReproIn/HeuDiConv (v0.5.3) with dcm2niix (v1.0.20181125 GCC6.3.0). HeuDiConv is programmed to automatically extract many acquisition and scanner operation parameters from DICOMs and place them alongside neuroimaging files in the BIDS dataset. For the purposes of this study, a subset of those parameters was selected as variables of interest for analysis based on prior knowledge regarding which variables could potentially affect the collected data (see Table 1). Furthermore, we added seasonal effects by using NumPy 1.18.4 inserting sine and cosine waves into the model with a period of one year to roughly estimate the four seasons; arguably, this was a very simplistic model due to our data’s short duration of under two years. Our data’s limited time range precluded us from using more elaborate seasonal models, and as noted in the introduction, seasonal effects may not exactly correspond to the four seasons. Still, we felt a simplistic representation of seasonal effects could help indicate the possibility of further investigation.\n\nWe used MRIQC (v0.14.2)7 on both the QA phantom and the human data from October 30, 2017 to August 28, 2018. MRIQC provided us with proxy measures of scanner operation characteristics, such as total SNR for anatomicals. Figure 1 presents a correlation structure between all variables of interest for phantom MRI data visualized using Seaborn 0.10.1. DataLad11 with the datalad-container extension12 was used for version control of all digital objects (data, code, singularity containerized environments), and all code and shareable data were made available on GitHub (see Data availability22 and Code availability21) with containers and data available from the ///con/nuisance DataLad dataset. At the moment we have concentrated on analysis of anatomical data, so only T1w images from phantom and human participants were used.\n\nRefer to Table 1 for explanation of abbreviated variables.\n\nWe used DataLad to run a modified version of the simple_workflow container and script13, which extracts certain segmentation statistics of the brain from real human MRI data. These include metrics relating to the accumbens area, amygdala, caudate, hippocampus, pallidum, putamen, thalamus proper, cerebrospinal fluid, and the gray and white matter in the brain. The original simple_workflow container is fully reproducible (frozen to the state of NeuroDebian as of 20170410 using nd_freeze) and uses FSL 5.0.9-3~nd80+1.\n\nThe free open source software facilitating our data preparation was Pandas 1.0.4.\n\nOrdinary least squares (OLS) regression, as implemented in StatsModels Python package (v 0.9.0)15, was used to model target variables of interest. As part of the modeling, certain interdependent variables were orthogonalized to account for possible covariance (in the order presented in Figure 1; seasonal variation data was orthogonalized last) using NumPy 1.18.4. The extent to which an independent variable affects the dependent variable was assessed using a t-test for single-valued variables, and using an F-test for arrayed values (such as patient position in the scanner). Subsequently, the explanatory power of the scanning parameters and characteristics on the phantom QA metric (snr_total) as the dependent variable was evaluated.\n\nNext, a segmentation statistic - gray brain matter on human participant data - was modeled using a proxy QA measure from phantom data (snr_total) and a scanner characteristic of the human participant scanning session (IOPD), demographics (age, gender), and seasonal effects. Gray brain matter was chosen because either gray or white brain matter were deemed likely to yield a statistically significant relationship. Because phantom QA data was acquired typically only each Monday, its value was interpolated in time to obtain values for the dates of human participants scanning. After modeling gray brain matter, other structures (such as white matter, cerebrospinal fluid, and subcortical regions) were analyzed. Our reasoning was that if gray brain matter yielded a significant result, then other brain segmentation statistics could also yield significant results, which could subsequently be investigated.\n\nThe free open source software facilitating the visualization of our model was Matplotlib 3.2.1.\n\n\nResults\n\nWe found that we could describe the total SNR of phantom data well with just a limited set of scanner operational characteristics. The R2 value of the model shown in Figure 2 was 0.533. Multiple variables (day time of acquisition, subject position, and SAR) were statistically significant and all survived false discovery rate (FDR) correction, as shown in Table 2.\n\nR2 value was 0.533, indicating scanning parameters have explanatory power.\n\nFDR, false discovery rate.\n\nGiven that certain scanning parameters and a QA metric were determined to have significant explanatory power in the phantom scans, we decided to proceed to model human participants’ gray brain matter volume using participants' basic demographic data, variables we found significant from phantom data (IOPD), and total SNR. Gray (or white) brain matter was deemed likely to be affected as they are two large “structures”.\n\nAfter correction, the phantom’s total SNR was found to be a statistically significant (p=0.002 and FDR-corrected=0.003) predictor of gray brain matter, as shown in Table 3. Other scanning parameters and subject characteristics found significant were subject age, sex, weight, as well as seasonal variation in data. Note that the orthogonalization carried out for the model in Table 3 was carried out in the order shown from top-to-bottom.\n\nFDR, false discovery rate; SNR, signal-to-noise ratio.\n\nAfter modeling gray brain matter, we modeled the other structures (background, subcortical regions, etc.), and assessed statistical significance for two independent variables of interest in each model: phantom’s total SNR, and seasonal variables. Results in Table 4 show no statistically significant results after FDR correction across structures except phantom total SNR in gray brain matter; notably, seasonal variables in gray brain matter were not significant.\n\nFDR, false discovery rate; SNR, signal-to-noise ratio; CSF, cerebrospinal fluid.\n\nGiven that scanning characteristics, a QA metric and seasonal effects seem to have a statistically significant effect on gray brain matter volume estimates (see Table 3), we decided to compare the fit of the model with and without those independent variables. We did so by removing one of either the QA metric or seasonal effects from the list of independent variables in the model for gray brain matter and observing the fit of the resulting model. From the initial R2 value of 0.242 (Akaike Information Criterion, AIC = 7044; Bayesian Information Criterion, BIC = 7091) with both QA metric and seasonal effects shown in Figure 3, removing the QA metric dropped the model’s R2 value to 0.208 (AIC = 7054, BIC = 7097), and removing seasonal effects resulted in an R2 value of 0.207 (AIC = 7052, BIC = 7092), thus showing that the QA metric and seasonal effects both have similar effects on the fit of the model. However, after removing both the QA metric and seasonal effects, the R2 value drops to 0.185 (AIC = 7058, BIC = 7093), which suggests that they are complementary in terms of explanatory power.\n\nR2 value was 0.242. The full fit plot (in red) shows the plot of all independent variables, whereas the partial fit plot (in pink) shows the plot of only the statistically significant variables. The black plot shows the actual fit of the real data.\n\n\nDiscussion\n\nOur results have indicated that the QA metric of the phantom data can be useful beyond routine monitoring of MRI scanner health. Specifically, our use case demonstrates the viability of using a QA metric to predict variance in estimates derived from human MRI scan data. Our results show also that the following scanning parameters: “patient” (in this case, phantom) position in scanner, day time of acquisition, and specific absorption rate; were statistically significant predictors of the phantom QA metric: total SNR. In turn, the phantom total SNR ratio was a statistically significant predictor of gray brain matter volume, even in the presence of actual data parameters relating to the patient such as age, sex, and weight. It seems that effects depend on the scale of data; in our data sample the uncorrected phantom QA metric provided an explanation for coarse “structures” (such as all of the gray brain matter) but failed to significantly explain any subcortical structure estimate.\n\nFurthermore, we provide further support for the idea that seasonal variations affect human data. The initial R2 value of the QA metric-human data model was 0.242; yet, by removing seasonal effects from the model, the R2 value dropped to 0.207, suggesting that including seasonal effect data is useful for attributing variance in MRI data. It should be noted that the effect of the proxy scanner health metric seems to have a similar magnitude as that of seasonal variation effects, given that the removal of the QA metric from the model also results in a similar decline in the R2 value to 0.208. However, we determined that this effect of seasonal variation is distinct from that from the QA metric, as can be seen by the fact that after removing both the QA metric and seasonal variation from the model, the R2 value declines further to 0.185. This result indicates that both the QA metric and seasonal variations may be important variables to account for when seeking to explain variance in MRI data, given that using the MRI scanner’s phantom QA metric was not sufficient to account for all seasonal variance (at least when gray brain matter was the dependent variable). This result weakly supports the idea that seasonal variation in human data is caused by biological, rather than scanner, effects due to the fact that seasonal variation was not significant for phantom data (i.e. scanner-only) but was significant for gray brain matter in human data.\n\nTo supplement our findings on seasonal variation effects, it should be noted that our seasonal model was very simplistic and was composed of sine and cosine waves. Given that we noticed that it affected our models to some extent, we anticipate developing a more sophisticated model of seasonal effects for a more accurate model.\n\nAn unsurprising observation we made is that positioning of the patient (or phantom) in the scanner accounted for some variance. In the case of the phantom, its significance did not pass the significance threshold after FDR correction, but was very significant for human participants. Meanwhile, patient weight was a very statistically significant predictor for gray matter volume (Table 2); conversely, the size of the phantom remained constant while SNR was affected by position in the scanner. However, prior studies have indicated that brain size strongly correlates with patient height and thus weight16. This result suggests that it is still beneficial to add patient position in the scanner into the models to account for position-specific variance in addition to patient size (weight).\n\nOne interesting negative side-effect of establishing a fully reproducible pipeline, as we have done, is that we cannot share even highly compressed derivatives of the human data, such as morphometric estimates, with the subjects’ participation dates. This information could potentially be used to cross-reference with datasets where such anonymized MRI data is fully shared, albeit with their dates stripped, and thereby used to violate the confidentiality of these subjects’ data. Unfortunately, to our knowledge, no large public datasets are accompanied with phantom QA data scans from the participating sites, which made it impossible to reuse publicly available datasets.\n\nOur use of ReproIn for “turnkey” collection of MRI data into BIDS datasets at DBIC was a highly beneficial methodology shown in our approach. An example of such a dataset is the phantom QA dataset we used in our study. The standardized structure of our dataset collection, from filenames to data format, facilitated our establishment of “meta-datasets” comprising data from multiple studies.\n\nOur investigation has used phantom and human data for the period from October 30, 2017 to August 28, 2018. We are going to compare the model’s predictions on additional data (from other studies and later dates) with actual data to check the generalizability of our established models on future data and feed new data into the model to make it more robust.\n\nAs mentioned in the Methods/Phantom section, we will consider using DBIC QA estimates such as T2* weighted EPI scans instead of MRIQC estimates to evaluate the significance of other sources of QA metrics in reducing variance.\n\nIn this study we used only anatomical (T1 weighted) data. We will investigate temporal SNR, which is a QA metric only available for functional data. Functional QA metrics are an interesting area of investigation in the future, as there is some notion of functional connectivity in resting state data, and statistical estimates from GLM on task data. Functional phantom QA and other scanner characteristics could provide explanatory power to analyses.\n\nThe software we used to derive morphometric estimates of the brain could have been affected by the software used, and an investigation into the effects of conversion software (e.g., FSL) and their versions on morphometric estimates could yield valuable insights.\n\n\nConclusions\n\nWe showed that the scanning parameters and QA metric of phantom data are useful for more than just QA. To maximize the statistical power of MRI studies, we propose using scanning parameters and a QA metric, total SNR, from an MRI scanner’s phantom data to reduce the unexplained variance that exists in MRI data.\n\nFurthermore, we have found that our simple representation of seasonal variation can help explain gray brain matter volume in human MRI data, and deserves further investigation to determine if this effect is truly of a biological origin, as our results weakly suggest. The incorporation of seasonal variables can also help reduce variance in MRI data.\n\n\nData availability\n\nThe human participants’ data used in this study cannot be shared, either in its anonymized form or in the form of derivative estimates of the brain structure volumes, due to the nature of the study which relies on the dates of scanning. As outlined in HIPAA, “The following identifiers of the individual or of relatives, employers, or household members of the individual must be removed to achieve the “safe harbor” method of de-identification: […](C) All elements of dates (except year) for dates directly related to the individual, including […] admission date, discharge date.” To apply for access to the human participant data, you may contact either of the authors (cheng1928c@gmail.com and yoh@dartmouth.edu) and request a Data User Agreement (DUA) to fill out; upon approval, you will be granted access subject to the conditions of the DUA.\n\nHarvard Database: Phantom MRI (Quality Assurance) Data From October 2017 to August 2018 at DBIC. https://doi.org/10.7910/DVN/PFH4FL22.\n\nThis project contains the following underlying data:\n\n- JSON files contain quality assurance metrics to be used as independent variables (AcquisitionTime, SAR, TxRefAmp, & ImageOrientationPatientDICOM) and as the dependent variable (snr_total) for logistic regression analysis\n\n- NII.GZ files that contain a compressed version of an MRI scan in the NIfTI-1 Data Format, and is the basis from which the quality assurance metrics of the JSON file were extracted. These nii.gz files were obtained from the original DICOM files using the HeuDiConv conversion program, which you may use to validate the JSON file’s metrics.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nCode availability\n\nCode available from: https://github.com/proj-nuisance/nuisance\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.386544121.\n\nLicense: Apache License 2.0\n\nThe aforementioned git repositories are also DataLad datasets that provide the complete computing environments used in the study (via git-annex), and contain full history of the analyses recorded in git commits history.", "appendix": "Acknowledgements\n\nWe would like to express our thanks to Professors Chang, Gobbini and Haxby and Dr. Jolly of Dartmouth College’s Psychological and Brain Sciences Department for making their data available for re-analysis, and to Chandana Kodiweera and Terry Sackett for their weekly collection of the phantom QA data, consultation on the details of the MRI hardware characteristics, and support in collecting all other datasets used in the study.\n\nBesides the tools we have already mentioned, we would like to acknowledge numpy, matplotlib, pandas, seaborn, statsmodels, and the other free open source software we used in our study.\n\n\nReferences\n\nDi X, Wolfer M, Kühn S, et al.: Estimations of the weather effects on brain functions using functional MRI – a cautionary tale. bioRxiv. 2019; 443. Publisher Full Text\n\nMeyer C, Muto V, Jaspar M, et al.: Seasonality in human cognitive brain responses. Proc Natl Acad Sci U S A. 2016; 113(11): 3066–3071. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu W, Dong K, Cui D, et al.: Quality assurance of human functional magnetic resonance imaging: a literature review. Quant Imaging Med Surg. 2019; 9(6): 1147–1162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlasser MF, Smith SM, Marcus DS, et al.: The Human Connectome Project’s neuroimaging approach. Nat Neurosci. 2016; 19(9): 1175–1187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEsteban O, Birman D, Schaer M, et al.: MRIQC: Advancing the automatic prediction of image quality in MRI from unseen sites. PLoS One. 2017; 12(9): e0184661. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalchenko Y, Dartmouth Brain Imaging Center: dbic/QA. Zenodo. 2020. Publisher Full Text\n\nVisconti di Oleggio Castello M, Dobson JE, Sackett T, et al.: ReproNim/reproin 0.6.0. 2020. Publisher Full Text\n\nLi X, Morgan PS, Ashburner J, et al.: The first step for neuroimaging data analysis: DICOM to NIfTI conversion. J Neurosci Methods. 2016; 264: 47–56. PubMed Abstract \| Publisher Full Text\n\nHalchenko YO, Hanke M, Poldrack B, et al.: datalad/datalad 0.11.6. 2019. Publisher Full Text\n\nHanke M, Meyer K, Halchenko YO, et al.: datalad/datalad-container 1.0.0 (Version 1.0.0). Zenodo. 2020. Publisher Full Text\n\nGhosh SS, Poline JB, Keator DB, et al.: A very simple, re-executable neuroimaging publication [version 2; peer review: 1 approved, 3 approved with reservations]. F1000Res. 2017; 6: 124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeabold S, Perktold J, Fulton C, et al.: statsmodels/statsmodels: Version 0.8.0 Release (Version v0.8.0). Zenodo. 2017. Publisher Full Text\n\nSkullerud K: Variations in the size of the human brain. Influence of age, sex, body length, body mass index, alcoholism, Alzheimer changes, and cerebral atherosclerosis. Acta Neurol Scand Suppl. 1985; 102: 1–94. PubMed Abstract\n\nChang LJ, Jolly E, Cheong JH, et al.: Endogenous variation in ventromedial prefrontal cortex state dynamics during naturalistic viewing reflects affective experience. bioRxiv. 2018; 487892. Publisher Full Text\n\nJolly E, Sadhukha S, Chang LJ: Custom-molded headcases have limited efficacy in reducing head motion during naturalistic fMRI experiments. Neuroimage. 2020; 222: 117207. PubMed Abstract \| Publisher Full Text\n\nJiahui G, Feilong M, di Oleggio Castello MV, et al.: Predicting individual face-selective topography using naturalistic stimuli. Neuroimage. 2020; 216: 116458. PubMed Abstract \| Publisher Full Text\n\nHaxby JV: Functional Anatomic Studies of Self-Affect: A Multimodal Approach. NIMH Data Archive. Reference Source\n\nCheng C, Halchenko Y: proj-nuisance/nuisance 0.20200520.0 (Version 0.20200520.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3865441\n\nCheng C, Halchenko Y: Phantom MRI (Quality Assurance) Data From October 2017 to August 2018 at DBIC. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/PFH4FL" }	[ { "id": "71690", "date": "06 Oct 2020", "name": "Xiangrui Li", "expertise": [ "Reviewer Expertise MRI" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 topic and has great practical application for the MRI studies. My major concern is the conclusion about effect of variables, such as day time of acquisition, subject position, and SAR, on the brain tissue volume. It is easy to understand the potential effect of these variables on the phantom SNR, while it sounds a big jump to claim the effect on tissue volume. Even if other biological variables are included as confounds, the variability among participants is too large to be accounted by the potential minor effect of the phantom SNR. A better approach may be to test the effect of the phantom SNR on the volume of the same brain, but it seems there is not enough repeats for a single brain in the dataset.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71693", "date": "22 Oct 2020", "name": "Blaise Frederick", "expertise": [ "Reviewer Expertise My area of expertise is fMRI and NIRS technique development", "in particular methods for characterizing and interpreting physiological \"noise\" effects", "and using them to quantify hemodynamics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article explores the relationship between various factors (SNR on a QA phantom, patient positioning, time of year, etc) and the results of quantitation of anatomic MRI images.\nThis is a solid piece of work, well executed and described, which demonstrates a useful method for reducing unexplained variance in MR data, in order to reveal underlying biological phenomena. The methods are very clearly described and reproducible, and all data is available for replication. I look forward to the companion paper on fMRI data. I would recommend accepting the paper as is (other than correcting Figure 2).\nSpecific comments: It would be worth explaining why different versions of dcm2niix were used for the phantom and human data. This is not a major issue - each dataset was processed consistently. It is a little odd however.\nFigure 2 shows a legend and y scale for a partial model fit, however no partial model fit appears in the graph, nor is there any mention of one in the text.\nWhile the correlation between gray matter volume and QA SNR is shown to be significant, it would be useful to mention the sign of the interaction - does apparent GM volume increase or decrease with increasing SNR? This would be an interesting thing to know.\nIt would be a nice addition to the paper to see if there were any correlations between day of the week and any of the patient measures (not possible for the phantom data, since it was done once a week) to complement the Time-of-day and season metrics.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75971", "date": "21 Dec 2020", "name": "Simon Duchesne", "expertise": [ "Reviewer Expertise Neuroimaging", "imaging protocols", "image processing", "biomarkers", "Alzheimer’s disease" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a great study addressing the subject of image quality and its influence on brain morphometric measurements. While most investigators assume that these measurements are indeed near infaillible, work of this kind serves to improve our collective ability at making these measurements ever more robust.\n\nThe authors have used a longitudinal series of AGAR phantom scans as well as human participants being recruited and scanned over the course of one year on a single (Siemens Healthcare Magnetom Prisma) machine. The statistical analysis is strong and demonstrate a possible effect of seasonal variations on the main outcome, namely grey matter segmentation.\nThe paper has a lot of merit, and hopefully these comments will serve to further improve it.\nMajor comments:\nThe choice of the segmentation technique matters a lot. The inherent reliability of the technique may be the biggest inherent contributor to the variability in the results; further, this can be nonlinearly influenced by the scan characteristics. Speaking to the first point, the authors have chosen to use FSL, by which it is assumed they used the FAST technique. The authors are encouraged to read (and cite!) our recent publication on this topic (Dadar et al., NeuroImage 20201 in which we compared various segmentation algorithms (FAST amongst them) on a human phantom dataset composed of repeat scans on multiple scanners of the same individual (albeit without correcting for time of day, SNR, and other metrics discussed here). In this work FAST was shown to be at the lower range of reliability, when compared to other publicly available techniques (an expedited review can be promised if the authors use our own technique!)(these open reviews allow for such clarity in reviewer motivations...). To speak to the second point, it becomes hard to dissociate without further experiments how a software segmentation tool may be more able (i.e. robust) than another at glossing over image quality variations in order to produce results with high reliability. Thus it may be that all of the quality metrics measured do influence the image; but the segmentation technique is able to remove these differences. Thus the agar phantom data is of greater importance here.\n\nTime of day seems indeed to matter a lot, possibly more than shown here. It would appear that most of the phantom scans were taken at the beginning of the week. It is rather typical to do these scans early on the first day of scans (i.e. Monday AM). Yet, multiple reports have shown that there is quite a lot of variability in image quality due to the scanner “warming up” after repeated activity, i.e. over the course of the day. Trefler and colleagues (NeuroImage 2016;2) have done a nice study of this (you could cite them too). Thus in this study this effect may not have shown up in the phantom data, but could possibly affect the human results. Scattering phantom scans could address this issue.\n\nMore generally, it is hard to draw conclusions on these effects with the choice of population. Studying GM in such a large number of different individuals on such a large age range is bound to be highly variable. It would be much better to focus on a subset of individuals (at the very least, only the young adults) and preferably only those with repeated measures, so that we can have a reasonable assumption that the scan-rescan variability should be very low.\n\nMinor comments:\nIt is unclear what kind of interpolation between time points was done on the phantom data QC metric to estimate its value at the time of human scanning;\n\nThe notion of IOPD is not quite clear. Does this mean some individuals were scanned 6 times? Otherwise, what goes into this metric? Patient position in the scanner is mentioned, but the position should always be the same (not like other techniques (e.g. chest x-ray) where you could have supine or prone, etc.)\n\nIt should be discussed that the choice of “seasonal effect” as a variable on participants being recruited over a year masks realities that may bias the study irrespective of the seasonal effect on the MRI scanner itself. For example, a competing hypothesis could be that seasons generated a recruitment bias, with young participants being more available in the summer, while older participants in the winter. One would need to better define seasonal variations and what they entail in terms of signal characteristics that are related to the scanner - yet as independent as possible to the SNR. For example, seasonal effects could serve as a proxy of room temperature, which can be measured (and may even be retrospectively available, depending on the building information system). The latter would affect cooling of the main field - which in turn would affect the main B0 but possibly other sources of thermal noise in the RF apparati. Thus a metric such as room temperature would be better suited than a sinusoidal model of season. Unpublished data from a colleague (J. Doyon, Montreal Neurological Institute) have showed a distinct relationship between room temperature and phantom SNR.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75970", "date": "04 Jan 2021", "name": "Jens Sommer", "expertise": [ "Reviewer Expertise QA procedures and improvement of MRI data in general" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 in a very reasonable way how to estimate previously unexplained variance of MRI studies using data from the regular QA procedure of the radiology department. As the QA procedure is only run once a week the authors interpolate QA data or QA parameters to fit the dates of subject measurements. The original subject data is not available as the combination of individual measurement dates and mri data could disclose details about the subjects. But all of the procedures are available and described in sufficient detail, so it is possible to reproduce the study with local mri data.\nRight now I expect that the article could benefit if you would include more details about the QA procedure of the radiology department. Is the procedure described somewhere? If not, what gel phantom is used (FBIRN)? Do you use a mount for reproducible positioning? Has the phantom been replaced during the study period?\nHave there been any modifications or replacement of parts of the scanner hardware or software?\nDid all subjects undergo the same structural scan protocol, i.e. MPRAGE with identical TR, TI, TE, bandwidth? What kind of filters were applied? Was the volume of interest automatically or manually aligned? As you use some of the positional data for your analysis, it would also be nice to get an idea of the value ranges. Could you add a table or histogram to display the positional data?\nFor the segmentation you used FAST from FSL 5.0.9 (described in Gosh et al., your reference #13). My former colleague L. Eggert found FAST to be more sensitive to varying image quality than other algorithms (Eggert et al.1). In his study he used FAST 4.1 from FSL 4.1.6. I did not check the FAST version used in the container you used but according the FSL version history FSL 5.0.9 should have also included FAST 4.1.\nI would be glad if in a further analysis the statistics would still remain significant when grey matter volume is based on a different segmentation algorithm. But even without this analysis your idea of QA data usage is interesting.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1131
https://f1000research.com/articles/9-1130/v1	14 Sep 20	{ "type": "Opinion Article", "title": "COVID-19 and beyond: a call for action and audacious solidarity to all the citizens and nations, it is humanity’s fight", "authors": [ "Charles Auffray", "Rudi Balling", "Niklas Blomberg", "Myrna C. Bonaldo", "Bertrand Boutron", "Samir Brahmachari", "Christian Bréchot", "Alfredo Cesario", "Sai-Juan Chen", "Karine Clément", "Daria Danilenko", "Alberto Di Meglio", "Andrea Gelemanović", "Carole Goble", "Takashi Gojobori", "Jason D. Goldman", "Michel Goldman", "Yi-Ke Guo", "James Heath", "Leroy Hood", "Peter Hunter", "Li Jin", "Hiroaki Kitano", "Bartha Knoppers", "Doron Lancet", "Catherine Larue", "Mark Lathrop", "Martine Laville", "Ariel B. Lindner", "Antoine Magnan", "Andres Metspalu", "Edgar Morin", "Lisa F.P. Ng", "Laurent Nicod", "Denis Noble", "Laurent Nottale", "Helga Nowotny", "Theresa Ochoa", "Iruka N. Okeke", "Tolu Oni", "Peter Openshaw", "Mehmet Oztürk", "Susanna Palkonen", "Janusz T. Paweska", "Christophe Pison", "Mihael H. Polymeropoulos", "Christian Pristipino", "Ulrike Protzer", "Josep Roca", "Damjana Rozman", "Marc Santolini", "Ferran Sanz", "Giovanni Scambia", "Eran Segal", "Ismail Serageldin", "Marcelo Bento Soares", "Peter Sterk", "Sumio Sugano", "Giulio Superti-Furga", "David Supple", "Jesper Tegner", "Mathias Uhlen", "Andrea Urbani", "Alfonso Valencia", "Vincenzo Valentini", "Sylvie van der Werf", "Manlio Vinciguerra", "Olaf Wolkenhauer", "Emiel Wouters", "Rudi Balling", "Niklas Blomberg", "Myrna C. Bonaldo", "Bertrand Boutron", "Samir Brahmachari", "Christian Bréchot", "Alfredo Cesario", "Sai-Juan Chen", "Karine Clément", "Daria Danilenko", "Alberto Di Meglio", "Andrea Gelemanović", "Carole Goble", "Takashi Gojobori", "Jason D. Goldman", "Michel Goldman", "Yi-Ke Guo", "James Heath", "Leroy Hood", "Peter Hunter", "Li Jin", "Hiroaki Kitano", "Bartha Knoppers", "Doron Lancet", "Catherine Larue", "Mark Lathrop", "Martine Laville", "Ariel B. Lindner", "Antoine Magnan", "Andres Metspalu", "Edgar Morin", "Lisa F.P. Ng", "Laurent Nicod", "Denis Noble", "Laurent Nottale", "Helga Nowotny", "Theresa Ochoa", "Iruka N. Okeke", "Tolu Oni", "Peter Openshaw", "Mehmet Oztürk", "Susanna Palkonen", "Janusz T. Paweska", "Christophe Pison", "Mihael H. Polymeropoulos", "Christian Pristipino", "Ulrike Protzer", "Josep Roca", "Damjana Rozman", "Marc Santolini", "Ferran Sanz", "Giovanni Scambia", "Eran Segal", "Ismail Serageldin", "Marcelo Bento Soares", "Peter Sterk", "Sumio Sugano", "Giulio Superti-Furga", "David Supple", "Jesper Tegner", "Mathias Uhlen", "Andrea Urbani", "Alfonso Valencia", "Vincenzo Valentini", "Sylvie van der Werf", "Manlio Vinciguerra", "Olaf Wolkenhauer", "Emiel Wouters" ], "abstract": "Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to a subgroup of coronaviruses rampant in bats for centuries. It caused the coronavirus disease 2019 (COVID-19) pandemic. Most patients recover, but a minority of severe cases experience acute respiratory distress or an inflammatory storm devastating many organs that can lead to patient death. The spread of SARS-CoV-2 was facilitated by the increasing intensity of air travel, urban congestion and human contact during the past decades. Until therapies and vaccines are available, tests for virus exposure, confinement and distancing measures have helped curb the pandemic. Vision: The COVID-19 pandemic calls for safeguards and remediation measures through a systemic response. Self-organizing initiatives by scientists and citizens are developing an advanced collective intelligence response to the coronavirus crisis. Their integration forms Olympiads of Solidarity and Health. Their ability to optimize our response to COVID-19 could serve as a model to trigger a global metamorphosis of our societies with far-reaching consequences for attacking fundamental challenges facing humanity in the 21st century. Mission: For COVID-19 and these other challenges, there is no alternative but action. Meeting in Paris in 2003, we set out to \"rethink research to understand life and improve health.\" We have formed an international coalition of academia and industry ecosystems taking a systems medicine approach to understanding COVID-19 by thoroughly characterizing viruses, patients and populations during the pandemic, using openly shared tools. All results will be publicly available with no initial claims for intellectual property rights. This World Alliance for Health and Wellbeing will catalyze the creation of medical and health products such as diagnostic tests, drugs and vaccines that become common goods accessible to all, while seeking further alliances with civil society to bridge with socio-ecological and technological approaches that characterise urban systems, for a collective response to future health emergencies.", "keywords": [ "COVID-19", "Pandemic", "Coalition", "Coronavirus", "SARS-CoV-2", "Solidarity", "Systemic crisis", "Systemic response" ], "content": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19)\n\nIn just a few short months, the new coronavirus SARS-CoV-2 causing COVID-19 has shaken planet Earth and its inhabitants in a chain of events starting in the megacity of Wuhan in China's Hubei Province whose speed and severity seem to have taken citizens and political and economic leaders by surprise. The COVID-19 epidemic turned into a pandemic affecting almost all of the 197 states recognized by the United Nations and their associated territories. The number of individuals affected, which is probably largely underestimated because of the large number of asymptomatic forms that are potentially contagious, exceeds 24 million and caused the death of more than 820,000 persons as of August 281–8. The pandemic shows no sign of slowing down globally, entering the range of casualties of the most severe influenza pandemics of the past 50 years. It has already provoked important secondary increases in chronic and psychiatric health conditions. This has locally saturated the health-care systems of the most affected countries, such as Italy, Spain, France, then Great Britain and the United States of America, from which the political leaders thought they were safe, and which were in turn severely impacted. This is particularly true in areas where major outbreaks of contagion have caused explosive spread of the SARS-CoV-2 coronavirus9–16. This phenomenon has started to reach into areas with poorly developed health infrastructures, with the risk of decimating entire populations, particularly in Africa, South America and South-East Asia. Most African countries, with their experience of recent epidemics such as Ebola, have been quick to take strict containment measures, and have been much less affected so far because of the youthfulness of their populations17. On the contrary, Mexico, Brazil and many other countries in South America are experiencing an epidemic outbreak, as is the case also in South Africa, the Middle-East, India and South-East Asia1,2.\n\nIn many of the structures where elderly, dependent people are cared for, such as the Établissement d'hébergement pour personnes âgées dépendantes (EHPADs; Accommodation facility for dependent elderly people) in France, the SARS-CoV-2 virus has caused large numbers of COVID-19 casualties. It is only recently that the victims who died in these facilities have been listed and included in a daily check-up that is constantly increasing, and we will only know the number of those who died undetected at home by comparing this year's mortality with that of previous years, as was the case during the 2003 heat wave in Western Europe. These various factors, combined with the very rapid worsening of the disease - which in its severe forms leads to the serial failure of different organs18–20, most often starting with the lungs and producing respiratory distress - have led to an underestimation of the severity and extent of the pandemic. In order to understand how the current health emergency came about, to imagine solutions to overcome this crisis, to prevent it from continuing and worsening in successive waves due to failure to comply with containment rules, or from reoccurring with other emerging pathogens, it is essential to re-examine with the necessary hindsight the conditions that have allowed the dazzling upheavals we are witnessing.\n\nAmong the factors that have accelerated the spread of SARS-CoV-2 and transformed the initial focus of the COVID-19 epidemic into a pandemic are the considerable increase in air and land transport or the swift expansion of megacities that have become unbreathable over the last two decades due to air pollution, as has been pointed out by many commentators. As the pandemic continues to develop, it is still difficult to identify its future course. It could resemble that of the previous flu epidemics with one or several additional waves of similar or increased intensity occurring after the current one due to insufficient immunity of the population, especially given the unequal distribution of COVID-19 within regions and countries1,2. Transfers of people from one region to another could lead to the emergence of new clusters of propagation, following one another with a temporary time lag. The pandemic may radiate to all the destinations permitted by passenger transport, and therefore unpredictably, without being subject to the physical constraints of atmospheric flows that we have gradually learned to model using massive data from measuring stations and satellite observations to feed our weather forecasts.\n\nThe SARS-CoV-1 epidemic in 2002–2003 was largely confined to China by the drastic measures taken by the Ministry of Health, affecting just over 8,000 people, 774 of whom died, mainly in the Canton region, but also in some 30 countries such as Canada, where it was carried by travellers21. The MERS-CoV epidemic mainly affected Saudi Arabia and Middle Eastern countries such as Jordan, before being spread to South Korea by a single traveller, causing an outbreak of contagion and the death of 186 people, then affecting some 30 countries. Between 2002 and 2005, this highly pathogenic virus caused the death of nearly 500 people among the 1,400 or so infected people who could be identified22.\n\nLike all these coronaviruses and viruses in general, SARS-CoV-2 is not a self-propagating unidentified flying object. It is an inert, newly identified viral object, which is only activated when it comes into contact with animal or human cells, from which it diverts its functions in order to multiply and transmit itself through contact first between animals and humans, and then similarly between humans. SARS-CoV-2 was identified and characterized as early as the beginning of 2020, which made it possible to rapidly develop tests to detect it.\n\nComparison of strains isolated from different affected regions and countries showed that SARS-CoV-2 is a distant cousin of SARS-CoV-1, their common ancestor dating back to the 13th century. These studies strongly suggest that SARS-CoV-2 originates from a reservoir of coronaviruses that have been prevalent in bats for at least 40–70 years, from which new coronaviruses may in turn emerge23. SARS-CoV-2 was detected initially in Wuhan. However, since the three different clads that are prevalent in Italy are distinct from the one that predominates in France, it is well possible that several closely-related variants have come to infect humans during the recent months and years, and burgeoned simultaneously in several locations, from which it irradiated to other close or distant places through infected travellers. Indeed, retrospective exploration of health records have identified early cases during the third trimester of 2019 that remained undetected or asymptomatic24.\n\nThis reversal of perspective allows us to understand that it is the organization of our economic and social life, as it has developed over the past few decades, that is at the root of the spread of these epidemics and the current pandemic. It affects a much larger number of people because of the higher contagiousness of SARS-CoV-2, but it causes proportionally fewer deaths in the population, because it is fortunately much less virulent than SARS-CoV-1 or SARS-MERS. A notable commonality between the development of these three epidemics of coronavirus disease is that their spread from the initial outbreak has occurred locally and remotely through human contact and has spread to other countries mostly through air and land travel. Epidemiological studies will help identify what other factors may have contributed to the high contagiousness of SARS-CoV-2, such as air pollution with fine particles, the simple diffusion of droplets produced by speech into ambient air, or even of the viral particles themselves25,26. Availability of population-based health status information provided by individuals in the field on real time through secure channels27,28, and platforms to provide access to virus and serocoversion testing29,30 will be essential for identifying local and regional outbreaks of SARS-CoV-2 and avoid further spread of COVID-19. Fast-prototyping and open-source approaches complement these actions by providing innovative, low-cost solutions that can be of particular interest for affected low-income countries.\n\n\nThe COVID-19 pandemic calls for safeguards and remediation measures through a systemic response\n\nThe COVID-19 pandemic due to the outbreak of SARS-CoV-2 thus impacts on biological, social, economic and human relations. It challenges the concepts of individuality and temporality, which are entangled at all scales. It forces us to change our relationship with nature, the built environment, education, health and death. It challenges our working habits and way of life, our understanding of living organisms and their relationships to the environment, as well as our political, social, economic, production and health organizations.\n\nOver the centuries brilliant human minds have explored the formation, organization and evolution of matter at the smallest scales, of galaxies at the largest scales, and of living systems at intermediate scales. In biology and medicine, we have not yet learnt sufficiently from the experience of astronomers and physicists who understood early on that complex systems require the generation of enormous amounts of data to deconvolute complexity, open science international collaborations supported by common infrastructures and open data sharing to enable diverse types of analyses and multiscale modeling. The web developed by the European Organization for Nuclear Research (CERN) to empower multiple centres to generate, access and analyze big data and exchange results, together with complementary efforts developing communication standards, enabled democratization of Internet. We need to organize similarly to understand complex and dynamic microbes-host-environment interactions, and resolve the pandemic threatening humanity31,32.\n\nThe complexity of SARS-CoV-2 and COVID-19 calls for a global, systemic approach. The coronavirus crisis, strikingly illustrates, as complexity theory proposes, that when a complex system is increasingly fragmented it eventually disintegrates33. But with proper integrative and analytical techniques the pieces can be assembled, to a certain extent, into a comprehensive and understandable whole. The current health crisis can thus be understood and resolved only by a systemic approach placing the analytical method in the context of each individual dynamic and longitudinal big data cloud34 to unveil dysfunctions and propose actionable solutions. The systemic approach links the complexities that interact within the same person in relation to her/his environment. This way of thinking transcends the barriers between different scientific and medical disciplines and the humanities.\n\nIt is the conjunction of the systemic and analytic approaches that will achieve the 17 sustainable development goals set by the United Nations responding to the great challenges of the 21st century: \"poverty, inequality, climate, environment, prosperity, peace and justice”35 while leaving no one on the side of the road, as “every life has similar value”36. Indeed, in accordance with article 27 of the Universal Declaration of Human Rights of 1948: \"Everyone has the right to freely participate in the cultural life of the community, to enjoy the arts and to participate in scientific progress and the resulting benefits.\" The third sustainable development goal is to “ensure healthy lives and ensure wellbeing for all at all ages”, with a special attention to the increasing burden of infectious and non-communicable diseases (NCDs) in low, middle, and high-income countries. It is clear that an infectious disease arising in any country can affect the economies of many other countries. To attain this ambitious goal and combat the current and future pandemics, a systemic approach to infectious diseases is essential to generate the data necessary for deciphering their complexity, develop global science collaborations with openly shared data, and to undertake massive joint investments for health system development that lead to intersectoral health interventions that are predictive, preventive, personalized and participatory37,38.\n\nCOVID-19 highlights human limitations at the individual, social and political levels. They have caused us to lose sight of these sustainable goals and could call into question our ability to achieve them. However, measures that seemed impossible to implement within one or more generations can actually be implemented in a very short time when inertia and bureaucracy are set aside for immediate action as shown by scientific and therapeutic programmes on COVID-19 performed in record time. We will no longer be able to cite as impossible the images of clear waters; animals that repopulate spaces left free by humans; deserted, pollution-free and silent cities. The COVID-19 crisis and the global response it triggered demonstrate that there can be the will to address all daunting challenges with global systemic approaches and strategic partnerships. Hopefully, history will mark the coronavirus health crisis as a turning point in moving forward towards solving others of the 21st century greatest challenges.\n\nThe health crisis is causing astounding and immediate changes. The aircraft carrier Charles de Gaulle, flagship of the French Navy, was forced to return to its home port due to a COVID-19 outbreak with more than half of the sailors infected39. Such is the case aboard cruise ships and in a number of nursing homes where elderly residents are among the most vulnerable to the disease and most likely to progress to death40. All these images and initiatives contrast with the short-term views and the unpreparedness of many political and economic leaders who suddenly contradicted the positions they had previously adopted. The coronavirus struck them like an electric shock, causing a painful awakening. They were accustomed to thinking and deciding in a binary manner without perceiving the complexity and uncertainty of reality. In several countries the coronavirus crisis has become bipolarizing, e.g. by demanding whether to take precautions or to ignore the disease and move on with normal life and supposedly immediate economic recovery, writing off those who die as casualties of financial success. The billions that have not been wisely invested in health research and logistics for prevention now necessitate mobilization of trillions to face the greatest economic crisis that occurred since 1870 according to the World Bank41, without a guarantee that this will quickly succeed in addressing COVID-19 challenges.\n\nVocal segments of the media nourished by these events feed a self-perpetuating loop of hype for exaggerated promises or miraculous remedies promising a rapid return to the conditions existing before the start of the epidemic. The lessons learnt from previous epidemics before globalization and social media are forgotten: who remembers the million deaths caused by the second wave of flu during the winter of 1969–1970, and the two million deaths of the 1956–1958 flu pandemic? Or the multiplicity of more devastating flu pandemic waves of 1918–1920? Antibiotics and antivirals have helped diminish the burden of these diseases, although their successful use is being increasingly compromised by the development of resistant strains. The celebration of the 40th anniversary of the eradication of smallpox was paradoxically obscured by a pandemic resulting from reduced alertness that led to the unfortunate reduction in will and dismantling of logistical, scientific and medical means to deal with infectious diseases outbreaks.\n\n\nThe Olympiads of Solidarity and Health: open and citizen science for collective advanced intelligence\n\nAll over the world, countless individual initiatives connect people, transcending social, cultural, religious or political barriers. Confined individuals applaud the extraordinary mobilization of the caregivers who are on the front lines of service and danger. This planetary movement of solidarity extends to all those who perform the ordinary but essential services of life: food, water, communication and energy supply, maintenance and waste management, transportation and security, and teaching. Businesses and citizens from all walks of life self-organize, providing ingenious solutions to the challenges of these services in a world stalled by COVID-19.\n\nWhat will be the course of this pandemic at global, regional, local and micro scales? Its development in insufficiently equipped settings could be devastating. Modelling COVID-19 data is using the same principles as those used in weather forecasting. From massive data, credible scenarios are identified in the short and medium term. Uncertainty remains, however, as to how the pandemic will unfold in different parts of the world, with the lack of context-aware data and knowledge gaps about the consequences of blunt public health interventions fueling real anxiety for the future.\n\nAs Charles Nicolle described nearly a century ago, all infectious diseases appear, grow and disappear42. They can also reappear later or in new forms if our vigilance decreases or conditions are suitable. While more than 7 million cases of SARS-CoV-2 infection and 400,000 COVID-19 deaths were recorded in the world when the initial version of this paper was completed on June 18, these numbers have increased rapidly to over 24 million recorded infections and 830,000 deaths as of August 281. WHO indicated the coronavirus may \"never disappear”, echoing signs that COVID-19 is becoming endemic43. It is therefore imperative that the current, overwhelming pandemic wave be used to understand how the virus is transmitted. So far, while there are some data pointing to risk factors for death, risk factors for acquiring the virus are largely unknown and their study is limited to superficial observational inferences. However, the epidemiology of the disease strongly suggests that there are gender, genetic as well as environmental risk factors44–46.\n\nA large part of the international scientific and medical community mobilized to accelerate understanding of SARS-CoV-2 and COVID-19. Researchers, engineers, biologists and doctors have united to facilitate the development of insights into the physio-pathology and epidemiology of COVID-19 in different environmental contexts to fight against SARS-CoV-2 and its devastating effects. A first map of interactions between the virus and human cells is available47. Lessons should be learnt from the experience acquired during this crisis from different parts of the world, and the same fast reaction capabilities should be implemented and maintained to prevent and contain new pandemics in non-pandemic times. Many scientific and clinical areas are challenged to rapidly provide industry and health systems with credible leads to design and implement, within a few weeks and months, research projects which formerly took many years to complete. These projects involve patients and their families, authorities from different sectors including health and environment, doctors, researchers, engineers, manufacturers and all stakeholders in economic and social life. Thus, the “Olympiads of Solidarity and Health\"48 have emerged spontaneously in response to this crisis as self-organized activities based on open and citizen science committed to the free dissemination of knowledge according to best practices for the conduct of ordinary life as well as research and the dissemination of the results. The COVID-19 crisis is providing a model for how to deal with the many other crises facing society today: to create citizen science-based Olympiads and use their integrated and collective ingenuity to solve the many intractable challenges.\n\n\nThe COVID-19 Olympiads: advanced collective intelligence to accompany the ongoing metamorphosis\n\nThe rapid use of “COVID-19 Olympiads” results by industrial players will serve as a basis for developing and providing doctors and sectors that influence health with effective treatments and interventions to prevent, detect, treat and cure COVID-19. The success of this endeavor will shorten the development time of treatments for other diseases and effective strategies to reduce vulnerability to disease, particularly in the context of cities where the high concentration of people and ecological disruptions increase the risk of emergence of new diseases. Such efforts will inform and contribute to the necessary transformation of our health and urban systems, reducing both healthcare costs that are becoming unmanageable and healthcare need. The power of systems medicine approaches to COVID-19 will be applied to many other diseases and ultimately to population health, thus gradually transforming 21st century medicine.\n\nMedical and computational technologies generate the data needed to make sense of massive medical imaging, genomic and functional studies, as well as the continuous streams of big data generated by connected devices recording human activities and health statuses in real time49. By combining human knowledge with the computing capabilities of machines, we are able to implement a collective \"advanced intelligence\"50 taking into account that SARS-CoV-2 affects multiple organs sensitive to environmental variations. This often begins with infection of the lungs, heart and brain, but the liver, kidneys, intestine or skin are other possible entry points for the virus. Old age and associated comorbidities such as diabetes and obesity are aggravating factors for COVID-1951. Understanding coronavirus-receptor interactions and susceptibilities as well as inflammatory cascades and organ damage will lead to new and more effective solutions to combat this disease, and support advocacy for whole-of-society interventions to prevent these comorbidities.\n\nThis health-digital convergence underlies the emergence of a systemic approach to medicine, and a shift from reactive medicine, intervening after the appearance of symptoms or pathogens, to proactive medicine capable of detecting warning signs that can lead to illness. Intervention at multiple scales from individual to environmental becomes possible early to counteract the development of the disease, thus maintaining health and wellbeing of each person. The attention focused on early diagnosis, prevention and resistance will become the norm, and will require unprecedented collaborations, bridging systems biology data with data from diverse disciplines and sectors to inform intervention development tailored to different contexts.\n\nOne concrete example of the response to the COVID-19 challenge within two weeks of the first reported coronavirus infections is the clinical trial initiated by the Institute for Systems Biology (ISB) together with Swedish Hospital within the Providence Saint Joseph Health (PSJH) system, the 3rd largest, non-profit healthcare systems in the United States, a speed made possible by the coronavirus crisis52. This study is generating longitudinal high-dimensional data for each patient to deconvolute COVID-19 complexity by collecting billions of measurements on each individual. The study protocol includes genome/phenome analyses, very deep analyses of each individual’s immune responses53 and is pioneering a new approach to viral diagnosis employing saliva with an assay that is simple, rapid, inexpensive, and easy to implement (Figure 1).\n\nDepth of the data collected and the analyses performed for each individual. The Institute for Systems Biology (ISB)/Providence St Joseph Health (PSJH) COVID-19 clinical trial started with 200 patients divided into four categories ranging from mild to most severe disease. Blood is collected at 3 times: diagnosis, 7–10 days later and after release to home; and also on patients that recovered at home. Viral infection is measured for all major organ systems. The study will be eventually extended to 600 or more COVID-19 patients at PSJH and at different locations. It employs high-dimensional analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected individuals: genome/phenome, electronic health records, organ-specific blood proteins to determine damaged organs, and deep analyses of both adaptive and innate immune responses to identify disease trajectories as well as protective epitopes key to successful vaccines. Each patient’s coronavirus RNA genome will be sequenced and their variation correlated with the patient’s disease phenotypes and genome sequence. Autopsy materials from patients who have died allow identification of the various sites (and cell types) of virus infection. One thousand individuals that experienced almost two years of scientific wellness monitoring through genome/phenome analyses serve as healthy controls. These individuals provide a unique opportunity for visualization of the transition from wellness to COVID-19 for the presumptive 20-30 individuals that will have contracted COVID-19. The study will use saliva-based, high-throughput coronavirus assays for reentry in ordinary life: one very simple device and two technicians make it possible to analyze 20,000 saliva samples a day rapidly and for $2 a test. Moreover, a simple, cheap and reliable home microfluidic device is under development to determine whether one is infected with coronavirus. BCR: B-Cell Receptor; TCR: T-Cell Receptor; TBD: To Be Determined. This image is reproduced under the terms of the Creative Commons Attribution 4.0 International licence (CC-BY 4.0).\n\nThis approach has attracted partners from academia, pharmaceutical, data generation, technology and diagnostic companies representing virtually all aspects of healthcare, already including 13 different companies and academics from 5 different institutions joining forces to address a hard societal problem. This is creating a COVID-19 ecosystem with two components: 1) a platform containing a very large amount of data from COVID-19 patients and 2) partners that have agreed to immediate data release for all to be able to analyze these big data, and no initial intellectual property constraints, remarkable industrial concessions driven by the COVID-19 emergency. The same study protocol is being implemented in partnership with ISB and the European Institute for Systems Biology and Medicine at Policlinico Gemelli and San Filippo Neri Hospital in Rome, Italy, supported by an ecosystem of 6 academic and 10 industrial partners, and by the network of translational research centers coordinated by the Jiao Tong and Fudan universities in Shanghai, China, supported by an ecosystem of 14 academic and 12 industrial partners. These COVID-19 ecosystems will, after the crisis abates, evolve to take on similar systems-driven clinical trials on major infectious and NCDs and then quickly move seamlessly to the systems medicine analysis of millions of patients then healthy citizens. When extended to other sites, this approach will lead to a transformation of 21st century medicine and healthcare globally. We recognise the inequities in health experienced globally arise not only from inequities in access to healthcare advances but also from exposures in the natural and built environments that determine health. Therefore, a critical role of this evolving ecosystem is to contribute to building stronger health systems for primary prevention54.\n\nAnother concrete example is the OpenCovid19 Initiative on Just One Giant Lab55, an online platform designed for open science, responsible innovation and continuous learning, empowering partners with academic labs, companies, startups, foundations, non-governmental organizations and public services to create participatory research programs for understanding and solving health, environmental, social and humanitarian issues. It currently coordinates the OpenCovid19 program fostering open-source and low-cost tools and methodologies that are safe and easy to use in response to the COVID-19 pandemic. Launched in early March 2020, powered by a global community of more than 4000 volunteers and experts who create solutions to better prevent, detect, and treat COVID-19, and to help forecast evolution of the pandemic. Over the course of three months, the community created 60 projects, 19 of which were awarded micro-grants through an open community peer-review system (Figure 2). To enable this collaborative citizen science process, researchers at the Center for Research and Interdisciplinarity (CRI)56 work with the Open Source Pharma Foundation (OSPF;57), that is providing an open research platform to support the development of devices, tests, drugs and vaccines to fight the coronavirus pandemic, leveraging the Open Source Discovery community57,58. Together, they launched an inclusive electronic survey that allows everyone not only to contribute their medical and well-being data in a distributed, anonymous fashion but also to debate and to contribute their own survey queries throughout the lifetime of the study, allowing participants to play an active role and tailor the survey to their own concerns and communities. Further, all raw anonymized data will be openly shared with the participants, allowing them to draw their own conclusions, discuss collectively and make informed suggestions for policy implementations locally and globally.\n\na. Overview of popular projects on the Just One Giant Lab online platform designed for open science, responsible innovation and continuous learning. Launched early March 2020, its OpenCovid19 Initiative develops open-source and low-cost tools and methodologies that are safe and easy to use in response to the Coronavirus disease 19 (COVID-19) pandemic. The OpenCovid19 program is powered by a global community of 4000+ volunteers and experts who create solutions to better prevent, detect, and treat COVID-19, and to help forecast evolution of the pandemic. These images are reproduced under the terms of the Creative Commons Attribution 4.0 International licence (CC-BY 4.0). b. Skill map of the OpenCovid19 community, visualized using Gephi 0.9.2 (https://gephi.org). Skills are linked if they appear in a common project. Node size represents degree. Topological modules are colored using the modularity algorithm. Skills encompass a wide range of disciplines, from education and game design to public health, from project management to data science, or from web development to bioinformatics and biology.\n\nThe experience acquired during the current crisis will thus be extremely useful for the treatment of other communicable diseases, as well as for many NCDs or genetic diseases. All biomedical research domains will be transformed through foundations for a \"World Alliance for Health and Well-being\"59. These will be developed in partnership with United Nations organizations such as WHO2, UNESCO3, UN Habitat60, their members, industrial partners and civil society. Although health is a right to all just as is freedom from hunger, we need to recalibrate the balance between health as a right and health as a business. In the last few decades, we have seen the balance tilting in the wrong direction of ever-increasing commodification of health services. Indeed, for decades, the advantages and disadvantages of an emphasis on the open commons or restricted anti-commons has fueled intense debates61–65. We need to proactively embrace open source development of tests, drugs and vaccines as common goods of humanity, supported through innovative and realistic mechanisms to ensure their economic sustainability57.\n\nThere will be no “day after” when all organizations and people confined voluntarily or by necessity resume their activities and habits as if nothing had happened. Our humanity, which is responsible for a cascade of events that turned into a global cataclysm, has responded by a collective attack on COVID-19. As the result, it is undergoing a metamorphosis from which a different world is emerging. Its success and future depend on us, for the better or for the worse.\n\nSince Antiquity, every doctor has taken the Hippocratic oath at the end of medical studies. Its universally recognized principles are not to harm patients, but to relieve them while respecting their autonomy, will, integrity, dignity, and privacy in full confidentiality, without discrimination and without abusively prolonging the agonies or deliberately causing death. The goals are to promote health in all its dimensions, to be faithful to the laws of honor and probity, and to preserve necessary independence in decision making with the active participation of the patients for their benefit. This requires dedication to public education, starting in primary schools66.\n\nThe oath of the scientists echoes and complements it prominently at the end of the luminous introduction by Michel Serres of Le Trésor - Dictionnaire des Sciences67: \"For what depends on me, I swear: not to serve my knowledge, my inventions and the applications that I could draw of these to violence, destruction or death, the growth of misery or ignorance, enslavement or inequality, but to devote them, on the contrary, to equality between men, their survival, their elevation and their freedom.\"\n\nThe world that is being born from the metamorphosis caused by the COVID-19 pandemic will depend on our ability to combine these two oaths, a requirement to leverage the power of systems approaches to complex problems and the incredible transformation potential of the collective of the concerned. We hope that the generations that follow us will grow and flourish in this uncertain world and make it more fraternal, harmonious and human than the one we built, leading to the activation and the rampant spread of the new and destructive coronavirus. This will be the case only if we always remain united and vigilant during and after the health crisis that we are all going through together. This is the fight of all humanity, and we must give primary attention to the most fragile of our citizens, while making concerted efforts to reduce modifiable vulnerabilities, to preserve our most precious common good, health.\n\nAs the poet Antonio Machado put it so well, the trajectory that we will follow to embark on this extraordinary human adventure depends primarily on us: \"Caminante, no hay camino, se hace camino al andar\" (wanderer, there is no path, the path is made by walking68).\n\n\nA World Alliance for Health and Wellbeing takes action through a coalition in response to the COVID-19 pandemic\n\nThe path is not drawn in advance, we will have to adapt wisely to future circumstances. Let us show audacious and determined solidarity in taking the systems-driven measures that are essential to overcome the obstacles that hinder us. Let us bring a lasting systemic response to the global systemic crisis that challenges our certainties. There is no alternative but action. Meeting in Paris in 2003, we set out to \"rethink research to understand life and improve health\"69. We now meet virtually through links consolidated over decades and, as a first step, have formed an international coalition to thoroughly characterize patients with COVID-19 during the pandemic using a shared study protocol and informed consent form (Figure 1). All results will be publicly available with no initial claims for intellectual property rights. This World Alliance for Health and Wellbeing has thus entered into concrete action with the mission to take a systems approach to understanding COVID-19 and to catalyze the creation of medical and health products such as diagnostic tests, drugs and vaccines that become common goods accessible to all on a sustainable basis. In addition, we recognise that many factors that determine health lie outside healthcare and that a growing majority of the world population live in cities, where the interwoven relationship between people, their environments, and emergence of disease is most apparent. As such, we further seek alliances with diverse sectors adopting systems approaches across disciplines, bridging biomedical lessons learned with the complex socio-ecological and technological systems that characterise urban systems, harnessing human and social capital for a collective response to future health emergencies.\n\n\nData availability\n\nNo data are associated with this article", "appendix": "Acknowledgements\n\nWe thank Bertrand De Meulder (EISBM) for help in formatting the references, colleagues and citizens for useful comments on the manuscript.\n\nA previous preprint version of this manuscript is available from SSRN (https://dx.doi.org/10.2139/ssrn.3630412)\n\n\nReferences\n\nUniversity CfSSaEaJH: COVID-19 Dashboard. 2020. Reference Source\n\nWHO: COVID-19 Pandemic. 2020. Reference Source\n\nUNESCO: COVID-19 Open Science and Reinforced Scientific Cooperation. 2020. Reference Source\n\nEMBL-EBI: COVID-19 Data Portal. 2020. Reference Source\n\nGovernment U: Public Health Information - Coronavirus. 2020. Reference Source\n\nOpenAIRE: COVID-19 Gateway. 2020. Reference Source\n\nZenodo: Coronavirus Disease Research Community. 2020. Reference Source\n\nNIH: Health Information - Coronavirus. 2020. Reference Source\n\nGambaro F, Behillil S, Baidaliuk A, et al.: Introduction and early spread of SARS-CoV-2 in France. medRxiv. PrePrint. 2020. Publisher Full Text\n\nSalje H, Tran Kiem C, Lefrancq N, et al.: Estimating the burden of SARS-CoV-2 in France. Science. 2020; 369(6500): 208–211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBi Q, Wu Y, Mei S, et al.: Epidemiology and transmission of COVID-19 in 391 cases and 1286 of their close contacts in Shenzhen, China: a retrospective cohort study. Lancet Infect Dis. 2020; 20(8): 911–919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGudbjartsson DF, Helgason A, Jonsson H, et al.: Spread of SARS-CoV-2 in the Icelandic Population. N Engl J Med. 2020; 382(24): 2302–2315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones TC, Mühlemann B, Veith T, et al.: An analysis of SARS-CoV-2 viral load by patient age. medRxiv. 2020. Publisher Full Text\n\nXu B, Gutierrez B, Mekaru S, et al.: Epidemiological data from the COVID-19 outbreak, real-time case information. Sci Data. 2020; 7(1): 106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerretti L, Wymant C, Kendall M, et al.: Quantifying SARS-CoV-2 transmission suggests epidemic control with digital contact tracing. Science. 2020; 368(6491): eabb6936. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang X, Tan Y, Ling Y, et al.: Viral and host factors related to the clinical outcome of COVID-19. Nature. 2020; 583(7816): 437–440. PubMed Abstract \| Publisher Full Text\n\nSenghore M, Savi MK, Gnangnon B, et al.: Leveraging Africa's preparedness towards the next phase of the COVID-19 pandemic. Lancet Glob Health. 2020; 8(7): e884–e885. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou J, Li C, Liu X, et al.: Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med. 2020; 26(7): 1077–1083. PubMed Abstract \| Publisher Full Text\n\nBarnes BJ, Adrover JM, Baxter-Stoltzfus A, et al.: Targeting potential drivers of COVID-19: Neutrophil extracellular traps. J Exp Med. 2020; 217(6): e20200652. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilk AJ, Rustagi A, Zhao NQ, et al.: A single-cell atlas of the peripheral immune response to severe COVID-19. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWikipedia: Wikipedia entry on Severe acute respiratory syndrome coronavirus. 2020. Reference Source\n\nWikipedia: Wikipedia entry on: Middle east repiratory syndrome - related coronavirus. 2020. Reference Source\n\nBoni MF, Lemey P, Jiang X, et al.: Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. bioRxiv. 2020. Publisher Full Text\n\nDu Z, Javan E, Nugent C, et al.: Using the COVID-19 to influenza ratio to estimate early pandemic spread in Wuhan, China and Seattle, US. EClinicalMedicine. 2020; 100479. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnfinrud P, Stadnytskyi V, Bax CE, et al.: Visualizing Speech-Generated Oral Fluid Droplets with Laser Light Scattering. N Engl J Med. 2020; 382(21): 2061–2063. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStadnytskyi V, Bax CE, Bax A, et al.: The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission. Proc Natl Acad Sci U S A. 2020; 117(22): 11875–11877. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossman H, Keshet A, Shilo S, et al.: A framework for identifying regional outbreak and spread of COVID-19 from one-minute population-wide surveys. Nat Med. 2020; 26(5): 634–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSegal E, Zhang F, Lin X, et al.: Building an International Consortium for Tracking Coronavirus Health Status. medRxiv. 2020. Publisher Full Text\n\nAarestrup FM, Albeyatti A, Armitage WJ, et al.: Towards a European health research and innovation cloud (HRIC). Genome Med. [Research Support, Non-U.S. Gov't]. 2020; 12(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabhive: We align efforts for more SARS-CoV2 tests. 2020. Reference Source\n\nWolkenhauer O, Auffray C, Brass O, et al.: Enabling multiscale modeling in systems medicine. Genome Med. 2014; 6(3): 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAuffray C, Noble D, Nottale L, et al.: Progress in integrative systems biology, physiology and medicine: towards a scale-relative biology. Eur Phys J A. 2020; 56: 88. Publisher Full Text\n\nMontuori A: Edgar Morin's Path of Complexity. 2008. Reference Source\n\nPrice ND, Magis AT, Earls JC, et al.: A wellness study of 108 individuals using personal, dense, dynamic data clouds. Nat Biotechnol. 2017; 35(8): 747–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNations U: 17 Goals to Transform Our World. 2020. Reference Source\n\nHood LE, Lazowska ED: Every life has equal value. Cell. 2013; 154(6): 1178–9. PubMed Abstract \| Publisher Full Text\n\nAuffray C, Chen Z, Hood L: Systems medicine: the future of medical genomics and healthcare. Genome Med. 2009; 1(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHood L, Balling R, Auffray C: Revolutionizing medicine in the 21st century through systems approaches. Biotechnol J. 2012; 7(8): 992–1001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAFP: Coronavirus: l'équipage du porte-avions \"Charles-de-Gaulle\" probablement contaminé lors d'une escale à Brest. Le Monde. 2020. Reference Source\n\nAFP: Coronavirus: les contaminations diminuent en Chine, mais progressent ailleurs dans le monde. Le Monde. 2020. Reference Source\n\nBouissou J: La crise économique provoquée par le coronavirus pourrait être la plus dévastatrice depuis 150 ans. Le Monde. 2020. Reference Source\n\nNicolle C: Naissance, vie et mort des maladies infectieuses. Paris: Librairie Félix Alcan. 1930. Reference Source\n\nWHO: Coronavirus disease may never go away. 2020. Reference Source\n\nWolrdbank: Gender and COVID-19 (coronavirus). 2020. Reference Source\n\nWilingham E: Genes may influence COVID-19 risk, new studies hint. 2020. Reference Source\n\nSpencer EA, Brassey J, Jefferson T, et al.: Environmental weather conditions and influence on transmission of SARS-CoV-2. CEBM; 2020 [updated 3 July 202027/08/2020]. Reference Source\n\nOstaszewski M, Mazein A, Gillespie ME, et al.: COVID-19 Disease Map, building a computational repository of SARS-CoV-2 virus-host interaction mechanisms. Sci Data. 2020; 7(1): 136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAuffray C: The Olympiads of Solidarity and Health. 2020. Reference Source\n\nAuffray C, Balling R, Barroso I, et al.: Making sense of big data in health research: Towards an EU action plan. Genome Med. 2016; 8(1): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKitano H: Artificial Intelligence to Win the Nobel Prize and Beyond: Creating the Engine for Scientific Discovery. AI Magazine. 2016; 37(1): 39. Publisher Full Text\n\nCaussy C, Pattou F, Wallet F, et al.: Prevalence of obesity among adult inpatients with COVID-19 in France. Lancet Diabetes Endocrinol. 2020; 8(7): 562–564. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nISBscience: COVID-19 Immune response study. 2020. Reference Source\n\nSu Y, Chen D, Lausted C, et al.: Multiomic immunophenotyping of COVID-19 patients reveals early infection trajectories. bioRxiv. 2020. Publisher Full Text\n\nOni T, Mogo E, Ahmed A, et al.: Breaking down the silos of Universal Health Coverage: towards systems for the primary prevention of non-communicable diseases in Africa. BMJ Glob Health. 2019; 4(4): e001717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJOGL: Open COVID-19 platform. 2020. Reference Source\n\nCRI: Center for Interdisciplinary Research. 2020. Reference Source\n\nOSPFoundation: Open Research Platform. 2020. Reference Source\n\nOSDD: Open Source Drug Discovery. 2020. Reference Source\n\nAuffray C, Sagner M, Abdelhak S, et al.: Viva Europa, a Land of Excellence in Research and Innovation for Health and Wellbeing. Progress in Preventive Medicine. 2017; 2(3): e006. Reference Source\n\nEnvironment CCaHWt: Integrating health in urban and territorial planning: a sourcebook. Geneva, Switzerland. 2020. Reference Source\n\nHeller MA, Eisenberg RS: Can patents deter innovation? The anticommons in biomedical research. Science. 1998; 280(5364): 698–701. PubMed Abstract \| Publisher Full Text\n\nHardin G: The Tragedy of the Commons. Science. 1968; 162(3859): 1243–8. PubMed Abstract \| Publisher Full Text\n\nMayor F: The Universal Declaration on the Human Genome and Human Rights. C R Biol. 2003; 326(10–11): 1121–5. PubMed Abstract \| Publisher Full Text\n\nAuffray C: Sharing knowledge: a new frontier for public-private partnerships in medicine. Genome Med. 2009; 1(3): 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnoppers BM, Thorogood A, Chadwick R: The Human Genome Organisation: towards next-generation ethics. Genome Med. 2013; 5(4): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCesario A, Auffray C, Russo P, et al.: P4 medicine needs P4 education. Curr Pharm Des. 2014; 20(38): 6071–2. PubMed Abstract \| Publisher Full Text\n\nSerres M, Farouki N: Le Trésor: dictionnaire des sciences. Paris: Flammarion; 1997. Reference Source\n\nWikipedia: Wikipedia entry on Antonio Machado. 2019. Reference Source\n\nAuffray C, Chen Z, Hood L, et al.: Foreword: from the TRANSCRIPTOME conferences to the SYSTEMOSCOPE international consortium. C R Biol. 2003; 326(10–11): 867–75. PubMed Abstract \| Publisher Full Text" }	[ { "id": "74650", "date": "25 Nov 2020", "name": "Ruth Chadwick", "expertise": [ "Reviewer Expertise Biomedical ethics", "gene-ethics", "Health care policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting piece giving a high-level overview perspective on the implications of the COVID-19 pandemic for the future of medicine, medical ethics and health care systems. Apart from the description of features of the pandemic, it is written in a very broad sweep way, which is necessary, given the predictions made about the future of health care. Specific points are, however, made about the avoidance of initial intellectual property claims in the context of the global cooperation that is going to be needed to address health care challenges such as the one currently faced across the world.\nThere are interesting points made about ethical implications. Given that it is claimed that what is needed is a transcendence of disciplinary boundaries and collaboration between science and the humanities, it would be good to see these fleshed out a little. For example, what does it mean to say that concepts of individuality will be challenged? The concept of solidarity is appealed to, and towards the end an interesting point is made about the link between the Hippocratic Oath and scientific ethics.\n\nIt is particularly important that these points are elaborated in order to avoid the possible perception that the claims about a future in which 21st century medicine is transformed constitute an ideal only, which may not be realised as a model for dealing with other future crises. Can we be sure that business as usual will not be resumed? What practical steps need to be taken so that a new ethic is internalised?\n\nIn what ways, precisely, does research need to be rethought - is it envisaged that current international principles of research ethics need to be rewritten?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "74443", "date": "25 Nov 2020", "name": "Hossein Khalili", "expertise": [ "Reviewer Expertise Medical-surgical", "healthcare system and policy development", "interprofessional collaborative practice", "higher education", "research and scholarship." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this article. The article is well written and very needed. Here are a few comments for the authors to consider in order to further improve the quality of their work:\nThe authors could consider and highlight/call the COVID-19 pandemic as not just a health crisis, rather both health and social crises that effect every aspect of our lives.\nWhen referring to the need for global systemic response to the pandemic, the authors could discuss concepts like the Whole-of-Government Approach, Health in All Policies, Interprofessional Collaborative Practice. In line with those when referring to treatments of COVID, it would be best if they use languages like 'healthcare providers' (rather than doctors as there are number of different professionals providing care to patients and families), and 'health care' (rather than medicine).\n\nWhen discussing the equity and access to COVID-19 treatments, the authors could refer to WHO’s Universal Health Coverage, Triple Billion Targets, and Sustainable Development Goals.\n\nLast, but not least, the authors should also highlight the need for resilience (training/development) among healthcare providers, students, teachers/educators, researchers, and the society for a success transition/overcome of the pandemic-related challenges.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "77874", "date": "22 Feb 2021", "name": "Tommaso Mazza", "expertise": [ "Reviewer Expertise computational and systems biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn a period when all readings get deeper into details of infection, mechanisms of molecular interaction, and strategies of analysis, it was a pleasure reading a commentary on humanity, solidarity, and shared science against COVID-19. Even if some of the authors are eminent representatives of research areas that intersect mines, I must admit that their community was unknown to me. It was interesting to read that already in 2003, they have joined their forces to rethink the way health research is done. However, I must also say that it is not clear how they are doing it in practice in this particular emergency time.\n\nThe manuscript goes through a long, clear-headed introduction to the history of the virus and its ancestors. I would somehow focus more on low-income countries and their difficulties to screen the population and enabling prevention actions. I’d refer here to two of the 17 UN’s sustainable goals, e.g., poverty and inequality referred to later in the text. Another point in this section regards the relationships between CoV-1 and CoV-2. CoV-2 is indeed much less virulent than CoV-1 and SARS if virulence is meant as the extent of damages caused to hosts. However, I’d remark that in the face of similar transmissibility, CoV-2 doubles the CoV-1 incubation time (2-7 days), thereby advantaging its spreading. Enhanced spreading is precisely the reason why we are going to live with this virus for a long time.\nThe web developed by CERN is mentioned as a spur to organize other similar initiatives. I’d liked to read more on the CERN’s web and what has been done in this pandemic. Throughout the manuscript, the authors speak several times about the need for proper integrative and analytical techniques and initiatives to better tackle health emergencies. I have noticed that this claim, which I share personally, is cluttered through the manuscript, and any time I bumped against it, I felt like it was only a claim and a general thought. Only at the end of the manuscript, when a couple of successful projects (ISB’s clinical trial and OpenCovid19) were briefly discussed, I could reconcile the feeling of missing information on systems biology/medicine that accompanied the whole reading. In this regard, I could not understand what’s the study protocol that ISB, together with the European Institute of Systems Biology and the Gemelli and S. Filippo Neri hospitals have implemented. I was not aware of this collaboration, neither I’ve read anything in the local newspapers. Being this reviewer Italian, he would be particularly interested to know how these two important hospitals are contributing to the project.\n\nFigure 1 is a sort of mixed graph with directed and undirected edges. This is fine when the aim is illustrative, as in this case. However, it is not clear what the multi-edge between some nodes, e.g., PROTEOMICS and Aptamer proteomics, do they mean. I like Figure 2B. However, it is not high resolution, and the text of small nodes is unreadable. It aims at representing the skills of the members of the OpenCovid19 community. In the figure legend, the authors refer to a broad range of skills; however, the high-degree nodes, which are the only readable, curiously report mostly computational and technical skills. Graphic design is the highest degree node. This tickles my curiosity about the composition of this community.\n\nI look forward to reading more about it and its activities. I thank all the authors for this piece of science.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1130
https://f1000research.com/articles/9-1124/v1	11 Sep 20	{ "type": "Method Article", "title": "Extraction of CPRD additional clinical data using R", "authors": [ "Anthony Nash", "M. Zameel Cader", "M. Zameel Cader" ], "abstract": "The Clinical Practice Research Datalink is a nation-wide database of primary healthcare data records in England (UK) linked to several health services. A visit to a health practitioner can result in the digital storing of diagnostic and prescription therapeutic information. Access to patient primary care and linked service data depends on the research in mind; however, typically several flat files that describe patient interactions with a health practitioner are delivered. Some of these files will describe additional data such as the result of medical tests and patient lifestyles, denoted collectively into entity values. This data is used to supplement the medical notes recorded by a general practitioner. We have made available a set of R scripts that reads the clinical flat files, additional clinical flat files and entity values, and returns patient clinical data linked with the requested additional data. We have also included medcode descriptions associated with several entities along with instruction of how to extend the code for additional entities. The code is free to download under the MIT license: https://github.com/acnash/CPRD_Additional_Clinical", "keywords": [ "CPRD", "Primary Care", "Electronic Healthcare Records", "Epidemiology", "R" ], "content": "Introduction\n\nThe Clinical Practice Research Datalink (CPRD) is an NHS primary care service that stores clinical, referral, therapeutic data and linked medical services such as imaging and hospital records of patients enrolled in England (UK). In its current form, CPRD has been active for over twenty years and holds up to 11 million records1. CPRD data has been widely used to improve medical practice and to further our understanding of drug efficacy and drug safety and disease mechanism2. As with all longitudinal data, understanding the outcome of a disease is usually confounded by several factors3. Patient comorbidities, therapy data and social habits (to name but a few) are all potential confounders that should be treated appropriately (see 4 for a research example of common patient characteristics using a CPRD data release). However, the curation and manipulation of CPRD data can cause significant barriers for many researchers, especially those less familiar with manipulating text-based large datasets or programming.\n\nData released from the CPRD Gold database data is presented as a series of flat files. A patient’s longitudinal data is presented across several rows, with each column denoting a record property. Patient records are linked between flat files using an anonymized patient identifier and several additional identifiers depending on the data at hand. Clinical diagnosis and prescribed therapeutics is encoded as a medcode and a prodcode, with a corresponding description available using the CPRD data dictionary. Most requests for CPRD Gold primary care data will usually return several clinical flat files. These are primary care records added by the patient’s General Practitioner (GP). The GP may also record additional clinical information, such as whether the patient smokes, how much they weigh, how often they drink, or the results from medical tests. This data is contained in a linked additional clinical set of flat files.\n\nWe present an R script that we have used to retrieve additional clinical data for patients. The script contains the code necessary to retrieve several patient characteristics (smoking, alcohol consumption, etc.) and is relatively straight forward to extend. We aim to continue updating our GitHub release with further additional clinical properties and any corresponding medcode descriptions whilst our projects remain active. In this manuscript, we outline the link between clinical and additional clinical patient data, the execution flow of the R scripts, and how to expand on the existing code. The R script is available for free and to download under an MIT license on GitHub: https://github.com/acnash/CPRD_Additional_Clinical\n\n\nMethods\n\nWe describe the link between patient clinical data and additional clinical data in cases where such a CPRD data release has been made available.\n\nBoth clinical data and additional clinical data are stored in two separate sets of files, often denoted as head_Extract_Clinical_##.txt and head_Extract_Additional_###.txt, respectively (this may depend on the service provided by those who extract the CPRD data). Additional clinical data, stored in the second set of files, is typically accompanied by a lookup data table with lookup codes stored in text files. Additional clinical data is identified by a unique entity (enttype, the entity type) value, for example, smoking status has an entity value of 4. Both clinical files and additional clinical files will have an enttype column. A researcher can retrieve all patients with a smoking status using this column. Then, depending on the particular additional information of interest, data on that entity can be retrieved from up to seven data fields, for example, whether they smoke, how many packs of cigarettes per week, whether they smoke a pipe, and how many ounces of tobacco. Some values are encoded and require the corresponding lookup files which share the same names as the entry in the data lookup column. For example, smoking status is defined in the first data column and the values are found in the YND.txt lookup file as denoted in the corresponding first data lookup column.\n\nWe have built an extensible R script which parses the clinical and additional clinical text files for an entity and then returns all clinical data with the corresponding additional clinical data for those patients with an entity of interest on record. As this is longitudinal data, patients may have several additional clinical rows on record for one entity. Therefore, the adid value (present in both sets of records) is used to match the clinical data with additional clinical data. If there are additional clinical records without a matching clinical record, the code ignores and moves to the next patient. We have also added medcode descriptions for those entities we are currently using. As our research expands so too will this list.\n\nThe R code is easy to expand and allows the researcher to treat each entity according to their needs. As the code stands, it currently supports our active research and we have been able to retrieve the additional clinical values and paired clinical medcode descriptions for smoking, alcohol consumption, exercise, and weight/BMI. A user need only execute several short lines of R code to retrieve any of the entities. For example, to retrieve patient data on smoking habits:\n\n\n\nThe idList is an R list or vector of patids (patient identifiers) of those patients of interest. The additionalFileList list contains the dedicated location of both sets of files. The content of these files is loaded into data frames and stored in the R environment. Ensure that there is enough system RAM available to open CPRD data files.\n\nThe entity value smoking is used to instruct the algorithm to look for all clinical and additional clinical data with an entity value of 4. A list of currently supported entities can be retrieved by executing getEntityValue(). All matching clinical data with additional clinical data (matched using the adid identifier if there are multiple rows per patient) are returned as a data frame with an additional column for medcode description. The medcode descriptions have been added into the R script and are used by the entity values currently supported.\n\nThe user executes one function and the existing R framework steps through a series of predefined functions before entering the user’s bespoke function (Figure 1). To extend the code to support additional clinical data, the researcher need only expand the if() statement in the function getEntityValue() with a function call to their own get<enttype>Data() function, following the format presented in the functions getExerciseData(), getSmokingData(), etc. Whether the researcher wishes to expand the results with a description of the medcodes is controlled by calling the defined function addMedcodeDescription(resultDF, <medcode_description_list>) within the if() statement. Both areas are illustrated below, with the potential code extension commented and a user defined entity name denoted with the place holder new_entity:\n\nThe function getSmokingData() in the orange square can be replaced by a user defined function with the same function arguments for other entities.\n\n\n\nUnfortunately, it is not entirely possible to automate the decoding of the additional clinical data for every entity. There currently 501 unique entities (CPRD Gold release February 2018) and 91 unique lookup text files. As presented, we have used this code to return the decoded entity data along with a corresponding clinical medcode description. However, depending on the research, a user can return a result bespoke to their needs by simply defining a new function. Up to that stage, the existing code finds and retrieves all data with a matching enttype value.\n\nThe script requires the R data.table package.\n\n\nResults\n\nWe present a sample of a use-case where clinical records were linked with smoking patient data. The data presented in this manuscript and uploaded to the GitHub repository has been fabricated in the interest of patient confidentiality. A test data set of 2,000 clinical primary care records were parsed for additional data concerning smoking. Of those patients, 1,993 had a record for smoking. Having matched those patients, retrieved the data and decoded their smoking status for either “Data not entered”, “Yes”, “No” or “Ex smoker”, each medcode from a corresponding clinical record was populated with a readable description. A snapshot of fabricated patient records is presented in Figure 2.\n\nAll dates and data have been fabricated.\n\n\nConclusion\n\nWe present an R script that searches for and retrieves additional clinical entity values and combines the results with patient clinical data. Although access to CPRD data requires an entrusted license holder, not all institutes will be fortunate enough to have access to software necessary to perform CPRD data curation and analysis5. At the time of writing, the software can support smoking, alcohol consumption, exercise and weight/BMI entity lookups. The script has been designed in such a way that most R users can extend the code and provide unique behaviour for a lookup entity of interest. We will continue to support the scripts, adding in new entities and medcode descriptions as our research requires. This script is part of a family of R packages and scripts currently under development.\n\n\nData availability\n\nZenodo: CPRD_Additional_Clinical, http://doi.org/10.5281/zenodo.39896346.\n\nThis project contains the following underlying data:\n\n- smokingDF.rda - a fabricated result R data frame, as presented in the Results section.\n\n\nSoftware availability\n\nR script available from: https://github.com/acnash/CPRD_Additional_Clinical\n\nArchived R script as at time of publication: http://doi.org/10.5281/zenodo.39896346.\n\nLicense: MIT\n\nRRID:SCR_018959\n\nThe script also holds medcode descriptions taken from the CPRD data dictionary for several entities. Samples of the clinical data and additional clinical data have not been provided as access to this data is only permitted following execution of the appropriate license agreements. If access to any of this data is required, we can provide medcode lists and the criteria used to extract the dataset (ISAC project 17_255R2) used to create this software, and we recommend contacting CPRD directly.", "appendix": "References\n\nHerrett E, Gallagher AM, Bhaskaran K, et al.: Data Resource Profile: Clinical Practice Research Datalink (CPRD). Int J Epidemiol. 2015; 44(3): 827–836. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhosh RE, Crellin E, Beatty S, et al.: How Clinical Practice Research Datalink data are used to support pharmacovigilance. Ther Adv Drug Saf. 2019; 10: 2042098619854010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkelly AC, Dettori JR, Brodt ED: Assessing bias: the importance of considering confounding. Evid Based Spine Care J. 2012; 3(1): 9–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWright AK, Welsh P, Gill JMR, et al.: Age-, sex- and ethnicity-related differences in body weight, blood pressure, HbA1c and lipid levels at the diagnosis of type 2 diabetes relative to people without diabetes. Diabetologia. 2020; 63(8): 1542–1553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDenaxas SC, George J, Herrett E, et al.: Data Resource Profile: Cardiovascular disease research using linked bespoke studies and electronic health records (CALIBER). Int J Epidemiol. 2012; 41(6): 1625–1638. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNash A: acnash/CPRD_Additional_Clinical: Initial release in line with F1000 submission and Zenodo link. (Version v0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3989634" }	[ { "id": "84116", "date": "12 May 2021", "name": "Zhi Yang", "expertise": [ "Reviewer Expertise Genomics", "Bayesian", "Machine learning." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author described an R script to quickly extract patient clinical data from CPRD for given patients. As a result, this R script is easy to use and extend based on users' interests. In general, the description is clear and straightforward to follow. However, due to errors in loading the test datasets, it is infeasible to validate whether the R script works as expected fully. Hopefully, the authors can address that issue soon.\n\nI tried to download the code from GitHub, and it seems that the smokingDF.rda is corrupted. The error message is shown as \"bad restore file magic number (file may be corrupted) -- no data loaded\". This error remained after trying with different R versions (3.6 and 4.0) and different operating systems (Windows and OS).\n\nIn the README file, since users don't have access to the example files in Line 36 & 37, it is unlikely to reproduce the same results and examine whether the script works as expected or not. Is it possible to provide some mock datasets for both additionalFiles and clinicalFiles to test the script?\n\nThe following comments are specific to the R script without actually running it. I should be able to provide more concrete suggestions after having access to the test datasets. - 3a. the as.data.table function needs to be added to @importFrom. From Line 283 to Line 291 of the R script, please explain why those variables are hard-coded. - 3b. In the R script, some documentations are missing for Title, @param, @return, etc., for example, getEthnicityData. - 3c. In the R script, idList is not used in the getEthnicityData function. - 3d. When generating the file names (Line 327), please use functions like fs::path rather than paste0 with \"\\\\\" which might throw an error at a different operating system.\n\nThis is just a suggestion for improvement. But it is highly recommended to make an R package rather than providing a single R script. It is easy for authors to maintain and for users to use and navigate. Especially, it is convenient for R developers to contribute in a consistent way like other R packages. To adapt the single R script into the R package structure, the main R script can be stored in the /R folder. All the medcode from Line 9 to 278 can be saved under the /inst folder as internal data. The example script can be provided as a vignette.\n\nThe authors mentioned about few scripts are available for researchers to obtain the requested information from CPRD. How about another R package called rEHR (https://github.com/rOpenHealth/rEHR)? Are there any additional features provided by this R script described here but not by rEHR?\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "142507", "date": "18 Jul 2022", "name": "Jessica Pinaire", "expertise": [ "Reviewer Expertise Health Data", "Machine Learning", "Text Mining" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents a set of scripts, developed in R, to explore clinical data from CRPD for a given patient.\nThis data is a big database of primary care in the UK, it contains various information such as a visit to a general practitioner, drug prescriptions, etc. Such a database is a complex database, hard to manage for the researchers not aware of its structure. To bridge the gap between clinical knowledge and data manipulation, the authors suggest a solution through their scripts.\nThe article is easy to follow and well written.\nThe article is interesting and highlights the difficulties faced by many biomedical researchers in various fields when they have to use databases with heterogeneous data and complex frameworks. This requires skills in various areas which they do not always have. However, since there isn't a specific method - like in data analysis - with results to present, I wonder if that kind of structure is adapted for this topic instead of a demonstration article.\nI have some remarks:\nIntroduction part: - Where can we find this data CRPD and who can access it? - How many researchers use it? - The article does not present a state of the art on the subject, are there equivalent works for other types of databases?\nMethod part: - The author refers to CRPD data throughout the article, but we don't see the data. As there are confidential data, it might be nice to provide a dummy dataset to be referred to throughout the article to make it easier to follow. - Without data it is impossible to check if the scripts work, in this case, the dummy dataset could be used to test the different scripts. - \"Ensure that there is enough system RAM available to open CPRD data files.\" What is the minimum RAM resource needed? Can we do everything? What is the limit? - \"Unfortunately, it is not entirely possible to automate the decoding of the additional clinical data for every entity.\" Can you explain more? What are the obstacles? - \"There currently 501 unique entities (CPRD Gold release February 2018) and 91 unique lookup text files.\" I think the verb is missing...\nConclusion part: - What are the limitations of this work? - What are the prospects for this work?\nPerhaps, it would be better to plan to put all the scripts in a package that is in the usual use of R.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1124
https://f1000research.com/articles/9-1122/v1	11 Sep 20	{ "type": "Research Article", "title": "ACE inhibitor, captopril, attenuates cytopathic effects of herpes simplex virus 1 in SH-SY5Y cells", "authors": [ "Amina S. Wofford", "Adam Standiford", "Kennedy Harris", "Ahmad B. Salam", "Cynthia A. Jackson", "Chastity N. Bradford", "Gerald D. Griffin", "Adam Standiford", "Kennedy Harris", "Ahmad B. Salam", "Cynthia A. Jackson", "Chastity N. Bradford", "Gerald D. Griffin" ], "abstract": "Background: Over 60% of the United States population is infected with herpes simplex virus 1 (HSV-1). Current HSV-1 treatment regimens exert their antiviral effects through a common mechanism of action and suffer from high dosing frequencies, which may contribute to patient noncompliance and the subsequent development of antiviral resistance. Although primarily known for their functions in maintaining homeostatic control of arterial blood and osmotic pressures, components of the Renin-Angiotensin Aldosterone System (RAAS) have been implicated in viral replication and some components demonstrated antiviral properties. However, the antiviral properties of RAAS components have not been well characterized in reference to HSV-1. Methods: To address this gap in knowledge, we evaluated the antiviral effects of captopril, an Angiotensin Converting Enzyme 1 (ACE-1) inhibitor, on HSV-1 infection in SH-SY5Y neuroblastoma cells. We demonstrated that captopril attenuates HSV-1-induced cytopathic effects (CPE) via cell-based and morphological assays. To investigate the potential mechanism, we conducted molecular modeling studies and identified the ability of captopril to interact with and bind HSV-1 glycoprotein D. To determine where in the virus life cycle captopril exerts its protective effects, we performed experiments observing the effect of captopril on both viral entry and replication utilizing a green fluorescent protein-tagged virus and subsequent quantitative Polymerase Chain Reaction. Results: Results suggest captopril protects cells from HSV-1-induced CPE through its effect on viral replication by increasing cell viability in infected cells and decreasing virus replication, which we propose is modulated through the decreased expression of ICP0. Conclusions: Collectively, the results presented here support further evaluation of captopril, and other RAAS components, as a basis for potential novel therapeutic interventions for the treatment of HSV-1, its associated pathologies, and potentially other virus infections.", "keywords": [ "herpes simplex virus 1", "renin angiotensin aldosterone system", "angiotensin converting enzyme-1", "captopril", "antiviral" ], "content": "Introduction\n\nHerpes simplex virus 1 (HSV-1) is a neurotropic virus that infects over 60% of the United States population and 90% globally1. HSV-1 is responsible for a diverse array of biological effects and physical manifestations, including cold sores and encephalitis. HSV-1 remains the leading infectious cause of corneal blindness and encephalitis2,3. Moreover, HSV-1 infection is a significant contributing factor in Human Immunodeficiency Virus (HIV) transmission and pathology3. Although HSV-1 infection is common, there is neither vaccine nor cure. Over 75% of HSV-1 antiviral drugs are nucleoside analogues that inhibit virus genome replication. Antivirals are effective in reducing the duration and frequency of outbreaks, but their efficacy is limited to active infections and doesn’t mitigate reactivation, which occurs from latent infections. Current therapeutic targets have major limitations, such as the requirement for an active infection, high dosing frequencies, and the development of antiviral resistance. Targeting a common mechanism of action contributes significantly to the development of antiviral resistance, an emerging problem for treating HSV-1 and other virus infections. The poor bioavailability of most HSV-1 antivirals requires high dosing frequencies, up to three to five times per day, to maintain therapeutic levels. However, Valacyclovir, a pro-drug of acyclovir, is the exception with an improved bioavailability profile compared to its parent drug and thus allows a reduced dosing frequency. Acyclovir, the mainstay of treatment for HSV-1 infection, consists of a synthetic nucleoside analogue that targets viral DNA polymerase and functions by inhibiting viral replication due to its affinity for viral thymidine kinase4,5. Acyclovir’s clinical use in immunocompromised patients can lead to the development of antiviral resistance6. HSV-1-induced pathologies, the global burden that results from HSV-1 infection, and the lack of effective treatments highlights a need for the identification of novel antiviral strategies and therapeutics. Thus, there is an urgent medical need to identify novel drug targets and subsequently develop and evaluate novel therapeutic strategies and interventions that can exert their action through a distinct mechanism.\n\nComponents of the Renin-Angiotensin Aldosterone System (RAAS), known for their function in the homeostatic control of arterial blood and osmotic pressures, have been implicated in regulating virus activity. Human cytomegalovirus, Human Herpesvirus-5 (HHV-5), has been demonstrated to cause an increase in arterial blood pressure7, further supporting our investigation into the relationship between viral infections and the RAAS system. The ongoing Severe Acute Respiratory Coronavirus 2 (SARSCoV2)-induced pandemic further highlights the interaction between viruses and RAAS components as it utilizes Angiotensin Converting Enzyme-2 (ACE-2), a cellular protein, as its primary entry receptor8. These findings support our hypothesis that components of RAAS may attenuate HSV-1 infection. Losartan, an angiotensin type 1 receptor blocker, and des-aspartate-angiotensin 1, both inhibitors of RAAS, have demonstrated antiviral effects in coxsackievirus and rhinovirus infections, respectively9,10. The work described here evaluated the hypothesis that Angiotensin Converting Enzyme-1 (ACE-1) inhibition is protective against HSV-1-induced cytopathic effects (CPE). We have demonstrated that the ACE-1 inhibitor captopril attenuates CPE in HSV-1-infected SH-SY5Y neuroblastoma cells. The results reported here are the first, to our knowledge, to show that captopril protects neuroblastoma cells from HSV-1-induced CPE by impairing virus genome replication.\n\n\nMethods\n\nThe neuroblastoma SH-SY5Y cell line (ATCC, CRL2266, lot # 11C016) was purchased from Sigma-Aldrich (St. Louis, MO). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis, MO) and 7% Penicillin and Streptomycin (Sigma Aldrich, St. Louis, MO). Cells were seeded into flasks containing supplemented medium and maintained at 37°C in saturated humidity with 5% CO2. For assays, SY5Y cells were sub-cultured into 96-well plates at a seeding density of 1× 105 cells per well.\n\nThe F strain of HSV-1 was graciously contributed by the Fraser Lab at The University of Pennsylvania. Cells were infected at a multiplicity of infection (MOI) of 0.01. During infection, virus was delivered to cells in serum-free DMEM and cells were administered either captopril (Sigma Aldrich, St. Louis, MO; administered at various concentrations (0, 1pM, 10pM, 1nM, 10nM, 1µM)) or vehicle control (0.1% dimethyl sulfoxide (DMSO)) post-infection. One-hour post-infection, inoculum was aspirated and an equal volume of complete media was added to the cells. Cells were incubated until the cell viability assay or imaging analysis was performed. Andrea Bertke (Virginia Tech) donated the green fluorescent protein-tagged HSV-1 (GFP-HSV-1).\n\nCell viability was quantified via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay (Sigma-Aldrich, M5655). Cells were cultured with cDMEM in 96-well plates until they were 90% confluent. Next, subsets of wells were infected with HSV-1 (F strain) at a MOI of 0.01 for 24 hours. MTT solution (5mg/ml) was added to each well and incubated for four hours at 37°C. After removing the supernatant from wells, 100 µl DMSO was added to each well. The absorbance was read at 570 nm with a 630nm reference wavelength via a Benchmark Plus (BIO RAD) spectrophotometer. All experiments were repeated in triplicate and processed in parallel.\n\nTime course analysis was evaluated via the previously described methods used in the cell viability protocol, utilizing the MTT assay at 3, 9, and 24 hpi. All experiments were repeated in triplicate and processed in parallel.\n\nPhotomicrographs were obtained utilizing an Olympus IX81 microscope equipped with a Hamamatsu camera. MetaMorph software (version 6) was used to image cells, and photomicrographs were taken with the differential interference contrast (DIC) filter at a final magnification of 20x (Micro-Manager is a free alternative if access to MetaMorph is not available). Three distinct fields of view were imaged per well.\n\nGreen fluorescent protein-tagged HSV-1 (GFP-HSV-1) was used to infect cells, as previously described. There were three treatment groups: Mock, Infected/Vehicle, and Infected/Captopril. A Mock/Captopril group was not used in this experiment as we wanted to examine the effect of captopril of HSV-1 replication and previous data demonstrated that captopril was non-toxic. Photomicrographs were taken with a DIC filter at 8, 24, and 48 hours post infection (hpi).\n\nAutoDock Vina (version 1.1.2), a docking program, was used to investigate the interaction between HSV-1 glycoprotein D (gD) (Protein Data Bank (PDB) ID: 1P7C) and captopril (PDBID: 2X8Z). Captopril was collected from the bound crystal structure of AnCE using PyMOL V1.8.6.2. AutoDock Vina was used to approximate the favorability of HSV-1 gD binding with captopril. The crystal structure of both molecules were obtained from the PDB and used to generate 3D structures. To perform the docking simulations, AutoDock Vina generated a grid box to enclose the active site with dimensions of 126 Å × 126 Å × 126 Å and a grid spacing of 0.375 Å using Autodock Tools. The results are represented by free energy binding.\n\nTo render the Ligand Interactions Diagram, Schrodinger Maestro V10.6 was used. Initially, crystal structure of HSV1 gD (receptor) and captopril (ligand) were obtained from the PDB as mentioned above. The imported protein structure obtained from the PDB was not suitable for immediate use in the molecular docking study as it contained excess material (i.e., water molecules, metal ions, cofactors, etc.). To overcome this obstacle, the protein structure was prepared using the protein preparation wizard (preprocessed, optimized, and minimized) in Maestro v10.611,12. The ligand structures of the data set were prepared by LigPrep module of Schrodinger v10.612.\n\nSH-SY5Y neuroblastoma cells were treated with either vehicle (0.1% DMSO), the F strain of HSV-1 (MOI=0.01), or captopril and HSV-1 (F strain, MOI=0.01) together in media without serum for one hour. After this, an equal volume of complete media was added to the cells. Next, RNA was isolated from treated cells according to the manufacturer’s instructions (Qiagen AllPrep DNA/RNA Mini Kit; Cat. No. 80204, Lot # 154018161). Following RNA isolation, the purity and concentration of resulting RNA were measured using the Thermo Scientific Nano Drop 2000 Spectrophotometer. All samples were stored at -80°C for future use.\n\ncDNA synthesis. The protocol used to synthesize first-strand complementary DNA from purified RNA was followed using the Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher, Cat. No. 18080-051). RNA (100ng) isolated from SH-SY5Y cells was placed in Bio-Rad Low-Profile PCR Tubes containing 10mM dNTP mix and 50ng of random hexamer primers. The final volume in each tube was 10 uL. Positive (HeLa RNA) and negative control (tube containing HeLa RNA but no reverse transcriptase) reactions were also prepared. Samples were placed in a Bio-Rad T100 Thermal Cycler and incubated at 65°C for 5 minutes, cDNA synthesis mix (according to the manufacturer’s instructions) was added to each tube including the positive and negative controls. After following subsequent steps detailed in the SuperScript III manual, the resulting cDNA was stored at -20°C.\n\nqPCR cycle settings. The protocol for qPCR was adapted from Thermo Fisher Scientific PowerUp SYBR Green Master Mix (Catalog No., lot# 1601009). Reactions were run in triplicate in Bio-Rad 96-well PCR plates. Primers for each gene of interest were synthesized by Integrated Data Technologies and diluted to 10uM concentrations with DEPC-treated water. Human GAPDH was used as a reference gene, and ICP0 was utilized to measure HSV-1 viral gene expression. The primer sequences for human GAPDH were: 5’ TGG GCT ACA CTG AGC ACC AG 3’ (forward) and 5’ GGG TGT CGC TGT TGA AGT CA 3’ (reverse). The forward primer sequence for ICP0 was 5’ CCC ACT ATC AGG TAC ACC AGC TT 3’. The sequence for ICP0 reverse primer was 5’ CTG CGC TGC GAC ACC TT 3’. Each analyzed well of the PCR reaction plate contained the following: forward primer (500nM), reverse primer (500nM), PowerUp SYBR Green Master Mix (2X), DEPC-treated water, and cDNA (10ng). The PCR plate was sealed and centrifuged, and inserted into the CFX96 Real-Time System. The qPCR cycling mode was as follows: two minutes at 50°C followed by two minutes at 95°C. Next, a total of 40 denature (15 seconds at 95°C) and anneal (60°C) cycles were utilized. Melt curve analyses were performed with each reaction. ICP0 gene expression levels were normalized to human GAPDH levels.\n\nAll analyses for statistically significant differences were performed utilizing a two-way analysis of variance (time x infection status or concentration x infection status). The alpha value was set at 0.05. If warranted, a Tukey’s post-hoc test was performed. For the concentration-response curve, a Dunnett’s multiple comparisons post-hoc test was used, comparing the different concentrations to the vehicle (0) control. Data are expressed as means (± SEM). GraphPad Prism (version 6) statistical analysis software was utilized to perform statistical comparisons and prepare graphical representations of quantitative data.\n\n\nResults\n\nTo determine the concentration dependent response of captopril on SH-SY5Y neuroblastoma cells, we first assessed cell viability and cellular morphology via the MTT assay and DIC microscopy, respectively. SH-SY5Y cells were treated with a range of concentrations (0, 1pM, 10pM, 1nM, 10nM, 1uM) of captopril. To investigate if captopril treatment alone adversely affected cell viability, the experiment was first performed in uninfected cells. The concentration response data in uninfected SH-SY5Y cells demonstrated that captopril (Figure 1) did not impact cell viability as viability remained approximately 100% with no statistically significant differences observed at any concentration.\n\nThe response of uninfected and HSV-1 infected SH-SY5Y neuroblastoma cells to varying concentrations (0, 1pM, 10pM, 1nM, 10nM, 1uM) of captopril was measured via MTT cell viability assay at 24 hours post infection and treatment. Data from uninfected and captopril treated cells showed no significant change in cell viability. Cells infected with the F strain of HSV-1 (MOI=0.01) demonstrated a statistically significant reduction in viability, compared to their uninfected counterparts (F(1,98)=10.22, p < 0.005). HSV-1 infected, captopril-treated cells had a statistically significant, dose-dependent increase in cell viability from 10pM to a maximum efficacy observed at 10nM, compared to infected, untreated cells (F(6,98)=23.82, p<0.0001). The asterisk denotes that the HSV-1-infected group was statistically significant from the uninfected group at the 0 concentration. The plus signs represent groups (infected with HSV-1 and treated with captopril) statistically significant from their counterparts that were infected the F strain of HSV-1 but received no captopril treatment. Each bar represents the average of six cell culture replicates.\n\nAlongside the uninfected cells, HSV-1 infected SH-SY5Y cells were treated with a range of concentrations of captopril (0, 1pM, 10pM, 1nM, 10nM, 1uM). Results from the MTT cell viability assay demonstrated captopril treatment caused a significant increase in viable cells at 10pM, 1nM, 10nM, and 1μM (F(6,98)=23.82, p<0.0001), when compared to the infected, untreated group. In subsequent experiments, we chose to use the 10nM dose of captopril as our results indicated it offered maximum protection, compared to the other doses evaluated, and was less than the 1μM dose.\n\nExperiments were conducted to measure cell viability at different time points (3, 9, and 24 hpi). Cell viability measurements were equivalent among all the groups at 3 hpi. At 9 hpi (F strain of HSV-1, MOI=0.01), the Virus/Vehicle group had 20% fewer viable cells (F(3,8) =23.27; p<0.0005) compared to every other treatment group (Figure 2). At 24 hpi there were approximately 25% less viable cells in the Virus/Vehicle group compared to all other groups. At 9 and 24 hpi, the only significant difference observed was between the Virus/Captopril group and the Virus/Vehicle group. Differences due to time alone were not statistically significant.\n\nCell viability was evaluated via MTT assay at 3, 9, and 24 hours post infection (hpi) with the F strain of HSV-1 (MOI=0.01). At 9 and 24 hpi, the Virus/Vehicle group was statistically significantly different compared to the all other groups. Each bar represents the average of six biological replicates. The asterisks indicate groups that were statistically significant from the other groups (not denoted with an asterisk) at their respective time points.\n\nPhotomicrographs were taken as a qualitative analysis to corroborate the MTT results (Figure 3). The experimental groups (Mock, Mock/Captopril, Virus/Vehicle, and Virus/Captopril) were exposed to the same conditions (24 hpi and 10 nM captopril) prior to obtaining photomicrographs. Cells in Figure 3a (Mock) are representative of healthy SH-SY5Y cells indicated by the confluence and the appearance of intact cellular extensions. Cells in the Mock/Captopril group (Figure 3b) were also very confluent and exhibited similar structural characteristics to the Mock group (Figure 3a). In both of these groups, there were no indicators of substantial cell death such as changes in cellular morphology (i.e., cell rounding) and loss of anchorage dependence (i.e., floating cells). HSV-1 infected cells (Virus/Vehicle) exhibited a reduction in confluence (Figure 3c). Cells in this group also demonstrated multiple instances of cytopathy such as changes in morphology, the loss of anchorage dependence, and a decrease in neurite extensions on cells that remained attached. HSV-1 infected cells treated with captopril (Virus/Captopril, Figure 3d) were more confluent compared to the Virus/Vehicle group and maintained normal characteristic extensions of neuroblastoma cells.\n\nPhotomicrographs show morphological changes 24 hours after captopril (10nM) administration. Uninfected cells administered vehicle (0.1% DMSO) (3a) or captopril alone (3b) were indistinguishable in confluence and structure. In the absence of captopril, HSV-1 infected cells were rounded, an indicator of adverse cell health (3c). Captopril treated, HSV-1 infected cells (3d) maintained their neurite extensions (as seen in 3a and 3b) and were more confluent when compared to HSV-1 infected, untreated cells (3c). (Arrows are intended to illustrate morphological changes.) Additionally, cells treated with captopril preserved the neurite extensions associated with healthy SH-SY5Y neuroblastoma cells. Images were taken at a final magnification of 200x using an IX Olympus 81 inverted microscope and MetaMorph microscopy imaging software.\n\nCells infected with a GFP-expressing HSV-1 strain were analyzed at 1, 3, 8, 24, and 48 hpi. In the Virus/Vehicle group, GFP expression was not observed until 8 hpi. After this time point, the percentage of GFP-positive cells increased in a time-dependent manner. At 8 hpi, an average of 13% of cells were GFP-positive in the Virus/Vehicle group compared to only 1% in the Virus/Captopril group (F (7,11) =3.58; p<0.05; Figure 4.). At 24 hpi, 30% of cells were GFP-positive in the Virus/Vehicle group compared to 25% of GFP-positive cells in the Virus/Captopril group. This pattern continued at 48 hpi, with 61% of GFP-positive cells observed in the Virus/Vehicle group versus 27% of GFP-positive cells in the Virus/Captopril group (F (7,11) =2.72; p<0.05).\n\nHSV-1 used was constructed to express GFP as a measure of replication. The percentage of GFP positive cells was quantified via manual image analysis at 1, 3, 8, 24, and 48 hpi. At 1 and 3 hpi there were no GFP-positive cells in any of the treatment groups. At later time points (8, 24, and 48 hpi), there was a time-dependent increase in percentage of cells expressing GFP in both the Virus/Vehicle and Virus/Captopril group. Each bar represents the average of six biological replicates. The statistically significant differences in the percentage of GFP positive cells were observed between the Virus/Vehicle and the Virus/Captopril groups at 8 and 48hpi (F(2,30) =12.82; p<0.0001).\n\nMicrographs of cells infected with the GFP-HSV were imaged at 20X magnification. Cells receiving the Uninfected/Vehicle treatment did not exhibit any GFP fluorescence at any time point (Figure 5a–c). With the administration of the GFP-expressing virus (MOI=0.01), cytopathic effects (e.g., cellular rounding) were noted as well as fluorescent imaging at all three time points (8, 24, and 48 hpi; Figure 5d–f). Co-administration of captopril (10nM) with the GFP-HSV reduced the occurrence of both cytopathy and green fluorescence (Figure 5g–i).\n\nPhotomicrographs from cells in the Uninfected/Vehicle, Virus/Vehicle, and Virus/Captopril groups were imaged at a final magnification of 200X using an Olympus IX81 microscope. An HSV-1 virus fused with GFP was utilized in this experiment. GFP expression, an indicator of viral replication, was not detected in any cells in the Uninfected/Vehicle group (a–c). GFP expression increased from over time in cells infected with GFP-HSV (MOI=0.01, d–f). With co-administration of captopril (10nM), there was a decrease in green fluorescence and dying cells (indicated by the rounded morphology) in HSV-1 infected cells (g–i).\n\nWe conducted molecular modeling studies to determine the free binding energy (∆G) between the ACE inhibitor captopril and HSV-1 gD. Results demonstrated that the free binding energy between captopril and HSV-1 gD was favorable and calculated to be -3.9 kCal/mol (Figure 6A). Further evaluation of the interaction between HSV-1 gD and captopril identified the specific amino acid residues responsible for the interaction (Figure 6B).\n\n(A) A ribbon diagram of HSV-1 gD is shown in complex with captopril (small molecule). Molecular modeling via AutoDock Vina determined that captopril has a favorable interaction with HSV-1 gD, indicated by a predicted free binding energy (ΔG) of -3.9 kCal/mol. (B) Captopril-HSV-1 gD predicted interaction with specific amino acid residues of gD color coordinated by charge.\n\nTranscripts of the viral gene ICP0 were detected at 2 hpi by SH-SY5Y neuroblastoma cells infected with the F strain of HSV-1 (Virus/Vehicle Group). qPCR results demonstrated that captopril co-administration to HSV-1 infected cells decreased ICP0 gene expression by approximately 34%, compared to infected cells not administered captopril (p<0.0001; Figure 7).\n\nTwo hours after being infected with the F strain of HSV-1 (MOI=0.01), cells in the Virus/Vehicle group had transcript levels of the viral gene ICP0 statistically significantly greater than those in the Uninfected/Vehicle condition (F(2,23)=41.59; p<0.0001). ICP0 was not detected in uninfected SY5Y neuroblastoma cells. Each treatment group consisted of eight biological replicates. qPCR was executed in a parallel fashion and each bar represents the average of three replicates. Transcript levels of the viral gene ICP0 were normalized to levels of human GAPDH.\n\n\nDiscussion\n\nTo strengthen the work relating RAAS components and antiviral activity, we studied the anti-HSV-1 effects of captopril in SH-SY5Y neuroblastoma cells. Based on previous findings from Zhang et al.9, we hypothesized that ACE inhibition would confer protective effects induced by HSV-1. We have demonstrated that captopril attenuates HSV-1induced CPE in SH-SY5Y cells. Results of cell viability assays indicate captopril protects cells from HSV-1-induced cellular death by 18% when compared to untreated counterparts. This finding was further substantiated by a qualitative assessment of photomicrographs in which GFP fluorescent intensity was used as a marker for virus entry and replication. Experiments using GFP-HSV-1 and subsequent qt-PCR demonstrated that captopril blunted HSV-1 replication. These results support our hypothesis that captopril reduces the cytopathic effects and replication of HSV-1.\n\nInfection with HSV-1 results in life-long infection with initial infection typically occurring in childhood. HSV-1 can cross the blood brain barrier, promote inflammation and damage the central nervous system. There are few drugs licensed for the treatment of HSV-1 infections. Acyclovir remains the “gold standard” in HSV-1 therapy, more than thirty years after its development4. Extensive clinical use of acyclovir and other approved treatments has contributed to the emergence and persistence of resistant viral strains, particularly in immunocompromised individuals13. Treatments effective against HSV-1 are nucleoside analogues with the exception of foscarnet, a pyrophosphate analogue. Foscarnet is primarily restricted to individuals whose virus infections are resistant to acyclovir14. Thus, few treatment options coupled with the emergence of resistant viral strains contribute to a growing need of novel antiviral targets and strategies13–15. Components of the RAAS have been implicated and shown to play a role in regulating virus activity and select RAAS inhibitors cross the blood brain barrier, leading us to hypothesize that captopril could attenuate the cytopathic effects of HSV-1 in SH-SY5Y cells9,10.\n\nSARS-CoV-2 that caused a worldwide pandemic also has links to RAAS components. SARS-CoV-2 utilizes ACE-2 as a receptor to facilitate entry into host’s cells8. This finding further demonstrates the potential significant relationship between RAAS components and viruses.\n\nIn this study, we investigated the role of ACE-1 and its inhibitors in HSV-1 replication. We determined the most effective captopril concentration for reducing the cytopathic effects of HSV-1 in SH-SY5Y cells was 10nM. According to Henda et al.16, captopril has an IC50 range of 1.79-15.1nM when used with synthetic substrates and an IC50 around 16.71µM when used with Angiotensin I. Thus, the concentration needed to reduce HSV-1 replication is well below its active range in inhibiting ACE. Other RAAS inhibitors, such as losartan, have been evaluated in vivo for antiviral effects using a concentration of 12mg/kg17,18 against coxsackievirus B3 which induces myocarditis. Des-aspartate-angiotensin I, an angiotensin nonapeptide, has also been shown to have an antiviral effect against rhinovirus when used between a 10-10 and 10-12 nanomolar range10. At such a low concentration, 10nM, we do not anticipate any significant non-specific effects. Collectively, our quantitative and qualitative data indicates that captopril does not significantly alter cell viability or induce any visual cytopathic effects in uninfected cells.\n\nTime course experiments were conducted at 3, 9, and 24 hpi to evaluate if there were time-dependent changes in cell viability. Time points were selected based on reported times for the initiation of viral gene expression. Immediate-early (α) genes, such as infected cell protein 0 (ICP0), are typically expressed within 1 hpi; early genes (β), such as thymidine kinase, are usually expressed within 3hpi; and late (γ) genes, such as glycoprotein C (gC), are typically expressed between six and eight hpi; and Latency Associated Transcripts are expressed upon initial infection and are continuously abundantly expressed following initial infection. Based on relevant literature and our preliminary findings, we decided to observe the effect of viral infection at 24 hpi. Significant differences were observed between the Virus/Vehicle group when compared to the Virus/Captopril groups at 9 and 24 hpi.\n\nComputational approaches to drug discovery are used extensively to determine the potential of candidate and novel drugs. The current COVID-19 crisis reinforces the use of computational approaches in drug discovery. We used a computational approach to evaluate the interaction between ACE inhibitors and HSV-1 via molecular modeling and found that captopril favorably interacts with HSV-1 gD with a negative free binding energy (ΔG) of -3.9 kCal/mol while the other evaluated ACE inhibitors (lisinopril and fosinopril) do not interact with HSV-1 gD.\n\nThe primary limitation to this study is the use of only one cell line. Future studies to determine the effects of captopril in alternative cell lines infected with HSV-1 are warranted. Moreover, it would be prudent to investigate how other methods of ACE inhibition or Angiotensin II receptor antagonism affect HSV-1 replication. Future work will also determine how well captopril impairs HSV-1 replication at higher MOIs. Yet, the work detailed here provides a firm basis for captopril to be further evaluated as a novel antiviral effective against HSV-1.\n\nThe overall goal of this study was to evaluate the anti-HSV-1 properties of captopril. Our results, both quantitative and qualitative in nature, demonstrate that captopril exerts a protective effect against HSV-1. Thus, these results strengthen work addressing antiviral effects of RAAS components and offers potential for a novel strategy for combatting HSV-1 infection. As a commonly prescribed and relatively safe blood pressure medication, captopril has demonstrated promising potential as a therapeutic intervention for the treatment of HSV-1. Problems associated with current HSV-1 treatment regimens are largely centered around antiviral resistance and high dosing frequencies due to low oral bioavailability. Acyclovir is reported to have a bioavailability between of 20%, requiring a dosing frequency of five times per day19 to maintain therapeutic levels. Valaciclovir, a prodrug of acyclovir, also used for the treatment of herpes virus infections increases bioavailability to 55%20, which causes a decrease in dosing frequency from five to three times per day to be effective, but is accompanied by a price increase which may affect which treatment options are prescribed. Captopril has an oral bioavailability of approximately 65%21, which if effective as an antiviral, as data suggest, may result in a further decrease in dosing frequency, potentially leading to an increase in patient compliance and is relatively inexpensive. Additionally, captopril crosses the blood brain barrier (BBB), and thus could decrease the CNS inflammatory response and prevent further damage caused by viral-mediated BBB permeability.\n\nCollectively, our findings warrant further studies that will test the mechanisms underlying the antiviral effect demonstrated in this work. Future work will delineate any possible direct interactions between the drug and HSV-1 itself as well as any effects of captopril on viral entry. This work highlights a critical area of study that could provide a novel approach to reducing HSV-1 entry and reducing the pathologies associated with its infection and reactivation while providing a much-needed novel therapeutic alternative.\n\n\nConclusions\n\nCurrent treatment options for HSV-1 and HSV-1-associated neurological complications largely rely on high dosing frequencies of nucleoside analogues, such as acyclovir, famciclovir and ganciclovir, which can have damaging side effects on multiple organ systems20,21. Previous studies have made connections between RAAS and viral activity, demonstrating that RAAS components may have antiviral abilities13,16. Here, we studied antiviral properties of the ACE inhibitor, captopril, against HSV-1. By combining qualitative analysis of cellular structure with quantitative measurements of cellular viability and viral replication, and molecular modeling, we have demonstrated that captopril effectively decreases the cytopathic effects induced by HSV-1 by mediating viral replication. Due to the difficulty of treating viral infections, this work has the potential to be applicable across a broad spectrum of other viruses and raises additional questions regarding systemic effects of HSV-1 infection, such as hypertension and other cardiovascular diseases in general. This work provides a platform to further investigate the mechanisms of captopril, an already approved drug, as an effective therapeutic alternative for common HSV-1 infection.\n\n\nData availability\n\nFigshare: Quantitative and qualitative data assessing the antiviral effects of Captopril, an ACE-1 inhibitor, on HSV-1, https://doi.org/10.6084/m9.figshare.12881441.v122.\n\nThis project contains the underlying data files:\n\n- Captopril Dose-Response data.xlsx [Captopril dose-response data]\n\n- HSV-1 & Captopril Time Course Data.xlsx [HSV-1 and captopril time course data]\n\n- JPG files - Qualitative comparison of cell morphology of different treatment groups\n\n- Virus Replication Assay-GFP+ Cells.xlsx [HSV-1 viral replication quantitation based on GFP+ cells]\n\n- PNG files - Qualitative evaluation of GFP+ cells in different treatment groups at different time points\n\n- Captopril-HSV-1 Interaction (dragged).pdf [Molecular modelling: Captopril has a favorable interaction with HSV-1 gD]\n\n- ICP0 gene expression data.csv [Captopril treatment down-regulates HSV-1 ICP0 expression]\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors acknowledge the kind support of the Fraser Laboratory (University of Pennsylvania), the Bertke Laboratory (Virginia Polytechnic Institute and State University), the Martinez Laboratory (Tuskegee University), and the Yates Laboratory (Tuskegee University).\n\n\nReferences\n\nLooker KJ, Magaret AS, May MT, et al.: Global and Regional Estimates of Prevalent and Incident Herpes Simplex Virus Type 1 Infections in 2012. PLoS One. 2015; 10(10): e0140765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffiths SJ, Koegl M, Boutell C, et al.: A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication. PLoS Pathog. 2013; 9(8): e1003514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCowan FM, Johnson AM, Mindel A: Herpes Simplex Virus Infection as a Risk Factor for Human Immunodeficiency Virus Infection in Heterosexuals. J Infect Dis. 1993; 167(3): 772. PubMed Abstract \| Publisher Full Text\n\nGreco A, Diaz JJ, Thouvenot D, et al.: Novel Targets for the Development of Anti-Herpes Compounds. Infect Disord Drug Targets. 2007; 7(1): 11–18. PubMed Abstract \| Publisher Full Text\n\nLuganini A, Nicoletto SF, Pizzuto L, et al.: Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers. Antimicrob Agents Chemother. 2011; 55(7): 3231–3239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStrasfeld L, Chou S: Antiviral Drug Resistance: Mechanisms and Clinical Implications. Infect Dis Clin North Am. 2010; 24(2): 413–437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng J, Ke Q, Jin Z, et al.: Cytomegalovirus Infection Causes an Increase of Arterial Blood Pressure. PLoS Pathog. 2009; 5(5): e1000427. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffman M, Kleine-Webber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2): 271–280.e8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang YY, Li JN, Xia HX, et al.: Protective effects of losartan in mice with chronic viral myocarditis induced by coxsackievirus B3. Life Sci. 2013; 92(24–26): 1186–1194. PubMed Abstract \| Publisher Full Text\n\nAng LT, Tan LY, Chow VT, et al.: Des-aspartate-angiotensin I exerts antiviral effects and attenuates ICAM-1 formation in rhinovirus-infected epithelial cells. Eur J Pharmacol. 2012; 683(1–3): 310–315. PubMed Abstract \| Publisher Full Text\n\nTahlan S, Kumar S, Ramasamy K, et al.: In-silico molecular design of heterocyclic benzimidazole scaffolds as prospective anticancer agents. BMC Chem. 2019; 13(1): 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchrödinger Release 2017-3: Maestro. Schrödinger, LLC. New York, NY. 2017. Reference Source\n\nBacon TH, Levin MJ, Leary JJ, et al.: Herpes Simplex Virus Resistance to Acyclovir and Penciclovir after Two Decades of Antiviral Therapy. Clin Microbiol Rev. 2003; 16(1): 114–128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaijo M, Suzutani T, Morikawa S, et al.: Genotypic Characterization of the DNA Polymerase and Sensitivity to Antiviral Compounds of Foscarnet-Resistant Herpes Simplex Virus Type 1 (HSV-1) Derived from a Foscarnet-Sensitive HSV-1 Strain. Antimicrob Agents Chemother. 2005; 49(2): 606–611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGnann JW Jr, Barton NH, Whitley RJ: Acyclovir: Mechanism of Action, Pharmacokinetics, Safety and Clinical Applications. Pharmacotherapy. 1983; 3(5): 275–283. PubMed Abstract \| Publisher Full Text\n\nHenda YB, Labidi A, Arnaudin I, et al.: Measuring Angiotensin-I Converting Enzyme Inhibitory Activity by Micro Plate Assays: Comparison Using Marine Cryptides and Tentative Threshold Determinations with Captopril and Losartan. J Agric Food Chem. 2013; 61(45): 10685–10690. PubMed Abstract \| Publisher Full Text\n\nChen L, Re RN, Prakash O, et al.: Angiotensin-converting enzyme inhibition reduces neuroblastoma cell growth rate. Proc Soc Exp Biol Med. 1991; 196(3): 280–283. PubMed Abstract \| Publisher Full Text\n\nYang YH, Chang HW, Chang WC, et al.: Pharmacogenetic Mechanism of ACE I/D Polymorphism Adversely Responding to ACE Inhibitors in Regulating the ACE Promoter Activity in Neurons. J Pharm Pharmacol. 2016; 4: 419–431. Publisher Full Text\n\nde Miranda P, Blum MR: Pharmacokinetics of acyclovir after intravenous and oral administration. J Antimicrob Chemother. 1983; 12 Suppl B: 29–37. PubMed Abstract \| Publisher Full Text\n\nPerry CM, Faulds D: Valaciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in herpesvirus infections. Drugs. 1996; 52(5): 754–772. PubMed Abstract \| Publisher Full Text\n\nDuchin KL, McKinstry DN, Cohen AI, et al.: Pharmacokinetics of Captopril in Healthy Subjects and in Patients with Cardiovascular Diseases. Clin Pharmacokinet. 1988; 14(4): 241–259. PubMed Abstract \| Publisher Full Text\n\nWofford A, Jackson CA, Griffin G, et al.: Captopril Dose-Response Data.xlsx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12881441.v1" }	[ { "id": "71286", "date": "25 Sep 2020", "name": "Maureen Knabb", "expertise": [ "Reviewer Expertise cell physiology", "virology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study demonstrates that captopril, an angiotensin converting enzyme-1 inhibitor, attenuates HSV-1 infection in SY5Y neuroblastoma cells. Strengths of the study include evidence that captopril at low doses modestly reduces HSV-1 infection using multiple techniques including cell viability, microscopy, GFP-replication assay, qPCR, and cDNA synthesis. Weaknesses of the work include lack of experimental evidence for the mechanism of action of captopril, especially in light of the molecular modeling prediction that captopril binds to gD, an important viral protein responsible for HSV-1 binding and cell-to-cell spread. Major and minor recommendations for acceptance are outlined below.\n\nMajor revisions:\nGiven the results of molecular modeling showing that captopril binds to glycoprotein D, the authors must address whether this binding pocket represents a nectin or HVEM binding domain and whether captopril acts to prevent HSV-1 binding or cell-to-cell spread.\n\nPLoS Pathog. 2011 Sep; 7(9): e1002277. Structure of Herpes Simplex Virus Glycoprotein D Bound to the Human Receptor Nectin-11\n\nHSV-1 spread from one cell to another requires gD and its receptors, enabling membrane fusion and multinucleated syncytia formation. Photomicrographs (Fig 5) suggest that this may be the case in this experiment. Since the authors only investigated the expression of ICP0, an early viral gene product made immediately upon HSV-1 entry, reduced expression in the presence of captopril suggests that captopril’s mechanism may be to reduce viral entry. The authors should perform additional experiments, such as a neutralization assay or viral titer analysis to address the mechanism of action of the captopril effect.\n\nCaptopril reduces growth in Sy5Y cells which could be responsible for reduction in HSV-1 replication.\n\nProc Soc Exp Biol Med . 1991 Mar;196(3):280-3. doi: 10.3181/00379727-196-43189. Angiotensin-converting enzyme inhibition reduces neuroblastoma cell growth rate2\n\nIn the discussion, the authors state that the primary limitation of this study is the use of only one cell line. If there are no previous studies investigating the use of this cell line and HSV-1 infection, the authors should provide a rationale as well as perform a growth curve to address the use of this cell line.\n\nThe authors state that other evaluated ACE inhibitors (lisinopril and fosinopril) do not interact with HSV-1 gD. Using another ACE-1 inhibitor to test the potential mechanism of captopril interaction with gD would be an appropriate control.\n\nDifferent studies provide conflicting evidence about whether captopril crosses the blood brain barrier (BBB). The authors provide no references for this statement in the discussion. Cerebrovascular effects of the converting enzyme inhibitor captopril. PMID: 64001122 J O Jarden, D I Barry, S Strandgaard, D I Graham, O B Paulson\n\nMinor revisions:\n\nPage 1 “some components demonstrated antiviral properties demonstrate”- provide specific examples\n\ncorneal blindness and encephalitis - repetitive\n\nHuman cytomegalovirus, Human Herpesvirus-5 (HHV-5), has been demonstrated to cause an increase in arterial blood pressure7—add by increasing expression of renin and Ang II\n\nAuthors need to provide additional information about captopril in the introduction.\n\nFig 1- there are no asterisks or plus signs in the figure- why 140-160% better viability?\n\nFig 3- any difference in the microscopy technique used for the photomicrographs a and d versus b and c- the light transmitted through the specimens looks different.\n\nFig 4- not much change at 24 hr but drastic change at 48- could this effect be due to captopril preventing reinfection via gD?\n\nNeed scale bars in micrographs- some of the micrographs look to be at different magnifications- is there any way to improve contrast? The cells are difficult to see with the dark background.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "71289", "date": "29 Sep 2020", "name": "Donald Alcendor", "expertise": [ "Reviewer Expertise Herpesvirus biology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reviewed manuscript summarizes ACE inhibitor, captopril, attenuates cytopathic effects of herpes simplex virus 1 in SH-SY5Y cells.\n\nOverall: The manuscript is fairly well written however the manuscript is poorly referenced, details needed in the introduction and methods sections, there is only a marginal effect of Captopril on HSV-1 replication, there are no direct PCR results for early and late gene markers of HSV-1 replication, and there are strong assumptions made on effects of virus replication and ICPO.\n\nIt is my opinion that the manuscript be 'Not Approved' based on the lack of rigor, the marginal effects of Captopril on HSV-1 replication that is not likely to be biologically relevant in preventing infection, and the lacks appropriate controls.\n\nMain Comments:\n\nThe implications of treating HSV-1 which is a latent virus that induces recurrent disease with an ACE-1 inhibitor could result in side effects related to blood pressure control and this intervention would not be curative. If ACE-1 inhibitors could disrupt viral latency, then this would be novel.\n\nThe authors state “Moreover, HSV-1 infection is a significant contributing factor in Human Immunodeficiency Virus (HIV) transmission and pathology”. It should be known that it is HSV-2 that predisposes individuals to HIV-1 acquisition and not HSV-1.\n\nThe manuscript is poorly referenced.\n\nThe level of HSV-1 clinical resistance and the resistance in the general population after acyclovir therapy should be stated in the introduction.\n\nI agree with the authors that novel therapies for HSV-1 are warranted but this has to include therapies that disrupt or prevent latency. Suppression of ICPO has to correlate with virus replication over a time course that includes appropriate controls.\n\nThere is no mention of the 8 different herpesviruses that infect humans in the introduction. There is no mention of HSV-2 and how it differs from HSV-1 in terms of clinical disease.\n\nThe authors state Components of the Renin-Angiotensin Aldosterone System (RAAS), known for their function in the homeostatic control of arterial blood and osmotic pressures, have been implicated in regulating virus activity. There is no reference to support this statement.\n\nThe authors should also consider that cytomegalovirus is a beta-herpesvirus that is known to be implicated in vascular disease whereas HSV-1 is an alpha-herpesvirus that goes latent in trigeminal ganglia.\n\nThe authors describe the attenuation of CPE in HSV-1-infected SH-SY5Y neuroblastoma cells. What is the rationale for using transform cell lines and not primary cells in these experiments?\n\nA brief description of the HSV-1 F strain is warranted in the materials and methods section of the manuscript.\n\nThe authors state “Cells were incubated until the cell viability assay or imaging analysis was performed. The exact time of incubation should be included.\n\nLab adapted strains behave differently than primary clinical isolates and this distinction should be made clear in the manuscript.\n\nThere is no information on how Captopril was obtained the source and how it was sterilized for cell culture in the infection assays. The rationale for the concentrations of Captopril was not mentioned in the manuscript.\n\nIn figure 1, there still significant infection in the cultures treated with Captopril at all concentrations with a viability increase of 30-40% for all concentrations of Captopril and there is no dose-response trend regarding viability.\n\nThe values found in Figure 2 are statistically significant but unlikely to be biologically relevant in suppressing HSV-1 infection in vivo.\n\nFigure 3 does not show if these cells are infected because staining for an HSV-1 marker was not performed here. It is also unclear what the arrow is pointing at.\n\nThe authors state “qPCR results demonstrated that captopril co-administration to HSV-1 infected cells decreased ICP0 gene expression by approximately 34%, compared to infected cells not administered captopril.” Only one immediate early gene was examined in this assay and the claims being made for ICPO are not supporter here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1122
https://f1000research.com/articles/8-1877/v1	07 Nov 19	{ "type": "Opinion Article", "title": "The de.NBI / ELIXIR-DE training platform - Bioinformatics training in Germany and across Europe within ELIXIR", "authors": [ "Daniel Wibberg", "Bérénice Batut", "Peter Belmann", "Jochen Blom", "Frank Oliver Glöckner", "Björn Grüning", "Nils Hoffmann", "Nils Kleinbölting", "René Rahn", "Maja Rey", "Uwe Scholz", "Malvika Sharan", "Andreas Tauch", "Ulrike Trojahn", "Björn Usadel", "Oliver Kohlbacher", "Bérénice Batut", "Peter Belmann", "Jochen Blom", "Frank Oliver Glöckner", "Björn Grüning", "Nils Hoffmann", "Nils Kleinbölting", "René Rahn", "Maja Rey", "Uwe Scholz", "Malvika Sharan", "Andreas Tauch", "Ulrike Trojahn", "Björn Usadel", "Oliver Kohlbacher" ], "abstract": "The German Network for Bioinformatics Infrastructure (de.NBI) is a national and academic infrastructure funded by the German Federal Ministry of Education and Research (BMBF). The de.NBI provides (i) service, (ii) training, and (iii) cloud computing to users in life sciences research and biomedicine in Germany and Europe and (iv) fosters the cooperation of the German bioinformatics community with international network structures. The de.NBI members also run the German node (ELIXIR-DE) within the European ELIXIR network. The de.NBI / ELIXIR-DE training platform, also known as special interest group 3 (SIG 3) ‘Training & Education’, coordinates the bioinformatics training of de.NBI and the German ELIXIR node. The network provides a high-quality, coherent, timely, and impactful training program across its eight service centers. Life scientists learn how to handle and analyze biological big data more effectively by applying tools, standards and compute services provided by de.NBI. Since 2015, more than 250 training courses were carried out with more than 5,200 participants and these courses received recommendation rates of almost 90% (status as of October 2019). In addition to face-to-face training courses, online training was introduced on the de.NBI website in 2016 and guidelines for the preparation of e-learning material were established in 2018. In 2016, ELIXIR-DE joined the ELIXIR training platform. Here, the de.NBI / ELIXIR-DE training platform collaborates with ELIXIR in training activities, advertising training courses via TeSS and discussions on the exchange of data for training events essential for quality assessment on both the technical and administrative levels. The de.NBI training program trained thousands of scientists from Germany and beyond in many different areas of bioinformatics.", "keywords": [ "de.NBI", "de.NBI Cloud", "Life Sciences", "Bioinformatics", "ELIXIR", "Training", "Education", "Germany" ], "content": "Introduction\n\nIn the last decade, researchers in the life sciences have been early victims of the ‘Big Data Problem’ because of technical improvements in the so-called ‘omics’ and image analysis fields including the challenges of the five ‘V’s of big data: volume, veracity, velocity, variety, and value1. Here, large and complex datasets are rapidly generated to analyze various biological levels in living cells per day. Even small benchtop sequencing machines are now capable of producing terabytes of data, but many life scientists neither possess the skills to analyze the data properly nor the knowledge to use existing analysis resources. Therefore, the true bottleneck of the current ‘Big Data Problem’ in the life sciences is often not the storage or compute power, but the knowledge and skills how to use existing bioinformatics services and tools. An important way to solve these deficiencies in the field of life sciences is by means of bioinformatics training. The need for such training was described recently: The majority (> 95%) of life scientists located in Europe work or plan to work with large datasets, but less than 35% possess the bioinformatics and statistical skills to handle the huge amount of generated data2.\n\nIn 2012, the German Bioeconomy Council published the recommendations ‘Requirements for a Bioinformatics Infrastructure in Germany for future Research with bioeconomic Relevance’, coming to the conclusion that expertise centers in Germany should have permanent structures for training users from life sciences3.\n\nAccordingly, the German Network for Bioinformatics Infrastructure (de.NBI) program was launched by the Federal Ministry of Education and Research (BMBF) in March 2015, and it now includes 40 projects that are operated by 30 research institutes organized in eight service centers3. de.NBI offers a large repertoire of high-quality training courses to support life scientists with different expertise levels in bioinformatics and from various research fields. These training activities of de.NBI are focused on teaching life scientists in Germany and Europe the proper handling and analysis of their biological big data more effectively by applying tools, standards and compute services provided by the service centers. Therefore, de.NBI trainers work together and within different existing German and European (training) communities, e.g. the Galaxy Training Network2, The Carpentries4, FAIRDOM5 etc., to connect de.NBI to researchers and to the most important topics in the life sciences community (Figure 1). These topics were introduced in the de.NBI training activities, which range from basic skills to advanced data analysis and expert hackathons including 1–14 day training courses, webinars, mentoring, online training and one- week summer schools3.\n\nOn the left side, the service centers are shown. These centers organize specific training events, create online training material and provide mentoring. In the middle, the map of Germany includes all de.NBI locations. The different colors represent the corresponding service center (see logos on the left side).\n\nThe special interest group 3 (SIG 3) ‘Training & Education’ is centrally coordinating these training courses and the education efforts within the network. It is composed of training experts from each service center. SIG 3 collects all planned de.NBI training activities and provides a structured process of reporting, documenting, and monitoring all training events. The general goal of de.NBI is to develop a national bioinformatics training program for Germany.\n\nThe following topics are currently in the focus of SIG 3:\n\nImprovement of the quantity and quality of training courses.\n\nIntroduction of online training/e-learning courses/platforms.\n\nStandardization of training course monitoring.\n\nIntegration into the ELIXIR training platform and TeSS.\n\nCoordination of training events and standards with ELIXIR.\n\nThe strategic planning of SIG 3 includes following aspects:\n\nClosing of gaps in the topics covered by the training courses.\n\nImprovement of the quantity and quality of e-learning material.\n\nQualification of more trainers.\n\nThe training activities of each de.NBI service center are described in the following sections.\n\n\nBioinformatics training in Germany by de.NBI\n\nThe Heidelberg Center for Human Bioinformatics (HD-HuB) unites bioinformatics expertise from research groups at four established research institutions in Heidelberg and Berlin: European Molecular Biology Laboratory (EMBL), the German Cancer Research Center (DKFZ), Heidelberg University and Berlin Institute of Health (BIH) at Charité. Since November 2016, a partner project on computational epigenetics (de.NBI-Epi) has been added to HD-HuB, which is coordinated by the researchers of Saarland University in association with DKFZ.\n\nResearch focus. HD-HuB service center focuses on delivering computational resources to support the development and maintenance of bioinformatics tools in four main application areas within the organizational framework of de.NBI:\n\n1. High-throughput sequencing (HTS) data analysis to study human genetics and genomics6.\n\n2. Taxonomic and functional microbiome profiling, genome and metagenome annotation and association of microbiome composition with host phenotypes to facilitate metagenomic studies7.\n\n3. Automated analysis and visualization of large-scale high-content phenotype screening data for phenotyping of human cells8,9.\n\n4. Analysis workflows for genome wide DNA methylation data for computational epigenetics research10.\n\nIn addition, HD-HuB members established two of the six de.NBI Cloud sites in Heidelberg and Berlin.\n\nTraining and outreach activities. The HD-HuB consortium has offered training courses addressing the computational needs of early stage and advanced researchers with a wide variety of research interests. The de.NBI Cloud compute resources are used in different HD-HuB training activities. Below, HD-HuB’s training activities are listed under different categories based on the different learner-profiles:\n\n1. Programming courses for novice and advanced learners. HD-HuB members from EMBL organize several beginner and advanced courses on programming skills every year in Python, R, Unix/Shell and version control. These courses have been positively received by the course participants who constitute ~50% of our overall learners every year. Such courses have been vital to the computational skill development of biologists and wet-lab scientists who lack formal training in computation and bioinformatics. In addition, The Carpentries courses have been very popular among the novice learners in our communities that allow them to acquire the basic building blocks of software development at the Software Carpentry workshops and get hands-on experience of data analysis at the Data Carpentry workshops.\n\n2. Training on specialized bioinformatics topics. In addition to offering general training courses, the HD-HuB service center offers specialized courses as per demand from the researchers with different research backgrounds. These so far include courses on protein bioinformatics, bioimage analysis, DNA methylation, experimental data analysis and RNA-Seq and Bisulfite sequencing data analysis in cancer research.\n\n3. Courses for advanced computational biologists. To support computational needs of advanced learners in our community we offer hands-on training on data analysis skills such as machine learning, workflow management, cloud computing and statistics. These workshops also provide an opportunity for peer-based learning by bringing scientists with similar research interests together who can potentially collaborate with each other in the future.\n\n4. Train the trainer courses. Trainers at our workshops are often volunteers from the respective scientific community, who spend time in creating useful resources to teach at these events. These volunteers are highly valuable to our mission of training computational skills to the future learners. In order to facilitate such knowledge transfer effectively, HD-HuB has started to train experienced researchers, who can become trainers in future courses at their respective institutes. These trainings are offered under the title ‘train the trainer’ where they can gain theoretical and practical knowledge of useful teaching techniques used in best teaching practices.\n\n5. Local outreach activities. HD-HuB hosted the first international de.NBI symposium ‘Bioinformatics for Human Health and Disease’ in 2016 at the German Cancer Research Center (DKFZ) in Heidelberg. Attended by 140 participants and over 20 international speakers, the conference provided an opportunity for the researchers to discuss their research and plans within the de.NBI framework. HD-HuB has also co-hosted local event series like de.NBier technical seminars and the Heidelberg Unseminar in Bioinformatics, which are mainly targeted at non-expert scientific communities.\n\nBiGi consists of three partners: the Genome Informatics group at Bielefeld University, the Bioinformatics & Systems Biology group at Justus-Liebig-University Gießen and the partner project at Otto-von-Guericke-University Magdeburg.\n\nResearch focus. The BiGi service center offers tools, services and training for microbial genome11–14, metagenome15, and postgenome research16 that is complemented by a large-scale hardware infrastructure. The focus in Gießen is on genomics (assembly, annotation and comparative analysis and short-read-mapping data evaluation) and read-based metagenomics research. Bielefeld focuses on assembly-based metagenomics and post-genome analysis, such as transcriptomics, proteomics and metabolomics. The partner project, located in Magdeburg, develops solutions for the analysis of metaproteome data. The BiGi service center concentrates on the computational analysis of isolated microbes as well as of microbial communities. It is equipped with large-scale computing and storage resources, and the institutes in Bielefeld and Gießen were involved in establishing a dedicated de.NBI Cloud infrastructure.\n\nBesides general bioinformatics consulting services, different tools are provided by the BiGi service center, e.g. EDGAR12–14, Fusion17, MetaProteomeAnalyzer18 and many more. Since the establishment of the de.NBI Cloud19,20, all partners work on different cloud-based applications such as tailor-made images for specific bioinformatics analysis (Metagenomics, Nanopore sequencing, ASA3P21) that also play a key role in many of the workshops provided.\n\nTraining and outreach activities. Researchers who are interested in the analysis of genomic, metagenomic or postgenomic data or who want to get familiar with the use of the de.NBI Cloud19,20 are welcome to join one of the workshops offered by BiGi.\n\n1. Nanopore best practice workshop. To keep up with recent developments in sequencing techniques, a Nanopore best practice workshop has been launched in Bielefeld in 2017 with the support of the service center GCBN that will be held annually due to high demand of the scientific community. The aim of this workshop is to familiarize the participants with the Nanopore sequencing technology, its applications and the ‘Best Practice’ bioinformatics workflow. The Nanopore technology has greatly facilitated the assembly of prokaryotic and eukaryotic genomes. Therefore, the workshop concentrates on the establishment of finalized genome sequences.\n\n2. Training on genomics tools. Since the start of the de.NBI network, Gießen offers an annual genome analysis workshop. Topic of this three-day-workshop is microbial sequence data analysis including quality control, assembly, genome annotation and comparative genomics with a focus on the usage of the BiGi software tools ASA3P21 and EDGAR12–14 as well as the BiGi Galaxy Server.\n\n3. Training on metagenomics tools. A further annual course organized by Bielefeld and Gießen together is the introduction into targeted and untargeted metagenome analysis. This course teaches best practices for targeted (16S rDNA operon gene amplicons) as well as untargeted (whole-genome shotgun) metagenome analysis based on high throughput next-generation sequencing data.\n\n4. Training on postgenomics tools. For post-genomic analyses, there is a workshop on the analysis, visualization and integration of multi-level -omics data, which introduces the web-based platform Omics-Fusion17. Aim of this workshop is to give answers to typical questions in omics data analyses, e.g. regarding data normalization strategies and handling of missing values, the detection of groups of transcripts, proteins and/or metabolites with similar patterns of expression/abundance using cluster analyses, the visualization of multi-omics data in the context of metabolic pathways, or the identification of differentially regulated transcripts, proteins, and metabolites.\n\n5. Training on metaproteomics tools. A workshop on the MetaProteomeAnalyzer18, also including a wet-lab part in some courses, is provided at least once a year in Magdeburg. The learning goal is to apply a complete workflow starting from design of experiments via sample preparation to measurement and bioinformatics analysis of high-resolution mass spectrometry (MS) data.\n\n6. Introduction to the de.NBI Cloud. There are basic courses on the use of the de.NBI Cloud19,20, which are offered in Bielefeld and Gießen or also as dedicated workshops on scientific conferences. In these courses, participants learn to setup a research project on the de.NBI Cloud, to work with virtual instances, to efficiently utilize cloud computing resources, about networking and security issues and means of deploying bioinformatics tools in the cloud.\n\nThe Bioinformatics for Proteomics (BioInfra.Prot) service center is located within the medical bioinformatics group at the Medizinisches Proteom-Center at Ruhr-University Bochum and at the Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V. in Dortmund. Within the Lipidomics Informatics for Life Sciences (LIFS) partner project of the center, three members are involved: the Biological Mass Spectrometry group at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, the bioanalytical chemistry group at Forschungszentrum Borstel - Leibniz Lungenzentrum, and the lipidomics group at ISAS in Dortmund.\n\nResearch focus. The main topic of BioInfra.Prot is proteomics data standardization and conversion, protein inference, quality standards, expression analysis and bioinformatics and statistical consulting22. BioInfra.Prot develops and maintains tools such as PIA23 for protein inference and identification, PAA24 for biomarker detection from protein microarray experiments, and SearchGUI25,26 and PeptideShaker26,27 for combined scoring of proteins from multiple search engines to enable automated high-throughput processing of large-scale proteomics data from clinical and general life sciences contexts.\n\nThe LIFS partner project mainly works on bioinformatics for targeted and comparative lipidomics with applications in cardiovascular and platelet-related disease characterization including lipid identification and discovery28. A further focus is placed on lipidomics data standardization and the establishing of a lipid-centric repository for high-resolution mass spectrometry data. LIFS develops and maintains the tools LipidXplorer29 for shotgun and LipidCreator for LC-MS lipidomics, as well as LipidCompass as a quantitative reference database for tissue and organism-specific lipidomes, and LUX Score30 for comparative visualization of lipidomes based on their shared lipid structural space.\n\nTraining and outreach activities. BioInfra.Prot offers courses mainly focused on mass-spectrometric data analysis and integration in proteomics and lipidomics. Therefore, the courses are mainly aimed either at researchers coming from life or analytical science and working with mass spectrometry data, who want to learn the basic tools and workflows of data preprocessing, statistical analysis and integration, or at bioinformaticians, who want to extend their knowledge on available tools and workflows in these areas.\n\n1. Training on proteomics tools. BioInfra.Prot offers regular courses on bioinformatics for proteomics at the annual German Society for Mass Spectrometry (DGMS) meeting and at Ruhr-University Bochum. These courses are generally open to anyone, providing material for beginners and more advanced participants alike. The online announcement of each course includes information on recommended prerequisites.\n\n2. Training on biostatistics / statistical analysis. BioInfra.Prot also offers trainings for differential analysis of quantitative proteomics data and for general introductions to biostatistics and statistical analysis with R. These courses also target beginners and more advanced participants. New courses are regularly announced via the de.NBI portal and on the BioInfra.Prot website.\n\n3. Training on lipidomics tools. LIFS offers an annual course for lipidomics bioinformatics tools at the Lipidomics Forum conference (ISAS Dortmund and FZ Borstel). This course is generally targeted at beginners and intermediate users with wet lab or bioinformatics background who want to learn about the analytical and bioinformatics challenges in lipidomics, how to apply the tools developed by LIFS within a lipidomics workflow, and which data formats to use for reporting of their results.\n\n4. Winter and summer schools. Both BioInfra.Prot and LIFS have (co-)organized summer schools, e.g. the de.NBI Summer School 2016 - From Big Data to Big Insights, the EUBIC winter schools 2017 and 2019 and the e:Med LipoSysMed summer school 2019. These events help to connect and deepen the interaction between the biological, analytical chemistry, clinical medicine and bioinformatics communities by providing more in-depth, audience-specific hands-on tool trainings.\n\nThe Center for Integrative Bioinformatics (CIBI) joins the three projects OpenMS31, SeqAn32 and KNIME33 that are developed and maintained by researchers at three well established research institutes within Germany: Eberhard Karls University of Tübingen, Free University of Berlin and the University of Konstanz. In addition, the two partner projects MASH (Leibniz Institute of Plant Biochemistry, Halle/Saale) and DAIS (Max Planck Institute for Molecular Cell Biology and Genetics, Dresden) joined CIBI in November 2016.\n\nResearch focus. CIBI delivers cutting-edge software solutions covering a large domain in the field of computational biology, biomedicine and bioimaging as well as their integration into the workflow system KNIME Analytics platform33 to enable data-driven innovations and achievements in these fields. Concrete, the service center develops state-of-the-art and strongly competitive tools in the fields of proteomics and metabolomics, genomics, image processing, data mining and workflow integration, with the goal of reducing time and cost expenses for various bioinformatics and biomedical data analysis tasks.\n\nIn the following section, a general overview of the key components of each project is shown:\n\n1. OpenMS31 and MetFrag34 provide essential tools for mass spectrometry data analysis, the key technology in proteomics and metabolomics. In addition, OpenMS offers a software library, which makes the efficient algorithms and data structures available for the broad community.\n\n2. SeqAn32 provides various applications for alignments, read mapping and error correction, variant detection, local alignment of genomic and proteomic data and metagenomics. The additional software library implements state of the art algorithms and data structures with parallelization and vectorization support, giving bioinformaticians all the resources they need to write modern and scalable software applications.\n\n3. DAIS develops and maintains a powerful, stable, and user-friendly open source software stack for bio-image analysis. Its flagship project is Fiji35, which among others also integrates state-of-the-art software libraries and components (e.g., ImgLib236, BigDataViewer) that power not only Fiji, but also ImageJ237 and KNIME image processing38, as well as other applications (e.g., Labkit, Mastodon, CARE image restoration).\n\n4. KNIME Analytics Platform is an open source workflow-based data analysis platform with a strong focus on data mining in many areas of research and commerce. Its role as the tool of choice for bioinformatic and biomedical data exploration has been strengthened through the tight integration of the OpenMS, SeqAn and DAIS tools, and thus enables a large community to effectively perform and evaluate life science data analysis on a cross-domain level.\n\nTraining and outreach activities. Within CIBI, a broad spectrum of training activities is offered to cover on the one hand the large domain the service center is actively contributing to and secondly to offer distinct training events for different target groups, such as data scientists dealing with big omics-data or application developers and bioinformaticians developing new tools using our resourceful libraries.\n\n1. Training on mass spectrometry data. CIBI offers various courses to introduce mass spectrometry data analysis with a focus on proteomics and metabolomics. In these workshops, concepts such as non-targeted label-free analysis are taught and users learn how to implement complex analysis workflows based on OpenMS tools with subsequent visualisation of the results based on real-life data. In addition to quantification analysis, metabolite identification with MetFrag and MetFamily are taught on several events. The target audience are mainly beginners and intermediate users of proteomics and metabolomics that want to learn how to efficiently process their mass spectrometry data.\n\n2. Training on sequencing data. Another major field in which CIBI offers various training activities relates to sequencing data analysis. Training courses are focused on two user groups. The first are bioinformaticians developing new tools. In dedicated hands-on sessions on the annual user meeting, bioinformaticians are taught how to use the SeqAn software library to write new competitive tools using state-of-the-art algorithms and data structures. The offered trainings mostly focus on beginners but require an intermediate degree of programming knowledge. Knowledge in C++ is helpful but not required. For advanced SeqAn users, we host an annual developer meeting where we tackle specific problems of the participants and add new components to the library. The second target group are data scientists. In these courses, data scientists explore typical analysis pipelines using SeqAn and external tools integrated into the KNIME analytics platform.\n\n3. Training on bioimage data. Annual hands-on courses introduce participants to develop within and for the Fiji, ImageJ2, and KNIME ecosystems. In addition, CIBI offers a deep learning course especially for image-based problems.\n\n4. Training on workflows and tool integration. CIBI organizes life science workshops on the KNIME Spring Summit and teaches the integration of our tools in KNIME to produce strong and efficient multi-omics pipelines on various meetings (either on international conferences or on joint user meetings). Hence, many of the CIBI events are accompanied with experts in KNIME and users are always welcome to bring their own data so CIBI experts can develop an efficient solution for their problem at hand.\n\n5. Training on conferences. CIBI offers one-day workshops on international conferences, where the CIBI members can get in touch with life scientists around the world and discuss the topical research subjects and how to tackle them with our software portfolios.\n\n6. Online training. CIBI made most of the training materials available online as self-paced training. These include written tutorials and videos with many practical tips and examples. The materials are intended for self-regulated learning to study and explore the capabilities of our software and tools in a comfortable way. The target audience of these materials ranges from novices to advanced users.\n\n7. Hackathons and developer meetings. For all projects and topics, CIBI offers annual hackathons and developer meetings. During these meetings, specific problems and future directions of our software are discussed and implementation of feature requests are tackled together with the community.\n\nThe RNA Bioinformatics Center (RBC) brings together all major RNA bioinformatics groups in Germany located at three sites: University of Freiburg, University of Leipzig and Max Delbrück Center in Berlin. Since November 2016, RBC includes two partner projects called de.STAIR (Structured Analysis and Integration of RNA-Seq experiments) and de.NBI-epi (Computational Epigenetics).\n\nResearch focus. The RNA Bioinformatics Center (RBC) deals with all RNA-related data not limited to transcriptome analysis but also RNA structure analysis, prediction of targets of RBPs (CLIP-Seq) and non-coding RNAs, definition and classification of RNA transcripts and the analysis of protein-RNA and RNA-RNA interactions. The RBC-Freiburg focuses on the analysis of RNA-RNA and RNA-protein interactions39, RBC-Leipzig on the analysis of non-coding RNAs and RNA structure40 and RBC-Berlin on RNA-binding proteins and post-transcriptional regulation41. The partner project de.STAIR consists of three sites: de.STAIR-Freiburg working on the regulatory RNA interaction and integration, de.STAIR-Jena on the causes and effects of quantitative and qualitative expression changes and de.STAIR-Rostock on RNA-Seq workflow specification and technical integration42. The second partner project de.NBI-epi is located in Berlin and in Freiburg and is focusing on Computational Epigenetics. The mission of the RBC is to support all RNA-related research within de.NBI, Germany, Europe and beyond. One important aspect is the integration of RNA-related data with other types of data such as transcriptome or epigenetic data. For that reason, RBC developed the RNA workbench, which allows the analysis of RNA-related data in an integrated form.\n\nTo achieve its mission, the center provides training and tools for life science researchers, with a special focus on RNA and the analysis of high-throughput data. It maintains and develops the largest Galaxy instance in Europe with more than 9000 users, responsible for more than 6 million submitted jobs. The Galaxy server is freely open to all researchers via useGalaxy.eu. RBC maintains an integrated, easily accessible RNA analysis workbench43, based on Galaxy, which can be downloaded and installed locally, or deployed on HPC-like environments or clouds. It includes more than 50 RNA tools, multiple workflows, interactive tours and training data.\n\nTraining and outreach activities. With its wider focus on the analysis of high-throughput data in Galaxy, an important goal of the RBC is to support researchers by educating them in big data analysis, programming, data management, Galaxy server administration and more. The RBC members believe that sharing of knowledge and the open science movement are the key points for the future success in the analysis of life science data. For that reason, the RBC and its partner projects provide training events covering diverse topics (data analysis, programming, tool development, containers, etc.) and targeting a diverse audience at different levels of experience (scientists, developers, administrators, trainers - from beginners to experts).\n\n1. Training on HTS and RNA-related data. For example, RBC-Freiburg is offering a full-week hands-on HTS data analysis workshop in Freiburg twice per year: Introduction to Galaxy and HTS, RNA-Seq, ChIP-Seq, Exome-Seq, MethylC-Seq, etc. Similar courses are also available at the RBC-Berlin site. Many other training workshops are also given in collaboration with other de.NBI centers and ELIXIR, not only in Germany and Europe but also around the world.\n\n2. Galaxy Training Network (GTN). As face-to-face workshops do not fit to the scale of demand, the RBC has put a lot of effort into developing and offering online training material. RBC-Freiburg is a major contributor to the community-driven development of Galaxy training material2. All of this material is freely accessible under a Creative Commons license at https://training.galaxyproject.org. It contains tutorials with hands-on, slides and interactive tours, designed for both self-training and workshops, as well as the technical support with tools, data, virtualized instances, etc. RBC-Freiburg via useGalaxy.eu is also offering a special service for Galaxy trainers: Training Infrastructure as a Service (TIaaS: https://galaxyproject.eu/tiaas), completely dedicated compute resources for the duration of a workshop training.\n\n3. Mentorship. Mentorship is also a practice in place at RBC. For example, RBC-Freiburg has guests regularly, who would like to learn a technology or gain specific knowledge by immersion for a few days. The mentorship also works remote via support on real-time chat and also via online meeting with other instructors (as in the Carpentries) or developers. Mentorship programs can be requested via an online booking system. RBC, in collaboration with HD-HuB and ELIXIR-UK, is also building a mentoring program for life-scientists on open science as part of the Mozilla Open Leader program.\n\n4. Hackathons. To combine forces, RBC regularly organizes hackathons and contribution fests: short events (usually few days) where people work together to develop new, or improving existing techniques, tools, training materials, etc. Several have been organized per year on site or online, in close cooperation with de.NBI, ELIXIR, and worldwide communities, like Bioconda.\n\n5. Administrator training. RBC is offering multiple workshops dedicated to administrators during a year. That involves administering servers, creating and setting up Virtual Research Environments (VRE), deploying VREs, managing cloud deployments, containers and monitoring. In a weeklong workshop, administrators can also learn how to deploy production ready Galaxy servers that scale to multiple thousands of users.\n\n6. Local outreach activities. RBC is also involved in an outreach program, Street Science Community, supported by de.NBI. The aim of this program is to bring science, in particular life science, to schools and to the general public via workshops during which participants can extract and sequence DNA of yeast from beers.\n\nThe German CropBioGreenformatics Network (GCBN) is a collaboration between three partners: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, German Research Center for Environmental Health Munich and Forschungszentrum Jülich.\n\nResearch focus. GCBN provides crop plant-related bioinformatics services in the field of green bioinformatics. GCBN provides transparent access to germplasm data, it provides tools to annotate plant genes, genomes and transposons and is collaborating with plant phenotyping centers (e.g. the ESFRI structure EMPHASIS)44. GCBN uses this interaction to help in bridging genotypes with phenotypes, i.e. to predict plant phenotypes from genotypes or to analyze plants, which are preserved in genebanks45. As such, GCBN is also involved in plant phenotype standardization together with the EMBL-EBI and all GCBN partners are involved in the ELIXIR plant community activities.\n\nTraining and outreach activities. Together with other de.NBI partners, GCBN organizes different training courses. GCBN consults and provides help to users from plant sciences and breeding companies. Given the expertise within GCBN, the main focus is on large scale data processing in the plant sciences, focusing on plant omics data such as metabolomics and transcriptomics data, genome assemblies and annotation, plant phenotyping data and the interaction between the plant genome(s) and the resulting phenotype.\n\n1. Computational biology starter. GCBN offers introductory courses into computational plant biology. These courses usually introduce the basic use of Linux and R as well specific tools for plant phenotyping and data analysis.\n\n2. Training on RNA-Seq for plants. RNA-Seq courses for plants focus on the quantitative analysis of plant RNA-Seq data from read mapping to data visualization using e.g. MapMan46,47. The courses focus on the specific issues of plant transcriptomics data such as polyploid genome and ancient gene duplications and their effect on expression data.\n\n3. Training on phenotyping analysis. Plant phenotyping data and analysis courses are usually run in conjunction with the German Plant Phenotyping Network DPPN or the EMPHASIS pan-European Infrastructure. Here, scientists learn about data standards, extraction of data from images and data normalization.\n\n4. Training on FAIR Data. GCBN contributes to FAIR (Findable, Accessible, Interoperable, Reusable) data workshops held by other service centers and mainly by the BioData service center. Furthermore, it is contributing the plant specific topics and FAIRification in the plant sciences especially for data provision in repositories like e!DAL-PGP48.\n\n5. Training on handling Nanopore data. GCBN contributes to the Nanopore best practice workshops of BiGi (see earlier subsection on BiGi). Here, GCBN provides its expertise on long read data generation, cleaning and assembly for complex, repetitive plant genomes.\n\nThe BioData service center is a collaboration of five partners: Jacobs University Bremen, Alfred-Wegener-Institut - Helmholtz Zentrum für Polar- und Meeresforschung, MARUM/University of Bremen, Leibniz Institute DSMZ, Technical University of Braunschweig and the partner project at the University of Hamburg.\n\nResearch focus. The BioData service center consists of the information systems SILVA49, PANGAEA50, BacDive51, BRENDA52 and ProteinsPlus. BioData provides highly curated reference datasets and related services for users in academia and industry. The BioData service center facilitates access to services for ribosomal RNA genes (SILVA), georeferenced data from earth system and environmental research (PANGAEA), detailed strain-linked information on the different aspects of bacterial and archaeal biodiversity (BacDive), comprehensive information on all aspects of enzyme functions (BRENDA), as well as easily accessible protein structure data (ProteinsPlus). SILVA and BRENDA have been identified as ELIXIR Core Data Resources underlining their fundamental importance to the wider life-science community and the long-term preservation of biological data in Europe and beyond53,54.\n\nTraining and outreach activities. The BioData service center concentrates on data products as well as on research and services. It is well known for its reference databases for taxonomy, phylogeny, biotechnology, biochemistry, pharmacy, medicine, quality control, diagnostics, environmental and biodiversity research. BioData provides analysis services for enzyme structures and functions, classification of next-generation sequencing data. In addition, BioData is highly active in data mobilization and publishing as well as research data management following the FAIR55 (Findable, Accessible, Interoperable, Reusable) data principles. Therefore, the targeted audience are researchers in the life- or medical sciences, who like to use data and data products as references in their research or applications and/or manage and publish their data in the spirit of science 2.0 aka open-science.\n\n1. Training on FAIR data. The BioData training courses include the topic ‘FAIR data’, especially within the de.NBI summer school 2018. Here, the courses raise awareness for the importance of proper research data management in general, but also provide a practical toolbox for the acquisition, curation, documentation, archiving and publication of research data following the FAIR data principles. Furthermore, practical training on accessing the data for re-use and integration are part of their training courses.\n\n2. Training on databases. The BioData service center bundles large databases and provides the corresponding training courses for these databases. For instance, in the training course on the rDNA reference database SILVA, in addition, all necessary steps researchers need to take when performing amplicon-based investigations using the rDNA as a marker gene are explained. The course starts with experimental design, including an overview of relevant sequencing technologies, and the selection, design, and evaluation of primers for the amplification of rDNA. It provides examples and best practice solutions for data pre-processing and quality assurance up to the contextualized submission of the NGS data to public repositories. It provides a framework for statistical analysis of the data including a short Linux and R crash course. In addition, this course also combines an introduction to the BacDive database (Bacterial Diversity Metadatabase) as a tool for further data integration to better understand the biology of sequences/organisms analyzed.\n\nde.NBI Systems Biology service center (de.NBI-SysBio) is the service center supporting particularly the systems biology community by providing data and model management tools, manually curated reaction kinetics data and world-wide recognized tools for mathematical modeling of biological networks. The data management part of de.NBI-SysBio is provided by the HITS (Heidelberg Institute for Theoretical Studies). The partner project de.NBI-ModSim (Heidelberg University and the Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg) deliver support in model construction.\n\nResearch focus. The data management part of de.NBI-SysBio has the mission to serve customers with standardized high-quality scientific data and data services. The data management system “SEEK for Science” has been developed by the transnational FAIRDOM project5 as a sharing space for projects fitted to the needs of systems and synthetic biology as well as systems medicine. Research partners can easily upload, enrich and exchange all kinds of data types, models, simulations and processes arising in an interdisciplinary project and at the same time make them FAIR (Findable, Accessible, Interoperable, Reusable). The database SABIO-RK provides structured, standardized and annotated kinetic data with a focus on supporting the computational modelling community to create models of biochemical reaction networks as well as on allowing experimentalists to gain further knowledge about enzymatic activities and reaction properties. The data coming mainly from literature are expert-curated and can be easily exported for direct use, e.g. in modeling tools like COPASI54 to run simulations.\n\nThe modeling part of de.NBI-SysBio is provided by Heidelberg University and the Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg and their tools COPASI56 and CellNetAnalyzer57. CellNetAnalyzer (CNA) is a MATLAB toolbox serving a graphical user interface and various computational methods and algorithms for exploring structural and functional properties of metabolic, signaling, and regulatory networks. The software includes numerous methods and algorithms from stoichiometric and constraint-based modeling (e.g. metabolic flux analysis, flux balance analysis (FBA), flux variability analysis, elementary-modes and pathway analysis, network and strain design). COPASI is a widely used software tool for creating, simulating and analysing models of biochemical reaction networks. It is open source free software and available for all major operating systems. Its features include deterministic and stochastic simulation, steady state analysis, stoichiometric analysis, optimisation and parameter estimation, time scale analysis, sensitivities and metabolic control analysis, Lyapunov exponents and linear noise approximation.\n\nTraining and outreach activities. The de.NBI-SysBio training courses, workshops and tutorials are suitable for beginners as well as for advanced users. For instance, the three-days training course ‘Tools for systems biology modeling and data exchange: COPASI, CellNetAnalyzer, SABIO-RK, SEEK’ with extensive hands-on sessions annually takes place alternately in Heidelberg or Magdeburg. Additionally, online tutorials are offered to introduce the main tools (e.g. FAIRDOMHub5, SABIO-RK58, COPASI56, CellNetAnalyzer57).\n\nAs regular international outreach activities de.NBI-SysBio is involved in the organization of satellites of the annual ICSB conference (International Conference on Systems Biology) with the ‘COMBINE Tutorial - Modelling and Simulation Tools in Systems Biology” and “Advanced Modeling with COPASI’.\n\n1. Training on data management and FAIR data. de.NBI-SysBio was involved in organization of the de.NBI summer school on FAIR data and data management in 2018. The data management training courses are open to everyone interested in producing FAIR data in project driven scientific research. de.NBI-SysBio offers special training courses for experimentalists or for modelers but also more general workshops and tutorials with breakout sessions to delve into the topic from a modeler’s, experimentalist’s or developer’s point of view (e.g. annual Systems Biology Developers Foundry).\n\nFurthermore, de.NBI-SysBio offers on demand visits to support customers in installing their projects or local SEEK instance.\n\n2. Training on modeling. The trainings for the modeling tools CellNetAnalyzer and COPASI offered by de.NBI-ModSim aim at modelers or those who want to become one. Attendees learn basic model construction and analysis techniques for kinetic modeling of biochemical systems (illustrated and exercised with COPASI) and principles of stoichiometric and constraint-based modeling of metabolic networks (coupled with hands-on exercises using CellNetAnalyzer).\n\n\nde.NBI Cloud-based training\n\nIn order to perform analysis on datasets available in the life sciences, an appropriate compute infrastructure is necessary. The de.NBI Cloud was created to fill this gap and offers storage and compute resources for researchers in life science in Germany. Through a cloud federation setup, the six cloud sites including Bielefeld, Gießen, Freiburg, Heidelberg, Berlin and Tübingen (Figure 2) are integrated into a single cloud platform and offer more than 15,000 cores and 38 PB of storage capacity in total. A single sign-on (SSO) mechanism enables the user to access any de.NBI Cloud service via their home institution account using the ELIXIR authentication and authorization infrastructure system (ELIXIR AAI) and in particular Perun, the identity and access management system of ELIXIR19.\n\nCurrently, the de.NBI Cloud offers the project types SimpleVM and OpenStack19. Both project types are using virtual machines (VM) where a VM is an emulation of an operating system and represents one of the main building blocks of the cloud. OpenStack is an Infrastructure as a Service (IAAS) system that allows a developer to configure any computational resources like networking, storage and VM settings for running large-scale analyses or offering of a web service in the cloud for other researchers. SimpleVM is a beginner friendly project type that streamlines the handling of VMs and does not demand any particular knowledge in Cloud Computing. While OpenStack is suitable for training in the context of Big Data like running workflows for metagenome analysis, the SimpleVM project type can be used for command line, Linux or any other trainings where running tools on one virtual machine is sufficient.\n\nThe procedure for applying, using and accessing computational resources for a de.NBI Cloud training course is the following. First, a principal investigator (PI) of a German university or research institution must apply for a project through the de.NBI Cloud portal. As soon as the access committee approves the application, the PI can add the actual trainer and set him as an administrator of the project. The trainer first starts a VM by choosing an appropriate Linux system, e.g. Ubuntu or CentOS, and then installs any software needed by the course participants. Once the trainer has installed the required software, the VM can be snapshotted and made accessible to all course participants. At the beginning of the training course, the PI adds further course participants, who can use exactly the same tools based on the initial VM created by the trainer. Finally, the trainer and the participants can directly start with the actual course.\n\nSince the beginning of 2018, the de.NBI Cloud is in production and multiple workshops, tutorials and even a user meeting have been organized. The given workshops range from research related topics like Nanopore workshops to more cloud computing related topics.\n\n1. Training on sequencing data. In Nanopore best practice workshops participants are taught about Nanopore sequencing technology, its applications and the “Best Practice” bioinformatics workflow (see subsections on BiGi and GCBN). Nanopore tools such as long read assemblers demand many cores and especially a lot of RAM that can be made available by the de.NBI Cloud.\n\n2. Training on metagenomics tools. Other training events like metagenome workshops are demonstrating how to store and analyze large datasets using state of the art bioinformatic tools in the cloud (see subsection on BiGi). These workshops highlight one of the main use cases of the cloud, which is the handling of Big Data.\n\n3. Summer schools and user meetings. Cloud Computing related tutorials and lectures are given during Summer Schools and the de.NBI Cloud User Meeting. In September 2018, de.NBI Cloud organized the first de.NBI Cloud User Meeting in order to teach users best practices in Cloud Computing and to allow them to meet Cloud Computing experts. The user meeting offered presentations about use cases and hands-on sessions for learning new technologies and best practices in handling data, tools and workflows. Workshops ranged from introductions into OpenStack but also covered service-oriented areas like e.g. Kubernetes. Due to the positive feedback received from the attendees, the second user meeting took place in Heidelberg in September 2019.\n\nIn 2016, Germany officially joined ELIXIR, the European life sciences infrastructure for biological information. The ELIXIR-DE node is operated by de.NBI and consequently, de.NBI and SIG 3 joined the ELIXIR training platform. SIG 3 already started to establish collaborations with ELIXIR in training activities and discussion on the exchange of data for training events on both the technical and administrative level, e.g. ELIXIR-DE co-organized the European Galaxy Developer Workshop 2017 in collaboration with other ELIXIR nodes. All de.NBI training course belong to the ELIXIR training program and are available on ELIXIR’s Training Portal, TeSS. Therefore, the user feedback of these courses in form of a standardized survey is collected by ELIXIR and included into the general ELIXIR training feedback. In addition, SIG 3 and the ELIXIR Germany training coordinators are part of a new e-learning working group and of the FAIR training working group. They participate in two implementation studies (Software/Data Carpentries & Cloud for Training), the annual ELIXIR BioHackathon and in different training activities of ELIXIR Communities. With the ELIXIR Staff Exchange Program for Galaxy Train-the-Trainer (TtT) events, SIG 3 can exchange knowledge and training skills with the different ELIXIR training communities and aim to build and provide high quality training resources. In association with members from the ELIXIR training network, the Galaxy Training Network and The Carpentries, SIG 3 also contribute to building a full curriculum on the computational analysis of high throughput sequencing data and runs the two corresponding hybrid workshops. As part of the Mozilla Open Leader program and in collaboration with the ELIXIR Training Platform, SIG 3 is also building a mentoring program on open science for early life-scientists in Europe. With the involvement in the e-learning working group, the e-learning support for bioinformatics in Germany will be improved by integrating different methods and other implementations studies within ELIXIR.\n\n\nFuture plans and conclusion\n\nTo show the success of the de.NBI training program for Germany, the progress in different types of training / education activities as well as the performance has to be summarized. First, the de.NBI summer schools provided training courses (primarily for undergraduate and graduate students) in specific topics related to one or several de.NBI service centers. The de.NBI summer schools on Microbial Bioinformatics organized by BiGi, RBC and de.NBI-SysBio in September 2015, on Proteomics by BioInfra.Prot, CIBI and BiGi in September 2016 and on Computational genomics and RNA biology by RBC in September 2017 were very successful in terms of applications (applicants 2015: 36; 2016: 53; 2017: 60). In addition, de.NBI also organized a summer school on cloud computing in Gießen in June 2017 and a winter school on metabolomics in March 2018. A summer school on FAIR data and data management ‘Riding the Data Life Cycle’ was organized in September 2018 (applicants 2018: 27), whereas in 2019, a summer school on (Bio)Data Science took place in September (applicants in 2019: 30) in Gatersleben.\n\nTool- and topic-specific training was also extended in the last few years (Figure 3). In 2015, 17 training courses with 329 participants were organized by de.NBI. In 2016, the network organized 40 training events with 882 participants. The new established training courses closed gaps in the topics covered in 2015 and were adapted to cover a broader range of qualification levels (from beginner to expert). In 2017, de.NBI further raised in the number of courses and participants (69 training courses with 1482 participants). In 2018, the number of training events plateau at 1520 participants and 77 courses. For the year 2019, a similar number of courses is planned to keep the high-quality standards of de.NBI training courses. Based on new external partners in the training field, a slight increase in training courses and participants is expected.\n\nRegarding performance, de.NBI and ELIXIR-DE guarantee high quality standards (~88% recommendation rate, ~82% very good or excellent votes based on about 1800 feedback responses) above ELIXIR average (Figure 4). However, a general comparison of the training programs provided by each ELIXIR node is very difficult. Every country has a specific teaching focus and different amounts of training courses. In general, ELIXIR UK and ELIXIR Switzerland offer a broad portfolio of training courses similar to de.NBI / ELIXIR-DE59,60, whereas smaller ELIXIR nodes, like Estonia, are only able to offer a few courses including the main research aspects in their countries.\n\nIn addition to face-to-face training, online training was introduced on the de.NBI website in 2016. Further, online hackathons for different software packages were established by the service center RBC in 2016. In 2018, the repertoire of online material was increased to 38 items and guidelines for online training were developed by SIG 3. In 2019, further online material will be provided by the different service centers to reach about 50 items - and thus a larger audience that cannot be covered by the training courses alone.\n\nTo sum up the effort of the de.NBI / ELIXIR-DE training platform: Since 2015, de.NBI service centers, the de.NBI Administration Office and SIG 3 worked very successfully together to establish, coordinate and expand bioinformatics training courses across Germany and in ELIXIR. In total, more than 4800 participants (Status July 2019) were trained in different bioinformatics disciplines to tackle the challenges of the five Vs of the big data problem.\n\nNevertheless, training still has a high priority for de.NBI / ELIXIR-DE and significant future efforts will be put into the creation of more e-learning material, the recruitment of new external de.NBI training partners and the qualification of trainers to keep pushing training activities forward and to continue participating in the ELIXIR training platform.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nHigdon R, Haynes W, Stanberry L, et al.: Unraveling the Complexities of Life Sciences Data. Big Data. 2013; 1(1): 42–50. PubMed Abstract \| Publisher Full Text\n\nBatut B, Hiltemann S, Bagnacani A, et al.: Community-Driven Data Analysis Training for Biology. Cell Syst. 2018; 6(6): 752–758. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTauch A, Al-DIlaimi A: Bioinformatics in Germany: toward a national-level infrastructure. Brief Bioinform. 2019; 20(2): 370–374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevens SLR, Kuzak M, Martinez C, et al.: Building a local community of practice in scientific programming for life scientists. PLoS Biol. 2018; 16(11): e2005561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWittig U, Rey M, Weidemann A, et al.: Data management and data enrichment for systems biology projects. J Biotechnol. 2017; 261: 229–237. PubMed Abstract \| Publisher Full Text\n\nReisinger E, Genthner L, Kerssemakers J, et al.: OTP: An automatized system for managing and processing NGS data. J Biotechnol. 2017; 261: 53–62. PubMed Abstract \| Publisher Full Text\n\nSchmidt TSB, Raes J, Bork P: The Human Gut Microbiome: From Association to Modulation. Cell. 2018; 172(6): 1198–1215. PubMed Abstract \| Publisher Full Text\n\nRauscher B, Valentini E, Hardeland U, et al.: Phenotype databases for genetic screens in human cells. J Biotechnol. 2017; 261: 63–69. PubMed Abstract \| Publisher Full Text\n\nWollmann T, Erfle H, Eils R, et al.: Workflows for microscopy image analysis and cellular phenotyping. J Biotechnol. 2017; 261: 70–75. PubMed Abstract \| Publisher Full Text\n\nDurek P, Nordström K, Gasparoni G, et al.: Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development. Immunity. 2016; 45(5): 1148–1161. PubMed Abstract \| Publisher Full Text\n\nMeyer F, Goesmann A, McHardy AC, et al.: GenDB--an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 2003; 31(8): 2187–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlom J, Albaum SP, Doppmeier D, et al.: EDGAR: A software framework for the comparative analysis of prokaryotic genomes. BMC Bioinformatics. 2009; 10: 154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlom J, Kreis J, Spänig S, et al.: EDGAR 2.0: an enhanced software platform for comparative gene content analyses. Nucleic Acids Res. 2016; 44(w1): w22–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu J, Blom J, Glaeser SP, et al.: A review of bioinformatics platforms for comparative genomics. Recent developments of the EDGAR 2.0 platform and its utility for taxonomic and phylogenetic studies. J Biotechnol. 2017; 261: 2–9. PubMed Abstract \| Publisher Full Text\n\nJünemann S, Kleinbölting N, Jaenicke S, et al.: Bioinformatics for NGS-based metagenomics and the application to biogas research. J Biotechnol. 2017; 261: 10–23. PubMed Abstract \| Publisher Full Text\n\nHeyer R, Schallert K, Zoun R, et al.: Challenges and perspectives of metaproteomic data analysis. J Biotechnol. 2017; 261: 24–36. PubMed Abstract \| Publisher Full Text\n\nBrink BG, Seidel A, Kleinbölting N, et al.: Omics Fusion - A Platform for Integrative Analysis of Omics Data. J Integr Bioinform. 2016; 13(4): 296. PubMed Abstract \| Publisher Full Text\n\nMuth T, Behne A, Heyer R, et al.: The MetaProteomeAnalyzer: A powerful open-source software suite for metaproteomics data analysis and interpretation. J Proteome Res. 2015; 14(3): 1557–65. PubMed Abstract \| Publisher Full Text\n\nBelmann P, Fischer B, Krüger J, et al.: de.NBI Cloud federation through ELIXIR AAI. F1000Research. 2019; 8: 842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinden M, Prochazka M, Lappalainen I, et al.: Common ELIXIR Service for Researcher Authentication and Authorisation. F1000Research. 2018; 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwengers O, Hoek A, Fritzenwanker M, et al.: ASA3P: An automatic and scalable pipeline for the assembly, annotation and higher level analysis of closely related bacterial isolates. bioRxiv. 2019. Publisher Full Text\n\nTurewicz M, Kohl M, Ahrens M, et al.: BioInfra.Prot: A comprehensive proteomics workflow including data standardization, protein inference, expression analysis and data publication. J Biotechnol. 2017; 261: 116–125. PubMed Abstract \| Publisher Full Text\n\nUszkoreit J, Maerkens A, Perez-Riverol Y, et al.: PIA: An Intuitive Protein Inference Engine with a Web-Based User Interface. J Proteome Res. 2015; 14(7): 2988–97. PubMed Abstract \| Publisher Full Text\n\nTurewicz M, Ahrens M, May C, et al.: PAA: an R/bioconductor package for biomarker discovery with protein microarrays. Bioinformatics. 2016; 32(10): 1577–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarsnes H, Vaudel M: SearchGUI: A Highly Adaptable Common Interface for Proteomics Search and de Novo Engines. J Proteome Res. 2018; 17(7): 2552–2555. PubMed Abstract \| Publisher Full Text\n\nKopczynski D, Sickmann A, Ahrends R: Computational proteomics tools for identification and quality control. J Biotechnol. 2017; 261: 126–130. PubMed Abstract \| Publisher Full Text\n\nVaudel M, Burkhart JM, Zahedi RP, et al.: PeptideShaker enables reanalysis of MS-derived proteomics data sets. Nat Biotechnol. 2015; 33(1): 22–4. PubMed Abstract \| Publisher Full Text\n\nSchwudke D, Shevchenko A, Hoffmann N, et al.: Lipidomics informatics for life-science. J Biotechnol. 2017; 261: 131–136. PubMed Abstract \| Publisher Full Text\n\nHerzog R, Schwudke D, Shevchenko A: LipidXplorer: Software for Quantitative Shotgun Lipidomics Compatible with Multiple Mass Spectrometry Platforms. Curr Protoc Bioinforma. 2013; 43(1): 14.12.1-30. PubMed Abstract \| Publisher Full Text\n\nMarella C, Torda AE, Schwudke D: The LUX Score: A Metric for Lipidome Homology. PLoS Comput Biol. 2015; 11(9): e1004511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPfeuffer J, Sachsenberg T, Alka O, et al.: OpenMS - A platform for reproducible analysis of mass spectrometry data. J Biotechnol. 2017; 261: 142–148. PubMed Abstract \| Publisher Full Text\n\nReinert K, Dadi TH, Ehrhardt M, et al.: The SeqAn C++ template library for efficient sequence analysis: A resource for programmers. J Biotechnol. 2017; 261: 157–168. PubMed Abstract \| Publisher Full Text\n\nFillbrunn A, Dietz C, Pfeuffer J, et al.: KNIME for reproducible cross-domain analysis of life science data. J Biotechnol. 2017; 261: 149–156. PubMed Abstract \| Publisher Full Text\n\nMeier R, Ruttkies C, Treutler H, et al.: Bioinformatics can boost metabolomics research. J Biotechnol. 2017; 261: 137–141. PubMed Abstract \| Publisher Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPietzsch T, Preibisch S, Tomancák P, et al.: ImgLib2--generic image processing in Java. Bioinformatics. 2012; 28(22): 3009–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRueden CT, Schindelin J, Hiner MC, et al.: ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics. 2017; 18(1): 529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDietz C, Berthold MR: KNIME for Open-Source Bioimage Analysis: A Tutorial. Adv Anat Embryol Cell Biol. 2016; 219: 179–97. PubMed Abstract \| Publisher Full Text\n\nBackofen R, Engelhardt J, Erxleben A, et al.: RNA-bioinformatics: Tools, services and databases for the analysis of RNA-based regulation. J Biotechnol. 2017; 261: 76–84. PubMed Abstract \| Publisher Full Text\n\nFallmann J, Will S, Engelhardt J, et al.: Recent advances in RNA folding. J Biotechnol. 2017; 261: 97–104. PubMed Abstract \| Publisher Full Text\n\nWreczycka K, Gosdschan A, Yusuf D, et al.: Strategies for analyzing bisulfite sequencing data. J Biotechnol. 2017; 261: 105–115. PubMed Abstract \| Publisher Full Text\n\nLott SC, Wolfien M, Riege K, et al.: Customized workflow development and data modularization concepts for RNA-Sequencing and metatranscriptome experiments. J Biotechnol. 2017; 261: 85–96. PubMed Abstract \| Publisher Full Text\n\nGrüning BA, Fallmann J, Yusuf D, et al.: The RNA workbench: best practices for RNA and high-throughput sequencing bioinformatics in Galaxy. Nucleic Acids Res. 2017; 45(W1): W560–W566. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmutzer T, Bolger ME, Rudd S, et al.: Bioinformatics in the plant genomic and phenomic domain: The German contribution to resources, services and perspectives. J Biotechnol. 2017; 261: 37–45. PubMed Abstract \| Publisher Full Text\n\nMascher M, Schreiber M, Scholz U, et al.: Genebank genomics bridges the gap between the conservation of crop diversity and plant breeding. Nat Genet. 2019; 51(7): 1076–1081. PubMed Abstract \| Publisher Full Text\n\nSchwacke R, Ponce-Soto GY, Krause K, et al.: MapMan4: A Refined Protein Classification and Annotation Framework Applicable to Multi-Omics Data Analysis. Mol Plant. 2019; 12(6): 879–892. PubMed Abstract \| Publisher Full Text\n\nUsadel B, Poree F, Nagel A, et al.: A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize. Plant Cell Environ. 2009; 32(9): 1211–29. PubMed Abstract \| Publisher Full Text\n\nArend D, Junker A, Scholz U, et al.: PGP repository: a plant phenomics and genomics data publication infrastructure. Database (Oxford). 2016; 2016: pii: baw033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlöckner FO, Yilmaz P, Quast C, et al.: 25 years of serving the community with ribosomal RNA gene reference databases and tools. J Biotechnol. 2017; 261: 169–176. PubMed Abstract \| Publisher Full Text\n\nDiepenbroek M, Schindler U, Huber R, et al.: Terminology supported archiving and publication of environmental science data in PANGAEA. J Biotechnol. 2017; 261: 177–186. PubMed Abstract \| Publisher Full Text\n\nReimer LC, Söhngen C, Vetcininova A, et al.: Mobilization and integration of bacterial phenotypic data-Enabling next generation biodiversity analysis through the BacDive metadatabase. J Biotechnol. 2017; 261: 187–193. PubMed Abstract \| Publisher Full Text\n\nSchomburg I, Jeske L, Ulbrich M, et al.: The BRENDA enzyme information system-From a database to an expert system. J Biotechnol. 2017; 261: 194–206. PubMed Abstract \| Publisher Full Text\n\nDrysdale R, Cook CE, Petryszak R, et al.: The ELIXIR Core Data Resources: fundamental infrastructure for the life sciences. bioRxiv. 2019. Publisher Full Text\n\nJeske L, Placzek S, Schomburg I, et al.: BRENDA in 2019: a European ELIXIR core data resource. Nucleic Acids Res. 2019; 47(D1): D542–D549. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarjes J, Triebel D, Link A, et al.: FAIR data in meta-omics research: Using the MOD-CO schema to describe structural and operational elements of workflows from field to publication. Biodivers Inf Sci Stand. 2019; e37596. Publisher Full Text\n\nBergmann FT, Hoops S, Klahn B, et al.: COPASI and its applications in biotechnology. J Biotechnol. 2017; 261: 215–220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Kamp A, Thiele S, Hädicke O, et al.: Use of CellNetAnalyzer in biotechnology and metabolic engineering. J Biotechnol. 2017; 261: 221–228. PubMed Abstract \| Publisher Full Text\n\nKrebs O, Golebiewski M, Kania R, et al.: SABIO-RK: A data warehouse for biochemical reactions and their kinetics. J Integr Bioinform. 2007; 4(1). Publisher Full Text\n\nLarcombe L, Hendricusdottir R, Attwood TK, et al.: ELIXIR-UK role in bioinformatics training at the national level and across ELIXIR. F1000Res. 2017; 6: pii: ELIXIR-952. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGerritsen VB, Palagi PM, Durinx C: Bioinformatics on a national scale: an example from Switzerland. Brief Bioinform. 2019; 20(2): 361–369. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "56336", "date": "14 Nov 2019", "name": "Roland Krause", "expertise": [ "Reviewer Expertise Bioinformatics", "training" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review of the training activities in the de.NBI network focuses on the description of the courses delivered as well as the contributing centers. Partially owing to the complex set-up of the centers it is a bit short on evaluation of what has worked and what was learned in the process. Including more experiences - courses that were not oversubscribed and those that were would improve the manuscript significantly. I cannot believe that no other lessons were learned after delivering ~200 courses. The discussion of lessons learned is vary valuable to course providers in other infrastructures (such as me). I acknowledge that such critical reviews might not find universal support across the many people involved in the training activities in de.NBI.\nThe structure of the overview can be improved by shortening the sections devoted to the individual centers and instead discussing items that are taught across all centers, such as basic statistics courses.\n\nThe contribution from the individual centers are too long and frequently include vague or obvious statements (existence of an HPC facility, the online announcement of course and requirements). A stronger editing mandate would be advised.\n\nSoftware developed in a particular center for which training is provided should only be mentioned in the section that discusses the training. There is not usually need to repeat them multiple times in the text.\nFigures:\nFigure 1. The composition of the individual centers by cities is difficult even for someone acquainted with the geography of Germany. There is space to include the names of the centers, which could then receive less coverage in the text.\n\nFigure 2. Has no content, would recommend to delete. The geographic spread of de.NBI nodes using AAI provides no insight beyond the names.\n\nFigure 3. The course title is repeated in caption and should be removed from the graphics. Altogether the whole figure could be represented on a easy to comprehend 3x4 table with a higher data to ink ratio. It would be more interesting to see the number of open seats per course and/or a resolution, maybe at the level of quarters.\n\nFigure 4. Pie charts. 4B is only repeating information already included in full in the text. Again, it would be more interesting to see the distribution of the courses at a different level, e.g. to see if some courses are doing very well or whether the numbers are similarly overall. It's not important to learn which courses exactly under perform but more what the course provider thought about the cause and what was done to rectify it.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5872", "date": "11 Sep 2020", "name": "Daniel Wibberg", "role": "Author Response", "response": "Reviewer 1: Roland Krause The review of the training activities in the de.NBI network focuses on the description of the courses delivered as well as the contributing centers. Partially owing to the complex set-up of the centers it is a bit short on evaluation of what has worked and what was learned in the process. Including more experiences - courses that were not oversubscribed and those that were would improve the manuscript significantly. I cannot believe that no other lessons were learned after delivering ~200 courses. The discussion of lessons learned is vary valuable to course providers in other infrastructures (such as me). I acknowledge that such critical reviews might not find universal support across the many people involved in the training activities in de.NBI. Thank you for the comment. Well, almost all courses are oversubscribed and in 5 years of de.NBI, we had to cancel only four courses because of a low amount of participants. Our learned lessons are Not to organize courses in the summer or Christmas holidays Provide a high diversity of training course topics Focusing of beginners, but don’t forget advance and expert users More day courses are more interesting than one day courses In our point of view, these points are not so interesting for the paper. We want to focus the paper on the training activities of de.NBI / ELIXIR-DE The structure of the overview can be improved by shortening the sections devoted to the individual centers and instead discussing items that are taught across all centers, such as basic statistics courses. Thank you for the comment. We shortened the service center chapters. Based on the topics of each service center, there is almost no overlap in the training program of the service centers. There are a few collaboration within service centers in training course, e.g. the summer school or the annual Nanopore workshop, but normally one service center provide one specific type of training. The contribution from the individual centers are too long and frequently include vague or obvious statements (existence of an HPC facility, the online announcement of course and requirements). A stronger editing mandate would be advised. Thank you for the comment. The chapters were edited and shortened. Software developed in a particular center for which training is provided should only be mentioned in the section that discusses the training. There is not usually need to repeat them multiple times in the text. Thank you for the comment. Sometimes, the software needs to be mentioned to underline the research focus of the center. Repeats were reduced. Figures: Figure 1. The composition of the individual centers by cities is difficult even for someone acquainted with the geography of Germany. There is space to include the names of the centers, which could then receive less coverage in the text. The names of the service centers are mentioned on the left side of the figure. The color underlines the belonging to a service center Figure 2. Has no content, would recommend to delete. The geographic spread of de.NBI nodes using AAI provides no insight beyond the names. Thank you for the comment. The figure was deleted. Figure 3. The course title is repeated in caption and should be removed from the graphics. Altogether the whole figure could be represented on a easy to comprehend 3x4 table with a higher data to ink ratio. It would be more interesting to see the number of open seats per course and/or a resolution, maybe at the level of quarters. Thank you for the comment. The figure was replaced by a table (s. table 1). Figure 4. Pie charts. 4B is only repeating information already included in full in the text. Again, it would be more interesting to see the distribution of the courses at a different level, e.g. to see if some courses are doing very well or whether the numbers are similarly overall. It's not important to learn which courses exactly under perform but more what the course provider thought about the cause and what was done to rectify it. Thank you for the comment. We included here a new figure showing the similar performance of all course providers (s. Figure 2)." } ] }, { "id": "56339", "date": "09 Dec 2019", "name": "Loredana Le Pera", "expertise": [ "Reviewer Expertise Bioinformatics", "bioinformatics teaching and training", "learning processes", "training platform coordination" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is a quite detailed description of the research foci and training activities carried out at service centres belonging to the German Network for Bioinformatics Infrastructure (de.NBI).\nGeneral comments:\n1) As the paper is presented as a description of the \"de.NBI / ELIXIR-DE training platform\", the relationship between de.NBI and ELIXIR-DE should be clearly described at the beginning of the paper (instead of being a sub-section of the Cloud section at the end of the paper). Indeed, except for a vague statement in the abstract (\"The de.NBI members also run the German node (ELIXIR-DE) within the European ELIXIR network\". Who are de.NBI members? What does it mean that they run ELIXIR-DE?), the reader becomes aware of the nature of this relationship only immediately before the conclusions. Confusion also arises from the following sentence in the Introduction: \"The following topics are currently in the focus of SIG 3: [...]\nIntegration into the ELIXIR training platform and TeSS\n\nCoordination of training events and standards with ELIXIR.\nBoth \"topics\" are rather obscure: What does it mean \"Integration (of what?) into the ELIXIR training platform and TeSS\"? and \"Coordination of training events and standards (which standards?) with ELIXIR\"? Results are presented as de.NBI activities and the role of ELIXIR-DE is not made explicit throughout the paper. In particular, Germany has become an ELIXR member in 2016, but data presented also include 2015, which further increases reader's confusion about the relationship between de.NBI and ELIXIR-DE.\n2) Currently, the article is a quite detailed description of research focus and training activities carried out at each service centre belonging to de.NBI and NOT of a \"Training Platform\". Therefore, it is not clear what the authors mean by \"de.NBI / ELIXIR-DE training platform\". Ideally, in a Training Platform (TP):\nCourses/events are presented as part of a coherent programme run at different locations/centres, not as an aggregation of courses that each individual centre/institution runs with (apparently) no coordination with other centres/institutions;\n\nThe structure of the TP (including the role of ELIXIR-DE) is described, including who coordinates it and how; who is actively involved in training activities; how many people are involved; what are their roles; how decisions are taken; etc.\n\nVision, pedagogical model(s), teaching approaches, goals, future plans should be discussed/explained, as well as how trainers are recruited/selected and how course planning and dissemination is managed; etc...\nBased on these considerations, if the authors really mean to present a de.NBI / ELIXIR-DE TP, they should adopt a different perspective, which means try to display courses in a more structured way. For example, grouped by topic and by providing summary information about each type of course. Our suggestion is to use a table with, e.g., the following columns:\nType of course/course category (e.g. sequencing data analysis).\n\nExample courses of each category (e.g. microbial sequence data analysis, RNA-Seq and Bisulfite sequencing data analysis in cancer research).\n\nBrief overview of the content (better if written in the form of general learning outcomes).\n\nAverage number of courses of each type run yearly/monthly/etc.\n\nLocation(s) (service centres/institutes) where courses are delivered.\n\nAverage number of participants.\n\nDelivery mode (F2F, e-learning, hackathon, mentoring).\n\nEtc...\nNarrative could be used to describe key features of the platform (as listed above) and of courses. e-learning model(s) also could be better described (synchronous?asynchronous? Blended? etc). A description of service centres and their research foci could be provided very succinctly in Figure 1 or in a separate table (listing, for each centre, the research focus and member institutes) and then leave further details to the literature reference.\n3) Figure 1 could be made way more informative and clear:\nWhat is the meaning of the circle's size?\n\nHow should the reader interpret two adjacent circles of the same colour?\n\nThe title of the figure reads: \"Training topics and locations of the eight de.NBI service centres in Germany and their connections with existing German and European communities\", but:\nTraining topics do not appear in the figure. For clarity, location of main German cities should be shown. The figure shows existing communities and event types but not their connection with service centres.\n\nSpecific comments/questions:\nWhat do you mean by \"Big Data Problem\"? Could you add a reference and explain what characterises it as a \"problem\"?\n\nIn the abstract, we suggest that you replace \"European ELIXIR network\" with \"European ELIXIR infrastructure\".\n\nWhat do you mean by \"standardisation of training course monitoring\"?\n\nA sentence on what is TeSS should be written before mentioning it in the Introduction.\n\np. 4: Re: \"Qualification of more trainers.\" How do you qualify trainers? What is a \"qualified trainer\"?\n\nAs SIG3 is for \"Training & Education\", do you also have programs for undergraduates and high school students?\n\nIn the abstract, the overall number of participants is updated to October 2019 (\"since 2015, more than 250 training courses were carried out with more than 5,200 participants\"), whereas in the paper it is updated to July 2019 ([...] more than 4800 participants (Status July 2019)). This data should be made consistent.\n\nRegarding performance, authors claim that de.NBI and ELIXIR-DE guarantee standards [...] above ELIXIR average. This statement should be supported by quantitative data.\n\np.11: re the sentence: \"ELIXIR Germany training coordinators\", does Germany have more than one ELIXIR TrC?\n\np.11: what is the \"general ELIXIR training feedback\"? Do you mean Impact study? If so, you should cite the paper on impact currently under revision at PLoS Computational Biology.\n\np.11: Re: \"new e-learning working group and of the FAIR training working group\". You should provide a reference/link to ELIXIR working groups. For example, FAIR training WG: https://github.com/elixir-europe/FAIR-Training.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "5873", "date": "11 Sep 2020", "name": "Daniel Wibberg", "role": "Author Response", "response": "Reviewer 2: Loredana Le Pera & Allegra Via The paper is a quite detailed description of the research foci and training activities carried out at service centres belonging to the German Network for Bioinformatics Infrastructure (de.NBI). General comments: 1) As the paper is presented as a description of the \"de.NBI / ELIXIR-DE training platform\", the relationship between de.NBI and ELIXIR-DE should be clearly described at the beginning of the paper (instead of being a sub-section of the Cloud section at the end of the paper). Indeed, except for a vague statement in the abstract (\"The de.NBI members also run the German node (ELIXIR-DE) within the European ELIXIR network\". Who are de.NBI members? What does it mean that they run ELIXIR-DE?), the reader becomes aware of the nature of this relationship only immediately before the conclusions. Confusion also arises from the following sentence in the Introduction: \"The following topics are currently in the focus of SIG 3: [...] Integration into the ELIXIR training platform and TeSS Coordination of training events and standards with ELIXIR. Both \"topics\" are rather obscure: What does it mean \"Integration (of what?) into the ELIXIR training platform and TeSS\"? and \"Coordination of training events and standards (which standards?) with ELIXIR\"? Results are presented as de.NBI activities and the role of ELIXIR-DE is not made explicit throughout the paper. In particular, Germany has become an ELIXR member in 2016, but data presented also include 2015, which further increases reader's confusion about the relationship between de.NBI and ELIXIR-DE. Thank you for the comment. We included an additional introduction to the ELIXIR-DE to describe the difference between de.NBI and ELIXIR-DE. Nevertheless, SIG3 is coordinating the training program of both networks resulting in a 100% overlap. 2) Currently, the article is a quite detailed description of research focus and training activities carried out at each service centre belonging to de.NBI and NOT of a \"Training Platform\". Therefore, it is not clear what the authors mean by \"de.NBI / ELIXIR-DE training platform\". Ideally, in a Training Platform (TP): Courses/events are presented as part of a coherent programme run at different locations/centres, not as an aggregation of courses that each individual centre/institution runs with (apparently) no coordination with other centres/institutions; The structure of the TP (including the role of ELIXIR-DE) is described, including who coordinates it and how; who is actively involved in training activities; how many people are involved; what are their roles; how decisions are taken; etc. Vision, pedagogical model(s), teaching approaches, goals, future plans should be discussed/explained, as well as how trainers are recruited/selected and how course planning and dissemination is managed; etc... Based on these considerations, if the authors really mean to present a de.NBI / ELIXIR-DE TP, they should adopt a different perspective, which means try to display courses in a more structured way. For example, grouped by topic and by providing summary information about each type of course. Our suggestion is to use a table with, e.g., the following columns: Type of course/course category (e.g. sequencing data analysis). Example courses of each category (e.g. microbial sequence data analysis, RNA-Seq and Bisulfite sequencing data analysis in cancer research). Brief overview of the content (better if written in the form of general learning outcomes). Average number of courses of each type run yearly/monthly/etc. Location(s) (service centres/institutes) where courses are delivered. Average number of participants. Delivery mode (F2F, e-learning, hackathon, mentoring). Etc... Narrative could be used to describe key features of the platform (as listed above) and of courses. e-learning model(s) also could be better described (synchronous?asynchronous? Blended? etc). A description of service centres and their research foci could be provided very succinctly in Figure 1 or in a separate table (listing, for each centre, the research focus and member institutes) and then leave further details to the literature reference. Thank you for the comment. In general, our courses are on an annual basis. Therefore, we provide in 80 courses almost 80 different types of courses. Therefore, such a table would be too large to present in the paper. The service centers have unique topics in de.NBI / ELIXIR-DE. Therefore, there is almost no topic overlap in the training courses between the different service centers. That is the reason, why we choose this strategy to present the training program. To describe the training platform in detail, we included an additional part introducing the training platform. 3) Figure 1 could be made way more informative and clear: What is the meaning of the circle's size? How should the reader interpret two adjacent circles of the same colour? The title of the figure reads: \"Training topics and locations of the eight de.NBI service centres in Germany and their connections with existing German and European communities\", but: Training topics do not appear in the figure. For clarity, location of main German cities should be shown. The figure shows existing communities and event types but not their connection with service centres. Thank you for the comment. The circle size is now explained in the figure legend. The service centers represent the training topics. As explained before, each service center has its topic and there is almost no overlap with another center. Specific comments/questions: What do you mean by \"Big Data Problem\"? Could you add a reference and explain what characterises it as a \"problem\"? Was added In the abstract, we suggest that you replace \"European ELIXIR network\" with \"European ELIXIR infrastructure\". Was replaced What do you mean by \"standardisation of training course monitoring\"? We developed a standardized form that must be filled out after every course and user survey based the ELIXIR survey questions. A sentence on what is TeSS should be written before mentioning it in the Introduction. A sentence on TeSS was added. p. 4: Re: \"Qualification of more trainers.\" How do you qualify trainers? What is a \"qualified trainer\"? We plan to organize more Train-the-Trainer and Software Carpentry Instructor course. In addition, a trainer community meeting is planned. As SIG3 is for \"Training & Education\", do you also have programs for undergraduates and high school students? No, we don’t have a special program for undergraduates and high school students. About 15% of all participants are undergraduates and we support a summer school for high school students. In the abstract, the overall number of participants is updated to October 2019 (\"since 2015, more than 250 training courses were carried out with more than 5,200 participants\"), whereas in the paper it is updated to July 2019 ([...] more than 4800 participants (Status July 2019)). This data should be made consistent. Was updated to July 2020. Regarding performance, authors claim that de.NBI and ELIXIR-DE guarantee standards [...] above ELIXIR average. This statement should be supported by quantitative data. Quantitative data was added. p.11: re the sentence: \"ELIXIR Germany training coordinators\", does Germany have more than one ELIXIR TrC? Yes, one chair and two deputies p.11: what is the \"general ELIXIR training feedback\"? Do you mean Impact study? If so, you should cite the paper on impact currently under revision at PLoS Computational Biology. Citation was added p.11: Re: \"new e-learning working group and of the FAIR training working group\". You should provide a reference/link to ELIXIR working groups. For example, FAIR training WG: https://github.com/elixir-europe/FAIR-Training. Reference was added" } ] } ]	1	https://f1000research.com/articles/8-1877
https://f1000research.com/articles/9-1116/v1	10 Sep 20	{ "type": "Research Article", "title": "Association between blood pressure, race, ethnicity and likelihood of admission to the hospital from United States emergency departments – A cross sectional study", "authors": [ "Jessie Kue", "William Meurer", "Jessie Kue" ], "abstract": "Background: The emergency department (ED) has emerged as the primary portal for entry to the hospital for most patients with health care problems, including hypertension. Hypertension is the most important risk factor for heart disease. Disparities may exist in access to hospitalization across race/ethnicity. Our objective was to estimate how the likelihood of hospital admission based on blood pressure (BP) was modified by race/ethnicity. Methods: We used data from the 2014 National Hospital Ambulatory Medical Care Survey, a representative sample of non-federal, U.S. emergency department visits. We plotted probability of admission by blood pressure stratified by race/ethnicity to assess for a linear relationship. We then fit logistic regression models that adjusted for other potential confounders including patient-, visit-, and hospital-level factors. All analyses were conducted with relevant SURVEY functions in SAS to account for design. Results: Just over 21,000 visits were included in the study, representing approximately 1.4 million U.S. ED visits. We included the range of systolic blood pressure from 110 to 180 mmHg based on the linear relationship with probability of admission. We found the odds ratio for admission was 1.11 [95% CI: (1.06, 1.18)] for each 10 mmHg rise in systolic blood pressure in the unadjusted analysis. In the final adjusted model accounting for confounders, we found that the relationship between BP and admission was no longer significant 0.96 [0.91 to 1.01]. Whites were substantially more likely to be admitted compared to Blacks and Hispanics at odds ratio 1.5 [1.2 to 2]. Conclusions: The relationship between BP and hospital admission is complicated. Blacks and Hispanics appear less likely to be admitted to the hospital from the ED at a given level of blood pressure even after accounting for triage severity, and other individual and hospital level factors. Further research is needed to better understand this disparity.", "keywords": [ "emergency department", "hypertension", "health disparities" ], "content": "Introduction\n\nThe emergency department (ED) has emerged as the primary portal for entry to the hospital for most patients with acute health care problems and acute manifestations of underlying chronic diseases, including hypertension1. According to the Center for Disease Control and Prevention, a striking 45% of U.S. adults have been diagnosed with high blood pressure, defined as a systolic blood pressure (SBP) of at least 130 mmHg or a diastolic blood pressure of at least 80 mmHg2. In addition, studies have demonstrated an increase in emergency department utilization for hypertension. One study reports that from 2006 to 2012, U.S. emergency departments saw a 30% increase in the proportion of adult visits related to hypertension and a 16% increase in the proportion of adult ED visits with a primary diagnosis of hypertension1. Hypertension is not only a known risk factor for cardiovascular disease, but an estimated 1-2% of hypertensive patients will develop a hypertensive crisis, which can lead to a variety of critical conditions, including stroke, heart attack, heart and kidney failure3.\n\nDespite known recommendations to repeat blood pressure values above 140/90 and refer to outpatient facilities for follow up care4, the protocol for when to admit patients presenting to the ED with high systolic blood pressure is currently undefined. Nevertheless, patients with significantly raised SBPs are routinely admitted into the hospital for administration of anti-hypertensive drugs and monitoring. In this retrospective study, using the National Hospital Ambulatory Medical Care Survey, we investigated the odds of admission for patients with various degrees of elevated systolic blood pressure. We hypothesized that increasing systolic blood pressure increases the likelihood of hospital admission. In addition to the severity of SBP, we elucidated the effect of race/ethnicity on admission. Finally, our sensitivity analysis assessed the odds of admission for a subpopulation of patients presenting to the ED with a primary complaint of chest pain.\n\n\nMethods\n\nData for this study was extracted from the raw data from the National Hospital Ambulatory Medical Survey (NHAMCS). This cross-sectional database collects information annually from a representative sample of emergency departments, outpatient clinics, and ambulatory surgery centers, excluding federally associated medical centers, military and veterans’ hospitals5. Further details regarding the methods for sampling and including visits are available at the NHAMCS website, maintained by the United States Centers for Disease control. The website also provides code for extracting the raw data into common statistical packages. We included all adult (age 18 and older) visits from 2014.\n\nOur pre-specified hypothesized biological relationship that presenting systolic blood pressure is related to the likelihood of hospital admission was based on the positive correlation between elevated SBP and cardiovascular disease (CVD). Although many indicators of heart disease have been identified, studies show that systolic hypertension is one of the leading risk factors6. Thus, it follows that patients with high SBP are more likely to be admitted into the hospital. We were also interested in how race/ethnicity modified this relationship. In addition, we believed a priori, that some very low and very high blood pressures represented different conditions (shock and hypertensive crisis, respectively).\n\nFor all analyses, we used the SURVEY functions of SAS (version 9.3) to account for the complex survey design when determining point estimates and standard errors. In the first step of our analysis, we plotted the probability of admission based on 10 mm Hg intervals of presenting systolic blood pressures (≤70, 71–80, etc.). In addition, we wished to determine the functional form of this relationship to see if we should include this as a linear predictor or apply some transformation. We then created a stratified plot by race ethnicity. Our plan was to visually inspect this stratified graph for an area of interest for multivariable models. After determining the area of interest, we excluded patients with very high and very low blood pressures. We characterized baseline visit, demographic, patient medical, and hospital level variables by reported race/ethnicity (including how many visits are represented in the data set).\n\nIn order to adjust for patient, hospital and regional related factors, we pre-specified a series of nested, logistic regression models that predicted the likelihood of admission. In the first model (Model 1), we included only race-ethnicity categorically as a predictor along with continuous systolic blood pressure. In addition, we scaled the variable to be equivalent to a 10 mm Hg increase in systolic blood pressure by dividing the observed SBP by 10. We used the odds ratio estimates in the first model to determine which race/ethnic groups to combine to determine whether we would have multiple groups, or two categories (non-Hispanic white versus all other) based on similar odds ratios. We pre-specified that we would either exclude or collapse race/ethnic categories with low numbers in the dataset. In subsequent models, we evaluated whether the point estimate for the odds ratio for admission based on race/ethnicity changed substantially with the addition of potentially confounding variables. In the second model (Model 2), we added the patient level variables that we believed a priori had a relationship with admission (age, sex, hypertension history, and insurance).\n\nIn the third model (Model 3), we added visit level variables describing the reason for presentation and the perceived acuity (immediacy with which patient should be seen, whether visit is related to injury/trauma or other, mode of arrival [ambulance vs other]). In the fourth and final model, we added hospital level characteristics (size, teaching status, region, urban vs rural, and hospital ownership). We pre-specified that if there was no evidence of confounding by the 10% rule (that the odds ratio for the covariate estimate for race/ethnicity did not change more than 10% when accounting for confounders) that we would accept that model for evaluating interactions (i.e. if adjusting for patient, visit, and hospital level predictors did not change the observed relationship between race and admission then we would use model 1.) We planned to determine whether an interaction term of race/ethnicity*blood pressure was significant at the p=0.05 level, and if it was we would report that as our final model.\n\nWe did not pre-specify a sample size, and focused on effect size estimation within a single year of NHAMCS data.\n\nNHAMCS analyses have not been validated using the complex survey packages in the open source statistical package R. However, the SAS datasets we created from the NHAMCS data can be imported into R using the haven package.\n\nSince it is possible that patients with different race/ethnic backgrounds may describe symptoms differently and have different thresholds for using the emergency department, we repeated the entire above analysis plan, but we limited the included population to patients with a chief complaint of chest pain. Chest pain is among the most common complaints in the ED and several potential causes of chest pain are the end result of long-standing hypertension.\n\n\nResults\n\nIn total, 23,844 observations were included in this study, representing approximately 140 million ED visits. We restricted our cohort to patients 18 years or older who had systolic blood pressures in the range concurrent with the positively linear aspect of the relationship between SBP and admission into the hospital. This cohort represented 13,119 encounters representing 80 million visits. Within this cohort, 1774 encounters (13.5%) were admitted to the hospital, representing 9.2 million visits. The mean age of this subpopulation was 45.4 years with a standard deviation of 0.44 year. 55.5% of these patients were female [95% CI (53.9, 57.1)], while 44.5% were male [95% CI (42.9, 46.1)]. In addition, 59.6% of patients identified as non-Hispanic White, 24.9% as non-Hispanic Black, 12.9% as Hispanic, and 2.7% as Other. Private insurance was the primary health care payer (29.2%) followed closely by Medicaid insurance (23.9%). Approximately 22% of patients had Medicare as primary insurance, and another 14% were self-pay. We provide the statistical package output and data files at the University of Michigan Deep Blue Institutional Repository (see underlying data7).\n\nThe unstratified graph between SBP and admission to the hospital demonstrates an initial negative relationship at the lower extremes of blood pressure (Figure 1). This association, however, becomes positive beginning with the categories of SBP that represent values ranging from 110 mmHg to greater than 200 mmHg. We decided to narrow our analysis to this positively linear aspect of the relationship (SBP categories 2-8), in accordance with our hypothesis that elevated systolic blood pressures warrant admission to the hospital for intervention and further monitoring and our a priori belief that extremely low or extremely high blood pressures would have high admission probability and not inform the modeling.\n\nProportion of adult patients admitted to hospital from the emergency department, based on triage blood pressure.\n\nFigure 2 shows the relationship between SBP and hospital admission stratified by race. The graph indicates that the rate of hospital admission for non-Hispanic Whites is generally higher for the same systolic blood pressure values than non-Hispanic Blacks, Hispanics, and those who identified as Other. Patients in the Other category were admitted to the hospital at the second highest rate, followed by non-Hispanic Blacks and Hispanics. Rate of hospital admission notably clusters around the same frequency among the last three race groups for elevated SBP. At these elevated blood pressures, a prominent gap between the last three racial groups and non-Hispanic Whites is quite evident.\n\nProportion of adult patients admitted to hospital from the emergency department by race and ethnicity, based on triage blood pressure within the region of interest for blood pressure.\n\nOur unadjusted logistic regression model is represented in Table 1. The odds ratio of this primary model is 1.12 (95% CI: 1.06, 1.18), which indicates that the odds of being admitted into the hospital is 1.12 times higher for every 10 mmHg increase in systolic blood pressure. In Model 1, we included race/ethnicity, for which we created a new, binary variable where non-Hispanic Black and Hispanic were binned together, and non-Hispanic White was the reference group. After holding systolic blood pressure constant, non-Hispanic Whites have approximately 1.9 times greater odds of being admitted than non-Hispanic Black or Hispanic patients [OR = 1.88, 95% CI (1.45, 2.42)]. Model 2 includes the two aforementioned variables as well as individual and visit factors we believed a priori may influence the odds of admission. These variables included age, sex, insurance type (a manually created binary variable in which private insurance, Medicare, Medicaid/CHIP, worker’s compensation, and other was coded as insured = 1), history of hypertension, arrival by ambulance, visit due to injury/trauma, overdose, poisoning, or some other adverse effect of medical care, and triage level. Counterintuitively, this model suggests that the odds of hospital admission decrease by about 5% with every 10-mmHg increase in SBP everything else held constant [OR = 0.96 95% CI (0.91, 1.005)]. Meanwhile, the odds of hospital admission for non-Hispanic Whites continues to be 1.5 times higher than the two minority groups [OR = 1.53, 95% CI (1.18, 1.98)]. In our fourth and final model, we combined the individual and visit level variables with hospital-level factors, including region, urbanicity, and whether the patient was seen by a resident, nurse practitioner, or physician’s assistant. Table 1 demonstrates the odds ratio estimates for this model and shows that elevated SBP continues to have lower odds [OR = 0.956, 95%CI (0.909, 1.005)] of admission and that non-Hispanic Whites have greater odds of being admitted [OR = 1.54, 95% CI (1.179, 2.007)] into the hospital than their non-White counterparts.\n\nResults of the models predicting hospital admission. Model 0 used only SBP to predict hospital admission, Model 1 added in race and ethnicity, Model 2 added in age, sex, hypertension history, and insurance, and Model 3 additionally adjusted for hospital-level factors, including region, urbanicity, and whether the patient was seen by a resident, nurse practitioner, or physician’s assistant.\n\nSBP = systolic blood pressure, CI = confidence interval, mm Hg = millimeters of mercury, OR = odds ratio and AOR = adjusted odds ratio.\n\nIn the third phase of our study, we repeated the above analysis with a subgroup of the study population comprised of those with a chief complaint of chest pain (Table 2). The odds ratio for the unadjusted model was 1.2 [95% CI (1.03, 1.41)], indicating that the odds of hospital admission are 1.2 times greater for every 10-mmHg increase in SBP in patients with chest pain. The odds ratio for hospital admission in Model 1 is the same as in the unadjusted model. Although more attenuated than in the first part of our analysis, non-Hispanic Whites continue to have greater odds of being admitted into the hospital than their non-Hispanic Black and Hispanic counterparts, even among those presenting with chest pain [OR = 1.12, 95% CI (0.65, 1.93)]. The third model includes age, sex, history of hypertension, and insurance status in addition to the variables in the first two models. Holding everything constant, odds of admission are slightly more attenuated but still greater than one [OR = 1.06, 95% CI (0.90, 1.24)] for every 10-mmHg increase in SBP among those with chest pain. Interestingly, Whites have a 13% lower odds of hospital admission in this final model [OR = 0.87, 95% CI (0.5, 1.53)]. We did not conduct a fully adjusted model with hospital factors given the smaller sample size of this subset, as the estimates became unstable.\n\nResults of the models predicting hospital admission. Model 0 used only SBP to predict hospital admission, Model 1 added in race and ethnicity, Model 2 added in age, sex, hypertension history, and insurance; we did not adjust for hospital factors in the final model given the smaller sample size of this subset.\n\nSBP = systolic blood pressure, CI = confidence interval, mm Hg = millimeters of mercury, OR = odds ratio and AOR = adjusted odds ratio.\n\n\nDiscussion\n\nThe unadjusted analysis of the relationship between admission to the hospital and systolic blood pressure demonstrates an overall upgoing trend – as blood pressure increases, the proportion of patients who are admitted into the hospital also increases. There is a portion of this graph in Figure 1 that indicates a negative association, however, blood pressures at the lower extreme are generally concerning for life-threatening conditions (e.g. shock, hemorrhage), and these patients are also likely to be hospitalized from the ED.\n\nThe positive association between increasing blood pressure and hospital admission was confirmed in our first two regression models (Table 1 and Table 2). In the fully adjusted model (Table 1), however, the odds of hospital admission unexpectedly decreased. Our sensitivity analysis with chest pain indicated higher odds of admission with race, age, and sex held constant, in accordance with Model 0 and Model 1 in our primary analysis. Previous studies have demonstrated an increase in ED utilization and hospitalization due to hypertension1,8. The results of our study appear to be generally consistent with these previous studies, however, the slightly reduced odds of admission in the fully adjusted model (Table 1) may simply speak to the complexity involved in the decision to hospitalize a patient.\n\nAccording to the stratified unadjusted graph (Figure 2), the same positive trend of hospital admission exists among all four race groups. Nevertheless, a consistently higher percentage of non-Hispanic Whites are admitted to the hospital compared to their non-Hispanic Black and Hispanic counterparts for the same range of SBP values. This was also confirmed in our regression models. After accounting for a variety of factors that we believed a priori could be associated with hospital admission, non-Hispanic Blacks and Hispanics consistently had lower odds of admission compared to non-Hispanic Whites. Similarly, in our analysis of patients presenting with chest pain, non-White patients still had lower odds of admission.\n\nThere is extensive literature identifying known patterns of racial disparity in cardiovascular health – namely that non-Hispanic Blacks have higher rates of hypertension and worse hypertension control9,10. Studies show that despite an overall decrease in cardiovascular mortality, African-Americans are 30% more likely to die from heart disease compared to non-Hispanic Whites9. Moreover, non-Hispanic Black individuals have higher rates of risk factors that contribute toward cardiovascular disease and are disproportionately affected by manifestations of heart disease, such as myocardial infarction (MI), stroke, and heart failure9. Health disparities are not only fueled by patient and provider behavior but health care systems as well9. While certainly a complicated relationship, our study identified a racial gap in hospital admission after accounting for a number of potential confounders, which may suggest an important area of improvement. Knowledge of the increased cardiovascular morbidity and mortality among African American and Hispanic patients should help to improve admission practices for minority patients and thus reduce the existing gap of cardiovascular outcomes between non-Hispanic Whites and non-Whites.\n\nThis study does contain a few limitations. First, we used only one year of NHAMCS data (2014), which prevented us from being able to establish temporal hospital admission trends for hypertension. Second, we recognize that patients with elevated systolic blood pressure could present to the ED for a number of reasons ranging from trauma to anxiety to headache and blurred vision. Patient presentation could significantly influence whether or not patients are admitted, thus making the association between hospital admission and increased SBP non-linear. Nevertheless, we attempted to mitigate this by conducting a sensitivity analysis with chest pain, and the results demonstrated similar patterns identified in the broader study. In addition, this sample was limited to non-Federal emergency department visits in the United States and how these findings would generalize to other locales is unknown.\n\nIn summary, elevated systolic blood pressure is a known risk factor for cardiovascular disease, and left unmanaged, can lead to a series of health consequences including heart disease, stroke, and even death. Our study highlights two important findings: 1) While patients with elevated systolic blood pressure are generally more likely to be admitted into the hospital, this relationship becomes more complex when considering patient-, visit-, and hospital-level factors. 2) Non-Hispanic Whites consistently have a higher likelihood of hospital admission for hypertension compared to non-Hispanic Black and Hispanic patients. This presents a crucial area of improvement, particularly given the worse cardiovascular outcomes in the minority patient population.\n\n\nData availability\n\nThe data utilized in the above study were from a publicly accessible data set found on the Center for Disease Control and Prevention’s website for the National Center for Health Statistics https://ftp.cdc.gov/pub/Health_Statistics/NCHS/Datasets/NHAMCS/ED2014.zip Instructions for formatting the raw data for common statistical packages are available at https://www.cdc.gov/nchs/ahcd/datasets_documentation_related.htm\n\nUniversity of Michigan - Deep Blue: Dataset derived from 2014 National Hospital Ambulatory Care Survey - Emergency Department. https://doi.org/10.7302/9c2f-gr80\n\n- nhamcs14.sas7bdat ( SAS datafile that contains all visits from the 2014 NHAMCS with SAS formats and labels)\n\n- will24July2017.sas (SAS syntax file that imports data from raw NHAMCS file and performs analyses– later version)\n\n- jessie14June2017.sas (SAS syntax file for analysis and import, preliminary version)\n\n- ed14for.txt ( SAS formats from NHAMCS for variables)\n\n- ed14lab.txt (SAS label file from NHAMCS for variables)\n\n- SAS_Output_July25.html (raw SAS output from final analysis that generated data for this manuscript, including all parameter estimates and fit statistics of logistic models)\n\n- SAS_Output_july24.htm (raw SAS output from preliminary analysis)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThis paper was presented in abstract form at the annual meeting of the Society for Academic Emergency Medicine in May 2018 in Indianapolis, Indiana, U.S.A.\n\n\nReferences\n\nMcNaughton CD, Self WH, Zhu Y, et al.: Incidence of Hypertension-Related Emergency Department Visits in the United States, 2006 to 2012. Am J Cardiol. 2015; 116(11): 1717–1723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCDC: Hypertension Prevalence in the U.S. Million Hearts®. Cent Dis Control Prev. 2020. [accessed 2020 Apr 25]. Reference Source\n\nChan SSW, Graham CA, Rainer TH: Hypertension in the Emergency Department. Curr Hypertens Rep. 2016; 18(5): 37. PubMed Abstract \| Publisher Full Text\n\nBrody A, Janke A, Sharma V, et al.: Public Health, Hypertension, and the Emergency Department. Curr Hypertens Rep. 2016; 18(6): 50. PubMed Abstract \| Publisher Full Text\n\nNAMCS/NHAMCS - About the Ambulatory Health Care Surveys. 2019. Reference Source\n\nSaladini F, Palatini P: Isolated Systolic Hypertension in Young Individuals: Pathophysiological Mechanisms, Prognostic Significance, and Clinical Implications. High Blood Press Cardiovasc Prev. 2017; 24(2): 133–139. PubMed Abstract \| Publisher Full Text\n\nKue J, Meurer W: Dataset derived from 2014 National Hospital Ambulatory Care Survey - Emergency Department. [Data set]. University of Michigan - Deep Blue. 2020. http://www.doi.org/10.7302/9c2f-gr80\n\nLiu L, An Y, Chen M, et al.: Trends in the Prevalence of Hospitalization Attributable to Hypertensive Diseases Among United States Adults Aged 35 and Older From 1980 to 2007. Am J Cardiol. 2013; 112(5): 694–699. PubMed Abstract \| Publisher Full Text\n\nGraham G: Disparities in Cardiovascular Disease Risk in the United States. Curr Cardiol Rev. 2015; 11(3): 238–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerdinand KC, Yadav K, Nasser SA, et al.: Disparities in hypertension and cardiovascular disease in blacks: The critical role of medication adherence. J Clin Hypertens (Greenwich). 2017; 19(10): 1015–1024. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "78792", "date": "23 Feb 2021", "name": "Stephen Pitts", "expertise": [ "Reviewer Expertise Health services research", "focused on acute care visits." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 uncomplicated analysis of one year of the publicly available US national emergency department survey. The methods used were appropriate for a complex survey, and the analysis plan was well described and results carefully presented. The tables were simple and well formatted. The two figures were ok, but figure two might have been better constructed as overlaid line graphs, or simplified by collapsing non-white categories as in the analysis. There is no way to assess the precision of point estimates in this figure, and especially in the older groups I imagine sample size was pretty scant. An alternative might be two scatter plots (W/non-W) with best fit lines using mean admission rates in linear regression, perhaps weighted by inverse variance, though such a figure might be too cluttered.\nThis is really a study of physician behavior, a social science hypothesis, rather than a study of hypertension, but the findings are interesting, though not too surprising. The racial disparity in admission rates has been examined in other reports. I would not put too much energy into trying to understand the complex causal path to hospital admission given the limited number of covariates available in the scanty national survey data, whose biggest role is in estimating national burdens rather than classic etiologic research. It is hard to know how much HTN alone contributed to the admission decision, given the huge variety and number of other potential influences on admission, and the wide variation in practice between the 300 or so hospitals surveyed. There are big known race/ethnicity differences in use of the ED. Despite the overall larger burden of cv disease among blacks, and lower quality of care, public hospital EDs have had a very low prevalence of MI compared with other hospitals (including the original Goldman/Lee papers), presumably because the ED is serving a safety net function for surrounding communities. Increased awareness of hypertension may not be among the solutions to reverse these historical differences, even though it tracks pretty well with the admission decision.\n\nThanks for the care you've taken in this analysis, and the great attention given to replicability. My main recommendation is to simplify the discussion. I don't think that you need to make any policy recommendations on the basis of your findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "123619", "date": "25 Feb 2022", "name": "Ji-Guang Wang", "expertise": [ "Reviewer Expertise Hypertension" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed analysis on admission blood pressure of over 21000 emergency department (ED) visits. A major finding is that at a given blood pressure level, Hispanics and Blacks were underrepresented in the ED visits. The analysis and finding are quite interesting. The reviewer has several suggestions for revision of the manuscript.\nThe authors may need to further clarify the clinical significance of the analysis. Is there any between ethnicity or race inequality or inequity for ED emergency in the US?\n\nThe authors analyzed blood pressure. Although the data are from an available database and the study is retrospective, it is still important to report the quality control of the blood pressure data, such as the blood pressure measuring devices, number of readings, etc.\n\nThe authors reported systolic blood pressure ranging from 60 mm Hg in Figure 1 and 100 mm Hg in Figure 2. The choice is reasonable. However, the authors may need to provide some further information on these low blood pressure patients. Who were they?\n\nHypertensive crisis may lead to ED visit. Is it possible to know the proportion of those patients with hypertensive crisis?\n\nThe authors compared various ethnic groups in the US. To strengthen generalizability of the findings, the authors may need to explain the differences between these ethnicity and races.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1116
https://f1000research.com/articles/8-2022/v1	28 Nov 19	{ "type": "Brief Report", "title": "Mortality risk factors among National Football League players: An analysis using player career data", "authors": [ "Justin Ehrlich", "Brittany Kmush", "Bhavneet Walia", "Shane Sanders", "Brittany Kmush", "Bhavneet Walia", "Shane Sanders" ], "abstract": "In general, National Football League (NFL) players tend to live longer than the general population. However, little information exists about the long-term mortality risk in this population. Frequent, yet mild, head trauma may be associated with early mortality in this group of elite athletes. Therefore, career playing statistics can be used as a proxy for frequent head trauma. Using data from Pro Football Reference, we analyzed the association between age-at-death, position, and NFL seasons-played among 6,408 NFL players that were deceased as of July 1, 2018. The linear regression model allowing for a healthy worker effect demonstrated the best fit statistics (F-statistic = 9.95, p-value = 0.0016). The overall association of age-at-death and seasons-played is positive beginning at the 10.75 and 10.64 seasons-played point in our two models that feature seasons-played and seasons-played squared as explanatory variables. Previous research that does not account for the healthy worker effect may not adequately describe mortality risk among NFL players.", "keywords": [ "CTE", "concussions", "football", "gridiron football", "NFL", "chronic traumatic encephalopathy", "sports" ], "content": "Introduction\n\nVery little information exists about mortality and long-term health outcomes among National Football League (NFL) players. Elite football players tend to have a lower overall mortality rate than the general population, often attributed to routine physical activity1,2. However, this occupational group cannot be directly compared to the general population3. Several studies in small numbers of NFL players have found an association between traumatic brain injuries with depression, suicide, dementia, and chronic traumatic encephalopathy4–6. There is mounting evidence that even sub-clinical head impacts, especially when they occur frequently, can also lead to these adverse health outcomes7,8. However, these relationships are difficult to study systematically due to few cases, challenges with diagnostics, and long lag time from the injury to symptom onset. Yet, there exists a rich repository of data surrounding NFL career playing statistics9. We hypothesize that certain player career attributes, including position-of-play and seasons-played, are likely to be strong predictors for mortality from repeated, yet mild, head trauma. Here, we study the association between mortality and NFL seasons-played, while controlling for playing position.\n\n\nMethods\n\nData was collected from Pro Football Reference, a free online database maintained by Sports Reference LLC that includes playing statistics from every player in NFL history, over 25,000 in total, with meticulously recorded data beginning in 19229. Merged and transformed data is available from an open repository10. Variables of interest include birthdate, death date, position, and seasons-played. Individuals with any missing data were eliminated, leaving 24,740 players. Of those, 6,408 (25.9%) had died according to Pro Football Reference, as of July 1, 2018. Playing position was divided into three standard categories according to previous literature11.\n\nCategory 1: defensive back, quarterback, wide receiver, and kicker: 1,600 dead/8,415 players (19%).\n\nCategory 2: running back, linebacker, tight end: 1,690 dead/7,228 players (23%).\n\nCategory 3: offensive and defensive linemen: 3,118 dead/9,097 players (34%).\n\nExpected age-at-death was calculated from the 2017 National Vital Statistics Report12 using average years of life remaining at 20 years of age for the decade of the 20th year plus 20. Age-at-death residuals were calculated as observed age-at-death minus expected age-at-death. This analysis was completed in Stata Version 1413, and data was visualized using R 3.6.114. Associations were assessed using linear regression models with a quadratic term for seasons-played. Specifically, we use (position) fixed-effect ordinary least squares modeling to determine whether associations exist between age-at-death residual, number of NFL seasons-played (squared), and position category fixed effects. In these models, we seek to assess whether career duration exposure relates significantly to age-at-death residual conditional on position-of-play. The healthy worker turning point was calculated using standard differential calculus techniques (i.e., calculating the minimum point of a best fit surface).\n\nBase Model:\n\nAge at Death Residual i,t = β0 + β1Number of Seasons Playedi,t + εi,t\n\nSeasons-played Squared Model:\n\nAge at Death Residual i,t = β0 + β1Number of Seasons Playedi,t + εi,t + β2Number of Seasons Played2i,t + εi,t\n\nPosition Category Fixed Effects Model\n\nAge at Death Residual i,t = β0 + β1Number of Seasons Playedi,t + εi,t + β2Number of Seasons Played2i,t + εi,t + β3Position Categoryi + εi,t\n\n\nResults and discussion\n\nTable 1 indicates substantial demographic sample variation between players of different position categories in height, weight, BMI, and age-at-death. Figure 1a-Figure 1b indicate a possible healthy worker effect among players of Category I and II. Certain healthy or durable players can play an increased number of seasons without a corresponding reduction in expected age-at-death as compared to players of shorter career duration3.\n\nBMI – body mass index\n\nDots represent individual players; Solid line represents a quadratic trend.\n\nDots represent individual players; Solid line represents a quadratic trend.\n\nThe Seasons-played Squared and Position Category Fixed Effects models specify a quadratic term for number of NFL seasons-played. For both models, the coefficient for this variable is significant and improves the model’s explanatory power according to an Anova F-test for difference in overall model significance (F-statistic = 9.95, p-value = 0.0016; F-statistic=10.98, p-value<0.001) (Table 2). We calculate that overall association of age-at-death residual and seasons-played is positive beginning at 10.75 and 10.63 seasons-played for the Seasons-played Squared and Position Category Fixed Effects model, respectively. This demonstrates a healthy worker effect, where certain players are not as prone to play-related mortality risk. For this player subset, the healthy worker effect is sufficiently strong to dominate an observed mortality risk effect, where the latter effect drives the negative relationship between seasons-played and age-at-death residual for those playing fewer than 10.75 (10.63) seasons. The healthy worker effect and the mortality risk effect hold conditional upon position category control variables, where controls exhibit substantial variation in effect upon age-at-death residual as in previous literature11. However, dividing players into three position categories may not sufficiently capture the differing on-field exposures that may contribute to mortality.\n\n\nConclusion\n\nThis paper finds evidence of both player health risk (in terms of age-at-death residual) for increasing NFL seasons played and healthy worker effects among NFL players. For Category I and II players, the latter effect dominates the former for NFL players with sufficient career survivorship. This effect holds conditional upon position-of-play control variables. Previous research not accounting for the healthy worker effect may not adequately describe mortality risk among NFL players.\n\n\nFuture work\n\nWe are pursuing additional research to examine the association of on-field playing characteristics with mortality and cause of death among NFL players.\n\n\nEthics\n\nThis study was determined by the Syracuse University Institutional Review Board to not be human subjects research and therefore, not to require review and oversight.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Mortality Risk Factors among National Football League Players: An Analysis using Player Career and Biometric Data. https://doi.org/10.7910/DVN/WHU4KC10\n\nThis project contains the following underlying data:\n\nFootball1922_2018.tab (Includes player name, position, year of birth, year of death, death residual, position, position category, and the number of seasons-played.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nTeramoto M, Bungum TJ: Mortality and longevity of elite athletes. J Sci Med Sport. 2010; 13(4): 410–416. PubMed Abstract \| Publisher Full Text\n\nOwora AH, Kmush BL, Walia B, et al.: A systematic review of etiological risk factors associated with early mortality among national football league players. Orthop J Sports Med. 2018; 6(12): 2325967118813312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi CY, Sung FC: A review of the healthy worker effect in occupational epidemiology. Occup Med (Lond). 1999; 49(4): 225–229. PubMed Abstract \| Publisher Full Text\n\nKerr ZY, Marshall SW, Harding HP, et al.: Nine-year risk of depression diagnosis increases with increasing self-reported concussions in retired professional football players. Am J Sports Med. 2012; 40(10): 2206–2212. PubMed Abstract \| Publisher Full Text\n\nIverson GL: Chronic traumatic encephalopathy and risk of suicide in former athletes. Br J Sports Med. 2014; 48(2): 162–5. PubMed Abstract \| Publisher Full Text\n\nShively S, Scher AI, Perl DP, et al.: Dementia resulting from traumatic brain injury: What is the pathology? Arch Neurol. 2012; 69(10): 1245–1251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMontenigro PH, Alosco ML, Martin BM, et al.: Cumulative head impact exposure predicts later-life depression, apathy, executive dysfunction, and cognitive impairment in former high school and college football players. J Neurotrauma. 2017; 34(2): 328–340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlosco ML, Tripodis Y, Jarnagin J, et al.: Repetitive head impact exposure and later-life plasma total tau in former National Football League players. Alzheimers Dement (Amst). 2016; 7: 33–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPro-Football Reference.com: Football encyclopedia of players. 2018; Accessed July 1, 2018. Reference Source\n\nEhrlich J, Sanders S, Kmush BL, et al.: Replication data for: Mortality risk factors among national football league players: An analysis using player career data [data set]. 2019. http://www.doi.org/10.7910/DVN/WHU4KC\n\nBaron SL, Hein MJ, Lehman E, et al.: Body mass index, playing position, race, and the cardiovascular mortality of retired professional football players. Am J Cardiol. 2012; 109(6): 889–896. PubMed Abstract \| Publisher Full Text\n\nMartin JA, Hamilton BE, Osterman MJ, et al.: National vital statistics reports. National Vital Statistics Reports. 2017; 66(1).\n\nStata statistical software [computer program]. Version 14. College Station, TX: StataCorp; 2015.\n\nR: A language and environment for statistical computing [computer program]. Version 3.6.1. R Core Team; 2019. Reference Source" }	[ { "id": "57258", "date": "16 Dec 2019", "name": "Adam M. Finkel", "expertise": [ "Reviewer Expertise quantitative risk assessment", "epidemiology", "regulatory policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis was a frustrating paper to review: on the one hand, I definitely applaud any analysis of athletes that compares athletes TO athletes and thus avoids the healthy worker effect HWE). (See Nguyen et al. 20191 for a good recent example of avoiding this pitfall; see footnote 2 in this article for a good put-down of the HWE fallacy2). On the other hand, I don’t think this analysis actually involves the HWE at all; as I will discuss, it actually purports to find a “frailty” issue within this worker population, which is a very different phenomenon and one with rather different research and policy implications than a true HWE finding.\n\nIn this context, a true HWE occurs when reach the mistaken conclusion that X is not riskier than not-X (as in “football players don’t die more often of cancer than general population”), or even that X is safer than not-X, but what’s really happening is that being in class X shows you were less likely to have the risk than general to begin with. This is important to find and point out, because then you either have to adjust for it (see Choi et al.3) or do a different study that compares apples to apples. But what these researchers may have found (see below) is that within subpopulation of NFL players, some are less frail than others. This is heterogeneity-dynamics at work (see Manton, K.G., E. Stallard, and J.W. Vaupel. 1986. Alternative models for the heterogeneity of mortality risks among the aged. J. Am. Stat. Assoc. 81:635-6444). Heterogeneity-dynamics means that you may need to adjust the slope of an observed dose-response function to account for the fact that there are 2 or more subpopulations within the subpopulation—everyone can have an individual positive association between seasons and risk, but the group may have a flat (null) slope simply because those who played the most seasons were not “lucky” but more immune.\n\nThe difference between a finding of an HWE between football and the general public, versus the finding of two of more differentially-susceptible subgroups within the NFL population, is far from merely semantic, because the practical implications of the two situations are so different. Finding an HWE allows the researcher to correct for it, either by discarding a flawed analysis in favor of an apples-to-apples comparison, or by taking the existing analysis and trying to re-estimate the odds ratio (see, for example, Joffe, 20125). Even without correction, researchers who appreciate the pitfalls of the HWE can simply say that it leads to false negatives preferentially: comparing, for example, the cognitive performance of 60-year-old retired NFL players against the general population of that age would tend to divert attention away from the consequences of repeated head trauma (RHT) simply because the general population includes many persons who were never fit enough to work, let alone work in this taxing occupation.\n\nBut finding a negative-then-positive relationship between length of (NFL) career and age at death, because the cohort under study has, by definition, more fit persons remaining as more and more of the cohort dies early, may have no empirical or policy implications whatsoever. The authors say nothing about how we might even identify who among incoming NFL players might “benefit” from longer careers and whose dose-response is the most steeply negative—if we could, and IF we had the means and the will to discourage the latter group from choosing this occupation, THEN perhaps we could make use of the “finding” that some players are not at risk to the extent that others are (there is an extensive literature about the gap between identifying a powerful interindividual risk factor in a working population—these are usually genetic factors, such as “slow acetylators” who are more at risk from certain occupational chemicals—and the wisdom of trying to exclude these people from the workplace. Generally, policy analysts prefer interventions that can make the workplace safe for everyone who participates—imagine a policy of not allowing people with hemophilia to become carpenters, as opposed to OSHA regulations that mandate guards on saws so that no one will be cut by them).\n\nSo this article, at most, finds an “expected curiosity”—that once you have had a long career, you probably are revealing to an epidemiologist that you have been more “immune” to the harmful effects of that career than the average person—and then says nothing about how we could use the finding to adjust scientific conclusions or policy responses. By invoking the HWE throughout the paper, the authors are not only using the wrong term, but inviting statistical corrections or policy responses that have already been made or that would not respond to what they may have actually found.\n\nBut all the foregoing assumes that the authors have actually found evidence of a “resistant subpopulation” within the NFL cohort, and I’m not sure that’s the case. The authors don’t mention alternative explanations for the slight upslope in Figures 1a and 1b, including: (1) reverse causation—if a significant number of players died on the field or soon after sustaining football-related injuries early in their careers, then of course the remaining population would not have “negative death residuals” that large; and (2) effect modification—similarly, if physical inactivity leads to earlier death, then players who sustained career-ending injuries early on would die earlier than others.\n\nMore problematic is the use of rote statistics without also applying “common sense” to the finding. How robust, in particular, is the slight upslope obtained by regression to the presence of outliers? The interactive Figures show that the five players with the longest careers were Sammy Baugh, Johnny Unitas, George Blanda, Earl Morrall, and YA Tittle. Some of these (Morrall) I believe played long careers but were backups much of the time, so an index based on number of games rather than seasons might have shown something different. It would be important to explore what happens to the upslope if outliers were trimmed—I say this in part because visual exploration of the Figures does not present a compelling “common sense” picture of a positive slope among the longer careers; I believe the numbers, but the visual impression, especially excluding outliers, is one of a rather FLAT dose-response that one might be convinced is slightly negative and then very slightly positive. Similarly, I accept (p. 3) that the quadratic model fits slightly better than the linear one, but the authors don’t let on how well the linear model fit in the first place. Could they be “finding” something more sophisticated simply by over-fitting? Also, I dispute that the fixed-effects model is correct, with respect to the kinds of effects (CTE, dementia) the authors clearly are trying to shed light on. The three categories they use seem out-of-place here: other studies have shown that the three positions with the greatest cumulative amount of RHT (instances times g-forces) are tight ends, quarterbacks, and defensive linemen, and yet these are in three different categories in this model!\n\nFinally, the authors focus on mortality, which is fine, but completely ignore quality of life. Just consider the case of Earl Morrall, who lived to age 79 (death residual of > 10) but who reportedly had Stage 4 CTE and a reduced QOL. Someone who comes away from the paper concluding that “once you play 11 seasons, you may live longer than your cohort” may not realize that age at death is not the most relevant outcome…\n\nIn summary, the authors should replace “HWE” with “interindividual variability in susceptibility,” explore the implications of THAT analysis, consider how robust their analyses of statistical significance actually are, and ground this paper in terms of the other more ambitious studies that have already explored the relationship between better indices of lifetime intensity of play and mortality/morbidity (especially Montenigro et al., 20176). They might also consider this recent paper by Mez et al.7 and explore if and how their quadratic model might better explain (or be contradicted by) these prior findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5179", "date": "29 Jul 2020", "name": "Justin Ehrlich", "role": "Author Response", "response": "Thank you for these important points; they will lead to improvements in our paper. This was a very helpful review even though it started off in a negative sense.We will change the way the HWE is described with respect to the data/results. Specifically, we will frame the HWE as a longitudinal survivorship effect where people are removed from the cohort. We agree there are heterogeneity dynamics at work and will term this as a “survivorship effect” throughout. We thank the reviewer for suggestions regarding the policy implications and will clarify our policy section accordingly. To make the game safer, we must identify which players are at an elevated risk. While the reviewer has valid points about outliers, we would prefer to keep them in our analysis. To address outliers, we specified robust standard errors to measure risk factors for mortality in a manner consistent with valid derivation of t-statistics. We disagree with the reviewer and prefer to keep outliers in the dataset. We did not eliminate the outliers so as not to introduce selection bias. Furthermore, this a complete census of the players and we calculated population parameters, not sample statistics; therefore, we prefer to keep all players in the analysis. We will be more clear in the revised draft that we are analyzing the population of NFL players. As such, there is no need to worry about outliers that have an unrepresentative influence vis-à-vis the underlying population. However, we will add in a statement in the limitations about our choice to keep the outliers.We agree that quality of life and cause of death are important considerations. Here, we analyze all-cause mortality, not CTE specific mortality. Therefore, the papers the reviewer suggested may not be appropriate because we have all deaths in this population, not a selected sub-sample. While in our future research, we hope to focus on quality of life and cause-specific mortality, that is not possible with this data. We will add in statements about these limitations and future directions." } ] }, { "id": "63530", "date": "03 Jun 2020", "name": "Thomas R. Sadler", "expertise": [ "Reviewer Expertise Economics", "Environment", "Professional Sports", "Pandemics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research analyzes the impact of the number of years played in the NFL and position on life expectancy. The question is excellent. The model concisely addresses the question, and the data are comprehensive. This is an important research study that could be replicated for other professional sports. An additional study could break the regressions down by individual position, instead of three categories, to see if there is a further distinction. But that is not necessary for this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/8-2022
https://f1000research.com/articles/9-773/v1	27 Jul 20	{ "type": "Research Article", "title": "An assessment of the extent to which the contents of PROSPERO records meet the systematic review protocol reporting items in PRISMA-P", "authors": [ "Alison Booth", "Alex S. Mitchell", "Andrew Mott", "Sophie James", "Sarah Cockayne", "Samantha Gascoyne", "Catriona McDaid", "Alex S. Mitchell", "Andrew Mott", "Sophie James", "Sarah Cockayne", "Samantha Gascoyne", "Catriona McDaid" ], "abstract": "Background: PROSPERO is an international prospective register for systematic review protocols. Many of the registrations are the only available source of information about planned methods. This study investigated the extent to which records in PROSPERO contained the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P). Methods: A random sample of 439 single entry PROSPERO records of reviews of health interventions registered in 2018 was identified. Using a piloted list of 19 PRISMA-P items, divided into 63 elements, two researchers independently assessed the registration records. Where the information was present or not applicable to the review, a score of 1 was assigned. Overall scores were calculated and comparisons made by stage of review at registration, whether or not a meta-analysis was planned and whether or not funding/sponsorship was reported. Results: Some key methodological details, such as eligibility criteria, were relatively frequently reported, but much of the information recommended in PRISMA-P was not stated in PROSPERO registrations. Considering the 19 items, the mean score was 4.8 (SD 1.8; median 4; range 2-11) and across all the assessed records only 25% (2081/8227) of the items were scored as reported. Considering the 63 elements, the mean score was 33.4 (SD 5.8; median 33; range 18-47) and overall, 53% (14,469/27,279) of the elements were assessed as reported. Reporting was more frequent for items required in PROSPERO than optional items. The planned comparisons showed no meaningful differences between groups. Conclusions: PROSPERO provides reviewers with the opportunity to be transparent in their planned methods and demonstrate efforts to reduce bias. However, where the PROSPERO record is the only available source of a priori reporting, there is a significant shortfall in the items reported, compared to those recommended. This presents challenges in interpretation for those wishing to assess the validity of the final review.", "keywords": [ "Systematic review", "protocol", "reporting", "registration" ], "content": "Introduction\n\nDetailing the planned methods for conducting a systematic review in advance of commencing the review is essential in order to minimise a range of potential biases1,2. The plan, set out in a protocol, should ideally be made available in the public domain to facilitate transparency3,4. In addition, registration of key protocol details is encouraged as best practice in reporting guidelines5,6 by publishers like the British Medical Journal (BMJ), Public Library of Science (PLoS), and BioMed Central (BMC), and is mandated in their instructions to authors by journals such as BMC Systematic Reviews, BMJ, BMJ Open, PLoS One, and National Institute for Health Research (NIHR) journals.\n\nThere are a number of options for putting systematic review protocols into the public domain, such as publication in open access journals like BMC Systematic Reviews and uploading to open data repositories like the Open Science Framework (OSF) (https://osf.io/registries/discover?q=protocols). PROSPERO (https://www.crd.york.ac.uk/prospero/) is a facility for registering key methodological details in advance of carrying out a review. Registration on PROSPERO requires completion of an internationally agreed minimum dataset for a systematic review protocol7,8. Registrants also have the option of uploading their protocol or providing a hyperlink to it.\n\nPROSPERO remains the only free, open access registry of systematic review protocols, making it a single searchable source of the protocols of on-going and completed reviews. Uptake of registration has increased exponentially and by the end of 2019 there were over 60,000 registrations in PROSPERO. There is evidence that considerably more systematic reviews are registered in PROSPERO than have peer-reviewed protocols published. In 2016, 1058 records were accepted by PROSPERO; in the same time period, only 404 published systematic review protocols were identified3. Another study reported identifying 20,814 non-Cochrane systematic review protocols from web scraping PROSPERO and bibliographic database searches. Of these, 924 were only published in journals, 807 were published in journals and registered in PROSPERO and 19,890 were only available as a record in PROSPERO9. There is further evidence from Ge et al (2018) that of the non-Cochrane reviews registered in PROSPERO, only 3% or 4% have a published protocol9,10. This means that for a large number of reviews a PROSPERO record is likely to be the only source providing details of the planned methods.\n\nPublished protocols and registration records aim to provide transparency in the review process by allowing public access to the key pre-specified elements for the conduct of a review. One of the stated aims of PROSPERO is to facilitate comparison between planned review methods and reported results8. Such a comparison enables peer reviewers and other readers of the final review to assess for themselves the potential for bias in the findings. There is also a steadily growing body of research using PROSPERO records to assess the risk of biases in final review reports10–15. Given this reliance on the information provided in PROSPERO records, it is important to understand the level of detail provided in records. The focus of this study was on the stated aim of PROSPERO to reduce the opportunity for bias by enabling comparison of the completed review with what was planned in the protocol8.\n\nThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Protocols (PRISMA-P) were developed through expert consensus using internationally compiled datasets such as PROSPERO and SPIRIT4,6.\n\nKey methodological aspects of a protocol are mandated for registration in PROSPERO; other items, mainly administrative fields, are optional7,8. Submissions for registration are not subject to any form of peer review or critical appraisal, they are simply checked for sense but not methodological rigor. Therefore, there is the possibility that PROSPERO records do not provide all the necessary information identified by the PRISMA-P guidelines to enable comparison with the completed systematic review. The registration record may be the only place where a priori methods are available for users, in particular peer reviewers, to check for potential issues such as selection, outcome reporting and publication biases. This study investigated the extent to which records in PROSPERO, where no protocol or other information was available, comply with each of the items for reporting of protocols set out in the PRISMA-P guidelines.\n\n\nMethods\n\nA random sample of PROSPERO registration records were assessed against the systematic review protocol reporting criteria set out in the PRISMA-P 2015 checklist4. Key methods are provided here with further details available in the protocol for this study, which was prepared and made publicly available on the OSF, 17 March 2020 (Extended data16).\n\nA dataset of non-Cochrane PROSPERO records was provided by Metaxis, the software managers of PROSPERO. Records of reviews defined by the record holder as a health intervention registered on or between 1 January 2018 and 31 December 2018, were identified.\n\nCochrane reviews, reviews of animal studies, non-intervention reviews as identified in PROSPERO, i.e. Diagnostic accuracy, Prognostic factors, Prevention, Epidemiological reviews relevant to health and social care, Public health, Service delivery in health and social care, Methodological reviews, reviews of reviews, and synthesis of qualitative studies, were all excluded as PROSPERO and PRISMA-P were developed for reviews of interventions. Only records with no evidence from the registration record of other protocol related information, for example in a published protocol or other links in the PROSPERO record, were included and we restricted the data set to those records with a single registry entry.\n\nRecords from the calendar year 2018 were used to allow time for dissemination and adoption of the PRISMA-P guidelines published in 2015. A sample of 20% of these records was randomly selected using simple random sampling for assessment against the PRISMA-P reporting criteria.\n\nThe PRISMA-P checklist recommends 17 numbered items, with nine subdivisions, totalling 26 items be reported in a systematic review protocol4. Seven of the 26 items were excluded from the assessment as they would always or never meet registration requirements in PROSPERO. For example, registration is implicit for a record accepted in PROSPERO, and there is no field for author contributions or sponsor role so these would never be reported. The study assessment tool, developed specifically for this study as a Google Form, therefore contained 19 of the PRISMA-P items. Where the PRISMA-P description for an item specified more than one piece of information, the individual elements were listed as subsets of the items4,6. This list contained 63 elements to be reported.\n\nWhere an item was reported or not applicable, a score of 1 was assigned. Where the information was not reported this scored 0. The maximum possible overall score for the PRISMA-P listed items was 19 per record. Scores for the breakdown of individual elements within the items was also reported, the maximum possible score was 63 per record.\n\nThe researchers undertaking the assessments (AB, ASM, AM, SJ, SC, SG) familiarised themselves with both PRISMA-P papers4,6. All had previously received training in systematic review methods and/or authored at least one systematic review. The draft assessment form and accompanying guidance notes were revised and finalised during a training session and piloted with the aim of achieving greater than 90% agreement.\n\nTwo researchers independently compared the information provided in each PROSPERO record with the relevant items in the study assessment tool. Options for decisions were: Reported (information provided as per PRISMA-P requirements); Not reported (some or all information not provided); and, Not applicable (where an item was not relevant to an individual record, e.g. a meta-analysis was not planned).\n\nRecords were randomly assigned to assessors by first creating a list of the sampled record unique identification numbers and dividing the list into 14 blocks of approximately equal size, with each block being assigned a colour. A copy of this list together with the block configuration was then placed alongside the original list. Seven sub-lists were then created by randomly selecting a block from the first list and a block from the second list, such that blocks of the same colour were not in the same sub-list, and each colour appeared in two sub-lists. Each sub-list was then randomly assigned to an assessor.\n\nIt was not feasible to blind the researchers to the authors of registrations in PROSPERO. None of the assessors were authors of included registrations. On completion of the pilot assessments and the full set of records, disagreements were resolved through discussion or recourse to a third researcher.\n\nThe assessment form and the guidance notes are available on the OSF (Extended data16).\n\nThe primary outcome for this study was the compliance of PROSPERO registration records to PRISMA-P reporting items. This was measured by the total mean score allocated by the two independent assessors to each of the 19 items assessed (maximum possible score 19) for each record and by the total mean score for the individual elements within items (maximum possible score 63). Overall scores for the assessed dataset, scores by the 19 PRISMA-P items and by the 63 elements were the planned outcome measures.\n\nFor the eligible 2018 records that were assessed and those not assessed, demographic data for month of registration, funding/sponsor, planned meta-analysis, number of authors, stage of review at registration, topic and country of review were to be reported. Comparisons to identify any association between records registered before or after screening started; whether a meta-analysis was planned or not; and whether a review was funded/sponsored or not and completeness of reporting of items were planned.\n\nDuring piloting of the assessment form, it became clear that it would not be possible to assess records for PRISMA-P item 5a Sources and 5b Sponsor. This would have required separating sources of financial support from sponsorship or any other form of support as reported in the single PROSPERO field, which was not possible. This item was therefore removed from the assessment form. Instead, a series of regular expression patterns was compared to the list of eligible records to identify those where the record contained any indication of funding/ sponsorship/support or indicated there was none. These data were used in the presentation of demographics and subgroup comparison.\n\n\nResults\n\nThe PROSPERO dataset contained 5,313 records for reviews of health interventions first accepted in 2018 (excluding Cochrane and reviews of animal studies). Applying the other study inclusion/exclusion criteria resulted in 2,194 eligible registration records. The randomly selected sample of 20% for assessment included 439 records. During assessment, six records were excluded, for not meeting the inclusion criteria (4), being a duplicate (1) or no longer available on PROSPERO (1). Assessments were therefore carried out on 433 PROSPERO records. A flow chart of record selection is shown in Figure 1.\n\nAgreement following initial piloting of the assessment form was 87%; after further discussions and revision of the assessment guidance notes and form a second pilot achieved 92% agreement. For all the records assessed, agreement between researchers was 90%, all differences were resolved through discussion or referral to a third researcher.\n\nDemographic details of the sample of PROSPERO records selected for assessment and those not assessed are provided in Table 1. The number of authors listed ranged between one and 17, with the exception of a single record, included in the assessed sample, where 47 authors were listed. The eligible sample for 2018 included records from 67 different countries: 20 records listed two countries and 15 listed between three and nine countries involved in the review. There were no substantial differences between the data sets in the month of registration; whether any details of funding and/or sponsorship were provided; whether a meta-analysis was planned or not; the number of authors listed per record; stage of review at registration; topic of review or country involved in undertaking the review.\n\n* the record with 47 authors was a single outlier: range excluding this record was 0-15\n\n details for three records were not available on PROSPERO\n\n* all items reported by authors included; therefore totals are more than the number of records\n\nNone of the PROSPERO records assessed against the eligibility criteria reported on all elements in each of the items recommended for a systematic review protocol in the PRISMA-P guidelines. The mean total score for individual PROSPERO records, where 1 point was gained for each of the 19 items in the PRISMA-P checklist, was 4.8, the standard deviation 1.8, the median 4, and range 2 to 11. Considering all items across all the assessed records, only 25% (2081/8227) of the items were scored as reported.\n\nThe mean total score for individual PROSPERO records where 1 point was gained for each of the 63 elements of the PRISMA-P reporting guidelines was 33.4, the standard deviation 5.8, the median 33 and the range 18–47. Overall, 53% (14,469/27,279) of the elements were considered as reported.\n\nThe highest scoring item was PRISMA-P 1b which requires the protocol to be identified as to whether it is an update of a review; the high score was the result of this being a not-applicable item for 423 (98%) of the 433 records (Table 2). Eligibility criteria (study design, setting, population, intervention, comparator, outcomes) was the next highest scoring item with 386 (89%) reporting all of these elements. Selection process (214, 49%), describing the criteria under which study data will be quantitatively synthesized (200, 46%), and describing the type of summary planned if quantitative synthesis is not appropriate (227, 52%) were the next highest scoring of the 19 items assessed.\n\nThe scores by PRISMA-P item and by breakdown of items are presented in Table 2. The full dataset with assessment outcomes and scores for individual records, and the subgroup analyses scoring are available on the OSF (Underlying data16).\n\n* Item/element required in PROSPERO Item/element identified in PROSPERO but as optional\n\nThe score for some of the 19 items was reduced as a result of just one or two of the constituent elements being omitted from reports while others were relatively regularly identified.\n\nAlthough overall the review question (item 7) was not found to contain all the expected elements, most did specify the elements of population (397, 92%) and the intervention (416, 96%) and just over half included the outcomes (237, 55%). The comparator was less frequently included (142, 33%); this may have been because of the intention of the review but where this was clear, the item was scored as not applicable (6%).\n\nInformation sources (item 9) was scored as completed in only two records (1%) overall; however, for the individual elements 431 (99%) did name the electronic databases to be searched, 289 (67%) said whether they planned to search study registries, and 238 (55%) indicated search dates. In item 10, provision of a draft search strategy (91, 21%) or search terms (100, 23%) was poor; but restrictions such as to English language papers were reported in 332 (77%).\n\nReporting of item 13, outcomes, scored badly overall (3, 1%) as, although the outcomes were included in most records (Primary 418, 97%; Secondary 430, 99%) only 8 (2%) were assessed as having provided a rationale for their choice of outcomes. Similarly, in item 14, the absence of information on how the risk of bias would be used in the synthesis, detracted from the high rate of inclusion of risk of bias tools and use. Reporting of the details for a quantitative synthesis, item 15b, had one element with a very low score (handling missing data, 14, 3%), the other six elements scored between 89 (20%) and 204 (47%).\n\nIn three items, the overall score reflected the general picture from the included elements. In item 6, rationale, both the reason for undertaking the review and the context were infrequently identified. PRIMSA-P items 16, meta-bias(es) and 17, confidence in cumulative evidence, were rarely reported. Only context is classified as optional information in PROSPERO, the remainder of these elements are not explicitly requested.\n\nThere appears to be a trend towards higher frequency of reporting of elements that are mandatory in PROSPERO, for example, in the eligibility criteria (item 8) and risk of bias (item 14). The trend is also seen in item 13, the required specification of primary and secondary outcomes, both frequently reported, but with a drop in specifying measures, which was optional.\n\nThe subgroup comparisons investigated the stage of review at registration; whether or not information was reported on source of funding, sponsorship or support and where none was indicated; and whether or not the relevant box in the registration form had been ticked to indicate a meta-analysis was planned.\n\nThere were no differences in total scores for the 19 PRISMA-P items or the 63 elements, between those records registered before screening against eligibility criteria had started and those records registered after screening had commenced. This held true for the mean, standard deviation, median and range of scores.\n\nA 6% difference was seen in the total score achieved for the meta-analysis (23%) vs no meta-analysis (29%) groups in the assessment of the 19 PRISMA-P items. The difference was reduced to 2% when considering the breakdown of 63 elements within the reported items (52% vs 54%). At both item and element level, the group of records with no planned meta-analysis scored slightly higher, but with a higher standard deviation from the mean and wider range of scores achieved.\n\nAcross all results for both the 19 items and 63 elements, the group with funding, sponsorship or support, scored slightly higher than those not receiving funding, sponsorship or support.\n\nThe results of the subgroups investigated are presented in Table 3. The subgroup scores by individual PRISMA-P reporting item are available on the OSF (Underlying data16).\n\nWe present the scores by the 19 PRISMA-P items and by the breakdown of 63 elements for the ten countries and topics with the highest number of assessed records, and for number of authors listed in Table 4. None of these factors appear to have a marked influence on the number of PRISMA-P items or elements reported in PROSPERO records.\n\nnumbers differ from Table 1 because of the record(s) excluded at assessment\n\n\nDiscussion\n\nPublication and registration of a systematic review protocol provides transparency in the review process, allowing readers to see the efforts made to minimise biases and where biases may still have influenced the final review findings. There is empirical evidence that few of the protocol registrations in PROSPERO have a corresponding published report9. Where there is no protocol, the registration provides the only public record of what was originally planned. This study set out to establish to what extent PROSPERO registrations of systematic review protocols of healthcare interventions reported on items in the PRISMA-P reporting guidelines.\n\nUsing a random sample of 433 PROSPERO records from 2018, two researchers independently assessed the frequency of reporting of 19 PRISMA-P items, with 63 individual elements. The results show that while some key methodological details are relatively frequently reported, much of the information recommended in PRISMA-P is missing. Reporting was unsurprisingly more frequent for items that are mandatory in PROSPERO than those that are optional. Comparisons by stage of review at registration, whether meta-analysis was planned and whether funding or sponsorship was reported showed no meaningful differences between groups. The slight difference between groups with a planned meta-analysis or none may be because in PRISMA-P more details are specified for the reporting of a meta-analysis than for a descriptive, narrative or qualitative analysis.\n\nEligibility criteria and type of analysis planned were most frequently reported and are all separate required fields in PROSPERO. However, study selection process, which is optional, was also a higher frequency reported item. This may be explained by considering that some elements of items, such as eligibility criteria, study selection and risk of bias have what might be considered a standard, recognisable format that facilitates reporting. Other items need a more nuanced approach underpinned by a clear understanding of systematic review methods, and therefore may be associated with being less frequently reported due to a lack of confidence or experience with these aspects of review methods. For example, how risk of bias will be used in the synthesis, data handling in a meta-analysis, meta-biases and confidence in cumulative evidence, all had low scores. Part of the problem may be the uncertainty of what the searches will find when designing a systematic review but needing to know so the design is appropriate. For example, the intention may be to perform a meta-analysis, this may not be possible once the studies for inclusion have been identified. While, both PROSPERO and PRISMA-P acknowledge that protocols are iterative documents and may need to be amended, changes should be documented, justified and the stage of review at the time of the amendment made clear. Therefore, it is better to record alternative options for activities such as how data will be analysed and the conditions for selection of option when finalising the protocol.\n\nDifferences in frequency of reporting may also reflect where researchers considered items to be less or more important than others. For example, naming the software used for data management may not be seen as crucial, whereas the eligibility criteria and approach to synthesis are.\n\nThere are strengths and limitations to this study. The assessed sample of 433 records was representative of all the eligible 2018 non-Cochrane intervention reviews registered in PROSPERO. As a result, the findings may reasonably be generalised to other registrations of healthcare interventions, but not necessarily other types of registered reviews excluded from our sample.\n\nPRISMA-P is a reporting guideline and not a rating scale, so judgements about whether sufficient information had been provided for some items carried a degree of subjectivity. The assessment guide and form developed for the study aimed to maximise objectivity but in accordance with PRISMA-P did not weight importance of items. Although two researchers independently carried out the assessments, achieving an overall agreement rate of 90%, subjectivity was minimised but not eliminated.\n\nPROSPERO was developed in 2011 to record key protocol details and does not necessarily accord with everything subsequently recommended in the 2015 PRISMA-P reporting guidelines. Some registration items are mandatory and others optional. However, this study looked at records that had no other protocol output and arguably should therefore have provided PRISMA-P level detail. The evidence that protocol details are only available in PROSPERO for around 96% of non-Cochrane reviews makes the infrequency of reporting of items a concern9,10. Based on the findings of other studies, promoting improved reporting of protocol details may help increase the quality of systematic reviews17,18.\n\nProtocols are iterative documents and even after a review has started there may be legitimate reasons for amendments. Such changes should and can be reported in a registration record, with their justification and timing. Just over two thirds of PROSPERO records have more than one version (Figure 1). While focussing on single entry records to be certain that any changes were not made after completion of the review this may have excluded records where more complete information was added to the record over time at key points in the review process.\n\nThis study simply looked at whether items were reported and not at the level of detail or suitability/appropriateness of the planned methods. The option of ‘partially reported’ could have been used at assessment but was avoided to minimise subjectivity. The focus was on simply establishing whether items were reported or not. The assessors focussed on whether the information was reported or could reasonably be inferred from what was reported. Assessing the quality of planned methods in protocol registrations needs to be the subject of further research.\n\nThis study shows that there is work to be done to promote the complete reporting of items recommended in the guidelines for systematic review protocols when the registration in PROSPERO is the only place they can be accessed. This is in line with other research that has identified issues with the quality of reporting, publication and outcome reporting biases in systematic review protocols in general3,9,11,13,19,20. As proposed in the PRISMA-P statement paper, actions and potential benefits to encourage adherence to PRISMA-P will take a joint effort on the part of a host of stakeholders, including reviewers, registries, and journal editors5,21.\n\n\nConclusions\n\nPROSPERO provides reviewers with the opportunity to be transparent in their planned methods and demonstrate efforts to reduce bias. However, where the PROSPERO record is the only available source of a priori reporting, there is a significant shortfall in the items reported, compared to those recommended in PRISMA-P. This presents peer reviewers and others wishing to assess the validity of the final review with challenges in interpretation. PROSPERO records are not peer reviewed or assessed for methodological quality, it is the responsibility of those registering their review to complete the registration form fully or provide access to a complete protocol. There are several areas requiring particular attention when completing the registration form. These include explaining the rationale for undertaking the review in the context of what is known; providing information sources beyond a list of databases to be searched; and reporting reproducible process methods for data management, study selection and risk of bias assessment. In addition, defining variables for data extraction, how specified outcomes will be measured, and the planned analyses, with criteria for undertaking a quantitative synthesis should all be included in detail.\n\nThis study only looked at whether recommended items were reported or not in PROSPERO records. Further research is needed to assess the quality of the planned methods in systematic review protocol registrations.\n\n\nData availability\n\nOpen Science Framework: PROSPERO and PRISMA-P, https://doi.org/10.17605/OSF.IO/7PW4G16.\n\nOpen Science Framework: PROSPERO and PRISMA-P, https://doi.org/10.17605/OSF.IO/7PW4G16.\n\nThis project contains the following underlying data:\n\n- Study protocol\n\n- Items, scoring options and guidance/rules for assessment of PROSPERO records compared to PRISMA-P reporting requirements\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank Fiona Rose for her assistance with the assessment of records.\n\n\nReferences\n\nCentre for Reviews and Dissemination: Systematic reviews: CRD’s guidance for undertaking reviews in health care. University of York, 2009. Reference Source\n\nHiggins JPT, Thomas J, Chandler J, et al.: Cochrane Handbook for Systematic Reviews of Interventions version 6.0 (updated September 2019). 2nd ed.: Wiley-Blackwell, 2019. Publisher Full Text\n\nAllers K, Hoffmann F, Mathes T, et al.: Systematic reviews with published protocols compared to those without: more effort, older search. J Clin Epidemiol. 2018; 95: 102–110. PubMed Abstract \| Publisher Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015; 350: g7647. PubMed Abstract \| Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015; 4(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth A, Clarke M, Dooley G, et al.: The nuts and bolts of PROSPERO: an international prospective register of systematic reviews. Syst Rev. 2012; 1: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth A, Clarke M, Ghersi D, et al.: Establishing a minimum dataset for prospective registration of systematic reviews: an international consultation. PLoS One. 2011; 6(11): e27319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViguera-Guerra I, Ruano J, Aguilar-Luque M, et al.: Evolution of international collaborative research efforts to develop non-Cochrane systematic reviews. PLoS One. 2019; 14(2): e0211919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGe L, Tian JH, Li YN, et al.: Association between prospective registration and overall reporting and methodological quality of systematic reviews: a meta-epidemiological study. J Clin Epidemiol. 2018; 93: 45–55. PubMed Abstract \| Publisher Full Text\n\nKhaleel S, Sathianathen N, Balaji P, et al.: Quality of urological systematic reviews registered in PROSPERO. BJU Int. 2019; 124(2): 195–196. PubMed Abstract \| Publisher Full Text\n\nParsons R, Golder S, Watt I: More than one-third of systematic reviews did not fully report the adverse events outcome. J Clin Epidemiol. 2019; 108: 95–101. PubMed Abstract \| Publisher Full Text\n\nTricco AC, Cogo E, Page MJ, et al.: A third of systematic reviews changed or did not specify the primary outcome: a PROSPERO register study. J Clin Epidemiol. 2016; 79: 46–54. PubMed Abstract \| Publisher Full Text\n\nKelly SE, Moher D, Clifford TJ: Quality of conduct and reporting in rapid reviews: an exploration of compliance with PRISMA and AMSTAR guidelines. Syst Rev. 2016; 5: 79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters JPM, Hooft L, Grolman W, et al.: Reporting Quality of Systematic Reviews and Meta-Analyses of Otorhinolaryngologic Articles Based on the PRISMA Statement. PLoS One. 2015; 10(8): e0136540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth A, James S, Cockayne S, et al.: PROSPERO and PRISMA-P. 2020. http://www.doi.org/10.17605/OSF.IO/7PW4G\n\nPage MJ, Moher D: Evaluations of the uptake and impact of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) Statement and extensions: a scoping review. Syst Rev. 2017; 6(1): 263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSideri S, Papageorgiou SN, Eliades T: Registration in the international prospective register of systematic reviews (PROSPERO) of systematic review protocols was associated with increased review quality. J Clin Epidemiol. 2018; 100: 103–110. PubMed Abstract \| Publisher Full Text\n\nRuano J, Gomez-Garcia F, Gay-Mimbrera J, et al.: Evaluating characteristics of PROSPERO records as predictors of eventual publication of non-Cochrane systematic reviews: a meta-epidemiological study protocol. Syst Rev. 2018; 7(1): 43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRunjic E, Rombey T, Pieper D, et al.: Half of systematic reviews about pain registered in PROSPERO were not published and the majority had inaccurate status. J Clin Epidemiol. 2019; 116: 114–121. PubMed Abstract \| Publisher Full Text\n\nMoher D, Stewart L, Shekelle P: Implementing PRISMA-P: recommendations for prospective authors. Syst Rev. 2016; 5: 15. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68072", "date": "31 Jul 2020", "name": "Dawid Pieper", "expertise": [ "Reviewer Expertise Research methods", "clinical epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is an analysis of how PROSPERO records adhere to the PRISMA-P guideline. The Analysis is based on a random sample of 439 PROSPERO records published in 2018. The authors conclude that reporting in PROSPERO should be improved given the fact that the PROSPERO record is often the only available source of a priori reporting.\nThe manuscript is methodologically sound and well written. What I think can be improved is the discussion. I wonder what is the implication of this study. Do the authors want to make the point that PROSPERO records should follow PRISMA-P? To the best of my knowledge PRISMA-P is even not mentioned in the PROSPERO guidance. If this would be the intention then wy not allign PROSPERO with the PRISMA-P items. I admit that PRISMA-P has been primarily designed for SRs of healthcare interventions, but most items are General and would be applicable to other review types as well. I do not want to make the point that this is a great idea, but it is somehow a logical question resulting from your manuscript and this should be mentioned in the discussion. Registries and protocols should be seen as different entities, and thus I think that a perfect result of all PROSPERO records meeting all PRISMA-P items cannot be what we aiming for. If this would be the case, this would probably dilute the difference between a PROSPERO record and a protocol.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5882", "date": "10 Sep 2020", "name": "Alison Booth", "role": "Author Response", "response": "We thank you for your peer comments and agree this is an important point. We have added the following paragraph to the discussion section: The review protocol is a detailed record of the planned methods developed through an iterative process. Once finalised or close to finalising, the key methodological details should be registered in PROSPERO. These are two separate but inter-related activities. PROSPERO was launched in 2011, a time when there were few opportunities to publish protocols, however, registration is not meant to be a substitute for preparation of a protocol. PROSPERO and PRISMA-P 2015 requirements are not aligned as they serve different purposes. However, a stated aim of registration is to facilitate comparison of what was planned with what is reported. Even if limited information were registered, we would expect the mandatory fields in PROSPERO to be fully completed. This was not the case, particularly for details related to outcome measures, assessment of risk of bias and quantitative analysis methods. It would not be reasonable to expect that PROSPERO records meet all the PRISMA-P recommended items, given the differences in purpose between a protocol and registration, but it is important to understand what information is available where registration is the only public source." } ] }, { "id": "68890", "date": "17 Aug 2020", "name": "Xin Sun", "expertise": [ "Reviewer Expertise Clinical Epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study conducted a methodological survey to assess the extent to which the contents of PROSPERO records meet the systematic review protocol reporting items in PRISMA-P. This paper addresses an important research question, and the findings may have implications for the reporting of systematic review protocols. However, there are a few issues for authors to consider:\nOne aim of PRISMA-P is to aid authors in transitioning their systematic review protocols prepared in accordance with PRISMA-P into full text, while the authors used records from PROSPERO (i.e., not full-text) to assess the compliance to PRISMA-P reporting items, which may be a limitation that should be discussed in this paper.\n\nIn the methods part, it could be desirable that the authors could clearly report how the 17 numbered items of PRISMA-P were broken down into 63 elements.\n\nThe author should clearly report whether the subgroup analyses reported in table 3 were pre-planned.\n\nThe use of a scoring scheme for PRISMA-P and the 63 elements may not be optimal, given the potential difference in item importance, which should be added to the discussion part as a limitation.\n\nIn table 2, values in parentheses are percentages, which should be indicated in the table.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5883", "date": "10 Sep 2020", "name": "Alison Booth", "role": "Author Response", "response": "We thank you for your peer review and give our responses as follows:1. One aim of PRISMA-P is to aid authors in transitioning their systematic review protocols prepared in accordance with PRISMA-P into full text, while the authors used records from PROSPERO (i.e., not full-text) to assess the compliance to PRISMA-P reporting items, which may be a limitation that should be discussed in this paper.Thank you for raising this point. We agree and have addressed this point in the addition of the following to the discussion: PROSPERO and PRISMA-P 2015 requirements are not aligned as they serve different purposes. However, a stated aim of registration is to facilitate comparison of what was planned with what is reported. Even if limited information were registered, we would expect the mandatory fields in PROSPERO to be fully completed. This was not the case, particularly for details related to outcome measures, assessment of risk of bias and quantitative analysis methods. It would not be reasonable to expect that PROSPERO records meet all the PRISMA-P recommended items, given the differences in purpose between a protocol and registration, but it is important to understand what information is available where registration is the only public source. 2. In the methods part, it could be desirable that the authors could clearly report how the 17 numbered items of PRISMA-P were broken down into 63 elements.We have added an example from the study protocol to illustrate the description of how the elements were derived from the 19 PRISMA-P items, as follows:Where the PRISMA-P description for an item specified more than one piece of information, the individual elements were listed as subsets of the items. For example, item 14. Risk of bias in individual studies, says: “Describe anticipated methods for assessing risk of bias of individual studies, including whether this will be done at the outcome or study level, or both; state how this information will be used in data synthesis.” Scoring for this item will be for each of the following separate elements: No risk of bias assessment planned and justification provided; Risk of bias tools named for all study types included; Outcome or study level or both; Domains/outcomes for risk of bias assessment stated; Risk of bias assessment process described; How risk of bias findings will be used in synthesis. Applying this approach to the 19 items resulted in a list containing 63 elements to be reported.3. The author should clearly report whether the subgroup analyses reported in table 3 were pre-planned.We can confirm they were all included in the study protocol, available at https://doi.org/10.17605/OSF.IO/7PW4G. We have added ‘which were all pre-defined’ to the section on Subgroup comparisons in the manuscript. 4.The use of a scoring scheme for PRISMA-P and the 63 elements may not be optimal, given the potential difference in item importance, which should be added to the discussion part as a limitation.This is an important point thank you, which we have incorporated into the discussion as follows:This study simply looked at whether items were reported and not at the level of detail or suitability/appropriateness of the planned methods. Use of a scoring system particularly as all items and elements carried the same weight is a limitation of this study. The scoring does not accord with the PROSPERO dataset which identifies information as either mandatory or optional. For this reason we have indicated the mandatory/optional fields in Table 2. The scoring only relates to the presence or absence of information. The option of ‘partially reported’ could have been used at assessment but was avoided to minimise subjectivity. The focus was on simply establishing whether items were reported or not. The assessors focussed on whether the information was reported or could reasonably be inferred from what was reported. Assessing the quality of planned methods in protocol registrations needs to be the subject of further research.We have also amended how the mandatory/optional fields are indicated in Table 2 so the difference is clearer. 5. In table 2, values in parentheses are percentages, which should be indicated in the table.Apologies for this omission, this has been corrected in the revised version." } ] } ]	1	https://f1000research.com/articles/9-773
https://f1000research.com/articles/9-1115/v1	10 Sep 20	{ "type": "Research Article", "title": "Coping strategies of students for anxiety during the COVID-19 pandemic in China: a cross-sectional study", "authors": [ "Mohammad Nurunnabi", "Syed Far Abid Hossain Hossain", "Karuthan Chinna", "Sheela Sundarasen", "Heba Bakr Khoshaim", "Kamilah Kamaludin", "Gul Mohammad Baloch", "Areej Sukayt", "Xu Shan", "Syed Far Abid Hossain Hossain", "Karuthan Chinna", "Sheela Sundarasen", "Heba Bakr Khoshaim", "Kamilah Kamaludin", "Gul Mohammad Baloch", "Areej Sukayt", "Xu Shan" ], "abstract": "Background: COVID-19 has severely affected university students everywhere in the world. Due to fear of infection, government and local authorities in China immediately closed academic institutions and tried to find survival techniques to cope with market turbulence. COVID-19 was present in China at the end of 2019. However, little attention has been paid by researchers to coping strategies during the COVID-19 pandemic, and few measures were taken to assess the coping strategies of university students, specifically following the closure of their institutions. To address this gap, this study attempted to discover the coping strategies of Chinese students during the COVID-19 pandemic in China. Methods: We conducted an online survey using a semi-structured questionnaire with a simple random sampling technique and received 559 responses. The survey questions captured information about students’ lives during the COVID-19 outbreak, actions to control anxiety, and what students care about during the pandemic. The associations between coping strategies used and levels of anxiety were tested using analysis of variance (ANOVA) procedures. SPSS Statistics v27 was used for statistical analysis in this study. Results: The university students reported that coping strategies and survival techniques were required due to high levels of anxiety and psychological pressure during the COVID-19 pandemic. Most of the respondents reported the prompt closure of their academic institutions due to COVID-19. Psychological concerns, such as lack of sleep, emotional support, mental support and social appeal, were also reported. Conclusions: This is one of the very first studies on coping strategies for anxiety in China. The study reveals that university students employ a number of coping strategies in relation to COVID-19, but also suggests a need to strengthen such strategies in this population. However, the study was limited to a small number of provinces in China, which may affect the generalizability of the research.", "keywords": [ "Coping strategy", "Students", "University", "China", "COVID-19", "Corona virus diseases" ], "content": "Introduction\n\nThe COVID-19 outbreak infected mainland China and the surrounding regions drastically at the end of 2019. To cope with the infection, the Chinese government executed severe control measures, including an expansion of the national holiday, immediate lockdown and strict post-lockdown measures as part of an effort to prevent a second coronavirus wave. In addition, the mobility of the population was controlled with severe restrictions at a national level, and social distancing was monitored as well as encouraged with a compulsory quarantine period of two weeks for returning migratory personnel. Reflecting these containment measures, the economy contracted by 6.8% in the first quarter (CNBC 20201). The increasing number of cases of COVID-19 in China between January and June 2020 can be seen in Figure 1.\n\nThis chart has been reproduced under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nIn the middle of February, the Chinese government gradually decided to remove restrictions on mobility of the population and other activities, prioritizing some provinces and various groups of people based on risk calculations. Most trade and universities reopened countrywide with careful observation during the stages of reopening. However, social distancing rules remained in place as did the mandatory use of a facial mask. Entering the country from overseas also remained restricted in some situations to control the importation of cases. The danger of new infections from imported cases has been highlighted by the local community and the government has taken the matter seriously and restricted foreign entry to China1. Apart from that, regular temperature checking and personal health QR codes were imposed on a compulsory basis for traveling locally and entering public places in order to cope with the pandemic (WHO 20202).\n\nFigure 2 shows the Chinese government’s response stringency index. The government response included the closure of universities and other academic institutions, factories and offices, and travel restrictions. The responses are measured on a scale of 10 to 100, and the graph in Figure 2 shows a very high rate of restrictions from March to May, 2020.\n\nThis chart has been reproduced under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThis chart has been reproduced under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigure 3 shows the daily confirmed cases of COVID-19 in China; February had the highest number of confirmed cases. Even though the daily confirmed cases has decreased recently, strategies for coping with the pandemic are still under the control of the Chinese government. For example, no international students have been allowed to return to university. If a student returned, they would receive serious penalties, as announced by some universities3. As a result, students are struggling to complete their studies on time, and many students are affected. Even if students are not infected with the virus, they may be depressed as their studies have been disrupted and they cannot finish their studies on time. Online teaching and graduation opportunities have been provided by some universities, but many students are not capable of finishing their studies in this way. Therefore, this study aimed to shed light on the coping strategies used by university students in China during the COVID-19 outbreak. A coping strategy in this article is defined as the behaviors, thoughts, and emotions that students use to adjust to the changes that occur in their life, e.g. writing issues down in order to process them during COVID-19.\n\n\nLiterature review\n\nChinese people have been familiar with various pandemics over the years. However, COVID-19 is the deadliest pandemic in China’s recorded history. In 2003, Severe Acute Respiratory Syndrome (SARS) affected China, especially in Beijing4. Although SARS was not as dangerous as COVID-19, it was considered as a “warning”5 to society, and the infection was controlled well within the country.\n\nMental health has been severely affected by the COVID-19 infection owing to fear of the pandemic, and various coping strategies are observed6, affecting mental health care, human care, psychological crisis control measures, and intervention in COVID-197. Previous research surrounding coping strategies has focused mainly on SARS; however, as COVID-19 is novel, the socio-psychological impacts of COVID-19 are still under investigation. Coping strategies are not only urgently required, but also need to be adapted to university students requiring an emergency strategy to be decided on by policy makers8.\n\nCoping strategies for COVID-19 cannot be short term. There may be a long time frame, since subsequent waves of COVID may arise. While researchers are continuously working to provide the right medicine, vaccinations and recovery measures, little research has been conducted on the coping strategies of students studying in China. SARS killed hundreds of people in China about ten years ago, and people struggled to cope with the situation9; the same situation arose in Hong Kong with Middle East respiratory syndrome (MERS)9. Anxiety among college students in Hong Kong was investigated by previous researchers10 during the SARS outbreak; however, the coping strategies of Chinese students currently studying in mainland China are still elusive.\n\nOne of the key aspects associated with coping strategies during COVID-19 in China is mental health. Different age groups are observed to face different mental challenges in coping with the pandemic. The way in which the unpredictable effects of COVID-19 were dealt with in China provide a lesson in how to cope with pandemic situations calmly. Among the general population in China, immediate action was taken by the local and central authority. This is known as an immediate psychological response, and can help people to cope with the pandemic situation11. However, there is little research into socio-psychological effects and the relevant coping strategies. So far, personal reactions have been examined in the context of community action taken to tackle the Wuhan COVID-19 outbreak7. Coping strategies are hard to implement anywhere; however, in China, the people supported and responded well to measures to cope with the pandemic during COVID-1911. The fact is, if a person’s mental health can be supported, coping strategies for fighting against COVID19 will be comparatively flexible as the COVID-19 outbreak is treated as a psychological problem. In order to implement coping strategies, psychiatrists have played an important role in society. The role of telehealth services has been revealed as an ideal coping strategy recently12. The coping strategies are ongoing, and based on industry and situation; as a result, the socio-psychological impact of the coping strategies on students studying in China should be investigated.\n\nChinese educational institutions played an exemplary role in controlling local as well as international students. Coping strategies depend on the mental health of students, which has varied a lot during the COVID-19 epidemic. Not only local Chinese students but also international students struggled to cope with the pandemic in order to achieve their desired academic performance. Different coping strategies are observed among both Chinese and international students. Local students mostly returned to their hometown as long as the local transport was operating. On the other hand, university authorities did not decide whether or not international students should go back to their own country; however, the students were instructed to be safe and not to travel anywhere without informing the responsible teacher. Following this decision, students in different cities in China faced mental upset and couldn’t decide what to do, as revealed on various social media13. The students exposed their problems and coping difficulties in specific cities like Wuhan, where the situation was unsettling from the end of January until the end of February. Coping strategies are also negatively associated with psychosocial problems, as stated in recent research14 due to the high risk of infection and anxiety among health workers. Finally, coping strategies in China during the COVID-19 outbreak are utilized differently by two different groups of students. According to a longitudinal study15, one group of students decided to stay in the dormitory and not to move anywhere during the epidemic. This coping strategy was not based on students’ concern only for themselves; rather, this group thought about others such as their parents and other family members. Another group of students assumed that they were safe and they decided to return to their homes. This is another coping strategy observed among students studying in China at the moment.\n\n\nMethods\n\nInitially, the questionnaire was piloted and validated by seven professors from various countries. There were no changes during the validation stage. An online semi-structured survey was developed by the authors. The survey was administered using a Chinese website called wjx.cn forms, which are similar to Google or Microsoft forms. The questionnaire was designed in English and then translated into Chinese with the help of a Chinese native speaker, who is also expert in English. The authors decided to use the Chinese version as native speakers feel more comfortable answering in Chinese. The authors re-translated the Chinese version using Google Translate to make sure that the original meaning was retained. Four coping strategies were assessed: “Seek social support,” “Avoidance,” “Mental disengagement” and “Humanitarian” were tested. The items were measured on a scale of 1 to 4; 1 = never/rarely, 2 = sometimes, 3 = often and 4 = very often/always. This is similar to the self-rating anxiety scale (SAS)16. The survey is available as Extended data17.\n\nThe survey was developed by the authors and was in two parts. Part 1 collected socio-demographic variables, including age group, gender, field of study, level of study, current accommodation and current stay (with whom). Part 2 contained the following sections related to anxiety: level of anxiety; coping strategies; and anxiety level with coping strategies. There were 20 multiple choice questions in the personal situation section that were rated on a scale from “Never” to “Always”; or “Not applicable” to “More than ever.”\n\nThe link to the survey was sent to students in various academic and study groups who are mainly research focused. A simple random sampling technique and various social networking sites (SNSs), including WeChat, were used. Inclusion criteria were that the students had to have access to the internet, were adults (> 18 years old), were able to understand Chinese, and were willing to give informed consent to participate.\n\nOn receiving and clicking the survey link, the students accessed information about the survey purpose, which included consent information. After they agreed, using a ‘yes’ button to consent to participating in the survey, they filled in their demographic information, and then the next set of questions appeared. The participants had to answer each section’s questions in order to proceed to the next section.\n\nThe participants were stimulated to join the survey with a reward offered (a total of 1,000 Chinese Yuan, or 2 RMB per participant) via red packet 红包 (hóngbāo). The red packet is a system well-known in China for distributing random amounts of money to people on SNSs.\n\nData collection was initiated from May 26th to June 3rd, 2020. Initially, the survey was sent to students in the Shaanxi province only; however, owing to a slow response, this was expanded to various provinces of China, including Shaanxi, Hubei, Beijing, Heilongjiang and Guangdong.\n\nThe associations between coping strategies used and levels of anxiety were tested using ANOVA procedures. In particular, SPSS v27 was used for statistical analysis in this study.\n\nAll work involving human participants was approved by the Prince Sultan University (PSU)’s Institutional Review Board Committee (approval reference: PSU IRB-2020-04-0038).\n\n\nResults\n\nWe received 559 responses. The demographic details of the participants are represented in Table 1. Among the 559 respondents, 40.4% were women and 50.3% were in the 18–22 age group. In total, 58.5% of respondents were economic management students and more than half were undergraduate students. While 26.8% of respondents stayed in the university dormitory, 17.9% lived in a rented apartment without family members.\n\nIn this study, we wanted to know what strategies the students used in coping with anxiety during the COVID-19 pandemic. In total, 66.90% of the students reported experiencing “normal” anxiety, and 23.80% reported it as “severe to extreme” (Figure 4). Table 2 shows the results showing how much each of the four coping strategies was used by the respondents. Overall, the usage of all four strategies was moderate to low. The distributions were fairly normal (skewness < 2, kurtosis < 7). Overall, the students practiced more mental engagement strategies and fewer social support strategies.\n\nIn the ANOVA analyses, the variances were similar (Table 3). All four strategies were significantly associated with levels of anxiety. The usage of all four coping strategies was higher in the “severe to extreme” group compared to the “normal” and “minimal to moderate” groups.\n\nNote: a, b pairwise differences\n\n\nDiscussion\n\nThe findings of the study indicate that the mental or cognitive health condition of university students in China needs to be further ascertained in order to elucidate the actual psychological impact due to the COVID-19 pandemic.\n\nThe outbreak of COVID-19 may be stressful and significantly affect individuals’ mental health. Symptoms of mental health issues, as well as social stressors such as uncertainty due to COVID-19, affect the coping tendency of students. The results of this study reveal that coping strategies are diverse based on demographic and regional difference in China.\n\nThe world has occasionally observed numerous life-threatening epidemics, such as COVID-19. It is worth noting that a failure to adopt coping strategies may seriously affect students’ academic achievement due to the unpredictability of COVID-1918. Financial problems, a lack of health care facilities and safety issues are also major concerns affecting coping strategies in China. Current research19 has conducted a parallel study based on Indian citizens. In that study in India, the authors mentioned a high demand for and shortages of hand sanitizers, hand wash, and facial masks, indicating people’s growing concern to avoid COVID-19 infection. Every epidemic has its unique characteristics in terms of causality, risk and control measures. COVID-19 is not different; however, socio-psychological pressure is acute. Back in 2015, during the Ebola virus outbreak in Ethiopia, coping measures and awareness were less satisfactory than the results of this study found in China.\n\nCoping strategies are directly associated with anxiety levels. The most extreme anxiety level in this study is reported by 20.9% of students, which is more than one-fifth of the total sample size. Students’ ability to seek social support, their isolation and mental disengagement and responsiveness to humanitarian issues are all assessed when measuring their copying strategies. For all the four cases, p-value is <0.001 between coping strategies and anxiety levels. This result indicates a strong positive relationship between coping strategies and the anxiety level of students. The contributors in this study had a satisfactory level of awareness concerning COVID-19 and the majority of them took the recommended preventive measures20.\n\nIn China, local government and community members emphasized preventive measures such as movement control, temperature checks, wearing respiratory masks, and so on. The study participants reported that most of their school or academic unit were using online teaching during the epidemic. A total of 93.92% reported that their academic institution conducted online lectures, training sessions, video conferencing, competitions and award ceremonies and so on via online links. A total number of 246 participants (44.01%) reported that their academic institutions started online classes in the second month of the outbreak. Due to uncertainty about future study continuation, students reported more anxiety than usual (more than 63%). Similarly, sleeping problems and nightmares were also reported at a higher percentage than before (more than 40%). In this study, 68.87% of students reported unclear learning tasks in the current academic year or semester due to COVID-19. Results from the univariate analysis indicate that female students were more affected than male students in terms of anxiety and stress. However, it is interesting to note that 66.9% of students reported their anxiety level as normal during COVID-19. This is consistent in the sense that knowledge can be recovered from any disruption21 like a pandemic. On the other hand, mental disengagement seems crucial in the analysis. Students from business and management majors have a higher level of anxiety, which contradicts previous studies claiming that music students were more anxious22. Those staying in rented apartments were prone to severe anxiety compared to those staying at home, which indicates that students may feel psychologically worse if they did not stay with their families. The discussion is consistent with the result of the overall study in terms of level of anxiety and coping strategies presented in Table 3.\n\n\nLimitations, conclusion and future research\n\nThe study has a few limitations that can be addressed in order to further investigate the phenomenon. First, the study is limited to students in China from different universities and majors. Also, the respondents are selected randomly due to online data collection from various provinces that were not affected equally. The respondents are Chinese students mostly from various recognized universities. Thus, findings should not be generalized to the overall student group. Future studies should investigate a proportional study among school, college and university students. In addition, the most affected and least affected areas should be separately investigated to fully analyze the phenomenon. In the future, a mixed methodology approach may further help with in-depth investigation of the phenomenon in a bibliometric context. This research is based on empirical analysis, which may affect the generalizability of the research. A more in-depth technique, such as interviews or other qualitative measures, in the future may assist a more thorough investigation of the students’ coping strategies. Furthermore, if the university authority, administrative staffs and teachers could be selected as respondents, the study might produce more interesting findings. An epidemiologically robust survey on a specific group of people can discover the phenomenon further.\n\nDespite some limitations, the study indicates numerous implications for society. First, the university authority should be aware of the students’ coping strategies. In particular, students who live without parents or relatives should be taken care of properly during the outbreak. Second, to help students cope with the mental pressure, university authorities may think about arranging or organizing programs such as an online experience-sharing competition, and encourage students by offering rewards or financial aids. Finally, required food and healthcare materials should be supplied to ensure the students’ safety.\n\nThe study sheds light on the coping strategies of Chinese students during the COVID-19 pandemic. Due to the lockdown policy, many students have had to stay in university hostels with no permission to go out. The coping strategy was as challenging for students at various universities in China for COVID-19 as for any other pandemic5. The overall findings of the study clearly indicate a significant relationship between university students and their coping strategies during the COVID-19 in China. Increasing psychological pressure on students – consistent with our study finding that 48.3% of respondents believed that during the epidemic period everything would collapse – affect the students’ usual coping strategies and also their regular academic activities such as class attendance and assignments. As a result, ideal and safe coping strategies should be identified for the students in order to face any epidemic challenges in the future10, which may help to ensure sustainable educational development in the world. Prioritizing research on mental health, anxiety and students’ coping strategies along with psychological effects is necessary.\n\n\nData availability\n\nFigshare: COVID-19 and Coping Strategies of Anxiety in China, https://doi.org/10.6084/m9.figshare.12801395.v317.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThis project contains the following extended data:\n\n- Respondent answers concerning their lives during the COVID-19 pandemic\n\n- Survey sent to students", "appendix": "Footnotes\n\n1https://www.cnbc.com/2020/04/17/china-economy-beijing-contracted-in-q1-2020-gdp-amid-coronavirus.html#:~:text=China Economy-,China says its economy shrank by 6.8 in the first,as the country battled coronavirus&text=China reported that its first,the world's second largest economy.\n\n2https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf.\n\n\nAcknowledgments\n\nThe authors would like to thank the Global Education Policy Network (GEPN), Prince Sultan University, Riyadh, Saudi Arabia.\n\n\nReferences\n\nIMF: Policy Responses to COVID-19. 2020. Reference Source\n\nRoser M, Ritchie H, Ortiz-Ospina E, et al.: Coronavirus Pandemic (COVID-19). 2020. Reference Source\n\nSouth China Morning post: Rule-breaking foreign students in China will be punished, education ministry warns. 2020. Reference Source\n\nMain A, Zhou Q, Ma Y, et al.: Relations of SARS-related stressors and coping to Chinese college students' psychological adjustment during the 2003 Beijing SARS epidemic. J Couns Psychol. 2011; 58(3): 410–23. PubMed Abstract \| Publisher Full Text\n\nde Jong JC, Claas EC, Osterhaus AD, et al.: A pandemic warning? Nature. 1997; 389(6651): 554. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrnell F, Schuch JB, Sordi AO, et al.: \"Pandemic fear\" and COVID-19: mental health burden and strategies. Braz J Psychiatry. 2020; 42(3): 232–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi W, Yang Y, Liu ZH, et al.: Progression of Mental Health Services during the COVID-19 Outbreak in China. Int J Biol Sci. 2020; 16(10): 1732–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdhikari SP, Meng S, Wu YJ, et al.: Epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (COVID-19) during the early outbreak period: a scoping review. Infect Dis Poverty. 2020; 9(1): 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu H, Stratton CW, Tang Y: Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle. J Med Virol. 2020; 92(4): 401–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWong TW, Gao Y, Tam WWS: Anxiety among university students during the SARS epidemic in Hong Kong Stress Health. 2007; 23(1): 31–5. Publisher Full Text \| Free Full Text\n\nWang C, Cheng Z, Yue XG, et al.: Risk Management of COVID-19 by Universities in China. J Risk Financial Manag. 2020; 13(2): 36. Publisher Full Text\n\nKeshvardoost S, Bahaadinbeigy K, Fatehi F: Role of Telehealth in the Management of COVID-19: Lessons Learned from Previous SARS, MERS, and Ebola Outbreaks. Telemed e-health. 2020; 26(7): tmj.2020.0105. Publisher Full Text\n\nGao J, Zheng P, Jia Y, et al.: Mental health problems and social media exposure during COVID-19 outbreak. Hashimoto K editor. PLoS One. 2020; 15(4): e0231924. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang W, Wang K, Yin L, et al.: Mental Health and Psychosocial Problems of Medical Health Workers during the COVID-19 Epidemic in China. Psychother Psychosom. 2020; 89(4): 242–250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Pan R, Wan X, et al.: Immediate Psychological Responses and Associated Factors during the Initial Stage of the 2019 Coronavirus Disease (COVID-19) Epidemic among the General Population in China. Int J Environ Res Public Health. 2020; 17(5): 1729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZung WW: A rating instrument for anxiety disorders. Psychosomatics. 1971; 12(6): 371–9. PubMed Abstract \| Publisher Full Text\n\nNurunnabi M, Hossain SFA, Chinna K, et al.: COVID-19 and Coping Strategies of Anxiety in China. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12801395.v3\n\nHossain SFA, Shan X, Nurunnabi M: Is M-Learning a Challenge?: Students Attitudes Toward the Sustainable Learning and Performance. International Journal of e-Collaboration. 2019; 15(1): 21–37. Publisher Full Text\n\nRoy D, Tripathy S, Kar SK, et al.: Study of knowledge, attitude, anxiety & perceived mental healthcare need in Indian population during COVID-19 pandemic. Asian J Psychiatr. 2020; 51: 102083. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQiu J, Shen B, Zhao M, et al.: A nationwide survey of psychological distress among Chinese people in the COVID-19 epidemic: implications and policy recommendations. Gen Psych. 2020; 33(2): e100213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNESCO: Education: From disruption to recovery. 2020. Reference Source\n\nSahu P: Closure of Universities Due to Coronavirus Disease 2019 (COVID-19):Impact on Education and Mental Health of Students and Academic Staff. Cureus. 2020. Reference Source" }	[ { "id": "71187", "date": "23 Sep 2020", "name": "Delia Deliu", "expertise": [ "Reviewer Expertise accounting and financial audit", "corporate governance", "CSR and sustainability in times of crisis", "financial auditor’s responsibilities and liabilities in a sensitive socio-economic context", "quality of (non-)financial reporting and external auditing", "the impact of Blockchain on the accounting and auditing profession", "impact of technology integration in education", "quality of education" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComment 1 – General comment:\nThe manuscript “Coping strategies of students for anxiety during the COVID-19 pandemic in China: a cross-sectional study” by Mohammad Nurunnabi, Syed Far Abid Hossain Hossain, Karuthan Chinna, Sheela Sundarasen, Heba Bakr Khoshaim, Kamilah Kamaludin, Gul Mohammad Baloch, Areej Sukayt and Xu Shan explores a theme of interest referring to the negative outcomes and consequences of remote studying (in general), respectively coping strategies of university students (in particular) in China, in the context of the new Coronavirus (Covid-19) pandemic.\nThe topic approached by the authors is a hot topic these days and of major importance to all actors on the market.\nThe paper has a very up-to-date approach, as tackling the Covid-19 negative impact is one of the most important concerns of worldwide economics. Moreover, having solutions for this time of crisis means having an efficient sustainable university management and a healthy governance, thus authors have approached this hot issue from the most pertinent point of view.\n\nComment 2:\nIn section 1. Introduction, you efficiently succeeded in creating reader interest in the topic and did well in hatching the broad foundation for the problem that leads to the study, the study being adequately placed within the larger context of the scholarly literature, as well as within the sensitive socio-economic context generated by the Covid-19 pandemic. Since you are working within a particular theoretical framework, the line of inquiry is introduced and discussed early, in the introduction, as well as in the literature review.\nThe purpose of the research is clearly outlined in the Introduction and, therefore, provides a specific and accurate synopsis of the overall purpose of the study, since you briefly define and delimit the specific area of the research.\nThe literature review provides the background and context for the research problem and is successful in establishing the need for the research and sharing the results of other studies that are closely related to the research.\nHowever, a more enhanced literature review (comprising more references) – are needed, in order to better place the study in the broad context and highlight why it is relevant, as well as to outline the factors to be taken into consideration in regards to psychological factors affecting individuals during endemics/pandemics/social crises.\nTherefore, in order to strengthen and improve this study, I suggest adding a more comprehensive literature review on the debated concepts.\n\nComment 3:\nRegarding the research materials and methods, the authors have provided a well thought-out rationale for your decision to use the design, methodology, and analyses you have selected.\n\nComment 4:\nSection 3. Results clearly provide a concise and precise description of this research, as well as the experimental conclusions that can be drawn from the performed analysis. The authors clearly discuss the results and explain how they can be interpreted in perspective of initial research assumptions and previous studies.\n\nComment 5:\nThe paper reached valuable conclusions regarding the negative implications of remote studying in the context of the new Coronavirus (Covid-19) pandemic, namely those induced by anxiety, also outlining strategies used by students in relation to coping with all the negative psychological effects.\nComment 6 – Final comment:\nIn conclusion, the study, which is focused on a very relevant topic, presents a convincing structure and is written fluently, the research methodology being adequate and robust.\nConversely, I consider the authors could improve it by adding more bibliographic references in order to outline the intercorrelation between the debated concepts.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71184", "date": "07 Oct 2020", "name": "Nizar Alsharari", "expertise": [ "Reviewer Expertise Accounting and Finance" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is still not sufficiently analyzed through the theoretical framework discussed in the early parts of the paper. Hence, I believe contribution to the literature is still limited. The paper has potential, but you need to refocus your literature review, provide more detail on the new ideas and analyze further your findings through your suggested theoretical framework. The literature should be updated as well.... The discussion and results are not presented clearly and analyzed appropriately. The conclusions are not adequately linking together the other elements of the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "75079", "date": "20 Nov 2020", "name": "Dario Salerno", "expertise": [ "Reviewer Expertise Banking", "Financial Markets", "Risk of Bankrupcty", "Corporate Governance", "CSR." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComment 1: Using an amazing dataset, the authors examine the coping strategies of Chinese students during the COVID-19 pandemic in China. The analyzed sample consists of 559 responses to an online survey in which they captured information about students’ lives during the COVID-19 outbreak, actions to control anxiety, and what students care about during the pandemic. They show that coping strategies and survival techniques were necessary due to high levels of anxiety and psychological pressure during the COVID-19 pandemic. In addition, during the period considered, the authors argue that the students analyzed also had psychological problems such as lack of sleep, emotional support, mental support, and social appeal. The topic analyzed is of particular interest and extremely current, given the changes that students have had in their lives during the pandemic period. Additionally, the paper is well organized and provides some helpful insights into our understanding of how institutions can manage the negative effects of the pandemic crisis and what the consequences may be on very young people. It also opens very interesting ideas for future research. It also sheds light on the effectiveness of the measures taken during the pandemic by the institutions.\nComment 2: The authors make some reasonable theoretical insights into the introduction of the paper. In particular, they explain well theoretically the channel by which coping strategies and survival techniques are necessary during the pandemic period. Also, the motivation and contribution are clear and the structure/writing is mostly easy to follow.\nComment 3: The methodology is articulate in most of its parts. The results are in-depth analyzed and discussed. In their current form, they have a substantive discussion in relation to the relevant specialized literature.\nComment 4: The implications for research and study limitations are addressed and depict and integrate the original outputs of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1115
https://f1000research.com/articles/9-1114/v1	10 Sep 20	{ "type": "Software Tool Article", "title": "MSclassifier: median-supplement model-based classification tool for automated knowledge discovery", "authors": [ "Emmanuel S. Adabor", "George K. Acquaah-Mensah", "Gaston K. Mazandu", "George K. Acquaah-Mensah", "Gaston K. Mazandu" ], "abstract": "High-throughput technologies have resulted in an exponential growth of publicly available and accessible datasets for biomedical research. Efficient computational models, algorithms and tools are required to exploit the datasets for knowledge discovery to aid medical decisions. Here, we introduce a new tool, MSclassifier, based on median-supplement approaches to machine learning to enable an automated and effective binary classification for optimal decision making. The MSclassifier package estimates medians of features (attributes) to deduce supplementary data, which is subsequently introduced into the training set for balancing and building superior models for classification. To test our approach, it is used to determine HER2 receptor expression status phenotypes in breast cancer and also predict protein subcellular localization (plasma membrane and nucleus). Using independent sample and cross-validation tests, the performance of MSclassifier is evaluated and compared with well established tools that could perform such tasks. In the HER2 receptor expression status phenotype identification tasks, MSclassifier achieved statistically significant higher classification rates than the best performing existing tool (90.30% versus 89.83%, p=8.62e-3). In the subcellular localization prediction tasks, MSclassifier and one other existing tool achieved equally high performances (93.42% versus 93.19%, p=0.06) although they both outperformed tools based on Naive Bayes classifiers. Overall, the application and evaluation of MSclassifier reveal its potential to be applied to varieties of binary classification problems. The MSclassifier package provides an R-portable and user-friendly application to a broad audience, enabling experienced end-users as well as non-programmers to perform an effective classification in biomedical and other fields of study.", "keywords": [ "Breast cancer", "protein subcellular localization", "machine learning", "software package", "HER2 receptor status", "classification." ], "content": "Introduction\n\nMachine learning tools are required to solve binary classification problems for optimal decision making in medicine and other fields of study. In recent times, they have been used to predict subcellular localization of proteins to assist in the functional annotation of gene products and protein secondary structure1,2. As the identification of the subcellular location of any given protein provides insights into its function, this prediction task is highly valuable. This is more so as the specific functions of many proteins remain to be fully characterized. In other contexts, for instance medicine, classifications of patients in breast cancer and other diseases are important for administering therapies. There are five molecular sub-types of breast cancer identified: basal-like, Luminal A, Luminal B, human epidermal growth factor receptor 2- (HER2-) enriched, and normal-like3. The prognosis and administration of therapies in breast cancer is aided by the determination of molecular subtype phenotypes4.\n\nHowever, for various reasons, occasionally immunohistochemistry and other methods for establishing the presence or absence of these receptors do not necessarily cover all available samples. For example, results can be equivocal for some samples. Machine learning techniques can be trained with data from those samples that have been definitively characterized to correctly classify other uncharacterized samples’ phenotypes based on gene expression profiles. Machine learning methods rely on availability of large datasets to infer accurate outcomes for appropriate decisions concerning problems. With the advent of DNA microarray and next generation sequencing technologies, huge amounts of data are increasingly becoming available for use by these machine learning methods. These have permitted machine learning methods to be applied to characterize prognostic breast cancer samples for constructing patient-specific networks and disease groupings in precision medicine5–7.\n\nMachine learning methods based on Random Forest have been used to identify a gene regulatory program of human breast tumour progression8. Other methods such as Support Vector Machine and Naive Bayes, have all been applied to studies in breast cancer9. Other methods applicable to such problems are Logistic Regression, Bayesian Networks, K-nearest neighbours and tree-based methods10–12. In general, binary classification problems, such as breast cancer classification, commonly occur in nature and they rely on these machine learning methods for effective grouping, and the classification of multiple outcomes.\n\nThese methods are implemented in software packages/applications. For instance, several of these methods are implemented in the Weka package13. In R, implementations are provided as fitting functions as well as packages such as randomForest14, ISLR15 and e107116 among others. Unlike linear regression models, which predict quantitative response variables, these methods infer models to predict qualitative response variables.\n\nRecently, median-supplement approaches were introduced and found to outperform the traditional machine learning methods in binary classification models involving classification of receptor status phenotypes in breast cancer17. More importantly, these approaches achieve accuracies that compare favourably with other protein/mRNA-based procedures to decipher hormone and HER2-receptor status phenotypes in as much as they outperform traditional machine learning methods17. This implies that irrespective of the performance of the traditional methods, enhanced approaches provide better results in binary classification problems. However, none of the existing packages (implementations) supports the new median-supplement approaches to the binary classification problems.\n\nHere, we aim to provide a median-supplement based tool, MSclassifier, for automated knowledge discovery from data and illustrate its applicability to both breast cancer and other binary classification problems in broader contexts of study. This provides an effective binary classification tool, preventing biases that may originate from requirements of traditional tools which generally influence the classification decisions. It enhances the capacities of both Naive Bayes and Random Forests to infer models that provide more accurate predictions of classes of observations. This package is implemented in R under free software (GNU General Public Licence).\n\nIn performing an effective binary classification, MSclassifier introduces a predetermined number of supplementary instances based on the median of each attribute (feature) of the training sets for binary classification problems involving unequal members of classes. The supplementary instances along with the training instances form a new set from which a Naive Bayes or a Random Forest model is inferred to predict new instances. The provision of additional instances introduced by the new methods increases their prediction accuracies. This is because the effectiveness of the learning methods is improved whenever the training instances are more18. This has necessitated the design of the software package presented in this report. There are existing tools in R, namely randomForest14 and e107116, which implement both Random Forest and Naive Bayes algorithms, respectively. The Random Forest algorithm is based on the method described in 19. These packages are compared with the MSclassifier as the median-supplement approaches represent enhancements in these methods implemented in R. In addition, these provide an objective evaluation of the tool.\n\n\nMethods\n\nThe package implements median-supplement approaches to machine learning, robust machine learning techniques that have the advantage of supporting complete compliance efforts by not missing sensitive sub-datasets or allowing certain sub-datasets to escape the classification process when balancing overall datasets. They are applicable to datasets with unequal numbers of instances associated with each class (group).\n\nMedian-supplement machine learning algorithms. They involve the following steps:\n\n1. Find the median of each attribute among all the samples (instances).\n\n2. Find the scalar multiplication of the median of each attribute and a corresponding column vector of an m by n matrix of uniformly distributed random numbers between 0 and 1. m is the difference between the numbers of groups of samples, and n is the number of attributes. These form a supplementary set.\n\n3. The supplementary set is added to the expression profiles to form the new balanced, median-supplement data set.\n\n4. Finally, classification models are inferred from the median-supplement data.\n\nThere are two kinds of median-supplement approaches, namely, median-supplement Random Forest and median-supplement Naive Bayes methods. Each approach is distinguished by the kind of model constructed from the median-supplement data. For a ’median-supplement Random Forest’, a Random Forest classifier is inferred from the median-supplement data to assign classes to instances. To obtain a ’median-supplement Naive Bayes classifier’, a Naive Bayes model is developed from the median-supplement data to classify instances. The overview of the underlying principles of median-supplement approaches as implemented in MSclassifier is shown in Figure 1.\n\nA set of medians of attributes and a randomly generated matrix with uniformly distributed values are initially derived from the training sample. The result of a scalar multiplication of medians and corresponding column vectors of the random matrix is obtained and aggregated to the initial training sample to form a median-supplemented dataset. Finally, median-supplemented models are inferred from the median-supplemented data to predict new instances.\n\nNaive Bayes model. This model applies the Bayesian framework to predict classes of new instances. Any classes having the highest posterior probability becomes the class of a new test instance. Let G be a set of attributes. Then, the probability that any instance belongs to any class/category, Cj, is given by:\n\n\n\nwhere P(G\|Cj) is the probability of G given class Cj, P(Cj) is the probability of Cj and P(G) is the probability of G occurring. In this model, the attributes of each class are presumed to be independent distributions if the class is known. Thus, for each i-th attribute of n attributes, gi, the probability is given by:\n\n\n\nRandom Forest model. Random Forest is advancement in multistage decision making. It is a collection of Decision Trees. This typically involves constructing a collection of trees from bootstrap samples each of which consists of a subset of variables of the training sets. This approach of inferring trees from bootstrap samples involves recursively repeating the following20:\n\nSelecting m variables from the full set of attributes, n, at random.\n\nSelecting the best split among the variables.\n\nSplit the nodes into two nodes.\n\nOnce all desired trees have been achieved in those steps (which repeats after reaching a putative node size), a classification is determined by a majority vote. Assume Cb(x) is the class prediction of the b-th random forest tree. Then the classifier is given by:\n\n\n\nTypically,m=n. Using random forest spans from the fact that it improves predictive accuracies of tree-based methods19,20.\n\nMSclassifier, implemented in R, can be installed and run on most operating systems. The sole requirement is the availability of a recent version of R (https://cran.r-project.org/). The package is organized as a programme with the flexibility of selecting a median-supplement Random Forest or a median-supplement Naive Bayes method. The overview of the package follows the structure presented in Figure 1. The Documentation of the package has detailed instructions for installation and usage as well as other descriptions of the package.\n\nMSclassifier does not require any special programming skills of the user. It accepts a tabular dataset in which the attributes and instances are in columns and rows respectively. In this way, the class of each instance is stored in the last column. At any time, two different datasets, training and test sets, may be supplied and the programme returns the predicted classes of instances of the test set. The training set comprises of characterized (labelled) samples whereas the test set is not characterized. In the absence of a test set, the user can specify only the training set to obtain a model for further analysis. Furthermore, the user specifies the desired median-supplement method. If no method is specified, a median-supplement Random Forest is automatically applied. Summary descriptions of arguments of MSclassifier function is described in Table 1. Samples of training and test sets are provided with the package. They are used in the illustration of the MSclassifier in the next section.\n\nIn order to illustrate the use of the package, we use HER2 datasets included in the package. These datasets were obtained from an earlier study that explored the use of machine learning techniques to determine hormone and receptor status phenotypes in breast cancer17. The training data consists of 86 HER2 receptor-negative and 14 HER2 receptor-positive samples while the independent test set consists of 51 HER2 receptor-negative samples and 11 HER2 receptor-positive samples. The illustration shows how to use the MSclassifier after installation.\n\n\n\nData sets. In order to illustrate the performance of the package, we use two real datasets. Particularly, the first data, obtained from previous study17 describes gene expression measurements in breast cancer. In this illustration, median-supplement models are inferred with the MSclassifier package to assign classes to new instances of the test set. In the case of the HER2 data, the class of each instance is the expressed receptor status phenotype while attributes are the relevant gene expression profiles. The data consists of 86 HER2 receptor-negative and 14 HER2 receptor-positive samples. These are samples included in the MSclassifier package.\n\nThe second (larger) dataset was derived from a study that characterized amino acid sequences of human proteins localized in nine cellular compartments21. Code written in LISP was used to determine values of physicochemical properties of proteins known to be primarily localized in the designated subcellular locations were used. Protein properties used are based on the amino acid composition (including hydrophobicity, normalized van der Waals volume, polarity, polarizability, and charge), transitions and distribution as detailed21. For instance, \"PERCENT-R\" is a reference to the percentage of arginine residues in the primary sequence of amino acids of a protein; \"HYDROPHOBICITY-PERCENT-GROUP1\" is a reference to the percentage of polar amino acids in the primary sequence of amino acids (i.e. group 1 amino acids are polar, group 2 amino acids are neutral, and group 3 amino acids are hydrophobic); \"POLARITY-GP1-GP3-TRANSITIONS\" is a reference to the frequency of transitions between low polarity residues (L, I, F, W, C, M, V, and Y) and high polarity residues in a given protein’s primary sequence of amino acids (H, Q, R, K, N, E, D). The data comprised of 2635 instances and 126 attributes. Among the instances, 1589 were associated with (localized in) the plasma-membrane and 1046 were associated with the nucleus22. In its usage to illustrate the package, instances of the dataset were classified as \"nucleus\" and \"plasma-membrane\".\n\nPerformance measures of packages. The performance of each method is determined by its classification rate: proportion of correctly classifying instance given by the ratio of correctly classified test instances to the total number of test instances23. In general, the classification rates agree with measures of accuracies of such classification methods. Higher classification rate of a method indicates that the package has higher chances of making accurate assignments of samples to their respective classes. Therefore, it is desirable to have higher classification rate. For instance, a higher classification rate for classifying receptor status phenotypes in breast cancer indicates the method has high sensitivity for deciphering the particular receptor status. This is because the sensitivity is also a proportion of correctly classified instances among characterized instances as exemplified in unsupervised learning systems24. Furthermore, Mann-Whitney tests are performed to evaluate differences among classification rates of the methods. Both independent and cross-validation testing methods are used to evaluate the packages22. While a 10-fold cross-validation is applied to the HER2 data, a 5-fold cross-validation is applied to the subcellular localization of proteins data22.\n\n\nResults and discussion\n\nIn this experiment, HER2 training and test sets made available in the package were used. It was found that the median-supplement Naive Bayes (MNB) implemented in MSclassifier outperformed all the other methods considered in this case (Figure 2). This was to be expected since the MSclassifier implements median-supplement methods, which have been shown to outperform the traditional machine learning methods17. Higher performance of this package on this test example is the result of the enhanced median-supplement training set from which MSclassifier infers models. Thus the enhancement makes more instances available to train models.\n\nNB is Naive Bayes, RF is Random Forest, MRF is median-supplement Random Forest and MNB median-supplement Naive Bayes. While both MRF and MNB are implemented in MSclassifier, NB is implemented in e1071 and RF is implemented in randomForest packages.\n\nThe classification rates of conventional methods, implemented in existing packages, ranged between 83% and 91%, methods implemented in the MSclassifier had values with minimum of 87% and maximum of 91%. Particularly, it was found that conventional random forest was significantly higher than the Naive Bayes (mean classification rate of 89.83% versus 85.43%, p = 1.48e-11). However, the median-supplement Naive Bayes implemented in MSclassifier achieved the highest classification rates among all the methods22. More importantly, it had significantly higher classifications rates than the random forest method (mean is 90.30% versus 89.83%, p = 8.62e-3). These results are consistent with performance of median-supplement methods on HER2 classifications studied earlier17.\n\nWith regards to the prediction of subcellular localization of proteins, although both MSclassifier and the other packages could attain equally high classification rates (94%) in this test, the minimum classification rate achieved by the median-supplement Naive Bayes was lower compared to the conventional Naive Bayes method (mean of 69% versus 86%, p = 4.55e-14). However, this observation was different in other studies17. The difference is attributable to the differences in data and prediction tasks. Nevertheless, these performances are suboptimal when compared to the random forest-based methods which achieved mean classification rates of 93%22. Specifically, the performances of both the random forest and the median-supplement random forest were statistically indistinguishable (mean of 93.42% versus 93.19%, p = 0.06). These results are indicative that tree-based random forest methods have better performances on larger datasets. However, the superiority of median-supplement methods over several other machine learning methods when applied to predict hormone and HER2 receptor phenotypes underpinned in the literature17. These results demonstrate the potential of MSclassifier to better predict instances of binary classifications problems.\n\n\nConclusion\n\nWe have presented the MSclassifier package to implement median-supplement approaches for machine learning to support medical decisions. The package was shown to decipher HER2 receptor status phenotypes in breast cancer and also predict subcellular localizations of proteins. MSclassifier compares favourably well with existing packages because it implements enhanced methods which offer effective approach to machine learning. Finally, MSclassifier can be installed and run on most operating systems. The sole requirement is the availability of a recent version of R. MSclassifier, steps for installation and other supplementary information are freely available at https://nweb.gimpa.edu.gh/schools/school-of-technology/software/MSclassifier/. Furthermore, the MSclassifier package and every other supporting data for this work have also been made publicly available at https://doi.org/10.5281/zenodo.394667522.\n\n\nSoftware availability\n\nSoftware available from: https://nweb.gimpa.edu.gh/schools/school-of-technology/software/MSclassifier/\n\nSource code available from: https://github.com/esadabor/MSclassifier.git\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.394667522\n\nLicense: GPL-3\n\n\nData availability\n\nDatasets used in Use Case:\n\n- Gene expression measurements in breast cancer, obtained from previous study17. Dataset available here: http://doi.org/10.5281/zenodo.396451425 (permission to reuse this dataset and to republish on Zenodo has been granted by Oxford University Press).\n\n- Characterized amino acid sequences of human proteins localized in nine cellular compartments, obtained from previous study21. Dataset available here: http://doi.org/10.5281/zenodo.396450326. Protein subcellular localisation dataset available here: https://doi.org/10.5281/zenodo.394667522.\n\nZenodo: Supporting information and data for MSclassifier: Median-Supplement model-based Classification tool for automated knowledge discovery, https://doi.org/10.5281/zenodo.394667522.\n\nThis project contains the following extended data:\n\n- Cross-Validation Testing information\n\n- Table S2: Performance of methods on HER2 dataset\n\n- Table S3: Performance of methods on plasma-membrane and nucleus classification dataset", "appendix": "References\n\nHua S, Sun Z: Support vector machine approach for protein subcellular localization prediction. Bioinformatics. 2001; 17(8): 721–728. PubMed Abstract \| Publisher Full Text\n\nDing CH, Dubchak I: Multi-class protein fold recognition using support vector machines and neural networks. Bioinformatics. 2001; 17(4): 349–358. PubMed Abstract \| Publisher Full Text\n\nOnitilo AA, Engel JM, Greenlee RT, et al.: Breast cancer subtypes based on ER/PR and Her2 expression: comparison of clinicopathologic features and survival. Clin Med Res. 2009; 7(1–2): 4–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoss JS, Hatzis C, Symmans WF, et al.: Commercialized multigene predictors of clinical outcome for breast cancer. Oncologist. 2008; 13: 477–493. PubMed Abstract \| Publisher Full Text\n\nNagarajan R, Upreti M: An approach for deciphering patient-specific variations with application to breast cancer molecular expression profiles. J Biomed Inform. 2016; 63: 120–130. PubMed Abstract \| Publisher Full Text\n\nDettling M, Bühlmann P: Boosting for tumor classification with gene expression data . Bioinformatics. 2003; 19(9): 1061–1069. PubMed Abstract \| Publisher Full Text\n\nBen-Dor A, Bruhn L, Friedman N, et al.: Tissue classification with gene expression profiles. J Comput Biol. 2000; 7(3–4): 559–583. PubMed Abstract \| Publisher Full Text\n\nLi R, Campos J, Iida J: A Gene Regulatory Program in Human Breast Cancer. Genetics. 2015; 201(4): 1341–1348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVural S, Wang X, Guda C: Classification of breast cancer patients using somatic mutation profiles and machine learning approaches. BMC Syst Biol. 2016; 10 Suppl 3(Suppl 3): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJames G, Witten D, Hastie T, et al.: An Introduction to Statistical Learning with Applications in R. Springer, New York, 2013; 103. Publisher Full Text\n\nLangley P, Iba W, Thompson K: An analysis of bayesian classifiers. In: Proceedings of Tenth National Conference on Artificial Intelligence. Menlo Park, 1992; 223–228. Reference Source\n\nFriedman N, Geiger D, Goldszmidt M: Bayesian network classifiers. Journal of Machine Learning. 1997; 29: 131–163. Publisher Full Text\n\nHall M, Frank E, Holmes G, et al.: The WEKA Data Mining Software: An Update. SIGKDD Explorations. 2009; 11(1): 10–18. Publisher Full Text\n\nLiaw A, Wiener M: Breiman and Cutler’s Random Forests for Classification and Regression, randoForest package version 4.6-12. 2015. Reference Source\n\nJames G, Witten D, Hastie T, et al.: Data for an Introduction to Statistical Learning with Applications in R, ISLR version 1.2. 2017.\n\nMeyer D, Dimitriadou E, Hornik K, et al.: Misc Functions of the Department of Statistics, Probability Theory Group (Formerly: E1071), TU Wien, e1071 package version 1.6.8. Reference Source\n\nAdabor ES, Acquaah-Mensah GK: Machine learning approaches to decipher hormone and HER2 receptor status phenotypes in breast cancer. Brief Bioinform. 2019; 20(2): 504–514. PubMed Abstract \| Publisher Full Text\n\nWitten IH, Frank E, Hall MA, et al.: Data Mining: Practical Machine Learning Tools and Techniques. Morgan Kaufmann, Burlington, MA, 4th edition, 2017.\n\nBreiman L: Random Forests. Journal of Machine Learning. 2001; 45(1): 5–32.\n\nHastie T, Tibshirani R, Friedman J: The Elements of Statistical Learning. Springer, New York, 2nd edition, 2009. Reference Source\n\nAcquaah-Mensah GK, Leach SM, Guda C: Predicting the subcellular localization of human proteins using machine learning and exploratory data analysis. Genomics Proteomics Bioinformatics. 2006; 4(2): 120–133.\n\nAdabor ES, Acquaah-Mensah GK, Mazandu GK: Supporting information and data for MSclassifier: Median-Supplement model-based classification tool for knowledge discovery (Version 1.0.0). Zenodo. 2020.http://www.doi.org/10.5281/zenodo.3946675\n\nMartinez WL, Martinez AR: Computational Statistics Handbook with MATLAB. Chapman & Hall/CRC, Boca Raton, FL, 2002.\n\nAdabor ES, Acquaah-Mensah GK, Oduro FT: SAGA: a hybrid search algorithm for bayesian network structure learning of transcriptional regulatory networks. J Biomed Inform. 2015; 53: 27–35. PubMed Abstract \| Publisher Full Text\n\nAdabor ES, Acquaah-Mensah GK, Mazandu GK: HER2 data used in the article entitled \"MSclassifier: Median-Supplement model-based Classification tool for automated knowledge discovery\" [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3964514\n\nAdabor ES, Acquaah-Mensah GK, Mazandu GK: Protein Subcellular localization prediction data used in the article entitled \"MSclassifier: Median-Supplement model-based Classification tool for automated knowledge discovery\" [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3964503" }	[ { "id": "74679", "date": "06 Jan 2021", "name": "Gregory Hart", "expertise": [ "Reviewer Expertise My postdoc was focused on machine learning and applying it to cancer diagnosis. The biology/medicine is often over my head by the software/methods are not." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would suggest that (and assume it would be easy) you allow for your package to run regular Random Forest and Naive Bayes in addition to the MS versions. While that functionality is already available in other packages it would be nice to have it all in one place.\nIn addition to the accuracy comparison it would be nice to get a feel for the computational cost of adding MS as well as how the algorithm scales.\nLastly, any reason to only do Random Forest and Naive Bayes? Would there be interest in including other classifiers in the future?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "76091", "date": "06 Jan 2021", "name": "Alper Uzun", "expertise": [ "Reviewer Expertise I focus on using genomics and bioinformatics to understand the genetic architecture of complex diseases. Our research group is managing large scale data (whole-genome genotyping data", "RNA-Seq", "targeted sequencing) and developing bioinformatics methods to identify causal variants. We use machine-learning approach to extract disease associated genes from published biomedical literature. We develop bioinformatics tools for both analyzing and visualizing protein protein interaction networks and in addition we develop tools to visualize variants." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors developed a median-supplement based tool, MSclassifier to automate knowledge discovery from available data sets. They demonstrated that it can be applied to binary classification problems. In this regard they used two datasets to demonstrate their application. The authors implemented the application in R which is freely accessible.\nI recommend the manuscript for indexing with minor revision: - Area Under the Receiver Operator Characteristic (AUROC) is a very useful metric to evaluate models. There are packages available to use AUROC. The manuscript will be benefited by adding this metric into the results.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1114
https://f1000research.com/articles/9-1113/v1	09 Sep 20	{ "type": "Case Report", "title": "Case Report: Ramipril and microscopic colitis; a necessary tool of cardiologists can rarely be devastating for patients", "authors": [ "Saad Hasan", "Haseeb Ur Rahman", "Stephen Hutchison", "Haseeb Ur Rahman", "Stephen Hutchison" ], "abstract": "Angiotensin converting enzyme inhibitors could lead to severe diarrhoea related to microscopic colitis. Few of such cases have been reported before and this serious problem, from a widely used class of drugs in hypertension and heart failure, needs to be more recognised. We describe the case of collagenous colitis related to ramipril use in the following case report. A 74-year-old farmer who had a history of triple vessel coronary artery disease was admitted to district general hospital with non-ST elevation myocardial infarction. He had known alcohol-related chronic pancreatitis with chronic diarrhoea as a complication, which was managed with pancreatic enzyme replacement therapy. However, he developed severe worsening of diarrhoea causing bowel incontinence and nocturnal symptoms during his admission to hospital. The explosive and watery nature of diarrhoea with urgency was so troublesome that it delayed coronary revascularisation and lead him to have significant psychological distress and low mood while nocturnal bowel motions meant he was unable to sleep. He was compliant with his pancreatic enzyme replacement therapy during this period. Infective causes were ruled out by stool microbiology examination and coeliac disease by oesophagogastroscopy and biopsy. It was noticed that he was recently prescribed ramipril that was later stopped as a possible diarrhoea trigger. Diarrhoea started settling immediately and resolved to his baseline within a week. A colonoscopy was performed in the meantime and biopsies demonstrated microscopic colitis (MC). He did not tolerate budesonide well so was stopped. However, a follow-up colonoscopy with biopsy in two months showed resolution of MC.", "keywords": [ "Ramipril", "microscopic colitis", "diarrhoea", "bowel incontinence" ], "content": "Introduction\n\nAngiotensin converting enzyme inhibitors (ACE-I) are widely used in the treatment of hypertension and heart failure and considered as a safe option. They are usually well tolerated in up to 90% of patients1 while diarrhoea is noticed in up to 8%2. We report a case of severe diarrhoea associated with ramipril use.\n\n\nCase report\n\nA 74-year-old man, farmer by profession, admitted from the A&E department where he presented with non-specific upper abdominal pain involving epigastrium and right upper quadrant. He had been drinking excessive alcohol for the previous three days. There was a previous history of alcohol excess, but he had been abstinent for ten years. The patient’s past medical history also included triple vessel coronary artery disease, for which he was awaiting revascularisation planned in three weeks time. He also had alcohol-related liver cirrhosis, chronic pancreatitis, diverticular disease, hypothyroidism and asthma. His regular medications included bisoprolol, lansoprazole, clopidogrel, isosorbide mononitrate, levothyroxine, atorvastatin, spironolactone, citalopram, amlodipine, thiamine and pancreatic enzyme replacement. He lived with his wife and was independent with activities of daily living.\n\nOn examination, the patient’s blood pressure was 79/51 mmHg, pulse 84/min, respiratory rate 17 and he was afebrile. Abdominal and chest examinations were within normal limits. Electrocardiography showed normal sinus rhythm with heart rate of 85 per minute. No ischemic changes were noticed. Computed tomography (CT) scan of abdomen and pelvis did not conclude acute pancreatitis or bowel obstruction but already known diverticular disease.\n\nBlood tests revealed raised high sensitivity troponin of 422 nanogram/litre (ng/L) with subsequent values at 3 hours and 6 hours being 403 ng/l and 385 ng/L respectively (normal range 0 – 34 ng/L). Serum amylase was normal. He was treated as non-ST elevation myocardial infarction with standard therapy. Ramipril was also commenced at 2.5 mg once daily dose. He was then planned for inpatient revascularisation.\n\nIn about 7 to 10 days of starting ramipril, the patient developed severe exacerbation of his ongoing chronic diarrhoea when he started opening his bowels fifteen to twenty times from about four to five times a day. The bowel motions were watery and explosive in nature with associated severe urgency leading to bowel incontinence episodes, which occurred on a daily basis. As a part of work up of this diarrhoea, stool samples to look for Clostridium difficile toxin, salmonella, shigella, Campylobacter, Escherichia coli 0157, ova, cysts, parasites and Cryptosporidium were sent, which all came back as negative. Faecal elastase could not be tested because of excessively watery stool, as artificially low elastase levels are expected in such samples. Oesophagogastroscopy with duodenal biopsies did not explain the cause of diarrhoea.\n\nDuring the period in which diarrhoea was being investigated, which had also delayed the planned cardiac intervention, the patient continued to suffer with severe debilitating symptoms. He was unable to get good sleep at night due to the sudden urges to open bowels, which often lead to incontinence. He would have to change his trousers about two to three times on average every night due to this. It also understandably resulted in severe psychological distress and low mood. This was a cause of concern amongst medical team looking after him and a sense of urgent need to get to the bottom of this issue surfaced.\n\nThe patient was then planned for repeat CT scan of the abdomen and pelvis after specialist gastroenterology input. It did not show any interval changes in comparison to the previous CT scan performed 4 weeks ago. Comments were made on intra-abdominal vasculature noting heavily calcified abdominal aorta. The origins of the superior mesenteric artery and celiac artery, although calcified, were widely patent.\n\nDrugs were reviewed and lansoprazole withheld to no avail. Afterwards ramipril was stopped approximately a month after it was commenced, which was one of the newly started medications after patient’s admission to the hospital, and this immediately lead to some improvement of symptoms. It was followed up by flexible sigmoidoscopy with biopsies taken from distal sigmoid colon, which showed diffuse increase in chronic inflammatory infiltrate. There was also patchy increase in basement membrane thickening and focal epithelial sloughing as features of collagenous colitis. A trial of budesonide was commenced after the biopsy results came back by gastroenterologist at 3 mg three times daily for two weeks with a plan to reduce it to 3 mg twice daily for two weeks and then 3 mg once daily for two weeks and then to stop. Budesonide caused significant abdominal discomfort and nausea, so it was stopped 15 days later, when the patient was seen in the outpatient clinic by gastroenterologist.\n\nThe diarrhoea had continued to settle, and bowel motion frequency had gone back to his pre-hospital admission baseline within one to two weeks of stopping ramipril. He underwent successful staged multi-vessel percutaneous coronary intervention and was discharged home after remaining an inpatient for a total of two months.\n\nThe patient was followed up as outpatient after two months with colonoscopy and biopsies taken again which came back as negative. Histology showed normal mucosal crypt architecture and no active inflammation which meant that MC had resolved.\n\n\nDiscussion\n\nACE-I have been known to cause diarrhoea owing to different aetiologies, including eosinophilic gastroenteritis3 and bowel angioedema4. MC is less commonly associated with ACE-I5.\n\nMC is a known cause of chronic watery diarrhoea, which can also cause faecal incontinence and abdominal pain. On histology, there is increased intraepithelial lymphocytes in lymphocytic colitis with loss of cell architecture and collagen band in sub- epithelium in collagenous colitis6.\n\nDrugs are often implicated in MC. Common ones are proton pump inhibitors, non-steroidal anti-inflammatory drugs, selective serotonin reuptake inhibitors and beta blockers. ACE-Is also increase the risk of MC7.\n\nUsually conservative measures, such as withdrawing the offending drug, can help resolve mild to moderate MC. To reach a conclusion that a drug is responsible, a thorough history should be taken to establish a relationship between starting the drug and development of symptoms6. Obviously, clinical context needs to be taken on board and more common causes should be ruled out first.\n\n\nConclusion\n\nAlthough ACE-Is are less commonly associated with MC, they are extensively used and hence can affect a large number of patients. Cardiologists need to be aware of this potential problem.\n\n\nConsent\n\nWritten informed consent for the publication of the manuscript and any associated images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAmadio P Jr, Amadio PB, Cummings DM: ACE inhibitors. A safe option for hypertension and congestive heart failure. Postgrad Med. 1990; 87(1): 223–6, 231–2, 235–43. Review. PubMed Abstract \| Publisher Full Text\n\nMangrella M, Motola G, Russo F, et al.: [Hospital intensive monitoring of adverse reactions of ACE inhibitors]. Minerva Med. 1998; 89(4): 91–7. Italian. PubMed Abstract\n\nBarak N, Hart J, Sitrin MD: Enalapril-induced eosinophilic gastroenteritis. J Clin Gastroenterol. 2001; 33(2): 157–8. PubMed Abstract \| Publisher Full Text\n\nGabriel JG, Vedantam V, Kapila A, et al.: Recognizing a Rare Phenomenon of Angiotensin-Converting Enzyme Inhibitors: Visceral Angioedema Presenting with Chronic Diarrhea-A Case Report. Perm J. 2018; 22: 17–030. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLucendo AJ: Drug Exposure and the Risk of Microscopic Colitis: A Critical Update. Drugs R D. 2017; 17(1): 79–89. Review. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark T, Cave D, Marshall C: Microscopic colitis: A review of etiology, treatment and refractory disease. World J Gastroenterol. 2015; 21(29): 8804–10. Review. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasclee GM, Coloma PM, Kuipers EJ, et al.: Increased risk of microscopic colitis with use of proton pump inhibitors and non-steroidal anti-inflammatory drugs. Am J Gastroenterol. 2015; 110(5): 749–59. Epub 2015 Apr 28. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75085", "date": "14 Dec 2020", "name": "Mujeeb Ullah Makki", "expertise": [ "Reviewer Expertise Gastroenterology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my opinion, content in this case report is well structured and organized. Appropriate history, examination and investigations were ordered. They have excluded other causes which could contribute to diarrhea in this patient. Author has also provided histological evidence of MC which improved after stopping ACEI.\n\nThere are few points/editions which I will recommend for this case report. Case report is acceptable for indexing with these minor editions/changes:\nComputed tomography (CT) scan of abdomen and pelvis did not conclude acute pancreatitis or bowel obstruction but already known diverticular disease.(please rephrase sentence).\n\nAny explanation for complete resolution of chronic diarrhea which was complicated by chronic pancreatitis? Did ACEI settled chronic pancreatitis and lead to complete resolution of chronic diarrhea or patient was still having few episodes of loose stool but had histologically improved MC?\n\nThe patient was then planned for repeat CT scan of the abdomen and pelvis after specialist gastroenterology input. It did not show any interval changes in comparison to the previous CT scan performed 4 weeks ago(please mention what was CT report 4 weeks ago? Any comments on pancreas?)\n\nPlease recheck references and write according to journal policy.\n\nOnce author has done these changes and discussed these points in his report, it can be accepted for indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "125826", "date": "14 Mar 2022", "name": "Syed Mujtaba Hasnain Nadir", "expertise": [ "Reviewer Expertise Gastroenterology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case is described in adequate detail and common differentials are excluded by relevant investigations. Furthermore, there is a histology proven diagnosis of Microscopic Colitis.\nAs per standard of care, the patient must have been given Aspirin as a treatment for his NSTEMI. Aspirin is a commoner cause of MC. Please specify in your case report whether Aspirin was continued after revascularization? Furthermore, the patient is on a PPI which is also a common cause of MC, was this discontinued?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1113
https://f1000research.com/articles/9-304/v1	28 Apr 20	{ "type": "Software Tool Article", "title": "GFF Utilities: GffRead and GffCompare", "authors": [ "Geo Pertea", "Mihaela Pertea", "Geo Pertea" ], "abstract": "Summary: GTF (Gene Transfer Format) and GFF (General Feature Format) are popular file formats used by bioinformatics programs to represent and exchange information about various genomic features, such as gene and transcript locations and structure. GffRead and GffCompare are open source programs that provide extensive and efficient solutions to manipulate files in a GTF or GFF format. While GffRead can convert, sort, filter, transform, or cluster genomic features, GffCompare can be used to compare and merge different gene annotations. Availability and implementation: GFF utilities are implemented in C++ for Linux and OS X and released as open source under an MIT license (https://github.com/gpertea/gffread, https://github.com/gpertea/gffcompare).", "keywords": [ "gene annotation", "transcriptome analysis", "GTF and GFF file formats" ], "content": "Introduction\n\nMany biomedical research applications employ pipelines to systematically analyze the gene content in a genome. Due to the explosion in transcriptomic data available, these pipelines routinely involve processing enormous amounts of data, and therefore require efficient bioinformatics tools that can handle multiple annotation and sequence files in order to speed up the genomic analysis. Such tools usually exchange and employ information about genes, transcripts or other genomic features in a tab-delimited text file format commonly known as GFF (General Feature Format). This format describes the exact coordinates and attributes of genes, transcripts, and other features such as start and stop codons, coding sequences etc. GFF has many versions, including its latest version GFF31 and the older GTF (Gene Transfer Format), sometimes also referred to as GTF22. While the older GTF format is limited to the representation of gene and transcript locations and their structures, the newer GFF3 format can represent many more genomic features and annotations in a hierarchical fashion. Some transcript data or genome annotation is available from the source in only one of these formats, but an application may require the other format as input. The GffRead and GffCompare utilities can automatically recognize and work with both these file formats seamlessly, extract and select transcript features from rich GFF3 annotation files, perform conversions from one from to another, and even convert files from and to other formats such as BED3 or FASTA4.\n\nAnnotation data from different sources may use different naming conventions for chromosomes and contigs, and GffRead can help with mapping such genomic sequence names and thus converting annotation from one reference naming convention to another. Gene prediction programs and transcript (RNA-Seq) assembly programs usually output their results in GTF or GFF3 format, and in such cases there is often a need to assess the accuracy of the predicted/assembled transcripts. GffCompare is designed to systematically compare one or more sets of transcript predictions to a reference annotation at different levels of granularity (base level, exon level, transcript level etc.), and in the process to provide a way to \"annotate\" such transcript predictions based on their overlaps or proximity to reference annotation transcripts. When multiple transcript files (samples) are provided, GffCompare generates a non-redundant combined set of transcripts, tracking structurally equivalent transcripts across multiple samples and classifying them according to their relationship to reference transcripts.\n\nDue to their efficiency and user-friendly nature, both GffRead and GffCompare have already been used in many bioinformatics projects as integral parts of pipelines for genome annotation5–7, novel gene discoveries and characterizations8–18, gene structure reconstruction accuracy19–21, and gene annotation comparisons22–25 among others. In this paper we provide detailed descriptions of the specific functions provided by our GFF utilities.\n\n\nMethods\n\nBoth our utilities share a code base built around a C++ class called GffObj that implements many of the common GFF parsing and indexing functions. Because the GFF format has no requirements for grouping and sorting of hierarchically linked genomic features (e.g. a transcript feature can have one of its exons at the beginning of the file and another at the end of the file), the parser has to keep transcript data in memory until the whole file is parsed. Feature identifiers (like transcript IDs) are kept in string hashes for fast identification of hierarchical relationship between features. Reference sequence names and GFF attribute names are also stored in global string hashes with numeric IDs associated, while pointers to the genomic feature objects (GffObj) are stored in dynamic arrays sorted by the genomic location such that a binary search can be used for quick overlap verification. The code shared by these utilities also implements functions to test and classify the structural similarities and overlaps between transcripts in the same location on the genome.\n\nGffRead. We initially implemented the GffRead utility as a fast tool for verification, filtering and conversion of the most popular annotation file formats, GTF and GFF3, and for quick extraction of transcript sequences from the genome sequence. With its many features added over time, GffRead is now a complex and versatile tool that can sort, filter, remap and even cluster transcripts into loci (based on exon overlaps) while optionally discarding \"redundant\" transcripts from an input GFF data. Different examples for the command lines used to perform all these functions are offered in the Use Cases section below.\n\nGffRead parses the input records given in GTF, GFF3 or BED format, and stores them into an internal collection of GffObj data structures that can be easily sorted and filtered according to different criteria. For instance, GffRead can output only the subset of the input transcripts that are multi-exonic, or do not belong to pseudogenes (see Table 1 for a complete set of filtering options). Besides conversions between different GFF formats, GffRead has many additional output options (see Table 2). Among these is a user-defined tab-delimited format, with a line for each transcript and the columns defined by a custom list of some of the GFF columns and attributes in the input annotation file. If a genome sequence is provided, GffRead can also generate multiple additional sequence data files in FASTA format such as: (1) a file with the transcript sequences produced by extracting and concatenating all of the exon sequences of each transcript; (2) a file with all the protein-coding sequences in each transcript; or (3) a file with the amino-acid translations of the coding sequence of each transcript. If a FASTA index file (such as the one created by the samtools utility26) is not present in the same directory with the genomic sequence, GffRead will first create one in order to accelerate the retrieval of the specific transcript sequences. If the transcripts in the annotation file have coding sequences (represented as CDS features in the file), GffRead can check their validity and add specific annotations to the output file, indicating if either the START or the STOP codons are missing in these transcripts or if there are in-frame STOP codons.\n\nThe transcript clustering functions of GffRead can group each set of input transcripts into a locus, where all transcripts in a locus are on the same strand, and any two transcripts in that locus have at least one exonic interval overlap. When clustering is enabled, the GFF output will have a new 'locus' feature for each cluster with attributes listing all the transcript IDs (and gene IDs, if available) that belong to that cluster. Optionally, GffRead can identify transcripts that are structurally \"matching\" or \"equivalent\", defined as transcripts that share all their introns, or have more than 80% of their length overlap in the case of single exon transcripts. GffRead can also discard redundant transcripts (either matching or contained within other transcripts) from the output, providing the user with the ability to choose among merging strategies with different levels of stringency when assessing redundancy in such cases.\n\nGffCompare. GffCompare is a generic, standalone tool for merging and tracking transcript structures across multiple samples and comparing them to a reference annotation. Initially written based on the CuffCompare utility program included with the Cufflinks suite27, GffCompare has the following main functions:\n\n1) merge structurally equivalent transcripts and transcript fragments (transfrags) across multiple samples;\n\n2) assess the accuracy of the assembled transcripts from an RNA-seq sample by comparing it to known annotation; and\n\n3) track, annotate, and report all structurally distinct transfrags across multiple samples.\n\nThe last two purposes require the user to provide a known reference annotation file that GffCompare then uses to classify all the transcripts in the input samples according to the reference transcript that they most closely overlap (Figure 1). To assess the accuracy of transcriptome assemblies, GffCompare reports several accuracy metrics previously employed for gene prediction evaluation28. These metrics include sensitivity and precision as well as the number of novel or missed features, and the metrics are computed at various levels (base, exon, intron chain, transcript, or locus). More details about how to obtain the different reports provided by GffCompare can be found in the Use Cases section.\n\nReference exons and transcripts are shown in black, transcripts to be classified are shown in blue, and hashed regions represent repeated regions in the genome. For example, the transcript in blue on the uppermost left panel is labeled “=” because all of its introns precisely match the annotation in black.\n\nSome pipelines can produce a very large number of transcripts that need to be evaluated; e.g. when merging the transcript assemblies from tens or hundreds of RNA-seq experiments. Because GffCompare always loads the entire transcript data into memory for clustering, running GffCompare on such large GTF/GFF files could be slow and memory intensive. One may be interested only in how these transcripts overlap the reference annotation, and then only wish to further analyze those transcripts that have specific types of overlaps with the reference annotation transcripts. GffCompare also only produces the best match of a transcript to a reference annotation, but for each transcript we might want to know all possible reference matches. In order to address these needs, we built TrMap (\"Transcript vs. reference Mapping\"), a program that we distribute along with GffCompare and that was designed to avoid using a large amount of memory by streaming the input transcript data. TrMap first loads the reference annotation into an interval tree data structure29, and then for each query transcript it reports all the reference transcripts that overlap it, along with their overlap classification codes. These are the same classification codes described in Figure 1, with the exception of codes p, r, and u which are reserved for transcripts that do not overlap reference transcripts and represent transcripts that are single exon and nearby genes (p), repeats outside of genes (r), and intergenic (u).\n\nThis software can be built on a Linux or MacOS system with no other library dependencies. A GNU C++ compiler (g++) is required for compilation (on Linux at least g++ version 4.5 is required). The release packages on Github include precompiled binaries for Linux and MacOS that can be used directly instead of having to build the programs from source. Linux compatibility goes back as far as RedHat Enterprise Linux 5, while on MacOS the programs can run on systems as old as OS X 10.7 (Lion). We also provide the gffread, gffcompare and trmap executables. These are supposed to be used as command line programs, in a Linux/Unix shell, in a terminal or a script. All programs take GFF3, GTF or BED files as their (main) input files. Both packages require the shared code provided in GCLib (https://github.com/gpertea/gclib30).\n\n\nUse cases\n\nThe following sections illustrate different use cases for our utilities. All the files used in the examples below as well as their output are included in the gffread and gffcompare Github release packages (https://github.com/gpertea/gffread31, https://github.com/gpertea/gffcompare32) so that the interested user can try these examples for themselves.\n\nThe program GffRead can be used to validate, filter, convert and perform various other operations on GFF files (see Table 1 and Table 2 for the full list of usage options). For instance, GffRead can be used to simply read an annotation file in a GFF format, and print it in either GFF3 (default) or GTF2 format (with the -T option), while optionally discarding any non-essential attributes, and fixing some potential issues with the input file. The command line for such a quick cleanup and a quick visual inspection of a given GFF file would be:\n\n\n\nThis will show the minimalist GFF3 re-formatting of the transcript records found in the input file (annotation.gff in this example) which could be given in either GFF3 or GTF2 format. The -E option directs GffRead to \"expose\" (display warnings about) any potential issues encountered while parsing the input file.\n\nIn order to obtain the GTF2 version of the same transcripts, the -T option should be added:\n\n\n\nGffRead can be used to generate a FASTA file with the DNA sequences for all transcripts in a GFF file. For this operation a fasta file with the genomic sequences have to be provided as well. This can be accomplished with a command line like this:\n\n\n\nThe file genome.fa in this example would be a multi-fasta file with the chromosome/contig sequences of the target genome. This also requires that every contig or chromosome name found in the 1st column of the input GFF file (annotation.gtf in this example) must have a corresponding sequence entry in the genome.fa file.\n\nThe program GffCompare can be used to compare, merge, annotate and estimate accuracy of one or more GFF files (the “query” files), when compared with a reference annotation (also provided as GFF). A basic command line to compare a list of GTF files to a reference annotation file is:\n\n\n\nThe reference annotation is specified in the annotation.gff file and transcripts.gtf represents the query file (more than one query file can be provided). Unless the -o option was provided, the output will be found in multiple files with the prefix “gffcmp.”. A list of the more important options for the GffCompare utility is provided in Table 3.\n\nGffCompare can be used to assess the accuracy of transcriptome assemblies produced by programs like StringTie19 in respect to a known reference annotation. To this end, GffCompare reports various statistics related to the accuracy of the input transcripts compared to the reference annotation in the <outprefix>.stats file. Among these statistics are sensitivity and precision values computed at various levels (base, exon, intron chain, transcript, locus), which are calculated as:\n\nwhere TP stands for \"true positives\", or query features (bases, exons, introns, transcripts, etc.) that agree with the corresponding reference annotation features; FN means \"false negatives\", i.e. features that are found in the reference annotation but are not present in the input data; FP (“false positives”) are features present in the input data but not confirmed by any reference annotation data. Notice that FP+TP amounts to the whole input set of query features in the input file. If multiple query GTF/GFF files are given as input, these metrics are computed separately for each sample.\n\nSensitivity and Precision values are estimated at various levels, which are largely an increasingly stringent way of evaluating the accuracy/correctness of a set of predicted transcripts (transfrags), when compared to the reference annotation provided with the -r option. The six different levels that GffCompare uses are described below:\n\n1) Base level. At the base level, TP represents the number of exon bases that are reported at the same coordinate on both the query transcripts and any reference transcript, FN is the number of bases in reference data exons that are not covered at all by any of the query exons, and FP is the number of bases which are covered by predicted transcripts' exons but not covered by any reference transcript exons.\n\n2) Exon level. We define the TP, FN, and FP values at the exon level similar to the base level, but now the unit of comparison is the exon interval on the genome, i.e. if an exon of the predicted transcript overlaps and matches the boundaries of a reference transcript exon, then it is counted as a TP.\n\n3) Intron Level. Intron intervals are the units that are matched at the intron level, therefore each intron of the predicted transcript is checked against any introns of the reference transcripts in the same region and if there is one with the same exact start-end coordinates, it is counted as a TP.\n\n4) Intron chain level. At this level we count as a TP any query transcript for which all of its introns can be found, with the same exact intron coordinates as in a reference transcript that has the same number of introns. Matching all the introns at this level implies that all the internal exons also match, but this might not be true for the external boundaries of the terminal exons.\n\n5) Transcript level. Note that intron chain level values are calculated only by looking at multi-exon transcripts, so it completely ignores the single-exon transcripts, which can be quite numerous in a RNA-Seq experiment (possibly due to a lot of transcriptional and alignment noise). The transcript level considers single-exons as well. A TP at this level is defined as a full exon chain match between the predicted transcript and a reference transcript, where all internal exons match and the outer boundaries of the terminal query exons can only slightly differ from the reference exons (with at most 100 bases by default). Also GffCompare considers single-exon transcripts as matching an overlapping single-exon reference transcript if there is a significant overlap between the two (more than 80% of the longer transcript by default).\n\n6) Locus level. At this level GffCompare considers that an observed locus, defined as a cluster of exon-overlapping transcripts, matches a similarly built reference locus if at least one predicted transcript has a transcript level match with a reference transcript in the corresponding reference locus.\n\nOther statistics reported by GffCompare are the number of missed or novel exons, missed or novel introns and missed or novel loci. Note that in order to properly evaluate precision and sensitivity when comparing two sets of transcripts, special care must be taken for duplicated (or redundant) entries within each set. GffCompare uses different levels of stringency of what to consider duplicated transcripts, depending on the option given in its input (see options -D, -S, -C, -A, -X in Table 3).\n\nWhen multiple input GTF/GFF files are provided, GffCompare reports a GTF file named <outprefix>.combined.gtf containing the union of all transfrags in each sample. If a transfrag with the same exact intron chain is present in both samples, it is thus reported only once in the output file.\n\nThe \"super-locus\" concept\n\nA super-locus is a region of the genome where predicted transcripts and reference transcripts get clustered together by exon overlaps. When multiple GFF files with are provided as input to GffCompare, this clustering is performed across all the input files. Due to the transitive nature of this clustering, these super-loci can occasionally get very large, sometimes merging a few distinct reference gene regions together, especially if there is a lot of transcription or alignment noise around the individual gene regions. For each super-locus, GffCompare assigns a unique identifier with the XLOC_ prefix.\n\nOne can run GffCompare on a single GTF/GFF input file using with the -r option (which provides a reference annotation), and without any specific options to remove redundant transfrags (such as the -D, -S, -C, -A, -X options) to produce an GTF file called <outprefix>.annotated.gtf that contains all the input transcripts annotated with several additional attributes: xloc, tss_id, cmp_ref, and class_code. The xloc attribute specifies the super-locus a specific transcript belongs to. The tss_id attribute uniquely identifies the transcription start for that transcipt, and using this value the user can quickly see which transcripts use the same transcription start, or how many different transcription starts are present in a locus. The cmp_ref gives the closest reference transcript (where applicable), while the relationship to this reference transcripts is given by the class_code attribute. The possible values for the class_code attribute are listed in Table 4.\n\nGffCompare can also be used to track all transcripts that are structurally equivalent among the different input files. GffCompare considers transcripts matching (or structurally equivalent) if all their introns are identical. Note that matching transcripts are allowed to differ on the length of the first and last exons, since these lengths can usually vary across samples for the same biological transcript. A list of all matching transcripts is reported in a file called <outprefix>.tracking in which each row represents a transcript. The first column in this file represents a unique id assigned to that transcripts. The second file represents the super-locus that contains that transcript. If GffCompare was run with the -r option, the 3rd and 4th columns contain the reference annotation transcript that was found to be closest to the transcript and the classification code (as specified by Table 4) that specifies the relationship between these two transcripts, respectively. The rest of the columns show the corresponding transcript from each input file in order. An example and a brief description for each column are given in Table 5.\n\nIn order to quickly see which reference transcripts match which transcripts from a sample file, two other files, called <outprefix>.<input_file>.refmap and <outprefix>.<input_file>.tmap are also created for each query <input_file>. The <outprefix>.<input_file>.refmap file is a tab-delimited file that has a row for each reference transcript that either fully or partially matches a transcript from the given input file. Its columns are described in Table 6. Conversely, the <outprefix>.<input_file>.tmap file has a row for each input transcript, while the columns in this file (as detailed in Table 7) describe the most closely matching reference transcript for that transcript.\n\nThe utility TrMap was designed for large scale overlap analysis of streaming transcript prediction data (millions of transcripts) with a reference annotation data set. Particularly, TrMap performs detection and classification of all the overlaps found between the streamed transcripts and the reference annotation transcripts.\n\nThe program trmap is distributed with GffCompare and a basic usage for it is shown below:\n\n\n\nThe default output is a pseudo-FASTA format showing a record for each query transcript that had at least one reference overlap. The query transcript is shown in the header of the record, with space delimited fields showing the genomic location and strand. Each reference overlap follows, as a line with tab delimited fields, starting with the \"classification code\" for the overlap and then providing the genomic location of the transcript (chromosome, strand, transcript-start, transcript-end, reference_transcriptID, exons).\n\nThe exons for both query and reference transcripts are shown as comma delimited lists of intervals. These are all 1-based coordinates like in the GTF/GFF format (even when input is BED).\n\n\nConclusions\n\nGffRead and GffCompare provide comprehensive features for converting, filtering, manipulating, clustering, combining and classifying transcript data from GFF files. Due to their ability to process hundreds or even thousands of transcript files at the same time, they can be used for large scale genome data analysis by many bioinformatics analysis pipelines.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nThe source packages for the latest release, with precompiled binaries and online manuals, are available at http://ccb.jhu.edu/software/stringtie/gff.shtml.\n\nSource code available from: https://github.com/gpertea/gffread\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.375568631\n\nLicense: MIT\n\nSource code available from: https://github.com/gpertea/gffcompare\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.375571532\n\nLicense: MIT\n\nSource code available from: https://github.com/gpertea/gclib\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.375874130\n\nLicense: Artistic License 2.0", "appendix": "Acknowledgments\n\nAuthors want to thank Cole Trapnell for direction and guidance leading to the writing of CuffCompare - the initial version of GffCompare. They also thank Steven Salzberg for proofreading the manuscript.\n\n\nReferences\n\nGeneric Feature Format Version 3 (GFF3). Reference Source\n\nGTF2 format (Revised Ensembl GTF). Reference Source\n\nBED File Format. Reference Source\n\nWhat is FASTA format? Reference Source\n\nPertea M, Shumate A, Pertea G, et al.: CHESS: a new human gene catalog curated from thousands of large-scale RNA sequencing experiments reveals extensive transcriptional noise. Genome Biol. 2018; 19(1): 208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshimura J, Ichikawa K, Shoura MJ, et al.: Recompleting the Caenorhabditis elegans genome. Genome Res. 2019; 29(6): 1009–1022. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimin AV, Cornish AS, Maudhoo MD, et al.: A new rhesus macaque assembly and annotation for next-generation sequencing analyses. Biol Direct. 2014; 9(1): 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoschiero C, Lundquist PK, Roy S, et al.: Identification and Functional Investigation of Genome-Encoded, Small, Secreted Peptides in Plants. Curr Protoc Plant Biol. 2019; 4(3): e20098. PubMed Abstract \| Publisher Full Text\n\nChang TC, Mendell JT: High-Throughput Characterization of Primary microRNA Transcripts. Methods Mol Biol. 2018; 1823: 1–9. PubMed Abstract \| Publisher Full Text\n\nHan L, Li L, Muehlbauer GJ, et al.: RNA Isolation and Analysis of LncRNAs from Gametophytes of Maize. Methods Mol Biol. 2019; 1933: 67–86. PubMed Abstract \| Publisher Full Text\n\nJain P, Sharma V, Dubey H, et al.: Identification of long non-coding RNA in rice lines resistant to Rice blast pathogen Maganaporthe oryzae. Bioinformation. 2017; 13(8): 249–255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Ma Y, Yin K, et al.: Long non-coding and coding RNA profiling using strand-specific RNA-seq in human hypertrophic cardiomyopathy. Sci Data. 2019; 6(1): 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLv Y, Liang Z, Ge M, et al.: Genome-wide identification and functional prediction of nitrogen-responsive intergenic and intronic long non-coding RNAs in maize (Zea mays L.). BMC Genomics. 2016; 17: 350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSablok G, Sun K, Sun H: NAMS: Noncoding Assessment of long RNAs in Magnoliophyta Species. Methods Mol Biol. 2019; 1933: 257–264. PubMed Abstract \| Publisher Full Text\n\nSong F, Wang L, Zhu W, et al.: Long noncoding RNA and mRNA expression profiles following igf3 knockdown in common carp, Cyprinus carpio. Sci Data. 2019; 6: 190024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSreenivasamurthy SK, Madugundu AK, Patil AH, et al.: Mosquito-Borne Diseases and Omics: Tissue-Restricted Expression and Alternative Splicing Revealed by Transcriptome Profiling of Anopheles stephensi. OMICS. 2017; 21(8): 488–497. PubMed Abstract \| Publisher Full Text\n\nStroehlein AJ, Young ND, Korhonen PK, et al.: The small RNA complement of adult Schistosoma haematobium. PLoS Negl Trop Dis. 2018; 12(5): e0006535. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun HX, Chua NH: Bioinformatics Approaches to Studying Plant Long Noncoding RNAs (lncRNAs): Identification and Functional Interpretation of lncRNAs from RNA-Seq Data Sets. Methods Mol Biol. 2019; 1933: 197–205. PubMed Abstract \| Publisher Full Text\n\nKovaka S, Zimin AV, Pertea GM, et al.: Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol. 2019; 20(1): 278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManchanda N, Portwood JL 2nd, Woodhouse MR, et al.: GenomeQC: a quality assessment tool for genome assemblies and gene structure annotations. BMC Genomics. 2020; 21(1): 193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao M, Kingsford C: Accurate assembly of transcripts through phase-preserving graph decomposition. Nat Biotechnol. 2017; 35(12): 1167–1169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzlan A, Obeidat SM, Yunus MA, et al.: Systematic identification and characterization of Aedes aegypti long noncoding RNAs (lncRNAs). Sci Rep. 2019; 9(1): 12147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChow EY, Zhang J, Qin H, et al.: Characterization of Hepatocellular Carcinoma Cell Lines Using a Fractionation-Then-Sequencing Approach Reveals Nuclear-Enriched HCC-Associated lncRNAs. Front Genet. 2019; 10: 1081. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiknafs YS, Pandian B, Iyer HK, et al.: TACO produces robust multisample transcriptome assemblies from RNA-seq. Nat Methods. 2017; 14(1): 68–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVadnal J, Granger OG, Ratnappan R, et al.: Refined ab initio gene predictions of Heterorhabditis bacteriophora using RNA-seq. Int J Parasitol. 2018; 48(8): 585–590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract \| Publisher Full Text \| Free 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): 511–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurset M, Guigó R: Evaluation of gene structure prediction programs. Genomics. 1996; 34(3): 353–67. PubMed Abstract \| Publisher Full Text\n\nCormen T, Leiserson C, Rivest R, et al.: Introduction to Algorithms, 3rd Edition. The MIT Press. 2009. Reference Source\n\nPertea G: gpertea/gclib: v0.11.9 (Version v0.11.9). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3758741\n\nPertea G, Rozenberg A: gpertea/gffread: v0.11.8 (Version v0.11.8). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3755686\n\nPertea G, Kirchner R: gpertea/gffcompare: v0.11.6 (Version v0.11.6). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3755715" }	[ { "id": "62865", "date": "12 May 2020", "name": "Andreas Stroehlein", "expertise": [ "Reviewer Expertise Genomics", "Bioinformatics", "Parasitology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a concise and useful summary of two widely-used tools for genomic analysis, GffRead and GffCompare. The paper is written well and provides clear guidelines and examples on how to use those tools. I only have very minor suggestions and overall have no objections to this work being approved for indexing:\n\nRef. 1: This is a great reference that describes the GFF3 format in detail. Nevertheless, I think the present paper would benefit from a concise, technical summary of the format in the Introduction section and maybe a sentence about issues surrounding different definitions of the format and the availability of GFF3 validators.\n\nPage 2, first paragraph, “rich GFF3 annotation files”: Does this mean “data-rich”? Maybe reword to clarify. What does “rich” entail?\n\nThe same paragraph, “perform conversions from one from to another”: I think this is meant to read” from one format to another”.\n\nPage 6, “Basic usage example of the GffCompare utility” This paragraph states that query input can be GFF or GTF (text says GFF and example uses a GTF file), but Table 3 states for the -i option “provide a text file with a list of (query) GTF files”. Could you please clarify whether the -i option also accepts GFF files (similar to the description for -r option)?\n\nPage 8, “The “super-locus” concept”, “When multiple GFF files with are provided as input to GffCompare”: This seems to be a typo, or there is a word missing in the sentence? Remove “with”?\n\nPage 8, “Annotating transcripts with GffCompare”, 3rd line: Change “produce an GTF file called” to “produce a GTF file called”.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5890", "date": "10 Sep 2020", "name": "Mihaela Pertea", "role": "Author Response", "response": "Thank you for reading our manuscript and appreciate all your comments. This new version contains minor text edits to address observed typos and clarify the meaning of some wordings as suggested." } ] }, { "id": "62863", "date": "13 May 2020", "name": "Michael I. Love", "expertise": [ "Reviewer Expertise RNA-seq bioinformatics", "biostatistics", "open source scientific software development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis software tool article motivates and describes the use of GffRead and GffCompare, two utility programs for manipulating GFF and GTF files, including a comparison of one set of transcripts to a reference. The article is well written and clearly describes the use cases for the software. I have downloaded the two programs onto a laptop running OS X and have used the precompiled Mac binaries as well as building the tools from source. I ran GffRead to change a Gencode GTF file to GFF, and to extract spliced transcript sequences from a genome FASTA. I ran GffCompare to compare two Gencode releases of a GTF file. The programs were easy to run, took at most a few minutes on a laptop (for comparison), and are both well documented. I have only minor comments (spelling/formatting):\nRNA-Seq is used twice, while RNA-seq is used once.\n\nTable 1 has \"(strand )\" with a space (angle brackets not allowed in the report, so I use parens).\n\nTable 2 row two, column two has a line break.\n\nTransfrags are defined in this article but not in the online documentation.\n\nUnder \"Use Cases\", there is a \"fasta\" not capitalized.\n\nUnder \"Transcript accuracy estimation with GffCompare\", perhaps \"with respect to a known reference annotation\".\n\nUnder \"Annotating transcripts with GffCompare\", should be \"this reference transcript\".\n\nUnder \"Tracking transcripts with GffCompare\", \"(input_file)\" is italicized.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5891", "date": "10 Sep 2020", "name": "Mihaela Pertea", "role": "Author Response", "response": "Thank you for reading our manuscript and appreciate all your comments. This new version contains minor text edits to address observed typos and clarify the meaning of some wordings as suggested." } ] }, { "id": "62867", "date": "02 Jun 2020", "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 describe the GFF Utilities suite, which is comprised of the GffRead, GffCompare, and trmap utilities. These tools long predate the article and have been in relatively widespread use for some time. For example, GffRead is commonly used to convert files to proper GFF3 format (and to record or catalog formatting errors), as well as to extract the sequences of transcripts (or related features) from a GFF file and associated genome. GffCompare is commonly used as a standard tool in the evaluation of genome-guided assembly, to compare predicted transcripts against an annotation, as well as to merge transcriptome assemblies across multiple samples while discarding duplicates. It is valuable, I think, to have an article describing these tools that can also be cited as they are used and built upon in the community. I found the visual description of the different transcript classification codes (Figure 1) particularly useful. I was able to download and build the source easily according to the instructions in the respective README files. In addition to the source and pre-compiled builds, it is worth noting that both GffRead and GffCompare are available through Bioconda, though I am not sure if the authors of the current manuscript are the Bioconda maintainers for these packages.\n\nOverall, the article is clear and describes the tools well. I have only very minor comments.\n\npg 6: \"produced by programs like StringTie in respect to\" -> \"produced by programs like StringTie with respect to\"\n\nI tried compiling both GffRead and GffCompare under multiple versions of G++. With G++5.5.0, everything worked well with only two warnings (a strict overflow warning in gfoAdd() and an unused variable warning for isCodonTableReady). However, when I compiled with G++9.2.0, there were multiple warnings in the GVec class about potentially incorrect uses of memset, which should actually not be an issue as the uses are present in a branch that first checks that the type being set is \"primitive\". Peeking into the Makefile, I noticed that a particular flag is set to disable this warning under newer versions of G++ (presumably, it does not occur in earlier versions). However, in the line of the makefile that checks this, `g++` is hard-coded, rather than making use of the potentially modified `${CXX}` variable. Changing `g++` to `${CXX}` in the lines of the makefiles defining the `GCCV8` variable resolved this behavior.\n\nFinally, this is not an issue with the current manuscript, but rather a general note. In addition to powering the GffRead and GffCompare tools, the Gff utilities source code provides a powerful programmatic interface (API) to parse, manipulate and process GFF and GTF files in the C++ language. If the authors feel it appropriate, a similar note or article describing the features and capabilities of that library may be of interest to a related (though admittedly smaller) part of the community.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5892", "date": "10 Sep 2020", "name": "Mihaela Pertea", "role": "Author Response", "response": "Thank you for reading our manuscript and appreciate all your comments. This new version contains minor text edits to address observed typos and clarify the meaning of some wordings as suggested. Thank you for the compiling suggestions too. We will make sure to update our makefile." } ] } ]	1	https://f1000research.com/articles/9-304
https://f1000research.com/articles/9-283/v1	23 Apr 20	{ "type": "Brief Report", "title": "Predicting the evolution and control of the COVID-19 pandemic in Portugal", "authors": [ "Ricardo J. Pais", "Nuno Taveira" ], "abstract": "Coronavirus disease 2019 (COVID-19) is a worldwide pandemic that has been affecting Portugal since 2 March 2020. The Portuguese government has been making efforts to contradict the exponential growth through social isolation measures. We have developed a mathematical model to predict the impact of such measures in the number of infected cases and peak of infection. We estimate the peak to be around 2 million infected cases by the beginning of May if no additional measures are taken. The model shows that current measures effectively isolated 25-30% of the population, contributing to some reduction on the infection peak. Importantly, our simulations show that the infection burden can be further reduced with higher isolation degree, providing information for a second intervention.", "keywords": [ "COVID-19", "Pandemic Control", "Predictive modeling", "Simulation", "Social Isolation", "Mathematical model" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is already considered a world pandemic which is starting to have dramatic effects in Europe, where, as of 27 of March, 265,421 cases have been reported1,2. COVID-19 infection in Portugal has been growing exponentially with an average rate of 34±13% new cases per day from 2 March and is far from reaching the peak by the end of March. As of March 27, 4268 infection cases and 76 deaths have been reported2. The highest infection burden is found in Porto (317 cases, 7.4%) and in Lisbon (284 cases, 6.7%) but the disease is present throughout the entire country. As in other countries, infection occurs mostly in individuals’ with ≥40 years of age (71.9% males; 69.3% females). Death occurs mostly in males (64.5%) all with ≥50 years of age.\n\nPredictive models estimate that the peak of COVID-19 infection globally will be between mid-April and May, with an estimated total of 48 million people infected3. As with most other countries, the Portuguese national health care system cannot deal with the increasing demand of care due to limited ventilators and care units3. Therefore, the Portuguese government together with the National Health Directorate (DGS) declared a state of emergency and adopted interventive populational measures (IM) on 18 March 2020 in an attempt to drop the peak of infections even if at the cost of prolonging the infection time. These measures are based on the isolation of people at home, social distancing and adopting protective antiseptic policies. Most forecasting models are based on the number of cases reported and do not take into account the effects of these government-imposed measures and behavioral change. Thus, how these measures impact the evolution of the COVID-19 infection and can prevent the expansion of the epidemic is unknown. Recently published mathematical modelling studies of COVID-19 transmission have already provided useful insights that can be used to guide public health measures and resource allocation to better control this pandemic4,5. However, most parameters of statistical models have been estimated with high degree of uncertainty, resulting in predictions with wide intervals of confidence4. Compartmental models such as susceptible, infected and resistant (SIR) models are deterministic approaches that have been successful in describing the dynamics of virus infection in populations, including COVID-195,6. Here, we provide a simple SI model that describe the dynamics of transition of COVID-19 in Portugal during the first 21 days and predicts the impact of isolation measures towards the expected peak of infection.\n\n\nMethods\n\nBasic transmission dynamics of COVID-19 was modelled using a simple mathematical model based on a system of two ordinary differential equations (ODE) developed specifically for this purpose (Equation 1 and Equation 2). The equations reflect the number of people infected (I) and susceptible (S) to infection per unit of time (dI/dt and dS/dt). In this model, we accounted for the reported average time of duration of infection (τ) of 14 days4,7. The model was calibrated by adjusting the rate constant (k) to approximate the total infection value reported by the DGS at 17 March. No further fitting was performed in this model. The effect of isolating different fractions of the population was modelled through the variation of parameter α in Equation 1 and Equation 2. We assumed that protective measures were 99% effective, accounted through model parameter β. The ODEs were encoded and solved using PLAS software version 1.2.0.120, where a series of simulations were carried scanning various values of the α parameter8. Simulations were carried with the initial two cases reported by the DGS and considering only the population of the grand Lisbon and Porto areas (total of 6.5 x 106) since they represent most of the susceptible population (see Figure 2). Further analysis, computations and plots were conducted using Python 3 in the Jupiter Notebook ipython 7.8.0 programing environment under Anaconda distribution version 4.7.12. Data regarding the daily evolution of number of total infected in Portugal by COVID-19 was collected from the DGS web site (https://covid19.min-saude.pt/ponto-de-situacao-atual-em-portugal/) from 2 to 27 March 2020 (see Source data, Table S1)9. The model is available as Extended data10.\n\n\nResults and discussion\n\nSimulation of the first 18 days with our model was able to describe the exponential increase of the number of confirmed cases reported by the DGS between 2 and 18 March 2020 (Figure 1). The predicted peak time for this scenario was 49 days which would be by the 21 of April. This is within the estimated range predicted by statistical modelling of US, Italy and Korea scenarios3. Further, the predicted numbers of cases for the end of March if no measures were taken would be around 42,000. This is also in agreement with the number released by the DGS to the social media based on statistical modelling. Thus, the model presented here is consistent with the forecasting made by conventional models, reinforcing the confidence on our model capacity to generate predictions. Importantly, our results show that the isolation measures had an immediate impact on diminishing the exponential increase of the number of infected cases and this depends on the percentage of the population that is isolated (Figure 2). This is evident by the increasing deviation of the reported number of cases relative to the unperturbed simulation (0%) with time. The evolution of the number of cases reported by DGS between 18 and 25 March fit between the simulation curves corresponding to 20% and 30% population isolation. This suggests that the estimated percentage of the population that have been effectively isolated is between these percentages. Interestingly, the results shows a slight gradual shift of the obedience towards isolation with time, starting from a predicted 20% isolation and reaching 30% isolation at March 27. From simulations, we identify other intervals (e.g. 50–60% and 70–75%) that suggest further isolation percentages may be more effective and still withing a plausible of pandemic time. Based on the fraction of hospitalized and mortality reported by the DGS on 27 March 2020, together with our model predictions, we computed several infection indicators for these intervals (Table 1).\n\nLeft, the distribution of confirmed cases on 19 March are depicted in the map. Right, evolution of the cases between 2 and 19 of March. Lines indicate simulation using the mathematical model and blue dots correspond to the confirmed cases reported by DGS.\n\nAbove, predicted total infected population in the month of March. The starting of the isolation measure is depicted by IM and the arrow indicates the time of change. Below, Predicted peak of infection.\n\nOur model analysis indicates that current government-mandated measures may shift the expected peak of infections towards the beginning of May and can cause a substantial reduction in the infection numbers (Figure 2, Table 1). Thus, the predicted peak in the number of cases without any isolation measures would be around 2 million, whereas the intervention measures have decreased it to around one half of the cases (Table 1). In addition, the estimated reduction of hospitalized patients and death cases on peak would be predicted around 59,000 and 12,000 people, respectively. Our simulations also indicate that the peak of infection can be further reduced by ~3.5-fold with a delay to November if 70% of the population were isolated at home and follows the government recommendations. For higher percentages of isolation (>75%), our model predicts a substantial reduction in the number of infections and delay of peaks, stopping the COVID-19 epidemic. These solutions would result in much less total mortality and hospitalization requirements on peak in comparison to the current trend (Table 1, Figure 2. Meanwhile, this comes with the burden of prolonging the time of pandemic to almost a year, which can be economically unbearable. In alternative, further isolation to 50–60% of the population may be also a solution that substantially reduce most pandemic indicators and shifts the ending of the pandemic to September, with the peak between June and July. The results obtained during simulations are available as Extended data, Table S29.\n\nAlthough our model precisely described the exponential curve and explains the shift in the temporal evolution of DGS data, it has limitations that may compromise the exact values of predictions. The fact that we only assume two compartments (susceptible and infected) considering the main populated cities (Lisbon and Porto) as one is huge approximation that neglects regional dynamics. Thus, the model is just an approximation that reflects an average trend and may fail to explain regional observations. In this model we also neglected many important parameters of infection transmission such as age groups, types of social interactions, contact dependent probability, and viral load dependent probability10. The inclusion of these parameters would definitely make the model more realistic. However, this data is not available for the Portuguese case and these models require accurate processing of data curation for suitable validation. We have bypassed these limitations by aggregating all of these parameters into one constant, which was fitted to the available data. Overall, the predictions shown here should be taken as semi-quantitative estimates within an upper and lower case-scenarios.\n\n\nConclusions\n\nIn this work we demonstrate the potential of modelling COVID-19 dynamics of infection as a useful support tool for predicting the impact of corrective measures. Government-mandated measures to isolate the Portuguese population at home effectively prevented COVID-19 from reaching dramatic numbers in Portugal but still can be substantially improved to reduce the infection peak Our estimates may help guiding additional measures to control the COVID-19 epidemic in Portugal.\n\n\nData availability\n\nFigshare: Modelling COVID-19 evolution and control in Portugal: Code and data from 2 to 27 of March 2020. https://doi.org/10.6084/m9.figshare.12136446.v19.\n\nThis project contains the following source data used in the present study:\n\nTable S1 (CSV). (The number of confirmed cases in Portugal officially reported by the DGS.)\n\nFigshare: Modelling COVID-19 evolution and control in Portugal: Code and data from 2 to 27 of March 2020. https://doi.org/10.6084/m9.figshare.12136446.v19.\n\nThis project contains the following extended data:\n\nmodel_code (TXT). (Code used for the model.)\n\nTable S2 (CSV). (Results obtained during simulation.)\n\nPython-code (MD). (Python code used with this model.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nEng. Pedro Fernandes for the critical discussions’ and advices on the modelling approach. The CiiEM (Egas Moniz) for hosting institution and supporting publication fees.\n\n\nReferences\n\nWHO: Coronavirus disease 2019 (COVID-19) Situation Report - 62. 2020. Reference Source\n\nWHO: Coronavirus disease 2019 (COVID-19) Situation Report - 67. 2020. Reference Source\n\nChristopher JLM: Forecasting COVID-19 impact on hospital bed-days, ICU-days, ventilator-days and deaths by US state in the next 4 months. 2020. Reference Source\n\nKucharski AJ, Russell TW, Diamond C, et al.: Early dynamics of transmission and control of COVID-19: a mathematical modelling study. Lancet Infect Dis. 2020; 3099: 1–7, pii: S1473-3099(20)30144-4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen TM, Rui J, Wang QP, et al.: A mathematical model for simulating the phase-based transmissibility of a novel coronavirus. Infect Dis Poverty. Infectious Diseases of Poverty; 2020; 9(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuppert A, Katriel G: Mathematical modelling and prediction in infectious disease epidemiology. Clin Microbiol Infect. European Society of Clinical Infectious Diseases; 2013; 19(11): 999–1005. PubMed Abstract \| Publisher Full Text\n\nLi Q, Guan X, Wu P, et al.: Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N Engl J Med. 2020; 382(13): 1199–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEberhard OV: Computational Analysis of Biochemical Systems: A Practical Guide for Biochemists and Molecular Biologists. Cambridge University Press; 2000.\n\nPais RJ, Taveira Nuno: Modelling COVID-19 evolution and control in Portugal: Code and data from 2 to 27 of March 2020. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12136446.v1\n\nDel Valle SY, Hyman JM, Hethcote HW, et al.: Mixing patterns between age groups in social networks. Soc Networks. 2007; 29: 539–54. Publisher Full Text" }	[ { "id": "63480", "date": "09 Jun 2020", "name": "Elves Heleno Duarte", "expertise": [ "Reviewer Expertise Genetics", "bioinformatics", "epidemiology", "medical entomology", "Wolbachia", "and host-microorganism interactions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presented by Pais & Taveira is entitled Predicting the evolution and control of the COVID-19 pandemic in Portugal and it aims to describe the spread of SARS-CoV-2 during the first 21 days. The authors used a simple mathematical model assuming 14 days infection period.\nAccording to the authors, the model 'predicts the impact of isolation measures towards the expected peak of infection'. I am not convinced this is accurate. I believed the authors used the model to estimate the strength of the measures (i.e. percentage lockdown) and not to estimate the impact of the measures on the spread of SARS-Cov-2. This would imply a more elaborated study (e.g. case-control study). Indeed the consistently wrote 'transmission dynamics of COVID-19' and not the correct form 'transmission dynamics of SARS-CoV-2'.\nThe authors predicted 2 million cases, which I find surprisingly high. Using the optimal isolation percentage (70-75%), the model still predicted over 150,000 cases which are approximately 5-fold higher than the current number of cases in Portugal (~35,000). I would like to see the model's prediction using even higher percentages of isolation. This should be also discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5854", "date": "09 Sep 2020", "name": "Ricardo Pais", "role": "Author Response", "response": "We would like to acknowledge the reviewer for the relevant comments and suggestions. We have considered them all and have revised the manuscript accordingly. Comment 1\"According to the authors, the model 'predicts the impact of isolation measures towards the expected peak of infection'. I am not convinced this is accurate. I believed the authors used the model to estimate the strength of the measures (i.e. percentage lockdown) and not to estimate the impact of the measures on the spread of SARS-Cov-2\"Reply 1We agree with the suggestion and have changed the manuscript to convey the idea of estimating the strength of measures, which include the percentage of lockdown, social isolation and usage of masks (see revised version). Comment 2\"the consistently wrote 'transmission dynamics of COVID-19' and not the correct form 'transmission dynamics of SARS-CoV-2\"Reply 2We agree with the suggestion and have changed the manuscript replacing COVID-19 with SARS-CoV-2 (see revised version).Comment 3\"The authors predicted 2 million cases, which I find surprisingly high. Using the optimal isolation percentage (70-75%), the model still predicted over 150,000 cases which are approximately 5-fold higher than the current number of cases in Portugal (~35,000)\"Reply 3Indeed, our predictions are higher than the reported values. This is because only a small fraction of SARS-COV-2- infected individuals are tested for the virus has only a few patients show symptoms. However, our results are in agreement with the recent serologic study conducted by the National Institute of Health (INSA) from Portugal that found that a total of 300.000 people were exposed to the virus, 6-fold higher than the number of reported cases. By the time we conducted the model calibration and analysis, these results were not known, resulting in the deviation between predictions and reported values. Thus, we have corrected the model accounting for the asymptomatic fraction not tested and other recent data. This resulted in novel results which were compared with new data up to August and presented in the revised manuscript. Comment 4\"I would like to see the model's prediction using even higher percentages of isolation. This should be also discussed.\"Reply 4We have simulated the model with higher percentages and discuss the results (see revised manuscript)." } ] }, { "id": "64347", "date": "09 Jul 2020", "name": "Kamal Shah", "expertise": [ "Reviewer Expertise Applied mathematics", "Numerical solutions. Mathematical modeling and analysis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCoronavirus disease 2019 (COVID-19) is a worldwide pandemic that has been affecting Portugal since 2 March 2020. The Portuguese government has been making efforts to contradict the exponential growth through social isolation measures. In this regard, the authors have developed a mathematical model to predict the impact of such measures in the number of infected cases and peak of infection. They have estimated the peak to be around 2 million infected cases by the beginning of May if no additional measures are taken. The model shows that current measures effectively isolated 25-30% of the population, contributing to some reduction in the infection peak. Importantly, their simulations showed that the infection burden can be further reduced with a higher isolation degree, providing information for a second intervention. The considered study is interesting in this regard and has the potential to give some sound information about COVID-19.\nThe work is good but there are some issues to be addressed:\nProvide the existence of the model.\n\nWhat is the feasible region for the considered model?\n\nAlso, simulate the model for a long time that is months, 40 days, etc.\n\nWhat techniques for numerical simulation have been used?\n\nSome relevant and recent work in this regard also must be included. Please see these examples of recent work: On a comprehensive model of the novel coronavirus (COVID-19) under Mittag-Lefer Chaos, Solitons and Fractals xxx (xxxx) 1098671 Qualitative Analysis of a Mathematical Model in the Time of COVID-19 , BioMed Research International 2020, Article ID 5098598, 11 pages2 Statistical analysis of forecasting COVID-19 for upcoming month in Pakistan, Chaos, Solitons and Fractals 138 (2020) 1099263 Study of Transmission Dynamics of Novel COVID-19 by Using Mathematical Model.\" (2020). Archive.4\nI recommend its publication in this journal strongly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5853", "date": "09 Sep 2020", "name": "Ricardo Pais", "role": "Author Response", "response": "We would like to acknowledge the reviewer for the relevant comments and suggestions. We have considered them all and have revised the manuscript accordingly. Comment 1\"According to the authors, the model 'predicts the impact of isolation measures towards the expected peak of infection'. I am not convinced this is accurate. I believed the authors used the model to estimate the strength of the measures (i.e. percentage lockdown) and not to estimate the impact of the measures on the spread of SARS-Cov-2\"Reply 1We agree with the suggestion and have changed the manuscript to convey the idea of estimating the strength of measures, which include the percentage of lockdown, social isolation and usage of masks (see revised version). Comment 2\"the consistently wrote 'transmission dynamics of COVID-19' and not the correct form 'transmission dynamics of SARS-CoV-2\"Reply 2We agree with the suggestion and have changed the manuscript replacing COVID-19 with SARS-CoV-2 (see revised version).Comment 3\"The authors predicted 2 million cases, which I find surprisingly high. Using the optimal isolation percentage (70-75%), the model still predicted over 150,000 cases which are approximately 5-fold higher than the current number of cases in Portugal (~35,000)\"Reply 3Indeed, our predictions are higher than the reported values. This is because only a small fraction of SARS-COV-2- infected individuals are tested for the virus has only a few patients show symptoms. However, our results are in agreement with the recent serologic study conducted by the National Institute of Health (INSA) from Portugal that found that a total of 300.000 people were exposed to the virus, 6-fold higher than the number of reported cases. By the time we conducted the model calibration and analysis, these results were not known, resulting in the deviation between predictions and reported values. Thus, we have corrected the model accounting for the asymptomatic fraction not tested and other recent data. This resulted in novel results which were compared with new data up to August and presented in the revised manuscript. Comment 4\"I would like to see the model's prediction using even higher percentages of isolation. This should be also discussed.\"Reply 4We have simulated the model with higher percentages and discuss the results (see revised manuscript)." }, { "c_id": "5855", "date": "09 Sep 2020", "name": "Ricardo Pais", "role": "Author Response", "response": "We would like to acknowledge the reviewer for the relevant comments and suggestions. We have considered them all and have revised the manuscript accordingly. Comment 1\"Provide the existence of the model\"Reply 1According to F1000 Research rules for supplementary data, we included the model code, data and the analysis code in python as extended on figshare (see revised version references). Comment 2\"What is the feasible region for the considered model?\"Reply 2The recommended feasible region for this model is 108 > (S+I) > 104 where initial S + I = city population. Comment 3\"Also, simulate the model for a long time that is months, 40 days, etc.\"Reply 3We have performed simulations for 500 days for predictions and 17 days for model calibrations. By the time we submitted the paper no more than 27 days of data was available. Indeed, we only used a 17 days simulation for model calibration to estimate a rate constant in the absence of control. Unfortunately, it is not possible to use more data for model calibration since an uncontrolled lockdown was implemented in Portugal immediately after these 17 days of infection. Using more data would actually result in wrong estimates since many people follow the DGS recommendations but others do not. This is why we have simulated a total of 500 days with multiple % of lockdown scenarios towards estimating how much % of lockdown during the evolution of COVID pandemic. However, we now include relevant reported DGS data (up to August) for contrasting with model simulations and predictions (see revised version). Comment 4\"What techniques for numerical simulation have been used?\"Reply 4The numerical solver was based on the Adams/BDF method, implemented in the LSODA routine of PLAS software. This is a general-purpose stiff, variable-step and variable-order solver. We add this information in the methods section (see revised version). Comment 5 \"Some relevant and recent work in this regard also must be included. Please see these examples of recent work\"Reply 5We have read your suggestions of new modelling work which are quite relevant and innovative for citing as examples of similar modelling approaches applied in the forecasting of SARS-CoV-2 infection dynamics in Pakistan and Wuhan. Thus, we have included these in the new revised version and other recent work as well." } ] } ]	1	https://f1000research.com/articles/9-283
https://f1000research.com/articles/8-1032/v1	09 Jul 19	{ "type": "Research Article", "title": "One-year test-retest reliability of ten vision tests in Canadian athletes", "authors": [ "Mehdi Aloosh", "Suzanne Leclerc", "Stephanie Long", "Guowei Zhong", "James M. Brophy", "Tibor Schuster", "Russell Steele", "Ian Shrier", "Mehdi Aloosh", "Suzanne Leclerc", "Stephanie Long", "Guowei Zhong", "James M. Brophy", "Tibor Schuster", "Russell Steele" ], "abstract": "Background: Vision tests are increasingly being suggested for use in concussion management and baseline testing. Concussions, however, often occur months after baseline testing and reliability studies generally examine intervals limited to days or one week. Therefore, our objective was to determine the one-year test-retest reliability of these tests. Methods: We assessed one-year test-retest reliability of ten vision tests in elite Canadian athletes followed by the Institut National du Sport du Quebec. We included athletes who completed two baseline (preseason) annual evaluations by one clinician within 365±30 days. We excluded athletes with any concussion or vision training in between the annual evaluations or presented with any factor that is believed to affect the tests (e.g. migraines, etc.). Data were collected from clinical charts. We evaluated test-retest reliability using Intraclass Correlation Coefficient (ICC) and 95% limits of agreement (LoA). Results: We examined nine female and seven male athletes with a mean age of 22.7 (SD 4.5) years. Among the vision tests, we observed excellent test-retest reliability in Positive Fusional Vergence at 30cm (ICC=0.93) but this dropped to 0.55 when an outlier was excluded. There was moderate reliability in Negative Fusional Vergence at 30cm (ICC=0.78), Phoria at 30cm (ICC=0.68), Near Point of Convergence break (ICC=0.65) and Saccade (ICC=0.56). The ICC for Positive Fusional Vergence at 3m (ICC=0.56) also decreased to 0.45 after removing one outlier. We found poor reliability in Near Point of Convergence (ICC=0.47), Gross Stereoscopic Acuity (ICC=0.03) and Negative Fusional Vergence at 3m (ICC=0.0). ICC for Phoria at 3m was not appropriate because scores were identical in 14/16 athletes. 95% LoA of the majority of tests were ±40% to ±90%. Conclusions: Four tests had moderate one-year test-retest reliability. The remaining tests had poor reliability. The tests would therefore be useful only if concussion has a moderate-large effect on scores.", "keywords": [ "concussion", "vision tests", "binocular", "saccades", "reliability" ], "content": "Introduction\n\nConcussion, a form of mild traumatic brain injury is a growing public health concern1. Estimates suggest up to 3.8 million sport-related concussions occur annually in the United States, with 50% going unreported2. United States emergency department visits for sports-related traumatic brain injuries have increased 60% over 2001–20093. Concussions can be associated with headaches, dizziness, visual disturbances, and other symptoms that can negatively affect performance in sport, school, and work and negatively impact quality of life2,4,5.\n\nDiagnosis of concussion and decisions to return-to-play are based on symptoms, signs, physical examination and special tests6. Previous research has shown an association between concussion and eye movement1. Concussion may therefore affect multiple aspects of vision, including saccades, pursuit, convergence, accommodation, and vestibulo-ocular reflex7. Some studies reported 50% to 90% incidence of visual symptoms, such as blurred vision and diplopia in individuals with concussion8. Therefore, vision testing may be helpful in the assessment and management of patients with concussion.\n\nEach vision test measures a function that is linked to a particular brain structure or pathway. Vision tests are noninvasive tests with rapid administration and scoring. Understanding test variability, independent of changes in pathology or recovery (i.e. reliability), is required to assess their clinical utility. However, only a limited number of reliability studies have assessed binocular vision tests9–16. In addition, these reliability studies measured a specific aspect of the vision. These studies are not uniform in their method and they are diverse in their population.\n\nPrevious investigations of the test-retest reliability of these vision tests have used short test-retest time intervals ranging from 1 day to 45 days9–17. For test-retest reliability to be useful in clinical management (e.g. return-to-play), the time intervals must reflect the time frame in which they would be used18. The previous studies have provided information on the usefulness of these tests when following improvement or deterioration of patients over short periods of time. However, concussions usually occur several months and up to one year after annual baseline testing, and not as 1 day to 45 days as in the previous studies. Therefore, we examined one-year test-retest reliability of ten vision tests in Canadian athletes over one year period of time.\n\n\nMethods\n\nThe study population included athletes over 16 years of age followed by the Institut National du Sport du Quebec (INSQ) in Canada from 2015–2018. Many of these athletes had a yearly examination done by a sports medicine physician and vision tests done by a clinician trained in orthoptic testing.\n\nWe only included athletes who had completed two baseline (preseason) annual evaluations within a 365-day (± 30 days) time period. We excluded athletes who suffered a concussion in between annual evaluations or had received preventive orthoptic training between the baseline measures. We also excluded athletes with a history of strabismus or treated strabismus, or were medically treated for depression, anxiety or psychiatric conditions that may affect binocular vision and saccades. Data were collected from electronic medical charts of one clinician trained in orthoptic measures and one sports medicine physician.\n\nAt the beginning of each season, athletes underwent baseline testing of ten vision tests by a single orthoptic-trained clinician (industry partner). The vision tests were Gross Stereoscopic Acuity, Near Point of Convergence (NPC), Near Point of Convergence break (NPCb), near (30cm) and far (3m) Positive Fusional Vergence, near (30cm) and far (3m) Negative Fusional Vergence, near (30cm) and far (3m) Phoria, and Saccades.\n\nA detailed description of each test including the procedures of each test and the theoretical range of scores is provided in Table 1. We will briefly describe each vision test here. We used a horizontal prism bar with the base-out for Positive Fusional Vergence and base-in for Negative Fusional Vergence, at both 30cm and 3m10. Phoria was measured at 30cm and 3m using the prism and alternate cover test using the procedures described by the Pediatric Eye Disease Investigator Group19. To perform NPC and NPCb, we followed the Maples et al., protocol13. We measured Gross Stereoscopic Acuity with the Randot Stereotest (Stereo Optical Co., Inc., Chicago, IL) according to the manufacturer’s instructions20. Evaluation of Saccades was done using the test procedures developed by the orthoptic-trained clinician. Participants assumed a tandem stance an arm’s length away from a screen attempting to fixate on appearing and disappearing lights on the screen, while trying to keep their head still. Light flashes appeared at a rate of 100 per minute for two minutes. This test was scored by the clinician based on quality (bad, medium, good), synchronization (bad, medium, good), and saccadic corrections (many, few, none). These three components were then combined into an overall percentage saccade score, based on an unpublished proprietary algorithm developed by the clinician who performed the testing.\n\nWe report the mean (SD) for continuous variables at baseline. We evaluated test-retest reliability using Intraclass Correlation Coefficient (ICC)21 and 95% limits of agreement (LoA)22. We considered ICC of ≤0.5 as poor, 0.51-0.74 as moderate, 0.75-0.89 as good, and ≥0.90 as excellent reliability23. We report the LoA in the raw units of the scale used by clinicians. To compare LoA across tests, we also standardized the scores and reported them as percent differences, [(T1- T2)/ mean(T1&T2)]*10022,24. Additionally, we summarized LoA graphically with Bland-Altman plots for each vision test using the standardized score for the y-axis to provide an overview of all vision tests. The raw scale measures are provided in parentheses to provide clinicians with information for individual patient assessment. Finally, we conducted a sensitivity analysis for the vision tests by excluding outliers that may have augmented the ICC results. We defined an outlier as a data point that was 1.5 interquartile ranges below the first quartile or above the third quartile.\n\nDue to the limited sample size (n=16) and to avoid being overly conservative in our evaluation, we followed the practical solution for addressing multiple testing proposed by Saville, the unrestricted least significant difference procedure (or multiple t-test)34. Formal multiplicity correction of confidence levels was not performed but we thoroughly reported all statistical assessments enabling an informal type-I error assessment by the reader. The data were analyzed using R statistical software 3.4.335. This study was approved by the McGill University Faculty of Medicine Institutional Review Board.\n\n\nResults\n\nOf the 199 athletes measured for the vision tests, only 16 individuals met our inclusion criteria (Figure 1). There were nine female and seven male athletes with a mean age of 22.7 (4.5) years at the baseline (preseason) measurement. Participants were athletes of water polo (n=6) and short-track speed skating (n=10). A second measurement was conducted between 335 and 372 days (mean of 356.4 (17.3) days) after the initial baseline.\n\nThe range of scores observed for each vision test can be found in each of the reliability figures (Figure 2–Figure 4)36. Our analysis suggested one-year test-retest reliabilities ranging from poor to excellent among the ten vision tests. We observed excellent one-year test-retest reliability in Positive Fusional Vergence at 30cm with ICC of 0.93 (Figure 2). In this test, 4 out of 16 pairs of measurements were identical after 1 year. The range of measurements was between 14 and 45 diopters with one outlier at 90 diopters. LoA of the test was ±41.9%. Given the very high ICC and the presence of an outlier that greatly increased the range of x-axis (known to increase ICC), we conducted a sensitivity analysis excluding the outlier. This decreased the ICC from 0.93 to 0.55, and increased the LoA to ±43.5%.\n\n(A) Scatter plot of test-retest reliability for Positive Fusional Vergence at 30cm. Identity line represents perfect agreement between the test-retest values; ICC refers to the Intraclass correlation coefficient and 95%CI refers to the 95% Confidence Interval. “n (1,2,3,4)” refers to the number of participants represented by each dot when scores exactly overlapped. (B) Bland-Altman plot with the mean of the test-retest on the x-axis and the difference between test-retest on the y-axis. Solid line represents the bias and dotted lines represent the 95% LoA. The y-axis represents a standardized LoA using percentage difference on the plot to allow one to compare the different tests to each other. The LoA in the units of measure, which are familiar to clinicians, are provided in the parentheses.\n\n(A) Scatter plot of test-retest for Negative Fusional Vergence at 30cm, Phoria at 30cm, Near Point of Convergence break (NPCb), Positive Fusional Vergence at 3m, and Saccade. (B) Bland-Altman plot related to each test. See Figure 2 for explanation of abbreviations and scales.\n\n(A) Scatter plots of test-retest for near point of convergence (NPC), Gross Stereoscopic Acuity, and Negative Fusional Vergence at 3m. (B) Bland-Altman plots related to each test. See Figure 2 for explanation of abbreviations and scales.\n\nFive tests showed moderate one-year test-retest reliability (Figure 3), including Negative Fusional Vergence at 30cm (ICC=0.78, LoA=41.2%), Phoria at 30cm (ICC=0.68, LoA=119.2%), NPCb (ICC=0.65, LoA=49.4%), Positive Fusional Vergence at 3m (ICC=0.56, LoA=59.8%), and Saccades (ICC=0.57, LoA=24.3%). There was also one outlier for Positive Fusional Vergence at 3m, 1.5 interquartile range above the third quartile. When removing this outlier, the ICC dropped from 0.57 to 0.21. In this case, the two scores from the outlier were quite different. Although one might anticipate that the ICC would increase by removing such an outlier, the ICC actually decreased because the range of the x-values decreased substantially.\n\nThree of the remaining four tests showed poor one-year test-retest reliability (Figure 4). These include NPC (ICC=0.47, LoA=73.9%), Gross Stereoscopic Acuity (ICC=0.03, LoA=92.5%) and Negative Fusional Vergence at 3m (ICC=0.0, LoA=48.4%). For Phoria at 3m, 14/16 athletes had identical scores on the two measures. In this context, the ICC and LoA were not appropriate measures of reliability and are not presented.\n\n\nDiscussion\n\nWe found that the one-year test-retest reliability for 10 vision tests in young elite athletes ranged from excellent to poor. Positive Fusional Vergence at 30cm showed excellent reliability, while that of the others appeared to be moderate to poor. The majority of the vision tests had standardized 95% LoA in the range of 40–90%, which indicates that repeated scores of an individual over time may vary by 40–90% of the mean score even without any actual change in vision function.\n\nThere are a limited number of test–retest reliability studies on non-vision neurocognitive tests over a one year period in teenage athletes. For instance, the ICC for different components of Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT), a computerized brain injury measurement tool, ranges from 0.50 to 0.8237. However, we could not find any research examining the stability of the vision tests over a one year period, in athlete or non-athlete populations. It is important that test-retest reliabilities fall within a range needed for clinical interpretation of concussion assessment and for discussion about return-to-play. In the context of comparing results after a concussion to annual baseline tests conducted in the pre-season, the time-frame for reliability comparisons should be up to one year18.\n\nAlthough there are no long-term reliability studies on vision tests, a number of studies have reported short term test-retest reliability of individual tests using various methods among various groups of individuals, including children and healthy adults9–16. For instance, one study reported an ICC of 0.65 for NPC in healthy individuals15, while other studies have reported excellent reliability for NPCb in healthy school children (ICC=0.94)16 and concussed athletes with normal vision (ICC = 0.92 to 0.97)12. In addition, we recently examined one-week test-retest reliability of the same ten vision tests with the same methods as this current study in 20 young non-elite athletes. We found one-week test-retest reliability ranging from poor (ICC = 0.34) to good (ICC = 0.88), with five out of ten tests showing moderate reliability (ICCs = 0.54 to 0.69)17. This suggests that these vision tests can only be useful if a concussion has a moderate to large effect on scores. Overall, the ICCs in the current study were generally smaller than those reported in our one-week study, suggesting increased temporal variability. Unexpectedly, the 95% LoA for one-year test-retest was smaller or equal to the 95% LoA of the one-week test-retest for all vision tests except NPC (±73.9 vs. ±57.9) and Gross Stereoscopic Acuity (±92.5 vs. ±55). In addition, in both the one-week and one-year intervals, almost all individuals had the same value in Phoria 3m, which leads to uninformative LoA.\n\nIn one-year test-retest, Positive Fusional Vergence showed excellent reliability at 30cm (ICC=0.93) and moderate at 3m (ICC=0.56), initially. These values were better than the one-week test-retest reliability (ICC=0.54 and 0.49, respectively)17. It is difficult to understand how test-retest reliability over one year could be better than test-retest reliability over one week. When we explored the data further, we noticed one outlier that greatly increased the range of values along x-axis in Figure 2 and Figure 3. Increasing the x-axis range is known to increase the ICC. This is because ICC is based on the results of an analysis of variance which separates the error into variability between individuals (x-range of values) and variability within an individual. Therefore, if variability between persons increases, indicated by a larger range of values on the x-axis, ICC will increase. We explored how removing the outlier in our data would affect the results even though we have no reason to believe the data are inaccurate. When we removed the outlier, the ICC dropped below the value found for the one-week test-retest reliability but it did not affect LoA. A similar effect was observed when we removed an outlier from Positive Fusional Vergence at 3m, even though this particular outlier had a large difference between the two test scores, which would normally be considered to decrease ICC (Figure 3). This finding indicates that as expected, if the range of values among the populations is similar, the one-year test-retest reliability for Positive Fusional Vergence is likely less than the one-week test-retest reliability.\n\nAside from outliers, there are other theoretical reasons that might explain why ICC is better at one-year than at one-week. First, it is possible that the non-athletes in our one-week test-retest study had less motivation to perform well on the repeat tests. If true, their scores would be less than the motivated athletes performing during the one-year test-retest. Second, there is a potential learning effect in retest measurements that could affect results. A learning effect, however, is unlikely in our study because the athletes were tested only twice, with a one-year interval between tests. Third, the increased ICC could have occurred simply by chance because of sampling variation.\n\nNegative Fusional Vergence tests showed good and poor one-year test-retest reliability, at 30cm and 3m (ICC=0.78 and 0.0, respectively). These results were similar to our one-week test-retest study17. Our results were also similar to those of another study examining one-week test-retest reliability of Positive Fusional Vergence reporting ICCs of 0.53–0.5916, which is similar to the ICC we found at 3m. The different results might occur because of minor differences in application of tests which are not explicitly mentioned in the Methods.\n\nOur measurements of Phoria at 30cm had moderate reliability for near (ICC=0.68) consistent with our one-week retest reliability study (ICC=0.69)17. Other studies in adults and children with strabismus38 or esotropia19 have not reported ICC. Therefore, comparing between studies is not possible. Moreover, our analytical methods differed slightly from those studies. We evaluated all angles of deviation together, and other authors analyzed smaller (2–20 Prism Diopter) or larger (>20 Prism Diopter) angles of strabismus separately because of different prism increments measured38. For Phoria at 3m, we found that the ICC and LoA were not appropriate measures of reliability because most of the population reported identical scores of zero for both measurements. One may consider that if we had a wider range of scores, ICC might provide meaningful information.\n\nOne-year test-retest reliability of NPC and NPCb (0.47 and 0.65, respectively) were similar to the results in our one-week reliability study (0.54 and 0.64, respectively)17. Brozek et al. reported a similar ICC of 0.65 for NPC in healthy adults15,16. However, other studies have reported excellent reliability for NPCb in school children (ICC=0.94)16 and in concussed athletes with normal vision (ICC=0.92 to 0.97)12. The discrepancies in results are most likely due to differences in testing procedures. For instance, we used the Maples method13 which is a non-accommodative test, whereas other studies used an accommodative target, such as RAF rule28, or Astron International Accommodative Rule12,16. Giffard et al. found that RAF rule had good test-retest reliability (ICC=0.84) for NPC using within a one-week interval. The Astron International Accommodative Rule for NPCb had excellent one-week test-retest reliability (ICC=0.94-0.98) among healthy children16,39.\n\nOur one-year test-retest results for Gross Stereoscopic Acuity in young athletes showed poor reliability (ICC=0.03; 95% LoA= ±92.5%) even though our previous one-week test-retest results reported good reliability in non-athlete young adults (ICC=0.86; 95% LoA = ± 55%)17 and another study using Titmus stereo fly and Frisby stereo tests in children revealed an excellent reliability (ICC=1.0)40. In addition, one study reported that 82.0% of their participants had identical results at test and retest taken on the same day in 100 healthy adult and children11. With an ICC of 0.03 and LoA of 92.5%, Gross Stereoscopic Acuity cannot be considered a reliable test to assess the vision function over one year, although it may still be appropriate for use in shorter time intervals, such as one week11,17,40.\n\nFinally, our clinician’s test of Saccades showed moderate reliability (ICC=0.57) with the smallest LoA (in percentage) of other tests, similar to the one-week study17. These results are similar to other findings in healthy adults over a two-month period (ICC=0.59)41. With a moderate reliability and the smallest LoA amongst the other vision tests, the results of the test of Saccades could be considered stable over a one year period assessing athletes.\n\nIn this study, four vision tests (Negative Fusional Vergence at 30cm, Phoria at 30cm, Saccade and NPCb) had moderate one-year test-retest reliability. The one test with identical scores in 14/16 athletes was Phoria at 3m. Therefore we cannot comment on the reliability of this test. This level of reliability would be useful in conditions where the concussion leads to a moderate change in vision function. The remaining five vision tests, including Positive Fusional Vergence at 30cm and 3m, NPC, Negative Fusional Vergence at 3m, and Gross Stereoscopic Acuity may be useful to detect the effect of concussion with a large change on vision function. Further studies are therefore required to assess the effect of concussion on vision test scores of the five vision tests. If it can be shown that the concussion has moderate to large effect on the test scores then these vision tests may still be useful clinically.\n\nSeveral studies have previously evaluated the inter-rater reliability of some vision tests19,38. However, inter-rater reliability is less important in the context of clinical care when patients are followed by one clinician over time. Our study evaluated the test-retest reliability of the ten vision tests over an interval that allows for the normal variation over time expected in clinical practice between baseline measures and subsequent concussions. The ICC represents how much of variability in scores is due to differences between subjects. For instance, the ICC of 0.78 for near Negative Fusional Vergence at 30cm suggests that 78% of the variability in the measurements was due to differences between participants, and 22% was due to normal variations within the measurement. Furthermore, the 95% LoA for each test in our study provides the magnitude of the normal variation that can be expected with repeated measurements. Differences in test results between baseline and diagnosis of a concussion likely represent a true signal of a change in vision function within the patient if these differences are larger than the noise (LoA). In addition, we conducted sensitivity analysis to evaluate the effect of outliers. This analysis suggested that our initial ICC results may have been artificially high for two tests.\n\nThis is a historical cohort observational study, a study design which has inherent limitations. In addition, the sample size was relatively small and composed of healthy athletes, which will limit the generalizability of these findings to other populations. Although we started with a pool of 199 athletes, many athletes were excluded because they only had one baseline test, a concussion occurred in between the two baseline tests, or the second baseline test occurred outside the testing window of 365±30 days.\n\n\nConclusion\n\nWe found that four out of the ten vision tests (Negative Fusional Vergence at 30cm, Phoria at 30cm, NPCb and Saccade) had moderate one-year test-retest reliability. This level of reliability is useful in conditions which produce a moderate change in vision function. The remaining six vision tests may be useful in detecting large effects on vision function. If further studies suggest that the effect of concussion on test scores is moderate to large, these vision tests may still be useful clinically.\n\n\nData availability\n\nOpen Science Framework: Vision Tests in Concussion. https://doi.org/10.17605/OSF.IO/VB4W836\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nDemographic data are not available. With only 9 males and 7 females from our clinical source, any demographic information would immediately allow some participants to be identified and therefore this information cannot be shared in order to preserve participant confidentiality.", "appendix": "Grant information\n\nThis project was funded by government sources (MITACS [IT08159] and MEDTEQ [G245120], a non-profit organization (Institut National du Sport du Quebec) and private industry (Apexk Inc. and Varitron Technologies Inc.)\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe would like to thank Isabel Pereira for her help throughout the course of this work. In addition, we would like to thank David Tinjust, from Apexk for examining the athletes.\n\n\nReferences\n\nSussman ES, Ho AL, Pendharkar AV, et al.: Clinical evaluation of concussion: The evolving role of oculomotor assessments. Neurosurg Focus. 2016; 40(4): E7. PubMed Abstract \| Publisher Full Text\n\nLanglois JA, Rutland-Brown W, Wald MM: The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil. 2006; 21(5): 375–8. PubMed Abstract \| Publisher Full Text\n\nCenters for disease control and prevention: Nonfatal traumatic brain injuries related to sports and recreation activities among persons aged ≤19 years--United States, 2001-2009. MMWR Morb Mortal Wkly Rep. 2011; 60(39): 1337–42. PubMed Abstract\n\nDikmen S, Machamer J, Fann JR, et al.: Rates of symptom reporting following traumatic brain injury. J Int Neuropsychol Soc. 2010; 16(3): 401–11. PubMed Abstract \| Publisher Full Text\n\nMcCrory P, Meeuwisse WH, Aubry M, et al.: Consensus statement on concussion in sport: The 4th international conference on concussion in sport held in zurich, november 2012. Br J Sports Med. 2013; 47(5): 250–8. PubMed Abstract \| Publisher Full Text\n\nMcCrory P, Meeuwisse W, Dvořák J, et al.: Consensus statement on concussion in sport-the 5th international conference on concussion in sport held in Berlin, October 2016. Br J Sports Med. 2017; 51(11): 838–47. PubMed Abstract \| Publisher Full Text\n\nVentura RE, Balcer LJ, Galetta SL: The neuro-ophthalmology of head trauma. Lancet Neurol. 2014; 13(10): 1006–16. PubMed Abstract \| Publisher Full Text\n\nTalavage TM, Nauman EA, Breedlove EL, et al.: Functionally-detected cognitive impairment in high school football players without clinically-diagnosed concussion. J Neurotrauma. 2014; 31(4): 327–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOberlander TJ, Olson BL, Weidauer L: Test-retest reliability of the king-devick test in an adolescent population. J Athl Train. 2017; 52(5): 439–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoss DA, Becker E: Comparison of near fusional vergence ranges with rotary prisms and with prism bars. Optometry. 2011; 82(2): 104–7. PubMed Abstract\n\nWang J, Hatt SR, O'Connor AR, et al.: Final version of the Distance Randot Stereotest: normative data, reliability, and validity. J AAPOS. 2010; 14(2): 142–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPearce KL, Sufrinko A, Lau BC, et al.: Near Point of Convergence After a Sport-Related Concussion: Measurement Reliability and Relationship to Neurocognitive Impairment and Symptoms. Am J Sports Med. 2015; 43(12): 3055–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaples WC, Hoenes R: Near point of convergence norms measured in elementary school children. Optom Vis Sci. 2007; 84(3): 224–8. PubMed Abstract \| Publisher Full Text\n\nAntona B, Barrio A, Barra F, et al.: Repeatability and agreement in the measurement of horizontal fusional vergences. Ophthalmic Physiol Opt. 2008; 28(5): 475–91. PubMed Abstract \| Publisher Full Text\n\nBrozek J, Simonson E, Bushard W, et al.: Effects of practice and the consistency of repeated measurements of accommodation and vergence. Am J Ophthalmol. 1948; 31(2): 191–8. PubMed Abstract \| Publisher Full Text\n\nRouse MW, Borsting E, Deland PN, et al.: Reliability of binocular vision measurements used in the classification of convergence insufficiency. Optom Vis Sci. 2002; 79(4): 254–64. PubMed Abstract \| Publisher Full Text\n\nLong S, Leclerc S, Tinjust D, et al.: Determining consistency and agreement of scores across two measurements of the visual system: Test-retest reliability. Med Sci Sports Exerc. 2018; 50(5S): 664. Publisher Full Text\n\nBroglio SP, Ferrara MS, Macciocchi SN, et al.: Test-retest reliability of computerized concussion assessment programs. J Athl Train. 2007; 42(4): 509–14. PubMed Abstract \| Free Full Text\n\nPediatric Eye Disease Investigator Group: Interobserver reliability of the prism and alternate cover test in children with esotropia. Arch Ophthalmol. 2009; 127(1): 59–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStereo optical co: Randot stereotest. In: Stereo optical co., ed.; 1995.\n\nShrout PE, Fleiss JL: Intraclass correlations: uses in assessing rater reliability. Psychol Bull. 1979; 86(2): 420–8. PubMed Abstract \| Publisher Full Text\n\nBland JM, Altman D: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986; 1(8476): 307–10. PubMed Abstract \| Publisher Full Text\n\nKoo TK, Li MY: A guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med. 2016; 15(2): 155–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnston BC, Thorlund K, Schünemann HJ, et al.: Improving the interpretation of quality of life evidence in meta-analyses: the application of minimal important difference units. Health Qual Life Outcomes. 2010; 8(1): 116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowe F: Clinical orthoptics. 3rd ed. Chichester, West Sussex: Wiley-Blackwell; 2012. Reference Source\n\nD'Agostino D: Basic examination: Physiology of eye movements - measurement of ductions, versions, and vergences. In: Scott W, D'Agostino D, Weingeist Lennarson L, editors. Orthoptics and ocular examination techniques. Baltimore: Williams & Wilkins; 1983.\n\nHurtt J, Rasicovici A, Windsor C: Comprehensive review of orthoptics and ocular motility: Theory, therapy, and surgery. 2nd ed. Saint Louis: The C.V. Mosby Company; 1977.\n\nBishop A: Convergence and convergent fusional reserves - investigation and treatment. In: Doshi S, Evans BJW, editors. Binocular vision and orthoptics: Investigation and management. Oxford: Butterworth-Heineman; 2001; 28–33. Publisher Full Text\n\nScheiman M, Gwiazda J, Li T: Non-surgical interventions for convergence insufficiency. Cochrane Database Syst Rev. 2011; (3): CD006768. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHayes GJ, Cohen BE, Rouse MW, et al.: Normative values for the nearpoint of convergence of elementary schoolchildren. Optom Vis Sci. 1998; 75(7): 506–12. PubMed Abstract\n\nSutter P, Harvey L: Vision rehabilitation: Multidisciplinary care of the patient following brain injury. Boca Raton: Taylor & Francis Group; 2011. Reference Source\n\nBirch E, Williams C, Drover J, et al.: Randot preschool stereoacuity test: Normative data and validity. J AAPOS. 2008; 12(1): 23–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiano ME, Tidbury LP, O'Connor AR: Normative Values for Near and Distance Clinical Tests of Stereoacuity. Strabismus. 2016; 24(4): 169–72. PubMed Abstract \| Publisher Full Text\n\nSaville DJ: Multiple comparison procedures: The practical solution. Am Stat. 1990; 44(2): 174–80. Publisher Full Text\n\nR core team: R: A language and environment for statistical computing [internet]. Vienna, austria: R foundation for statistical computing; 2015. 2015.\n\nShrier I: Vision Tests in Concussion. 2019. http://www.doi.org/10.17605/OSF.IO/VB4W8\n\nMoser RS, Schatz P, Grosner E, et al.: One year test-retest reliability of neurocognitive baseline scores in 10- to 12-year olds. Appl Neuropsychol Child. 2017; 6(2): 166–71. PubMed Abstract \| Publisher Full Text\n\nde Jongh E, Leach C, Tjon-Fo-Sang M, et al.: Inter-examiner variability and agreement of the alternate prism cover test (APCT) measurements of strabismus performed by 4 examiners. Strabismus. 2014; 22(4): 158–66. PubMed Abstract \| Publisher Full Text\n\nGiffard P, Daly L, Treleaven J: Influence of neck torsion on near point convergence in subjects with idiopathic neck pain. Musculoskelet Sci Pract. 2017; 32: 51–6. PubMed Abstract \| Publisher Full Text\n\nMoganeswari D, Thomas J, Srinivasan K, et al.: Test Re-Test Reliability and Validity of Different Visual Acuity and Stereoacuity Charts Used in Preschool Children. J Clin Diagn Res. 2015; 9(11): NC01–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEttinger U, Kumari V, Crawford TJ, et al.: Reliability of smooth pursuit, fixation, and saccadic eye movements. Psychophysiology. 2003; 40(4): 620–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "60656", "date": "10 Mar 2020", "name": "M Nadir Haider", "expertise": [ "Reviewer Expertise Statistical design", "physiological and biochemical markers of concussion", "autonomic regulation of cerebral blood blow." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this manuscript. It measures the retest reliability of common ocular/oculomotor tests over one year. The sample size is 16 college-aged athletes. Intra-class correlation is performed and presented. I have read through the entire manuscript and it is exceptionally well-written, it shows that it has gone through several internal, and even some external, reviews and revisions already. The statistical analysis are correctly described and the appropriate tests and graphs are used to present data.\n\nThe most obvious downside of this study is the small sample size, there is so much within-subject variation among these test due to the natural process of aging and ocular adaptations which could be due to insignificant events like getting a new monitor for work. Future studies should be performed on larger sample sizes, etc.\nBut I believe that there is merit in having your study indexed for a couple of reasons. The research protocol and analysis are well explained and could be used for design future oculomotor retest reliability studies. Secondly, I am glad that you had concussion as your exclusionary criteria since there are a hundred different publications showing abnormalities in vision function tests after concussion, yet present no retest reliability without the presence of a concussive head injury. I think this paper provides some preliminary evidence which should be made available to other researchers and I think this is a citable manuscript. I do not have any sentence by sentence suggestions, but my only major suggestion is to remove the pre-outlier ICC of Positive Fusional Vergence at 30cm value of 0.93 and say that it is 0.55 (moderate). And I think Negative Fusional Vergence at 30cm should be classified as Good ICC (not moderate since it is between 0.75 and 0.9).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5332", "date": "31 Mar 2020", "name": "Ian Shrier", "role": "Author Response", "response": "REVIEWER #1Comment: Thank you for giving me the opportunity to review this manuscript. It measures the retest reliability of common ocular/oculomotor tests over one year. The sample size is 16 college-aged athletes. Intra-class correlation is performed and presented. I have read through the entire manuscript and it is exceptionally well-written, it shows that it has gone through several internal, and even some external, reviews and revisions already. The statistical analysis are correctly described and the appropriate tests and graphs are used to present data. Answer: We thank the reviewer for the kind comments. ________Comment: The most obvious downside of this study is the small sample size, there is so much within-subject variation among these test due to the natural process of aging and ocular adaptations which could be due to insignificant events like getting a new monitor for work. Future studies should be performed on larger sample sizes, etc. Answer: In this paper, we used all eligible participants from a clinical database. Therefore, we could not calculate an a priori sample size. Our primary approach to sample size requirements is to estimate precision rather than use hypothesis testing. We have tried to provide information for both approaches in the current version, and the new final paragraph of the limitations section is provided below. \"This is a historical cohort observational study, a study design which has inherent limitations. In addition, the sample size was relatively small and composed of healthy athletes, which will limit the generalizability of these findings to other populations. Although we started with a pool of 199 athletes, many athletes were excluded because they only had one baseline test, a concussion occurred in between the two baseline tests, or the second baseline test occurred outside the testing window of 365±30 days. With an effective sample size of 16, the anticipated precision of ICC estimates was +/- 0.25 and the study had 80% power to detect ICC values >= 0.6 and more than 90% power to detect ICC values >=0.7 i.e. rejection of the null hypothesis (Table 1a in 42). Note that a total of >60 individuals were required to exclude ICC values <=0.5 with 80% power and an anticipated true ICC>0.7 (Table 2b in 42).\"Reference: Bujang MA, N. B. A simplified guide to determination of sample size requirements for estimating the value of intraclass correlation coefficient: a review. Arch Orofac Sci. 2017; 12(1): 1-11. ______Comment: But I believe that there is merit in having your study indexed for a couple of reasons. The research protocol and analysis are well explained and could be used for design future oculomotor retest reliability studies. Secondly, I am glad that you had concussion as your exclusionary criteria since there are a hundred different publications showing abnormalities in vision function tests after concussion, yet present no retest reliability without the presence of a concussive head injury. I think this paper provides some preliminary evidence which should be made available to other researchers and I think this is a citable manuscript. Answer: We again thank the reviewer for the kind comments. ________Comment: I do not have any sentence by sentence suggestions, but my only major suggestion is to remove the pre-outlier ICC of Positive Fusional Vergence at 30cm value of 0.93 and say that it is 0.55 (moderate). Answer: We thank the reviewer for the comment. Recommended practice is to only delete data points if you have a very good reason to believe they are inaccurate. Otherwise, one should keep the original analysis intact and apply sensitivity analyses. For example, if deleting a point improved the ICC, we are confident the reviewer would agree that we should not delete the data point. For this reason, we have not changed our results as suggested. However, we have modified the text to further emphasize the importance of the sensitivity analysis. _________ Comment: And I think Negative Fusional Vergence at 30cm should be classified as Good ICC (not moderate since it is between 0.75 and 0.9). Answer: We thank the reviewer for pointing out this oversight. We have now indicated that Figure 3 shows results for good to moderate reliability tests, and made the associated changes in the abstract and manuscript as well." } ] } ]	1	https://f1000research.com/articles/8-1032
https://f1000research.com/articles/9-6/v1	06 Jan 20	{ "type": "Research Article", "title": "High vaccination coverage, inadequate knowledge and high vector density: Findings from a community-based cross-sectional study on Japanese Encephalitis in Yangon, Myanmar", "authors": [ "Pyae Phyo Kyaw", "Hemant Deepak Shewade", "Nang Thu Thu Kyaw", "Khaing Hnin Phyo", "Htar Htar Lin", "Aye Mon Mon Kyaw", "Mg Mg Mya", "Sein Thaung", "Yan Naung Maung Maung", "Hemant Deepak Shewade", "Nang Thu Thu Kyaw", "Khaing Hnin Phyo", "Htar Htar Lin", "Aye Mon Mon Kyaw", "Mg Mg Mya", "Sein Thaung", "Yan Naung Maung Maung" ], "abstract": "Background: Japanese encephalitis (JE) is a mosquito-borne disease with high case fatality and no specific treatment. Little is known about the community’s (especially parents/guardians of children) awareness regarding JE and its vaccine in Yangon region, which bears the highest JE burden in Myanmar. Methods: We conducted a community-based cross-sectional study in Yangon region (2019) to explore the knowledge and perception of parents/guardians of 1-15 year-old children about JE disease, its vaccination and to describe JE vaccine coverage among 1-15 year-old children. We followed multi-stage random sampling (three stages) to select the 600 households with 1-15 year-old children from 30 clusters in nine townships. Analyses were weighted (inverse probability sampling) for the multi-stage sampling design. Results: Of 600 parents/guardians, 38% exhibited good knowledge of JE, 55% perceived JE as serious in children younger than 15 years and 59% perceived the vaccine to be effective. Among all the children in the 600 households, the vaccination coverage was 97% (831/855). Conclusion: In order to reduce JE incidence in the community, focus on an intensified education program is necessary to sustain the high vaccine coverage in the community.", "keywords": [ "JE vaccine", "knowledge and perception", "community-based survey", "caregiver’s knowledge", "SORT IT" ], "content": "Introduction\n\nJapanese encephalitis (JE) is a zoonotic disease caused by Japanese encephalitis virus (JEV). JEV exists in a transmission cycle between mosquitoes, pigs and/or water birds and is transmitted to humans through bites from infected mosquitoes of the Culex species (mainly Culex tritaeniorhynchus)1. JE usually presents as acute encephalitis syndrome (AES) and is confirmed by serology.\n\nJE is a disease of public health importance as billions of people are at risk of getting infected by JEV and children below 15 years are more susceptible1. A systematic review (2011) reported that 67,900 clinical cases of JE occur annually in 24 Asian and western Pacific countries despite the widespread availability of the vaccine, with approximately 13,600 to 20,400 deaths. While the overall incidence of JE is 1.8 per 100,000 per year in endemic countries, it is 5.4 among 1–15 year-old children2. The infection can lead to severe complications with high case fatality. There is no specific treatment to date. JE is not easily prevented by protection from mosquito control and mosquito bites. Hence, vaccination is the most effective form of prevention1. Globally, live attenuated SA 14-14-2 JE vaccine is the most commonly used JE vaccine. Vaccine efficacy is reported to be between 80% and 99% following single-dose vaccination and 98% or greater with two doses3.\n\nIn Myanmar, Culex tritaeniorhynchus is the main JE vector4. In 2012, there were only 14 confirmed JE cases, which increased to 151 in 2015 and more than 380 in 2016 and 2017, indicative of improved surveillance in the country5.\n\nIn 2017, the Expanded Programme on Immunization (EPI) under the Department of Public Health carried out a nationwide JE vaccination catch-up campaign supported by the GAVI, WHO, the United Nations Children’s Fund and PATH. In November and December, nearly 14 million children (aged nine months to 15 years) were targeted and vaccinated with the WHO live attenuated (SA 14-14-2) JE vaccine. Alongside the campaign there were extensive advocacy and sensitization sessions provided to schools and communities6. Since January 2018, immediately after the JE campaign, routine immunization in Myanmar has included JE live attenuated vaccine, given at the age of nine months together with the measles-rubella vaccine.\n\nAdequate knowledge of JE and a positive perception of the JE vaccine are important for the adoption of preventive measures7. In addition, high coverage of the JE vaccine in populations at risk of disease is required to reduce the JE cases because JE vaccination would not provide herd immunity8. A study conducted in one township in northern Shan state in Myanmar (2018) showed the level of awareness of JE and its vaccine was low but the perception towards knowledge of JE was generally positive9. The vaccination coverage was 93% among 391 study participants.\n\nLittle is known about the community’s (especially parents/guardians of children) awareness regarding JE and its vaccine in Yangon region, which bears the highest JE burden in the country6. This data, alongside vaccination coverage data for children, may help the regional vector borne disease control (VBDC) programme and EPI to develop a new coordinated strategic plan to successfully reduce JE transmission in Yangon region.\n\nTherefore, in Yangon region, we aimed to describe the i) knowledge and perception of the parents/guardians of children (1–15 years old) towards JE disease and vaccine, and ii) JE vaccine coverage among 1–15 year-old children.\n\n\nMethods\n\nThe Ethics Review Committee, Department of Medical Research, Myanmar (Ethics/DMR/2018/102EA/2019/028) and the Union Ethics Advisory Group of the International Union against Tuberculosis and Lung Disease (The Union), Paris, France (EAG 04/19) approved the study. Written informed consent for participation in the survey was taken from the parents/guardians of children aged 1–15 years old and the consent process was approved by the ethics committees.\n\nThis was a community-based cross-sectional survey involving primary data collection.\n\nMyanmar is located in the Southeast Asia region, neighboring Laos to the east, Bangladesh to the west, Thailand to the southeast, the Republic of China to the north and northeast and India to the northwest. Myanmar has a population of 51 million with an urban:rural population ratio of 30:70. The country has 14 states and regions including Nay Pyi Taw council territory. It consists of 74 districts, 330 townships, 398 towns, 3065 wards, 13,619 village tracts and 64,134 villages10.\n\nYangon is the economic capital of Myanmar with four districts, 46 townships, 743 wards and 628 village tracts. The population of Yangon region is the highest in size when compared with other states and regions in the country. The urban:rural population ratio is 70:3010.\n\nThe study population included parents/guardians of children (1–15 years old) living in Yangon region for the first objective and children (1–15 years old) for the second objective (February to June 2019).\n\nFor the first two objectives, assuming that the prevalence of community awareness of JE and its vaccination among parents/guardians of children (1–15 years old) and vaccination coverage of children (1–15 years old) in Yangon being 50% with 5% precision at 95% CI, the calculated sample size was 384. Assuming a non-response rate of 5% and a design effect of 1.5, the final sample size was 600 households with children (1–15 years old) We used the conservative assumption of 50% prevalence as there is no previous data on community awareness and vaccination coverage in Yangon region and 5% non-response rate based on field experience.\n\nWe used three stage random sampling to sample the 600 households with children (1–15 years) from 30 clusters (ward or village tract) in nine townships in Yangon region (see Figure 1 and Figure 2). In the first two stages, we used stratified random sampling and in the third stage, we used systematic random sampling. First, we randomly selected the nine townships after stratifying them into urban, rural and mixed based on the classification used in population census in Myanmar10. Then, in the second stage, we randomly selected 30 clusters after stratifying them into wards and village tracts in each selected township. Within each township, trained field assistants went directly to the general administration department for a list of the households and map of the selected wards/village tracts. The trained field assistants conducted systematic random sampling to select 20 households with children aged 1–15 years old within each cluster. The trained field assistants chose a random starting point using the map and then selected the first household. Then, they went to next household using the sampling interval until the required sample size was reached. Sampling interval was calculated by dividing the total number of household in the selected wards or village tracts by the required sample size for that selected wards or village tracts. If the selected household did not have any children aged 1–15 years old, field assistants went to the next adjacent household with a child in this age range. If the selected house was locked or there was no parent or guardian, field assistants followed the same procedure as above. In case the selected house was an apartment building, we selected one household randomly. We interviewed the parent or guardian of the child available at the time of survey, preferably the mother.\n\nPredominantly urban (>70% urban population); Predominantly rural (>70% rural population).\n\nDuring February-June 2019, at the selected households, field assistants conducted face to face interviews with parents/guardians using a pretested structured questionnaire (see Annex 1 and Annex 3, Extended data)11,12. The field assistant asked the questions verbally to the participants and completed the questionnaires. The structured questionnaires were tested during a pilot survey and were revised according to feedback received during the pilot survey.\n\nThe questionnaire consisted of four sections. The first section highlighted the socio-demographic information. The second section was comprised of 12 questions that assessed the knowledge of participants about JE disease and prevention (including vaccination). The third section assessed perception. The fourth section included information about vaccination status of the children. If the selected household had more than one child aged 1–15 years old, we asked the parent/guardian about the vaccination status of all the children in this age range. Vaccination status was based on parents’ or guardians’ recall. Field assistants also asked about the presence or absence of a vaccination card.\n\nData from the survey forms were double-entered and validated using EpiData entry software (version 3.1, EpiData Association, Odense, Denmark). Data were analyzed using STATA (version 12.1, STATA Corp., College Station, TX, USA). There were no missing data in the study.\n\nWe provided weighted estimates as the analyses were weighted (inverse probability sampling) for the multi-stage sampling design. We used frequency and proportion to summarize the characteristics of the study participants. We assigned a knowledge score to each participant based on the number of correct or appropriate responses. Each appropriate answer was assigned one point and incorrect responses or “do not know” were assigned zero points. The scores were further dichotomized into poor or good (0–6 as poor and 7–12 as good). No overall score was calculated for perception. Vaccine coverage was calculated by the number of children that received JE vaccination (either during campaign or routine immunization) divided by the total number of children and presented as proportion and 95% confidence interval (CI).\n\nThe socio-demographic characteristics of 600 parents/guardians are presented in Table 113. Among them, 1% were aged ≤ 20 years, 4% were aged >60 years and 74% were female. A total of 50% had a high school or graduate level education and 29% had a monthly family income of more than $285 USD.\n\nJE, Japanese encephalitis; USD, United States dollar; one USD = 1525 Myanmar Kyats.\n\nParent or guardian of the 1–15 year-old children available at time of survey, preferably mother. Weighted estimates given taking into account the sampling design.\n\n* Column percentage.\n\nOverall, 37.6% exhibited good knowledge of JE. We have depicted the knowledge of respondents regarding cause, transmission, symptoms, prevention and treatment in Table 2. Among 600 parents/guardians, 49.3% had correct knowledge that JE is a fatal disease. Although 65.3% knew that the JE vaccine was available locally, only 26.8% correctly answered that vaccination is the most effective means to protect against JE.\n\nJE, Japanese encephalitis.\n\nParent or guardian of the 1–15 year-old children available at time of survey, preferably mother. Weighted estimates given taking into account the sampling design.\n\n** Column percentage.\n\nIt was found that 23% did not know the symptoms of JE, 16% responded incorrectly that JE has specific treatment and 58.8% responded that they did not know whether there is a treatment for JE or not. Participants responded that they used mosquito nets (31.3%), mosquito coils (14.1%) and spray or fumigation methods (10.7%) to avoid mosquito bites.\n\nOver half (55%) of participants perceived JE as serious in children younger than 15 years, 59% perceived the vaccine to be effective and 25% perceived JE to be harmful for pig farmers (Figure 3). Health care staff (25%) and television (17%) were the main sources of information about JE disease (Figure 4). The source of information on JE routine vaccination or campaigns was from school (35.1%), health worker visits (33.1%), announcements made using microphones in the neighborhood (13.9%) and volunteer visits (5.3%).\n\nThe variable “JE is serious if it occur in you” was included because sometimes adults think that some kinds of mosquito borne diseases such as Japanese encephalitis and Dengue are more serious in children and not serious when those diseases occur in them. Researchers in this study wanted to know whether participants had these perceptions. JE, Japanese encephalitis. Y axis as percentage (responded ‘yes’). Parent or guardian of the 1–15 year-old children available at time of survey, preferably mother. Weighted estimates given taking into account the sampling design.\n\nY axis as percentage (responded ‘yes’). Parent or guardian of the 1–15 year-old children available at time of survey, preferably mother. **Weighted estimates given taking into account the sampling design.\n\nAmong all the children in the 600 households (n=855), 831 were vaccinated. The vaccination coverage was 97.2% (95% CI: 95.9-98.1). Of 831 vaccinated children, 423 (50.9%) were cross-checked through a vaccination card. Of 831 children, 516 (62.1%) received the vaccination during the JE campaign, 234 (28.2%) during routine immunization and 67 (7.8%) received the vaccine twice during both campaign and routine vaccination. Among the 24 children that did not receive vaccination, the main reasons were: parents or guardians did not realize the importance of JE vaccination (n=4), the child was sick at the time of immunization (n=3), the parents/guardians did not know about the JE vaccination (n=2) and travel (n=6).\n\n\nDiscussion\n\nIn this region-wide survey on JE and its vaccine coverage in Yangon, Myanmar, the majority of parents or guardians did not have good knowledge of JE. Perception of the seriousness of JE disease was poor in half of participants. Vaccination coverage was excellent.\n\nData were robust as double entry and validation minimized data entry errors. Only one attempt was made to visit each household, which may impact the generalizability of the results as the households with parents and guardians who were working during the survey visit might be missed. Half of parents/guardians did not produce a vaccination card (JE routine immunization or campaign) and recall bias cannot be ruled out. However, as JE vaccination is through an injection (subcutaneous) at a specific site (in the right upper arm) and the vaccination campaign was implemented only once in 2017, we do not think this is a serious limitation.\n\nWe found that parents/guardians had poor knowledge of JE. This finding is similar to the study conducted in a slum of Kolkata by Dasgupta et al. (2016), which showed that 56.7% of mothers had poor knowledge14. Our study showed that 39.5% of participants knew that JE is caused by mosquito bites, which is higher than the 25.6% reported by Dasgupta et al.14. In our study, the majority of participants showed a lack of knowledge about treatment of the disease, which is similar to a study from India (2015) by Ahmad et al.7.\n\nMore than half of the participants perceived JE as serious in children younger than 15 years old, but they did not perceive pig farming as contributing to the threat of JE. This is similar to the study in India, where respondents did not perceive pig farming as contributing to the threat of JE15. Possible reasons for poor knowledge and perception of participants was that communities did not think of JE as a possible threat like other vector-borne diseases such as dengue and malaria.\n\nIn our study, although only 55.2% of participants knew that JE is a preventable disease and 65.3% knew the most effective means to protect JE (vaccination), almost all parents/guardians vaccinated their children. It reflects their reliance on existing health care delivery systems and EPI. JE vaccine coverage was high in this study and an effective JE vaccination catch-up campaign by EPI might have contributed to the high vaccine coverage. Coverage is similar to the study from the district of Ambala, Haryana in north India, which demonstrated a high level (93.9%) of JE vaccine coverage in the study area16. Similarly, JE vaccine coverage in Hsipaw Township, Northern Shan State, Myanmar was 93.0% in 20189.\n\nThe most important vector is Culex tritaeniorhynchus, which feeds on vertebrate hosts, primarily pigs in preference to humans17. Both larva and adult mosquito of Culex tritaeniorhynchus can spread JEV and were collected for an entomological and vector bionomics study of JE transmission done in four villages of Letpadan Township, Bago region (Myanmar), showing a high proportion (69.8%) of Culex tritaeniorhynchus in areas with high pig farming18.\n\nJE cases in Yangon region have declined in 2018 and 2019 compared to earlier years. In January – August 2019, there were only 244 AES cases and 12 confirmed JE cases19. To sustain this decline, high vaccination coverage, health education on JE and effective vector control activity should be maintained. There is a possibility that the high coverage may not be maintained in the long term. This is because within 16–20 months of JE campaign, many were not aware of JE or its vaccine. Hence, steps should be taken in this direction.\n\nAuthorities should encourage retention of cards or records of vaccination so unvaccinated children can be identified and vaccinated in future.\n\n\nConclusions\n\nJE vaccination coverage was excellent in Yangon region, Myanmar, despite the majority of parents/guardians having poor knowledge and perception of JE disease, its prevention and vaccination. In order to reduce JE incidence in community, a focus on an intensified education program is necessary to sustain the high vaccine coverage in the community.\n\n\nData availability\n\nFigshare: Annex 2. https://doi.org/10.6084/m9.figshare.10548623.v113.\n\nFigshare: Annex 1. https://doi.org/10.6084/m9.figshare.10552088.v111.\n\nFigshare: Annex 3. https://doi.org/10.6084/m9.figshare.11458437.v112.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe acknowledge the regional health department, Vector Borne Disease Control Program, Expanded Programme on Immunization, staff in medical entomology research division, the township administrative offices and basic health staff in Yangon for their support while collecting data.\n\nDisclaimer: The views expressed in this paper are of the authors and do not represent the views of the authors’ organizations.\n\n\nReferences\n\nWorld Health Organization WHO: Japanese encephalitis, Immunization, Vaccines and Biologicals, estimate of the global disease burden. 2015. (accessed on Sep 20, 2019). Reference Source\n\nCampbell GL, Hills SL, Fischer M, et al.: Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ. 2011; 89(10): 766–74, 774A–774E. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Japanese encephalitis/live attenuated vaccine. 2006. (accessed on May 20, 2006). Reference Source\n\nWorld Health Organization: The sixth Bi-regional meeting on Prevention and control of japanese encephalitis. Bangkok, Thailand, 2014. Reference Source\n\nCentral Epidemiology Unit: Monthly Epidemiology Bulletin. 2018. Reference Source\n\nVector Borne Disease Control Unit: Annual VBDC report. NayPyiTaw, Myanmar, 2014.\n\nAhmad A, Khan MU, Gogoi LJ, et al.: Japanese Encephalitis in Assam, India: Need to Increase Healthcare Workers' Understanding to Improve Health Care. PLoS One. 2015; 10(8): e0135767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHegde NR, Gore MM: Japanese encephalitis vaccines: Immunogenicity, protective efficacy, effectiveness, and impact on the burden of disease. Hum Vaccin Immunother. 2017; 13(6): 1–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThinn T, Lin TP, Thaung Y, et al.: Awareness, Perception and Vaccination of Japanese Encephalitis Vaccine among Care Givers in Hsipaw Township, Northern Shan State. 2018. (accessed on Sep 20, 2019). Reference Source\n\nMinistry of Immigration and Population: The 2014 Myanmar Population and Housing Census. NayPyiTaw, 2015. Reference Source\n\nShewade HD: Annex 1. figshare. http://www.doi.org/10.6084/m9.figshare.10552088.v1\n\nShewade H: Annex 3. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.11458437.v1\n\nShewade HD: Annex 2. figshare. http://www.doi.org/10.6084/m9.figshare.10548623.v1\n\nAparajita D, Atanu D, Bobby P, et al.: Perception of Prevention of Japanese Encephalitis with Emphasis on Its Vaccination Programme: A Community Based Study In a Slum of Kolkata. Int J Public Heal Res. 2017; 7(2): 845–851. Reference Source\n\nChaturvedi S, Sharma N, Kakkar M: Perceptions, practices and health seeking behaviour constrain JE/AES interventions in high endemic district of North India. BMC Public Health. 2017; 17(1): 645. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar D, Singh AJ: Response of Various Stakeholders towards Newly Introduced Japanese Encephalitis Vaccine in a North Indian State. J Vaccines. 2014; 1–4. Publisher Full Text\n\nGupta N, Rao PV: Transcriptomic profile of host response in Japanese encephalitis virus infection. Virol J. 2011; 8: 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMya MM, Lin Z, Min Hein ZN, et al.: Vector bionomics of Culex species responsible for Japanese Encephalitis transmission in 4 villages of Letpadan Township Bago region, Myanmar. Myanmar Heal Res J. 2017; 48: 37–39.\n\nCentral Epidemiology Unit: AFP surveillance Indicators by State and Region, Fever with Rash Surveillance. 2019. Reference Source" }	[ { "id": "62066", "date": "27 Apr 2020", "name": "Jing An", "expertise": [ "Reviewer Expertise Interaction between viruses and hosts", "mosquito-borne flavivirus", "vaccine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 here conducted a set of survey data to reflect the public awareness on JEV in community. A great deal of data collection was presented. These contribute to show the significant progresses of the JE campaign in Yangon. However, there are several questions to be answered.\n\nSome Comments:\n\nThe whole article was simple as a scientific research. More in-depth analysis of data is recommended. More comparison should be conducted to reflect the details in multi-aspects. For example, \"parents/guardians had poor knowledge of JE\" is sentenced. Since the details of the guardians were investigated, the correlation should be conducted, like with age? or occupations? or else? or none. Besides, are there any differences in awareness between urban and rural places?...\n\nTo fully reflect the effect of public awareness in Yangon, authors should not choose nine townships randomly. Especially the locations are concentrated. The choices need to be more meaningful, like the widest vector distribution ones, the most severely suffered ones. OR, at least, the details of these nine townships should be presented, like incidence cases.\n\nThe complex relationship between JEV and congeneric members, like DENV and ZIKV, is widely proposed. Since dengue is prevalent locally, the vaccination against Flavivirus members or infections should be also considered in the questionnaire.\n\nIn table 1, (1) is single parent family excluded? (2) what does \"Do not know family members suffered JE\" exactly mean? do not know the symptoms or do not know the children's conditions?\n\n\"23% did not know the symptoms of JE\" is sentenced. So, how will these parents know their kids are not suffered from JE? Is the \"0\" morbidity presented by parents credible?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5658", "date": "30 Jun 2020", "name": "Pyae Phyo Kyaw", "role": "Author Response", "response": "1. The whole article was simple as a scientific research. More in-depth analysis of data is recommended. More comparison should be conducted to reflect the details in multi-aspects. For example, \"parents/guardians had poor knowledge of JE\" is sentenced. Since the details of the guardians were investigated, the correlation should be conducted, like with age? or occupations? or else? or none. Besides, are there any differences in awareness between urban and rural places?... Response : Thank you for your suggestion and we performed more in-depth analysis to determine the socio-demographic characteristics associated with good knowledge score and presented in the table-3. (line 218-234) We also performed an in-depth analysis to determine the good knowledge score with the child being vaccinated and presented in the table-4. (line 239-244) 2. To fully reflect the effect of public awareness in Yangon, authors should not choose nine townships randomly. Especially the locations are concentrated. The choices need to be more meaningful, like the widest vector distribution ones, the most severely suffered ones. OR, at least, the details of these nine townships should be presented, like incidence cases. Response : We believe that random selection of township would provide generalizable information for the Yangon region. If we selected based on the incidence rate of JE cases or vector distribution, the knowledge, perception and vaccination coverage would not be representative to the population in Yangon. We hope this is fine. (line 78-90) 3. The complex relationship between JEV and congeneric members, like DENV and ZIKV, is widely proposed. Since dengue is prevalent locally, the vaccination against Flavivirus members or infections should be also considered in the questionnaire. Response: In Myanmar, we have no vaccination programme against Dengue and Flavivirus infections and we did not have the opportunity to explore about this. 4. In table 1, (1) is single parent family excluded? (2) what does \"Do not know family members suffered JE\" exactly mean? do not know the symptoms or do not know the children's conditions? Response: (1) Single parent family was not excluded, considered under nuclear family. In type of family, Nuclear family means family which has father or mother with their children. Extended family means family which has either father, mother or grandfather, grandmother or uncle, aunty with children. We added this in the footnote of the table. (line 159-161) (2) \"Do not know family members suffered JE\" means parents/guardians did not know their children have previous history of infection with JE virus. (We removed that variable from table-1 as the author group feels it does not add valuable information) 5. \"23% did not know the symptoms of JE\" is sentenced. So, how will these parents know their kids are not suffered from JE? Is the \"0\" morbidity presented by parents credible? Response: The answer by the participant to the question on JE morbidity by asking whether the family member suffered JE before was based on the clinic or hospital record. So it is possible that the participant can report whether or not their children suffered JE even they did not know the symptoms of JE. Anyway, I removed that variable from table 1 as the author group feels it does not add valuable information." } ] }, { "id": "63433", "date": "29 May 2020", "name": "Babasaheb V. Tandale", "expertise": [ "Reviewer Expertise Epidemiology of virus diseases causing outbreaks." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 composed well and has been presented in a very simple way making it very easy to understand by the readers. As indicated by the other reviewer, more in-depth analysis and comparison to explain the similarities and differences in knowledge and perception of JE disease and JE vaccine may be considered along with the explanatory variables for coverage of JE vaccination. The comparisons would thus help identify the important aspects that could be targeted for improvements.\n\nThe sampling design could be better explained with sampling ratios planned at different phases of sampling.\n\nVector distribution / density has been indicated in title, however it is not dealt in abstract, objectives, methods and results. Therefore, this aspect may be dropped from title also. It could be mentioned in discussion section as the secondary data and indicated for its importance for understanding of study findings.\n\nThe higher coverage of vaccination in spite of low level of knowledge and perception of JE disease and JE vaccine is difficult to understand. It may be highlighted and discussed in details.\n\nThe objectives may be clearly mentioned at the end of background section.\n\nThe potential sources of bias in selection, verification of information and interpretation of information given by respondents needs to be critically appraised and discussed. The confounders also need to be addressed properly.\n\nThe verification of vaccination card for ascertaining vaccination recall needs to be addressed with efforts made for data quality.\n\nThe sample size and statistical analysis plan could be included with specific details on the basis of assumptions and proposed analysis. The sample size estimation could have been based on reported coverage of vaccination to be 93% in small survey findings earlier. The weighting approach needs to be provided clearly with its application in crude/unadjusted and adjusted/corrected coverage.\n\nThe survey currently presented is mostly descriptive in nature and needs to be made analytical with comparisons that could be made and presented.\n\nThe questionnaire validity may be presented with variable responses for similar aspects.\n\nNon-responses and its handling in analysis may be included.\n\nThe baseline characteristics of respondents may be compared with good or bad knowledge, acceptable perception or otherwise and vaccinated and unvaccinated.\n\nLimitations of study may be discussed with interpretations based on the same.\n\nGeneralisability of study findings may be considered and discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5659", "date": "30 Jun 2020", "name": "Pyae Phyo Kyaw", "role": "Author Response", "response": "1. The manuscript is composed well and has been presented in a very simple way making it very easy to understand by the readers. As indicated by the other reviewer, more in-depth analysis and comparison to explain the similarities and differences in knowledge and perception of JE disease and JE vaccine may be considered along with the explanatory variables for coverage of JE vaccination. The comparisons would thus help identify the important aspects that could be targeted for improvements. Response: Thank you for your suggestion and we performed more in-depth analysis to determine the socio-demographic characteristics associated with good knowledge score and presented in the table-3. (line 218-234) We also performed an in-depth analysis to determine the good knowledge score with the child being vaccinated and presented in the table-4. (line 239-244) 2. The sampling design could be better explained with sampling ratios planned at different phases of sampling. Response: We added a detailed explanation of the sampling design and procedure in the method section. (line 81-105) 3. Vector distribution / density has been indicated in title, however it is not dealt in abstract, objectives, methods and results. Therefore, this aspect may be dropped from title also. It could be mentioned in discussion section as the secondary data and indicated for its importance for understanding of study findings. Response: We have revised the title according to your suggestion. 4. The higher coverage of vaccination in spite of low level of knowledge and perception of JE disease and JE vaccine is difficult to understand. It may be highlighted and discussed in details. Response: We have added a discussion in the revised manuscript. (line 276-282) 5. The objectives may be clearly mentioned at the end of background section. Response: We revised the paragraph at the end of introduction section and stated the objectives clearly. (line 42-44) 6. The potential sources of bias in selection, verification of information and interpretation of information given by respondents needs to be critically appraised and discussed. The confounders also need to be addressed properly. Response: We have discussed the potential for recall bias as half the respondents could not produce the immunization or JE campaign card (for details see line 251-258). We performed more in-depth analysis to assess factors associated. (line 145-148) (line 218-226) 7. The verification of vaccination card for ascertaining vaccination recall needs to be addressed with efforts made for data quality. Response: We were able to verify 50% of the vaccinated children with their vaccination card. We acknowledged that the respondents who did not have vaccination card would subject to recall bias. (line 254-255) 8. The sample size and statistical analysis plan could be included with specific details on the basis of assumptions and proposed analysis. The sample size estimation could have been based on reported coverage of vaccination to be 93% in small survey findings earlier. The weighting approach needs to be provided clearly with its application in crude/unadjusted and adjusted/corrected coverage. Response: We agree that we should have calculated the sample size based on a previous estimate. But we decided to assume a coverage of 50% that would give a maximum sample size. We have added a detailed description on sample size calculation and statistical analysis in the revised manuscript. We provided weighted estimates as the analyses were weighted (inverse probability sampling) for the multi-stage sampling design, this was done both for the descriptive and analytical calculations (line 82-89, 136-137) 9. The survey currently presented is mostly descriptive in nature and needs to be made analytical with comparisons that could be made and presented. Response: We performed more in-depth analysis to determine the socio-demographic characteristics associated with good knowledge score and presented in the table-3. (line 218-240) We also performed an in-depth analysis to determine the good knowledge score with the child being vaccinated and presented in the table-4. (line 239-244) 10. The questionnaire validity may be presented with variable responses for similar aspects. Response: Thank you for the comment. Unfortunately, we do not have information on questionnaire validity. 11. Non-responses and its handling in analysis may be included. Response: There was no non-responses in our study because we went to the household using the sampling interval until the required sample size was reached. We added this information in the revised manuscript. (line 104-105) 12. The baseline characteristics of respondents may be compared with good or bad knowledge, acceptable perception or otherwise and vaccinated and unvaccinated. Response: We have added results on the association between the baseline characteristics of respondents with knowledge level and vaccination status. (line 218-240) 13. Limitations of study may be discussed with interpretations based on the same. Response: We have added this discussion in our revised manuscript. (line 250-258) 14. Generalisability of study findings may be considered and discussed. Response: We have added this discussion in our revised manuscript. (line 250-258)" } ] } ]	1	https://f1000research.com/articles/9-6
https://f1000research.com/articles/9-99/v1	10 Feb 20	{ "type": "Systematic Review", "title": "Comparison of major bleeding in patients with acute coronary syndrome that underwent coronary artery bypass grafting treated with clopidogrel or ticagrelor: a systematic review and meta-analysis", "authors": [ "Mohammad Saifur Rohman", "Yeni Purnamasari", "Muhammad Ilmawan", "Bagus Aulia Mahdi", "Fredo Tamara", "Aditya Indra Mahendra", "Mazen Mazen", "Teuku Heriansyah", "Muhammad Yamin", "Budi Susetio Pikir", "Jonny Karunia Fajar", "Mohammad Saifur Rohman", "Yeni Purnamasari", "Muhammad Ilmawan", "Bagus Aulia Mahdi", "Aditya Indra Mahendra", "Mazen Mazen", "Muhammad Yamin" ], "abstract": "Background: There is controversy among physicians regarding the use of dual antiplatelet therapy (DAPT) in acute coronary syndrome (ACS) patients treated with coronary artery bypass grafting (CABG). Moreover, the evidence of previous studies about this topic remained inconclusive. This study aimed to perform a meta-analysis concerning the relation between the risk of major bleeding and the use of different DAPT (clopidogrel or ticagrelor) in ACS patients treated with CABG. Methods: A meta-analysis was conducted during March to October 2019. Searches were carried out in Pubmed, Embase, Cochrane, and Web of Science. The predictor covariate in our present study was DAPT (clopidogrel or ticagrelor), and the outcome measure was the risk of major bleeding. Sub-group analysis was also performed, where data were classified into pre- and post-CABG. Furthermore, to determine the correlation and effect estimation, data were analyzed using fixed or random effect model. Results: A total of 13 studies consisting 34,015 patients treated with clopidogrel and 32,661 patients treated with ticagrelor was included in our study. Our pooled calculation revealed that the incidence of major bleeding was not different significantly between clopidogrel and ticagrelor. In pre- and post-CABG sub-groups, our results also found no significant difference in major bleeding incidence between clopidogrel and ticagrelor groups. Conclusions: Our meta-analysis clarifies that clopidogrel, compared to ticagrelor, or vice versa, is not associated with the risk of major bleeding in ACS patients treated with CABG.", "keywords": [ "major bleeding", "coronary artery bypass grafting", "clopidogrel", "ticagrelor" ], "content": "Introduction\n\nIn the last two decades, the management of acute coronary syndrome (ACS) has been well defined and periodically updated. Management has developed drastically over this period1. Management options are numerous, and they depend on the facilities of the hospital. Of these treatment options, coronary artery bypass grafting (CABG) is considered the most challenging and the final option when other treatment options, including percutaneous coronary intervention (PCI) and thrombolytic therapy, fail to restore blood flow in the infarct-related artery2. Moreover, the drugs used in ACS patients in all management options are complex, and dual antiplatelet therapy (DAPT) is commonly used. DAPT is globally used to treat patients with ACS. It was first reported in 19963, and was first recommended for treating ACS patients in 2007 in American College of Cardiology (ACC)/American Heart Association (AHA) guidelines4. Since then, DAPT has been widely used in the early management of ACS patients5,6.\n\nRecently, when performing DAPT, whether to use clopidogrel or ticagrelor (the choice between acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor) has remained controversial due to the current assumption that one of the two might provide higher risk of major bleeding7,8. In the Indonesian National Health Insurance drug catalog, in 2018 clopidogrel was withdrawn and substituted with ticagrelor. However, in the drug price list (https://e-katalog.lkpp.go.id/; website in Indonesian), ticagrelor is more expensive than clopidogrel. It is unclear whether the assumptions made about the risk of major bleeding caused by clopidogrel or ticagrelor were supported by the evidence or were possibly the result of conspiracy among pharmaceutical industries to increase their products marketing. Ticagrelor may provide a more potent platelet inhibition effect, therefore reducing the risk of a thrombotic event9. In the context of ACS, the greater effect may be accompanied more complications. Therefore, the benefits of DAPT and the risk of complications (bleeding) should balance. In the case of ACS patients undergoing CABG, to prevent major bleeding, it is recommended that DAPT should be discontinued for at least three and five days before elective CABG for ticagrelor and clopidogrel, respectively10. Furthermore, in the case of emergency or urgent CABG, DAPT should be discontinued prematurely11. The discontinuation of DAPT might increase the risk of a thrombotic event12. However, delay in CABG had also been shown to associate with poor clinical outcome and increased risk of mortality13. Therefore, identifying the appropriate DAPT, whether ticagrelor or clopidogrel, is crucial to prevent the risk of major bleeding. Although 2016 ACC/AHA guidelines recommended ticagrelor over clopidogrel because ticagrelor is considered to have a more potent anti-platelet effect than clopidogrel14, the evidence from previous studies regarding the association between the risk of major bleeding and the use of different DAPT using either clopidogrel or ticagrelor in ACS patients treated with CABG were inconclusive. Therefore, those inconclusive data of previous studies required clarification using a meta-analysis approach.\n\nTherefore, the present study aimed to perform a meta-analysis whether the use of different DAPT (clopidogrel or ticagrelor) might affect the risk of major bleeding or not in ACS patients treated with CABG. Our study outcome could clarify the real effect of the use of DAPT (clopidogrel or ticagrelor) to the risk of major bleeding in ACS patients treated with CABG. Moreover, we also expect that our current meta-analysis might correct previous assumptions concerning the use of different DAPT.\n\n\nMethods\n\nA Meta-analysis was performed during March to October 2019 to assess the association between the incidence of major bleeding and the use of DAPT either clopidogrel or ticagrelor in ACS patients treated with CABG. In effort to attain our goal, potentially relevant papers were identified and collected from PubMed, Embase, Cochrane, and Web of Science to calculate odd ratio (OR) and 95% confidence interval (95%CI) using either fixed or random effect model. A checklist adapted from Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) and the design of our previous meta-analyses15–20 were used to guide the meta-analysis protocols in our present study21. See Reporting guidelines for a completed PRISMA checklist for this study22.\n\nWe conducted a systematic search in PubMed, Embase, Cochrane, and Web of Science up to 20 September 2019. The search strategy, conformed to medical subjects heading (MeSH), involved the use of combination the following keywords: [\"Major Bleeding\"] AND [\"Coronary Artery Bypass Grafting\" OR \"CABG\"] AND [\"Dual Anti Platelet Therapy\" OR \"DAPT\"], and [\"Clopidogrel\" OR \"Ticagrelor\"]. In our searching strategy, language restrictions were not applied. We only used the study with the larger sample size and that was more up-to-date if we found the same data among studies. Moreover, we also searched the potential papers from the reference list of relevant or eligible studies. We also employed the \"related article\" option in PubMed to broaden our searching strategy. The potentially relevant papers were identified by two independent investigators (Y.P., M.I.). Disagreement between two independent investigators was resolved by discussion and/or by consulting to the senior investigator (J.K.F.).\n\nThe inclusion criteria for this study were (1) retrospective studies, (2) prospective studies, (3) randomized controlled trials (RCTs), (4) evaluating the association between the incidence of major bleeding and DAPT either using clopidogrel or ticagrelor in ACS patients treated with CABG, (5) providing sufficient data for calculation of OR with 95% CI. While, articles were excluded if the following criteria were found: (1) irrelevant topic, (2) review, (3) conference presentation, and (4) having low quality (see Quality assessment). For data extraction, information related to (1) name of the first author, (2) year of publication, (3) country of origin, (4) sample sizes of case and controls, and (5) the incidence of major bleeding were extracted from each study. To prevent human errors, data extraction was performed by two independent authors. If discrepancy occurred, a consensus or discussion was established.\n\nThe predictor covariate in this study was DAPT either using clopidogrel or ticagrelor. While, the main outcome measure was the incidence of major bleeding in patients receiving both clopidogrel and ticagrelor. The major bleeding included in our analysis was restricted to thrombolysis in myocardial infarction23 and platelet inhibition and outcomes criteria24. Moreover, to confer a comprehensive analysis, we also performed sub-group analysis. Data were classified into the incidence of major bleeding in ACS patients treated with DAPT (clopidogrel or ticagrelor) before and after CABG.\n\nTo ensure the quality of each study and to avoid the potential bias in each study, the quality of retrieved studies was controlled and collected by two independent investigators (Y.P., M.I.). The quality and risk of bias of each study was assessed using Methodological Index for Non-Randomized Studies (MINORS) score25. The MINORS score ranged from 0 to 24, and consisted of 12 items. Each item was assessed as 0 if the item was not reported, 1 if the item was inadequate reported, and 2 if the item was adequate reported. Each study was interpreted as having low quality if the score was less than or equal to 12, moderate if the score was less than or equal to 16 and more than 12, and high quality if the score was more than 1625. If disagreement was found between two independent authors, consensus was achieved through discussion between the two investigators. If the disagreement was not resolved, a consultation to senior researcher (JKF) was conducted.\n\nThe comparison and effect estimation of major bleeding between DAPT with clopidogrel and ticagrelor were determined using the Z-test. The pooled calculation and effect estimation were described using forest plots. The model of forest plot for describing the comparison and effect estimation was conformed with a Q test. Before analysis using the Z-test, we evaluated heterogeneity and potential publication bias. A Q-test was employed to evaluate heterogeneity. P-value of less than 0.10 was considered to indicate heterogeneity. If we found heterogeneity, a random effect model was used. While, if heterogeneity was not found, a fixed effect model was used. For testing publication bias, an Egger test was used. A P-value of less than 0.05 was considered significantly having publication bias. All analyses in our study were carried out using Review Manager version 5.3 (RevMan Cochrane, London, UK) and Comprehensive Meta-Analysis (CMA, New Jersey, US) version 2.1.\n\n\nResults\n\nA flowchart of article searches and study selection is shown in Figure 1. Initially, 37 articles were identified from the literature search. However, eight of them were excluded because they did not have relevance to the topic, leaving a total of 29 articles. The full text of these articles was retrieved and reviewed; it was found that 16 studies did not meet the eligibility criteria because they were reviews (n=5), commentaries (n=4), family-based studies (n=3), included the same study data (n=2), and not providing sufficient data for calculation of OR and 95%CI (n=2). Finally, a total of 13 studies were eligible for our meta-analysis. Baseline characteristics of studies included in our analysis are provided in Table 1.\n\nMB, major bleeding; n, sample size; CABG, coronary artery bypass grafting; RCT, randomized controlled trial; MINORS, Methodological Index for Non-Randomized Studies; STEMI, ST-Elevation Myocardial Infarction; NSTEMI, Non-ST-elevation myocardial infarction; ACS, acute coronary syndrome.\n\nA total of 13 studies2,26–37, consisting 34,014 patients treated with clopidogrel and 32,661 patients treated with ticagrelor, were included in our study. Of those, the correlation between the use of DAPT (either clopidogrel or ticagrelor) and the risk of major bleeding was found in only three studies29,30,37. A further ten studies failed to clarify the association2,26–28,31–36. Our calculation revealed (Figure 2A) that the incidence of major bleeding was not significantly different between clopidogrel and ticagrelor (OR = 1.10, 95%CI = 0.98–1.24, p = 0.0990). Moreover, in pre-CABG sub-group, we included nine studies28–33,35–37 consisting of 15,109 patients treated with clopidogrel and 13,939 patients treated with ticagrelor. Our results found (Figure 2B) that no significant different of major bleeding incidence was observed between clopidogrel and ticagrelor (OR = 1.19, 95%CI = 0.97–1.45, p = 0.0910). While, in the post-CABG sub-group, a total of four papers2,26,27,34 consisting of 18,905 patients treated with clopidogrel and 18,722 patients treated with ticagrelor was enrolled for our analysis. Our pooled data (Figure 2C) confirmed no significant different in major bleeding incidence between clopidogrel and ticagrelor (OR = 1.00, 95%CI = 0.93–1.08, p = 0.9230). The summary of correlation and effect estimation between the risk of major bleeding and the use of different DAPT is provided in Table 2.\n\n(A) Overall analysis. (B) Pre-coronary artery bypass grafting (CABG) sub-group. (C) Post-CABG sub-group.\n\nCABG, coronary artery bypass grafting; MB, major bleeding; OR, odds ratio; CI, confidence interval; pHet, p heterogeneity; pE, p Egger.\n\nEvidence of heterogeneity was assessed using the Q-test. Our analysis found that evidence of heterogeneity (p <0.10) was observed in overall analysis and pre-CABG sub-group. Therefore, random effect model was applied to determine the correlation and effect estimation. While, for post-CABG sub-group, we used fixed effect model to assess the correlation and effect estimation because we did not find the evidence of heterogeneity. Furthermore, potential publication bias was assessed using an Egger test. Our analysis confirmed that potential publication bias was found in post-CABG sub-group (p <0.05). In overall analysis and pre-CABG sub-group, we found no publication bias. The summary of study heterogeneity and potential publication is described in Table 2.\n\n\nDiscussion\n\nIn our present meta-analysis, we included 13 papers consisting of 34,014 and 32,661 patients treated with clopidogrel and ticagrelor following CABG, respectively. Our current findings confirmed that neither clopidogrel nor ticagrelor was associated with risk of major bleeding among ACS patients treated with CABG. To our knowledge, no previous meta-analysis has reported the comparison between clopidogrel and ticagrelor in the context of CABG. Therefore, we were unable to perform a direct comparison. However, in other case settings, meta-analyses have been conducted in the case of PCI and thrombolytic for treating ACS patients. In the case of PCI for treating ACS patients, the reports from previous meta-analyses remained conflicting. A study conducted by Fan et al.38 found that clopidogrel was associated with increased risk of major bleeding compared to ticagrelor. On the other hand, Guan et al.39 revealed that ticagrelor was proven to correlate with increased risk of major bleeding compared to clopidogrel. A meta-analysis conducted by Westman et al.40 involved 15 papers, consisting of 26,093 patients treated with clopidogrel and 7,192 patients treated with ticagrelor. The authors revealed that although ticagrelor was associated with increased risk of minor bleeding compared to clopidogrel, the incidence of major bleeding was not significantly different between ticagrelor and clopidogrel. Moreover, in the case of fibrinolytic, a meta-analysis involving three RCTs showed that neither ticagrelor nor clopidogrel was correlated with the risk of major bleeding41. Furthermore, in the case of ACS, a meta-analysis involving 10 studies revealed that the risk of bleeding was not significantly different between patients receiving clopidogrel and ticagrelor42. Therefore, it makes sense that in our current meta-analysis, no association was observed between the use of DAPT either clopidogrel or ticagrelor and the risk of major bleeding.\n\nTo provide a comprehensive analysis, we also performed subgroup analysis, divided into pre- and post-CABG. Our findings in sub-group analysis were consistent with our main findings, we emphasized that the incidence of major bleeding either in pre- and post-CABG was not significantly different between clopidogrel and ticagrelor. To our knowledge, until now the major bleeding effect of clopidogrel and ticagrelor in the setting of before and after CABG has not been well defined. Besides the existence of no previous meta-analysis concerning this subject, reports in other case settings did not assess this effect in the pre- or post-intervention context of. Hence, the possible direct and indirect explanations was difficult to clarify. To date, the major bleeding effect of DAPT therapy before and after CABG remained conflicting. A previous study revealed that discontinuation of DAPT therapy 24–72 hours before emergency CABG was proven to increase the risk of major bleeding35. Moreover, Deo et al.43 also reported that increased risk of major bleeding was observed in post CABG patients treated with ASA and clopidogrel. However, a study by Solo et al.44 might support our findings. They evaluated the incidence of major bleeding between ASA and clopidogrel and ASA and ticagrelor. Although statistical analysis was not directly performed, they confirmed that the risk of major bleeding in post CABG patients among different anti-platelets had no strong evidence. Therefore, due to inconclusive reports regarding the risk of major bleeding and DAPT therapy before and after CABG, further studies are required to clarify our current findings.\n\nThe theory underlying the risk of major bleeding due to clopidogrel or ticagrelor is not well defined. However, some theories have been proposed. To stimulate inhibition of platelet aggregation, both clopidogrel and ticagrelor are P2Y12 antagonists. However, associated with the risk of major bleeding, each agent has a different mechanism. Clopidogrel is known to irreversibly induce bleeding by inhibiting P2Y12 receptors, and may cause persistent blockade of the adenosine diphosphate (ADP) binding site. Those inhibitory effects may persist until the platelets are renewed in 7–10 days45. Therefore, as it has a longer inhibitory effect than ticagrelor, those treated with clopidogrel may be more vulnerable to risk of bleeding than ticagrelor42. Ticagrelor is a reversible P2Y12 receptor antagonist. It works directly on P2Y12 receptors, and therefore may produce rapid inhibition effects and provide rapid recovery of platelet function46. It has been already reported that ticagrelor has faster onset and offset than clopidogrel47. As a result, when each drug is stopped, the effect of ticagrelor may disappear faster than clopidogrel. In animal subjects, a study proposed that clopidogrel was found to have 3.5-fold associated with higher bleeding risk compared to ticagrelor48. Clopidogrel is metabolized by cytochrome P2C19 enzyme49, and recent gene-disease interaction studies reported that cytochrome P2C19 CYP2C19*20 C-889T>G (SNP rs11568732) was associated with the risk of bleeding in ACS patients treated with clopidogrel50–52. Therefore, theoretically, the risk of major bleeding with clopidogrel should be higher than with ticagrelor. However, the evidence from previous large-scale studies, including our present meta-analysis, are conflicting and have not clarified the association. Hence, because it was not supported by the evidence, in our opinion, the risk of major bleeding due to different DAPT, for this time being, might be considered as a hypothesis. In the near future, we expected that more complex study designs might be applied to elucidate the real association between the risk of major bleeding and the use of different DAPT.\n\nTo the best of our knowledge, our present study was the first meta-analysis assessing the association between the risk of major bleeding and the use of different DAPT in ACS patients treated with CABG. Our current meta-analysis might clarify the inconclusive findings of previous studies regarding this topic, and we emphasized that clopidogrel, compared to ticagrelor, or vice versa, was not associated with the risk of major bleeding in ACS patients treated with CABG. In the last decade, the use of DAPT, either clopidogrel or ticagrelor, has brought about a dilemma for physicians due to the assumption that one of them was considered to trigger the risk of major bleeding. This dilemma was worsened owing to drug marketing competition among pharmaceutical industries to recommend ticagrelor over clopidogrel. However, our present study indicates that the dilemma was not supported by evidence, and therefore the dilemma might be considered as \"the ocean without the waves\". The present meta-analysis emphasizes the safety of DAPT administration, either clopidogrel or ticagrelor, in the context of the risk of major bleeding, and hence we expect that our present meta-analysis might reduce the dilemma regarding the risk of major bleeding due to the use of DAPT either clopidogrel or ticagrelor among physicians. The management of ACS patients using CABG has developed in the last decade, and therefore the use of DAPT in CABG management should conform with the adequate evidence. Furthermore, we hope that our current meta-analysis might be involved in the future revision of CABG management for treating patients with ACS.\n\nIn our present study, several crucial limitations were observed. First, some factors that might contribute to the risk of major bleeding, such as coagulation factors, history of stroke, chronic kidney disease, hyperglycemia, and anemia53, were not included and controlled for. Second, our current findings should be interpreted with caution due to relatively small sample size. Third, more than a half of our included studies were cross-sectional studies. Therefore, further studies with higher study design are required to obtain better evidence. Fourth, human factors (skills) were not involved in the analysis. Fifth, other drugs that might govern the risk of bleeding were not analyzed.\n\n\nConclusion\n\nOur meta-analysis reveals that the use of different DAPT either clopidogrel or ticagrelor is not associated with the risk of major bleeding in ACS patients treated with CABG. Our sub-group analysis also fails to confirm this association both in pre- and post-CABG sub-groups. Our findings may provide the clarification of previous conflicting studies in the context of the risk of major bleeding and the use of different DAPT in ACS patients treated with CABG. We also expect that our findings may contribute to the future recommendation of the use of DAPT among ACS patients treated with CABG.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Comparison of major bleeding in patients with acute coronary syndrome that underwent coronary artery bypass grafting treated with clopidogrel or ticagrelor: a systematic review and meta-analysis’. https://doi.org/10.6084/m9.figshare.11688525.v122.", "appendix": "Acknowledgements\n\nWe thank MIT-Indonesia Research Alliance (MIRA), Lembaga Pengelola Dana Pendidikan (LPDP), and DSKF publishing campus for supporting our project.\n\n\nReferences\n\nBurlacu A, Tinica G, Nedelciuc I, et al.: Strategies to Lower In-Hospital Mortality in STEMI Patients with Primary PCI: Analysing Two Years Data from a High-Volume Interventional Centre. J Interv Cardiol. 2019; 2019: 3402081. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang HW, Kim HJ, Yoo JS, et al.: Clopidogrel versus Ticagrelor for Secondary Prevention after Coronary Artery Bypass Grafting. J Clin Med. 2019; 8(1): pii: E104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchomig A, Neumann FJ, Kastrati A, et al.: A randomized comparison of antiplatelet and anticoagulant therapy after the placement of coronary-artery stents. N Engl J Med. 1996; 334(17): 1084–9. PubMed Abstract \| Publisher Full Text\n\nAnderson JL, Adams CD, Antman EM, et al.: ACC/AHA 2007 guidelines for the management of patients with unstable angina/non ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 2002 Guidelines for the Management of Patients With Unstable Angina/Non ST-Elevation Myocardial Infarction): developed in collaboration with the American College of Emergency Physicians, the Society for Cardiovascular Angiography and Interventions, and the Society of Thoracic Surgeons: endorsed by the American Association of Cardiovascular and Pulmonary Rehabilitation and the Society for Academic Emergency Medicine. Circulation. 2007; 116(7): e148–304. PubMed Abstract \| Publisher Full Text\n\nAmsterdam EA, Wenger NK, Brindis RG, et al.: 2014 AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014; 64(24): e139–e228. PubMed Abstract \| Publisher Full Text\n\nRoffi M, Patrono C, Collet JP, et al.: 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC). Eur Heart J. 2016; 37(3): 267–315. PubMed Abstract \| Publisher Full Text\n\nArora S, Shemisa K, Vaduganathan M, et al.: Premature Ticagrelor Discontinuation in Secondary Prevention of Atherosclerotic CVD: JACC Review Topic of the Week. J Am Coll Cardiol. 2019; 73(19): 2454–64. PubMed Abstract \| Publisher Full Text\n\nLi B, Jin X, Wang L, et al.: Loss of dominance of ticagrelor over clopidogrel in East Asian patients with acute coronary syndrome. Int J Clin Exp Med. 2019; 12(5): 4528–39. Reference Source\n\nJiang Z, Zhang R, Sun M, et al.: Effect of Clopidogrel vs Ticagrelor on Platelet Aggregation and Inflammation Markers After Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction. Can J Cardiol. 2018; 34(12): 1606–12. PubMed Abstract \| Publisher Full Text\n\nValgimigli M, Bueno H, Byrne RA, et al.: 2017 ESC focused update on dual antiplatelet therapy in coronary artery disease developed in collaboration with EACTS: The Task Force for dual antiplatelet therapy in coronary artery disease of the European Society of Cardiology (ESC) and of the European Association for Cardio-Thoracic Surgery (EACTS). Eur Heart J. 2018; 39(3): 213–60. PubMed Abstract \| Publisher Full Text\n\nWallentin L, Becker RC, Budaj A, et al.: Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009; 361(11): 1045–57. PubMed Abstract \| Publisher Full Text\n\nGimbel ME, Minderhoud SCS, Ten Berg JM: A practical guide on how to handle patients with bleeding events while on oral antithrombotic treatment. Neth Heart J. 2018; 26(6): 341–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSobolev BG, Levy AR, Kuramoto L, et al.: The risk of death associated with delayed coronary artery bypass surgery. BMC Health Serv Res. 2006; 6: 85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevine GN, Bates ER, Bittl JA, et al.: 2016 ACC/AHA Guideline Focused Update on Duration of Dual Antiplatelet Therapy in Patients With Coronary Artery Disease: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines: An Update of the 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention, 2011 ACCF/AHA Guideline for Coronary Artery Bypass Graft Surgery, 2012 ACC/AHA/ACP/AATS/PCNA/SCAI/STS Guideline for the Diagnosis and Management of Patients With Stable Ischemic Heart Disease, 2013 ACCF/AHA Guideline for the Management of ST-Elevation Myocardial Infarction, 2014 AHA/ACC Guideline for the Management of Patients With Non-ST-Elevation Acute Coronary Syndromes, and 2014 ACC/AHA Guideline on Perioperative Cardiovascular Evaluation and Management of Patients Undergoing Noncardiac Surgery. Circulation. 2016; 134(10): e123–55. PubMed Abstract \| Publisher Full Text\n\nFajar JK, Andalas M, Harapan H: Comparison of Apgar scores in breech presentations between vaginal and cesarean delivery. Ci Ji Yi Xue Za Zhi. 2017; 29(1): 24–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFajar JK, Harapan H: Socioeconomic and attitudinal variables associated with acceptance and willingness to pay towards dengue vaccine: a systematic review. Arch Clin Infet Dis. 2017; 12(3): e13914. Publisher Full Text\n\nFajar JK, Heriansyah T, Rohman MS: The predictors of no reflow phenomenon after percutaneous coronary intervention in patients with ST elevation myocardial infarction: A meta-analysis. Indian Heart J. 2018; 70(Suppl 3): S406–S18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFajar JK, Mahendra AI, Tamara F, et al.: The Association Between Complete Blood Count and the Risk of Coronary Heart Disease. Turkiye Klinikleri J Med Sci. 2019; 39(1): 56–64. Publisher Full Text\n\nFajar JK, Taufan T, Syarif M, et al.: Hip geometry and femoral neck fractures: A meta-analysis. J Orthop Translat. 2018; 13: 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrihatiningsih S, Fajar JK, Tamara F, et al.: Risk factors of tuberculosis infection among health care workers: a meta-analysis. Indian J Tuberc. 2019. Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFajar JK: PRISMA: Comparison of major bleeding in patients with acute coronary syndrome that underwent coronary artery bypass grafting treated with clopidogrel or ticagrelor: a systematic review and meta-analysis. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11688525.v1\n\nMehran R, Rao SV, Bhatt DL, et al.: Standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the Bleeding Academic Research Consortium. Circulation. 2011; 123(23): 2736–47. PubMed Abstract \| Publisher Full Text\n\nHusted S, James SK, Bach RG, et al.: The efficacy of ticagrelor is maintained in women with acute coronary syndromes participating in the prospective, randomized, PLATelet inhibition and patient Outcomes (PLATO) trial. Eur Heart J. 2014; 35(23): 1541–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlim K, Nini E, Forestier D, et al.: Methodological index for non-randomized studies (minors): development and validation of a new instrument. ANZ J Surg. 2003; 73(9): 712–6. PubMed Abstract \| Publisher Full Text\n\nBecker RC, Bassand JP, Budaj A, et al.: Bleeding complications with the P2Y12 receptor antagonists clopidogrel and ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial. Eur Heart J. 2011; 32(23): 2933–44. PubMed Abstract \| Publisher Full Text\n\nDery J, Dagenais F, Mohammadi S, et al.: Risk of bleeding complications in patients treated with ticagrelor undergoing urgent coronary artery bypass grafting surgery: a single center experience. Can J Cardiol. 2014; 30(10): S328. Publisher Full Text\n\nDiNicolantonio JJ, D'Ascenzo F, Tomek A, et al.: Clopidogrel is safer than ticagrelor in regard to bleeds: a closer look at the PLATO trial. Int J Cardiol. 2013; 168(3): 1739–44. PubMed Abstract \| Publisher Full Text\n\nGajanana D, Weintraub WS, Kolm P, et al.: The impact of in-hospital P2Y12 inhibitor switch in patients with acute coronary syndrome. Cardiovasc Revasc Med. 2018; 19(8): 912–6. PubMed Abstract \| Publisher Full Text\n\nHansson EC, Jideus L, Aberg B, et al.: Coronary artery bypass grafting-related bleeding complications in patients treated with ticagrelor or clopidogrel: a nationwide study. Eur Heart J. 2016; 37(2): 189–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHansson EC, Rexius H, Dellborg M, et al.: Coronary artery bypass grafting-related bleeding complications in real-life acute coronary syndrome patients treated with clopidogrel or ticagrelor. Eur J Cardiothorac Surg. 2014; 46(4): 699–705. PubMed Abstract \| Publisher Full Text\n\nHeld C, Asenblad N, Bassand JP, et al.: Ticagrelor versus clopidogrel in patients with acute coronary syndromes undergoing coronary artery bypass surgery: results from the PLATO (Platelet Inhibition and Patient Outcomes) trial. J Am Coll Cardiol. 2011; 57(6): 672–84. PubMed Abstract \| Publisher Full Text\n\nHolm M, Biancari F, Khodabandeh S, et al.: Bleeding in Patients Treated With Ticagrelor or Clopidogrel Before Coronary Artery Bypass Grafting. Ann Thorac Surg. 2019; 107(6): 1690–8. PubMed Abstract \| Publisher Full Text\n\nKang HJ, Clare RM, Gao R, et al.: Ticagrelor versus clopidogrel in Asian patients with acute coronary syndrome: A retrospective analysis from the Platelet Inhibition and Patient Outcomes (PLATO) Trial. Am Heart J. 2015; 169(6): 899–905.e1. PubMed Abstract \| Publisher Full Text\n\nRusso JJ, James TE, Ruel M, et al.: Ischemic and bleeding outcomes after coronary artery bypass grafting among patients initially treated with a P2Y12 receptor antagonist for acute coronary syndromes: Insights on timing of discontinuation of ticagrelor and clopidogrel prior to surgery. Eur Heart J Acute Cardiovasc Care. 2008; 2048872617740832. PubMed Abstract \| Publisher Full Text\n\nSchaefer A, Sill B, Schoenebeck J, et al.: Preoperative Ticagrelor administration leads to a higher risk of bleeding during and after coronary bypass surgery in a case-matched analysis. Interact Cardiovasc Thorac Surg. 2016; 22(2): 136–40. PubMed Abstract \| Publisher Full Text\n\nVarenhorst C, Alström U, Scirica BM, et al.: Factors contributing to the lower mortality with ticagrelor compared with clopidogrel in patients undergoing coronary artery bypass surgery. J Am Coll Cardiol. 2012; 60(17): 1623–30. PubMed Abstract \| Publisher Full Text\n\nFan ZG, Zhang WL, Xu B, et al.: Comparisons between ticagrelor and clopidogrel following percutaneous coronary intervention in patients with acute coronary syndrome: a comprehensive meta-analysis. Drug Des Devel Ther. 2019; 13: 719–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuan W, Lu H, Yang K: Choosing between ticagrelor and clopidogrel following percutaneous coronary intervention: A systematic review and Meta-Analysis (2007–2017). Medicine (Baltimore). 2018; 97(43): e12978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWestman PC, Lipinski MJ, Torguson R, et al.: A comparison of cangrelor, prasugrel, ticagrelor, and clopidogrel in patients undergoing percutaneous coronary intervention: A network meta-analysis. Cardiovasc Revasc Med. 2017; 18(2): 79–85. PubMed Abstract \| Publisher Full Text\n\nKheiri B, Osman M, Abdalla A, et al.: Ticagrelor versus clopidogrel after fibrinolytic therapy in patients with ST-elevation myocardial infarction: a systematic review and meta-analysis of randomized clinical trials. J Thromb Thrombolysis. 2018; 46(3): 299–303. PubMed Abstract \| Publisher Full Text\n\nWang D, Yang XH, Zhang JD, et al.: Compared efficacy of clopidogrel and ticagrelor in treating acute coronary syndrome: a meta-analysis. BMC Cardiovasc Disord. 2018; 18(1): 217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeo SV, Dunlay SM, Shah IK, et al.: Dual anti-platelet therapy after coronary artery bypass grafting: is there any benefit? A systematic review and meta-analysis. J Card Surg. 2013; 28(2): 109–16. PubMed Abstract \| Publisher Full Text\n\nSolo K, Lavi S, Kabali C, et al.: Antithrombotic treatment after coronary artery bypass graft surgery: systematic review and network meta-analysis. BMJ. 2019; 367: l5476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker RC, Gurbel PA: Platelet P2Y12 receptor antagonist pharmacokinetics and pharmacodynamics: A foundation for distinguishing mechanisms of bleeding and anticipated risk for platelet-directed therapies. Thromb Haemost. 2010; 103(3): 535–44. PubMed Abstract \| Publisher Full Text\n\nVAN Giezen JJ, Nilsson L, Berntsson P, et al.: Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation. J Thromb Haemost. 2009; 7(9): 1556–65. PubMed Abstract \| Publisher Full Text\n\nGurbel PA, Bliden KP, Butler K, et al.: Randomized double-blind assessment of the ONSET and OFFSET of the antiplatelet effects of ticagrelor versus clopidogrel in patients with stable coronary artery disease: the ONSET/OFFSET study. Circulation. 2009; 120(25): 2577–85. PubMed Abstract \| Publisher Full Text\n\nvan Giezen JJ, Berntsson P, Zachrisson H, et al.: Comparison of ticagrelor and thienopyridine P2Y(12) binding characteristics and antithrombotic and bleeding effects in rat and dog models of thrombosis/hemostasis. Thromb Res. 2009; 124(5): 565–71. PubMed Abstract \| Publisher Full Text\n\nSangkuhl K, Klein TE, Altman RB: Clopidogrel pathway. Pharmacogenet Genomics. 2010; 20(7): 463–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWallentin L, James S, Storey RF, et al.: Effect of CYP2C19 and ABCB1 single nucleotide polymorphisms on outcomes of treatment with ticagrelor versus clopidogrel for acute coronary syndromes: a genetic substudy of the PLATO trial. Lancet. 2010; 376(9749): 1320–8. PubMed Abstract \| Publisher Full Text\n\nNovkovic M, Matic D, Kusic-Tisma J, et al.: Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel. Eur J Clin Pharmacol. 2018; 74(4): 443–51. PubMed Abstract \| Publisher Full Text\n\nMirabbasi SA, Khalighi K, Wu Y, et al.: CYP2C19 genetic variation and individualized clopidogrel prescription in a cardiology clinic. J Community Hosp Intern Med Perspect. 2017; 7(3): 151–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulman S, Kearon C, Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis, et al.: Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemost. 2005; 3(4): 692–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "61678", "date": "23 Jun 2020", "name": "Jinesh Bahubali Nagavi", "expertise": [ "Reviewer Expertise Drug interaction studies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well explained with all the data and results.\n\nThe conclusion is very short and precise.\n\nSearch strategy should be elaborated.\n\nObjectives should be mentioned clearly in the abstract.\n\nThe discussion part is explained well in detail.\n\nStatistical analysis and its interpretation is appropriate\n\nChoice of references is very opt and good.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5879", "date": "07 Sep 2020", "name": "Jonny Fajar", "role": "Author Response", "response": "Thank you for reviewer comments. We have revised our manuscript in some part to increase the consistency of the text without changing the meanings or findings.1. The article is well explained with all the data and results.Response: Thank you.2. The conclusion is very short and precise.Response: Thank you.3. Search strategy should be elaborated.Response: The search strategy has been provided in methods.\"Search strategyWe conducted a systematic search in PubMed, Embase, Cochrane, and Web of Science up to 20 September 2019. The search strategy, conformed to medical subjects heading (MeSH), involved the use of combination the following keywords: [\"Major Bleeding\"] AND [\"Coronary Artery Bypass Grafting\" OR \"CABG\"] AND [\"Dual Anti Platelet Therapy\" OR \"DAPT\"], and [\"Clopidogrel\" OR \"Ticagrelor\"]. In our searching strategy, language restrictions were not applied. We only used the study with the larger sample size and that was more up-to-date if we found the same data among studies. Moreover, we also searched the potential papers from the reference list of relevant or eligible studies. We also employed the \"related article\" option in PubMed to broaden our searching strategy. The potentially relevant papers were identified by two independent investigators (Y.P., M.I.). Disagreement between two independent investigators was resolved by discussion and/or by consulting to the senior investigator (J.K.F.).\"4. Objectives should be mentioned clearly in the abstract.Response: The objective of our study has been outlined in abstract.\"This study aimed to perform a meta-analysis concerning the relation between the risk of major bleeding and the use of different DAPT (clopidogrel or ticagrelor) in ACS patients treated with CABG.\"5. The discussion part is explained well in detail.Response: Thank you.6. Statistical analysis and its interpretation is appropriate.Response: Thank you.7. Choice of references is very opt and good.Response: Thank you." } ] }, { "id": "69370", "date": "25 Aug 2020", "name": "Sheng-Hu He", "expertise": [ "Reviewer Expertise Interventional cardiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis systematic review and meta-analysis aimed to compare the risk of major bleeding between clopidogrel and ticagrelor in ACS patients treated with CABG. It is a meaningful clinical topic that needs more support from evidence-based medicine. According to the results of this study, ticagrelor should be used more confidently in ACS patients treated with CABG.\nHowever, there are disadvantages in some issues:\nThe characteristic summary of the 13 included trials was not shown in the manuscript. It is an important part of a meta-analysis, which should contain the data of age, gender, race, doses of medication, and bleeding risk scores.\n\nBecause only 2 of them are RCTs, most of them are case-control trails, the methodological bias of the included studies should be analyzed. There are many methods that can estimate the qualities of the included studies, at least one of them should be used in this study to remind the readers interpreting the results with caution.\n\nAlthough this study has analyzed the main observation, the risk of major bleeding, and did subgroup analysis for pre-surgery and post-surgery patients, other factors should be considered using subgroup analysis, for example, race, region, dose and time, bleeding risk scores if possible.\n\nThe logic of the discussion is not very clear. The structure of this part should be modified, some points should be simplified, and some duplicate expressions should be removed to make readers understand quicker and easier.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5880", "date": "07 Sep 2020", "name": "Jonny Fajar", "role": "Author Response", "response": "1. This systematic review and meta-analysis aimed to compare the risk of major bleeding between clopidogrel and ticagrelor in ACS patients treated with CABG. It is a meaningful clinical topic that needs more support from evidence-based medicine. According to the results of this study, ticagrelor should be used more confidently in ACS patients treated with CABG. However, there are disadvantages in some issues: The characteristic summary of the 13 included trials was not shown in the manuscript. It is an important part of a meta-analysis, which should contain the data of age, gender, race, doses of medication, and bleeding risk scores.Response: We have provided the additional characteristics in Table 1.2. Because only 2 of them are RCTs, most of them are case-control trails, the methodological bias of the included studies should be analyzed. There are many methods that can estimate the qualities of the included studies, at least one of them should be used in this study to remind the readers interpreting the results with caution.Response: The additional information regarding the possibility of methodological bias and to interpret our results with caution has been provided in study limitations.\"Third, more than a half of our included studies were cross-sectional studies, and might provide the methodological bias. Therefore, our results should be interpreted with caution.\"3. Although this study has analyzed the main observation, the risk of major bleeding, and did subgroup analysis for pre-surgery and post-surgery patients, other factors should be considered using subgroup analysis, for example, race, region, dose and time, bleeding risk scores if possible.Response: We have tried to perform sub-group analyses in accordance with ethnicity, region, dose, and bleeding risk score; however, the data were imbalanced, and therefore the calculation was unable to be performed.4. The logic of the discussion is not very clear. The structure of this part should be modified, some points should be simplified, and some duplicate expressions should be removed to make readers understand quicker and easier.Response: We have revised our manuscript in some part to increase the consistency of the text and to avoid the unnecessary repetition without changing the meanings or findings." } ] } ]	1	https://f1000research.com/articles/9-99
https://f1000research.com/articles/9-1100/v1	07 Sep 20	{ "type": "Brief Report", "title": "The enforcement of statewide mask wearing mandates to prevent COVID-19 in the US: an overview", "authors": [ "Philip Jacobs", "Arvi P. Ohinmaa", "Arvi P. Ohinmaa" ], "abstract": "Face masks have become the bulwark of COVID-19 prevention in the US. Between 10 April and 1 August, 2020, 33 state governors issued orders requiring businesses to require their customers and employees to wear face masks, and persons outdoors who could not social distance to do the same. We documented the policies and enforcement actions for these policies in each of the states. We used governors’ orders and journalists’ news reports as our sources. Our results show that the states used a variety of state and local (county and municipality) agencies to enforce business prevention behaviors and primarily local law enforcement agencies to enforce outside mask-wearing behaviours. In particular, law enforcement officers demonstrated a strong preference for educating non-mask wearers, and indicated a reluctance to resort to civil penalties that were enacted in the state orders. Businesses expressed a preference to have government agencies enforce non-mask wearing behaviours. But there was also a widespread reluctance on the part of local law enforcement to resort to legal remedies.", "keywords": [ "COVID-19", "face masks", "statewide mandates", "governor's order", "enforcement" ], "content": "Introduction\n\nIn the United States, the Centers for Disease Control first recommended the wearing of face masks on April 3, 2020. On April 8, The Governor of New Jersey was the first to issue a general, statewide mandate, which required “workers and customers to wear cloth face coverings while on the premises”. By August 1, 33 state governors had issued state-wide mask wearing orders. The resulting pattern of regulations regarding mask wearing across the states is a welter. There is wide variation in the design of the ordinances, including whose behavior is being targeted, what is expected of them, when they must observe the ordinances, where their behavior is to be regulated, why they must submit to these orders, and how violations will be enforced. In this paper we summarize the introduction of state policies in those states which issued statewide mandates, and how these mandates are being enforced.\n\n\nMethods\n\nOur subjects were the 33 states which had mask mandates in effect by August 1.\n\nWe used newspaper and broadcasting articles as our primary sources. We identified these articles using Google searches ending August 4 with the following keywords: ”COVID-19”, “State” (e.g., “Pennsylvania”) AND “mask order” or “mask mandate” AND “enforcement” OR “education”. We used newspaper or broadcast articles as the primary source because they contained essential information about the mandates, as well as additional information about enforcement. We identify primary sources for each state in the Underlying data, Appendix 11. Most news sources came from local broadcast stations, local and national news services, networks including the Public Broadcasting System, CBS, and ABC. We used the actual ordinances as secondary sources, but these often did not contain information about enforcement.\n\nWe abstracted the following items for each state:\n\nAre businesses given first-line responsibility for their customers and employees? By reading news articles, we determined if businesses were directly responsible for mask wearing behaviors of customers and employees on their premises. Answers were “Yes,” “No,” or in a few cases, “Unsure.”\n\nWhat authorities enforce whether businesses apply mask wearing orders on their premises? We determined from the news articles which government agencies were responsible for ensuring that businesses were applying the governors’ orders. This information might also be obtained from the governors’ orders. For each state, we listed these authorities: “LLE” indicates “Local Law enforcement,” “LPH” signifies “Local Public Health,” and “DOH” signifies “Department of Health.”\n\nWhat authorities enforce mask wearing behaviors outside of business premises? We identified the enforcement authorities from the news articles. Abbreviations are the same as the previous question.\n\nIs education viewed as a primary tool to encourage mask wearing behaviors? We obtained this information from news articles. Answers were “Yes” or “No”.\n\nDo local governments (counties and municipalities) pass their own orders? We obtained this information by searching news articles which identified additional county or municipal mask orders within individual states. Answers were “Yes” or “No”.\n\nHas local law enforcement shown any resistance to enforcing statewide orders? We obtained this information by searching for news articles that identified local law enforcers’ comments on “enforcement.” Answers were “Yes” or “No”.\n\n\nResults\n\nThe publications on which the analysis was based are shown in the Underlying data, Appendix 11. These data underlying the analysis are shown in the Data Availability section.\n\nWe identify the 33 states with statewide mandates (green) in Figure 1. We did not cover states with county or municipal orders (yellow), states with only municipal orders (brown) or states with no orders (red).\n\nThe first state order (New Jersey) came in effect on April 8, 2020. We followed trends until 1 August, 2020. State acronyms are as follows. Alabama (AL), Alaska (AK), Arizona (AZ), Arkansas (AR), California (CA), Colorado (CO), Connecticut (CT), Delaware (DE), Florida (FL), Georgia (GA), Hawaii (HI), Idaho (ID), Illinois (IL), Indiana (IN), Iowa (IA), Kansas (KS), Kentucky (KY), Louisiana (LA), Maine (ME), Maryland (MD), Massachusetts (MA), Michigan (MI), Minnesota (MN), Mississippi (MS), Missouri (MO), Montana (MT), Nebraska (NE), Nevada (NV), New Hampshire (NH), New Jersey (NJ), New Mexico (NM), New York (NY), North Carolina (NC), North Dakota (ND), Ohio (OH), Oklahoma (OK), Oregon (OR), Pennsylvania (PA), Rhode Island (RI), South Carolina (SC), South Dakota (SD), Tennessee (TN), Texas (TX), Utah (UT), Vermont (VT), Virginia (VA), Washington (WA), West Virginia (WV), Wisconsin (WI), Wyoming (WY).\n\nThe entire population, with exceptions, was included in the order. Persons exempted from the orders were persons with disabilities or medical conditions who were over specific ages. The age above which masks were mandatory were 2 (14 states), 4 (3 states), 5 (3 states), 6 (1 state), 7 (2 states), 9 (4 states), 10 (4 states), 11 (1 state) and 12 (1 state). See Table 1.\n\nIncludes date the order was in effect, fines and sentences, and maximum age for exemption.\n\nMandates became effective in the months of April (8 states), May (7 states), June (4 states), July (12 states) and August (2 states). See Figure 1.\n\nThe mandates covered indoor only (8 states: Colorado, Hawaii, Indiana, Maine, Montana, Minnesota, Montana, Oregon, and West Virginia), outdoor and indoor or “public” places usually where recommended spacing was not available or there were large crowds (25 states). Some states excluded certain industries (e.g., gyms) if they did not meet public health requirements. See Table 2 for individual state information.\n\nEnforcement includes business responsibility, the state authority that enforces both inside and outdoor subjects, whether states emphasize education regarding mask wearing, whether local governments also impose mask orders, and if local law enforcement resisted a role in enforcing the order. L=local, LE = law enforcement, PH=public health, OSH=occupational safety and health, DOH=department of health.\n\nIn 26 states, businesses and establishments formed the primary targeted group. Three states (Arkansas, Indiana, New Jersey) targeted only individual mask non-wearers. In two states (Pennsylvania, Wisconsin) we were unsure of the targeted group. See Table 2.\n\nTen states had provisions for fines only. The usual amount was between $100 and $200, but NY had a maximum of $1,000. Nine states had provisions for fines and/or jail sentences. The maximum fine in this group was $5,000 (Hawaii, Maryland) and the maximum jail sentence was one year (Maryland), but these amounts were outliers. More typical values were $500 fines and/or 30 days to six months in jail. In five states fines varied by county. The other states did not specify penalties, and some of these could have no penalty: in a number of states sheriffs expressed a strong preference for education over criminal or even civil proceedings. See Table 2 for individual state information.\n\nIn most of the states (27 states) the government relied on private businesses to enforce mask wearing behaviors. Three states (Arkansas, Indiana, New Jersey) focused directly on non-mask-wearers. We were unsure of the governors’ focuses in three other states (Massachusetts, Pennsylvania, Wisconsin). In addition, because many states extended the mandate to outdoor non-business settings, there was enforcement in these settings as well. The pattern of enforcement in the two settings was very different.\n\nAgencies which enforced businesses include state occupation safety and health agencies (Nevada, Ohio, Oregon, Washington, Kentucky); state or local public health agencies (Conneticut, Delaware, Indiana, Kentucky, Montana, New Mexico, New York, Ohio, Virginia, Wisconsin), city or county law enforcement (Connecticut, Hawaii, Kansas, North Carolina), alcohol and beverage control (California, Colorado), business regulators and licensers (Maine, Mississippi, Rhode Island). In some cases regulators were not specified, or the area was not enforced by the government. The enforcement of non-mask wearing behavior outside of businesses was usually assigned to local law enforcement (19 states) or public health (3 states). In other cases, there was limited to no enforcement outside the business setting. Many local sheriffs or local police chiefs stated their objections to enforcement on grounds that there were limited resources; a few also mentioned constitutional grounds.\n\n\nDiscussion/conclusions\n\nBased on public and journalist reports, we analyzed the introduction and enforcement of statewide mask wearing mandates in the 33 US states that imposed such orders between April 10 and August 1, 2020. Most states relied on businesses to enforce customer and employee mask wearing behavior. However, both the contents of the governors’ orders and the type and degree of enforcement varied widely between the states. Enforcement responsibility for personal (outdoor) mask wearing behavior was handed over to local law enforcement.\n\nThe business sector of the economy has been very active in COVID-19 prevention during this time period by introducing mask wearing regulations for its customers and employees. A survey of large US retail chains showed that 16 chains had introduced cross-country mask wearing policies in May, 2 in June, and 34 in July. Despite the adoption of storewide prevention policies, businesses, their trade associations, and employee trade unions have expressed concern at taking on primary enforcement roles. Private companies still relied on state laws to provide them with a rationale for requiring customers to wear masks. These businesses are subject to degrees of enforcement that have been inconsistent across states and that have often been lax. Many governors’ orders also covered outdoor areas, and most of these were nominally enforced by local law enforcement agencies. However, senior law enforcement officers in all states issued statements that they would not enforce mask wearing orders. For example, 38 sheriffs in Montana issued an op-ed which stated that a mask wearing directive “is not a mandate for law enforcement to issue citations and arrest violators.” Although such statements were not universal, examples can be found in every state.\n\nWe used both governors’ orders and news reports for our data sources. Policies like engaging in public education are subjectively described and data for them are not collected; instead we used sheriffs’ interviews with journalists to document the policies. Also, we could not obtain data on citations written by local law enforcement. Nevertheless, information on education and enforcement was widely reported in the press across the nation, and strongly suggests a national trend. It also indicates that mask wearing behavior, now considered a bulwark against the spread of COVID-19, is not being strongly enforced, especially in the outdoor sectors.\n\n\nData availability\n\nHarvard Dataverse: Press articles on statewide mask orders in 33 US states. https://doi.org/10.7939/DVN/SFIT0R1\n\nThis project contains the following underlying data:\n\n- Appendix 1 Press Publications. This file contains the news articles that we used to abstract the descriptive variables. It includes state, topic, and internet address.", "appendix": "References\n\nJacobs P: Press articles on statewide mask orders in 33 US states. UAL Dataverse, V1. 2020. http://www.doi.org/10.7939/DVN/SFIT0R" }	[ { "id": "75986", "date": "10 Dec 2020", "name": "Evelyn Forget", "expertise": [ "Reviewer Expertise Health economics", "health policy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article documents policies and enforcement actions in the 33 states that adopted face mask policies between 10 April and 1 August 2020, by examining news articles and broadcasting articles retrieved through Google on August 4. They found that states used a variety of state and local agencies to enforce policies, and that these agencies overwhelmingly focused on education rather than legal penalties.\nThe article is descriptive in nature. The methods are straightforward and clearly articulated, including search terms, databases and dates of access. The methodology is appropriate. The author has made the data retrieved available and therefore it is possible to replicate the study, reproduce it in the future to determine whether enforcement agencies or policies have changed over time as case positivity rates, vaccination availability and/or mortality change, and to augment the study by using additional search terms on the original or alternative databases. The article is clearly written, and the conclusions supported by the evidence.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75987", "date": "20 Jan 2021", "name": "Hina Shah", "expertise": [ "Reviewer Expertise Epidemiology", "public health systems", "public health policy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a descriptive study of state-level mask mandates in the U.S. that were implemented between April 10, 2020, and August 1, 2020, to mitigate the spread of COVID-19. The objective of the study is to document which states had issued mask mandates, to characterize the mask mandates and to assess how the mandates were being enforced. The study also assessed whether law enforcement officials had demonstrated resistance to enforcing mask mandates.\nThe study methodology involved conducting online searches using Google for newspaper and broadcast articles as the primary data source. The actual text of the orders (described as “ordinances”) was identified as a secondary data source. The documents were reviewed, and specific characteristics of interest were abstracted from the text.\nThe study found that policies varied substantially among the 33 states that had issued mask mandates, particularly with respect to enforcement and penalty provisions. State policies varied also by authority of local governments to issue mask mandates and exceptions (e.g., age, medical contraindications). While the degree to which law enforcement officials had demonstrated resistance to enforcement was not characterized in the study, the authors noted that examples of resistance could be found in every state.\nThe article clearly describes the methodology used to identify which states had mask mandates, including the specific online search terms and other parameters. While general descriptions of the abstract categories were provided (e.g., enforcement requirements for businesses, which authorities enforce business requirements, whether education is viewed as a primary tool, etc.), the article did not include detailed definitions for each category. The article also did not describe how the actual text of the orders was utilized in the abstraction process. These methodology details might provide additional clarity with respect to how the states were categorized. For example, Kansas is categorized as having a statewide mask law. While that is correct, the law also allows local boards of county commissioners to adopt orders that are less stringent than the state order, thereby giving counties the option to opt out, which many have done1,2. The article provides a link to the source data in the form of a Microsoft Word document with URLs for each article or order by state.\nReproducibility of the study is uncertain given the qualitative nature of the methods, the lack of coding method details and reliance on news articles as the primary data source. In addition, while URLs are provided for each source document, full citation details are not provided. As a result, links might be obsolete, or the content might have changed. In our own government mitigation policy research, we have found it challenging to interpret and consistently categorize aspects of public health orders and other official governmental mitigation policy actions. In addition, we have often found discrepancies between published news reports and the actual text of the orders. Nonetheless, the article is clearly written and well-organized, the conclusions appear sound, and the study makes an important contribution to understanding the challenges of implementing and enforcing population-level policies to control the spread of COVID-19.\n(1) Kansas Health Institute. A Kansas Twist—Reopening plans for Kansas counties. Available https://www.khi.org/policy/article/20-251\n(2) Van Dyke ME, Rogers TM, Pevzner E, Satterwhite CL, Shah HB, Beckman WJ, Ahmed F, Hunt DC, Rule J. Trends in county-level COVID-19 incidence in counties with and without a mask mandate – Kansas, June 1 – August 23 23, 2020. Morbidity and Mortality Weekly Report, 2020, 69(47), 1777-1781.2\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1100
https://f1000research.com/articles/8-530/v1	23 Apr 19	{ "type": "Data Note", "title": "RVDB-prot, a reference viral protein database and its HMM profiles", "authors": [ "Thomas Bigot", "Sarah Temmam", "Philippe Pérot", "Marc Eloit", "Sarah Temmam", "Philippe Pérot", "Marc Eloit" ], "abstract": "We present RVDB-prot, a database corresponding to the protein equivalent of the nucleic acid reference virus database RVDB. Protein databases can be helpful to perform more sensitive protein sequence comparisons. Similarly to its homologous public repository, RVDB-prot aims to provide reliable and accurately annotated unique entries, while including also an Hidden Markov Model (HMM) protein profiles database for distant protein searching.", "keywords": [ "virus", "genomes", "proteins", "hmm", "clusters", "annotations", "database" ], "content": "Introduction\n\nSequence assignation often uses similarity criteria to infer homology, and hence taxonomy and / or protein type. In order to search for this similarity, reliable, accurate and comprehensive databases are required. In the specific field of viruses, several solutions are available yet their ability to provide valid results is highly dependant on the goal of the study and on the available computer resources. Using a database with a high number of sequences, such as NCBI nr/nt may seem appropriate, but it implies an increased computation time and annotation quality is not always optimal. RefSeq on the other hand, is generally better curated, but it contains only full-length genomes and rarely includes the latest discoveries. Other specialized databases provide only specific groups of taxa for specific purposes, for instance, virus families responsible for infectious diseases like HIV or influenza.\n\nThus, the need for better, well-annotated and comprehensive public viral databases that can be used for the identification of viruses by high-throughput sequencing lead Goodacre et al. to propose their Reference Viral DataBase (RVDB)1. This database consists of a collection of all currently known viral genomes and virus-related nucleic sequences retrieved from NCBI nr or RefSeq, which includes a specific, both manual and computational reviewing process, as well as four updates of the contents per year. These features make RVDB quite attractive for the virology research community and in fact, in February 2018, version 15.1 was released.\n\nSince viral genomes mainly consist of coding sequences, the need for an equivalent reference database that provides the protein version of these sequences may prove quite advantageous.\n\nIndeed, protein sequences are useful when searching for distant homologs: their substitution rates are much lower than nucleic sequences. Additionally, proteins can also be efficiently clustered according to their similarity, and the resulting clusters can then be used to build Hidden Markov Model (HMM) Profiles in order to identify more evolutionary distant proteins. In fact, programs like HMMER2 allow the building of a HMM profile from a multiple protein sequence alignment. This profile can then be able to recognize proteins based on complex positionspecific models of sequence conservation and evolution, and it does so in a more accurate way than if a classic sequence alignment is used.\n\nThus, we propose a protein sequence version of RVDB whose update will be synchronized with the original nucleotide RVDB release. Here we describe the conversion from the nucleotide version of RVDB to the protein version RVDB-prot, as well as the clustering process leading to the HMM profiles.\n\n\nMethods\n\nThe current version of RVDB, v15.13 consists of a collection of 2 719 839 nucleic sequences1. The accession numbers were extracted in order to gather the corresponding database entries in genbank format. From these entries, coding domain sequences and the description of these sequences were located and copied into the protein collection. The resulting protein file contains the nucleic sequence reference, for traceability purposes. The sequence names are formatted in the following way:\n\n>acc\|<p_bank>\|<p_acc>\|<n_bank>\|<n_acc>\|<descr[sp]>, where:\n\np_bank is the bank in which the protein can be found\n\np_acc is the accession number corresponding to the protein sequence\n\nn_bank is the bank in which the original nucleotide sequence was found\n\nn_acc is the original information found in the nucleic database\n\ndescr is the description of the protein sequence as found in the database entry\n\nsp is the species name.\n\nThis process produces a 3 899 699 protein sequence file.\n\nThe HMM generation rationale was inspired from VFam (the database of profile HMM built from all the viral proteins present in RefSeq, discontinued from 2014)4, but was entirely re-coded as a Snakemake pipeline5, using different tools for some key steps (clustering, alignment). The proteins sequences were clustered with a 100% identity criteria to duplicates, using CDHit 4.7.06. Then, the sequences were processed using Blast 2.2.267 performing an all-against-all comparison. These comparisons allow Silix 1.2.68 to define clusters of sequences according to the sequence similarity. This step produces a file text in which each sequence is associated to one cluster. The information of each cluster containing at least four sequences was transformed into a fasta file containing all of its sequences. Then, we performed multiple alignment using Mafft 7.0239 in auto mode. The multiple sequence alignments were processed by HMMER 3.2.12 in order to obtain the HMM profiles. The HMM profiles were then put together in a single file.\n\nIn our pipeline, a cluster consists in a set of sequences, where each sequence belongs to a species, and each sequence is associated with a description. In order to characterize the clusters, these pieces of information and other indicators (such as cluster length and sequence number) are combined into an annotation database, in SQLite format. The schema of this database is shown in Figure 1.\n\nThe first type of data associated to a cluster is a set of keywords. These keywords correspond to the union of all the set of sequence names belonging to the cluster, weighted according to their frequencies, and excluding trivial words. For instance, for the cluster number 1, containing 588 sequences, the keywords and their frequencies, are: parvovirus(441), protein(423), Canine(359), capsid(345), VP2(233), virus(89), VP1(83), disease(48), Aleutian(48), mink(48) allowing to describe a cluster composed of Canine parvovirus capsid protein sequences. The database stores all these taxa, using NCBI TaxIDs. For each cluster, the taxonomic information is summarized by a Last Common Ancestor (LCA) that corresponds to the taxon in the tree of life to which all the sequence taxa belong. Finally, the database also provides the length (number of amino acids of the multiple sequences alignment) and the number of sequences in each cluster.\n\nThis database is available in SQLite format, and to provide more direct access, flat text files are proposed. A text file for each cluster, identified with its cluster number contains all the information related to it.\n\nThe different steps explained above are performed using a Snakemake pipeline5, available at Institut Pasteur’s Gitlab.\n\nPipeline available from https://gitlab.pasteur.fr/tbigot/rvdb-prot/.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.263059310\n\nLicence: GNU GPL v3.0\n\nSeveral tools are needed to run the pipeline, including: Python, Mafft, Golden, Hmmer, Snakemake, Silix, Blast+. The versions of these tools compatible with the pipeline are listed in the README file.\n\n\nData availability\n\nDatabase files are available at https://rvdb-prot.pasteur.fr/. Release 15.1 described in this manuscript is also available from Figshare.\n\nFigshare: U-RVDBv15.1 https://dx.doi.org/10.6084/m9.figshare.77459693.\n\nThis project contains the following underlying data:\n\nU-RVDBv15.1-prot.fasta (fasta file containing protein features of the original database: -prot.fasta)\n\nU-RVDBv15.1-prot.fasta-prot.hmm (the HMM profiles, generated with and for hmmer 3.2.1 (from 2019, 3.1b2 before))\n\nU-RVDBv15.1-prot.fasta-prot-hmm.sqlite (SQLite db containing annotations (please find a documentation below))\n\nU-RVDBv15.1-prot.fasta-annot.txt (a directory of annotations with plain text files (one per protein family))\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nTable 1 shows some summary metrics for the entries of this release and the different resources.\n\nUpdates are manually curated each time a new release of the main database (nucleic RVDB) is announced, i.e., four times a year. The following older versions are also available online: 14.0 (2018-09), 13.0 (2018-06), 12.2(2018-03), 11.5 (2017-10), 10.2 (2017-04).\n\nUsage HMMER can be used to search for all profiles in a fasta sequence file (sequences.fasta): hmmsearch U-RVDBv15.1-prot.fasta-prot.hmm sequences.fasta > result.out. Additional options are available in HMMER User’s Guide.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Peter Skewes-Cox, Jr., author of VFAM database for kindly providing his scripts which were an inspiration for the earlier versions of RVDB-prot. We thank Natalia Pietrosemoli for her help in the editing of the manuscript. This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris.\n\n\nReferences\n\nGoodacre N, Aljanahi A, Nandakumar S, et al.: A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection. mSphere. 2018; 3(2): pii: e00069-18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEddy SR: Accelerated Profile HMM Searches. PLoS Comput Biol. 2011; 7(10): e1002195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBigot T, Temmam S, Pérot P, et al.: U-RVDBv15.1. figshare. Fileset. 2019. http://www.doi.org/10.6084/m9.figshare.7745969.v1\n\nSkewes-Cox P, Sharpton TJ, Pollard KS, et al.: Profile hidden Markov models for the detection of viruses within metagenomic sequence data. PLoS One. 2014; 9(8): e105067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöster J, Rahmann S: Snakemake--a scalable bioinformatics workflow engine. Bioinformatics. 2012; 28(19): 2520–2522. PubMed Abstract \| Publisher Full Text\n\nLi W, Godzik A: Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006; 22(13): 1658–1659. PubMed Abstract \| Publisher Full Text\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J Mol Biol. 1990; 215(3): 403–410. PubMed Abstract \| Publisher Full Text\n\nMiele V, Penel S, Duret L: Ultra-fast sequence clustering from similarity networks with SiLiX. BMC Bioinformatics. 2011; 12(1): 116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatoh K, Misawa K, Kuma K, et al.: MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002; 30(14): 3059–3066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBigot T: RVDB-prot v15.1.0 (Version 15.1.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.2630593" }	[ { "id": "47715", "date": "16 May 2019", "name": "Philippe le Mercier", "expertise": [ "Reviewer Expertise Virus proteomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors present a RVDB-prot, a reference viral protein database and its HMM profiles. The purpose of this approach is providing a complete reference database of viral proteins to identify new sequences. Their database is based on nucleotide Reference Viral Database (RVDB). The rationale of this work is that protein sequences can be better than nucleotides for searching distant homologs.\nIn brief, their approach was as follows: RVDB database was converted to proteins, thereby creating a new dataset of 3,899,699 proteins. The protein were clustered, and these clusters used to create HMM. Words frequently present in sequence names of a cluster were used to annotate HMM profiles. The software, pipeline and final database are all available.\nThe final data are of good quality, will hopefully be maintained along with RVDB and offer a new approach for protein virus reference.\nWhile the article is well written and the method is well described, there are a number of issues that need to be addressed:\nThe database is described as facilitating sequence assignation. This seems a bit vague, a sentence describing possible applications could help.\n\nThe introduction may describe better the current state of research in the field. UniProtKB should be cited in “existing databases” for viral proteins, and authors may add citations of its use in virus detection. (ex: UniRef90 used with success to create synthetic human virome1 (PMID: 26045439)). This would also highlight new potential applications for RVDB-prot.\n\nUniProtKB provides data for 3,972,271 viral proteins, a bit more than RVDB-Prot (3,899,699). RVDB-prot data is based similarity gathering of sequences with viral RefSeq, which has the advantage to ignore any taxonomical issues. On the other hand, many RefSeq are provisional, and those are not free or errors. The authors may discuss the advantage of their method over existing protein dataset. Similarly, UniRef90 contains 577,105 clusters of proteins, which could be compared to the 489,207 unique proteins of RVDB-prot. Further discussion may help understanding the advantages of these two datasets.\n\nThe paper could provide more details on parameters used for defining clusters with Silix.\n\nMinor remark:\nThe naming system may be perfected. Although imaginative and automatic, it seems to be limited. For example cluster 77 in the -prot-hmm-txt.zip (v 15.1) contains 398 sequences, which are obviously rep proteins for ssDNA viruses circo, gemini and their satellites, but the names fished out by author’s method are not clear. This can be problematic for sequence assignation.\nKeywords for cluster 77: protein 329 replication 296 virus 183 alphasatellite 154 associated 138 putative 120 viral 103 CRESS 78 leaf 50 curl 49 initiator 49 Rep 41 yellow 39 Circoviridae 39\nActually, the name used to create RVDB-pro keywords is not clearly defined. The name of GenBank protein entry are not very consistent and this may explain these problems. Maybe using pfam or any other method of identification over the clusters may help naming them in a more consistent way.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5867", "date": "07 Sep 2020", "name": "Thomas Bigot", "role": "Author Response", "response": "We would like to thank the Reviewer. Please find below our line-by-line responses. The database is described as facilitating sequence assignation. This seems a bit vague, a sentence describing possible applications could help. We have added the following sentence to exemplify possible applications: “When trying to characterize sequences present in a metagenomics sample, searching first for related sequences in a viral database can lead to identify rapidly a known virus (high identity between the query sequence and the one in the database), or identify potential new species (low identity with any known sequence). Such hits must be further characterized on more comprehensive databases to increase the robustness of taxonomic assignations.” The introduction may describe better the current state of research in the field. UniProtKB should be cited in “existing databases” for viral proteins, and authors may add citations of its use in virus detection. (ex: UniRef90 used with success to create synthetic human virome1 (PMID: 26045439)). This would also highlight new potential applications for RVDB-prot. UniProtKB provides data for 3,972,271 viral proteins, a bit more than RVDB-Prot (3,899,699). RVDB-prot data is based similarity gathering of sequences with viral RefSeq, which has the advantage to ignore any taxonomical issues. On the other hand, many RefSeq are provisional, and those are not free or errors. The authors may discuss the advantage of their method over existing protein dataset. We have updated the introduction, introducing UniProtKB viral sequences: “UniProtKB11 contains numerous viral sequences (: 4 497 049 in total, including 17 008 (0.38%) reviewed ones) that could, as for NCBI/nr, increase computation time when thousands of sequences have to be analyzed concomitantly, which is routinely practiced in metagenomics analyses.” We have also updated the description of RefSeq (with updated contents) and better exemplified the benefit of RVDB over these two databases. Similarly, UniRef90 contains 577,105 clusters of proteins, which could be compared to the 489,207 unique proteins of RVDB-prot. Further discussion may help understanding the advantages of these two datasets. We stressed on the first asset of RVDB: the curation that is done on the sequences is unique and allows to raise confidence in the fact that all the sequences of the database are real viral sequences. The paper could provide more details on parameters used for defining clusters with Silix. Done. We used the default parameters of Silix. The naming system may be perfected. Although imaginative and automatic, it seems to be limited. For example cluster 77 in the -prot-hmm-txt.zip (v 15.1) contains 398 sequences, which are obviously rep proteins for ssDNA viruses circo, gemini and their satellites, but the names fished out by author’s method are not clear. This can be problematic for sequence assignation. Actually, the name used to create RVDB-pro keywords is not clearly defined. The name of GenBank protein entry are not very consistent and this may explain these problems. Maybe using pfam or any other method of identification over the clusters may help naming them in a more consistent way. We are grateful for this remark which helped us make the naming system clear. Indeed, the pipeline does now query PFAM to annotate sequences. As explained in the new version of the manuscript, for each cluster, we query PFAM with every sequences of this cluster, using --cut_ga option of HMMER (this option makes HMMER trust PFAM GA bitscore cutoff defined for each cluster). We kept the original system (using sequences descriptions) despite the fact they are inaccurate, since sometimes, we do not find homologs clusters in PFAM." } ] }, { "id": "47713", "date": "20 May 2019", "name": "Guy Perriere", "expertise": [ "Reviewer Expertise Phylogeny", "molecular evolution", "comparative genomics", "sequence databases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 resource that can be of use for people dealing with comparative genomics in viruses. There are some points that need to be clarified before this paper can be indexed though.\nIn order to ease reproducibility, the parameters used for the different programs (e.g. HMMER, SiLiX) of the pipeline should be provided. Why is it required to locally translate the Coding DNA Sequences (CDS) from the original RVDB nucleotide database instead of downloading them from the resource? I have a question for the taxonomic assignation to the Last Common Ancestor (LCA) when building the clusters. How are handled the possible contradictions within a cluster? More exactly, what is done exactly if sequences that belong to distantly related taxa are clustered together? If a strict LCA rule is applied, then it would be possible to have a really inprecise assignation (something like \"virus\" and that's it).\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5868", "date": "07 Sep 2020", "name": "Thomas Bigot", "role": "Author Response", "response": "We would like to thank the Reviewer. Please find below our line-by-line responses. In order to ease reproducibility, the parameters used for the different programs (e.g. HMMER, SiLiX) of the pipeline should be provided. Done. Parameters are now specified in the new version of the manuscript. Actually, for both of these programs, we use default parameters. Why is it required to locally translate the Coding DNA Sequences (CDS) from the original RVDB nucleotide database instead of downloading them from the resource? We have clarified this point in the new version: actually, we use translations provided in the entry of each nucleic sequence. They are provided in the raw data of the original database (Genbank, RefSeq) along with the accession number. What we do amounts to retrieve all protein sequences corresponding to a nucleic sequence from protein database with accession numbers, but doing it directly from the nucleic database is faster. I have a question for the taxonomic assignation to the Last Common Ancestor (LCA) when building the clusters. How are handled the possible contradictions within a cluster? More exactly, what is done exactly if sequences that belong to distantly related taxa are clustered together? If a strict LCA rule is applied, then it would be possible to have a really inprecise assignation (something like \"virus\" and that's it). Indeed, we use naïve LCA assignation, and it can lead to imprecise assignation (some clusters can be tagged as Viruses). As we do not have other information about the cluster we characterize, we chose not to avoid this possibility. We have added a precision about this case in the new version of the manuscript: “For each cluster, the taxonomic information is summarized by a Last Common Ancestor (LCA) that corresponds to the taxon in the tree of life to which all the sequence taxa belong; this LCA can be close to the root of the tree (Viruses), but is usually specific to a family.”" } ] } ]	1	https://f1000research.com/articles/8-530
https://f1000research.com/articles/8-1719/v1	04 Oct 19	{ "type": "Research Article", "title": "Frequencies of parasite infections among students of primary school in Al Kalakla Locality, Khartoum State, Sudan: a cross-sectional study", "authors": [ "Hala Abdalazim Hassan", "Ahmed Bakheet Abd Alla", "Tayseer Elamin Mohamed Elfaki", "Mohammed Baha Eldin Ahmed Saad", "Hala Abdalazim Hassan", "Tayseer Elamin Mohamed Elfaki", "Mohammed Baha Eldin Ahmed Saad" ], "abstract": "Background: Intestinal parasite spread in tropical countries is especially common among primary school students. This study aimed to determine the frequencies of the intestinal parasite in Alkalakla locality, Khartoum state. Methods: This study was conducted in school students in Al-kalakla locality in Khartoum state from period between 20th December 2016 to 5th May 2017. Stool samples were collected from 134 randomly selected students, of whom 67 were males and 67 were females. All samples were examined using the wet preparation technique, formal ether concentration technique and saturated sugar floatation technique. Results: The frequency of intestinal parasites was 35.5% overall in the students examined; females were more affected than males (38.8% and 32.8%, respectively). The more affected age groups were 12-14 years followed by 9-11 and 6-8 years old (53.8%, 36.3% and 26.4% respectively). The least frequent intestinal parasite was Taenia spp. (1.5%) followed by Giardia lamblia (3.7%), Schistosoma mansoni and Ascaris lumbricoides (5.2% each), Entamoeba coli (7.5%), Hymenolepis nana (10.4%), and Entamoeba histolytica (16.4%). In total, 20.9% were infected with single parasite while 14.9% were infected with more than one parasite. The frequency of parasite by formal ether concentration method was 35.8 %, by wet preparation method was 17.9 % and by the saturated sugar flotation method was 16.4%. Conclusion: Our data showed that intestinal parasites were common in school students; however, females were more affected than males and the 12-14-years age group was the most affected age group. The formal ether concentration method was the best method for detecting of intestinal parasite.", "keywords": [ "Al-kalakla", "E. histolytica", "H. nana", "formal ether concentration technique", "intestinal parasite", "frequencies", "Khartoum." ], "content": "Introduction\n\nIntestinal parasites, particularly in tropical and subtropical areas, are a significant health issue1. Approximately 3.5 billion individuals are estimated to be impacted in developing nations and 450 million are sick as a consequence of these diseases, the majority being children2. Approximately one-quarter of the world's population is infected with intestinal parasites and about 80% of all deaths in developing nations are caused annually by infectious and parasitic illnesses3. There is a powerful correlation between the elevated incidence of these diseases and poverty, bad environmental health and insufficient health facilities4. Also involved is poor personal hygiene, an unsafe water supply and an absence of health education5. The transmission of intestinal parasites is based on characteristics of the parasite, actions of the individual and ecological and biological factors6. Transmission occurs by ingestion of contaminated fecal food or water, by hands contaminated with fecal matter coming into contact with the mouth or by skin penetration by larval stage of the parasite following direct contact with contaminated fecal soil7. Children of school-age are especially prone to symptoms, sometimes carrying a greater burden of parasites than adults8. Diagnosis is routinely performed using a microscope, with fecal samples prepared for microscopy by direct wet mounting or concentration methods. Although direct wet mounting has low sensitivity9,10, it is still used in low- and middle-income countries10. There are many methods to concentrate the stages of intestinal parasites; cysts, eggs and larvae can be analyzed as specimens using techniques such as formal ether sedimentation and flotation11. These techniques are better than direct wet mounting since they identify more parasites12.\n\n\nMethods\n\nThis study is a cross-sectional study conducted in the locality of Al-Kalakla in the state of West Khartoum. This study was carried out between December 2016 and May 2017.\n\nThe study population was children at a primary school, between the ages of 6 and 14. The purpose of the study was explained to the guardians, students and head of school and a total of 134 participants agreed to participate in this study. The children were split into three age groups (6–8, 9–11, 12–14 years) and the same number of male and female participants was selected (n=67 of each). A labeled, large-mouth stool container was given to each selected student for collection of fecal samples. Samples were collected during the school day\n\nE. histolytica and G. lamblia were identified by presence of a cyst or trophozoite in the stool. Taenia spp., Schistosoma mansoni, Ascaris lumbricoides, Entamoeba coli and Hymenolepis nana were identified by presence of eggs of each helminth in the stool.\n\nWet preparation was achieved by blending a small part of the stool sample taken with a wooden applicator with a drop of normal saline on a slide. The sample was enclosed with a cover slip and systematically examined under a microscope using a 10X lens and elevated magnification 40X lens for further detail observation.\n\nApproximately 1 g of feces from separate areas of the specimen was gathered and emulsified in glass beaker in 5 ml of formal saline. A further 5 ml were added and mixed from the same solution. The resulting suspension was strained through a strainer. The filtered sample was poured back into a centrifuge tube and an equal quantity of ether was added. The tube was shaken by hand for 1 min and then centrifuged at 2000 rpm for 5 minutes. The upper three layers were removed and the sediment was transported to a slide covered with a cover slip and examined at 10X and 40X magnification under a light microscope.\n\nApproximately 100 ml of saturated sugar solution (dextrose) was put into a glass measuring cup. And approximately 1 g of stool sample was added. The sample of fecal was blended with the saturated sugar solution for flotation. The fecal debris was filtered into another cup and the remaining fluid was drawn out. The preparation that was filtered was poured into a glass test tube. To the top of the tube was added fecal flotation solution. A cover slip was placed on the top of the tube. The pipe remained unchanged for 15 to 30 minutes and the covering glass was closely put on a slide and microscopically examined13.\n\nData were analyzed using Statistical Package for Social Sciences (version 16). Using Chi square test, the significant obtained when P < 0.05. Data were presented in tables.\n\nThe sensitivity and specificity of each technique was calculated using the two formulae below:\n\nSensitivity=positivecasesoftestedtechniquepositivecasesofreferencetechnique×100\n\nSpecifity=negativecasesoftestedtechniquenegativecasesofreferencetechnique×10\n\nEthical clearance for this study was obtained from Committee of medical laboratory science, Sudan University of Science and Technology, ethical approval number (MLS – IEC – 08 – 16). Written informed consent for participation and publication of the data was obtained from the guardians of all participants included in this study.\n\n\nResults\n\nAnalysis showed that 48 of the 134 stool samples collected from participants were positive for gastrointestinal parasites in Al-kalakla locality, Khartoum state. This was an overall prevalence rate of 35.8%. Infection statuses of all participants, alongside the methods used to identify infection, are available as Underlying data14.\n\nThe research disclosed that in females the occurrence of gastrointestinal parasites was 38.8% while in males it was 32.8%. The difference in gender rates was found to be statistically insignificant (P=0.471; Table 1). Findings showed that age groups 6–8, 9–11 and 12–14 had parasite prevalence rates of 26.4%, 36.3% and 53.8%, respectively. These variations in rates at P = 0.057 (Table 2) were statistically insignificant. The prevalence of different parasites was found as follows: E. histolytica (16.4%), H. nana (10.4%), E. coli (7.5%), A. lumbricoides (5.2%), S. mansoni (5.2%), G. lamblia (3.7%) and Taenia spp. (1.5%) (Table 3). The results showed that 28 (20.9%) were infected with single parasite and that there were 20 infected with more than one parasite (14.9%) (Table 4).\n\nP = 0.471\n\nP = 0.057\n\nThe frequency of gastrointestinal parasites by various parasitological methods was as follows: 35.8% by formal concentration method, 17.9% by the wet preparation method and 16.4% by the saturated sugar flotation method. A comparison between the detection rate of each method found that detection rates were significantly different (P = 0.000; Table 5).\n\nP = 0.000\n\nAssuming that the formal ether concentration technique was the gold standard, the sensitivity and specificity of the wet preparation technique was 50% and 100% respectively, and the sensitivity and specificity of the saturated sugar flotation technique was 45% and 100%, respectively (Table 6).\n\n\nDiscussion\n\nIt was evident from the results that the overall prevalence of gastrointestinal parasites among children's schools in Al-kalakla was high (35.8%) and was found to be higher than the rate reported by Muhajir et al. (2017) in Alkalakal (30%)15. Nevertheless, our rate was found to be lower than that reported by Gabbad and Elawad (2014) in Elengaz, Sudan (64.4%)16. The study found that females had a slightly higher occurrence of gastrointestinal parasites (38.8%) than males (32.8%). This finding was not aligned with Muhajir et al. (2017) in Al-kalakla, which found higher rates of infection in males (16.5%) compared to females (13.5%)15.\n\nThe highest prevalence rate (53.8%) among the 12–14 age group was reported in this study. This rate did not agree with that of Magambo et al. in Southern Sudan, who reported the most affected age group was those 6–10 years17.\n\nThe findings of this study indicated that the common gastrointestinal parasites in children's schools were E. histolytica (16.4%), H. nana (10.4%), E. coli (7.5%), A. lumbricoids (5.2%), S. mansoni (5.2%), G. Lamblia (3.7%) and Taenia spp. (1.5%), while Muhajir et al. reported E. histolytica the most common gastrointestinal parasites in Al-kalakla (15.5%), G. lamblia (12.5%), H. nana (1.5%) and S. mansoni (0.5%)15. However, Gabbad and Elawad in Elengaz, Sudan, revealed that G. lamblia was the predominant gastrointestinal parasite (33.4%), followed by H. nana (26.4%), T. saginata (8.6%), Enterobius vermicularis (6.2%), S. mansoni (4.4%) and E. histolytics (3.6 %)16.\n\nAs far as the detection levels for the three methods used were concerned, it was evident that the best detection rate (35.8%) was recorded for the formal ether concentration method and the lowest rate (16.4%) was recorded for the saturated sugar flotation method, while the wet preparation method showed a detection rate of 17.9%. Our results on formal ether were not in agreement with Eisa in Keryab Village, Sudan, who reported a 90% detection rate18. However, the detection rate reported in our study was lower than the detection rate reported by Eman (44%)19.\n\nThe study found that the detection rate for wet preparation (17.9%) was lower than the detection rate reported by Eman (41.4%) in Southern Sudan19. Furthermore, detection rate for saturated sugar flotation technique (16.4%) was lower than the detection rate for Duria in Khartoum, Sudan, reported (58.6%)20.\n\nSurprisingly, the findings showed a sensitivity of 50% and a high specificity (100%) of the wet preparation method and a sensitivity of 45% and a high specificity (100%) of the saturated sugar flotation method. This could probably be attributed to severe intestinal parasite infections among subjects studied in this study.\n\n\nConclusion\n\nFrom the outcomes, it can be concluded that gastrointestinal parasites are common among children's schools in Al-kalakla, Khartoum state. Furthermore, prevalence rate was significantly greater among females, the highest rates of infection were recorded in the 12–14-years age group and, lastly, the formal ether concentration method showed the highest sensitivity level for the identification of distinct gastrointestinal parasites.\n\n\nData availability\n\nFigshare: Hala and Ahmed file.sav. https://doi.org/10.6084/m9.figshare.9804869.v114.\n\nThis project contains the following underlying data:\n\nHala and Ahmed file.sav (parasites detected in stool samples for each child, with method used to detect them)\n\nData dictionary (2).docx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to express our immense gratitude and appreciation to the department of parasitology and medical entomology staff of Sudan University of Science and Technology, also to the school manager and the all participant in this study.\n\n\nReferences\n\nDamen JG, Luka J, Biwan EI, et al.: Prevalence of Intestinal Parasites among Pupils in Rural North Eastern, Nigeria. Niger Med J. 2011; 52(1): 4–6. PubMed Abstract \| Free Full Text\n\nWegayehu T, Tsalla T, Seifu B, et al.: Prevalence of intestinal parasitic infections among highland and lowland dwellers in Gamo area, South Ethiopia. BMC Public Health. 2013; 13(1): 151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Braiken FA: Is intestinal parasitic infection still a public health concern among Saudi children? Saudi Med J. 2008; 29(11): 1630–5. PubMed Abstract\n\nAl-Braiken FA: Is intestinal parasitic infection still a public health concern among Saudi children? Saudi Med J. 2008; 29(11): 1630–1635. PubMed Abstract\n\nBarkhori Mahni M, Rezaeian M, Kia EB, et al.: Prevalence of Intestinal Parasitic Infections in Jiroft, Kerman Province, Iran. Iran J Parasitol. 2016; 11(2): 232–238. PubMed Abstract \| Free Full Text\n\nHarhay MO, Horton J, Olliaro PL: Epidemiology and control of human gastrointestinal parasites in children. Expert Rev Anti Infect Ther. 2010; 8(2): 219–234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdulkadir A, Ahmed M, Abubakar BM, et al.: Prevalence of urinary schistosomiasis in Nigeria, 1994–2015: Systematic review and meta-analysis. Afr J Urol. 2017; 23(4): 228–239. Publisher Full Text\n\nCook DM, Swanson RC, Eggett DL, et al.: A retrospective analysis of prevalence of gastrointestinal parasites among school children in the Palajunoj Valley of Guatemala. J Health Popul Nutr. 2009; 27(1): 31–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHailu T, Abera B: Performance evaluation of direct saline stool microscopy, Formol ether concentration and Kato Katz diagnostic methods for intestinal parasitosis in the absence of gold standard methods. Trop Doct. 2015; 45(3): 178–182. PubMed Abstract \| Publisher Full Text\n\nBayoumi M, Nykwac O, Kardaman M, et al.: Intestinal Parasitic Infections in School Students in Malakal City, Upper Nile State, South Sudan. SOJ Microbiol Inf Dis. 2016; 4(1): 1–5. Publisher Full Text\n\nMohamed MM, Ahmed AI, Salah ET: Frequency of intestinal parasitic infections among displaced children in Kassala Town. Khartoum medical journal. 2012; 2(1): 175–177. Reference Source\n\nScullion FT, Scullion MG: Gastrointestinal protozoal diseases in reptiles. J Exot Pet Med. 2009; 18(4): 266–278. Publisher Full Text\n\nAlla AA, Hassan HA: Hala and Ahmed file.sav. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9804869.v1\n\nMuhajir AEM, Hajissa K, Mohamed Z, et al.: Prevalence of Intestinal Parasitic Infection among Children in Al-kalakla, Khartoum, Sudan. World Appl Sci J. 2017; 35(2): 219–22. Reference Source\n\nGabbad AA, Elawad MA: Prevalence of intestinal parasite infection in primary school children in Elengaz area, Khartoum, Sudan. Academic Research International. 2014; 5(2): 86. Reference Source\n\nMagambo JK, Zeyhle E, Wachira TM: Prevalence of intestinal parasites among children in southern Sudan. East Afr Med J. 1998; 75(5): 288–290. PubMed Abstract\n\nEisa IM: The efficiency of different technique in the detection of intestinal parasites in school children in Keryab village, Khartoum state. M.Sc. thesis, AlzaimAlazhari University. 2005.\n\nAhmed HAH: Prevalence rate of intestinal parasites among schools children in Al-kalakla locality-Khartoum state (Doctoral dissertation, Sudan University of Science & Technology). 2017. Reference Source\n\nAhmed HAH: Prevalence rate of intestinal parasites among schools children in Al-kalakla locality-Khartoum state (Doctoral dissertation, Sudan University of Science & Technology). 2017. Reference Source" }	[ { "id": "55524", "date": "23 Oct 2019", "name": "Rama Paudel", "expertise": [ "Reviewer Expertise Infectious diseases (including parasitic diseases)", "antimicrobial resistance", "and medical education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study provides basic information about the frequencies of gastrointestinal parasites among children of a school in Sudan. The findings of this study may be useful to people of public health interest in the local region but can not be generalised. The following observations were noted while reviewing this manuscript:\nTitle:\nSince the study: a. detects only the intestinal parasites, b. does not reveal anything about the clinical symptoms of parasitic infections and c. involves children of only one school, the title should be changed to 'Frequencies of gastrointestinal parasites among students of a primary school in Al Kalakla Locality, Khartoum State, Sudan: a cross-sectional study'.\nMethods:\nStudy population: The authors should clearly mention how the sampling was determined and how each of the participants was selected (e.g, random sampling).\n\nIdentification of parasites: The information (whole paragraph) about identification of parasites should be mentioned/moved after the last concentration method (i.e just before data analysis).\n\nSaturated sugar flotation technique: The first and second sentences can be joined with removal of full stop after first sentence and use of 'and' 3rd line: 'sample of fecal' should be 'fecal sample'.\n\nData analysis: specificity should be determined by multiplication of the fraction by 100 (not by 10).\n\nThe authors should clearly mention (under 'Methods') whether all the samples were subject to examination by all the three methods in the study.\n\nResults\nThe term 'occurrence' would be better than 'prevalence' (which has its specific epidemiological meaning) in the title/contents of first two paragraphs.\n\n3rd line of 2nd paragraph: 'years' should be added after the age groups mentioned (i.e, 6–8, 9–11 and 12–14 years).\n\nDiscussion\nFirst sentence: The study done at one school can not be generalised. This sentence can be restructured as 'It was evident from the results that the overall occurrence of gastrointestinal parasites among children of a school (school name can be mentioned) in Al-kalakla was high........\n\nThe study needs to discuss the reasons for higher occurrence: a. of the parasites in this study compared to that of other studies, b.of the parasites in females compared in males c. of the parasites in 12–14 years of age group compared in other age groups.\n\nConclusion: The study done at one school can not be generalised. The authors can note the first comment made under 'discussion' and conclude accordingly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1719
https://f1000research.com/articles/9-92/v1	07 Feb 20	{ "type": "Research Article", "title": "Multidrug-resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school-going children in Kibera slum, Kenya", "authors": [ "Nduhiu Gitahi", "Peter B. Gathura", "Michael M. Gicheru", "Beautice M. Wandia", "Annika Nordin", "Peter B. Gathura", "Michael M. Gicheru", "Beautice M. Wandia", "Annika Nordin" ], "abstract": "Background: The objective of this study was to determine the prevalence of thermophilic Campylobacter spp. in asymptomatic school-going children and establish the antibiotic resistant patterns of the isolates towards the drugs used to treat campylobacteriosis, including macrolides, quinolones and tetracycline. Campylobacter spp. are a leading cause of enteric illness and have only recently shown resistant to antibiotics. Methods: This study isolated Campylobacter spp., including Campylobacter coli, Campylobacter jejuni and Campylobacter lari, in stool samples from asymptomatic school-going children in one of the biggest urban slums in Kenya. The disc diffusion method using EUCAST breakpoints was used to identify antibiotic-resistant isolates, which were further tested for genes encoding for tetracycline resistances using primer-specific polymerase chain reaction. Results: In total, 580 stool samples were collected from 11 primary schools considering both gender and age. Subjecting 294 biochemically characterized Campylobacter spp. isolates to genus-specific PCR, 106 (18.27% of stool samples) isolates were confirmed Campylobacter spp. Out of the 106 isolates, 28 (4.83%) were Campylobacter coli, 44 (7.58%) were Campylobacter jejuni while 11 (1.89%) were Campylobacter lari. Campylobacter jejuni had the highest number of isolates that were multi-drug resistant, with 26 out of the 28 tested isolates being resistant to ciprofloxacin (5 mg), nalidixic acid (30 mg), tetracycline (30 mg) and erythromycin (15 mg). Conclusions: In conclusion, a one-health approach, which considers overlaps in environment, animals and human ecosystems, is recommended in addressing campylobacteriosis in humans, since animals are the main reservoirs and environmental contamination is evident.", "keywords": [ "Multidrug", "resistance", "Campylobacter", "genes", "asymptomatic" ], "content": "Introduction\n\nCampylobacter spp. infection is a leading cause of enteric illness1,2, manifesting as mild-to-severe diarrhoea with watery loose stool that is often followed by bloody diarrhoea3. Infections also manifest as meningitis, pneumonia, miscarriage, severe form of Guillain-Barre syndrome (GBS) and reactive arthritis (ReA) and irritable bowel syndrome (IBS)3–5. Isolation of pathogenic Campylobacter spp. from asymptomatic children would be as a result of the pathogens not expressing the virulence factor cytolethal distending toxin, which is able to induce host cell apoptosis. Pathogenesis could also be influenced by host immune system and pathogens adaptation strategies6. Other factors like motility and chemotaxis affect effective Campylobacter colonization and pathogenesis; these have been shown to vary in mutants7.\n\nCampylobacter spp. are found in the intestinal tract of wild and domestic animals, particularly in birds, asymptomatically as temporal carriers but causing illness in humans3. The bacteria can survive up to five months at -20°C but die off in a few days at room temperature5,8,9. They are vulnerable to air exposure, drying, low pH and heating3. Three species, namely C. jejuni, C. coli and C. lari, account for 99% of human Campylobacter spp. isolates, with C. jejuni contributing 90% of the isolates. C. fetus and C. upsaliensis have also been isolated in humans10–12.\n\nDistinguishing between Campylobacter species using phenotypic methods is difficult; however, genotypic methods have been developed that are capable of distinguishing species. This has enabled more elaborate epidemiological understanding of Campylobacteriosis, thus identification of the sources and routes of infection13,14. The use of multiplex PCR methods has resulted in cheap, rapid and sensitive genetic identification procedures15.\n\nIt was not until recently that Campylobacter spp. was shown to exhibit multidrug resistance (MDR). Before that, the bacteria were considered to be susceptible16. Tetracycline is one of the antimicrobial agents against which Campylobacter spp. have showed resistance. In Campylobacter spp., tetracycline resistance has been reported to have been mediated by more than one tetracycline resistance (tet) genes. The tet(O) gene is the ribosomal protection protein and plays the primary part in tetracycline resistance in C. jejuni and C. coli17,18. This is transferred as a plasmid encoded gene19 or as non-self-mobile form when transferred through the chromosome17. Similarly, the tet(S) gene is a ribosomal protein gene which is transferable through the same channels like the tet(O) gene. The tet(A) gene encodes the 46 kDa membrane-bound efflux protein. This protein carries tetracycline from the cell membrane and its first known resistance role in Campylobacter spp. was reported in 201416.\n\nIn Campylobacter spp. resistance to the antibiotic quinolone is mainly due to a single point mutation in the quinolone resistance determination region of gyrA gene (QRDF)20,21, at amino acid 86 by replacement of Thr by Ile22. Occasionally, mutation in topoisomerase IV (ParC) results to resistance against quinolones. Other amino acids substitutions have been reported by Paddock et al. and others23–26. In Campylobacter there has been no documented mutational change to the gyrB subunit gene in relation to resistance against quinolones; however, Piddock et al.22, and Changkwanyeun et al.27 noted that resistance to ciprofloxacin in Campylobacter is mediated by mutations on the gyrA gene.\n\n\nMethods\n\nThe study was carried out at primary schools located at Kibera informal settlement, Nairobi County, Kenya in July 2015. Kibera is located at an altitude of 1670 m above sea level, at latitude 36°50’ east and longitude 1°17’ south, about 140 km south of the equator. Kibera is located 5 km South of Nairobi Central Business District (CBD), the Capital of Kenya. Kibera is divided into 9 official villages. The average living place is 3 m2, with an average of 5 persons per place. The study site presents a population with diverse enteric infections28. In total, 11 primary schools with pupil population ranging from 120 to 189 were randomly sampled and, 40 to 80 stool samples collected from pupils in each school, depending on the school population, making a total of 580 stool samples. With a known prevalence of 40.7% of soil transmitted helminths in school going children in urban Kenya, the formula by Martin et al. (1998) was used to determine the desired minimum sample size. The schools were distributed in five administrative villages, namely Lindi, Silanga, Laini Saba, Gatwekera and Mashimoni. Participants’ parents provided written consent through the care givers. This was done during parents’ school meetings, where parents were informed of the intended study and its benefits, those who agreed their children to participate were issued with consent forms for them to sign and return to their class teacher. Only those who their parents consented participated in the study.\n\nResearch clearances were given by National Commission for Science, Technology and Innovation (research clearance permit No. 3756) and ethical clearance (PKU/278/1274) was granted by Kenyatta University Ethical Review Committees.\n\nCampylobacter spp. culture. In the laboratory, 5 g of freshly collected faecal sample was pre-enriched by suspending the faeces in 45 ml buffered peptone water (BPW) (Oxoid, Hampshire, England) and incubating the suspension at 42°C for 18 hours in a 50-ml closed culture tube. The pre-enrichment was inoculated onto modified campylobacter charcoal-cefoperazone deoxycholate (mCCDA) agar plates with supplement (polymyxin B 2500IU, rifampincin 5 mg, trimethoprim 5 mg and cycloheximide 50 mg) using a sterile swab and the plates incubated at 45°C for up to 48 hours under anaerobic conditions. The mCCDA culture media (Oxoid, Hampshire, England) was prepared according to manufacturer’s instructions and stored at 4°C until use. Anaerobic conditions were achieved by adding a 21.3-g sachet of CampyGenTM 3.5 L (Oxoid, Hampshire, England) in an anaerobic jar with the cultures resulting to a maximum of 13.2% O2 within 24 hours and 9.5% CO2 in 1 hours. After 24 hours of incubation, the plates were checked for characteristic growth and plates without growth were re-incubated for an additional 24 hours. Characteristic colonies (grey/white or creamy grey in colour with moist appearance) were examined and counted. Distinct colonies were harvested and tested for oxidase and peroxidase breakdown, by picking a portion of distinct colony with a sterile wire loop and placing it on a drop of 30% hydrogen peroxide on a clean microscope slide. Production of effervescent air bubbles was recorded as peroxidases positive. The same colonies were tested for cytochrome oxidase enzyme production by placing a portion of the test colony onto oxidase paper impregnated with NNN’N’ tetramethyl-p-phenylene-diamine dihydrochloride (Oxoid, Basingstoke, UK). Purple colour change was recorded as positive reaction. Reactive colonies were processed for DNA and a portion stored in skimmed milk at -80°C for further characterization.\n\nDNA preparation from bacteria colonies and multiplex PCR. For DNA preparation, three distinct colonies from pure bacteria cultures were lifted with a sterile wire loop and suspended in 0.5 ml sterile, distilled water. The suspension was boiled for 30 minutes in a water bath. After cooling to room temperature, the preparation was centrifuged at 2000 x g and the supernatant harvested and stored at -20°C until analysis by polymerase chain reaction (PCR). PCR was first undertaken to confirm Campylobacter genus for the isolates after which three specific species were also identified: C. coli, C. jejuni and C. lari. The Campylobacter DNA preparation (2 µl) was amplified in a 25 µl reaction mix by mixing 2.5 µl 10X PCR buffer (Coraload), 0.5 µl dNTPs, 0.125 µl Taq DNA polymerase (Inqaba biotec, Pretoria, South Africa) and 0.1 µl of each specific primer to 10 pmole (Inqaba Biotec, Pretoria, South Africa), 2 µl DNA template and 18.657 µl DNAse/RNAse-free distilled water. The DNA was amplified using a program of initial heating at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 minutes, annealing at 56°C for 1 min, extension at 72°C for 1 min with a final extension of 72°C for 10 min using a Veriti 96 wells thermocycler, (Applied Biosystems, model 9902, Singapore) in 0.2-ml PCR tubes. The PCR products were kept at -20°C until gel electrophoresis was done.\n\nThe Campylobacter genus-specific primers, C412F and C1228 R, described by Linton et al.29 were used to amplify a 812 bp fragment within the 16S rRNA gene of Campylobacter species using forward primer C412F 5’-GGATGACACTTTTCGGAGC-3’ and reverse primer; C1228R 5’-R-CATTGTAGCACGTGTGTC-3’. Multiplex PCR was carried out for C. jejuni and C. coli with specific primers CjejlpxAF, CjejipxAR (shared by both species) and CcollpxAF, described by Klena et al.15 to amplify 331 bp and 391 bp fragment flanking the lpxA gene. The primer sequences were; CjejlpxAF (forward) 5’-ACAACTTGGTGACGATGTTGTA-3’, CjejipxAR (reverse, shared by CjejlpxA and CcollpxA) 5’-CAATCATGDGCDATATGASAATAHGCCAT-3’ for C. jejuni and for C. coli CcollpxAF (forward) 5’-AGACAAATAAGAGAGAATCAG -3’. The C. lari specific primers were forward primer lpxAC, 5’-AGACAATAAGAGAGAATCAG-3’ and reverse primer lpxARKK2M, 5’CAATCATGDGCDATATGASAATAHGCCAT-3’.\n\nThe PCR products were visualized by electrophoresis in a 1.5% agarose (Genetics analysis grade, Fisher Scientific, New Jersey) gel stained with 0.02% ethidium bromide and amplicons identified against molecular marker (50 bp DNA ladder, England Biolab) run alongside the samples.\n\nFor confirmation, the positively identified PCR products were submitted for sequencing. The PCR products were fist purified using exonuclease1, shrimp alkaline phosphatase mixture (ExoSAP mix) according to the manufacturer’s instructions. Briefly, this was done by adding 2.5 µl of ExoSAP mix to 10 µl PCR product. The mixture was then incubated at 37°C for 30 minutes and reaction stopped by heating at 95°C for 5 minutes. The clean PCR product was then quantified using a fluorimeter (Qubit 2.0, Invitrogen, USA). The clean DNA was first labelled with BigDye terminator v3.1kit (Applied Biosystem, CA, USA) according to the manufacturer’s instructions and loaded into Genetic Analyzer (ABI 3730 capillary analyser; Applied Biosystems, Foster City, CA, USA) for sequencing. Sequences were obtained in ABI files that were opened and edited to remove unspecific ends using BioEdit version 7.0.4 (Hall, CA, USA) software. Clean sequences were then submitted to NCBI GenBank database and BLASTn program used to test for homology and genetic identity of bacteria isolates.\n\nAntimicrobial sensitivity test (AST) for PCR-confirmed Campylobacter spp.. Campylobacter spp. isolates were phenotypically tested for resistance using selected antimicrobial agents according to European committee on antimicrobial susceptibility testing (EUCAST)30. Only those antibiotics with EUCAST established breakpoints were tested, namely tetracyclines (tetracycline 30 mg), quinolones (ciprofloxacin 5 mg, naladixic acid 30 mg) and macrolides (erythromycin 15mg). Mueller-Hinton agar plates plus 5% de-fibrinated horse blood with 20 mg/L β-nicotinamide adenine dinucleotide Mueller-Hinton fastidious (β-NAD (MH-F)); (Oxoid, Basingstoke, UK) were prepared and dried at 35°C, with the lid removed, for 15 min prior to inoculation to reduce swarming. Inoculum turbidity was adjusted to McFarland 0.5 prior to inoculation. The antibiotic discs were placed on the inoculated plates using a sterile multi-disc dispenser and incubated in a microaerobic environment at 41±1°C for 24 hours. Isolates with insufficient growth after 24 hours of incubation were re-incubated immediately and inhibition zones read after a total of 40–48 hours incubation. The inhibition zones were defined by the point showing no growth when viewed from the front of the plate with the lid removed and with reflected light.\n\nGenotypic characterization of Campylobacter spp. isolates for antimicrobial resistance. The 90 antibiotic resistant, PCR-characterized Campylobacter spp. isolates including; 11 C. lari, 30 C. coli and 49 C. jejuni were selected for demonstration of genes encoding for resistance to tetracyclines. Presence of four tetracycline resistance genes, tet(A), tet(B), tet(C) and tet(O), were tested. Multiplex PCR was carried out as described above. Primers used for amplification of products encoding for the resistant genes to tetracyclines are shown in Table 1.\n\n\nResults\n\nOf the 580 stool samples collected in 11 schools in Kibera, 294 (51%) were phenotypically characterized as suspect Campylobacter spp. When these isolates were subjected to PCR using genus and species-specific primers, 106 (18%) isolates were confirmed to be Campylobacter spp. Among the 106 isolates, 28 (4.8%) were C. coli, 44 (7.6%) C. jejuni while 11 (1.9%) were C. lari (Figure 1). In total, 23 (4.0%) Campylobacter isolates were not species identified as belonging to C. coli, C. jejuni or C. lari (Table 2).\n\nFrom left to right, lane 1 and 2 positive samples; mixture of Campylobacter jejuni and Campylobacter coli obtained from sequenced laboratory isolates (PHPT 1 &2). Lane 3 negative control: purified water. Lanes 4, 5, 9, 11 and 12: C. jejuni. Lanes 6, 8 and 15: Campylobacter coli. Lanes 7, 10, 13 and 14: negative samples. Lane 16: 100-bp ladder.\n\nThe EUCAST disk diffusion method was used to determine the resistance patterns of only the identified isolates, 68 (C. jejuni, C. coli and C. lari) confirmed by PCR (Table 2). Fifteen isolates were not recovered from storage culture after identification and thus not tested. All of the antibiotics studied had isolates showing resistance towards them, with 96% of isolates resistant to tetracycline (30 mg), 93% to naladixic acid (30 mg) and all the isolates tested resistant to erythromycin (15 mg). The antibiotic that most isolates were sensitive to was ciprofloxacin (5 mg) which still had 84% of the isolates showing resistance (Table 3). Of the four tet genes tested, tet(A) was most frequently identified in 20 (29.1%) of the isolates followed by tet(O) in 8 (11.7%) isolates and tet(C) in only 2 (2.9%) isolates. None of the isolates had more than one tet gene demonstrated. (Table 3).\n\nFour MDR profiles were observed. All of the tested isolates were resistant to two or more antimicrobial agents, but the majority of isolates (84%) were resistant to all the antibiotics studied (profile 3 and 4). Campylobacter jejuni had the highest number of isolates that were MDR with 25 (37%) isolates being resistant to all four antibiotics tested (profile 1). C. coli had 23 (34%) isolates resistant to all the four antibiotics while C. lari had 9 (13%) isolates resistant to the four antibiotics. One (1.5%) C. jejuni and C. lari isolates was resistant to drugs in profile 2, while three (3%) C. coli isolates were in this profile. However, profile 4 had only one (1%) C. coli isolate while profile 4 had 2 (3%) C. lari, 4 (6%) C. coli and 5 (7%) C. jejuni MDR isolates (Table 4).\n\n\nDiscussions\n\nA prevalence of 18% Campylobacter spp. in asymptomatic school going children was confirmed with PCR in this study. Campylobacter isolation from healthy children has been reported in developing countries34 at a prevalence of 15%, which closely agrees with this study’s findings. The authors attributed the infections with Campylobacter to close contact with reservoir animals like chickens, as well as poor sanitation20. Both of these factors are prominent in this study area, where chicken share housing with humans. The isolates were further characterized and C. jejuni was isolated more frequently (7.6%) as compared to C. coli (4.8%) and C. lari (2%), whereas 4% were none of the three species analysed. This distribution between Campylobacter species agrees with other reports from both developed and developing countries, as well as from Kenya34–36. Among the thermophilic Campylobacter species, C. upsaliensis was not characterised using PCR in this study. Phenotypic methods available for identification and differentiation of individual thermophilic Campylobacter spp. are non-conclusive and more sensitive methods are recommended37.\n\nThe Campylobacter spp. resistant to tetracycline had more tet(A) genes than tet(O) genes which were found in 20 (29%) and 8(12%) isolates respectively. This echoes Nguyen et al.20 who identified more tet(A) genes than tet(O) genes in Kenyan Campylobacter spp. isolates from chickens, at 35% and 13%. respectively. The high resistance rates obtained in this study, with 84% of isolates being resistant to all four agents, was in agreement with findings of Nguyen et al.20 and Coker et al.34 for chicken and human Campylobacter isolates, respectively. Both studies reported more than 70% resistance to ciprofloxacin, nalidixic acid and tetracycline. However, these results contrast with those of a report on human Campylobacter from diarrhoea cases in Western Kenya, where resistance towards ciprofloxacin were observed in 6% cases, towards nalidixic acid in 26%, and towards tetracycline in 18%. Erythromycin resistance in this study was also high, which contrasts the findings of Nguyen et al.20 in chicken-isolated Campylobacter. In the setting of the current study, with domestic animals hosted within the human settlements and poor sanitation, the possibility of cross-infection is very likely, as is horizontal transfer of antimicrobial resistance-encoding genes. Ciprofloxacin and erythromycin are the drugs of choice for Campylobacter treatment. These drugs are often used in Kenya for self-treatment of infections other than gastroenteritis, and resistance can be expected to increase in developing countries34.\n\nIn conclusion, the World Health Organization has recommended a multi-tiered and goal-oriented approach to control Campylobacter infections in both human and animals. Appropriate measures need to be taken on the various routes of transmission, including contaminated water and milk, through chlorination and pasteurization, respectively. Poultry, as the major reservoir, must be the main target4.\n\n\nData availability\n\nFigshare: Multidrug resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school going children in Kibera slum, Kenya.xlsx. https://doi.org/10.6084/m9.figshare.1130229238.\n\nFile ‘Multidrug resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school going children in Kibera slum, Kenya.xlsx’ contains the bacterial species identified from samples, the antibiotic zones of inhibition and the presence or absence of antibiotic-resistance genes in each sample.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors are grateful to the Grand Challenge Canada and the Swedish research Council VR for funding this study; the pupils, teachers and parents of the study schools; The contribution of the technical members of staff Department of Public Health Pharmacology & Toxicology and the staff of Peepoople Kenya is highly acknowledged.\n\n\nReferences\n\nHavelaar AH, Haagsma JA, Mangen MJ, et al.: Disease burden of foodborne pathogens in the Netherlands, 2009. Int J Food Microbiol. 2012; 156(3): 231–8. PubMed Abstract \| Publisher Full Text\n\nScallan E, Hoekstra RM, Angulo FJ, et al.: Foodborne illness acquired in the United States--major pathogens. Emerg Infect Dis. 2011; 17(1): 7–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNachamkin I, Blaser MJ, Tompkins LS: Campylobacter jejuni current status and future trends. American Society for Microbiology. Washington D.C. 1992. Reference Source\n\nWorld Health Organization: Antimicrobial resistance: global report on surveillance. 2014; 1–7. Reference Source\n\nBlaser MJ, Perez GP, Smith PF, et al.: Extra intestinal Campylobacter jejuni and Campylobacter coli infections: host factors and strain characteristics J Infect Dis. 1986; 153(3): 552–559. PubMed Abstract \| Publisher Full Text\n\nDasti JI, Tareen AM, Lugert R, et al.: Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int J Med Microbiol. 2010; 300(4): 205–11. PubMed Abstract \| Publisher Full Text\n\nvan Vliet AHM, Ketley JM: Pathogenesis of enteric Campylobacter infection. J Appl Microbiol. 2001; 90: 45S–56S. PubMed Abstract \| Publisher Full Text\n\nCastillo A, Escartin EF: Survival of Campylobacter jejunion sliced watermelon and papaya. J Food Prot. 1994; 57(2): 166–168. PubMed Abstract \| Publisher Full Text\n\nFricker CR, Park RWA: A two-year study of the distribution of 'thermophilic' campylobacters in human, environmental and food samples from the Reading area with particular reference to toxin production and heat-stable serotype. J Appl Bacteriol. 1989; 66(6): 477–490. PubMed Abstract \| Publisher Full Text\n\nLinton D, Owen RJ, Stanley J: Primer sequences for genus Campylobacter Rapid identification by PCR of the lettuce treated with spray-contaminated irrigation water. J Food Prot. 1996; 73: 1023–9.\n\nPatton DM, Shaffer N, Edmonds P, et al.: Human disease associated with \"Campylobacter upsaliensis\" (catalase-negative or weakly positive Campylobacter species) in the United States. J Clin Microbiol. 1989; 27(1): 66–73. PubMed Abstract \| Free Full Text\n\nKlein BS, Vergeront JM, Blazer MJ, et al.: Campylobacter Infection Associated With Raw Milk. An Outbreak of Gastroenteritis Due to Campylobacter Jejuni and Thermotolerant Campylobacter Fetus Subsp Fetus. JAMA. 1986; 255(3): 361–364. PubMed Abstract \| Publisher Full Text\n\nFrost JA: Current epidemiological issues in human Campylobacteriosis. Symp Ser Soc Appl Microbiol. 2001; 90(30): 85S–95S. PubMed Abstract \| Publisher Full Text\n\nWassenaar TM, Newell DG: Genotyping of Campylobacter spp. Appl Environ Microbiol. 2000; 66(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlena JD, Parker CT, Knibb K, et al.: Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol. 2004; 42(12): 5549–5557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdi-Hachesoo B, Khoshbakht R, Sharifiyazdi H, et al.: Tetracycline Resistance Genes in Campylobacter jejuni and C. coli Isolated From Poultry Carcasses. Jundishapur J Microbiol. 2014; 7(9): e12129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChopra I, Roberts M: Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev. 2001; 65(2): 232–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMazi W, Senok AC, Al-Mahmeed A, et al.: Trends in antibiotic sensitivity pattern and molecular detection of tet (O)-mediated tetracycline resistance in Campylobacter jejuni isolates from human and poultry sources. Jpn J Infect Dis. 2008; 61(1): 82–4. PubMed Abstract\n\nGibreel A, Wetsch NM, Taylor DE: Contribution of the CmeABC efflux pump to macrolide and tetracycline resistance in Campylobacter jejuni. Antimicrob Agents Chemother. 2007; 51(9): 3212–3216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen TNM, Hotzel H, Njeru J, et al.: Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya. Gut Pathog. 2016; 8(1): 39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEFSA: Scientific opinion of the panel on biological hazards on a request from the European Food Safety Authority on foodborne antimicrobial resistance as a biological hazard. The EFSA J. 2008; 765: 1–87. Reference Source\n\nPiddock LJV, Ricci V, Pumbwe L, et al.: Fluoroquinolone resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes. J Antimicrob Chemother. 2003; 51(1): 19–26. PubMed Abstract \| Publisher Full Text\n\nHakanen A, Jousimies-Somer H, Siitonen A, et al.: Fluoroquinolone resistance in Campylobacter jejuni isolates in travelers returning to Finland: association of ciprofloxacin resistance to travel destination. Emerg Infect Dis. 2003; 9(2): 267–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBachoual R, Ouabdesselam S, Mory F, et al.: Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli. Microb Drug Resist. 2001; 7(3): 257–61. PubMed Abstract \| Publisher Full Text\n\nZirnstein G, Li Y, Swaminathan B, et al.: Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol. 1999; 37(10): 3276–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuiz J, Goñi P, Marco F, et al.: Increased resistance to quinolones in Campylobacter jejunia genetic analysis of gyrA gene mutations in quinolone-resistant clinical isolates. Microbiol Immunol. 1998; 42(3): 223–6. PubMed Abstract \| Publisher Full Text\n\nChangkwanyeun R, Yamaguchi T, Kongsoi S, et al.: Impact of mutations in DNA gyrase genes on quinolone resistance in Campylobacter jejuni. Drug Test Anal. 2016; 8(10): 1071-1076. PubMed Abstract \| Publisher Full Text\n\nKaranja J, Wambari E, Okumu D, et al.: A study of awareness of malaria among Kibera population: Implication for community based intervention. J Natl Inst Public Health. 2002; 51(1): 51–55. Reference Source\n\nLinton D, Owen RJ, Stanley J: Primer sequences for genus Campylobacter Rapid identification by PCR of the lettuce treated with spray-contaminated irrigation water. J Food Prot. 1996; 73: 1023–9.\n\nThe European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 9.0. 2019. Reference Source\n\nRandall LP, Cooles SW, Osborn MK, et al.: Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK. J Antimicrob Chemother. 2004; 53(2): 208–216. PubMed Abstract \| Publisher Full Text\n\nVan TT, Chin J, Chapman T, et al.: Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes. Int J Food Microbiol. 2008; 124(3): 217–223. PubMed Abstract \| Publisher Full Text\n\nAbdi-Hachesoo B, Khoshbakht R, Sharifiyazdi H, et al.: Tetracycline Resistance Genes in Campylobacter jejuni and C. coli Isolated From Poultry Carcasses. Jundishapur J Microbiol. 2014; 7(9): e12129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoker AO, Isokpehi RD, Thomas BN, et al.: Human Campylobacteriosis in Developing Countries. Emerg Infect Dis. 2002; 8(3): 28–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooks JT, Ochieng JB, Kumar L, et al.: Surveillance for bacterial diarrhea and antimicrobial resistance in rural Western Kenya, 1997-2003. Clin Infect Dis. 2006; 43(4): 393–401. PubMed Abstract \| Publisher Full Text\n\nOberhelman RA, Taylor DN: Campylobacter infections in developing countries. (ed): Nachamkin I., Blaser M.J, Campylobacter, 2nd edition. Washington: American Society for Microbiology. 2000; 139–53.\n\nSteinhauserova I, Ceskova J, Fojtikova K, et al.: Identification and thermophilic Campylobacter spp. by phenotypic and molecular methods. J of App Micobiol. 2001; 90(3): 470–475. PubMed Abstract \| Publisher Full Text\n\nGitahi N, Gathura P, Gicheru M, et al.: Multidrug resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school going children in Kibera slum, Kenya.xlsx. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.11302292.v2" }	[ { "id": "59720", "date": "27 Mar 2020", "name": "Musafiri Karama", "expertise": [ "Reviewer Expertise Molecular epidemiology of Foodborne pathogens: Pathogenic E. coli", "Campylobacter and Salmonella and bacteriophages." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “Multidrug-resistant Campylobacter jejuni, Campylobacter coli and Campylobacter lari isolated from asymptomatic school-going children in Kibera slum, Kenya” investigated the occurrence and antimicrobial resistance of Campylobacter spp. among school-going children in a populous slum of Kenya. The study is well executed but will need wordsmithing to improve its readability.\nA weakness of this manuscript is the lack of recent citations in the reference list. I suggest the authors update the reference list by including at least more than 60% of references that have been published in the last 10 years or less. The conclusion will also have to focus on the topic of the research (Campylobacter prevalence and antimicrobial resistance in young children).\nI have inserted in the manuscript my corrections and suggestions to improve its clarity and style. This can be found here.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "65988", "date": "21 Jul 2020", "name": "Byeonghwa Jeon", "expertise": [ "Reviewer Expertise Antibiotic resistance of foodborne pathogens" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract:\nBackground: Should read: the antibiotic resistance patterns of\n\nResults: antibiotic concentrations, not the amount, need to be used. This applies to the entire manuscript.\n\nIntroduction:\n\"It was not until recently that Campylobacter spp. was shown to exhibit multidrug resistance (MDR). Before that, the bacteria were considered to be susceptible\" it may be difficult to say like this as the MDR was reported more than two decades ago.\n\nQuinolone resistance determination region (QRDR).\n\nCampylobacter does not have parC. Mutations in parC cause FQ resistance in E. coli and Salmonella; but this is not applied to Campylobacter.\n\nMethod:\nAntibiotic concentrations, not the amount, are to be made known in the supplement.\n\nCampylobacter is microaerophilic. 'Anaerobic conditions' --> 'Microaerobic conditions'.\n\nDid the authors include a quality control strain to the antibiotic susceptibility test?\n\nResults:\nFigure 1: There is no positive control for C. lari\n\nOthers:\nDid the asymptomatic children carrying Campylobacter have any previous experience of diarrhea? It may be an important point to discuss whether Campylobacter-positive children are completely asymptomatic carriers or were recovering from Campylobacter exposure without manifesting clinical symptoms.\n\nOverall, it reads well, but there are some minor English issues in some parts.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-92
https://f1000research.com/articles/9-63/v1	29 Jan 20	{ "type": "Software Tool Article", "title": "Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants", "authors": [ "Maxime Garcia", "Szilveszter Juhos", "Malin Larsson", "Pall I. Olason", "Marcel Martin", "Jesper Eisfeldt", "Sebastian DiLorenzo", "Johanna Sandgren", "Teresita Díaz De Ståhl", "Philip Ewels", "Valtteri Wirta", "Monica Nistér", "Max Käller", "Björn Nystedt", "Maxime Garcia", "Szilveszter Juhos", "Malin Larsson", "Pall I. Olason", "Marcel Martin", "Jesper Eisfeldt", "Sebastian DiLorenzo", "Johanna Sandgren", "Teresita Díaz De Ståhl", "Philip Ewels", "Valtteri Wirta", "Monica Nistér", "Max Käller" ], "abstract": "Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.", "keywords": [ "Analysis workflow", "Whole Genome Sequencing", "Germline variants", "Somatic variants", "Cancer" ], "content": "Introduction\n\nWhole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies opens up new avenues for research and for clinical applications, with many large initiatives launched worldwide. While much effort has been invested in novel sequencing analysis software, the importance of providing and maintaining workflows to combine software in an efficient and reproducible manner has been underestimated and too few resources are typically dedicated to address this issue. This is of particular importance for somatic variant analysis and especially for analysis of complex cancer genomes, where a combination of tools is still required for optimal sensitivity and specificity and to detect various types of gene mutations and other abnormalities (Alioto et al., 2015). Some encouraging solutions have been presented in recent years, including SeqMule (Guo et al., 2015), SpeedSeq (Chiang et al., 2015), Bcbio-nextgen, and DNAp (Causey et al., 2018). While all of the above represent commendable and important efforts, we have not found any workflow solution that in our opinion fulfils all of the following important user aspects: (i) easy installation, (ii) robust portability across different compute environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Here we present Sarek, an easy-to-install community-maintained workflow, offering a complete and scalable solution for germline and somatic variant detection, annotation and quality control. Sarek supports several reference genomes and can handle data from WGS, WES and gene panels, and is intended to be used both as a production workflow at core facilities and as a stand-alone tool for individual research groups. By using Docker or Singularity containers, Sarek installs easily on all POSIX compatible systems such as Linux and Mac OS X and is designed to work on compute environments dedicated to handle sensitive personal data without direct internet access—a situation expected to become increasingly common with growing data security awareness.\n\n\nMethods\n\nSarek offers a portable workflow for germline and somatic variant detection, annotation and quality control based on WGS, WES or gene panel data, using a range of state-of-the-art software and data resources in the field (Table 1, Figure 1). In the pre-processing step, sequence reads are aligned to the reference genome with BWA-MEM (Li, 2013), followed by deduplication and recalibration with GATK (McKenna et al., 2010). For germline samples, single-nucleotide variants and small insertion/deletions are detected with HaplotypeCaller (McKenna et al., 2010) and Strelka2 (Kim et al., 2018), and structural variations are detected with Manta (Chen et al., 2016) and TIDDIT (Eisfeldt et al., 2017). For somatic samples, somatic single-base mutations (SSM) and small somatic insertion/deletion mutations (SIM) are detected by GATK4 Mutect2 (Cibulskis et al., 2013) and Strelka2 (Kim et al., 2018). Somatic structural variants (including copy-number variation), as well as ploidy and sample purity are detected by Manta (Chen et al., 2016), ASCAT (Van Loo et al., 2010), and Control-FREEC (Boeva et al., 2012). All variants are annotated for potential functional effects with snpEff (Cingolani et al., 2012) and VEP (McLaren et al., 2016). Importantly, Sarek also generates a wide range of quality control metrics using FastQC, QualiMap (Okonechnikov et al., 2016), BCFtools (Li, 2011), Samtools (Li et al., 2009), and VCFtools (Danecek et al., 2011), visualized as an aggregated quality control review across samples with MultiQC (Ewels et al., 2016).\n\nA list of all the software required and currently implemented in Sarek. All analysis and quality metrics software are installed automatically when Sarek is launched.\n\nA schematic overview including some of the main analysis software implemented in the Sarek workflow. A more comprehensive list of the currently implemented software is given in Table 1.\n\nSarek is implemented in Nextflow (Di Tommaso et al. 2017), a workflow language designed specifically for bioinformatics applications. Nextflow has a transparent design, making the Sarek code easy to read, adjust and extend. Compared to the Bpipe workflow language (used in for example DNAp), Nextflow offers superior support for different execution environments, like Slurm, Sun Grid Engine, LSF and Kubernetes, and includes native support for cloud compute environments including Google Cloud and AWS. Support for AWS batch gives the possibility to easily distribute thousands of batch jobs on Amazon Web Services. Sarek is part of a rapidly growing community effort of well documented and community-tested Nextflow pipelines, and adheres to the nf-core portability and documentation guidelines (Ewels et al., 2019). To facilitate easy installation and to ensure reproducibility, all Sarek required tools are managed in Docker or Singularity (Kurtzer et al., 2017) containers, or a Conda environment. While Docker is a widely appreciated container solution, it is not always allowed at high-performance computing centers because of the involved security risks, making Singularity the preferred choice at these sites (Kurtzer et al., 2017). This is of particular importance for computer environments designed for handling sensitive personal data, where a high level of data security has to be maintained across multiple projects and users.\n\nSarek can be installed and executed on any POSIX-compatible computer system. To run a full WGS analysis, including both germline and somatic variants from a tumour/normal dataset with 90x/90x read coverage, we recommend a minimum of 16 cores on a node with 128 GB RAM, and at least 4 TB available free storage (in addition to the initial FASTQ files) in the input/output working directory. Of this, about 1.4 TB will be allocated for BAM files, annotated VCF files and CNV files, but excluding GVCF files (Table 2). At the end of the run, 2.3 TB temporary data can be removed, unless the user plans to perform re-runs from intermediate processing states. Many processes are distributed across cores by dividing the genome into smaller chunks, each being handled as a separate core job, with all the results being merged and sorted in a final step. Some of the used software are parallelized by design, while for others Sarek uses a scatter-gather approach to efficiently distribute the processing load across CPU cores and reduce the wall clock runtime.\n\nResource usage during a Sarek run on a WGS 90X/90X coverage medulloblastoma dataset on a 48-threaded computer node, starting from compressed FASTQ files. The storage resources refer to result files only. The total storage including all temporary data was 3.7 TB.\n\nGB, gigabyte; CPU, central processing unit; h, hours; m, minutes.\n\nSarek is run from a computer system with a local installation of Nextflow and support for either Conda environments, Docker or Singularity containers. Nextflow can automatically fetch the Sarek source code from GitHub. All software dependencies are encapsulated in Docker or Singularity containers which are downloaded from Docker Hub, or built in a new Conda environment using Bioconda (Grüning et al., 2018). As such, cumbersome software installations by the user are completely avoided. Configuration files allow tailoring to specific user needs and incomplete runs are easily restarted from any stage in the workflow process. Sarek comes with a small test dataset and a suite of tests to verify the installation. This is also used for Continuous Integration testing with GitHub Actions.\n\n\nResults\n\nTo test performance in terms of resource usage and biological results, Sarek was run on a medulloblastoma WGS tumour/normal dataset from a sample with high tumour cell content (∼98%), and with a curated “Gold Set” of verified somatic mutations from a previous benchmark study (Alioto et al., 2015). In line with the above benchmark study, Sarek (version 2.5.2) was executed with WGS germline and somatic variant calling using a 90X/90X tumour/normal dataset (accession number EGAD00001001859, read sets EGAR00001387019-24 and EGAR00001387025-32). Runs were performed on a single 48-thread node with a local direct attached storage (DAS): A Dell PowerEdge R740 server, with two Intel Xeon Gold 6126 with a total of 24 cores (48 threads) CPUs, 756 GB memory, and 100 TB SCv3020 Compellent Storage. The complete Sarek run including preprocessing followed by both germline and somatic variant calling and annotation took 48 hours and 21 minutes, and required about three times more storage than the original input data (Table 2). Notably, the complete Sarek run was executed by a single command, with fully automated installation, execution, and efficient job distributions of the more than 15 different software tools to complete the analysis and provide quality control metrics, without any manual intervention needed during the two-day run. To ensure that the Sarek output was biologically sound, we calculated precision, recall and F1 statistics for the Sarek output based on the “Gold Set” of somatic single-base mutations (SSM) and somatic insertion/deletion mutations (SIM) as previously defined (Alioto et al., 2015). Using the intersection of the output from the two somatic variant callers (GATK4 Mutect2 and Strelka2), Sarek provided accuracy measures for SSMs (F1 score = 0.80) and SIMs (F1 score = 0.58) in the top range of the 18 somatic variant calling procedures included in the original benchmarking study on this data set (Table 3), indicating that the workflow operates as intended. The sample purity was estimated to be 100%, as compared to 98% previously reported for this sample. For somatic structural variants and ploidy, no relevant benchmark data was available, and therefore no quantitative assessment beyond previously published results for the implemented software could be performed, but the integrity of the runs were checked by comparing the results of Manta, ASCAT, and Control-FREEC run within Sarek and as stand-alone.\n\nSummary of accuracy measures for the two somatic variant callers used in Sarek to detect somatic single-base mutations (SSMs) and somatic insertion/deletion mutations (SIMs), as well as their union and intersection.\n\n* The median accuracy measures across 18 somatic variant calling procedures as previously reported (Alioto et al., 2015)\n\n\nUse case\n\nSarek has been extensively tested and applied on various WGS datasets, including thousands of samples for germline variant analyses, and hundreds of paired tumour/normal samples for somatic mutation analyses. In addition, Sarek has also been successfully tested on WES data and gene panels. Below we present a standard use case with a tumour/normal WGS dataset as input, running both germline and somatic variant analyses.\n\nFor a somatic variant analysis, the user should provide the sequencing FASTQ files from both tumour and normal control tissue from the same individual, described in a tab-delimited TSV file (here: samples.tsv). Each line of the TSV file contains information about a sequence data file, including: The identifier of the individual, the gender (XX or XY), the status of the sample (0 for Normal or 1 for Tumour), the identifier of the sample, the sequencing lane (if samples are multiplexed across multiple lanes), and the paths to the FASTQ file of the first and second read in the read-pair. Relapse samples from the same individual are also supported.\n\nRunning Sarek with Singularity container on a computer system supporting Java 8 requires only installation of Nextflow and Singularity. A full analysis run starting from FASTQ files including mapping, recalibration, variant calling and annotation, as well as generating a full QC report can be invoked by a single Nextflow command:\n\n\n\nNextflow will recognize the workflow name and will download the specified version (2.5.2) of the pipeline from GitHub, including the corresponding container, as well as fetching the required reference files from AWS-iGenomes. The default reference genome is human GRCh38, but Sarek also supports GRCh37 and nearly 30 other genomes directly accessible from iGenomes. Alternatively, users can manually supply Sarek with other reference genomes. Non-default parameters and links to local reference files are handled in accordance with nf-core guidelines. User configuration profiles can be stored locally or centrally at https://github.com/nf-core/configs.\n\nA full Sarek run will produce a large number of output files, but the main results consist of (i) a set of annotated variants in VCF files from the various included tools for both germline and somatic variants, (ii) tumour sample purity and ploidy results for somatic samples, and (iii) a broad set of QC metrics. A detailed description of all output files is given at the Sarek documentation pages.\n\n\nDiscussion\n\nHuman WGS is transforming medical research, and provides a foundation to develop novel clinical applications and improve health care. An important aspect to harvesting the potential of WGS is however to empower the research community with adequate bioinformatics tools, and reproducible bioinformatics workflows are important drivers of scientific progress by making complex processing of large datasets feasible for a wide range of researchers. While we are highly appreciative of existing workflows for cancer and non-cancer variant detection, we argue that there is no one-size-fits-all solution and more initiatives are needed to serve the large and diverse research user community, especially for WGS data. Sarek builds on a philosophy of reasonably narrow, independent workflows, written in the domain-specific language Nextflow. In our experience, this is an effective strategy to simplify workflow maintenance at sequencing core facilities, and to allow easy deployment and modifications by individual research groups. Sarek efficiently utilizes cloud and high-performance compute clusters and installs easily across compute environments. Sarek provides annotated VCF files, CNV reports and quality metrics for germline and cancer samples from raw FASTQ sequencing data in about 48 hours for 90X/90X WGS data (as demonstrated here), in a few hours for WES data, and within minutes for gene panels (in-house data, not presented here). Ongoing efforts aim to develop add-on ranking and visualization modules and to efficiently extract clinically and biologically relevant findings, to help advance basic and translational research.\n\n\nConclusion\n\nSarek is a portable and reproducible workflow to detect germline and somatic variants from WGS, WES and gene panel data. It includes extensive analysis and quality control metrics, while still being limited to a relatively narrow scope to achieve optimal usability, functionality and transparency. Sarek is flexible with a low threshold for user modifications, and is thus well adapted to the current requirements in the research community. Thanks to its design, it installs easily and reproducibly on all POSIX compatible computer systems, including secure compute environments for sensitive personal data with indirect Internet access.\n\n\nData availability\n\nEuropean Genome-phenome Archive: A comprehensive assessment of somatic mutation detection in cancer using whole genome sequencing. https://www.ebi.ac.uk/ega/datasets/EGAD00001001859. Read sets EGAR00001387019-24 and EGAR00001387025-32 were analysed.\n\nThese data are held under restricted access. Readers wishing to apply for access to the data must first apply through the ICGC Data Access Compliance Office (https://icgc.org/daco) and complete the data access form. Access will be granted to those whose projects conform to the goals and policies of ICGC. Help with completing the data access form is available at https://icgc.org/daco/help-guide-section.\n\nThe workflow itself comes with a prebuilt profile with a complete configuration for automated testing, including links to a small test dataset.\n\n\nSoftware availability\n\nSarek is available at: https://nf-co.re/sarek.\n\nSource code available at: https://github.com/nf-core/sarek.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3579102 (Garcia et al., 2019).\n\nLicense: MIT License.", "appendix": "Author contributions\n\n\n\nMK, BN and MN conceived the idea for Sarek. MG and SJ led the project. MG, SJ, ML, PIO, MM, JE, and SDL designed and implemented the workflow. JS, TDS, VW, MN, BN, PE and MK performed testing and provided design feedback. MG, SJ and BN wrote the manuscript with the help from all authors.\n\n\nAcknowledgements\n\nWe are grateful for the valuable input from the Oslo University Hospital bioinformatics core facility (Oslo University Hospital), the T Martinsson lab (Gothenburg University), the A–C Syvänen lab (Uppsala University), and Alex Peltzer (Quantitative Biology Center, University of Tübingen). The National Genomics Infrastructure (NGI) and Uppsala Multidisciplinary Centre for Advanced Computational Science (UPPMAX) provided computational resources. Help with graphical design was provided by Dr. Jonas Söderberg (Uppsala university).\n\n\nReferences\n\nAlioto TS, Buchhalter I, Derdak S, et al.: A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing. Nat Commun. 2015; 6: 10001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoeva V, Popova T, Bleakley K, et al.: Control-FREEC: a tool for assessing copy number and allelic content using next-generation sequencing data. Bioinformatics. 2012; 28(3): 423–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCausey JL, Ashby C, Walker K, et al.: DNAp: A Pipeline for DNA-seq Data Analysis. Sci Rep. 2018; 8(1): 6793. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen X, Schulz-Trieglaff O, Shaw R, et al.: Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics. 2016; 32(8): 1220–1222. PubMed Abstract \| Publisher Full Text\n\nChiang C, Layer RM, Faust GG, et al.: SpeedSeq: ultra-fast personal genome analysis and interpretation. Nat Methods. 2015; 12(10): 966–968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCibulskis K, Lawrence MS, Carter SL, et al.: Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol. 2013; 31(3): 213–219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCingolani P, Platts A, Wang le L, et al.: A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6(2): 80–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDanecek P, Auton A, Abecasis G, et al.: The variant call format and VCFtools. Bioinformatics. 2011; 27(15): 2156–2158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Tommaso P, Chatzou M, Floden EW, et al.: Nextflow enables reproducible computational workflows. Nat Biotechnol. 2017; 35(4): 316–319. PubMed Abstract \| Publisher Full Text\n\nEisfeldt J, Vezzi F, Olason P, et al.: TIDDIT, an efficient and comprehensive structural variant caller for massive parallel sequencing data [version 2; peer review: 2 approved] F1000Res. 2017; 6: 664. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEwels P, Magnusson M, Lundin S, et al.: MultiQC: Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016; 32(19): 3047–3048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEwels PA, Peltzer A, Fillinger S, et al.: nf-core: Community curated bioinformatics pipelines. bioRxiv. 2019; 610741. Publisher Full Text\n\nGarcia M, Peltzer A, Alneberg J: nf-core/sarek: Sarek 2.5.2 - Jåkkåtjkaskajekna (Version 2.5.2). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3579102\n\nGrüning B, Dale R, Sjödin A, et al.: Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018; 15(7): 475–476. PubMed Abstract \| Publisher Full Text\n\nGuo Y, Ding X, Shen Y, et al.: SeqMule: automated pipeline for analysis of human exome/genome sequencing data. Sci Rep. 2015; 5: 14283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, Scheffler K, Halpern AL, et al.: Strelka2: fast and accurate calling of germline and somatic variants. Nat Methods. 2018; 15(8): 591–594. PubMed Abstract \| Publisher Full Text\n\nKurtzer GM, Sochat V, Bauer MW: Singularity: Scientific containers for mobility of compute. PLoS One. 2017; 12(5): e0177459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011; 27(21): 2987–2993. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv 1303.3997v2. 2013. Reference Source\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–1303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLaren W, Gil L, Hunt SE, et al.: The Ensembl Variant Effect Predictor. Genome Biol. 2016; 17(1): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkonechnikov K, Conesa A, García-Alcalde F: Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics. 2016; 32(2): 292–294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Loo P, Nordgard SH, Lingjærde OC, et al.: Allele-specific copy number analysis of tumors. Proc Natl Acad Sci U S A. 2010; 107(39): 16910–16915. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "59295", "date": "09 Mar 2020", "name": "Tony Håndstad", "expertise": [ "Reviewer Expertise Diagnostic bioinformatics (variant calling pipelines) and variant interpretation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSarek is a workflow for variant detection and analysis of sequencing data from WGS, WES and targeted panels. The workflow is comprehensive and versatile, allowing for variant detection in both germline and somatic samples, from WGS/WES/panel sequencing.\nIt includes variant calling of SNPs, indels, and structural variants, as well as annotation and extensive quality control.\n\nSarek is open source and part of the nf-core community effort which builds well-curated analysis pipelines in the Nextflow pipeline framework.\n\nSarek is very user friendly, and installation, configuration and execution is easy to perform, while the workflow is also flexible.\n\nImplementation manages to be clear, despite also being advanced with parallelization and ample choice of installation/execution. Many researchers would likely struggle to implement pipelines at this advanced level.\n\nThe documentation is excellent, and despite the comprehensive functionality, most users should find it easy to set up Sarek and get started. I have no doubt that Sarek will be a very valuable addition to the research community.\n\nThe paper is well written and fulfils all the reviewer criteria. As reviewer, I have only a few minor comments:\nSarek can use different Nextflow configuration profiles. In the Sarek documentation, it says that the test profile is a profile with a complete configuration for automated testing and that it includes links to test data so needs no other parameters.\nIt should be obvious to most users, but I would suggest that the authors make it clear that when using the test profile, a user must also supply conda, docker or singularity profile if not all the tools are installed in the PATH. This is clear from the general nf-core documentation (https://nf-co.re/usage/introduction) but less so from the Sarek documentation.\n\nWhereas the choice of Nextflow is justified, there is little argumentation for why the different tools (variant callers in particular) are selected other than that they represent state-of-the-art. Also why several tools for variant calling are combined is not mentioned, though the referred paper (Alioto et al., 2015) makes a clear case for this, at least for somatic variant calling.\n\nThe paper title makes it clear that Sarek is for whole genome sequencing analysis, but as stated in the text, Sarek is also applicable to exome and targeted panel analyses where the authors say it has been run successfully.\nMany researchers are using exome sequencing, so it could be of interest to know if the authors have an opinion or experience with how use of targeted sequencing data limit Sarek in terms of accuracy or utility, for example, are the tools used for structural variant calling able to handle exome data well (to the extent possible with targeted sequencing?)\nThe documentation has a small chapter stating that the authors recommend supplying a BED file with the targeted regions, but there is not so much explanation of what the effect of this is.\n\nThe authors demonstrate that Sarek is both fast and accurate by running it on a tumor/normal(germline) dataset from a previous benchmark study. I think this is acceptable/sufficient, but one could always wish for more; the paper could be strengthened by for example running the well-known public germline HG001 sample against the Genome In a Bottle gold standard dataset.\n\nAccurate somatic variant calling is difficult. But the included benchmark study demonstrates that Sarek performs well in comparison with other pipelines. The tool leaves it up to the user to decide whether to use output from a single variant caller or the union or intersection from all tools for increased sensitivity or precision.\n\nIn summary, I think Sarek is a great addition to the community and recommend the paper for indexing.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5674", "date": "04 Sep 2020", "name": "Björn Nystedt", "role": "Author Response", "response": "We are grateful to the reviewer for the positive and constructive comments! We have uploaded a revised version of the manuscript and included updated documentation for the workflow, including adjustments and improvements as suggested by the reviewer, and as described in the detailed comments below, marked in bold. Approved Sarek is a workflow for variant detection and analysis of sequencing data from WGS, WES and targeted panels. The workflow is comprehensive and versatile, allowing for variant detection in both germline and somatic samples, from WGS/WES/panel sequencing. It includes variant calling of SNPs, indels, and structural variants, as well as annotation and extensive quality control. Sarek is open source and part of the nf-core community effort which builds well-curated analysis pipelines in the Nextflow pipeline framework. Sarek is very user friendly, and installation, configuration and execution is easy to perform, while the workflow is also flexible. Implementation manages to be clear, despite also being advanced with parallelization and ample choice of installation/execution. Many researchers would likely struggle to implement pipelines at this advanced level. The documentation is excellent, and despite the comprehensive functionality, most users should find it easy to set up Sarek and get started. I have no doubt that Sarek will be a very valuable addition to the research community. The paper is well written and fulfils all the reviewer criteria. As reviewer, I have only a few minor comments: Sarek can use different Nextflow configuration profiles. In the Sarek documentation, it says that the test profile is a profile with a complete configuration for automated testing and that it includes links to test data so needs no other parameters. It should be obvious to most users, but I would suggest that the authors make it clear that when using the test profile, a user must also supply conda, docker or singularity profile if not all the tools are installed in the PATH. This is clear from the general nf-core documentation (https://nf-co.re/usage/introduction) but less so from the Sarek documentation. This is a good suggestion and we have highlighted and improved this information in the Sarek documentation. We are also working to revise the general documentation format in nf-core to make this more transparent throughout. Whereas the choice of Nextflow is justified, there is little argumentation for why the different tools (variant callers in particular) are selected other than that they represent state-of-the-art. Also why several tools for variant calling are combined is not mentioned, though the referred paper (Alioto et al., 2015) makes a clear case for this, at least for somatic variant calling. This is a good suggestion and we have clarified this point in the manuscript. In brief, we have included software we have judged being of high quality, well-maintained and robust. Additional software will be added to Sarek later on in a community effort, and this process is already ongoing. The paper title makes it clear that Sarek is for whole genome sequencing analysis, but as stated in the text, Sarek is also applicable to exome and targeted panel analyses where the authors say it has been run successfully. Many researchers are using exome sequencing, so it could be of interest to know if the authors have an opinion or experience with how use of targeted sequencing data limit Sarek in terms of accuracy or utility, for example, are the tools used for structural variant calling able to handle exome data well (to the extent possible with targeted sequencing?) The documentation has a small chapter stating that the authors recommend supplying a BED file with the targeted regions, but there is not so much explanation of what the effect of this is. This is a relevant comment, and we want to clarify that Sarek has been run on whole-exome sequencing data in pilot user projects, and has been reported to us to work well (personal communication), but no comprehensive benchmark has been performed by us to evaluate this. This has been clarified in the Sarek documentation and in the manuscript. The authors demonstrate that Sarek is both fast and accurate by running it on a tumor/normal(germline) dataset from a previous benchmark study. I think this is acceptable/sufficient, but one could always wish for more; the paper could be strengthened by for example running the well-known public germline HG001 sample against the Genome In a Bottle gold standard dataset. This is a good suggestion and we have run germline variant calling with Sarek on the HG001 sample and compared to the Genome In a Bottle gold standard dataset, and these results are now included in the manuscript. Accurate somatic variant calling is difficult. But the included benchmark study demonstrates that Sarek performs well in comparison with other pipelines. The tool leaves it up to the user to decide whether to use output from a single variant caller or the union or intersection from all tools for increased sensitivity or precision. In summary, I think Sarek is a great addition to the community and recommend the paper for indexing. Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes" } ] }, { "id": "61129", "date": "31 Mar 2020", "name": "Esa Pitkänen", "expertise": [ "Reviewer Expertise Bioinformatics", "cancer genetics", "machine learning" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes Sarek, a workflow for analyzing next-generation sequencing (NGS) data. Sarek is based on Nextflow, a popular tool for defining computational workflows. In order to process NGS data, i.e., generating annotated variant calls ready for downstream analyses, multiple complex software tools need to be executed. This is not only computationally demanding, but also labor-intensive due to operators having to install and maintain a complicated collection of software as well as diagnose failed analysis runs, often resulting in high management overhead compared to the total computation time (Yakneen and Waszak, 20201). Sarek aims to minimize the installation and management time overhead by building a NGS workflow on top of Nextflow, automatically installing the required software components. These software consist of some of the state-of-the-art tools in read mapping, variant calling and annotation and quality control. Sarek is a welcome addition to the toolkit of bioinformaticians looking for an NGS analysis workflow, which can be easily installed on a HPC cluster or cloud environment. The article is well-written and clear to understand. While I'm happy to recommend indexing of the manuscript also in the present form, I have a few suggestions how to improve it:\n\nMajor comments:\nWhile some of the existing NGS workflows are mentioned, I would appreciate it if Sarek was compared to these approaches in more detail. Is there functionality that is currently missing from Sarek that is present in one of the other workflows?\n\nTypically in NGS data analysis, a lot of time can be spent on debugging failed runs to find out whether one of the tools failed, if the data is corrupted/missing or if there was a hardware error. How does Sarek support run diagnostics and relaunching failed jobs?\n\nHow does Sarek combine variant calls when multiple callers are used for a variant type?\n\nSomatic single nucleotide and indel variant calls from Sarek were shown to match well with a previously defined gold standard callset. No benchmark data was available for more complex somatic variants and variant calling accuracy for germline variants was not evaluated. I am interested in seeing more comprehensive tests to cover all germline and somatic variant types.\n\nI would appreciate it if a minimal test dataset together with instructions and a suite of automated tests was provided with Sarek. This would make it easier for the user to test out an installation as well as raising issues in GitHub.\n\nMinor comments:\nHow easy it is for users to modify or extend Sarek by for example adding a new variant caller to the workflow? This could be explored in more detail in text.\n\nIt would be good to easily see which tools are used to call and analyze each variant type. This information could be added either to Fig 1, Table 1 or both.\n\nFreeBayes is included in Figure 1 but missing from Table 1.\n\nThe wording in “To facilitate easy installation and to ensure reproducibility, all Sarek required tools are managed in Docker or Singularity (Kurtzer et al., 2017) containers, or a Conda environment.” should be clarified -- are all tools being maintained in all the three systems?\n\nWhen running Sarek with default options, it crashes in the tool version check. It took me a while to figure out this was due to the default “-profile” argument of “standard” which seems to assume Singularity is available. It would be good to improve error messages so that it is easier to understand the underlying cause. A minimal installation and testing procedure mentioned above would help in this regard.\n\nTypo: “Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies opens…” -> “...open”.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5675", "date": "04 Sep 2020", "name": "Björn Nystedt", "role": "Author Response", "response": "We are grateful to the reviewer for the positive and constructive comments! We have uploaded a revised version of the manuscript and included updated documentation for the workflow, including adjustments and improvements as suggested by the reviewer, and as described in the detailed comments below, marked in bold. Approved This manuscript describes Sarek, a workflow for analyzing next-generation sequencing (NGS) data. Sarek is based on Nextflow, a popular tool for defining computational workflows. In order to process NGS data, i.e., generating annotated variant calls ready for downstream analyses, multiple complex software tools need to be executed. This is not only computationally demanding, but also labor-intensive due to operators having to install and maintain a complicated collection of software as well as diagnose failed analysis runs, often resulting in high management overhead compared to the total computation time (Yakneen and Waszak, 20201). Sarek aims to minimize the installation and management time overhead by building a NGS workflow on top of Nextflow, automatically installing the required software components. These software consist of some of the state-of-the-art tools in read mapping, variant calling and annotation and quality control. Sarek is a welcome addition to the toolkit of bioinformaticians looking for an NGS analysis workflow, which can be easily installed on a HPC cluster or cloud environment. The article is well-written and clear to understand. While I'm happy to recommend indexing of the manuscript also in the present form, I have a few suggestions how to improve it: Major comments: While some of the existing NGS workflows are mentioned, I would appreciate it if Sarek was compared to these approaches in more detail. Is there functionality that is currently missing from Sarek that is present in one of the other workflows? This is a relevant comment, but a detailed comparison of the current functionality of different workflows risk being quickly outdated as functionality will frequently change both in Sarek and in the other mentioned workflows. Also, the main purpose of Sarek is not to provide unique functionality per se, but to provide a workflow solution with some generally important features as detailed in the manuscript “..(i) easy installation, (ii) robust portability across different compute environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting.” Therefore, the main difference between Sarek and the other mentioned workflows is in practical usability rather than a particular functionality, as these can typically be tuned or added as needed. Typically in NGS data analysis, a lot of time can be spent on debugging failed runs to find out whether one of the tools failed, if the data is corrupted/missing or if there was a hardware error. How does Sarek support run diagnostics and relaunching failed jobs? This is a good comment, and NextFlow reports the failed process and the error-code from the underlying software, and Sarek is designed to make it very easy to resume failed jobs from the point of failure. This has now been better highlighted in the manuscript under the heading “Portability and reproducibility”. We will work continuously with the user community to further avoid run failures and to continuously improve the diagnostic capability and error-handling. How does Sarek combine variant calls when multiple callers are used for a variant type? Sarek will report variants from all callers include in the run, but it is up to the user to decide on how to combine results from different callers, since e.g. the optimal balance between high specificity versus high sensitivity differs across research projects. This has been clarified in the manuscript under the heading “Output”, and in the “Discussion”. Somatic single nucleotide and indel variant calls from Sarek were shown to match well with a previously defined gold standard callset. No benchmark data was available for more complex somatic variants and variant calling accuracy for germline variants was not evaluated. I am interested in seeing more comprehensive tests to cover all germline and somatic variant types. This is a good suggestion and we have run germline variant calling with Sarek on the HG001 sample and compared to the Genome In a Bottle gold standard dataset, and these results are now included in the manuscript. Benchmarking of complex somatic variants is very complex and difficult due to the lack of robust and relevant benchmark datasets making such testing beyond the scope of this publications, since Sarek is a workflow and does not provide any novel algorithms or methodology per se. For now, we refer users to the tests published along with the respective included variant calling software. This limitation is stated in the manuscript under the heading “Results”. I would appreciate it if a minimal test dataset together with instructions and a suite of automated tests was provided with Sarek. This would make it easier for the user to test out an installation as well as raising issues in GitHub. This is a good suggestion, and there is actually already a minimal test dataset and a suit of tests available and documented in Sarek, as detailed under the heading “Installation and testing”. We have improved the Sarek documentation to make this clear and easy to find. Minor comments: How easy it is for users to modify or extend Sarek by for example adding a new variant caller to the workflow? This could be explored in more detail in text. This is a good suggestion and we have clarified this point in the manuscript under the heading “Operation: Workflow overview and software”. In brief, we have started with software we have judged being of high quality, well-maintained and robust. Additional software will be added to Sarek later on in a community effort, and this process is already ongoing. It would be good to easily see which tools are used to call and analyze each variant type. This information could be added either to Fig 1, Table 1 or both. This is a good suggestion and we have added this information to Table 1. FreeBayes is included in Figure 1 but missing from Table 1. This is a good note, and we have adjusted the manuscript accordingly. FreeBayes can optionally be run in Sarek, and it is now included in Table 1. The wording in “To facilitate easy installation and to ensure reproducibility, all Sarek required tools are managed in Docker or Singularity (Kurtzer et al., 2017) containers, or a Conda environment.” should be clarified -- are all tools being maintained in all the three systems? This is a very useful comment, and this has now been clarified in the manuscript under the heading “Portability and reproducibility”. All tools are installed in Conda, and then pushed to DockerHub (https://hub.docker.com/). This way all tools are available directly from all three systems; Conda, Docker and Singularity. When running Sarek with default options, it crashes in the tool version check. It took me a while to figure out this was due to the default “-profile” argument of “standard” which seems to assume Singularity is available. It would be good to improve error messages so that it is easier to understand the underlying cause. A minimal installation and testing procedure mentioned above would help in this regard. This is a very useful comment, and we have improved the error message and the documentation regarding the “-profile” arguments. Typo: “Whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies opens…” -> “...open”. This typo has been corrected in the manuscript. Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes" } ] } ]	1	https://f1000research.com/articles/9-63
https://f1000research.com/articles/8-1843/v1	01 Nov 19	{ "type": "Clinical Practice Article", "title": "A case series analysing patients with dental anxiety: a patient-centred model based on psychological profiling", "authors": [ "Riccardo Tizzoni", "Laura Veneroni", "Alfonso D'Aloia", "Marta Tizzoni", "Carlo Alfredo Clerici", "Laura Veneroni", "Alfonso D'Aloia", "Marta Tizzoni", "Carlo Alfredo Clerici" ], "abstract": "Anxiety and distress can jeopardize dental care experience of patients and may affect the clinical result. Although a wide range of sedation and analgesia techniques are currently available to relieve distress and pain during dental procedures, operative models to choose the most effective sedation-analgesic strategies are lacking. This case series proposes a patient-centred model to optimize patients’ cooperation during dental care delivery. We describe how to achieve correct anaesthesia by using the least sedative procedure, accounting for the dental procedure needed and patient’s psychological profile. Five patients were considered as paradigmatic to show the balance between patients’ subjective experiences and the clinical procedures: a patient with low stress, good compliance (case 1); moderate stress and reduction in compliance (case 2); anxious patient (case 3); patient with acute anxiety and emotional distress (case 4); anguished patient (case 5). A multimodal treatment of emotional and behavioural condition and a patient-centred model approach contributed to achieve the best patient satisfaction in the five cases detailed here.", "keywords": [ "dental anxiety", "sedation", "analgesia", "compliance", "patient-centred model" ], "content": "Introduction\n\nAn increasing number of patients are undergoing day-case dental procedures or surgeries, and in some cases they may experience significant emotional upset for either consultation or dental therapy. Patients are often required to cooperate for a long time and stay in a coerced position: this may affect their psychological state and induce discomfort, fear, anxiety, and pain1. When patients can arduously cooperate during treatments, an appropriate analgesia should be achieved and a satisfactory anxiolysis should be accomplished. To this aim, a range of techniques, from the tell-show-do approach to conscious sedation and general anaesthesia, is currently available2,3.\n\nWhen fearful patients cannot be reassured by explanation and comforting professional behaviour, the clinician should consider a well-tolerated and effective sedative agent, associated with topical and/or injectable anaesthetics.\n\nThe plurality of strategies available enable the team to safely provide a comprehensive care to minimize patient discomfort. Treatments include anxiolysis with oral benzodiazepines, conscious sedation with nitrous oxide, intravenous conscious sedation with benzodiazepines (with lidocaine and prilocaine cream as an aid for venepuncture), premedication for anaesthesia induction, and general anaesthesia4–8.\n\nThese techniques should be tailored based on specific individual needs and safety, to minimize anxiety, pain and memory of the dental procedure. To date in the scientific literature, there is a lack of evidence based procedures to ensure patient’s compliance, considering both safety and cost-to-benefit ratio.\n\nIn this case series, five clinical cases (2 men and 3 women) and the procedures adopted to manage pain, behaviour and discomfort are analysed. We applied a patient-centred model to maintain patient’s cooperation and manage patient’s response to distress according to his/her psychological profile and the planned dental therapies.\n\n\nCase 1: a patient with low stress, good compliance\n\nA 38-year-old woman came to our private practice for long-term periodontal disease: she suffered spontaneous bleeding during the night and during dental hygiene, halitosis, and had difficulty in hygiene maintenance. At the first visit, the patient was relaxed and confident with the environment and professionals. After a comprehensive periodontal visit, the patient received all the information on her disease and house hygiene maintenance and two dental chair appointments for dental hygiene were scheduled. We discussed the available procedures for pain and distress control; however, she refused any medication as she exhibited very low stress and good compliance. She only accepted pre-anaesthetic liquid on the gums (Lidocaine 15% spray). Both cleaning procedures were performed without distress and the patient was successfully treated for an initial form of periodontitis. Afterwards, the patient was included in a six month-recall programme for dental hygiene. The patient reported in each follow-up visit satisfaction for the previous treatment.\n\nFor patients with low stress and good compliance and undergoing dental treatments or procedures, such as dental hygiene, X-ray examinations, impression taking, simple stages of prosthetic rehabilitation and restorative dentistry, dentist behaviour and the use of pre-anaesthetic paste or liquid are enough to control pain and manage the patient.\n\n\nCase 2: a patient with moderate stress and reduced compliance\n\nA 34-year-old woman was referred to our private practice by a psychologist: she had suffered panic attacks and agoraphobia and had been in psychological therapy for about five years. The patient needed professional dental hygiene and some fillings.\n\nAt the first visit, the patient revealed slightly uneasiness and was unsettled and tense: she disclosed her discomfort while being subjected to dental procedures and enduring long dental chair appointments and she was worried about potential pain. In agreement with her psychologist, we decided to supply her with local analgesia (Pre-anaesthetic liquid: Lidocaine 15% spray; injected local anaesthesia: Mepicain 2%, 1.8 ml, 1:100.000 adrenaline, or Mepicain 3%, 1.8 ml, without adrenaline) and benzodiazepine per os (10 drops of bromazepam were administered, 30 minutes before each dental procedure). Dental procedures were performed within a reasonable length of time with the patient remaining comfortable throughout the dental chair appointments. The patient reported to her psychologist satisfaction for the received treatments and, thus, the same regimen of anxiolysis was maintained during the following dental chair appointments.\n\nRestorative dentistry, endodontic therapies, scaling and root-planning therapies are generally well tolerated since they have a low intensity of physical discomfort and low grade of psychological effort. However, for certain patients, the use of per os benzodiazepine is advisable in addition to local anaesthesia.\n\n\nCase 3: an anxious patient\n\nA 54-year-old male needed the extraction of an upper wisdom tooth; the patient complained of pain, posteriorly, to the upper left side of the maxilla. After X-ray examination, the upper left wisdom tooth was diagnosed with a carious lesion with pulpal involvement, causing the pain.\n\nThe patient seemed to be cooperative, compliant and confident with the professionals and the surgery, even though the clinical history and medical record showed previous episodes of moderate anxiety related to dental procedures. Before surgical intervention, a pre-anaesthetic liquid (Lidocaine 15% spray) was used and local injected anaesthesia were administered on the vestibular and palatal aspects, for both pain control and vasoconstriction (Ecocain, 2x 20 mg/ml, 1,8 ml, 1:50:000 adrenaline). However, due to pulpal involvement and the presence of an infection, good pain control was not achievable9: immediately at the beginning of the procedure, the use of periosteals to break and cut gingival fibres and the use of forceps to move the tooth for dislocation caused intense pain. The patient showed clear signs and symptoms of distress and an inability to withstand that surgical situation. Thus, he rapidly became nervous and agitated. To not lose the confidence and trust of the patient, we administered benzodiazepine (bromazepam 15 drops per os) and nitrous oxide (Inhalation; Start: 10% nitrous oxide and 90% oxygen, progressively reaching 40% nitrous oxide and 60% oxygen). After 20 minutes of relaxation, we could perform the extraction with the perfect compliance of the patient and better control of intraoperative pain. At the end of the procedure, we administered 100% oxygen for 10 minutes for patient recovery. The patient reported satisfaction for previous treatment at the suture removal visit.\n\nWhen facing a patient with special needs (anxiety and reduced compliance), especially before procedures such as a planned minor surgery, or an extraction or a mucogingival surgery or an osseous periodontal surgery or a simple case of implantology, the dental team should preserve patient confidence, reduce anxiety and obtain compliance. In addition to local anaesthesia, the dentist should be able to manage benzodiazepine per os associated with nitrous oxide administration, even in absence of the specialist anaesthesiologist.\n\nThe association of benzodiazepine and nitrous oxide positively affects both the patient and the dental professional: in fact, a good disturbance of time and space perception and deep conscious sedation are achievable by adjusting nitrous oxide titration; the dentist is more relaxed and concentrated on the procedure. Analgesia is then well controlled by means of local anaesthesia.\n\n\nCase 4: a patient with acute anxiety and emotional distress\n\nA 58-year-old female in good general health status required some restorative dentistry and a complex implant-prosthetic rehabilitation on both arches.\n\nShe suffered from acute anxiety and emotional distress; she constantly asked for explanations before and after each planned dental treatment, with an extreme waste of time during dental chair appointments. The dental treatment presupposed sinus lift procedures on both sides of the maxilla and concomitant implant placement. Bone harvesting from the malar surface of both maxillary bones by a scraper was planned. Therefore, the planned procedure was complex and poorly accepted by patient . For this reason and accounting for her emotional status, the patient underwent intravenous sedation (Diazepam: starting dose 7mg; then 0.6 mg every 25 minutes were administered up to 20 minutes before the end of the procedure). A 2-hour observation period followed the surgery to allow full recovery of the patient. Administering sedation and post-operatively anti-inflammatory drugs intravenously was helpful. A good disturbance of time and space perception was obtained, with complete recovery and full patient’s satisfaction. The patient maintained a good compliance and report satisfaction during the next dental chair appointment. In following appointments, for prosthetic rehabilitation completion, the patient was managed by means of per os benzodiazepines (bromazepam 15 drops 30 minutes before each dental procedure) and local anaesthesia when requested by the procedure (pre-anaesthetic liquid: Lidocaine 15% spray; injected local anaesthesia: Mepicain 2%, 1.8 ml, 1:100.000 adrenaline).\n\nIntravenous sedation is an effective therapy for patients suffering acute anxiety and distress during dental procedures. It offers several advantages: the patient may become completely relaxed, depending on the deepness of sedation obtained by drug titration, compliant and with a good disturbance of time and space perception. In this condition, even major surgery can be accomplished, with a reduced duration of dental procedure and lower stress for the professional. Intravenous sedation has been successfully utilised for complex surgical clinical cases, such as bilateral sinus lifting and bone grafts and implants or bilateral vertical and lateral augmentation procedures with bone harvesting. Through the same vein utilised to administer conscious sedation drugs, it is advisable to administer also anti-inflammatory drugs in the immediate postoperative phase. The patient should be in good health and it is desirable to give the patient postoperative time for recovery and to have someone accompanying him/her back home. The dental office should have a good professional relationship with a specialist anaesthesiologist and higher costs should be expected for the patient.\n\n\nCase 5: an anguished patient\n\nA 50-year-old male in excellent general health condition needed to undergo fixed implant-supported dental rehabilitation. Despite the strong motivation to be rehabilitated and the awareness of the related surgeries, at the first visit the patient was stressed about dental therapies, exhibiting distress and anguish.\n\nFrom an oral point of view, the patient was afflicted by severe periodontal disease. He lacked all the teeth of the upper left hemi-maxilla, except the central incisor; bone reabsorption of the area was massive with the tongue interposing between the arches laterally. The periodontal disease was progressively well controlled by proper therapies and a bone grafting at the left side of the maxilla was planned. According to the literature, autogenous bone is considered as the gold standard for bone grafting procedures10; therefore, calvaria was selected as the donor site, to harvest bone blocks enough to perform vertical and horizontal bone augmentation in a large area for proper dental implants insertion. Sinus lifting was planned concomitantly with bone augmentation. In this case, the unique appropriate sedation technique was general anaesthesia (Propofol; Starting dose: 144 mg; then 640 mg per hour of continuous venous infusion up to 15 minutes before the end of the surgical procedure; then 320 mg per hour of continuous venous infusion up to 5 minutes before the end of the surgical procedure; then the infusion was stopped). Local anaesthesia was also administered in the mouth and in the parietal bone area of the patient, for both pain control and vasoconstriction (Ecocain, 6 × 20 mg/ml, 1.8 ml, 1:50:000 adrenaline). After 5 months, five endo-osseous dental implants were placed. In previous dental care procedures, the dental team gradually gained patient’s confidence, so that the surgical stage of implant placement and other prosthetic phases were performed with local anaesthesia (Pre-anaesthetic liquid: Lidocaine 15% spray; injected local anaesthesia: Mepicain 2%, 1.8 mm, 1:100.000 adrenaline) or, rarely, with benzodiazepine per os (bromazepam 15 drops, 30 minutes before each treatment). All the implants were rehabilitated four months after implant placement.\n\nGeneral anaesthesia is of paramount importance for the anguished patient while undergoing dental treatment. It may also represent the only chance to treat children under 4 years of age or to accomplish particular surgical procedures, such as bone harvesting from hip or calvaria for successive implant stabilisation or interventions in the extreme proximity to vascular or nervous anatomical structures.\n\nThe patient should be in good general health, hospitalisation is essential for the patient as well as the presence of a specialist anaesthesiologist. Generally, it is advisable to guarantee at least 24 hours for full recovery. Costs may eventually increase.\n\n\nDiscussion\n\nThis case series describes five scenarios that frequently occur in clinical practice, with the evident limit of showing only few of the main typology of distress and pain management during dental procedures. Facing a wide range of patients, from relaxed and collaborative patients to anguished ones, the dental team should optimize and tailor its approach, considering both patients’ psychologic profile and the planned procedures.\n\nThe applicability and, at the same time, the limit of the described approach is that the evaluation and treatment of anxiety and distress had been made by clinical observation and counselling and patient self-report, but was not based upon a mental health specialist evaluation and treatment (questionnaire of rating scale, psychotherapy or psychiatric consultation), since they are not available in a routine clinical dental practice.\n\nDuring treatments, patients are required to cooperate to achieve a degree of relaxation enough to maintain the fixed positioning necessary for the treatment. Therefore, in patients with a pre-existing psychiatric history, psychosocial maladjustment or psychological problems such as anxiety and previous traumatic experiences related to dental therapies, behavioural manifestations can hinder the correct execution of procedure, safety and clinical outcomes11.\n\nBefore choosing the type of sedative-analgesic approach, the preoperative diagnosis, patient psychological profile, and the planned dental procedure should be carefully evaluated. The final aim is to perform the therapies with relaxed and cooperating patients and in an uneventful and smooth induction of anaesthesia and sedation, if needed. If well-balanced, sedation can convert the behaviour of a patient uncooperative to undergo dental treatment.\n\nOur experience indicates that it is necessary to adopt the most appropriate combination of resources (Table 1), according to patient’s characteristics and needs. Therefore, we proposed a decision-making strategy for the dental clinician, as a tool to maintain an acceptable level of collaboration and, therefore, of patient comfort all the time. Clinical management strategy, analgesic agent and sedation therapy should be driven by the subjective perception of the patient. Besides generally utilised medication for the control of the pain, the dental clinician can discuss with the patient to determine the best approach to reduce distress and anxiety and properly manage the clinical scenario, considering all available techniques of sedation.\n\nTherefore, in this patient-centred model the clinical decision of the behavioural support, analgesia and/or sedation procedures is guided by patient complexity (Table 2). For example, a patient with strong anxiety, but with a very simple procedure may still require highly complex anxiolytic and analgesic procedures (as in cases 3 and 4). If a level is not feasible (e.g. local anaesthesia does not work for an infection), the next level of anxiolysis should be considered.\n\nAs an example, when it is not possible to use more analgesia or sedation, it is convenient to use all behavioural techniques to perform a complex procedure with a difficult patient.\n\n0: no increment of behaviour, analgesia or sedation; +: small increment of behaviour, analgesia or sedation; ++: medium increment of behaviour, analgesia or sedation; +++: high increment of behaviour, analgesia or sedation.\n\nAlong specific goals of sedation, the safety and prompt recovery to a state of consciousness must be considered. Furthermore, dentist’s preparation, expertise and experience can decrease the duration of the procedure, thereby limiting the need for sedation and analgesia12,13.\n\nNevertheless, from our experience we learnt that it is still a subjective process and each clinician must always be aware of how the patient is responding to sedatives. The more complex and/or as the length of the procedure increases, the more the dentist’s behaviour and/or analgesia and/or sedation need to match the new situation. A patient-centred approach still needs more studies to validate operative models to choose sedation and analgesic strategies to achieve the best patient satisfaction.\n\nIn conclusion, a patient-centred approach considering both clinical characteristics and psychological profile can help to achieve a tailored management of pain control and sedation in patients with emotional and behavioural problems during dental procedures.\n\n\nConsent\n\nAll patients gave their written informed consent to publish the data presented in this case series.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nFacco E, Zanette G: The Odyssey of Dental Anxiety: From Prehistory to the Present. A Narrative Review. Front Psychol. 2017; 8: 1155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarter AE, Carter G, Boschen M, et al.: Pathways of fear and anxiety in dentistry: A review. World J Clin Cases. 2014; 2(11): 642–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAraújo JO, Motta RHL, Bergamaschi CC, et al.: Effectiveness and safety of oral sedation in adult patients undergoing dental procedures: protocol for a systematic review. BMJ Open. 2018; 8(1): e017681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSebastiani FR, Dym H, Wolf J: Oral Sedation in the Dental Office. Dent Clin North Am. 2016; 60(2): 295–307. PubMed Abstract \| Publisher Full Text\n\nArmfield JM, Heaton LJ: Management of fear and anxiety in the dental clinic: a review. Aust Dent J. 2013; 58(4): 390–407. PubMed Abstract \| Publisher Full Text\n\nO'Halloran M: The use of anaesthetic agents to provide anxiolysis and sedation in dentistry and oral surgery. Australas Med J. 2013; 6(12): 713–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown TB, Lovato LM, Parker D: Procedural sedation in the acute care setting. Am Fam Physician. 2005; 71(1): 85–90. PubMed Abstract\n\nAppukuttan DP: Strategies to manage patients with dental anxiety and dental phobia: literature review. Clin Cosmet Investig Dent. 2016; 8: 35–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFleury AA: Local anesthesia failure in endodontic therapy: the acute inflammation factor. Compendium. 1990; 11(4): 210, 212, 214 passim. PubMed Abstract\n\nShamsoddin E, Houshmand B, Golabgiran M: Biomaterial selection for bone augmentation in implant dentistry: A systematic review. J Adv Pharm Technol Res. 2019; 10(2): 46–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Jongh A, Adair P, Meijerink-Anderson M: Clinical management of dental anxiety: what works for whom? Int Dent J. 2005; 55(2): 73–80. PubMed Abstract \| Publisher Full Text\n\nYamalik N: Dentist-patient relationship and quality care 3. Int Dent J. 2005; 55(4): 254–6. PubMed Abstract \| Publisher Full Text\n\nAmerican Society of Anesthesiologists Committee on Standards and Practice Paramet: Practice Guidelines for Moderate Procedural Sedation and Analgesia 2018: A Report by the American Society of Anesthesiologists Task Force on Moderate Procedural Sedation and Analgesia, the American Association of Oral and Maxillofacial Surgeons, American College of Radiology, American Dental Association, American Society of Dentist Anesthesiologists, and Society of Interventional Radiology. Anesthesiology. 2018; 128(3): 437–79. PubMed Abstract \| Publisher Full Text" }	[ { "id": "65974", "date": "07 Jul 2020", "name": "Mawlood Kowash", "expertise": [ "Reviewer Expertise Dental management of patients with special needs and behavioural problems." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe five-case series of adult patients with varying degrees of anxiety and dental treatment proposes a patient-centred model to optimize patients’ cooperation during dental care delivery. Dental treatment of the five patients was achieved with behaviour management and local analgesia alone or with oral and inhalation conscious sedation, intravenous sedation, or general anaesthesia. The reviewers suggest the following corrections to make the paper scientifically sound: English language revision, revising the introduction by adding: 1) a paragraph on definition and assessment of dental anxiety; 2) a paragraph on conscious sedation, patient’s assessment, and monitoring. The discussion may be improved by revising Table 2 and adding a paragraph to emphasize the importance of communication and utilizing the different behaviour management techniques to manage patient dental anxiety. Finally, revision of the conclusion is suggested.\n\nEnglish Language Revision: the whole paper needs English language editing. The authors should use the commonly used words/terms in academic writing.\n\nIn the introduction: 1st paragraph: change the word “for” to “from” either consultation or dental therapy. 3rd paragraph: use of topical analgesia prior to Local analgesia (LA) injection- and not “venipuncture” which means intra-venous access. In Case 1, line 7: “House hygiene maintenance” to be changed in to “oral hygiene maintenance”. The authors used the term (analgesia) in the abstract which is the correct term for dental local analgesia (LA). However, they also used (anaesthesia) throughout the paper to describe dental LA. Analgesia means the absence of pain sensation, while anesthesia means loss of all sensation including pressure and proprioception. Therefore, we suggest changing the term “anaesthesia” with ‘analgesia” for dental LA. The abbreviations should be defined when first used in the paper: e.g. Benzodiazepine per os: is OS mean by oral route? Use the word “restorations” instead of “fillings”\n\nIn case 2: First paragraph: define “agoraphobia”: extreme or irrational fear of entering open or crowded places, of leaving one's own home, or of being in places from which escape is difficult. In 2nd paragraph, line 5 ”we decided to supply her…should be changed to ”The local analgesia is applied Case 3: In the last paragraph: change the word “association” to “combination” of benzodiazepine and nitrous oxide positively affects ….\n\nCase 5: line 8: change “reabsorption” to “resorption”.\n\nRevision of Cases Case 1\nStart with the patient complaint or the reason for attending (e.g. refereed by..) and not by diagnosis. It is not clear how the stress level was evaluated or based on which criteria?\n\nCase 2\nConsultation with the patient’s psychologist should have been done prior to subjecting the patient to complex dental treatment. The following statement needs revision because the mentioned dental procedures are anxiety and pain-provoking procedures and generally are not tolerated by anxious patients:\n“Restorative dentistry, endodontic therapies, scaling and root planning therapies are generally well tolerated since they have a low intensity of physical discomfort and low grade of psychological effort. However, for certain patients, the use of per os benzodiazepine is advisable in addition to local anaesthesia.”\n\nCase 3\nNeeds language revision as shown:\nA 54-year-old male needed the extraction of an upper wisdom tooth; the patient complained of pain posteriorly, to (on) the upper left side of the maxilla. After X-ray clinical and radiographic examinations, the upper left wisdom tooth was found diagnosed with deeply a carious lesion with pulpal involvement, causing the pain.\nAs mentioned in case 2, for the patient presented in Case 3 proper treatment planning should have been carried out because “clinical history and medical record showed previous episodes of moderate anxiety related to dental procedures”. In addition, he required extraction of pulpally involved upper third molar. The infiltration LA should have been supplemented with inhalation sedation and/or benzocaine oral sedation.\n\nIn the second paragraph, line 11: following sentence needs revision:\n“immediately at the beginning of the procedure, the use of periosteals to break and cut gingival fibres and the use of forceps to move luxate the tooth for dislocation caused intense”\n\nCase 4\nThe following statement is inappropriate:\n“She suffered from acute anxiety and emotional distress; she constantly asked for explanations before and after each planned dental treatment, with an extreme waste of time during dental chair appointments”. The procedures should have been explained to the patient in simple language with empathy, taking into consideration her acute anxiety and emotional stress. Treatment under IV sedation should have been planned because she was diagnosed with acute anxiety and emotional stress.\n\nA good “disturbance” to be changed to “consciousness” of time and space perception…”\n\nIntroduction- Add the following: Definition and assessment of dental anxiety The term Dental Anxiety (DA) includes anxiety, fear and phobia which are used interchangeably (McDonnell-Boudra et al. 2014). DA is a reaction to unknown perceived dental danger especially when the treatment proposed was never experienced before. Dental fear is a reaction to a known perceived danger which involves a flight-or-fight response when provoked with the frightening stimulus, while dental phobia is an extreme, marked, and persistent fear of clearly visible defined objects or situations.\n\nUnderstanding the level of patient anxiety allows its appropriate management. However, anxiety is difficult to measure. There are several methods available for dentist to score patients dental anxiety for example the Modified Child Dental Anxiety Scale (MCDAS) (Wong et al. 1998).\n\nDefinition of conscious sedation, patient’s assessment, and monitoring Conscious sedation is a drug-induced depression of consciousness where the patient purposefully responds to verbal commands, either alone or by light tactile stimulation. No interventions are required to maintain a patent airway, and spontaneous ventilation is adequate. Patient’s cardiovascular function is usually maintained (Galeotti et al. 2016).\n\nMeticulous pre-sedation evaluation with respect to patient’s general health status, airway, fasting, and understanding about the pharmacodynamics and pharmacokinetics of the drugs must be recognized. Availability of airway management equipment, sedative drugs’ antidote,\n\nvenous access, suitable intraoperative monitoring such as pulse oximetry and well-trained staff must be ensured (Attri et al. 2017). Conscious sedation can be administered through various routes such as oral, intramuscular, intravenous, and inhalational.\n\nMcDonnell-Boudra D, Martin A, Hussein I. In vivo exposure therapy for the treatment of an adult needle phobic. Dent Update. 2014;41(6):533–40. Wong HM, Humphris GM, Lee GTR. Preliminary validation and reliability of the modifed child dental anxiety scale. Psychol Rep. 1998; 83:1179–86. Galeotti A, Garret Bernardin A, D’Antò V, Ferrazzano GF, Gentile T, Viarani V, et al. Inhalation conscious sedation with nitrous oxide and oxygen as alternative to general anesthesia in precooperative, fearful, and disabled pediatric dental patients: A large survey on 688 working sessions. Biomed Res Int 2016. 2016:7289310. Attri JP, Sharan R, Makkar V, Gupta KK, Khetarpal R, Kataria AP, et al. Conscious sedation: Emerging trends in pediatric dentistry. Anesth Essays Res. 2017; 11:277–81.\n\nDiscussion\nIn table 2 the highlighted sentence needs deletion from the title.\nTable 2. Modulating and balancing behaviour and/or analgesia and/or sedation in response to an increasing complexity and/or length of the procedure and increasing level of anxiety and distress. As an example, when it is not possible to use more analgesia or sedation, it is convenient to use all behavioural techniques to perform a complex procedure with a difficult patient.\nIn Table 2: it is not possible to have a profound analgesia (+++) in a patient with (0 increment) behaviour and (0 increment) sedation. Add the following at the end of the discussion:\nMost anxious patients can be managed by proper communication and utilizing different behaviour management techniques (e.g. Tell-Show-Do, Distraction, Modelling, etc.) tailored to the patient psychological status.The clinician should strive to perform dental treatment using proper communication, behaviour management and local analgesia with or without oral and inhalational conscious sedation.Intravenous sedation and general anaestheisa should be reserved to complex cases to avoid potential morbidities and mortalities and additional treatment costs.\n\nConclusion: May be improved as follows: In conclusion, a patient-centred approach considering both clinical characteristics and psychological profile can help to achieve high quality dental care through a tailored management of pain and anxiety in patients with emotional and behavioural problems. This can be achieved by proper behavior management and local analgesia with or without conscious sedation or in complex cases using intravenous sedation or general anaesthesia.\n\nIs the background of the cases’ history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [ { "c_id": "5761", "date": "04 Sep 2020", "name": "Riccardo Tizzoni", "role": "Author Response", "response": "Object: revisions to article A case series analysing patients with dental anxiety: a patient-centred model based on psychological profiling Dear Reviewer,Thank you for reviewing our manuscript. The comments and suggestions indicated by you have been precisely considered and we hope satisfactorily addressed.Enclosed please find the revised version that incorporates the reviewers’ comments as follows: REVIEWER: The language of the whole article was revised. We changed the word “for” with “from”. We have explained the use of topical analgesia for the skin prior to venipuncture. We specified the term “topical analgesia” prior to “local analgesia”. We changed “House hygiene maintenance” to “oral hygiene maintenance”. We changed the word, when appropriated, “anaesthesia” with “analgesia”. We specified the term “per os” (that means “by oral route”) when first used in the paper. We changed the world “fillings” with term “restorations” as suggested. We explainded the meaning of agoraphobia (extreme or irrational fear of entering open or crowded places, of leaving one’s own home, or of being in places from which escape is difficult) We modified the sentence “we decided to supply her” with “the local analgesia is applied” We changed the word “association” with “combination” in case 3 We changed the word “reabsorption” with “resorption” REVISION OF CASESCase 1 We started with the patient complaint or the reason for attending and not by diagnosis The stress level was evaluated by clinical observation and self report of the patient (and we added in Discussion this period: “The applicability and, at the same time, the limit of the described approach is that the evaluation and treatment of anxiety and distress had been made by clinical observation and counselling and patient self-report, but was not based upon a mental health specialist evaluation and treatment (questionnaire of rating scale, psychotherapy or psychiatric consultation), since they are not available in a routine clinical dental practice)”. Case 2 The Consultation with the patient’s psychologist has been done prior to subjecting the patient to complex dental treatment. We slightly modified the meaning of the sentence as requested. Case 3 We revised the first paragraph as indicated. For point 2 of case 3, please see point 2 of Case 1. For the patient presented in Case 3 also we clarified proper treatment planning with phrase “supplemental infiltration with inhalation sedation with nitrous oxide administration and / or oral sedation”. In the second paragraph, line 11 we deleted the indicated phrase, and we emphasized patient inability to withstand the surgical situation and surgical steps. Case 4 We modified the phrase correctly indicated as inappropriate: We changed to “consciousness” of time and space perception…” We added: a paragraph on definition and assessment of dental anxiety; 2) a paragraph on conscious sedation, patient’s assessment, and monitoring. We revised Table 2 and added a paragraph to emphasize the importance of communication and utilizing the different behaviour management techniques to manage patient dental anxiety. We added the suggested paragraph in discussion. We revised the conclusions. In table 2 we deleted, as requested, the highlighted sentence from the title and explained the same concepts in the text. We considered possible to add doses of local analgesia (+++) in patients in whom is not need an increment of behavioural effort and sedation. We hope that our responses and revisions satisfy your request for revisions.Best regards" } ] }, { "id": "65973", "date": "13 Jul 2020", "name": "Senem Yildirimturk", "expertise": [ "Reviewer Expertise Oral surgery", "sedation", "dental fear", "dental anxiety", "oral radiology", "cbct", "dental implantology", "facial fractures", "oral and maxillofacial surgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere are my comments to the authors of 'A case series analysing patients with dental anxiety: a patient-centred model based on psychological profiling [version 1; peer review: 1 approved with reservations]':\nIf this study is an original research/research article, then what is the null hypothesis of the study? If it is a review, the authors should better enhance the discussion and strongly support it with literature conducting dental anxiety, MDAS, sedation, etc. The examples given in the study (one in each type) are not enough to make a review!\n\nThe introduction and discussion parts are too short.\n\nThe authors should better use valid scales or questionnaires to avoid subjectivity. The dental history of the patient mostly affects the current state/mood on the day or even before the day of dental appointment regardless of operation type.\n\nThe grammar is poor.\n\nThe aim and methodology of the study should be revised and planned in detail.\n\nIs the background of the cases’ history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [] } ]	1	https://f1000research.com/articles/8-1843
https://f1000research.com/articles/9-1092/v1	04 Sep 20	{ "type": "Opinion Article", "title": "Reevaluation and correction of Maxwell’s Equations: a magnetic field has a source, a moving electric charge", "authors": [ "M.J. Koziol" ], "abstract": "Maxwell’s Equations are considered to summarize the world of electromagnetism in four elegant equations. They summarize how electric and magnetic fields propagate, interact, how they are influenced by other objects and what their sources are. While it is widely accepted that the source of a magnetic field is a moving charge, one of the equations instead states that the magnetic field has no source. However, it is widely accepted that a magnetic field cannot be created without a moving electric charge. As such, here, after carefully reevaluating how Maxwell derived his equation, a limitation was identified. After adjustments, a new equation was derived that instead demonstrates that the source of a magnetic field is a moving charge, confirming experimentally verified and widely accepted observations.", "keywords": [ "Maxwell Equation", "magnetism", "electromagnetism", "field" ], "content": "Introduction\n\nJames Clerk Maxwell [1831–1879] was a pioneer in studying the propagation of electromagnetic waves1–5. He unified his observations with Faraday’s, Gauss’s and Ampere’s Laws into a set of four equations, known as Maxwell’s Equations (Figure 1)1–9. Maxwell’s Equations summarize the world of electromagnetics in four elegant equations that state: (a) an electric charge is the source of an electric field; (b) a moving electric charge results in a magnetic field; (c) a changing magnetic field results in an electric field; (d) a magnetic field has no source (Figure 1)1. As such, Maxwell’s Equations summarize the source of electric and magnetic fields, and also describe how they are interlinked. Simultaneously, Maxwell’s Equations also demonstrated for the first time that electromagnetic waves and visible light have the same speed and are essentially the same1,2,6. As such, the equations provide also an equation for light and its origin, making them of fundamental importance to physics and life. To illustrate their importance, the biblical quote “And God said let there be light, and there was light”10 has been jokingly altered by physicists and is also found displayed on various merchandise: “And God said [Maxwell’s equations], and there was light”11.\n\na, an electric charge is the source of an electric field1–9 (E: external electric field, A: area; Q: electric charge; ε0: dielectricty constant of the vacuum, εr: dielectricty constant of the material). b, a moving electric charge results in a magnetic field1–9 (B: magnetic field intensity, x: distance from current, μ0: permeability constant of the vacuum, μr: permeability constant of material, ε0: dielectricty constant of the vacuum, εr: dielectricty constant of the material, I: electric current, t: time, E: external electric field, A: area). c, a changing magnetic field results in an electric field1–9 (E: external electric field, x: distance from current, t: time, B: magnetic field intensity, A: area). d, a magnetic field has no source1–9 (B: magnetic field intensity, A: area).\n\nHere, I revisit one of Maxwell’s Equations, which shows that a magnetic field has no source. By carefully reanalyzing how this equation was derived, I show that this equation ought to be adjusted as a substitution has not been made. After the revisions presented here, the equation now demonstrates that a magnetic field has a source, namely a moving electric charge. In fact, this adjusted equation confirms experimental observations. It is widely accepted and an undisputed experimental observation that a moving electric charge gives rise to a magnetic field5,6,12–14. Without a moving electric charge, or in other words without a source, no magnetic field exists. The calculations presented here identify that an essential substitution was not made in one of Maxwell’s Equations, which ought to be revised. When adjusted for the substitution, the equation shows a different conclusion: instead of showing that a magnetic field has no source, the equation now demonstrates that the magnetic field has an actual source, and that the source is a moving electric charge.\n\n\nElectric and magnetic fields can be described by different vectors\n\nIn physics, a field is a region in which each point is affected by a force5–7,9. For example, objects fall to the ground as they are affected by the force of a gravitational field15. An electrical field surrounds an electrical charge and it repels or attracts other charges5–7,9. An electromagnetic field is caused by the motion of an electric charge5–7,9. A stationary charge will only give rise to an electric field. Only if such charge is moving, a magnetic field is produced5–7,9.\n\nAn electric field can be described by two vectors: the external electric field E and the electric displacement field D, which is also known as the dielectric displacement field. D describes the vector field in a non-conducting medium, a dielectric, which is an electrically insulating material6. D is proportional to E, but they differ: External materials such as dielectrics change the effective electric field E, while D remains constant5,6. In other words, D can be regarded as a constant of the system being studied, while E is the variable vector of the electric field in that system. The relationship between E and D is summarized with the equation D = E ⋅ ε0 ⋅ εr, with ε0 being the dielectricity constant of the vacuum and εr the dielectricity of the material5,6.\n\nAnalogous to the electric fields E and D, two different vectors are used to describe magnetic fields: one is called magnetic flux density or magnetic induction, and is symbolized by B; the other is called magnetic field strength or magnetic field intensity, abbreviated as H. B is proportional to H, but they differ; external materials such as diamagnetics or ferromagnetics change the magnetic field intensity H, while B remains constant (Figure 2b)5,6. As such, B can be regarded as a constant of the system being studied, while H is the variable vector of the electric field in that system. The connection between B and H is represented by B = H ⋅ μ0 ⋅ μr, with μ0 being the permeability constant of the vacuum and μr the permeability constant of the material5,6.\n\na, Example of an electric field along a capacitor: external factors such as capacitors and dielectrics change the external electric field (E) vector, while the electric displacement field (D) vector remains constant5,6. b, Example of a magnetic field along a ring coil: external factors such as gaps in a coil, diamagnetics or ferromagnetics change the magnetic field intensity (H) vector, while the magnetic induction field (B) vector remains constant5,6.\n\nEven though E and B are the most commonly used field vectors to describe electric and magnetic fields, respectively, they are not equivalents. This is important to highlight. Instead, D and B are equivalents, as D and B both do not change upon the addition of external materials such as dielectrics or diamagnetics. Similarly, E and H are equivalents, as they are not influenced by such external materials (Figure 3)5,6.\n\nThe vectors D and E both describe electric fields, while the vectors B and H describe magnetic fields. D and B are equivalent vectors, as both do not change upon the addition of external materials such as dielectrics or diamagnetics, respectively5,6. In contrast, E and H are equivalent vectors, as they are not influenced by such external materials. The relationships between D and E, and between B and H are interlinked with permeability (μ0 and μr) dielectricty (ε0 and εr) constants, respectively. Both E and H depend on the electric charge Q, and its distance r away from that charge, and in the case of H also on how the electric charge Q changes over time t5,6.\n\n\nFlow definition of electric and magnetic fields through a surface are not equivalent\n\nTo describe the flow of an electric field through a given surface A, the electric flux 𝜓 is used. Similarly, the magnetic flux ϕ is a measure of a magnetic field through a given surface A5,6. Both depend on the field’s vectors: 𝜓 has been defined by E through 𝜓 = ∮E ⋅ dA, while ϕ has been defined through B, with ϕ = ∮B ⋅ dA5,6. Although these flux definitions might seem equivalent, they are not: 𝜓 has been defined using the electric field vector E that changes with external materials, while ϕ has been defined based on the magnetic field vector B, which remains constant if for example diamagenetics are introduced into the system. As such, within a given system, the flux of the electric field is defined by a variable vector of the electric field, namely E, which can change. However, the flux of the magnetic field has been defined by a constant vector of the magnetic field, B, which remains constant even if diamagenetics are introduced into the system. Hence, the flux of electricity and magnetisms are not equivalent, as 𝜓 is based on the variable vector E, while ϕ is based on a constant vector B5,6. However, B depends on the variable vector H, with B = H ⋅ μ0 ⋅ μr, and E on the constant vector D, with D = E ⋅ ε0 ⋅ εr5,6. As such, both 𝜓 and ϕ can also be described with constant or variable field vectors:\n\n\n\n\n\n\nMaxwell’s Equations: electric field source\n\nOne of Maxwell’s Equation focuses on 𝜓, and shows that ψ=∮E⋅dA=Qε0⋅εr,which demonstrates that an electric charge is the source of an electric field (Figure 1a)5,6. This equation is derived from 𝜓 = ∮E ⋅ dA, and is applied onto one electric point charge Q5,6: the electric field of an electric point charge is defined by radial coordinates. As such, E is proportionate to the point charge Q, but decreases with increased radial distance away from that charge. The electric field dependency on an electric charge is summarized by the following equation E=14πε0εr⋅Qr2 (Figure 3)5,6. Substituting E within the 𝜓 equation, gives rise to Maxwell’s equation that shows that an electric charge it the source of an electric field1,2,5–9.\n\n\n\n\nMaxwell’s Equations: magnetic field source\n\nAnalogous to the above, another of Maxwell’s equations focuses on ϕ, and states that ϕ = ∮B ⋅ dA = 05,6. This equation illustrates that a magnetic field has no source5,6. This equation is derived based on the argument that B is constant, and does not change. As such, B entering an area (B1) and B exiting (B2) the same area is the same. With B1 = B2, the following Maxwell equation was derived1,2,4–9:\n\n\n\n\n\n\nEffect on Maxwell’s equation when substituting variable vector with constant vector in electric flux calculation.\n\nBoth of the above equations by Maxwell are derived from the equation describing the flux of the electric or magnetic field through a surface A, as shown in Equation (3) and Equation (5)1,2,4–9. However, unlike 𝜓, which is usually represented by the variable vector E, ϕ is most commonly represented by the constant vector B1,2,4–9. However, both electric and magnetic flux can ultimately be described by variables of the electric or magnetic fields, as shown in Equation (1) and Equation (2). As such, they also ultimately depend on such variables and have to be taken into account. If not, relationships and dependence of variables are eliminated incorrectly.\n\nFor example, as outlined above in Equation (3), Maxwell has shown that 𝜓 can be expressed through ψ=∮E⋅dA=Qε0⋅εr1–9. To derive this equation, the variable E was replaced by the electric charge Q (Figure 3)1–9. He applied Coulomb’s law1–9. However, what if he instead of E=14πε0εr⋅Qr2 used Ε=Dε0⋅εr for the substitution, which is derived from the relationship D = E ⋅ ε0 ⋅ εr1–9. If E is substituted by Ε=Dε0⋅εr in the 𝜓 equation, the following relationship is obtained, in which ψ is only represented by constants and a constant vector.\n\n\n\nNext, following Maxwell’s argument that he used in deriving his Equation (4): D, as well as ε0 and εr are constants that do not change. As such, D entering an area (D1) and D exiting (D2) the same area is the same. Also, the material constants ε0 and εr remain the same. As such, D1ε0⋅εr=D2ε0⋅εr. Following Maxwell’s reasoning and calculation (4), the following conclusions could be made:\n\n\n\nThe above reasoning leading to calculation (7), which Maxwell applied to his calculations with ϕ1–9, one would obtain an equation shown below (8), which illustrates that an electric field has no source.\n\n\n\nAlthough Equation (8) might appear mathematically correct, it is not: 𝜓 is experimentally dependent on the variable E, which in turn is dependent on the electric charge Q, summarized in Coulomb’s law E=14πε0εr⋅Qr2 (Figure 3)1–9. Hence, because of E’s dependence on Q, 𝜓(Q), ∮E ⋅ dA ≠ 0, if Q is not zero.\n\n\nMaxwell’s equation changes when substituting constant vector with variable vector in magnetic flux calculation\n\nOverall, functions are defined by variables that cannot be ignored as otherwise wrong conclusions are drawn. However, this is what occurred when the equation ∮B ⋅ dA = 0 was derived1–9: Even though B is a constant, it is dependent on H, a variable of the magnetic field. This dependence was not considered in the calculation of one of Maxwell’s equations that analyzed the source of the magnetic flux ϕ1–9.\n\nAs such, here, the ϕ calculation is reanalyzed: ϕ is defined by B in a given area A, through ϕ = ∮B ⋅ dA1–9. The magnetic field and the vector B are known to depend on an electric current I, which is a variable of B and cannot be ignored5,6. Analogous to Coulomb’s law that describes E5,6, the Biot-Savart law, B=μ0⋅μr2πr⋅I, describes the dependence of B on I5,6: B is proportionate to the electric current I along its path x, but decreases with increased radial distance r away from I (Figure 4a)5,6. This is true for B along a straight current-carrying wire. For an arbitrary shape current, for example surrounding a moving charge, an integral calculation can be carried out (Figure 4b)5,6. Since the electric current I is defined by the rate of flow of an electric charge through I=Qdt5,6, B can also be defined as:\n\n\n\na, The magnetic induction field (B) is dependent on several variables: the electric current (I) and the radial distance away from I, as well as μ0, and μr, with μ0 being the permeability constant of the vacuum and μr the permeability constant of the material5–8,13. b, Illustration of the magnetic induction field going through a surface surrounding a point charge5–8,13: the magnetic induction field (B) going through a circular surface A perpendicular to B can be described by the electric current (I) and the vectors r and dx defining the circular surface A, with dA = 2π ⋅ r ⋅ dx. All vectors r, x and B are perpendicular to each other.\n\nIn the integral ϕ equation over an area, the area A can be also be replaced by two vectors representing the area, r and x, with x being the vector along the path of the electric charge Q, while r is the distance between Q and the position of B (Figure 4b). All vectors are perpendicular to each other. In case of one electric point charge, the magnetic field at that point can be best described by a circular surface area dA = 2π ⋅ r ⋅ dx (Figure 4b). Furthermore, since dxdt describe the velocity ν of the electric charge Q, the following equation can be derived:\n\n\n\nTherefore, the following relationship for the magnetic field can be derived:\n\n\n\nThe equation ∮B ⋅ dA = μ0 ⋅ μr ∫Q ⋅ dν illustrates that a charge in motion is the source of the magnetic field. As such, one could also write the magnetic field as ϕ(Q, ν), analogous to 𝜓(Q), which summarizes the electric field’s dependence on the electric charge Q. The above equation summarizes what is already well known in physics: a moving electric charge is the source of a magnetic field5–7,12–14: only when no electric charge is in motion, no magnetic field exists5–7,12–14. This is what has been also experimentally verified and is widely accepted5–7,12–14.\n\nAs a control for the calculations above that lead to ∮B ⋅ dA = μ0 ⋅ μr ∫Q ⋅ dν one could also consider Ampere’s law: Ampere’s law states ∫B⋅ds=μ0⋅μr⋅I=μ0⋅μr ⋅Qdt5,6. As a simple control, when adding an additional dimension to Ampere’s law through the addition of another vector dx, newly derived equation from above is derived, confirming Equation (11) in a different way ϕ = ∮B ⋅ dA = μ0 ⋅ μr ∫Q ⋅ dν.\n\n\nDiscussion\n\nMaxwell’s Equations summarize the electromagnetic field1–9. One of the equation’s, ∮B ⋅ dA = 0, deduces that a magnetic field has no source1. This is in contrast to what is experimentally known, namely that a moving electric charge gives rise to the magnetic field5–7,12–14. Also, a magnetic field cannot be created without this source, hence, it has a source, namely a moving electric charge. This is essential, as without this source, no magnetic field exists5–7,12–14.\n\nHere, after carefully reevaluating how Maxwell derived the equation ∮B ⋅ dA = 01, a shortcoming was identified: A substitution has not been made, eliminating an existing experimental finding1. Although ∮B ⋅ dA = 0 still applies when a charge is not in motion, Maxwell’s equation ignored the dependence of the magnetic field on variables1. Here, when taking into account such variables and making appropriate substitutions, the adjusted Maxwell’s equation instead demonstrates that the source of a magnetic field is a moving charge: ∮B ⋅ dA = μ0 ⋅ μr ∫Q ⋅ dν\n\nThis adjustment has consequences on science. For example, Schrödinger’s Equation that describes the probability waves of small particles, has been for example applied to describe the hydrogen atoms characteristics16–19. It used Maxwell’s Equation ∮B ⋅ dA = 0 to derive such characteristics16–19. However, with ∮B ⋅ dA = μ0 ⋅ μr ∫Q ⋅ dν, Schroedinger’s Equation ought to be revisited, as it is only true when a moving charge is not in motion.\n\nOverall, it is important to adjust one of Maxwell’s Equations1, to have a more complete understanding and representation of the world through formulas. After all, if “God said ∮B ⋅ dA = 01, there would be no light”.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nI am grateful to my colleagues in CIBR for their continuous support.\n\n\nReferences\n\nMaxwell JC: A Dynamical Theory of the Electromagnetic Field. Phil Trans R Soc Lond. 1865; 155: 459-512. Reference Source\n\nThomas EG, Meadows AJ: Maxwell's Equations and their Applications. Adam Hilger Ltd., Bristol and Boston, 1985. Reference Source\n\nMaxwell JC: A treatise on electricity and magnetism. Oxford, UK:Clarendon Press, 1873; 2. Reference Source\n\nFleisch DA: A Student’s guide to Maxwell’s equations. Cambridge University Press, 2008. Reference Source\n\nGriffiths DJ: Introduction to electrodynamics. 4th edition, Pearson, 2013. Reference Source\n\nFeynman R, Leighton R, Sands, M: The Feynman Lectures on Physics, Electromagnetism and Matter. Addison-Wesley Reading, MA ,1964; 2. Reference Source\n\nMaxwell JC: On Faraday’s lines of force. Trans Camb Philos Soc. 1864; 10: 27-83.\n\nMaxwell JC: On physical lines of force. Philos Mag. 1861; 90: 11-23. Publisher Full Text\n\nMunley F: Challenges to Faraday’s flux rule. Am J Phys. 2004; 72: 1478. Publisher Full Text\n\nGenesis 1: 3-5. Holy Bible: King James Version, Christian Art Publishers, 2016. Reference Source\n\nAmazon: God Said Maxwell Equations and Then There Was Light.2020. Reference Source\n\nLorrain P, Corson DL, Lorrain F: Fundamentals of Electromagnetic Phenomena. W. H. Freeman, New York, 2000. Reference Source\n\nFaraday M: Experimental researches in electricity. eds R. Taylor & W. Francis, 1839-1855; 1. Reference Source\n\nGilbert W: De Magnete. English transl. by Gilbert Club, Basic Books, New York, 1958. Reference Source\n\nFeynman R, Leighton R, Sands M: The Feynman Lectures on Physics, Mechanics, Radiation and Heat. Addison-Wesley Reading, MA, 1970. Reference Source\n\nSchrödinger E: An Undulatory Theory of the Mechanics of Atoms and Molecules. Physical Review. 1926; 28: 1049-1070. Publisher Full Text\n\nGriffiths DJ: Introduction to Quantum Mechanics. 2nd edition, Prentice Hall, 2004. Reference Source\n\nLaloe F: Do We Really Understand Quantum Mechanics. Cambridge University Press, 2012. Reference Source\n\nFeynman R, Leighton R, Sands M: The Feynman Lectures on Physics, Quantum Mechanics. Addison-Wesley Reading, MA, 1970. Reference Source" }	[ { "id": "100380", "date": "06 Dec 2021", "name": "Jonathan Gratus", "expertise": [ "Reviewer Expertise Mathematical Physics", "Electrodynamics", "Differential geometry", "General Relativity." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article states that, in their current form, Maxwell's equations state that the magnetic field does not have a source. This comes from the Div(B)=0 equation. Since moving charges can give rise to magnetic fields, the author claims that Maxwell's equations need to be modified. In particular the Div(B)=0 should be replaced by a source on the right hand side.\nThis article does not justify such a radical step.\n\nThe premise, that the magnetic field does not have a source is incorrect. The sources are the current, I, and the rate of change of D. They simply couple through the curl of H, instead of the divergence. They have to be consistent with the well established observation that there are no magnetic monopoles, i.e. Div(B)=0. As noted in equation (4) this simply states that any magnetic flux line which enters a closed surface must also leave.\nThere are numerous mathematical errors which makes following the maths difficult. The last equation on figure 3 H=1/(2πr)(Q/t) cannot be correct as this would make H equal infinity when t=0. It is possible the author meant H=1/(2πr)(dQ/dt). This would be consistent with Div(B) not equal zero. Equation (9) has a term (I=Q/dt). This is not meaningful. There are integral signs missing in equation (7).\nThe bibliography contains only one recent peer reviewed article and that is a pedagogical one. It is also not clear if F1000 is the correct journal for articles about fundamental physics.\nThis article is not acceptable for indexing.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [] }, { "id": "145680", "date": "01 Aug 2022", "name": "Qasem Al-Mdallal", "expertise": [ "Reviewer Expertise Computational Fluid Dynamics", "heat transfer", "nanofluids." ], "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 report presents some notes on Maxwell’s equations with emphasis on the source of magnetic field and moving electric charge. The contribution of the report to the body of knowledge is significant and novel. It is noteworthy that the objectives of the research study are within the scope of your Journal. But, the present form of the report needs a revision using the comments and the technical questions/observations listed below:\nConsidering the nature of the research methodology, it would not be appropriate to title the report re-evaluation and correction. Hence, revision of the title is needed. The author may or may not consider the title below:\nNotes on the moving electric charge and source of magnetic field: revised version of Maxwell equation.\n\nAbstract: It was written: \"They summarize how electric and magnetic fields propagate, interact, how they are influenced by other objects and what their sources are.\" The literature presents the influence of other objects, sources, propagation and interaction of electric and magnetic fields. The keywords were written as, “Maxwell Equation, magnetism, electromagnetism, field”. I suggest to increase this to five keywords.\nIt was written: \"After adjustments, a new equation was derived that instead demonstrates that the source of a magnetic field is a moving charge, confirming experimentally verified and widely accepted observations.\" The phrase, “after adjustments” is not appropriate in that context. Is the research methodology all about adjustments? Note that an effective Abstract is a global paragraph that prepares the reader for the rest of the document. It is expected that the abstract should present (1) the significance of the study, (2) the aim of the study, (3) the research methodology, (4) the conclusion drawn from the study.\n\nFigure 1 should be converted to written equations. Citation of figure 1 is not appropriate because of reference purposes.\n\nThere are some cases of wrong usage of pronoun in the manuscript which is a scientific report. For instance:\na) Page 2 of 9, it was written, “Here, I revisit one of Maxwell’s Equations, which shows that a magnetic field has no source.”\nb) Page 2 of 9, it was written, “By carefully reanalyzing how this equation was derived, I show that this equation ought to be adjusted as a substitution has not been made.”\nComment: I suggest revising this and avoid the use of the pronoun, “I”.\n\nAll the sections and subsections should be given a number. After Eq. 3 in page 4 of 9, it was written, “Maxwell’s Equations: magnetic field source”. Under this unnumbered section, it was written that, “Maxwell’s equations focus on ϕ,”. What is phi in that context?\nPage 4 of 9, before Eq. 6, it was written, “For example, as outlined above in Equation (3), Maxwell has shown that Ψ can be expressed through Ψ= ∮ E ⋅ dA =… equation, the following relationship is obtained, in which is only represented by constants and a constant vector”. This argument is not clear. Try to revise. More so, what do you mean by, “He applied Coulomb’s law1–9.” Do not use any form of pronoun in a scientific report. Also, why would you write, “However, what if he instead of…” Please revise this.\n\nThe argument presented in this report is not guided by the objectives of the research. This may be the reason the authors write out of point in many sections of the report. Could you please write all the objectives down? Then ensure you are guided by the actualization of these objectives.\n\nThe report has no conclusion section? Why? In a short conclusion, state the most important outcome of the work based on the interpreted discussion. Did you just come up with any conclusion as you deemed fit? What are the objectives? I suggest listing all the objectives of the study and then showing how the conclusion relate to the research objectives.\n\nAfter reading the entire report, it is worth noting that the author needs to revise the entire report to address the following points:\na) present the limitation of the Maxwell equation\nb) show that the source of a magnetic field is a moving charge.\n\nI suggest to update the introduction with the fact that the Maxwell equation is useful within the scope of fluid dynamics: The dynamics of various fluids on inclined magnetic field by Ref. A et al. (2020), subject to Lorentz force by Ref B et al. (2022), aligned magnetic field effects by Ref. C et al. (2017), and magnetic flux density by Ref. D et al. (2022) are some of the applications of Maxwell equation within the scope of fluid dynamics.(ref-1), (ref-2), (ref-3), (ref-4).\nSources:\nRef. A et al. (2020) Case Studies in Thermal Engineering 17, 100571, 2020.(ref-1)\n\nRef. B et al. (2022) Ratio of Momentum Diffusivity to Thermal Diffusivity: Introduction, Meta-analysis, and Scrutinization.(ref-2)\n\nRef. C et al. (2017) Journal Article published May 2017 in Physica E: Low-dimensional Systems and Nanostructures 89, 33 - 42.(ref-3)\n\nRef. D et al. (2022) Surfaces and Interfaces 28, 101654. https://doi.org/10.1016/j.surfin.2021.101654.(ref-4)\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1092
https://f1000research.com/articles/9-1087/v1	03 Sep 20	{ "type": "Systematic Review", "title": "The effects of dietary seaweed inclusion on growth performance of broiler chickens: a systematic review and meta-analysis", "authors": [ "Faizal Andri", "Nanung Danar Dono", "Heru Sasongko", "Zuprizal Zuprizal", "Faizal Andri", "Nanung Danar Dono", "Heru Sasongko" ], "abstract": "Background: There has been great interest in the use of seaweed as a functional feed ingredient for poultry in the last decade. This study aimed to assess the effects of dietary seaweed inclusion on growth performance of broiler chickens by using a systematic review and meta-analysis approach. Methods: A systematic search of published research articles related to seaweed, broiler chickens, and growth performance was conducted using three online databases (Scopus, PubMed, and SciELO). Mean values, standard deviation, and sample size were extracted from each eligible study. The estimated effect size was then quantified using Hedges’ g with a 95% confidence interval (CI). Data were pooled using a fixed-effect model due to the absence of heterogeneity after being pre-checked using the I2 statistic. Results: A total of six studies (nine comparisons) involving 2,257 broiler chickens were accommodated in this study. The seaweed type consisted of seaweed blend, Laminaria japonica, Undaria pinnatifida, Hizikia fusiformis, and Ulva lactuca. The inclusion dose ranged from 2 to 30 g/kg, while the intervention duration ranged from 21 to 42 days. No substantial heterogeneity among studies (I2 = 0.00%) was found for feed intake, body weight gain, and feed conversion ratio. Dietary seaweed had no significant effect on feed intake (Hedges’ g = 0.19; 95% CI = -0.22 to 0.60; P = 0.280). However, broiler chickens fed dietary seaweed had superior body weight gain (Hedges’ g = 0.64; 95% CI = 0.22 to 1.06; P = 0.000) and preferable feed conversion ratio (Hedges’ g = -0.53; 95% CI = -0.95 to -0.11; P = 0.004). Conclusions: The current investigation highlights that dietary seaweed had growth-promoting potency for broiler chickens. However, more research on this issue is still required to build more comprehensive evidence.", "keywords": [ "alginate", "body weight gain", "fucoidan", "fucoxanthin", "functional feed", "laminarin", "macroalgae", "poultry" ], "content": "Introduction\n\nThere has been great interest in the use of seaweed as a functional feed ingredient for poultry in the last decade. The primary functional compounds in seaweed are polysaccharides, peptides, fatty acids, phlorotannins, and carotenoids1–3. These compounds have antimicrobial, antioxidant, and immunomodulatory properties4–7, which are essential to support production performance.\n\nSeveral reviews have compiled studies regarding the effect of dietary seaweed inclusion on poultry performance8–13. However, those reviews were based on a narrative approach, which mostly led to an inconclusive epilogue due to the contradictory results among studies. The use of systematic review and meta-analysis has become popular in animal science14–18. This methodology can integrate and determine the overall effect of interventions from several studies to provide more accurate insight than the narrative review. Therefore, this study aimed to assess the effect of dietary seaweed inclusion on the growth performance of broiler chickens using a systematic review and meta-analysis approach.\n\n\nMethods\n\nThis study was reported based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines19. The PRISMA checklist is presented in Reporting guidelines20.\n\nResearch articles published in peer-reviewed journals between the years of 2000 to 2020 and written in English were eligible. Additionally, eligible studies also should fulfill the participants, interventions, comparisons, outcomes, and study design (PICOS) criteria given in Table 1.\n\nThe online search was conducted using three databases, namely Scopus, PubMed, and SciELO, with the queries in Table 2. The final search was on 25 June 2020. The references from the included studies were also screened to find additional eligible studies.\n\nFirstly, the duplicate reports were removed from the database in Microsoft Excel for Microsoft 365 software. After that, the title and abstract were examined. Irrelevant studies, non-English reports, and review articles were then excluded from the list. The full text was further evaluated according to the eligibility criteria.\n\nMean values, standard deviations, and sample sizes were extracted from each included study. The target variables in this study were feed intake (FI), body weight gain (BWG), and feed conversion ratio (FCR). When a study used the standard error of means as a variance measure, it was converted into standard deviation21. In the case of more than one seaweed type used in a study, each treatment was coded individually. On the other hand, the treatment was pooled when a study used more than one dose of the same seaweed type22. None of the authors were contacted for further clarification.\n\nData analysis was performed using Meta-Essential version 1.523. The estimated effect size (the difference between seaweed intervention and control) was quantified using Hedges’ g with a 95% confidence interval (CI)24. Data were pooled using a fixed-effect model due to the absence of heterogeneity after being pre-checked using the I2 statistic25. A significant effect was declared when the overall estimated effect size had P < 0.05. Publication bias was not evaluated because the number of the included studies was fewer than 1026.\n\n\nResults\n\nThe PRISMA flow diagram is shown in Figure 1. The search using three online databases identified 47 records. Of these, five studies met the eligibility criteria. Additionally, one study from reference screening also found to be eligible. Therefore, a total of six studies, with nine comparisons were included in the synthesis.\n\nThe details of the included studies are shown in Table 3. A total of 2,257 broiler chickens were involved in this study. The seaweed type used included seaweed blend27, Laminaria japonica28,30, Undaria pinnatifida29,31, Hizikia fusiformis31, and Ulva lactuca32. The inclusion dose ranged from 2 to 30 g/kg, while the intervention duration ranged from 21 to 42 days. The extracted data of target variables is presented as Extended data33.\n\nn: number of broiler chickens, SBM: soybean meal.\n\nAs shown in Figure 2, no substantial heterogeneity was found for any variables (I2 = 0.00%). Dietary seaweed had no significant effect (P > 0.05) on FI. However, this intervention significantly improves (P < 0.05) the BWG and FCR of broiler chickens. The overall estimated effect size values for BWG and FCR were 0.64 and -0.53, respectively, which were equivalent to the raw mean difference of 77.24 g and -0.07, respectively.\n\nFI: feed intake, BWG: body weight gain, FCR: feed conversion ratio, CI: confidence interval.\n\n\nDiscussion\n\nIn this study, the use of dietary seaweed had a beneficial impact on BWG and FCR of broiler chickens. According to Cohen34, the overall estimated effect size of BWG and FCR in the present study was categorized into the medium (0.5) to large (0.8) standardized effect size. In agreement with this finding, other studies also showed that the use of seaweed could improve production performance in laying hens35–37 and geese38. Seaweed contained numerous unique bioactive substances such as alginate, ulvan, laminarin, fucoidan, and fucoxanthin. Those compounds could inhibit the colonization of pathogenic bacteria (Escherichia coli and Salmonella Enteritidis), promote the growth of beneficial gut microbes (lactic acid bacteria), improve small intestinal architecture, antioxidant status, and immune response39–43. Together, those mechanisms could ultimately improve the growth performance of broiler chickens.\n\nNevertheless, this finding is accompanied by the limited number of included studies. It is possible that not all relevant studies were captured by the searching strategies. For those reasons, the current results should be elucidated with caution. Moreover, due to the enormous diversity of seaweed in nature (around twenty thousand species)44, future studies regarding seaweed intervention in broiler chickens are still open and strongly encouraged to provide a robust body of knowledge.\n\n\nConclusions\n\nThe current systematic review and meta-analysis highlight that dietary seaweed had no adverse effect on FI. Instead, they could improve BWG and FCR of broiler chickens. However, more research on this issue is still required to build more comprehensive evidence.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended data for ‘The effects of dietary seaweed inclusion on growth performance of broiler chickens: a systematic review and meta-analysis’. https://doi.org/10.6084/m9.figshare.12721454.v133.\n\nThis project contains the following extended data in DOC format:\n\n- Extended data 1 – extracted data of feed intake\n\n- Extended data 2 – extracted data of body weight gain\n\n- Extended data 3 – extracted data of feed conversion ratio\n\n- Extended data 4 – list of included studies\n\nFigshare: PRISMA checklist for 'The effect of dietary seaweed inclusion on growth performance of broiler chickens: a systematic review and meta-analysis'. https://doi.org/10.6084/m9.figshare.12721118.v120.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nHoldt SL, Kraan S: Bioactive compounds in seaweed: functional food applications and legislation. J Appl Phycol. 2011; 23(3): 543–597. Publisher Full Text\n\nCardoso SM, Pereira OR, Seca AM, et al.: Seaweeds as preventive agents for cardiovascular diseases: From nutrients to functional foods. Mar Drugs. 2015; 13(11): 6838–6865. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRengasamy KR, Mahomoodally MF, Aumeeruddy MZ, et al.: Bioactive compounds in seaweeds: An overview of their biological properties and safety. Food Chem Toxicol. 2020; 135: 111013. PubMed Abstract \| Publisher Full Text\n\nShannon E, Abu-Ghannam N: Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications. Mar Drugs. 2016; 14(4): 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoveda-Castillo GD, Rodrigo D, Martínez A, et al.: Bioactivity of fucoidan as an antimicrobial agent in a new functional beverage. Beverages. 2018; 4(3): 64. Publisher Full Text\n\nCorino C, Modina SC, Di Giancamillo A, et al.: Seaweeds in pig nutrition. Animals (Basel). 2019; 9(12): 1126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomez-Zavaglia A, Prieto Lage MA, Jimenez-Lopez C, et al.: The potential of seaweeds as a source of functional ingredients of prebiotic and antioxidant value. Antioxidants (Basel). 2019; 8(9): 406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEvans FD, Critchley AT: Seaweeds for animal production use. J Appl Phycol. 2014; 26(2): 891–899. Publisher Full Text\n\nRajauria G: Seaweeds: a sustainable feed source for livestock and aquaculture. In: Brijesh KT, Declan JT, eds. Seaweed Sustainability. San Diego, CA, USA: Academic Press. 2015; 389–420. Publisher Full Text\n\nAngell AR, Angell SF, de Nys R, et al.: Seaweed as a protein source for mono-gastric livestock. Trends Food Sci Technol. 2016; 54: 74–84. Publisher Full Text\n\nMakkar HP, Tran G, Heuzé V, et al.: Seaweeds for livestock diets: A review. Anim Feed Sci Technol. 2016; 212: 1–17. Publisher Full Text\n\nHaberecht S, Wilkinson S, Roberts J, et al.: Unlocking the potential health and growth benefits of macroscopic algae for poultry. Worlds Poult Sci J. 2018; 74(1): 5–20. Publisher Full Text\n\nØverland M, Mydland LT, Skrede A: Marine macroalgae as sources of protein and bioactive compounds in feed for monogastric animals. J Sci Food Agric. 2019; 99(1): 13–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelluco S, Barco L, Roccato A, et al.: Escherichia coli and Enterobacteriaceae counts on poultry carcasses along the slaughterline: A systematic review and meta-analysis. Food Control. 2016; 60: 269–280. Publisher Full Text\n\nVieira BS, Silva FG, Oliveira CF, et al.: Does citric acid improve performance and bone mineralization of broilers when combined with phytase? A systematic review and meta-analysis. Anim Feed Sci Technol. 2017; 232: 21–30. Publisher Full Text\n\nOzdemir M, Kopuzlu S, Topal M, et al.: Relationships between milk protein polymorphisms and production traits in cattle: a systematic review and meta-analysis. Arch Anim Breed. 2018; 61(2): 197–206. Publisher Full Text\n\nToledo TD, Pich CS, Roll AA, et al.: The effect of litter materials on broiler performance: a systematic review and meta-analysis. Br Poult Sci. 2019; 60(6): 605–616. PubMed Abstract \| Publisher Full Text\n\nMcCarthy KM, McAloon CG, Lynch MB, et al.: Herb species inclusion in grazing swards for dairy cows—A systematic review and meta-analysis. J Dairy Sci. 2020; 103(2): 1416–1430. PubMed Abstract \| Publisher Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndri F, Dono ND, Sasongko H, et al.: PRISMA checklist for 'The effect of dietary seaweed inclusion on growth performance of broiler chickens: a systematic review and meta-analysis'. 2020. http://www.doi.org/10.6084/m9.figshare.12721118.v1\n\nGreig JD, Waddell L, Wilhelm B, et al.: The efficacy of interventions applied during primary processing on contamination of beef carcasses with Escherichia coli: A systematic review-meta-analysis of the published research. Food Control. 2012; 27(2): 385–397. Publisher Full Text\n\nHiggins JPT, Li T, Deeks JJ: Choosing effect measures and computing estimates of effect. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA, eds. Cochrane Handbook for Systematic Reviews of Interventions. 2nd Edition. Chichester, UK: John Wiley & Sons. 2019; 143–176. Publisher Full Text\n\nSuurmond R, van Rhee H, Hak T: Introduction, comparison, and validation of Meta-Essentials: A free and simple tool for meta-analysis. Res Synth Methods. 2017; 8(4): 537–553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHedges LV, Olkin I: Statistical Methods for Meta-Analysis. San Diego, CA USA: Academic Press. 1985. Reference Source\n\nHiggins JPT, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med. 2002; 21(11): 1539–1558. PubMed Abstract \| Publisher Full Text\n\nSterne JA, Sutton AJ, Ioannidis JP, et al.: Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ. 2011; 343: d4002. PubMed Abstract \| Publisher Full Text\n\nMohammadigheisar M, Shouldice VL, Sands JS, et al.: Growth performance, breast yield, gastrointestinal ecology and plasma biochemical profile in broiler chickens fed multiple doses of a blend of red, brown and green seaweeds. Br Poult Sci. 2020; 1–9. PubMed Abstract \| Publisher Full Text\n\nBai J, Wang R, Yan L, et al.: Co-supplementation of dietary seaweed powder and antibacterial peptides improves broiler growth performance and immune function. Braz J Poult Sci. 2019; 21(2): eRBCA-2018–0826. Publisher Full Text\n\nShi H, Kim SH, Kim IH: Effect of dietary inclusion of fermented sea mustard by-product on growth performance, blood profiles, and meat quality in broilers. J Sci Food Agric. 2019; 99(9): 4304–4308. PubMed Abstract \| Publisher Full Text\n\nAhmed ST, Mun HS, Islam MM, et al.: Effects of fermented corni fructus and fermented kelp on growth performance, meat quality, and emission of ammonia and hydrogen sulphide from broiler chicken droppings. Br Poult Sci. 2014; 55(6): 745–751. PubMed Abstract \| Publisher Full Text\n\nChoi YJ, Lee SR, Oh JW: Effects of dietary fermented seaweed and seaweed fusiforme on growth performance, carcass parameters and immunoglobulin concentration in broiler chicks. Asian-Australas J Anim Sci. 2014; 27(6): 862–870. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbudabos AM, Okab AB, Aljumaah RS, et al.: Nutritional value of green seaweed (Ulva lactuca) for broiler chickens. Ital J Anim Sci. 2013; 12(2): e28. Publisher Full Text\n\nAndri F, Dono ND, Sasongko H, et al.: Extended data for ‘The effects of dietary seaweed inclusion on growth performance of broiler chickens: a systematic review and meta-analysis’. 2020. https://www.doi.org/10.6084/m9.figshare.12721454.v1\n\nCohen J: Statistical Power Analysis for the Behavioral Sciences. Hillsdale, NJ, USA: Lawrence Erlbaum Associates. 1988. Reference Source\n\nKulshreshtha G, Rathgeber B, Stratton G, et al.: Feed supplementation with red seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, affects performance, egg quality, and gut microbiota of layer hens. Poult Sci. 2014; 93(12): 2991–3001. PubMed Abstract \| Publisher Full Text\n\nChoi Y, Lee EC, Na Y, et al.: Effects of dietary supplementation with fermented and non-fermented brown algae by-products on laying performance, egg quality, and blood profile in laying hens. Asian-Australas J Anim Sci. 2018; 31(10): 1654–1659. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMandal AB, Biswas A, Mir NA, et al.: Effects of dietary supplementation of Kappaphycus alvarezii on productive performance and egg quality traits of laying hens. J Appl Phycol. 2019; 31(3): 2065–2072. Publisher Full Text\n\nMa WQ, Cheng HZ, Zhao DH, et al.: Effects of dietary Enteromorpha powder supplementation on productive performance, egg quality, and antioxidant performance during the late laying period in Zi geese. Poult Sci. 2020; 99(2): 1062–1068. PubMed Abstract \| Publisher Full Text\n\nYan GL, Guo YM, Yuan JM, et al.: Sodium alginate oligosaccharides from brown algae inhibit Salmonella Enteritidis colonization in broiler chickens. Poult Sci. 2011; 90(7): 1441–1448. PubMed Abstract \| Publisher Full Text\n\nZhu W, Li D, Wang J, et al.: Effects of polymannuronate on performance, antioxidant capacity, immune status, cecal microflora, and volatile fatty acids in broiler chickens. Poult Sci. 2015; 94(3): 345–352. PubMed Abstract \| Publisher Full Text\n\nSweeney T, Meredith H, Vigors S, et al.: Extracts of laminarin and laminarin/fucoidan from the marine macroalgal species Laminaria digitata improved growth rate and intestinal structure in young chicks, but does not influence Campylobacter jejuni colonisation. Anim Feed Sci Technol. 2017; 232: 71–79. Publisher Full Text\n\nGumus RE, Gelen SU, Koseoglu S, et al.: The effects of fucoxanthin dietary inclusion on the growth performance, antioxidant metabolism and meat quality of broilers. Braz J Poult Sci. 2018; 20(3): 487–496. Publisher Full Text\n\nLi Q, Luo J, Wang C, et al.: Ulvan extracted from green seaweeds as new natural additives in diets for laying hens. J Appl Phycol. 2018; 30(3): 2017–2027. Publisher Full Text\n\nTanna B, Choudhary B, Mishra A: Metabolite profiling, antioxidant, scavenging and anti-proliferative activities of selected tropical green seaweeds reveal the nutraceutical potential of Caulerpa spp. Algal Res. 2018; 36: 96–105. Publisher Full Text" }	[ { "id": "70757", "date": "28 Sep 2020", "name": "Samadi Samadi", "expertise": [ "Reviewer Expertise Animal Nutrition" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript aims to assess the effects of dietary seaweed inclusion on the growth performance of broiler chickens by using a systematic review and meta-analysis approach. The type of work is suitable for publication in the journal. However, the manuscript needs some correction before it can be fully accepted. The comments are as follows.\nIntroduction was too short and needs more explanation from the previous study (state of arts) relating to seaweed on broiler production.\n\nFrom PRISMA flow diagram, there were a lot of studies excluded for data analysis, out of 47 studies only 6 studies were continued for quantitative analysis. Is the data sufficient to make conclusions for this meta-analysis study?\n\nThere was a wide range of seaweed inclusion in broiler performance from the data (2-30 g/kg). Any explanation?\n\nDiscussion of this study was too short, it is better to extend more explanation regarding to the findings based on this study.\n\nBased on the literature, are there any different bioactive compound from various seaweed in nature (around twenty thousand species) #44\n\nConclusion of this study was too general, are there any recommendations for seaweed inclusion in the broiler diet based on the meta-analysis data, since the seaweed concentration from the literature of this study was too wide-ranging from 2-30 g/kg diet.\n\nI suggest it is better also to add one more table informing about FI, BWG, and FCR from 6 studies to get information for the readers about the broiler performance data.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "70759", "date": "05 Nov 2020", "name": "Mary Kivali Ambula", "expertise": [ "Reviewer Expertise Poultry nutritionist with interest in use of non-conventional feed ingredients in chicken diets" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe keywords listed are not mentioned much in the text which might make searching for the article a little difficult.\n\nClearly state if seaweed was used as a substitute for another ingredient or not.\n\nConclusions may not fully be drawn from the results since information on the type of basic diets is not provided e.g were they typical corn-soybean broiler diets or what?\n\nThe effect of seaweed on broilers may also depend on other feed ingredients used (alfalfa meal, corn gluten meal etc). Authors can clarify this to the reader to inform further research.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "92551", "date": "06 Sep 2021", "name": "Henny Akit", "expertise": [ "Reviewer Expertise I am an expert in broiler nutrition currently doing research in alternative protein ingredients in broilers. However", "i am not an expert in the statistic part." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSome of the keywords such as alginate, fucoidan, fucoxanthin and laminarin were not mentioned in the abstract and introduction. You may consider excluding these keywords.\nThe rationale for the systematic review was to provide a more accurate insight than the narrative review. The authors may add on how this insight could benefit researchers or related feed industries for seaweed application.\n\nIn discussion, the authors explained the possible mechanisms that led to improved broiler performance. I suggest that the authors include the year of broiler studies used. In addition, I think more points should be included emphasising how the results could benefit other researchers, for example, suggestions on the inclusion rate of seaweed, etc. Since this is a review paper, I feel that the discussion should be more extensive.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1087
https://f1000research.com/articles/8-615/v1	03 May 19	{ "type": "Research Article", "title": "Modifiable factors associated with weight regain after bariatric surgery: a scoping review", "authors": [ "Lisa Kaouk", "Amy T. Hsu", "Peter Tanuseputro", "Mahsa Jessri", "Lisa Kaouk", "Amy T. Hsu", "Peter Tanuseputro" ], "abstract": "Background: Although bariatric surgery is the most effective treatment for severe obesity, weight regain may still occur. While non-modifiable factors associated with weight regain have been explored, modifiable factors responsible for weight regain are understudied. This scoping review aimed to identify modifiable behaviors associated with weight regain after bariatric surgery. Methods: A systematic search was conducted in Medline, Google Scholar, Cochrane, National Collaborating Centre for Methods and Tools (NCCMT) and Practice-based Evidence in Nutrition (PEN) which included articles published between January 1990 and February 2 2017, for studies examining “weight regain” after bariatric surgery. A total of 293 citations were retrieved. Eligible articles must have examined modifiable factors and addressed weight regain, or a long-term post-operative phase in which weight regain may occur. After removing duplicates, 22 studies were included for thematic analysis. Results: Key modifiable factors associated with weight regain were identified and categorized under the following themes: poor dietary adherence (e.g. excessive calorie, carbohydrate, and alcohol intake), maladaptive eating behaviors (e.g. grazing, binging), lack of on-going follow-up with the bariatric team and insufficient physical activity. Conclusions: Health professionals and self-monitoring tools for patients who have undergone bariatric surgery may benefit from these findings to direct their education and interventions to target behavior change.", "keywords": [ "Bariatric surgery", "weight loss surgery", "weight regain", "weight recidivism", "modifiable behaviors" ], "content": "Abbreviations\n\nBMI: body mass index, RYGB: Roux-en-Y gastric bypass, EWL: excess weight loss, BED: binge eating disorder, LAGB: laparoscopic adjustable gastric band, AGB: adjustable gastric band, VBG: vertical banded gastroplasty, QOL: quality of life, CBT: cognitive behavioral therapy, DS: duodenal switch, ASMBS: American Society for Bariatric and Metabolic Surgery.\n\n\nIntroduction\n\nSevere obesity, measured by a body mass index (BMI) ≥35 kg/m2, is a complex, multifactorial disease that has been shown to significantly increase the risks of morbidity (e.g. cardiovascular diseases, type 2 diabetes, cancers) and mortality1. Bariatric surgery has been established as a promising treatment option for severe obesity and shown to be successful in achieving varying degrees of weight loss, health gain (including reduced morbidity and mortality), improved mental health, and quality of life2. However, the sustained health improvements following bariatric surgery are dependent on the individual’s adherence to long-term changes in lifestyle habits3. As a result, despite its effectiveness, weight regain after bariatric surgery is still possible.\n\nStudies have estimated an average of 56% of patients regain weight within two years of their surgery3, and about one in four fail to achieve the average expected weight loss and begin to regain weight from their lowest post-operative weight, following the first post-operative year. On average, individuals will achieve 20 – 30% of total weight loss at one to two years post-operative4, and can regain an average of 7% of their total body weight from their lowest post-operative weight over the course of 10 years2,5,6. Among patients who have had Roux-en-Y gastric bypass (RYGB), about 15% regain between 2 – 5% of weight from their lowest reported post-operative weight (nadir weight) within two years of surgery, which has been reported to increase to 70% of patients between two and five years, and 85% at over five years post-surgery3. The high prevalence of weight regain after bariatric surgery has resulted in a significant increase in revisional bariatric surgery6, which bears an increase in surgical risk and adverse outcomes to the patient7.\n\nDespite the prominence of weight regain following bariatric surgery, the underlying reasons for weight regain are not well-understood, but have been attributed to a number of surgical, biological and behavioral factors8. Although non-modifiable factors (e.g. hormonal, metabolic, surgery-related) have been identified in the literature8, less attention has been given to the modifiable behaviors and practices that could be implemented by patients and health care professionals. The primary objective of this scoping review was to identify the modifiable factors associated with weight regain following bariatric surgery. A secondary objective of this scoping review was to identify gaps and limitations of existing studies and evidence, which may provide guidance on areas of future research. We followed guidelines of Colquhoun et al.9, which is based on the Arksey and O’Malley framework10, for conducting and reporting of scoping reviews.\n\n\nMethods\n\nA systematic search of the literature was conducted in Medline, Google Scholar, Cochrane, National Collaborating Centre for Methods and Tools (NCCMT), and Practice-based Evidence in Nutrition (PEN). The most recent search was conducted on February 2, 2017. We included studies published in English between January 1990 and January 2017, using Boolean search terms—such as, “weight loss maintenance” OR “weight regain” AND “behavior” AND “English” AND “adult” (see Extended data) - that were identified by the research team11. An example of the search strategy used in Medline includes the following search terms: (\"weight loss maintenance\"[All Fields] OR \"weight regain\"[All Fields]) AND \"behavior\"[All Fields] AND ((\"1990/01/01\"[PDAT] : \"2017/02/02\"[PDAT]) AND \"humans\"[MeSH Terms] AND English[lang] AND medline[sb] AND \"adult\"[MeSH Terms]). Manual searches of cited references were also conducted to identify additional articles, as described in the eligibility criteria explained below. In total, 276 studies were identified using this search strategy once duplicates were removed.\n\nOur inclusion criteria were any published studies, reviews, practice guidelines and expert opinions involving adults (18+ years) in English between January 1, 1990 and January 31, 2017. All studies and reviews published after this date were excluded. To further narrow the number of papers for full-text review, only titles and/or abstracts containing the terms “bariatric surgery” or “weight loss surgery”, or an association of these terms, were included (n=32). All bariatric surgery types were considered, even those no longer being commonly performed (e.g. vertical banded gastroplasty, VBG). We retained studies that examined modifiable factors (such as diet, behavior/psychology, support, and physical activity) either exclusively or in conjunction with other influences. Studies that focused solely on non-modifiable factors including, but not limited to, gastric pouch size, age, sex, and pre-operative body mass index (BMI), were excluded. The included studies must have identified or referenced weight regain, or explored a period of possible weight regain or weight maintenance; however, no limit in time-frame was applied. Studies that only addressed insufficient weight loss without addressing weight regain were excluded. A total of 32 full-text reviews were performed and 22 articles were included for thematic analysis, after being assessed for relevancy and removing articles mentioned in the systematic reviews that were included (see Figure 1).\n\nThis PRISMA flowchart depicts the number of records identified through database searching and other sources, and the final number of articles – 22 articles – included in this scoping review.\n\nData was charted into an excel spreadsheet. This data was gathered, charted, and inputted independently, and reviewed by two other authors (AH, MJ). All authors discussed the themes that emerged in order to define the variables. Emerging themes that were factors in weight regain were documented and grouped into variables that summarized the themes, namely into behavioral, environmental, support, and exercise. All articles were assigned and coded to one, or more, of the stated variables.\n\n\nResults\n\nAfter removing duplicates (n = 17), 276 articles were screened by their titles, followed by their abstracts. After excluding the articles that did not meet our inclusion criteria, 32 articles remained. Articles were further excluded if they were not relevant to weight regain post-bariatric surgery, involved clinical practice guidelines that did not explicitly examine measurable outcomes, and if they were already mentioned in the included systematic reviews. A total of 22 full-text articles were included in the final synthesis of this scoping review. We extracted information on each study’s authors, location, design, scope of the evaluation (i.e. type of surgery, characteristics of the patient population, modifiable behaviors examined), outcome metrics and key findings (see Table 1).\n\nD = dietary, B = behavioral, avg = average, BMI = body mass index, BED = binge eating disorder, RYGB = Roux-en-Y gastric bypass, EWL = excess weight loss, E = exercise, LAGB = laparoscopic adjustable gastric band, S = support, VBG = vertical banded gastroplasty, QOL = quality of life, LGP = laparoscopic gastric plication, DS = duodenal switch\n\nThree of the authors (LK, AH, MJ) independently reviewed the studies and organized the findings into themes, based on the types of modifiable behavior examined (see Figure 2). Weight regain after bariatric surgery was found to be multifactorial, resulting from an interplay of different modifiable behaviors—including poor dietary adherence, behavior/psychological issues, lack of support and physical inactivity. The following sections provide a critical review of each of these themes in more detail. While weight regain was defined and expressed differently across each study, behavioral differences that were associated with weight regain were compiled into themes.\n\nThis conceptual framework depicts the factors associated with weight regain including dietary non-adherence, behavioral/psychological issues, lack of support, and physical inactivity, as well as the subgroups specific to each factor.\n\nOf the 22 studies included in our thematic analysis, eight (36%)8,12–18 suggested that diet was significantly associated with weight regain (p = 0.001-0.05) after bariatric surgery. Poor adherence to dietary guidelines - represented by higher carbohydrate intake16, higher alcohol intake8,15,16 and lower dietary quality8 - were key contributors.\n\nTwo studies reported that a higher energy intake (2000 vs. 1500 Kcal/day) was associated with a weight regain of 11 kg from nadir weight as of two years after surgery8,12 Similarly, Himes et al.17 reported a 10% weight loss of the total weight regained in as little as six weeks by reducing the frequency of eating episodes from 6.7 to 5.5 episodes, and 86% of participants in Faria et al.14 lost 54% of their weight regain in three months following a 1400 Kcal/day prescription. This suggests that a higher frequency of eating episodes and higher energy intake over time may have contributed to weight regain prior to the interventions.\n\nIn terms of dietary quality and alcohol intake, Reid et al.16 observed a 26% difference in weight outcomes (p ≤ 0.01), at 12 years post-operation, between people who maintained their weight and people who regained some weight. In this study, people who regained some weight consumed more carbohydrates than people who maintained their weight (222 vs. 162 g/day, p < 0.05); however, there was no difference in the percentage of energy intake from carbohydrates in both groups (43% vs. 42%, respectively)16 While people who regained some weight consumed more alcohol than people who maintained their weight (1.32 vs. 0.19 standard drinks/day; p < 0.05) in Reid et al.’s study16 reported consumption was still within the suggested limits for the general population. Other studies, however, have determined that alcohol misuse or abuse was associated with weight regain8,15. A higher median fat intake was observed in people who regained some weight (88.6 vs. 64.3 g/d, p < 0.05), although the percentage of energy intake from fats was similar in both groups (41.7% vs. 37.4%, respectively)12. Similarly, Karmali et al. refer to studies that reported a higher energy intake19 and poor dietary quality3, including higher sugar, sweets and fatty foods, were attributable to weight regain as of two years after RYGB, VBG, and adjustable gastric band (AGB).\n\nFinally, Bastos et al. examined the influence of having a food-related occupation on weight regain. They determined that working in food production - whether as a baker or working in a cafeteria, snack bar, restaurant, or grocery store - was correlated with significant weight regain (p = 0.003)13.\n\nOf the eight studies that observed diet as a factor associated with weight regain after bariatric surgery, seven of them observed calorie intake as a contributing factor. A higher calorie intake - whether from carbohydrates, alcohol, low nutritive value sweets, fatty foods, or as a result of a higher frequency of eating episodes - are associated with weight regain8,12–18. Only one study observed the association of working in the food industry with weight regain. Contrary to other studies, however, they did not observe alcohol intake as a contributing factor.\n\nThirteen studies (59%) identified post-operative diet-related behaviors, or eating habits, and psychological factors were associated with long-term post-operative weight regain.\n\nMaladaptive eating behaviors. Of the 12 studies that examined diet-related behaviors, nine studies (75%) found a significant association with post-operative weight regain8,18,20–27. Variations of these habits, including binge eating, disinhibited eating, picking and nibbling or grazing, and loss of control eating behaviors, have contributed to a weight regain ranging from 10 kg to 17 kg8,17, nearly 11% gain from nadir weight at two years after surgery22. At five years post-operative, weight regain was found to be as high as 44% from nadir weight, as a result of disinhibited eating, or the tendency to overeat, and unsuitable eating behaviors21. While most studies have demonstrated the association of maladaptive dietary behaviors with weight regain8,18,20–27, Himes et al.17 demonstrated that these behaviors improved with interventions; for example, a group behavioral cognitive behavioral therapy (CBT) intervention led to a 1.6 kg weight loss over six weeks in those who had been on a weight regain trend post bariatric surgery. Meanwhile, Mitchell et al.20 determined that grazing and binging habits, in addition to a lack of self-monitoring, accounts for 16% variability in weight outcomes. Pekkarinen et al.24 showed that although people who binged and people who did not binge had similar outcomes at one year post-VBG (55% vs. 57%, respectively), people who binged regained significantly more weight than people who did not binge two years after their operation (24% vs. 50%, respectively, p = 0.04). Finally, Hsu et al.27 showed that those who suffered from binge-eating disorder (BED) prior to surgery continued to struggle with this after bariatric surgery. This finding was further confirmed by Colles et al.,28 who found that grazing habits increased post bariatric surgery (26% pre-operation to 38% by one year post-operative), while Nicolau et al. determined that 72% of people who grazed gained weight vs. people who did not graze (p < 0.0001)23. However, Alvarez et al. did not find BED to be associated with post-operative weight regain.\n\nOf all 22 studies reviewed, only one had identified nocturnal eating as an important determinant of weight regain (p = 0.01)15.\n\nPsychological factors. Three articles determined that depression8,15, anxiety12, and alcohol and/or substance abuse8,12,15 were associated with post-operative weight regain.\n\nFive articles (23%) suggested poor post-operative support was associated with long-term weight regain8,29–32. Four of these studies examined the impact of follow-ups with a bariatric team; they determined that little to no post-operative follow-up can lead to poor long-term outcomes8,29–31. Those who maintained regular follow-up, for up to three years post-operation, had better long-term weight outcomes (74% excess weight loss, EWL)30,31 than those who were lost to follow-up within the first year of their surgery (56% EWL, p < 0.05)30. Among the 75% of patients who no longer received follow-up as of three years post-operation, they were observed to regain up to 14% more weight; in comparison, only 25% of patients who received an annual follow-up up to five to six years post-operation were found to experience weight regain29. In another study, regular follow-up represented 47% difference in %EWL at two years post-operation30.\n\nPost-operative support from health care professionals was found to be an important component in long-term success. Karmali et al.8 cited that, among those who failed surgery, 60% had never seen a dietitian and 80% had never seen a psychologist. Comparatively, Gould et al.31 reported greater long-term outcomes in patients who attended all post-operative follow-up visits with a multidisciplinary team. A post-operative bariatric surgery patient who received individual or group CBT sessions had a higher %EWL (90% vs. 43% EWL) at two years post-surgery and better weight loss outcomes than the controls who did not receive support30. Yet, despite the known benefits of post-operative support, there was little evidence in the literature in support group attendance and its influence on weight regain or weight maintenance. Liebl’s qualitative study32 described the experiences of post-operative bariatric patients who were successful at maintaining weight loss (average of 8% of EWL regained) at an average of 69 months post-surgery. The patients surveyed in the study reported that support from their family, peers and professionals from their bariatric surgery clinic had been necessary to achieve positive outcomes.\n\nSeven studies (32%) addressed the relationship between physical activity and weight regain. Questionnaires, which primarily captured moderate to vigorous intensity activity, were used to determine activity levels in three of the studies15,18,21. Four systematic reviews reinforced that lower physical activity levels were associated with poorer weight loss outcomes and higher weight regain, despite undergoing bariatric surgery8,18,29,33,34. Furthermore, Amundsen et al.21 observed that people who significantly regained weight (total weight regain ≥ 15% from nadir weight), were less active than people who regained a normal amount of weight (p=0.06). People who maintained their weight spent remarkably more time being moderately active (567 min per week) compared to people who regained weight (287 min per week).\n\n\nDiscussion\n\nIn this scoping review, we have presented a summary of the existing literature on modifiable factors associated with weight regain after bariatric surgery - namely, poor dietary adherence, behavioral and psychological issues, lack of support, and physical inactivity - and highlight their potential relevance to patients and practicing healthcare professionals in this field.\n\nStudies related to dietary adherence suggested that poor observance of the dietary guidelines -represented by higher carbohydrate intake, higher alcohol intake and lower dietary quality - were key contributors to weight regain in the long-term recovery from bariatric surgery.\n\nChanges in dietary adherence over the course of the post-operative phase may be associated with weight regain in patients after bariatric surgery8,12,16,18. Higher carbohydrate consumption appears to be the most evident dietary cause associated with weight regain16. Although the source of carbohydrates was not clearly defined in all studies, some have demonstrated that an increased consumption of liquid calories and sugar intake from non-nutritive sources were attributable to weight regain26. Thus, increases in patient’s non-nutritive, free- and added-sugar intake potentially explain some of the weight regain following bariatric surgery.\n\nAlcohol is a source of empty liquid calories which contributes significantly to one’s caloric intake, and some studies have found a positive association between weight regain and alcohol abuse or misuse8,15,16. This is particularly concerning because alcohol abuse has been shown to be higher among people who have had bariatric surgery, in comparison to the general population35. While there does not appear to be a consensus on post-operative weight regain and alcohol intake, one of the studies reported an association between intake levels at or beneath the suggested alcohol intake limits16,36. The discrepancies seen in the literature may be due to the nature of the data collection. Alcohol intake is known to be underreported by up to 50% when self-reported37. This is especially true among middle-aged women, which coincides with the demographic of the bariatric population37. Despite the inconclusive results, results from this review suggest alcohol intake should be more closely assessed and monitored in the people who have had bariatric surgery, even if their consumption is within the suggested limits according to the Centers for Disease Control and Prevention36.\n\nGiven the gastric restriction of bariatric surgery, increased calorie intake occurs in the form of more frequent eating episodes and/or in the consumption of more calorie-dense foods or liquid calories. This demonstrates that dietary patterns after bariatric surgery do not remain consistent; rather, there is a gradual onset of undesirable dietary habits that develops, and some patients may not be cognizant of the effects this will have on their future outcome. Therefore, bariatric surgery alone is not protective in the long term; patients will likely require the ongoing support and monitoring of dietitians and their bariatric team to find solutions and alternatives to the challenges to maintain adherence to dietary recommendations.\n\nBehavioral and psychological factors may impede one’s ability to comply with post-operative lifestyle recommendations. While restrictive and malabsorptive procedures may limit the amount of food consumed in a given sitting, it does not generally limit the ability to eat significant volumes over the course of a day. Grazing and binging were the most commonly identified eating behaviors associated with weight regain. All, except for one study, clearly observed this relationship. Although maladaptive eating habits do not negatively affect one’s weight outcomes at one year post-operation, people who continue to binge have a higher risk of regaining weight by the second year following surgery24. Furthermore, Himes et al.17 suggests that therapy aimed at reducing binge eating behaviors can lower the number of daily eating episodes and encourage weight loss following weight regain. Therefore, targeted therapy towards maladaptive eating behaviors provided early on in the patient’s recovery process may help to prevent occurrence of weight regain.\n\nGiven that maladaptive eating behaviors that exist prior to the surgery appear to remain an issue in the post-operative phase27, dietary behaviors are an important target point for discussion even before the weight loss surgery is performed. Health professionals should educate patients on the evolution of maladaptive eating behaviors after surgery to encourage therapy and treatment prior to the operation and on an ongoing basis. Professionals should continue to monitor and probe for various types of maladaptive eating behaviors long-term after surgery.\n\nMost of the articles that studied behaviors looked at maladaptive eating behaviors also found lower levels of physical activity as contributing factor to weight regain. This suggests that there may be a relationship―and, potentially, interactive or cumulative impacts―between eating behaviors, dietary intake and physical activity.\n\nThe American Society for Metabolic and Bariatric Surgery (ASMBS) has produced guidelines on post-operative follow-up care with a bariatric team, which emphasizes the importance of follow-up care on a patient’s progress and outcomes. Follow-up care helps to track the patient’s progress, ensure their adherence to recommendations made by their health care team and offers solutions for managing weight and lifestyle. With 50% of people who have had bariatric surgery regaining weight within two years of their surgery, it is not surprising that 60% of these patients had not received regular nutrition follow-up and 80% did not receive psychological follow-up38; in contrast, patients who attended all of their post-operative follow-up were more successful in the long-term31. Follow-up maintained for up to three years post-operation resulted in 18% higher EWL compared to a patient who was lost to follow-up within the first year of surgery31, which may infer a positive correlation between longer follow-up periods and improved health outcomes. The impact of support has been well studied post bariatric surgery, as evidenced by several systematic reviews on the subject. Unfortunately, studies included in this scoping review were only able to highlight the benefit of post-operative support with a bariatric team; it was not clear from the literature if there is a single member of the bariatric team that is pivotal in providing the follow-up, even though several studies observed follow-up with either a dietitian or a psychologist.\n\nFrom the patient’s perspective, positive family, peer, and professional bariatric support were identified as being vital in achieving long-term post-operative success. This offers important insight into the patients’ perspectives and the three prongs of support considered necessary to optimize their long-term outcomes. Based on these findings, a patient’s health care provider and social network - which may include family members and friends - should encourage the patient to present to follow-up appointments and, ideally, accompanied by their family, partners or friends. Given that patients usually forget the information they have been taught prior to surgery, which further worsens at one year post-operation39, inviting the patients’ support systems to follow-up appointments may encourage them to maintain regular contact with their bariatric team and also serve as an aid to remind patients of the positive interventions and behaviors discussed during consultations.\n\nBariatric surgery could greatly improve mobility and reduces the burden of osteoarthritis. As previously supported in the literature, participants with obesity tend to over-report their physical activity levels40. While 89% of patients self-reported to be regularly active at two years post-operation33, other studies observed that only about half of patients were engaged in moderate to vigorous activity for more than one session per week41, which is lower than the activity of adults in the general population (48% vs 53%, respectively)42. Furthermore, only 11%16 of people who have had bariatric surgery, as compared to 35%42 of the general population, actually achieve 10,000 steps per day when measured using accelerometers.\n\nAlthough there were several systematic reviews on the impact of physical activity on post-operative bariatric surgery outcomes, there is little information available on the subject of physical activity and weight regain; most articles cite that ‘low energy expenditure’ was associated with weight regain. It has also been suggested that current physical activity guidelines may be too low to prevent long-term weight regain after bariatric surgery21. However, it is observed that despite having similar moderate to vigorous physical activity habits to the general population, people who have had bariatric surgery are less active on a daily basis.\n\nThere were several identified gaps in the literature, which could limit the generalizability of the conclusions presented here. Firstly, there is not a consistent definition and measure for “weight regain”, or a clear indication of what is considered to be normal weight regain. This makes it difficult to compare the effect size across studies and modifiable factors in relation to weight regain. The ASMBS has suggested standardized outcomes reporting; however, the association has not offered guidelines on reporting magnitude of weight regain. In addition, although participants were stratified into groups, the cut-off points used in some studies to describe percentage of weight regain may not be sensitive enough (i.e. >50% EWL vs ≤50% EWL) to observe clinically significant changes. As a result, there may have been poor differentiation between outliers and participants who narrowly fell within the cut-off range12.\n\nSecondly, the surveys and questionnaires used were unique to each study, resulting in inconsistent metrics that are often not directly comparable. This is important since different surveys for assessing lifestyle habits may lead to varying results and conclusions. The use of different tools and questionnaires that have not been validated for the bariatric surgery population may also explain the discrepancies observed for the expected similar associations.\n\nThirdly, the majority of the studies relied on self-reported measures of modifiable behaviors. Dietary misreporting in the population affected by obesity is particularly concerning and has been well-documented in a previous study, such that people who under-reported their energy intake were more likely to be inflicted with obesity43. Similar results have been observed for self-reported physical activity, which is generally over-reported. All but one study included in this scoping review relied on questionnaires and self-reported data; hence, making it difficult to reach a robust conclusion on physical activity levels post-surgery. Therefore, self-reported data inevitably limits the external validity of the findings.\n\nAnother source of variability to consider is that this scoping review included all types of bariatric surgery, even though the VBG is no longer performed and the AGB is being phased out as a routine procedure. However, even though each procedure is different in the weight loss achieved and outcome experiences (with regards to tolerance of certain foods), the modifiable behaviors would be similar across surgery types.\n\nLastly, most of the studies included in this scoping review were observational. However, even among the interventional studies, the sample sizes were very small (n<30), making it difficult to determine the true cause and effect.\n\n\nConclusion\n\nAlthough effective, weight regain can still occur after bariatric surgery. Findings of this article support the notion that people who have had bariatric surgery need to be informed of the modifiable factors associated with weight regain in an effort to encourage long-term weight loss maintenance. Regular, routine and long-term follow-up with the bariatric team is essential to the long-term weight regain prevention. Follow-up support may act as a pivot to addressing poor dietary adherence, behavioral issues and physical inactivity that impact long-term weight outcomes in a timely manner.\n\nFuture research should identify a common definition and measurement for weight regain post-bariatric surgery and agree upon accepted surveys and questionnaires validated in the bariatric population. Future research should also identify the specific foods, eating frequency and type of physical activity that may be the most relevant to people who have had bariatric surgery to provide healthcare professionals with a better understanding of the types of foods to suggest to limit and the types of activities to reinforce. The literature can benefit from more randomized clinical trials targeting dietary protocols and patient support that include better controls. Finally, rigorous subgroup analyses to enable comparison of outcomes and relevant interventions among patients undergoing different procedures, as well as among those who suffer from severe obesity (BMI 35-49) and super-severe obesity (BMI ≥50), will be important for personalized care planning in this patient population.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: Modifiable Factors Associated with Weight Regain After Bariatric Surgery: A Scoping Review. https://doi.org/10.17605/OSF.IO/9Q78A11.\n\nThis project contains the following extended data:\n\n- Boolean Search Terms.docx\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis project was supported by a targeted donor grant to the Bruyère Foundation (The Big Data Research Fund), assigned to Dr. Peter Tanuseputro, Dr. Doug Manuel, Vivian Welch (PhD) and Dr. Peter Walker.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge the support of patients, who have generously given their time to inform the research team of their challenges and needs, as them embark on this journey towards a healthy lifestyle following a bariatric surgery. LK has received a Masters of Public Health Practicum Award from the School of Public Health and Health Systems at the University of Waterloo. ATH and MJ have received funding from the Canadian Institutes of Health Research for their postdoctoral training.\n\n\nReferences\n\nWHO: Obesity and Overweight: Fact sheet (2016). Accessed March 1 2017. Reference Source\n\nCourcoulas AP, Yanovski SZ, Bonds D, et al.: Long-term outcomes of bariatric surgery: a National Institutes of Health symposium. JAMA Surg. 2014; 149(12): 1323–1329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreire RH, Borges MC, Alvarez-Leite JI, et al.: Food quality, physical activity, and nutritional follow-up as determinant of weight regain after Roux-en-Y gastric bypass. Nutrition. 2012; 28(1): 53–58. PubMed Abstract \| Publisher Full Text\n\nHeber D, Greenway FL, Kaplan LM, et al.: Endocrine and nutritional management of the post-bariatric surgery patient: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2010; 95(11): 4823–4843. PubMed Abstract \| Publisher Full Text\n\nSjöström L: Review of the key results from the Swedish Obese Subjects (SOS) trial - a prospective controlled intervention study of bariatric surgery. J Intern Med. 2013; 273(3): 219–234. PubMed Abstract \| Publisher Full Text\n\nBrethauer SA, Kothari S, Sudan R, et al.: Systematic review on reoperative bariatric surgery: American Society for Metabolic and Bariatric Surgery Revision Task Force. Surg Obes Relat Dis. 2014; 10(5): 952–72. PubMed Abstract \| Publisher Full Text\n\nSpyropoulos C, Kehagias I, Panagiotopoulos S, et al.: Revisional bariatric surgery: 13-year experience from a tertiary institution. Arch Surg. 2010; 145(2): 173–177. PubMed Abstract \| Publisher Full Text\n\nKarmali S, Brar B, Shi X, et al.: Weight recidivism post-bariatric surgery: a systematic review. Obes Surg. 2013; 23(11): 1922–1933. PubMed Abstract \| Publisher Full Text\n\nColquhoun HL, Levac D, O’Brien KK, et al.: Scoping reviews: time for clarity in definition, methods, and reporting. J Clin Epidemiol. 2014; 67(12): 1291–1294. PubMed Abstract \| Publisher Full Text\n\nArksey H, O'Malley L: Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nKaouk L: Modifiable Factors Associated with Weight Regain After Bariatric Surgery: A Scoping Review. 2019. http://www.doi.org/10.17605/OSF.IO/9Q78A\n\nAlvarez V, Carrasco F, Cuevas A, et al.: Mechanisms of long-term weight regain in patients undergoing sleeve gastrectomy. Nutrition. 2016; 32(3): 303–308. PubMed Abstract \| Publisher Full Text\n\nBastos EC, Barbosa EM, Soriano GM, et al.: Determinants of weight regain after bariatric surgery. Arq Bras Cir Dig. 2013; 26 Suppl 1: 26–32. PubMed Abstract \| Publisher Full Text\n\nFaria SL, de Oliveira Kelly E, Lins RD, et al.: Nutritional management of weight regain after bariatric surgery. Obes Surg. 2010; 20(2): 135–139. PubMed Abstract \| Publisher Full Text\n\nYanos BR, Saules KK, Schuh LM, et al.: Predictors of Lowest Weight and Long-Term Weight Regain Among Roux-en-Y Gastric Bypass Patients. Obes Surg. 2015; 25(8): 1364–1370. PubMed Abstract \| Publisher Full Text\n\nReid RE, Oparina E, Plourde H, et al.: Energy Intake and Food Habits between Weight Maintainers and Regainers, Five Years after Roux-en-Y Gastric Bypass. Can J Diet Pract Res. 2016; 77(4): 195–198. PubMed Abstract \| Publisher Full Text\n\nHimes SM, Grothe KB, Clark MM, et al.: Stop regain: a pilot psychological intervention for bariatric patients experiencing weight regain. Obes Surg. 2015; 25(5): 922–927. PubMed Abstract \| Publisher Full Text\n\nMcGrice M, Don Paul K: Interventions to improve long-term weight loss in patients following bariatric surgery: challenges and solutions. Diabetes Metab Syndr Obes. 2015; 8: 263–274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSjostrom L, Lindroos AK, Peltonen M, et al.: Lifestyle, diabetes, and cardiovascular risk factors 10 years after bariatric surgery. N Engl J Med. 2004; 351(26): 2683–93. PubMed Abstract \| Publisher Full Text\n\nMitchell JE, Christian NJ, Flum DR, et al.: Postoperative Behavioral Variables and Weight Change 3 Years After Bariatric Surgery. JAMA Surg. 2016; 151(8): 752–757. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmundsen T, Strømmen M, Martins C: Suboptimal Weight Loss and Weight Regain after Gastric Bypass Surgery-Postoperative Status of Energy Intake, Eating Behavior, Physical Activity, and Psychometrics. Obes Surg. 2017; 27(5): 1316–1323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConceição E, Mitchell JE, Vaz AR, et al.: The presence of maladaptive eating behaviors after bariatric surgery in a cross sectional study: importance of picking or nibbling on weight regain. Eat Behav. 2014; 15(4): 558–562. PubMed Abstract \| Publisher Full Text\n\nNicolau J, Ayala L, Rivera R, et al.: Postoperative grazing as a risk factor for negative outcomes after bariatric surgery. Eat Behav. 2015; 18: 147–150. PubMed Abstract \| Publisher Full Text\n\nPekkarinen T, Koskela K, Huikuri K, et al.: Long-term Results of Gastroplasty for Morbid Obesity: Binge-Eating as a Predictor of Poor Outcome. Obes Surg. 1994; 4(3): 248–255. PubMed Abstract \| Publisher Full Text\n\nConceição E, Bastos AP, Brandão I, et al.: Loss of control eating and weight outcomes after bariatric surgery: a study with a Portuguese sample. Eat Weight Disord. 2014; 19(1): 103–109. PubMed Abstract \| Publisher Full Text\n\nSarwer DB, Dilks RJ, West-Smith L: Dietary intake and eating behavior after bariatric surgery: threats to weight loss maintenance and strategies for success. Surg Obes Relat Dis. 2011; 7(5): 644–651. PubMed Abstract \| Publisher Full Text\n\nHsu LK, Benotti PN, Dwyer J, et al.: Nonsurgical factors that influence the outcome of bariatric surgery: a review. Psychosom Med. 1998; 60(3): 338–346. PubMed Abstract \| Publisher Full Text\n\nColles SL, Dixon JB, O'Brien PE: Grazing and loss of control related to eating: two high-risk factors following bariatric surgery. Obesity (Silver Spring). 2008; 16(3): 615–622. PubMed Abstract \| Publisher Full Text\n\nLauti M, Kularatna M, Hill AG, et al.: Weight Regain Following Sleeve Gastrectomy-a Systematic Review. Obes Surg. 2016; 26(6): 1326–1334. PubMed Abstract \| Publisher Full Text\n\nRudolph A, Hilbert A: Post-operative behavioural management in bariatric surgery: a systematic review and meta-analysis of randomized controlled trials. Obes Rev. 2013; 14(4): 292–302. PubMed Abstract \| Publisher Full Text\n\nGould JC, Beverstein G, Reinhardt S, et al.: Impact of routine and long-term follow-up on weight loss after laparoscopic gastric bypass. Surg Obes Relat Dis. 2007; 3(6): 627–630; discussion 630. PubMed Abstract \| Publisher Full Text\n\nLiebl L, Barnason S, Brage Hudson D: Awakening: a qualitative study on maintaining weight loss after bariatric surgery. J Clin Nurs. 2016; 25(7–8): 951–961. PubMed Abstract \| Publisher Full Text\n\nSilver HJ, Torquati A, Jensen GL, et al.: Weight, dietary and physical activity behaviors two years after gastric bypass. Obes Surg. 2006; 16(7): 859–864. PubMed Abstract \| Publisher Full Text\n\nLivhits M, Mercado C, Yermilov I, et al.: Exercise following bariatric surgery: systematic review. Obes Surg. 2010; 20(5): 657–665. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNIH: Alcohol Facts and Statistics (on-line). 2017; Accessed March 1, 2017. Reference Source\n\nCDC - Centers for Disease Control and Prevention: Alcohol and public health: frequently asked questions (2017). Accessed March 1 2017. Reference Source\n\nLivingston M, Callinan S: Underreporting in alcohol surveys: whose drinking is underestimated? J Stud Alcohol Drugs. 2015; 76(1): 158–164. PubMed Abstract \| Publisher Full Text\n\nMagro DO, Geloneze B, Delfini R, et al.: Long-term weight regain after gastric bypass: a 5-year prospective study. Obes Surg. 2008; 18(6): 648–651. PubMed Abstract \| Publisher Full Text\n\nMadan AK, Tichansky DS: Patients postoperatively forget aspects of preoperative patient education. Obes Surg. 2005; 15(7): 1066–1069. PubMed Abstract \| Publisher Full Text\n\nWarner ET, Wolin KY, Duncan DT, et al.: Differential accuracy of physical activity self-report by body mass index. Am J Health Behav. 2012; 36(2): 168–178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerman KM, Carver TE, Christou NV, et al.: Keeping the weight off: physical activity, sitting time, and weight loss maintenance in bariatric surgery patients 2 to 16 years postsurgery. Obes Surg. 2014; 24(7): 1064–1072. PubMed Abstract \| Publisher Full Text\n\nColley RC, Garriguet D, Janssen I, et al.: Physical activity of Canadian adults: accelerometer results from the 2007 to 2009 Canadian Health Measures Survey. 2015; Accessed March 1 2017. Reference Source\n\nJessri M, Lou W, L’Abbé MR: Evaluation of different methods to handle misreporting in obesity research: evidence from the Canadian national nutrition survey. Br J Nutr. 2016; 115(1): 147–159. PubMed Abstract \| Publisher Full Text" }	[ { "id": "49560", "date": "24 Jun 2019", "name": "Robert Kushner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed a systematic review using multiple databases for modifiable factors associated with weight regain following all bariatric surgery procedures. The time frame of the search, search terms and selection process for the studies appears appropriate. Twenty-two studies were included in the analysis. All articles were from 2007 to 2016 except for one (a 1994 report with VBG – a procedure that is no longer performed). The authors categorized the articles into 4 general themes. The limitations cited are appropriate. Table 1 displays all studies with key information included (year, type of surgery and modifiable behaviour, study design and population, intervention and group comparisons, and results). Figure 1 is a PRISMA flowchart, figure 2 is a conceptual framework. All appropriate for a review article.\nComments:\nThe topic reviewed is important and useful for clinicians and clinical researchers. It also provides the most up-to-date studies. However, there is a noticeable dearth of studies that focus on prospective interventions. Past reviews have included modifiable factors and interventions.1, 2, 3 This is an important point since few, if any, interventions have been found to be effective in preventing or treating weight regain. So, simply laying out modifiable factors without including clinical research that has attempted to intervene is unsatisfying. This becomes somewhat problematic for the authors. There are several instances where they include interventions, such as citing the Hines et al (ref 17) or McGrice et al (ref 18), but few if any other interventions. It is also problematic when recommendations are given, e.g, “Regular, routine and long-term follow-up with the bariatric team is essential to the long-term weight regain prevention.” Although this is sound advice, there is a large body of literature that has attempted to understand which interventions should be provided, when to intervene, for how long and by whom. As noted earlier, interventions have been largely disappointing.\n\nIn the introduction, severe obesity is defined as a BMI ≥40 kg/m2, not ≥35 as stated. It is more clinically useful to express the impact of weight regain as ‘percent of weight regained from nadir’ versus ‘an average of x% of their total body weight…’ The statement that 85% of patients experience weight regain at 5 years post-surgery sounds dramatic, but has little meaning if it is not defined by more criteria. Essentially all people experience some weight regain regardless of the treatment modality.\n\nIt is also noteworthy that the 4 behavioural modifiable domains identified by the authors are the same that are associated with any intervention for obesity. These are not unique to the bariatric surgery patient. This may be mentioned in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "52485", "date": "30 Sep 2019", "name": "Boris Zevin", "expertise": [ "Reviewer Expertise Bariatric and Metabolic Surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for the opportunity to review this manuscript. The authors conducted a scoping review of the literature from 1990 to 2017 identify the modifiable factors associated with weight regain following bariatric surgery. I commend the authors on exploring this important topic as the issue of weight regain after bariatric surgery is gaining increased importance with over 215,000 primary bariatric procedures performed per year in USA 1.\n\nTo help situate this manuscript in the current literature, I would suggest for the authors to define two distinct patient populations following bariatric surgery: (1) individuals who were initially successful in losing weight and have regained some or all of weight previously lost, and (2) individuals who have failed to lose the appropriate percentage of weight post-op. These two patient populations are different and respond differently to additional weight loss interventions. Defining these would help the readers understand which patients this review is focused on (i.e. # 1).\n\nI would encourage the authors to better define weight regain in this manuscript. At present, it is unclear what cut-off is being used to define weight regain (i.e. gaining 5% of maximum weight lost vs regaining all of the weight previously lost). A recent study in JAMA suggested reporting weight regain as percentage of maximum weight lost as this parameter appeared to perform better for association with most clinical outcomes than the alternatives examined [2].\nIn addition, I would encourage the authors to write a paragraph in the introduction section of the manuscript discussing obesity as a chronic disease, which, as other chronic diseases, will require multiple interventions over the person's lifetime to treat.\n\nThe Obesity Medicine Association defined obesity as \"a chronic, relapsing, multifactorial, neurobehavioral disease, wherein an increase in body fat promotes adipose tissue dysfunction and abnormal fat mass physical forces, resulting in adverse metabolic, biomechanical, and psychosocial health consequences.\" Framing your discussion around this contemporary definition of obesity would help the readers understand why lifestyle, pharmacotherapy and surgery are all important treatments for patients with obesity.\n\nLastly, I would like to make a couple of suggestions for minor revisions to the manuscript:\n\nIntroduction: The authors note that \"Bariatric surgery has been established as a promising treatment option for severe obesity \"; however, I would argue that bariatric surgery is a proven treatment for severe obesity with over 20 years of outcomes data. I suggest revising the wording.\n\nIntroduction: The authors report that \"Studies have estimated an average of 56% of patients regain weight within two years of their surgery, and about one in four fail to achieve the average expected weight loss and begin to regain weight from their lowest post-operative weight, following the first post-operative year.\" This statement needs to be clarified in the context of my prior comments about the definition of weight regain. In the current form, it makes it seem that most of the patients regain weight after surgery, suggesting that surgery is not effective. This is false and can contribute to the biases among the health care providers that surgery is not effective.\n\nIn a recent study from Kothari and colleagues, reported on 10 year follow-up post bariatric surgery on > 70% of patients 3. They reported a 56% EWL at 10 years post gastric bypass - confirming durability of the results after this operation.\n\nIn the discussion section, please consider discussing why patients should be educated about abstinence from alcohol after malabsorptive bariatric operations (in addition to possible weight regain) and changes in alcohol metabolism post gastric bypass.\n\nDiscussion: Given the finding of frequency of follow-up being associated with weight loss outcomes, please consider discussing how the health care system and health care providers can address the need for ongoing follow-up of bariatric surgery patients in a resource constrained environment of a publicly funded Canadian health care system? Why should be following these patients? How long and how often?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5859", "date": "03 Sep 2020", "name": "Lisa Kaouk", "role": "Author Response", "response": "Dear F1000 Editors and Dr. Zevin, Thank you for facilitating the peer-review process. We have uploaded a revised copy of the manuscript and have addressed all of Dr. Zevin’s comments to further improve the clarity and quality of our paper. Please find our responses to each comment below. 1. “To help situate this manuscript in the current literature, I would suggest for the authors to define two distinct patient populations following bariatric surgery: (1) individuals who were initially successful in losing weight and have regained some or all of weight previously lost, and (2) individuals who have failed to lose the appropriate percentage of weight post-op. These two patient populations are different and respond differently to additional weight loss interventions. Defining these would help the readers understand which patients this review is focused on (i.e. # 1).” We have added the following in the introduction:There are two distinct patient populations observed with people who had bariatric surgery with suboptimal or poor outcomes. There are those who do not lose the expected or anticipated average percentage of weight following surgery, while there are others who lose a successful amount of weight but who regain some, or most, of the weight they had initially lost via bariatric surgery. This scoping review observes the latter group of people. 2. “I would encourage the authors to better define weight regain in this manuscript. At present, it is unclear what cut-off is being used to define weight regain (i.e. gaining 5% of maximum weight lost vs regaining all of the weight previously lost). A recent study in JAMA suggested reporting weight regain as percentage of maximum weight lost as this parameter appeared to perform better for association with most clinical outcomes than the alternatives examined [2].” We have added the following in the Inclusion and exclusion criteria of the Methods section.”Given that the research community has not yet reached consensus on how weight regain is defined or compared, all studies addressing weight regain either as a percentage of regain or in weight gained were included regardless of the extent of regain. 3. “In addition, I would encourage the authors to write a paragraph in the introduction section of the manuscript discussing obesity as a chronic disease, which, as other chronic diseases, will require multiple interventions over the person's lifetime to treat. The Obesity Medicine Association defined obesity as \"a chronic, relapsing, multifactorial, neurobehavioral disease, wherein an increase in body fat promotes adipose tissue dysfunction and abnormal fat mass physical forces, resulting in adverse metabolic, biomechanical, and psychosocial health consequences.\" Framing your discussion around this contemporary definition of obesity would help the readers understand why lifestyle, pharmacotherapy and surgery are all important treatments for patients with obesity.” We have added the following to the introduction:Before discussing bariatric surgery, it would be important to first address obesity as a complex chronic disease which requires several interventions over the course one a person’s lifetime in an effort to treat it. The Obesity Medicine Association (OMA) defines obesity as “a chronic, relapsing, multifactorial, neurobehavioral disease, wherein an increase in body fat promotes adipose tissue dysfunction and abnormal fat mass physical forces, resulting in adverse metabolic, biomechanical, and psychosocial health consequences”. As such, the treatment of obesity requires a multifaceted approach customized to each individual’s needs including treatment options such as lifestyle modifications, pharmacotherapy, and surgery, are valuable treatments. 4. “Introduction: The authors note that \"Bariatric surgery has been established as a promising treatment option for severe obesity \"; however, I would argue that bariatric surgery is a proven treatment for severe obesity with over 20 years of outcomes data. I suggest revising the wording.” The change has been made. 5. “Introduction: The authors report that \"Studies have estimated an average of 56% of patients regain weight within two years of their surgery, and about one in four fail to achieve the average expected weight loss and begin to regain weight from their lowest post-operative weight, following the first post-operative year.\" This statement needs to be clarified in the context of my prior comments about the definition of weight regain. In the current form, it makes it seem that most of the patients regain weight after surgery, suggesting that surgery is not effective. This is false and can contribute to the biases among the health care providers that surgery is not effective. In a recent study from Kothari and colleagues, reported on 10 year follow-up post bariatric surgery on > 70% of patients 3. They reported a 56% EWL at 10 years post gastric bypass - confirming durability of the results after this operation.” Upon reading our reference #3, it appears that we misread the statement, and in fact, what Dr. Zevin had suggested is correct. As such, we maintained the reference originally used in the scoping review and made the numerical correction to properly cite the reference originally used to indicate that 56% of people regain some weight after a period of 10 years. 6. “In the discussion section, please consider discussing why patients should be educated about abstinence from alcohol after malabsorptive bariatric operations (in addition to possible weight regain) and changes in alcohol metabolism post gastric bypass.” We added the following to the discussion section:Patients need to be educated on the effects of alcohol consumption after malabsorptive bariatric procedures due to changes in alcohol metabolism, particularly after a gastric bypass. Studies have demonstrated an acceleration in alcohol absorption after a gastric bypass such that it takes a shorter time to reach a maximum concentration. In addition, there is a higher maximum alcohol concentration achieved, as well as it taking longer to fully metabolize and eliminate alcohol.43 However, I did not address that patients need to be educated about abstinence of alcohol as ASMBS’ position statement on alcohol use prior to and after surgery does not recommend abstinence post-operatively. Rather they advise patient education to inform patients of the risks of alcohol misuse post-op. While several bariatric programs across North American advise abstinence of alcohol after surgery, given that this is not a recommendation of the ASMBS, we felt it best to follow their guidelines at this time. https://asmbs.org/app/uploads/2016/03/SOARD-AlcoholStatement_Published_Feb2016.pdf 7. “Discussion: Given the finding of frequency of follow-up being associated with weight loss outcomes, please consider discussing how the health care system and health care providers can address the need for ongoing follow-up of bariatric surgery patients in a resource constrained environment of a publicly funded Canadian health care system? Why should be following these patients? How long and how often?” We agree that this is an important discussion to have. However, given that our scoping review provides context not only in Canada but internationally, we feel it best not to acknowledge the public Canadian healthcare system, which differs from the US, Brazil, Europe, Australia, and Middle Eastern contexts. In addition, given that the provincial health ministries in Canada regulate their own healthcare systems, there are differences in practices, staffing, and follow-up availability across provinces. These differences are even seen within bariatric centers in the same provinces and even in the same cities. Therefore, we feel that keeping an open context to our scoping review may be of more interest to readers on an international level and are relevant across the globe, such that the references used in this scoping review were influenced by international studies." } ] } ]	1	https://f1000research.com/articles/8-615
https://f1000research.com/articles/9-633/v2	08 Jul 20	{ "type": "Systematic Review", "title": "Effects of vitamin D supplementation on 25(OH)D levels and blood pressure in the elderly: a systematic review and meta-analysis", "authors": [ "Farapti Farapti", "Chusnul Fadilla", "Niwanda Yogiswara", "Merryana Adriani", "Chusnul Fadilla", "Niwanda Yogiswara", "Merryana Adriani" ], "abstract": "Background: Hypertension and vitamin D deficiency are prevalent among the elderly. This study evaluated the effects of vitamin D supplementation on changes in serum 25-hydroxyvitamin D (25(OH)D) levels and blood pressure (BP) in the elderly (age > 60 years). Methods: Randomized controlled trials from electronic databases on the elderly taking oral vitamin D, until the end of March 2019, were selected. Two reviewers independently screened the literature on the basis of specific inclusion criteria. The primary outcomes were serum 25(OH)D level, systolic BP (SBP), and diastolic BP (DBP) changes. Results: Our analysis revealed significant differences in serum 25(OH)D level changes between the vitamin D and control groups (mean difference [MD] = 13.84; 95% confidence interval [CI] = 10.21–17.47; P < 0.000). There were no significant differences in SBP and DBP changes between the vitamin D and control groups. Subgroup analysis revealed significant differences in SBP changes between the hypertensive and vitamin D-deficient subgroups (MD = –4.01; 95% CI = –7.45 to –0.57; P = 0.02 and MD = –1.91; 95% CI = –3.48 to –0.34; P = 0.02, respectively), and DBP changes only in the hypertensive subgroup (MD = –2.22; 95% CI = –4.1 to –0.34; P = 0.02). Conclusions: Vitamin D supplementation significantly increases 25(OH)D levels and seems beneficial in lowering BP, specifically in the elderly with elevated BP and vitamin D deficiency.", "keywords": [ "vitamin D", "blood pressure", "elderly", "25(OH)D) levels" ], "content": "Introduction\n\nHigh blood pressure (BP), or hypertension, is still regarded as one of the most influential factors for cardiovascular diseases, especially in the elderly. An increasingly aging population and the increasing prevalence of hypertension emphasize the importance of proper treatment of hypertension. Nutrient supplementation is an alternative treatment since the elderly have multiple chronic diseases and take multiple drugs1,2. Vitamin D is one kind of steroid hormone and micronutrient synthesized in the skin by exposure to ultraviolet B rays and also obtained through dietary intake or supplementation3. Most vitamin D is distributed in the human body in the form of serum 25(OH)D4.\n\nVitamin D deficiency has become an important public health concern because it could take place at any age, and most countries report deficiency as high in the elderly3,5. Vitamin D has an essential part in metabolism regulation and has a significant role in the pathogenesis of hypertension4. However, the result of meta-analyses has revealed that the relationship between serum 25(OH)D levels and a decrease in BP is inconsistent. Qi et al. (2017) reported that low serum 25(OH)D levels are not significantly associated with a risk of hypertension6. In contrast, other studies demonstrated a significant relationship between low serum 25(OH)D levels and hypertension3,7. Another meta-analysis also proved that the serum level of 25(OH)D was significantly associated with the risk of incident hypertension on the general population3.\n\nThe elderly is an age group susceptible to deficiency of this fat-soluble vitamin. Skin aging reduces 7-dehydrocholesterol production to 75%, which is known to play a key role as the main source of vitamin D in the human body8. The impaired eating ability in the elderly may also contribute to low levels of vitamin D. Therefore, vitamin D deficiency is often associated with various geriatric syndromes. Low levels of vitamin D affects the activity of endocrine hormones as in sufferers of diabetes mellitus type 2 (T2DM) and cardiovascular functions, such as coronary artery disease, heart failure, stroke, and hypertension9. A recent meta-analysis in individuals with vitamin D deficiency showed oral vitamin D3 reduces both systolic BP (SBP) and diastolic BP (DBP) in individuals with hypertension and decreases SBP in individuals above 50 years. In contrast, another study has revealed that in younger women, there is a strong association between high serum 25(OH)D levels and the risk of hypertension3. Since the elderly, defined as individuals of more than 60 years of age, have a high risk of vitamin D deficiency and suffer hypertension1,2,10, it is important to provide a meta-analysis of randomized controlled trials gathering the evidence of the effects of vitamin D supplementation compared to placebo on serum 25(OH)D levels and BP, specifically in the elderly population.\n\n\nMethods\n\nA comprehensive search was performed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement11. All authors searched independently correlated studies in multiple electronic database including, PubMed, ClinicalTrials.gov, and the Cochrane Library from inception until 29th March 2019 using a combination of keywords and subject headings. One of our search strategies used on PubMed using the following keywords: (vitamin D) AND ((blood pressure) OR (hypertension)) AND (elderly). Further relevant articles were then obtained using manually searching the references of retrieved articles.\n\nAll titles and abstracts were screened using Mendeley reference software, and duplications were removed manually. Full text of relevant articles were examined for eligibility criteria. The inclusion criteria were as follows: randomized controlled trial (RCT) design; participants with average age > 60 years; primary outcomes SBP change, DBP change, and serum 25(OH)D level change; and only vitamin D (cholecalciferol) intervention. The exclusion criteria were as follows: nonrandomized study; assessment of the full text of the study not possible; the study control group was not placebo; outcomes relevant to our interest not reported; intervention with combined vitamin D and other nutrients supplementation; and non-English full text.\n\nData from each included article were extracted by three investigators (F.F., C.F., N.Y.) by utilizing a piloted form. If there is any disagreement between the authors, the final decision was made by discussion and majority vote. The following data were extracted: year of publication, year of study, geographic location, sample size, health status of participants, mean age, intervention dose, duration of the study, mean and standard deviation (SD) of serum 25(OH)D levels, SBP, and DBP in both intervention and placebo groups at the baseline and at the end of study, and changes from the baseline.\n\nEach RCT’s quality was evaluated using the risk of bias tools developed by Cochrane collaboration evaluating six domains. We assessed the selection bias by evaluating the study description on the method of the randomization, the method of allocation concealment, and evaluated if there is difference in baseline between the two groups. Performance and detection bias were evaluated by finding a description about the blinding method. Attrition bias was assessed by calculating the number of participants that withdrew from the study. Reporting bias and other bias were then evaluated if there found any concern not addressed in the other domain12.\n\nThe continuous data were presented as mean difference (MD) and SD. Where the change in mean (Δ Mean) was not available, we calculate the change by subtracting post intervention outcome with the baseline data. When a study did not report enough information of the change on SD (ΔSD), we calculated the data imputation applying the formula for imputing SD from baseline13:\n\ncorr = (SDbaseline2 + SDpost2 - SDchange2)/(2 × SDbaseline × SDpost)\n\nΔSD was then calculated as:\n\n\n\nTo calculate the estimated effect size on MD, we used random-effect model if there was heterogeneity found using X2 test and I2 test14. p value of <0.10 dan I2 > 50% were considered high. Otherwise, the fixed-effects Mantel–Haenszel model was used. We performed univariate meta-regression analyses to evaluate differences in the continuous outcome variable. Analyses of subgroups were conducted to assess predefined sources of heterogeneity. Dose of supplementation, duration of the study, treatment regimen, hypertension, and vitamin D status were considered as sources of heterogeneity. We assessed publication bias by visual assessment on graphical funnel plots with Egger’s regression test of asymmetry15.\n\nAll statistical analyses were performed using STATA 16.0 (STATA Corporation). P value < 0.05 was considered statistically significant.\n\n\nResults\n\nFigure 1 presents the flowchart of this study. We screened 980 articles. Of those, 28 were excluded because of duplicate publication, and 42 articles were assessed for eligibility criteria. Of those, 30 were not eligible to be included. Finally, 12 RCTs16–27 were included in the quantitative synthesis. The quality assessment demonstrated that almost all of the included studies has a low risk of bias. The results for quality assessment was summarized in Extended data: Figure S128.\n\nPRISMA, Preferred Reporting Items for Systematic Reviews and Meta-analyses.\n\nTable 1 summarizes the characteristics of the included RCTs. The RCTs were conducted in different continents: Asia23, Europe19–22,25–27, America17, and Oceania18. All were placebo controlled and published in English. The mean age of the participants was 65.5 years with differing health conditions. Only three RCTs included participants without certain medical criteria but with some conditions that indicated vitamin D deficiency, including postmenopausal women16,17 and those taking vitamin D supplements < 400 IU19. In addition, several RCTs targeted conditions related to blood sugar levels, such as T2DM21,26 and prediabetes24. Hypertension patients were also the subjects of several RCTs, which focused on isolated systole hypertension (ISH)22, arterial hypertension20,25, and essential hypertension23.\n\nHT, hypertension; 25(OH)D, 25-hydroxyvitamin D; BP, blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; ∆SBP/DBP, changes of SBP/DBP; wk, weeks; ISH, isolated systole hypertension; T2DM; type 2 diabetes mellitus; *, significant\n\nThe type of hypertension affected the baseline BP of the participants. The BP varied; some participants had hypertension, whereas others had normal BP. SBP in all participants ranged from 109.2 to 174 mmHg, whereas DBP ranged from 64.8 to 95 mmHg. High SBP usually occurred in ISH and was most commonly found in the elderly. However, on average, in each RCT, the participant’s BP was categorized as prehypertension or hypertension (>120/80 mmHg). Hypertension and diabetes experienced by the elderly made it difficult for the participants to be excluded on the basis of medications. Therefore, some of the RCTs had inclusion criteria that required participants not to change their medical treatment throughout the study duration20,26,27, because the use of different drugs affects vitamin D intervention. In their study at one of the general hospitals in Beijing, China, Chen et al. (2014) included participants who were taking 30 mg/dL of nifedipine23. Another factor that may influence the effectiveness of vitamin D supplementation was baseline 25(OH)D levels before the intervention. Some of the RCTs had set serum 25(OH)D limits to <2026, 3022,25,27, 4022, or 60 ng/mL17; all these values indicate deficiency in vitamin D.\n\nTable 1 shows that most participants revealed baseline data of mean 25(OH)D levels <20 ng/mL, and the maximum was 30 ng/mL17. Eight RCTs evaluated changes of 25(OH)D levels after giving vitamin D intervention. The 1293 participants were divided into treatment (n = 641) and control groups (n = 652). Pooling data revealed that the vitamin D group had a significant higher serum 25(OH)D levels compared to the control group (MD = 13.84; 95% CI = 10.21–17.47; P < 0.0001). We observed heterogeneity among the RCTs (I2 = 93%), so we selected a random-effects model (Figure 2).\n\nThe overall effect size estimate is represented by the red dashed line. SD, standard deviation; CI, confidence interval.\n\nThe change of SBP and DBP was synthetized from 12 RCTS. We did not observe heterogeneity among the RCTs (I2 < 50%), so we selected a fixed-effects Mantel–Haenszel model. Pooled analysis revealed that overall, the SBP change (MD = –0.83; 95% CI = –1.88 to 0.23; P = 0.12) and DBP changes (MD = 0.40; 95% CI = –1.00–0.19; P = 0.18) in vitamin D group were not significant compared with the control group. The effects of vitamin D on SBP and DBP were summarized as forest plots presented in Figure 3 and Figure 4. The funnel plots of SBP change and DBP change are summarized in Figure 5. Our analysis showed that there was no publication bias when examining funnel plots and the result of Egger’s test of asymmetry for SBP change and DBP change (p = 0.158; p= 0.069; respectively).\n\nThe overall effect size estimate is represented by the red dashed line. CI, confidence interval; SD, standard deviation.\n\nThe overall effect size estimate is represented by the red dashed line. CI, confidence interval; SD, standard deviation.\n\nFunnel plot assessing publication bias for the effect of (a) systolic blood pressure (BP) change and (b) diastolic BP change.\n\nTable 2 and Table 3 present the pooled estimated effect size of vitamin D on the change of SBP and DBP, on the basis of BP baseline, vitamin D status baseline, intervention dose, treatment duration, and treatment regimen. Our analysis indicate that vitamin D supplementation had no significant influence on SBP and DBP changes on the basis of dose, duration, and treatment regimen. However, subgroup analysis revealed a marginal trend toward significance in terms of DBP changes with treatment duration ≤ 6 months (MD = –0.82; 95% CI = –1.66 to –0.02; P = 0.05). Subgroup analysis by hypertensive and deficiency of vitamin D status indicated that vitamin D supplementation could significantly reduce SBP (MD = –4.01; 95% CI = –7.45 to –0.57; P = 0.02 and MD = –1.91; 95% CI = –3.48 to –0.34; P = 0.02, respectively). However, we found a significant difference in DBP changes only in the hypertensive subgroup (MD = –2.22; 95% CI = –4.1 to –0.34; P = 0.02) (Table 2 and Table 3). The forest plot of each subgroup analysis is available as Extended data: Figure S228. Our study provided enough observations to conduct univariate meta-regression, summarized in Table 2 and Table 3. The result for SBP change was presented as bubble plots in Figure 6.\n\nSBP, systolic blood pressure; BP, blood pressure; MD, mean difference; CI, confidence interval.\n\nDBP, diastolic blood pressure; BP, blood pressure; MD, mean difference; CI, confidence interval.\n\n(a) Participant blood pressure baseline and the mean difference (MD) in systolic blood pressure (SBP) change; (b) Participant 25(OH)D baseline and the MD in SBP change. Each circle characterizes a study and the size of the circle reflects the influence of that study on the model. The regression prediction is represented by the solid line.\n\n\nDiscussion\n\nSerum 25(OH)D levels has a major role as a marker for determining vitamin D status in humans. As mentioned before, most vitamin D circulates in human body in the form of 25(OH)D. This is a result of vitamin D metabolism from the skin and vitamin D intake and binds to vitamin D-binding protein, which has a half-life of 2–3 weeks. The clinical practice guidelines issued by the Institute of Endocrinology has defined vitamin D deficiency as levels of 25(OH)D below 20 ng/mL29. In addition, the average normal value for serum 25(OH)D levels for all ages is 30 ng/mL, whereas in the elderly it is >20 ng/mL or 50 nmol/L30.\n\nDeficiency in vitamin D could be caused by physiological and pathological factors in the elderly. One of the most common physiological factors is decreasing pre-vitamin D production in the skin. The reasons are that compared with young adults, the skin’s capacity to produce vitamin D decreases by 75% at 70 years29,31. In addition, the elderly has a tendency to wear closed clothing for fear of flu, thus causing minimal exposure to ultraviolet B rays9. Their food intake decreases because of a decrease in chewing ability and financial conditions30. Also, decreased calcium absorption results in impaired vitamin D metabolism and decreased kidney function29–34. Pathological factors are related to organs that play a role in the digestion and metabolism of vitamin D. Decreased bioavailability in the digestive tract (malabsorption due to disease) inhibits vitamin D metabolism. Patients with liver disease can suffer from vitamin D hydroxylation disorders. Kidney pathologies, such as nephrotic syndrome and chronic kidney disease, increase vitamin D activation disorders34. However, the 12 RCTs included in this study excluded participants with pathological conditions that might cause vitamin D deficiency.\n\nOne of the main findings of the present meta-analysis was vitamin D supplementation has significant effect on serum 25(OH)D levels among the elderly. It increases serum 25(OH)D levels in people that are older than 60 years old. Almost all studies included have revealed a significant increase in serum 25(OH)D levels from the baseline. The contradictory result was shown by Witham et al. (2010) that inconsistent with a previous study by Sugden et al. (2008), which has revealed a significant difference between the treatment and control groups with same doses and duration24. Another study has reported increasing serum 25(OH)D levels at follow-up in the vitamin D group, with no change in the placebo group18. The relationship between serum 25(OH)D levels and a decrease in BP is still debatable. A meta-analysis of observational cross-sectional and prospective studies on general populations has proven the relationship between serum 25(OH)D levels and the risk of incident hypertension3. However, a newer meta-analysis showed oral vitamin D3 has no significant effect on blood pressure in individuals with vitamin D deficiency5. To the best our knowledge, this is the first study that analyses the effects of vitamin D on serum 25(OH)D levels, the gold standard to measure vitamin D status in humans.\n\nThis study included research from four different continents. However, characteristically there are no specific differences for each continent. The results were random and more relevant to the baseline data and effect of the RCT itself. The present study provides evidence that although the supplementation could increase serum 25(OH)D levels, there was no significant difference in SBP and DBP changes compared with the control group. It means that the increasing serum 25(OH)D levels were not followed by decreasing BP among elderly. Several studies have revealed not only a relationship between an increase in serum 25(OH)D levels and a decrease in BP after vitamin D supplementation but also a significant change in other conditions, such as parathyroid hormone, serum calcium, renin, and angiotensin II levels16,20,23, indicating that vitamin D regulates a decrease in BP through various mechanisms. The effect of vitamin D supplementation on a decrease in BP is inconsistent in several studies. Some studies have reported that vitamin D supplementation can reduce BP, although only SBP, so vitamin D supplementation can be a supportive therapy for hypertension8,21,26. Similar to our result, a meta-analysis by Golzarand et al. has revealed that vitamin D supplementation is only associated with an increase in serum 25(OH)D levels, not SBP or DBP35. Meanwhile, according to Chen et al. (2014), vitamin D supplementation that complements 30 mg/dL nifedipine in patients with grade I or II essential hypertension can reduce SBP or DBP23. They were the first to look at the effectiveness of the interaction between vitamin D and specific drugs in contrast to other studies that only provide inclusion criteria in the form of no change in medication consumption.\n\nSeveral meta-analyses have been conducted to evaluate the association between vitamin D and BP. The findings of the meta-analysis of observational studies on general populations have proven the association between vitamin D status and the risk of incident hypertension3,7,8. However, the association between vitamin D and hypertension is not causal, so placebo-controlled RCTs are required in order to prove the effects of vitamin D on BP. Previous RCTs and meta-analyses have revealed that vitamin D might be beneficial in lowering BP, especially in vitamin D-deficient patients with hypertension, which is similar to our results4,18. In contrast to our findings, Ke et al. (2015) reported no increased risk of hypertension in the elderly or in vitamin D-deficient participants; however, their research involved a prospective study design3. The newest meta-analysis has revealed that vitamin D has no significant effect on BP in vitamin D-deficient people; it reduces SBP in vitamin D-deficient people older than 50 years and in people with both vitamin D deficiency and hypertension5. However, a previous meta-analysis involved subjects between 18 and 74 years old and serum 25-OHD are lower than 20ng/mL, and subgroup analysis used criteria older than 50 years old. Meanwhile, our study involved subjects with the mean age more than 60 years, according to WHO definition for elderly10.\n\nThe other important finding in our study was significant differences in SBP and DBP changes among the hypertensive subgroup. Previous meta-analyses have also revealed a significant effect of vitamin D on BP in patients with hypertension at the baseline and no significant decrease in normotensive patients at the baseline5,36. In contrast to our findings, a meta-analysis by Golzarand et al. (2016) showed that vitamin D showed hypotensive effects in both healthy and hypertensive subjects35. To the best of our knowledge, there has been no research that suggests that there are differences in the metabolism of vitamin D in the elderly with hypertension and norm tension, except secondary hypertension associated with kidney organs37. Decreased BP also occurs in arterial hypertension patients who have vitamin D deficiency. Patients with deficiency in vitamin D may acquire the effects of vitamin D supplementation20. However, administering vitamin D to participants who meet the same criteria can give zero results with regard to a decrease in BP because of the shorter time of administration (only 8 weeks with almost the same dose)25. In addition, an updated meta-analysis’s results were similar to our study in that subgroup analysis showed vitamin D supplementation may reduce SBP and DBP in patients with low vitamin D status and hypertension5. Searching articles until the end of March 2019, our study provides the most up-to-date meta-analysis and strongly supports that vitamin D supplementation significantly decreases BP in the elderly, specifically with elevated BP and deficiency in vitamin D.\n\nIn addition to hypertension patients, T2DM patients also exhibit a decrease in BP, although only SBP, after a high dose of vitamin D supplementation21,26. Again, serum 25(OH)D levels also contributed to the effects of vitamin D supplementation on lowering BP. Pre diabetic patients reveal absolutely no effect of the same dose of vitamin D on BP because they are not vitamin D deficient24. Most of the RCTs included in this study reveal an insignificant decrease in BP after given a dose supplementation of vitamin D. In the case of ISH, which is common among the elderly, vitamin D supplementation is less effective. The reason is probably because vitamin D cannot decrease blood vessel stiffness and is effective only during the early stages of the disease. Hypertension in the elderly does not increase renin levels22. Other studies have revealed no effect of vitamin D supplementation on lowering BP in postmenopausal women17, chronic peripheral arterial disease patients20, and the elderly without certain medical conditions16,18,19. The actual conditions suffered by the elderly due to multiple chronic diseases and therefore multiple drugs should be considered as an important aspect that may influence the effects of vitamin D on BP.\n\nThis study had a few limitations. First, although we conducted a systematic review of peer-reviewed research, we did not include agency reports, dissertations, and conference proceedings. Second, we included only English-language RCTs. Third, the RCTs were heterogeneous with respect to demographic characteristics of the participants, the duration, and treatment dose.\n\n\nConclusions\n\nVitamin D deficiency is prevalent among the elderly, and vitamin D supplementation significantly increases serum 25(OH)D levels. The use of vitamin D supplementation appears to be beneficial in lowering BP, specifically in the elderly with hypertension and vitamin D deficiency. We recommend vitamin D supplementation for elderly individuals with hypertension and serum 25(OH) D levels below the target values. The actual conditions suffered by the elderly because of multiple chronic diseases and therefore multiple drugs should be considered as important factors that influence the effects of vitamin D on BP. The homogenous duration, dose intervention, and treatment regimen need to be investigated in future studies in order to determine the proper treatment for hypertension in the elderly.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: Extended data for “Effects of vitamin D supplementation on 25(OH)D levels and blood pressure in the elderly: a systematic review and meta-analysis.” http://doi.org/10.17605/OSF.IO/EXF2628.\n\nThis project contains the following extended data:\n\nSpreadsheets in .sav format containing data for supplementation efficacy outcomes in 25(OH) levels, systolic blood pressure, and diastolic blood pressure.\n\nSupplementary figure in .doc format containing: Figure S1: results for quality assessment, Figure S2: forest plot of each subgroup analysis, Figure S3: Funnel plot and egger test results.\n\nOpen Science Framework: Extended data for “Effects of vitamin D supplementation on 25(OH)D levels and blood pressure in the elderly: a systematic review and meta-analysis.” http://doi.org/10.17605/OSF.IO/EXF2628.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nRobles N, Macias J: Hypertension in the Elderly. Cardiovasc Hematol Agents Med Chem. 2015; 12(3): 136–45. PubMed Abstract \| Publisher Full Text\n\nHandschin A, Henny-Fullin K, Buess D, et al.: [Hypertension in the elderly]. Ther Umsch. 2015; 72(6): 397–403. PubMed Abstract \| Publisher Full Text\n\nKe L, Mason RS, Kariuki M, et al.: Vitamin D status and hypertension: A review. Integr Blood Press Control. Dove Medical Press Ltd.; 2015; 8: 13–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPilz S, Tomaschitz A, Ritz E, et al.: Vitamin D status and arterial hypertension: A systematic review. Nat Rev Cardiol. 2009; 6(10): 621–30. PubMed Abstract \| Publisher Full Text\n\nHe S, Hao X: The effect of vitamin D3 on blood pressure in people with vitamin D deficiency: A system review and meta-analysis. Medicine (Baltimore). 2019; 98(19): e15284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQi D, Nie X, Cai J: The effect of vitamin D supplementation on hypertension in non-CKD populations: A systemic review and meta-analysis. Int J Cardiol. Elsevier Ireland Ltd; 2017; 227: 177–86. PubMed Abstract \| Publisher Full Text\n\nKunutsor SK, Apekey TA, Steur M: Vitamin D and risk of future hypertension: Meta-analysis of 283,537 participants. Eur J Epidemiol. 2013; 28(3): 205–21. PubMed Abstract \| Publisher Full Text\n\nQi D, Nie X, Wu S, et al.: Vitamin D and hypertension: Prospective study and meta-analysis. Slominski AT, editor. PLoS One. 2017; 12(3): e0174298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKweder H, Eidi H: Vitamin D deficiency in elderly: Risk factors and drugs impact on vitamin D status. Avicenna J Med. 2018; 8(4): 139–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgeing and health. Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Altman DG, Gøtzsche PC, et al.: The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ. 2011; 343(7829): d5928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBook Series C, Higgins JP, Green S: Cochrane Handbook for Systematic Reviews of Interventions. THE COCHRANE COLLABORATION ®. 2008. Reference Source\n\nHiggins JPT, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med. 2002; 21(11): 1539–58. PubMed Abstract \| Publisher Full Text\n\nEgger M, Smith GD, Schneider M, et al.: Bias in Meta-Analysis Detected by a Simple, Graphical Test. BMJ. 1997; 315(7109): 629–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWood AD, Secombes KR, Thies F, et al.: Vitamin D3 supplementation has no effect on conventional cardiovascular risk factors: A parallel-group, double-blind, placebo-controlled RCT. J Clin Endocrinol Metab. 2012; 97(10): 3557–67. PubMed Abstract \| Publisher Full Text\n\nGepner AD, Ramamurthy R, Krueger DC, et al.: A prospective randomized controlled trial of the effects of Vitamin D supplementation on cardiovascular disease risk. PLoS One. 2012; 7(5): e36617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSluyter JD, Camargo CA, Stewart AW, et al.: Effect of monthly, high-dose, long-term vitamin D supplementation on central blood pressure parameters: A randomized controlled trial substudy. J Am Heart Assoc. 2017; 6(10): e006802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomson J, Hin H, Emberson J, et al.: Effects of Vitamin D on Blood Pressure, Arterial Stiffness, and Cardiac Function in Older People After 1 Year: BEST-D (Biochemical Efficacy and Safety Trial of Vitamin D). J Am Heart Assoc. 2017; 6(10): e005707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarsen T, Mose FH, Bech JN, et al.: Effect of cholecalciferol supplementation during winter months in patients with hypertension: A randomized, placebo-controlled trial. Am J Hypertens. 2012; 25(11): 1215–22. PubMed Abstract \| Publisher Full Text\n\nWitham MD, Dove FJ, Dryburgh M, et al.: The effect of different doses of vitamin D3 on markers of vascular health in patients with type 2 diabetes: A randomised controlled trial. Diabetologia. 2010; 53(10): 2112–9. PubMed Abstract \| Publisher Full Text\n\nWitham MD, Price RJG, Struthers AD, et al.: Cholecalciferol treatment to reduce blood pressure in older patients with isolated systolic hypertension the VitDISH randomized controlled trial. JAMA Intern Med. 2013; 173(18): 1672–9. PubMed Abstract \| Publisher Full Text\n\nChen WR, Liu ZY, Shi Y, et al.: Vitamin D and nifedipine in the treatment of Chinese patients with grades I-II essential hypertension: A randomized placebo-controlled trial. Atherosclerosis. 2014; 235(1): 102–9. PubMed Abstract \| Publisher Full Text\n\nSollid ST, Hutchinson MYS, Fuskevåg OM, et al.: No effect of high-dose vitamin D supplementation on glycemic status or cardiovascular risk factors in subjects with prediabetes. Diabetes Care. 2014; 37(8): 2123–31. PubMed Abstract \| Publisher Full Text\n\nPilz S, Gaksch M, Kienreich K, et al.: Effects of Vitamin D on Blood Pressure and Cardiovascular Risk Factors: A Randomized Controlled Trial. Hypertension. 2015; 65(6): 1195–201. PubMed Abstract \| Publisher Full Text\n\nSugden JA, Davies JI, Witham MD, et al.: Vitamin D improves endothelial function in patients with Type 2 diabetes mellitus and low vitamin D levels. Diabet Med. 2008; 25(3): 320–5. PubMed Abstract \| Publisher Full Text\n\nStricker H, Tosi Bianda F, Guidicelli-Nicolosi S, et al.: Effect of a single, oral, high-dose vitamin D supplementation on endothelial function in patients with peripheral arterial disease: A randomised controlled pilot study. Eur J Vasc Endovasc Surg. 2012; 44(3): 307–12. PubMed Abstract \| Publisher Full Text\n\nYogiswara N, Farapti F, Fadilla C: Extended data for: “Effects of vitamin D supplementation on 25 (OH)D levels and blood pressure in the elderly: a systematic review and meta-analysis”. 2020. http://www.doi.org/10.17605/OSF.IO/NWUCA\n\nHolick MF, Binkley NC, Bischoff-Ferrari HA, et al.: Evaluation, treatment, and prevention of vitamin D deficiency: An endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96(7): 1911–30. PubMed Abstract \| Publisher Full Text\n\nBoucher BJ: The problems of vitamin D insufficiency in older people. Aging Dis. 2012; 3(4): 313–29. PubMed Abstract \| Free Full Text\n\nZittermann A: Vitamin D and disease prevention with special reference to cardiovascular disease. Prog Biophys Mol Biol. Elsevier Ltd; 2006; 92(1): 39–48. PubMed Abstract \| Publisher Full Text\n\nVera V, Setiati S, Roosheroe AG: Determinan Diagnostik Klinis Defisiensi Vitamin D pada Wanita Berusia Lebih dari 50 Tahun. J Penyakit Dalam Indones. 2015; 2(1): 38. Publisher Full Text\n\nRussell RM: Factors in aging that effect the bioavailability of nutrients. J Nutr. 2001; 131(4 Suppl): 1359S–61S. PubMed Abstract \| Publisher Full Text\n\nGallagher JC: Vitamin D and Aging. Endocrinol Metab Clin North Am. 2013; 42(2): 319–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGolzarand M, Shab-Bidar S, Koochakpoor G, et al.: Effect of vitamin D3 supplementation on blood pressure in adults: An updated meta-analysis. Nutr Metab Cardiovasc Dis. 2016; 26(8): 663–73. PubMed Abstract \| Publisher Full Text\n\nWitham MD, Nadir MA, Struthers AD: Effect of vitamin D on blood pressure: A systematic review and meta-analysis. J Hypertens. 2009; 27(10): 1948–54. PubMed Abstract \| Publisher Full Text\n\nVaidya A, Williams JS: The relationship between vitamin D and the renin-angiotensin system in the pathophysiology of hypertension, kidney disease, and diabetes. Metabolism. 2012; 61(4): 450–8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68253", "date": "05 Aug 2020", "name": "Alexandre S. Silva", "expertise": [ "Reviewer Expertise Nutrition", "exercise and Hypertension", "Nutrition", "exercise and weight loss", "Fresh food as ergogenic aid for athletes", "nut" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a review with a well-structured meta-analysis, following Cochrane principles. Compared to other previous meta-analyzes, the present review differs in that it performed the analysis in the elderly population. Although it has been noticed that this manuscript has already been revised, I still see some small issues that can be improved, especially to ensure the replicability of the methodology. This and other questions are presented below.\n\nIntroduction:\nSecond paragraph: I suggest the authors to present the biological rationale of hypovitaminosis D with arterial hypertension (before presenting the results of some meta-analyzes).\n\nMethods:\nThe authors presented the PubMed search strategy only. I suggest presenting the other strategies, so that the methodology is replicable.\n\nOne of the exclusion criteria was: \"assessment of the full text of the study not possible\". This actually happened? I suggest that the authors identify these studies and present the difficulties to access this study (s). Important for the replication of the methodology.\n\nDiscussion:\nI missed the clinical implications of this study. Do data provide evidence for the use of vitamin D for hypertensive elderly people? Although significant, does the observed reduction in blood pressure really have clinical relevance compared to other interventions?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5894", "date": "04 Sep 2020", "name": "Farapti Farapti", "role": "Author Response", "response": "We thank the editors for facilitating the peer-review process and we appreciate Professor Silva's positive opening remarks and his expert review and suggestions for improving the paper. We have uploaded a revised copy of the manuscript. The specific recommendations for improvement are four:First, the Reviewer suggested that the paper should “present the biological rationale of hypovitaminosis D with arterial hypertension before presenting the results of some meta-analyzes”. In the revised paper, we have now added the sentence to present the biological rationale of hypovitaminosis D with arterial hypertension in the introduction paragraph two. Second, the Reviewer suggested that the paper should present the strategy used in other databases. In our revision, we have added the information regarding other strategies used in Cochrane Library and Clinicaltrial.gov so that the methodology is replicable.Third, \"One of the exclusion criteria was: \"assessment of the full text of the study not possible\". This actually happened? I suggest that the authors identify these studies and present the difficulties to access this study (s). Important for the replication of the methodology\". We are sorry that we used a phrase that leads to sentence misconstruction. The sentence \"assessment of the full text of the study not possible\" should be written by \"no full text available.\" Fourth, \"I missed the clinical implications of this study. Do data provide evidence for the use of vitamin D for hypertensive elderly people? Although significant, does the observed reduction in blood pressure really have clinical relevance compared to other interventions?\"This is an important question and we have addressed it directly in the revision. In the revised paper, we now incorporate the study's clinical implication and cite the relevant papers regarding clinical relevance compared to other intervention in the discussion section. We thank Prof. Silva for calling our attention to these ideas.Finally, we thank Professor Silva for raising these points which we feel have improved the paper considerably.Farapti, et al." } ] }, { "id": "69387", "date": "18 Aug 2020", "name": "Barbara J. Boucher", "expertise": [ "Reviewer Expertise diabetes", "metabolism", "vitamin d and betel quid chewing." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review and meta-analysis of previous studies on the effects of vitamin D supplementation on vitamin D status and on blood pressure in the elderly is a topic of importance for population health since hypertension and vitamin D deficiency are both so common in elderly people globally, including isolated systolic hypertension. This manuscript has already been assessed by earlier peer review as being appropriate in design and analytical methodology. The present comments, therefore, are mainly directed at presentation, readability and the additional information requested by the previous reviewers. It is unfortunate that only 8 of the many RCTs considered for this study were suitable for meta-analysis. The use of Individual Participant Data, if obtainable, could be more revealing on the question of whether supplementation is more effective for reducing BP in older people in deficiency than in repletion, as suspected in other health problems and as the present data suggests. This would be a valuable follow-on study if it were to be possible.\nGeneral comments:\nThe English language usage is often awkward, which distracts from the readability of the text. Simple editing by a native English language speaker with a scientific/medical background would greatly improve the readability of the text throughout.\n\nVitamin D was not measured in any studies analysed, but 25-hydroxyvitamin D [25OHD], and this term should be used throughout. Lab data reports ‘concentrations’ not ‘levels’ and this term should be used while ‘levels’ is good for cut-offs and definitions.\n\nSpecific comments:\nTable 2, the heading ‘vitamin D’ would be clearer saying ‘baseline vitamin D status’.\n\nPage 10, column 1, line 2, do you mean decreases by 75% or decreases by 25% - please clarify; line 3, ..have..; 2nd column, line 6, do you mean ‘…reduce renal activation, [though not that in other target tissues], but such problems were amongst the exclusion criteria’?\n\nPage 10, 2nd column, para 2, the last sentence reads as if this is the 1st study to assess the effects of supplementation on serum 25OHD values but this cannot be true either for this submission or for the ‘newer’ meta-analysis mentioned in the previous sentence that needs a reference. These comments should be corrected - maybe you mean ‘...in elderly people for both vitamin D status and effects on blood pressure’?\n\nPage 11, 1st column, line 15, 'adjunctive' might be a better word than 'supportive'; Para 2, line 4, ‘....have confirmed the association’ might be better than proven & ‘ ….however associations are not proof of causality …..; column 2, 1st para, lines 6/7 are obscure, this section reads as if deficiency lowers blood pressure, which is not what is meant - maybe say, re. ref 24, ‘ ...because they were not vitamin D deficient’. Re ref 22 comment, it would be better to say that circulating renin concentrations are not raised in the elderly and not the other way around as renin raises the BP and is suppressed by vitamin D, [if you are sure that this is a general finding]. Para 3, do you mean that supplemental doses were heterogeneous or that treatments for hypertension were heterogeneous, or, as I suspect, that both of these were heterogeneous? Please clarify.\n\nPage 12, para 1, is unclear, if you mean that studies homogeneous for duration, etc, need to be carried out, please rephrase for clarity, perhaps concluding, ‘...so as to identify optimal treatment regimes for hypertension in the elderly that may need to include correction of vitamin D deficiency’?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "5899", "date": "07 Sep 2020", "name": "Niwanda Yogiswara", "role": "Author Response", "response": "We appreciate Professor Barbara Joan Boucher for her time, constructive comments, and for recommending approval of our paper. The major recommendations for improvement are two:First, \"The English language usage is often awkward, which distracts from the readability of the text. Simple editing by a native English language speaker with a scientific/medical background would greatly improve the readability of the text throughout.\" We have tried to revise some sentence misconstruction and also improve the English language. Every specific comment and suggestion indicated by the Reviewer has been precisely considered, and we hope satisfactorily addressed. Second, the Reviewer points out that the paper should use the term 'concentrations' not 'levels'. This is an important suggestion since lab data reports 'concentrations' not 'levels', we have replaced the term 'levels' with 'concentrations', except for cut-off and definitions.Again, we thank Prof. Boucher for giving her valuable insight with very detailed comments, which we feel have significantly improved paper readability." } ] } ]	2	https://f1000research.com/articles/9-633
https://f1000research.com/articles/9-1086/v1	02 Sep 20	{ "type": "Study Protocol", "title": "Classifying colorectal cancer or colorectal polyps in endoscopic setting using convolutional neural network: protocol for a systematic review and meta-analysis", "authors": [ "Kamyab Keshtkar", "Abbas Keshtkar", "Alireza Safarpour", "Abbas Keshtkar", "Alireza Safarpour" ], "abstract": "Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Although colonoscopy screening has been proven as an effective strategy for preventing CRC unfortunately, even conventional colonoscopy by expert gastroenterologists can miss adenomas or pre-cancerous lesions in up to 25% of cases. This systematic review aimed to classify colorectal polyps (CRP) or CRC in endoscopic clinic settings using a new machine learning method, convolutional neural network (CNN).\n\nMethods: We will search PubMed/MEDLINE, Scopus, Web of Science, IEEE, Inspec, ProQuest, Google Scholar, Microsoft Academic Search, ScienceOpen, arXiv, and bioRxiv from 1st January 2010 to the 31th of July 2020. Our search will not be restricted based on language or geographical area. The primary studies will be selected that have observational design (cross-sectional, case control or cohort); the study subjects will be adult patients (>= 18 years old) referred to colonoscopy clinics; and the results of their colonoscopy evaluation will be available in the form of images or videos. The extracted data will be combined using meta-analysis of prediction models. The primary data synthesis will be performed based on area under curve-receiver operating characteristic curve and/or accuracy measures. We will use Stata version 14.2 (Statacorp; College Station, TX) for primary and secondary data synthesis. Conclusion: The inferences of our secondary research will provide evidence to evaluate the prognostic role of CNN in discriminating CRP or CRC in colonoscopy settings.", "keywords": [ "Colorectal Polyp", "colorectal cancer", "convolutional neural network", "prediction model", "computer aided diagnosis" ], "content": "Introduction\n\nColorectal cancer (CRC) is the third most common cancer worldwide. Among the causes of cancer-related death, CRC ranks second1. Although the biological pathways that transform the normal colonic into malignant tissue are different, polyps are presumed to be the precursor lesions for malignant tumors in all cases2. Colorectal polyps (CRP) can be seen in various forms or shapes on colon endoscopy. CRP are histologically classified into two main categories based on growth pattern, namely serrated and adenomatous (adenomas) polyps2.\n\nA new systematic review and meta-analysis combined 70 primary population-based cross-sectional studies that used colonoscopy for assessing different colorectal neoplastic lesions. This study reported the worldwide prevalence of adenoma, advanced adenoma and CRC as 25.9%, 5.2% and 0.6% in patients older than 50 years, respectively3. Brenner et al. investigated almost 850,000 colonoscopies of German national screening of CRC. They projected that 10-year risk of progression of advanced adenomas to CRC varied between 25% and 40% in men and women older than 50 years of age4.\n\nThe role of colonoscopy screening has been proven as an effective strategy for preventing CRC development. This intervention can affect CRC mortality by two pathways; first, polypectomy or removing adenomas and second, detecting early stages of CRC5. Because removal of CRP does not reduce the risk of CRC to zero, current guidelines recommend a 5–10-year rescreening interval after a negative colonoscopy result6,7. Recently, a large-scale multi-center cohort study followed 200,000 persons who underwent baseline colonoscopies for more than eight years. This large cohort study showed that high-risk and low-risk adenomas resulted in 2.6 and 1.3 fold increase, respectively, in the risk of CRC development when compared to persons without adenoma8.\n\nDuring the last two decades, despite efforts made to screen CRC/CRP in target groups (e.g., population aged more than 50 years old), studies have shown that even conventional colonoscopy by expert gastroenterologists can miss adenomas or pre-cancerous lesions in up to 25% of cases9,10. Over the last few decades, several measures have been developed and recommenced to evaluate the quality of colonoscopy in the diagnosis of polyps and colon cancers11. An important quality measure is the Adenoma Detection Rate (ADR). A recent large-scale investigation on more than 300,000 colonoscopies performed by 136 colonoscopists during 13 years showed that an 1.0% increase in the ADR was associated with a 3.0% decrease in the risk of CRC12.\n\nIn recent years, several technologies have been combined with colonoscopy, leading to new endoscopic methods for improving detection rate of colorectal lesions. Image-Enhanced Endoscopy (IEE), cap-assisted colonoscopy, the Third Eye® Retroscope®, wide-angle colonoscope, Endocuff® device, and water-assisted colonoscopy are just a few of these new methods of colonoscopy that are available to augment the detection, diagnosis, and treatment of these subtle lesions13,14.\n\nIn the last few years, several adjunct techniques or devices are under investigation for improving ADR in colonoscopy settings. These are methods categorized under the generic term “Computer Aided Diagnosis” (CAD)15. The collection of models such as “Artificial Intelligence”, “Deep Neural Networks” and “Machine Learning” are under CAD. Recent investigations have shown that CAD-methods, along with colonoscopy data, have advantages; higher CRC/CRP detection, better histopathologic differentiation, decreasing overall healthcare costs, and decreasing operator independency15.\n\nConvolutional neural networks (CNNs) are a class of deep neural networks that are highly effective at performing image and video analyses. CAD–CNN models for colonoscopy could assist endoscopists in detecting polyps and performing optical diagnosis. CNNs are trained using thousands of colonoscopy images to identify and differentiate between hyperplastic and adenomatous polyps16.\n\nSince 2018, several systematic reviews have been published to diagnose different clinical outcomes using CNN methods. The assessed outcomes were in skin cancer17, breast cancer18, hepatocellular carcinoma or hepatic mass19, and ischemic brain strokes20.\n\nBased on our knowledge, no systematic review or meta-analysis to evaluate the diagnostic accuracy for discrimination of CRC from CRP has been published. Unfortunately, nearly all systematic reviews assessing the diagnostic accuracy of CNNs in detecting or differentiating different health outcomes, especially different cancers that have been published so far have some limitations. First, the papers had no study protocols and no evidence was provided regarding registration of the protocols. Second, all the published systematic reviews were performed without doing a meta-analysis. Third, in the systematic review methods, risk of bias assessment or critical appraisal was not designed. Therefore, we will aim to design and conduct a systematic review and meta-analysis using a standard and high-quality method to evaluate diagnostic accuracy for discrimination of CRC/CRP by CNN.\n\n\nProtocol\n\nThis protocol was first designed based on the “priori” approach and then registered in Open Science Framework21.\n\nAssessing the accuracy of CNN model (Intervention: I) for discrimination malignancy (cancer) and/or polyp (Outcome: O) from normal colorectal tissue (Comparison: C) in CRC or CRP probable patients (Participants: P) who attended colonoscopic clinics or centers, based on colonoscopic images or videos.\n\nType of primary studies. Primary studies will be included as follows: first, all of the study subjects have colonoscopy videos or images (at least one per subject); second, all of the study subjects have pathological or cytological diagnoses (as the gold standard or reference standard); third, the study design is cross-sectional, prospective, or retrospective approach. Therefore, all observational studies with case-control, cohort, or cross-sectional design will be included. Interventional studies (trials, experimental or quasi-experimental), reviews (secondary research), editorials, letters, and similar articles will be excluded.\n\nType of participants. Since CRPs are usually diagnosed in adults older than 50 years of age, all studies conducted on adult populations of either gender (i.e., patients older than 18 years) will be eligible to be included.\n\nReference standard. The histopathologic examination results of the colorectal lesions (CRC or CRP) will be used as the reference standard.\n\nIndex test (the output of prediction model). CNNs are a supervised learning method. They can learn and find the relationship between the input (images or videos) and the class labels. CNN layers are generally divided into two categories: hidden layers and fully connected layers. The task of the hidden layers is to extract the features. The fully connected layers are used for the classification and detection object in the input images, at the end of the CNN network. The different class labels of all the assessed images or videos should be reported.\n\nWe will search the following bibliographic databases: PubMed/MEDLINE, SCOPUS, Web of Science, IEEE (Institute of Electrical and Electronics Engineers), Inspec, ProQuest, Google Scholar, Microsoft Academic Search, ScienceOpen, arXiv, bioRxiv. Moreover, we will assess relevant conferences for content (Conference on Computer Vision and Pattern Recognition, International Conference of Computer Vision, European Conference on Computer Vision). We will search Gastroenterology, Pattern Recognition, Scientific Reports as key journals by hand-searching. The search time interval will cover the 1st of Jan 2010 up to the 31th of July 2020. Our search will not be restricted based on language or geographical area. The PubMed search syntax is provided as Extended data21.\n\nAfter searching in the sources mentioned above, we will screen all the primary studies based on titles or abstracts. A screening checklist will be developed using 4 to 6 criteria. The criteria will be selected based on the most common components reported in the abstracts of primary research. We will select included or potentially included studies for further assessment. Two reviewers will independently evaluate the considered studies based on full-text papers or documents. We will resolve any disagreement between the two reviewers by consensus.\n\nTwo reviewers will independently assess the methodological quality (risk of bias) of the included studies. The risk of bias checklist will be selected based on PROBAST tool22. The checklist has two main domains: risk of bias (ROB) and applicability domains. The ROB domains contain four items: participants, predictors, outcome, analysis. The applicability domain has three items: participants, predictors, and outcome. All seven items will be assessed according to one of three options: low risk of bias, high risk of bias and unclear risk of bias. The overall quality status (overall risk of bias status) will be determined based on defined guidelines (PROBAST guideline). We will resolve any disagreement between the two reviewers by consensus.\n\nWe will design an extraction form based on the study objectives. After testing of the extraction form (at least one primary study), we will finalize the data extraction form. Two reviewers will independently extract the required data from all the primary studies (papers or documents). We will resolve any disagreement in the extracted data between the two reviewers by consensus.\n\nThe quantitative primary measures are one of two performance indicators: area under curve-receiver operating characteristic curve (AUC-ROC) (C or Concordance Statistics) or accuracy measure. As primary data synthesis (meta-analysis), we will combine the AUC and/or accuracy measures.\n\nThe secondary data are the CNN architecture model (VGG23, AlexNet24, GoogLeNet25, etc.) or the features of CNN architecture, such as the number of layers, the size of layers (kernel (filter) and stride size), kind of pooling layers (max or average) and so on, the CNN model sensitivity, specificity, and diagnostic odds ratio.\n\nThe subgroup variable data will be transfer learning (existing or absence), learning rate, the features of CNN architecture (number of layers, size of the kernel and stride, and so on).\n\nCombining the primary and secondary data will be performed based on Debray et al guidelines26. We will use Stata version 14.2 (StataCorp. College Station, TX) and R 4.0.0 for conducting the meta-analysis method.\n\nWe will use a forest plot for presenting the accuracy measure pooling and I2 (inconsistency) measure and Q Cochrane test for assessing heterogeneity27. We will use the subgroup analysis based on the variables mentioned above. The subgroup analysis or meta-regression will be utilized for finding the potential sources of heterogeneity. The funnel plot, Begg's or Egger's tests28, and Fill and Trim methods29 will be used for evaluating publication or reporting bias.\n\nWe will apply sensitivity analysis for assessing the relation between the primary study quality and the accuracy amount, and a one-out remove method will be used too30.\n\nThe Grading of Recommendations, Assessment, Development and Evaluation (GRADE) tool to assess the quality of evidence and strength of recommendations was originally developed for interventional systematic review and meta-analysis, but the standard tool was changed later for diagnostic and predictive systematic reviews. However, we will use the modified version of GRADE31 for prediction model systematic review.\n\n\nDissemination\n\nWe will present the systematic review and meta-analysis findings at relevant conferences. Moreover, we will submit the manuscript to relevant scholarly peer-reviewed journal.\n\n\nStudy status\n\nThis systematic review is currently in the search phase for information sources.\n\n\nDiscussion\n\nCNN is a promising advanced method that can be useful in differentiating CRC from benign lesions, such as polyps. The results of this systematic review and meta-analysis will further our knowledge about the role of CNN in predicting colorectal lesions. As far as we know, no comprehensive systematic review has been published so far elaborating the role of CNN in predicting colorectal lesions.\n\nWe will try to conduct the study strictly adhering to the methods described. Any changes in the study conduction will be clarified in the final manuscript. Since heterogeneity is a common phenomenon in prediction model studies, we will try to address this concern by appropriate methods such as meta-regression and/or subgroup analysis.\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: Classification of different polyps in colorectal Endoscopy using Convolutional Neural Network; A Systematic Review Protocol Review title and timescale, https://doi.org/10.17605/OSF.IO/QJ7EU21.\n\nThis project contains the following extended data:\n\n- PubMed search syntax.\n\n- Protocol\n\nRepository name: PRISMA-P checklist32 ‘Classifying colorectal cancer or colorectal polyps in endoscopic setting using Convolutional Neural Network (CNN): protocol for a systematic review and meta-analysis’, https://doi.org/10.17605/OSF.IO/QJ7EU21.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nKeum NN, Giovannucci E: Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol. Nature Research, 2019; 16(12): 713–32. PubMed Abstract \| Publisher Full Text\n\nØines M, Helsingen LM, Bretthauer M, et al.: Epidemiology and risk factors of colorectal polyps. Best Pract Res Clin Gastroenterol. Bailliere Tindall Ltd, 2017; 31(4): 419–24. PubMed Abstract \| Publisher Full Text\n\nWong MCS, Huang J, Huang JLW, et al.: Global Prevalence of Colorectal Neoplasia: A Systematic Review and Meta-Analysis. Clin Gastroenterol Hepatol. W.B. Saunders, 2020; 18(3): 553–561.e10. PubMed Abstract \| Publisher Full Text\n\nBrenner H, Hoffmeister M, Stegmaier C, et al.: Risk of progression of advanced adenomas to colorectal cancer by age and sex: Estimates based on 840,149 screening colonoscopies. Gut. 2007; 56(11): 1585–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZauber AG, Winawer SJ, O’Brien MJ, et al.: Colonoscopic polypectomy and long-term prevention of colorectal-cancer deaths. N Engl J Med. 2012; 366(8): 687–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLieberman DA, Rex DK, Winawer SJ, et al.: Guidelines for colonoscopy surveillance after screening and polypectomy: A consensus update by the us multi-society task force on colorectal cancer. Gastroenterology. W.B. Saunders, 2012; 143(3): 844–57. PubMed Abstract \| Publisher Full Text\n\nWinawer SJ, Zauber AG, Fletcher RH, et al.: Guidelines for Colonoscopy Surveillance after Polypectomy: A Consensus Update by the US Multi-Society Task Force on Colorectal Cancer and the American Cancer Society. CA Cancer J Clin. 2006; 56(3): 143–59; quiz 184–5. PubMed Abstract \| Publisher Full Text\n\nLee JK, Jensen CD, Levin TR, et al.: Long-term Risk of Colorectal Cancer and Related Death After Adenoma Removal in a Large, Community-based Population. Gastroenterology. 2020; 158(4): 884–894.e5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarty TR, Aihara H: Role of image-enhanced endoscopy: how to improve colorectal polyp detection rates in the coming decade. Gastrointest Endosc. Mosby Inc, 2020; 91(1): 113–4. PubMed Abstract \| Publisher Full Text\n\nVan Rijn JC, Reitsma JB, Stoker J, et al.: Polyp miss rate determined by tandem colonoscopy: A systematic review. Am J Gastroenterol. 2006; 101(2): 343–50. PubMed Abstract\n\nLund M, Trads M, Njor SH, et al.: Quality indicators for screening colonoscopy and colonoscopist performance and the subsequent risk of interval colorectal cancer: A systematic review. JBI Database System Rev Implement Rep. Lippincott Williams and Wilkins; 2019; 17(11): 2265–300. PubMed Abstract \| Publisher Full Text\n\nCorley DA, Jensen CD, Marks AR, et al.: Adenoma detection rate and risk of colorectal cancer and death. N Engl J Med. 2014; 370(14): 1298–306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee TJW, Nair S, Beintaris I, et al.: Recent advances in colonoscopy [version 1; peer review: 2 approved]. F1000Res. 2016; 5: F1000 Faculty Rev-328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGill S, Soetikno R, Kaltenbach T: Image-enhanced endoscopy in practice. Can J Gastroenterol. 2009; 23(11): 741–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChao WL, Manickavasagan H, Krishna SG: Application of artificial intelligence in the detection and differentiation of colon polyps: A technical review for physicians. Diagnostics (Basel). MDPI AG, 2019; 9(3): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlagappan M, Brown JRG, Mori Y, et al.: Artificial intelligence in gastrointestinal endoscopy: The future is almost here. World J Gastrointest Endosc. 2018; 10(10): 239–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrinker TJ, Hekler A, Utikal JS, et al.: Skin cancer classification using convolutional neural networks: Systematic review. J Med Internet Res. 2018; 20(10): e11936. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGardezi SJS, Elazab A, Lei B, et al.: Breast cancer detection and diagnosis using mammographic data: Systematic review. J Med Internet Res. 2019; 21(7): e14464. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzer SA: Deep learning with convolutional neural networks for identification of liver masses and hepatocellular carcinoma: A systematic review. World J Gastrointest Oncol. 2019; 11(12): 1218–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurray NM, Unberath M, Hager GD, et al.: Artificial intelligence to diagnose ischemic stroke and identify large vessel occlusions: A systematic review. J Neurointerv Surg. 2020; 12(2): 156–64. PubMed Abstract \| Publisher Full Text\n\nKeshtkar K: Classification of different polyps in colorectal Endoscopy using Convolutional Neural Network; A Systematic Review Protocol Review title and timescale.2020. http://www.doi.org/10.17605/OSF.IO/QJ7EU\n\nWolff RF, Moons KGM, Riley RD, et al.: PROBAST: A tool to assess the risk of bias and applicability of prediction model studies. Ann Intern Med. 2019; 170(1): 51–8. PubMed Abstract \| Publisher Full Text\n\nSimonyan K, Zisserman A: Very deep convolutional networks for large-scale image recognition. arXiv Prepr arXiv14091556. 2014. Reference Source\n\nKrizhevsky A, Sutskever I, Hinton GE: Imagenet classification with deep convolutional neural networks. In: Advances in neural information processing systems. 2012; 1097–105. Publisher Full Text\n\nSzegedy C, Liu W, Jia Y, et al.: Going deeper with convolutions. In: Proceedings of the IEEE conference on computer vision and pattern recognition. 2015; 1–9. Publisher Full Text\n\nDebray TPA, Damen JAA, Snell KIE, et al.: A guide to systematic review and meta-analysis of prediction model performance. BMJ. 2017; 356: i6460. PubMed Abstract \| Publisher Full Text\n\nHiggins JPT, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med. 2002; 21(11): 1539–58. PubMed Abstract \| Publisher Full Text\n\nEgger M, Smith GD, Schneider M, et al.: Bias in meta-analysis detected by a simple , graphical test measures of funnel plot asymmetry. BMJ. 1997; 315(7109): 629–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuval S, Tweedie R: Trim and fill: A simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis. Biometrics. 2000; 56(2): 455–63. PubMed Abstract \| Publisher Full Text\n\nThe Handbook of Research Synthesis and Meta-Analysis - Google Scholar. [cited 2020 Jul 5]. Reference Source\n\nIorio A, Spencer FA, Falavigna M, et al.: Use of GRADE for assessment of evidence about prognosis: Rating confidence in estimates of event rates in broad categories of patients. BMJ. 2015; 350: h870. PubMed Abstract \| Publisher Full Text\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2016; 20(2): 148–60. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70722", "date": "15 Sep 2020", "name": "Iman Tahamtan", "expertise": [ "Reviewer Expertise Systematic review", "designing search strategies in academic databases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 protocol that aims to evaluate the diagnostic accuracy for discrimination of colorectal cancer (CRC) and colorectal polyps (CRP) using a convolutional neural network (CNN).\n\nThe authors have very well explained previous systematic reviews (and studies) related to the study's topic and their rationale for conducting this study. One rationale is mentioned to be methodological issues of previous research, such as not doing the risk of bias assessment in previous systematic reviews or studies being conducted without including a meta-analysis.\n\nI ask the authors to be unique and explicit in stating the objective of the study. In the 'abstract' of the protocol, they mention that the aim of the study is classifying CRP and CRC using CCN, while it is mentioned to be evaluating the diagnostic accuracy for discrimination of CRC and CRP using CNN. Please also explain, in the objectives (in both the abstract and text) that this study aims to investigate the accuracy of CNN in classifying CRC and CRP compared to its accuracy in identifying the normal colorectal tissue (Comparison: C).\n\nIn the last paragraph of the introduction, the authors mention that one limitation of previous studies is ‘not registering their protocols’. Although this may seem to be a limitation, does this justify the need for conducting this research? If the answer is yes, please explain how? However, the second and third provide a strong rationale for the need for conducting this research.\n\nAlthough it is clear to experts what type of primary studies will be included in this study, please make some language modifications to the first sentence to make it more transparent. For instance, you could change it as follows: \"The primary studies with all the following features will be included in this systematic review and meta-analysis: … \".\n\nType of participants: it is not clear why when CRPs are usually diagnosed in adults older than 50 years of age, the authors mention that 'patients older than 18 years will be eligible to be included' in this systematic review. Shouldn’t it have been 50 instead of 18?\n\nIndex test (the output of prediction model): please explain what you mean by 'class labels'? What are the features that the hidden layer will extract?\n\nPlease clarify the following sentence: “The fully connected layers are used for the classification and detection object in the input images, at the end of the CNN network\". Detection of which objects in the input images? It is not clear what the authors mean in 'at the end of the CNN network'. Does this mean the final thing that the CNN does is the detection of objects in the input images?\n\nIf you have already designed the screening checklist, please mention which criteria it includes?\n\nPlease explain whether you will include all studies with any risk of bias or if those with low and unclear risk of bias will be excluded.\n\nI also recommend that the authors explain CNN in non-jargon language, for example, by providing an example so that non-experts in machine learning would better understand how CNN works.\n\nBriefly explain the Debray et al. guidelines for combining the primary and secondary data.\n\nIn the following sentence, in parentheses specify the variables again: “We will use the subgroup analysis based on the variables mentioned above”.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1086
https://f1000research.com/articles/9-626/v1	18 Jun 20	{ "type": "Opinion Article", "title": "The evolution of universal adaptations of life is driven by universal properties of matter: energy, entropy, and interaction", "authors": [ "Irun R. Cohen", "Assaf Marron", "Assaf Marron" ], "abstract": "The evolution of multicellular eukaryotes expresses two sorts of adaptations: local adaptations like fur or feathers, which characterize species in particular environments, and universal adaptations like microbiomes or sexual reproduction, which characterize most multicellulars in any environment. We reason that the mechanisms driving the universal adaptations of multicellulars should themselves be universal, and propose a mechanism based on properties of matter and systems: energy, entropy, and interaction. Energy from the sun, earth and beyond creates new arrangements and interactions. Metabolic networks channel some of this energy to form cooperating, interactive arrangements. Entropy, used here as a term for all forces that dismantle ordered structures (rather than as a physical quantity), acts as a selective force. Entropy selects for arrangements that resist it long enough to replicate, and dismantles those that do not. Interactions, energy-charged and dynamic, restrain entropy and enable survival and propagation of integrated living systems. This fosters survival-of-the-fitted – those entities that resist entropic destruction – and not only of the fittest – the entities with the greatest reproductive success. The “unit” of evolution is not a discrete entity, such as a gene, individual, or species; what evolves are collections of related interactions at multiple scales. Survival-of-the-fitted explains universal adaptations, including resident microbiomes, sexual reproduction, continuous diversification, programmed turnover, seemingly wasteful phenotypes, altruism, co-evolving environmental niches, and advancing complexity. Indeed survival-of-the-fittest may be a particular case of the survival-of-the-fitted mechanism, promoting local adaptations that express reproductive advantages in addition to resisting entropy. Survival-of-the-fitted accounts for phenomena that have been attributed to neutral evolution: in the face of entropy, there is no neutrality; all variations are challenged by ubiquitous energy and entropy, retaining those that are “fit enough”. We propose experiments to test predictions of the survival-of-the-fitted theory, and discuss implications for the wellbeing of humans and the biosphere.", "keywords": [ "Evolution", "Energy", "Entropy", "Interaction", "Cooperation", "Niche", "Fitness", "Fittedness" ], "content": "Introduction\n\nEvolution may be defined as the transitions over time of living systems, which include cells, organisms, species and ecosystems, along with their component genes and other molecules. Evolution can take place when several conditions are fulfilled: variations among the evolving systems; interactions with the environment, both the organic and the inorganic; destruction of some variants and continuing propagation of others; and mechanisms that can select between the two fates. Any mechanisms proposed to drive the processes of selection and heritability would also have to explain, or at least not contradict, the reality of presently living systems; observed life constitutes a reality test for theories of evolution.\n\nThe generally accepted mechanism of evolution is Natural Selection resulting from survival-of-the-fittest1. The neo-Darwinian theory of evolution has expanded Darwin’s concept of Natural Selection to include modern genetics. Natural Selection proposes that variant individuals must struggle for resources in a necessarily limited environment; the individual who manifests superior adaptive fitness to its environment wins out in the struggle to survive and procreate. The winner’s offspring inherit the winner’s genes and so enrich the species with increased frequencies of the fittest genes. Fitness, or “being fittest” is equated with reproductive success2. Iteration of this process over generations eventually leads to an optimally adapted genotype for the species in the given environment3,4. Thus, species would be expected to evolve an optimum uniformity in a stable environment. Survival-of-the-fittest selection in a new environment generates a new species and restarts the optimization process.\n\nHowever, the rationale behind the theory of Natural Selection has been called into question by the recent discovery that all multicellular organisms —– plants, invertebrates and vertebrates—are holobionts composed of gene products of the eukaryote organism in cooperative interaction with great numbers of resident microbiome prokaryote genes and organisms5. In a word, there are no individual organisms to engage in classical Survival-of-the-Fittest struggle6–8. All multi-cellular organisms are groups.\n\nIndeed, microbiome organisms and their genes are acquired somatically after birth and are independent of host eukaryote genome reproduction to a significant degree. Consequently, the inheritance of eukaryote genes alone cannot endow holobiont offspring with the phenotypic fitness of a progenitor holobiont. For example, a mouse that has no microbiome, a germ-free mouse, develops an abnormal gut, a crippled immune system and other abnormalities9. If there be no genetically independent, procreating individuals, individual eukaryote reproductive success alone cannot be responsible for genetic progress.\n\nRecent publications have discussed the inheritance of the microbiome10 and controversy surrounds the definition of the holobiont: for example, whether the holobiont is an integrated individual or a community6,11. These matters raise questions critical to our understanding of the holobiont’s role in the process of evolution. The issue has yet to be resolved.\n\nAnother recent controversy surrounding the mechanism of evolution relates to the concept of niche construction – the co-evolution of the environmental niche with its resident species12.\n\nThe theory of Extended Evolutionary Synthesis (EES)13–15 reconsiders evolution in the light of niche construction, the holobiont and other challenges facing classical thinking. The issue is subject to debate16.\n\nWe shall not attempt to resolve these controversies; rather we draw a distinction between universal adaptations expressed by essentially all multicellular organisms and local adaptations expressed by particular species in a defined environment; we here focus our attention only on universal adaptations. We reason that these adaptations must have been driven and selected for survival by ubiquitous physical features of matter: Energy, Entropy and Interaction.\n\nWe shall first describe energy and entropy and then proceed to interaction and how their integration can account for universal adaptations. Along the way, we contrast our proposed material mechanism with the classical mechanism of survival-of-the-fittest. We term our theory survival-of-the-fitted. We do not deal directly with the evolution of uni-cellular eukaryotes or prokaryotes.\n\nThis paper has evolved from preliminary thoughts about the adaptive immune system17, the role of information in art, science and evolution18 and the evolutionary relationship between information and entropy19.\n\n\nEnergy and entropy\n\nDarwin did not refer to energy as a formative factor in evolution, and neo-Darwinian gene-centered discourse has paid little attention to either energy or entropy. In contrast, we shall show that energy and entropy (and networks of cooperative interaction) are more consequential to the evolution of universals than is the selfish Darwinian struggle.\n\nEnergy is defined by physicists in various ways using different mathematical formulations, each formulation suitable for a different situation20. Nevertheless, we can discuss the evolutionary role of energy without having to get into the mathematical details. Dictionaries define energy as the impetus behind all motion and all activity and as the capacity to do work21. Capacity to do work relates to energy in an organized or ordered form. Living systems, as we discuss below, exploit and are also damaged by energy, both ordered and disordered. The world is permeated with energy in many forms: irradiation from the sun, the earth and outer space; the force of gravity; impacts with asteroids; chemical interactions; electromagnetism; the weather—wind, rain, snow, hailstones, cold, heat; lightning; tides, rivers, floods, and droughts; fires; seismic movements and eruptions; to these inorganic elements we can add energy originating from living systems and from human technology.\n\nThe word entropy is formally defined as a measure of the number of possible micro-states of a system22. The term is informally used to refer to disorder per se, to a measurement of the amount of disorder, and to the tendency, processes or forces that spontaneously effect deterioration, destruction, or dissolution23. To avoid ambiguity24, we use the term to include all the circumstances and events that can lead to the destruction or dissolution of any specific arrangement.\n\nThe concept of entropy derives from the second law of thermodynamics, which states that disorder in a closed system will continuously increase spontaneously22,25. Entropy, as we said above, is formally defined as a statistical property of any system that contains many component parts or states - so called micro-states: the greater the number of parts or micro-states, the less likely will be any particular, given arrangement; randomness will prevail (Figure 1). Moreover, the internal energy inherent in all substances26 - seen, for example in Brownian motion - propels them, sooner or later, to fall apart—unless, as we discuss below, cooperative interactions and other processes hold that arrangement together or renew it.\n\nThe border rectangles mark a closed system. The \"I\" structure in the left frame will spontaneously dissolve into a random disorder of its component pixels, shown in the right frame; the spontaneous process is irreversible. The challenge facing life is to resist entropic dissolution of essential structure. The figure was inspired by P. W. Atkins29.\n\nLiving systems depend on the specific arrangements and interactions of molecules, cells, organs, individuals, species, and ecosystems28. Consequently, life has had to adapt to entropy, the metaphoric destroyer of specific arrangements. Ludwig Boltzmann, one of the founding fathers of thermodynamics, already made the point in 187529:\n\nThe general struggle for existence of animate beings is not a struggle for raw materials – these, for organisms, are air, water and soil, all abundantly available – nor for energy which exists in plenty in any body in the form of heat, but a struggle for [negative] entropy, which becomes available through the transition of energy from the hot sun to the cold earth.\n\n“Negative entropy” refers to “order”.\n\nIn his book What is Life, the physicist Erwin Schrödinger concluded that entropy—the decay of orderly arrangements into atomic chaos—is the ultimate challenge facing living systems25. Schrödinger did not attempt to relate evolution to entropy, and accepted Darwinian Natural Selection and survival-of-the-fittest as a fact (page 11). He made it clear, however, that living organisms have to pay for their order by exporting into the environment a due measure of entropy in the form of heat.\n\nThe mystery of life, says Schrödinger, is that “existing order displays the power of maintaining itself and of producing orderly events.” Schrödinger offers no mechanism that might maintain and produce order by restraining entropy, other than to state that “we, no doubt, draw on experience concerning social organization and other events which involve the activity of organisms. And so it might seem that something like a vicious circle is implied.” In other words, Schrödinger speculates that the orderly arrangements of life emerge from the order inherent in life’s ongoing activities—in a “vicious circle”; cooperative, “social” interactions somehow restrain the effects of entropy.\n\nDespite the physical definition of entropy as a statistical measure of microstates, physicists Boltzmann and Schrödinger both relate to entropy as a concrete threat to life.\n\nIt is clear that entropy is life’s nemesis, but how can entropy also function as evolution’s facilitator? Above, we pointed out that evolution requires variation, interaction, selection, destruction and propagation. Obviously, entropy plays important roles in variation and destruction, but, as we shall see, entropy will also turn out to be a major factor in selection.\n\nOver the years, many theoretical papers have been written to reconcile the evolution of life with entropy and the second law of thermodynamics (for example, see 30–32); but, as far as we know, none of these authors have challenged the paradigmatic neo-Darwinian mechanism of survival-of-the-fittest, and none of them have invoked entropy as a selective factor in evolution, as we do here.\n\nAdaptation is often described as the aim of evolution33: adaptation designates a state in which an organism or species fits the constraints and demands of its environment; to adapt is to solve a vital survival problem imposed by the environment. This concept of adaptation, however, is paradoxical; for example, wings are local adaptations for flying and fins for swimming. Local adaptations are limited to a species in a certain environment; wings and fins only solve fitness problems if one is already a bird or a fish; an elephant would only have more problems surviving with wings or fins.\n\nLevins and Lewontin state that; It is difficult to think of any physical force or universal physical law that represents a fixed problem to which all organisms must find a direct solution34. If you think about it, however, there is one universal physical law to which adaptation is required a priori for living – all creatures in any environment have to deal with entropy. Therefore, adaptations that restrain entropy are ubiquitous in living systems; extending Schrödinger, we propose that cooperative, “social” interaction is one such adaptation.\n\n\nInteractions\n\nThe physicist Richard Feynman referring to the atomic and subatomic scales, claimed that “all mass is interaction”35 (Prologue); similarly, Carlo Rovelli stated that all reality is interaction36. Since matter is made of atoms, everything made of matter is made of moving, interacting atoms. Feynman suggested a sentence that bears the most information for posterity: “all things are made of atoms—little particles that move around in perpetual motion, attracting each other when they are a little distance apart, but repelling upon being squeezed into one another”26. Here we apply dynamic interaction as a building block at higher, biological scales represented by molecules, cells, organisms, species and ecosystems.\n\nWe define an interaction as a relationship between two or more entities involving a transfer or exchange of matter, information and/or energy. Interactions include both struggle and cooperation: in a struggle, the participants each strive to win and dominate the others – who become the losers. In a cooperative interaction, there are no losers; the participants each gain some benefit, or at the least suffer no loss.\n\nLiving systems interact with other living systems, but they also interact with non-living matter like water, minerals and air. Interactions maintain life, and are critical to the evolution of life. Darwin proposed that evolution is driven by interactions expressed as individual struggles for survival. In a limited environment, one’s gain must be offset by others’ losses. Such a zero-sum game logically generates selfishness37–39. When, however, we look at the dominant characteristics of living systems, what we see is not only selfishness but also interactions that generate cooperation and symbiosis.\n\nSymbiotic cooperation—living and working together—seems to have been decisive in the evolution of complex life: the primordial development of a progenitor eukaryote cell, estimated to have occurred about two billion years ago, is now thought to have resulted from the symbiotic amalgamation of two or more prokaryote (bacteria or archaea) cell types to eventually evolve a new living entity – a eukaryote cell40,41. Prokaryote life was then, and still is consummately fit; the foray of life into a complicated eukaryotic endosymbiosis is a fortuitous meander of nature that has eventually led to us complex humans and to our effects on the biosphere.\n\nThe evolution of single-celled eukaryotes into multicellular organisms is also a cooperative enterprise. The phenotype of any multi-cellular individual is the expression of at least two internal cooperative processes: First, there is the developmental and physiological cooperation of the offspring’s eukaryote genes and cells, which initially emerged from the endosymbiosis of parental germ cells. One’s eukaryote cells share a common DNA genome, but differential epigenetic expressions of this shared genome are key to interactions resulting in growth, development, differentiation, immunity, repair, reproduction, physiology, behavior, metabolism, species and ecosystems. Indeed, all processes of multi-cellular life involve cooperative interactions: all depend on a variety of cooperative interactions between molecules, cells and organisms. Even the development of a mutated, lethal tumor can progress only through cooperative interactions of the tumor cells with a microenvironment of non-tumor cells and tissues, including angiogenesis and immune system complicity42.\n\nThe second level of eukaryote cooperative interaction is with a resident microbiome, which may include bacteria, archaea, yeasts, algae and viruses43. Research is yet in an early stage, but the microbiome is clearly an essential component of all multi-cellular invertebrate, vertebrate and plant organisms, which, as we mentioned, are holobionts. The gut microbiome in humans and other organisms performs a variety of significant functions: metabolism of otherwise indigestible foodstuffs; production of vitamins and other essential molecules; detoxification of harmful substances; neutralizing or blocking pathogens; assistance in developing the immune system; warming the body; influencing brain development and behavior; and other benefits8. The human species may actually benefit from the ridding of burdensome, aged humans by the timely evolution of “pathogenic” microbiome bacteria44. In general, microbiome-host metabolic interactions are critical in human health and disease; an abnormal microbiome (dysbiosis) is a hazard45. The microbiome dynamically changes with age, diet, gender, and other factors; for example, a change in diet can greatly expand or reduce particular components of a holobiont’s microbiome and, consequently, affect the holobiont’s health, adaptation to environmental changes and survival.\n\nHow the essential microbiome and the host organism genome are transmitted faithfully across generations is still a matter for debate10. As part of this discussion, Stencel and Wloch-Salamon propose that the holobiont is not inherited as a single entity, but is a cooperating group11. Hence, the discovery of the holobiont obliges evolutionary theory to reconsider group selection – a controversial idea that is incompatible with the concept of evolution driven by the heritable fitness of a single-genome individual46.\n\nBiology has recently discovered that all species of vertebrates bear genes encoding molecules like oxytocin that function to enhance cooperation, social bonding, and love, and activate brain reward centers while reducing conflict47; evolution for many millions of years seems to have generated mechanisms fostering cooperation and mutual benefit. Indeed, oxytocin-like genes are borne by many invertebrates48; and the products of microbiome bacteria stimulate oxytocin production in their cooperating host49.\n\nSocial bonding and empathy would also appear to be programmed in the systems of mirror neurons discovered in the brains of humans and other primates50; these neuronal centers are active in imitation and empathy for others51. Mirror neurons probably evolved even before the primates52.\n\nOf course, mutual identification and cohesion within a group may also give rise to prejudices and to episodic conflicts with others outside the group53. In any case, it is clear that evolution has generated species outfitted with innate aptitudes for bonding, cooperation, empathy and social interaction. Even the evolution of the dog from the wolf appears to have involved mutual oxytocin-related bonding with humans54.\n\nThe transfer of genetic material between existing species can be seen as a further example of non-Darwinian cooperation; a species receives genes from organisms of other species that have evolved in a different environment. Horizontal gene transfer occurs regularly among prokaryotes, but also has occurred in eukaryote evolution. A notable example is the development of the placenta, a formative adaptation in mammalian evolution; placenta function involves the syncytin gene, believed to have been transferred horizontally from a virus55. The survival-of-the-fittest theory, which is based on the evolution of genes internal to a single species, cannot easily account for the roles in evolution of horizontal gene transfer56,57.\n\nFigure 2 illustrates that cooperative interactions between the components of an arrangement can delay the entropic destruction of the arrangement.\n\nCompare this figure to Figure 1. Here, the pixels that form the \"I\" structure on the left are held together by cooperative interactions, pictured as zippers that link the pixels. These interactions slow down the deterioration of order and structure, depicted by the long and tortuous arrow from the left to the right frame, while both short arrows are blocked.\n\nDarwin was aware of the intrinsic conflict between selfish struggle and cooperation1 (Chapter 7); later evolutionary thinkers have attempted to explain cooperation as a result of an underlying selfish drive by theories of kinship selection or reciprocation agreements (see summary in 58), including the application of mathematical game-theory explanations59–61.\n\nThese proposed solutions have been widely accepted, but we believe that the evolution of cooperation as a universal characteristic is more easily understandable once we hedge selfish struggle and include entropy as a selective agent in the process of evolution. An empirical example of the ability of cooperation to restrain entropic degradation is provided by the DNA molecule.\n\nDNA is well known as the repository of genetic information shared by all forms of eukaryote life. Compare the fragility of single-stranded DNA (ssDNA) with the unparalleled stability of double-stranded DNA (dsDNA)62,63. The paired strands of dsDNA, bound together, are relatively resistant to random damage because each strand is in constant interaction with its sister strand; the two strands can be perceived to be in continuous cooperation; essentially all potentially reactive, non-covalent bonding energy is engaged in cooperative interactions between the strands. In marked contrast to the stable dsDNA dimer, ssDNA and single stranded RNA (ssRNA) are highly susceptible to destruction; reactive bonding energy in the single-strand configuration is not quenched by physiological interactions and is available for haphazard interactions with illicit target molecules, leading to degradation (Figure 3). Cooperative interaction explains why stable dsDNA can be recovered from mummies or woolly mammoths thousands of years old64, while unstable ssDNA and ssRNA survive for only days, at best. dsDNA illustrates how an exchange of energy between the interacting strands retards their entropic dissolution. dsDNA models the universal power of interaction to forestall entropy. We shall cite a higher scale molecular example in the section Metabolism below, when we discuss how dynamic metabolic interactions establish networks that restrain entropy.\n\nThe colored bars protruding horizontally from the DNA strands represent non-covalent bonding sites. Interactions between the bonding sites in dsDNA protect the joint molecule from illicit, potentially damaging interactions. DNA in the single stranded form—ssDNA—is not protected from non-physiological interactions and is thereby readily degraded. Image of ss and dsDNA reproduced from 72 under fair use licensing for nonprofit and educational purposes.\n\nWe have all observed the role of cooperative interactions in maintaining life at the macroscopic scale: physical isolation or enforced bed rest of an elderly person in a hospital can lead to sudden collapse and death65. Why did hospitalization kill the grandmother with the broken hip? She was receiving all the material resources needed for physical survival—food, fluids, oxygen, nursing care; what did she lack? It was not only the concentration of lethal bacteria and other such medical hazards in hospitals that killed the patient; deprivation of her customary daily activities and interactions with her familiar physical and human environments made her more susceptible to undesirable interactions66. There are many observations that social interaction itself is critical. Retirement or social isolation accelerates mental and physical decline67; newborns isolated from maternal interactions can sicken and die68. In contrast, relationships and responses to challenge delay entropic decline and rejuvenate and maintain body and soul. Life thrives on interaction69. As they say; use it or lose it.\n\nThe function of interaction in maintaining life may help us understand why some species of birds, and other organisms, have evolved to “waste” resources in elaborate courtship displays and bowers and to indiscriminately feed parasitic chicks (which may be obscenely large) of other bird species70. Such behaviors clearly contradict individual Darwinian fitness. We propose, however, that seemingly wasteful activities and interactions in themselves maintain cooperative fittedness; as long as a holobiont organism or group has enough matter and energy to act, evolution will not select against a fitted action that is merely inefficient or blatantly wasteful71. Interaction itself restrains entropy. Indeed, as we discuss below, living systems shield themselves from entropy, not by saving energy, but by deploying networks of energy to maintain interactions. Cooperation is its own reward.\n\nMicrobiologists, until recently, have studied prokaryotes isolated in “pure” cultures in the laboratory; in nature, however, prokaryotes, like eukaryotes, live in complex communities of cooperating organisms in microbiomes, in biofilms, in soil, in water, and in other collective environments73. Mutual group behaviors are adjusted by the exchange of molecular signals and genetic elements between prokaryotes and, in microbiomes, also between prokaryote and eukaryote cells74. Cooperative interactions support the prokaryote world too.\n\n\nMetabolism\n\nMetabolism refers to chemical interactions that produce and transform energy. The term is usually applied to the breakdown or synthesis of molecules that release, consume, or store energy.\n\nLife has evolved metabolic arrangements that organize energy: photosynthesis and other contrivances trap disorganized light, heat and chemical energy released by our exploding sun75 and our quaking earth76; living systems, through metabolic networks, transform these potentially destructive forces into organized molecular and cellular structures, processes and networks of interactions that build and maintain life. Living systems also exploit energy and matter, both ordered and disordered, obtained from other living systems – creating food chains and ecosystems77. The trapping of available energy, both disordered and ordered, and its conversion into networks of orderly arrangements and interactions, maintain life and its renewal in the face of entropy.\n\nThe subject of metabolism was thought to have been solved decades ago with the discoveries of the functions of vitamins and the major cycles, like the Krebs cycle, that control energy transformations. But metabolism has returned lately as a major subject of research. Metabolic pathways can differ in the substances used as fuels, the molecules consumed or synthesized, the activated enzymes, the involvement of aerobic or anaerobic pathways, the efficiency of the processes, and more. The details are beyond our present scope, but dynamic shifts in metabolic processes have been discovered not only to fuel life, but to actually signal and regulate key cellular activities and interactions. Metabolism regulates stem cell differentiation78, immune cell functions79, microbiome interactions80, brain cell signaling81, and the growth and development of cancer cells82, among other functions. It is no wonder that oxytocin, the hormone of cooperation and bonding, also affects metabolism47.\n\nThe flows of energy through the networks of a living system create a unified structure that resists entropic dissolution. Building a house of cards exemplifies the power of interactive energy: a single card will not stand on a tabletop for more than an instant; two cards interacting as a triangle can stand appreciably longer—the electrostatic interactions between the cards and their balanced support keep them up, and you can progress to build a stable house of cards. That is, until you open the window and the winds of entropy blow it down. If, however, you want your house of cards to survive casual entropic winds, you only have to add a drop of glue to the interfaces between the cards. The glue augments the electrostatic bonding interactions between the cards and the house of cards will hold up, at least until the dog demolishes it. Thus do ongoing metabolic interactions (largely electrostatic) between living systems create a jointly unified structure that can restrain destructive effects of entropy.\n\nAn ongoing interaction between two agents reduces the numbers of random microstates — and thus of the entropy— otherwise present in each of the agents when they are not interacting. For example, as stated in 83:\n\n“...A spark applied to the mixture [of hydrogen and oxygen] initiates a chemical reaction in which hydrogen and oxygen combine to form water. If the temperature of the system is held constant, the entropy of the system decreases because 3 moles of two differing reactants have been combined to form 2 moles of a single product. The gas now consists of a uniform set of indistinguishable molecules”.\n\nBonding between reacting entities reduces their combined micro-states and thus the total amount of entropy.\n\nThe reduction of internal entropy achieved by integrated energy networks accounts for the evolution and persistence of interactions that would appear to detract from zero-sum Darwinian fitness. In the eyes of Natural Selection, any arrangement that efficiently saves energy should win in a struggle with arrangements that wastefully squander resources. Nevertheless, the biosphere abounds with inefficient interactions72: people waste time and money on trinkets; birds, humans and other suitors invest energy on displays and rituals; butterflies, fish, birds and mammals undertake long migrations filled with peril; many species survive with energy-draining parasitisms. Only some of these “wasteful” arrangements can be explained by variants of a handicap principle84, an argument that has its share of criticism (see summary in 85).\n\nClearly, one can imagine mutations that could do away with such extravagances. But living systems regularly defy the logic of exclusively Darwinian bookkeeping. Cooperative interactions, like those that connect the two strands of dsDNA or a glued house of cards, can form a hedge against entropy, irrespective of efficiency or wastefulness. In other words, interaction itself resists entropy, as suggested by Schrödinger, and so Fittedness can account for arrangements and interactions that make little sense to survival-of-the-fittest thinking. Metabolism fuels the “preservative social” activities of life envisioned by Schrödinger to restrain entropy25.\n\n\nProgrammed renewal\n\nSurvival requires rebirth. The inevitable dissolution of structure implies that all livings beings are destined, in time, to fall apart and die (Figure 1). Life has had no alternative but to effect its own renewal. In fact, we might define a living entity as one that is able to exploit energy, by way of metabolism, to renew itself.\n\nDarwin founded Natural Selection on the assumption that living organisms strive to survive and reproduce as long and as much as they can1. The fittest individuals, according to classical survival-of-the-fittest thinking, should have the most offspring. After a century and a half of refinements, qualifications, and other added nuances, the underlying principle of present-day evolutionary theory is that “Natural selection is all about variation in reproductive success” and “ Natural selection is not ‘survival of the fittest,’ but rather ‘reproduction of the fittest.’2. The fittest should live the longest, provided that longevity does not compromise reproduction2,86.\n\nBut when we take a look at life as lived, we see that lifespans and birthrates are essentially programmed by an organism’s species more than by an organism’s success in struggles with competing organisms5,87. Each species has evolved its genetic endowment to encode a relatively standard lifespan for its member organisms – whether for days, seasons, or years. The apportionment of survival time, barring accidents, is fairly fixed86. Classical explanations based on Natural Selection attribute fixed lifespans to a delicately balanced optimization of factors, such as age of sexual maturity, numbers of offspring, and reproductive cycle timing. But superior or successful individuals do not necessarily live the longest or beget the most offspring.\n\nIndeed, a mindset focusing on biological survival may have delayed the recognition of programmed death88 until apoptosis89–92 and autophagy93 became important subjects of research.\n\nSimilar to lifespan, the time of birth and replication is closely linked to the style of life evolved by the species as a whole; the time of birth of a new organism, how long the organism lives and its numbers of offspring are associated with the way the species has evolved to make its living and manage its reproductive energy. Humans and elephants, for example, would never survive if they bore litters like rabbits; the rabbit species would never thrive if rabbits lived as long as elephants or people. Quite simply, birthrates, aging rates and spans of life are intrinsic to the genetic endowment of the species.\n\nThe human genetic disease progeria makes the point – persons bearing mutations in the LMNA gene manifest accelerated aging and usually die of “old age” in their teenage years94. Standard life times are clearly demonstrated in the turnover rates of the cells that compose our bodies – different cell types manifest half-lives in tune with their healthy functions95. A cell that competitively out-survives its fellows can grow into a tumor and kill the organism96; unbridled survival is destructive. Death, like reproduction, cannot be left to the whims of entropy – entropy is disorganized and death by entropy, although inevitable, is too disorganized to be adaptive. A lifespan is an adaptation87, not a prize for successful competition. We propose that programmed birth and death are advantageous to a species because they preempt entropic disorder by imposing order on the inevitable turnover of individuals. Death, the ultimate disorder, becomes ordered.\n\n\nDiversification, poly-determination, attractors and saltation\n\nReplicates of any single material entity are necessarily diverse—no two genetically identical cells, for example, can occupy the same place at the same time; each cell expresses its own history and resides, as it were, in a separate environment. Beyond this fundamental, physical diversity, living systems have evolved to express higher orders of biological diversity. The concept of survival-of-the-fittest would lead one to expect that evolution should produce a single, optimal plan, approaching “perfection”, in the words of Darwin1. But evolution does not produce a single optimum; evolution is marked by continuous diversification—living systems, including prokaryotes, eukaryotes, multi-cellular organisms, species and ecosystems, are composed of component parts and processes that are intrinsically variable and diverse. Diversification of genomes is evident in the tens of millions of single-nucleotide polymorphisms (SNPs) and other genetic variations found in healthy people97.\n\nTumors express the advantages, at least for the tumor, of diversification: a tumor that includes a great diversity of clones manifests relatively more aggressiveness, greater resistance to therapy, and enhanced fittedness when metastasizing to new organs98. Lethal tumors have no evolutionary future, neither for themselves nor for their hosts, but they do illustrate a short-term advantage of diversification, however selfish and ultimately self-defeating.\n\nAmazingly, living systems can utilize different combinations of their given component parts and processes to achieve a relatively uniform output. No single internal plan monopolizes a particular system. In other words, living systems can be said to be poly-determined—a given arrangement or behavior can be produced by multiple, diverse networks of interaction99. A goal in football (soccer) is an accessible example of poly-determination: a scored goal is a goal, but no two are ever achieved through precisely identical field play. The poly-determination of sports is obviously an invention of human game designers; in contrast, the poly-determination of living systems has been selected by the nature of evolution.\n\nNote that poly-determination differs from redundancy; a redundant structure or process is perceived as being a replicate of the one ideal solution; poly-determination, in contrast, is intrinsic to the multiple ways the system works to generate a given output.\n\nA telling example of poly-determined diversification can be seen in the formation of the human species; in how many different ways has evolution been able to devise a human? We can safely assume that no two humans have ever housed identical genomes, brains, immune systems and microbiomes—monozygotic twins may emerge from a single fertilized egg, but their brains and immune systems quickly diverge and their microbiomes and epigenetic landscapes also differ. Since billions of individual holobionts have populated the human species since its inception, we can conclude that evolution has been able to concoct diverse instantiations of the human species using billions of different component recipes.\n\nPoly-determination is also evident in the microbiome-host relationship: diverse combinations of different gut bacteria may perform similar metabolic or other functions in the host gut; in other words, a particular function may be realized by diverse combinations of microbes; as Doolittle and Booth have said: “the song is more important than the singer”100.\n\nDiversification is another name for individuality––no two are exactly the same; hence, evolution, as it were, has fostered individuality. Even artificially inbred mice caged together and fed identical diets manifest individual differences101. Genetically identical round worms, too, manifest individuality102.\n\nDiversification, poly-determination and individuality are consequential to disease and therapy: the basic biological differences between people frustrate the traditional assumptions of Western Medicine that a given disease will appear essentially the same in different people and that an effective treatment will work in essentially all patients; actually, different subjects diagnosed with diabetes, dementia, cancer or other conditions, or people infected with a given virus or bacterial pathogen each express different clinical manifestations and will respond differently to particular treatments. Personalized medicine is a necessary consequence of entropy-selected diversification.\n\nBiologic diversification enables living systems to keep ahead of entropy—the failure or loss of one arrangement or interaction can be compensated by alternatives. And, diversity is not a onetime thing; as we shall discuss shortly, ongoing diversification is one of the outcomes of sexual reproduction.\n\nAbove, we noted that a stable species, like the human species, is poly-determined: the species emerges from variable dynamic interactions between its varying component organisms; different human organisms with their different microbiomes and different states of structure and activity interact to constitute a single species. From this perspective, a species can be likened metaphorically to an attractor103; an attractor is mathematically defined as a set of number values towards which a dynamical system will tend to evolve. Similarly, despite their individual differences, the collective of organisms belonging to a given species will tend, on the whole, to exhibit a characteristic species profile; humans will be humans irrespective of their age, gender, ethnicity, and other individual differences; dogs will be dogs despite their various strains; different peach trees will bear peaches, and so forth. A poly-determined species remains stable despite the differences between its component organisms. Diversification and poly-determination endow a species with robust behavior and continuity that actually exploits entropic variation.\n\nSNPs and other diversifications intrinsic to healthy genomes suggest that single random mutations are not likely to change gene functioning; this resistance to phenotypic change indicates that one or a few mutations are not likely to affect an organism functionally. Indeed, healthy tissues tolerate large numbers of mutations97; the development of a tumor, as we pointed out above, arises from critical “driver mutations”104. Since healthy genomes are intrinsically diverse, a meaningful evolutionary change in a given species is also likely to withstand small genetic changes and respond primarily to a combination of many genetic changes or to a critical “driver” mutation affecting a key phenotypic character.\n\nAccording to this reasoning, evolution need not advance only in small steps, as taught by Darwin, but may also progress in large jumps, or saltations105. Darwin favored evolution by small increments for fear of encouraging creationist and intelligent design thinking. The roles of diversification and poly-determination in taming entropy, however, provide a physical rationale for saltatory evolution. Survival-of-the-fitted explains saltations as arising from the accumulation of multiple invisible changes that ‘finally’ exceed a certain quantum threshold; indeed, such changes can involve interactions with other organisms, materials or processes leading to entirely new entities. Phenotypical saltation poses no threat to evolution theories based on small changes.\n\n\nSexual reproduction and diversification\n\nSexual reproduction, a defining characteristic of multi-cellular species, entails the random mixing in the offspring of the gene alleles derived from different parental organisms. Gene mixing by sexual reproduction guarantees that no multi-cellular organism will sexually transmit its exact DNA genotype to the next generation. No matter how great the Darwinian fitness of one parent, the baby will never inherit that fit genotype alone – a new baby always inherits a random mix of half the alleles of each of its parents. Sexual reproduction ensures that genetic fitness is not transmissible intact from one generation to the next. By constantly reshuffling genomes, sexual reproduction frustrates the advancement of Darwinian perfection, as assumed to result from the reproductive success of the fittest individuals1.\n\nIn his book106, Graham Bell asserts that “Sex is the queen of problems in evolutionary biology”. The problem is still open; it is clearly expressed by Burke and Bonduriansky107:\n\n”Why sexual reproduction is so widespread despite its substantial costs is one of the most important unsolved problems in evolutionary biology. Because sex is associated with numerous short-term costs that asexual organisms mostly avoid, theory predicts that asexual or parthenogenetic lineages should outcompete and outnumber sexual lineages, all else being equal. However, paradoxically, sex is the dominant mode of reproduction in many lineages of complex eukaryotes.”\n\nThe community has not yet agreed on a mechanism consistent with survival-of-the-fittest struggle and the debate continues - see 108,109. However, sex, from the perspective of fittedness, is not a problem for evolution, but a solution.\n\nSexual reproduction eschews the optimum, while it creates continuous innovation – we dare say that the optimal response to entropy is to avoid a single optimum. Darwin himself grappled with the problem of explaining sex110. In any case, diversification is programmatically linked to the renewal of members of a species by sexual reproduction. An individual’s offspring automatically diversify the species in each generation. Quite simply, living systems are not constrained by a single “optimum” species genotype; they are programmed to avoid a single optimum; diverse arrangements retard destruction by entropy. Continuous diversification is obviously beneficial, but it is not an obvious outcome of a survival-of-the-fittest mechanism.\n\nOf course, we must not ignore the function of sex in bonding, cooperation, family, and pleasure47—evolution has programmed vertebrates like us to enjoy interactive life. Repeated sexual interactions thwart entropy even when they don’t necessarily generate renewal.\n\nSexual encounters between organisms of the same sex, widely observed in over 1,500 species of organisms, contradicts a basic assumption of Natural Selection108: homosexual interactions produce no offspring; hence there is no survival-of-the-fittest mechanism for transmitting their fitness to future generations of the species. Survival-of-the-fitted, however, can explain the existence of homosexual behavior as a social interaction that enhances cooperative bonding, contributes to the environmental niche of the evolving group and restrains entropy. Thus it is not surprising that a potential for homosexuality may indeed be genetically maintained within a breeding population111.\n\n\nThe theory of neutral evolution\n\nThe theory of neutral evolution aims to explain the development within a species of genetically encoded traits that do not seem to make significant contributions to survival and reproduction; neutral evolutionary characters somehow are free of selection by a survival-of-the-fittest mechanism. Most neutral evolution has been described at the molecular level and includes molecular polymorphisms, variations in protein or nucleic acid sequences between organisms112–114.\n\nKimura proposed that the mechanism of neutral evolution was based on random mutations that did not affect protein functions; contending selectionists argued that some sort of selection must have taken place115. Most evolutionary biologists today would agree with Kimura that neutral evolution results from random genetic mutation and random gene drift114.\n\nThe theory of neutral evolution marks a major distinction between Darwin’s theory of Natural Selection and our theory of entropic selection: clearly, there exist genetic traits that manifest no obvious impact on individual survival and reproductive success. However, we reason that all manifestations of evolution must have undergone selection by restraint of entropy. The existence of universal entropy obliges all outcomes of evolution to undergo selection—there can be no neutrality; all persisting living systems and their component parts must withstand entropic dissolution. Evolution cannot be neutral in the face of entropy; all existing variants must, in some way, help maintain stability, or at the very least not totally sabotage stability.\n\n\nSpecies and ecosystems\n\nA universal outcome of evolution is the organization of organisms into higher scale species and ecosystems. Species and ecosystems are like corporations – a corporation is defined as an organization whose continuous existence is independent of the turnover of its individual members116. Member organisms (and their molecules) come and go, but the species as a whole persists. Each organism is a transient subunit in the corporate body of a species and an ecosystem.\n\nThe organization of multi-cellular organisms into species is so plainly obvious that one might hesitate to enquire in public about its evolutionary origins. Darwin’s book The Origin of Species by Natural Selection1, despite its title, did not explore the origin of species as a universal adaptation but rather the origin of the local evolution of one species into another species. The existence of species was apparently seen as an unquestionable given – just as emperors are assumed to be wearing clothes, individuals are assumed to come dressed in species. Let’s penetrate the obvious to ask what might be the evolutionary advantage of the higher scale organization of individual creatures into many different species.\n\nThe word species originates from the Latin term designating entities that look alike (from specio – I see). Biologists, in former times, did not know about DNA. We now use the term species in eukaryote biology to designate a collective of creatures that can breed mutually by sexual reproduction thanks to their similar DNA117. Not all members of a species need to actually reproduce and they may reproduce only with certain other members of the species, and only at particular times; but they could do so at least potentially. The term species is also applied to prokaryotes, but only some prokaryotes reproduce sexually and there is no functional definition of prokaryote species; for practical purposes, similarity of 16S rDNA sequences is arbitrarily used to define bacterial species118. We shall not discuss here the effect of entropic selection on the evolution of prokaryote species.\n\nThe linking of species to sexual reproduction in eukaryotes is functional; a species thwarts entropy by the continuous rebirth of its diverse members despite their inevitable death; a species, by way of sexual reproduction, also reshuffles its renewal continuously. Furthermore, sexual reproduction, by species-specific exclusivity, constitutes a barrier against invasion of the species by members of a different species; thus sexual reproduction creates a boundary, as it were, that encloses the species.\n\nAnother defining function of a species is the ability of its member organisms to construct and thrive in a shared environmental niche; the members of a species, in principle, can each perform the interactions that characterize the species – the collective of members interacts to exert an amplified effect on their shared environment. In addition, malfunctioning members of the species are replaceable.\n\nMoreover, members of a species can specialize in particular functions: For example, male and female organisms perform different roles in social activities, group defense, habitat construction, and so forth; alpha males, for example, maintain a pack’s genetic vigor; experienced adults can educate the newborns; musically gifted persons help maintain social cohesion. So the packaging of organisms into species enables renewing, collective existence and functional similarity along with environmental continuity and individual specialization. A species can be understood as a stable attractor of diverse, continuously turning over organisms. Different species cannot reproduce across their borders, but they may link together to form ecosystems.\n\nAn ecosystem is a corporation of multiple species and environments that efficiently channels flows of energy through a biosphere network119. In an ecosystem, the byproducts of one species can serve as a resource for another species linked in the network. The fecal and urinary excretions of herbivores fertilize the earth and so enable the existence of all the creatures that make their living from soil or from plants; weak and dying prey animals sustain their predators, who, in turn, maintain the health of the prey species and of the environment; dead animals feed many other species along with myriads of prokaryotes who renew the flows of vital chemicals throughout the biosphere. Indeed, the dead body of one whale on the floor of the deep ocean – whale fall – can form the nucleus of an ecosystem that sustains many species including giant isopods, lobsters, shrimp, prawns, hagfish, crabs and others for decades if not for a century120. See the whale as a conveyance for harvesting the ocean surface for sun-dependent forms of life and bringing, in the molecules of its dead body, the energy-rich output of the sun to the darkness of the ocean depths. Ecosystems transform sunlight and other disordered energy sources into the staff of life of the biosphere as a whole. Recall that atmospheric oxygen, essential to many forms of life, is the toxic waste of photosynthesis121.\n\n\nMaintenance and repair\n\nIndividual living cells and multi-cellular organisms, in responding to entropy, have evolved subsystems that function to repair damage inflicted by accidents, noxious parasites, injury or just plain wear-and-tear. Much of the genetic endowment of individuals and species has evolved to encode molecules and processes directed to repair. Heat shock proteins and other stress responses have been conserved since the onset of prokaryote life122; DNA repair processes keep genomes healthy123; immune systems of various types have evolved in all species of organisms, including prokaryotes124. Life has been busy repairing itself since its beginning. We need not get into the details of these and other subsystems of maintenance; their very existence highlights the daily struggle of life with entropy.\n\n\nEnvironments and co-evolution\n\nAny discussion of evolution must include environmental niches. The niche is that segment of the environment within which a species lives and interacts; the niche includes the material, biological and behavioral factors that house the species125,126. The word niche derives from the Latin nidus, a nest.\n\nClassical Darwinian theory has tended to present environments as fixed arenas that provide only limited amounts of the energy, materials and space needed for survival; as a consequence, organisms are obliged to struggle amongst themselves for a livelihood1. Survival-of-the-fittest, as we said, is assumed to reward the individual winners with a larger piece of the fixed environmental pie and with a reproductive advantage that disseminates their more fit genes. Darwin’s “tangled bank” of life arises, he concludes, “from the war of nature, from famine and death.” However, we see a different picture when we view the environmental niche from the perspective of fittedness: a life-sustaining environment extends beyond serving as a mere source of energy, matter and living space; an enabling environment includes interacting networks of organisms and ecosystems – effectively, the essential environment is itself a tangled web-of-life126.\n\nIn contrast to Darwinian reasoning, survival does not depend merely on the ability of a cell, organism, or species to grab a maximal amount of physical energy, matter and space. Survival is not mere sustainability; survival requires that a living entity be integrated within biological and material networks that have evolved to convert entropic disorganization into organized construction. Failure to fit, or the loss of its niche, dooms the cell, organism or species to unrestrained dissolution by unrelenting entropy. Fittedness means fitting in.\n\nIn contrast to classical survival-of-the-fittest thinking, successful organisms and species find or actually construct their own tailored niches. Consider the human species: the human and the chimpanzee diverged from a common great-ape ancestor about 6 or 7 million years ago127. But we evolved without competing with great apes for the same environmental niche. An objective observer would conclude that we humans arose as failed apes. We simply left the apes to their natural environments and invented our own niche. The species of plants and animals that we have domesticated (wheat, maize, rice, fruits, vegetables, chickens, ducks, horses, camels, sheep, goats, and the rest) owe their success to having “found” us to serve as their environments. Deer, barn owls, raccoons, rodents, falcons, pigeons, bats, cockroaches and other feral creatures have constructed their unique niches in our belfries, homes, gardens, farms, suburbs, cities, golf courses, and more; various types of bacteria have adopted our hospitals and our bodies.\n\nWe humans evolved human culture as our niche; but niche building is not exclusive to us128,129: beavers are famous for damming lakes to build their own pools; soils are created by combined activities of prokaryotes of various types, plant roots, ants, worms, moles and other creatures. Predator and prey mutually signal each other and organize the kill to maintain both species130,131. Even bacteria build niches in particular organs in our bodies by their metabolic products and their adjustment of oxygen and acid concentrations132,133. Environment-building has been a feature of life on earth from earliest evolutionary times. Levins and Lewontin put it this way:\n\nIt is impossible to avoid the conclusion that organisms construct every aspect of their environment themselves. They are not the passive objects of external forces, but the creators and modulators of these forces. The metaphor of adaptation must therefore be replaced by one of construction. . .34\n\nIf, indeed, every species fashions its own environment, we may conclude that no two species ever occupy exactly the same environment. Different species may share a physical space in land, sea or air, but each species interacts in its own unique way with that space and with its other tenants. Life converts generic “space” into a tailored “place”, a private address, as it were (see discussion in 134). In other words, the attractor that stabilizes a living system is not the species alone, but the species together with its co-evolving niche.\n\nThe evolution of any species can take place only if its fittedness, selected by retarding entropy, can be passed on to future generations of offspring. The problem is that the microbiome component of a holobiont is acquired independently of host genetic reproduction; how then can the fittedness of a holobiont group be transmitted somatically to holobiont offspring? The answer, we reason, is by way of niche construction and co-evolution.\n\nThe niche of each species has co-evolved with the species in many ways, some of which enable faithful transmission of the collective of microbiome elements to the next generation—not genetically, but by a combination of somatic conditions. In plants, the seeds of a species germinate in particular types of soil suited to the species—suitable soils contain microbiomes needed by the species, within the soil itself or present in adult plants growing close to the seedlings; organisms living in water are bathed by flowing concentrations of suitable microbiomes; eggs and chicks are nested in close microbial contact with their mother birds; newborns emerging from birth canals acquire mother’s microbiomes in the course of being born8.\n\nThe environmental foods that have co-evolved with resident organisms can provide needed microorganisms: humans, for example, are attracted to fermented food stuffs rich in suitable organisms such as lactobacilli, which also help preserve the food; animals transmit microorganisms when they groom and fondle each other135; courting rituals and sexual interactions can transmit microbiomes; some species eat feces; attractant odor organs are usually located at oral and anal orifices; attractive odors of men and woman are produced though fermentation of secretions by skin bacteria. The reader may supply additional examples of environments and behaviors that transmit microorganisms among holobionts belonging to one or more species.\n\nOf course, changes in the environment of a species can lead to the loss of an essential element of a species microbiome: Martin Blaser has pointed out that the overuse of antibiotics has contributed to serious pandemics of chronic diseases (diabetes, obesity, heart disease, liver disease, and others) by eliminating from the human environment critical microbiome organisms136. Our discovery of antibiotics and their extensive over-use has changed for the worse the environment of the human species, and that of other species too. We are paying a high price, and, if we don’t act to characterize and preserve essential microbes, the price will only rise.\n\nSpecies construct collective niches that emerge from the individual niches constructed by the organisms that compose the species. Human culture exemplifies embedded niches: each of us (our microbiomes included) constructs our own dynamically evolving niche as we progress through life, from birth, development, education, occupation, family, and so forth. Each personal niche is fashioned with regard to collective cultural niches that precede, accompany and supersede us—language, systems of belief, values, polity, personality types, science, technologies, diet, and so forth. Niches are multi-scale fractals: niches within niches within niches. Life cannot rest at one scale; entropy must be dealt with at all habitable scales: molecular, genetic, metabolic, cellular, organismal, environmental, etc.\n\nThe orderly dynamics of the physical environment impose entropy-restraining order on life. Many of the most formative arrangements and processes of living systems are organized by biological clocks – including metabolic reactions, cycles of nutrients, courting and fertility, conception, growth, development, migrations, aging, regeneration and repair, illness and death. Life is organized by recurrent time and the clocks of life are tuned to the cycles of nature manifest in the repetitive movements of the earth, moon and sun, which are expressed through cycles of precipitation, tides, seasons, temperatures, weather, dark and light, and even the frequencies of light. Living systems are also tuned to long-term manifestations of physical reality such as the movements of land masses, volcanic activities, and magnetic pole migrations. The details are far beyond the scope of this paper; we only need note that living systems dance to organized cadences of physical reality. The interactions of life have thus evolved to restrain entropic disorder by their linkage to the enduring physical clocks and order of material nature.\n\n\nInvaders and extinctions\n\nEvolution, we said at the outset, requires selection of some variations for propagation and others for destruction. At the level of the organism, propagation results from fitting into a network of supporting interactions; the destruction of the unfitted is carried out by the inexorable process of unrestrained entropy. At the level of the species, propagation is effected by construction of a co-evolving environmental niche supported by a flow of ecosystem energy. What causes the extinction of a species? Species can go extinct by the loss of their niche. For example, the dinosaurs underwent extinction when their niches were deconstructed in the wake of the earth’s collision with an extra-terrestrial body137. The mammals have replaced the dinosaurs, not by struggling with them, but by constructing new niches with a variety of more effective technologies. The evolution of the organs of speech by H. sapiens may have enabled modern humans to replace the Neanderthals by constructing a more advanced social communications niche138. Likewise, Internet shopping is replacing big-box retail stores139 because Internet corporations like Amazon have evolved to exploit advanced Internet communications technology (a new mutation, as it were) to construct a new niche environment; the environments of many big-box stores are now drying up following “collision” with the Internet. Niches are found (or made) and niches are lost (or deconstructed). The loss of a niche means the loss of its species; the loss of a person’s home niche can generate a homeless person whose niche becomes a street corner.\n\nAn invader species is one that enters and thrives in a locale in which it had not previously evolved; often the success of an invading species leads to the decline or loss of other species at one time “native” to that locale. In Israel we have witnessed the spread of Grey Crows from the countryside into human neighborhoods, associated with the disappearance of the local populations of many birds including Bee-eaters; similarly, Myna birds, escaping from the cages of bird fanciers, have proliferated at the expense of house Sparrows, that themselves were invaders from England accompanying British soldiers. Perhaps the most successful invader of once natural habitats have been modern humans, who came out of Africa many tens of thousands, perhaps hundreds of thousands of years ago to take over the planet. Casting the observed events as a struggle between species followed by victory and conquest by one of them does not negate the broader view of this reality as a deconstruction of native networks of interactions in the wake of the construction of new niche interactions. Networks of interaction do not necessarily “compete” with each other, and many such networks in fact continue to coexist.\n\nObviously, niche deconstruction is not the only way to extinction; a species can be eradicated by a viral or bacterial pandemic, or by the dissemination of some toxic substance, or by predators, or by atomic reactions, and so forth. Entropic mishaps have no limitations.\n\nFittedness shoulders human culture with responsibility – we humans, who uniquely exercise conscious choice, are accountable, at least to ourselves, for our role in the state of the biosphere. Even when the human species numbered only some few millions of hunters and gatherers scattered about the earth, we appear to have eradicated many other species using only fire and weapons of stone and wood140. Now we number in the billions, and are armed with ever-advancing technologies. Without intending to deconstruct the \"natural\" environment, our domestication of a small number of species has markedly affected the biosphere: the biomass of chickens by now far outweighs the combined biomass of all wild birds and the biomass of cattle outweighs by many fold the biomass of all other mammals combined141. The states of the earth and the biosphere are in our hands.\n\nAccording to classical Darwinian thinking, to be fit is to win in the struggle to exploit an ever larger slice of a fixed environmental pie. The environment was viewed as a battle ground on which we, and the rest of the earth’s creatures, must fight to survive. Unfortunately, liability for undue exploitation is not inherent in the concept of Natural Selection. One may wonder whether survival-of-the-fittest thinking may have played a role in the ideology of unrestrained exploitation of the earth for economic and political “gain”. Our niche encompasses the world; unbridled “gain” will deconstruct us all.\n\n\nThe emergence of complexity\n\nComplexity is easier to recognize than it is to define. But however one defines complexity, it is evident that a biosphere that includes multi-cellular organisms is more complex than a biosphere composed of prokaryotes only. Indeed, increasing complexity seems to evolve relentlessly; see for example the quote “Everybody seems to know that complexity increases in evolution” stated by by McShea142. The general increase in complexity is balanced by evidence for particular reductions in complexity, as exemplified in genome reductions discussed by Wolf and Koonin143; indeed, salmon parasites show an entire loss of the mitochondrial genome and associated nuclear genes required for its transcription144.\n\nComplex species repeatedly have undergone mass extinctions145. Yet evolution, as it were, picks up the simple leftovers and starts anew accumulating complexity. Why is this so? Complex systems are not more fit in Darwinian terms than are simpler systems; simple systems seem in fact to be much fitter; bacteria have survived all the extinctions suffered by the more complex organisms and will be more likely than us to survive global warming or nuclear catastrophe146.\n\nEntropic selection is compatible with the idea that complexity itself fuels more complexity147. Prevailing energy and entropy, along with biological diversification, continuously drive the emergence of new mutations, combinations, interactions and functional variations. A given molecular entity will tend to increase its range of interactions over evolutionary time; the oxytocin molecule, for example, has evolved to perform a range of functions in bonding, sexual and social behavior, metabolism, inflammation and the control of stress and aggression47. In each case the oxytocin molecule has added new molecular partners and so, over time, it has participated in new and more complex network interactions. Heat shock stress proteins first evolved as chaperones in primitive bacteria; they have gone on to serve as chaperones in eukaryote cells and in multi-cellular organisms, and have evolved into important signal molecules for the mammalian immune system148.\n\nEvolution seems to have created increasing complexity by adding innovations to its existing molecules, cells, organisms and species. Certain aspects of this general process have been studied under the concepts of preadaptation or exaptation149. Round worms express some 20,000 genes150 compared to a similar number of genes expressed in humans151. These numbers are estimates subject to revision. Nevertheless, it is clear that evolution has proceeded by establishing increasingly more varied interactions and functions for a given number of genes already present in its primitive toolbox; chimpanzees and humans share more than 95% of their genes127—the greater complexity of humans, expressed in language and global culture, emerges from more complex uses for common genes. Advancing complexity seems to have emerged both from novel regulatory DNA sequences and even from gene loss and not necessarily from entirely new genes152,153.\n\nAutocatalysis of complexity is also expressed in the increased likelihood that a random variation or mutation in a gene, molecule, cell, or organism will by chance find a fit in a large landscape of different interacting entities. A complex infrastructure will tend to provide opportunities to increase its complexity; consider the ever-increasing complexity of the Internet’s evolution before our eyes. Indeed, the jump of viruses like Ebola, influenza and corona from animals into humans has resulted from the advancing complexity of human culture.\n\n\nThe question of a unit of evolutionary selection\n\nThe designation of a \"unit of evolutionary selection\" is an attempt to pinpoint the primary substrate upon which the process of evolution directly acts; the unit of evolution defines the seminal entity whose evolution generates the life we see around us. Which entity is the prime mover of evolution and which entities are secondary consequences? In the beginning, Darwin proposed that individuals are the primary units driving evolution; according to Darwin the character of any species is a secondary result of the procreative success of the fittest individuals engaged in the struggle for survival1. Dawkins, in contrast, argues that the gene is the primary replicator unit of evolution; individuals are merely transient phenotypic vehicles that bear the unit genes37. Lewontin includes the whole spectrum of biological entities as units of evolution – genes, individuals, groups and species; according to this view, life at all its scales evolves together154. Zilber-Rosenberg and Rosenberg proposed the holobiont as a unit of selection6. Views and debates on the issue are reviewed by Lloyd in 155; the discussions nevertheless continue 11,156. These designations of a unit or units of evolution assume that survival-of-the-fittest is the driving mechanism of evolutionary change; how do we define the unit of evolution from the perspective of survival-of-the-fitted?\n\nAbove, we have argued that the mechanism of universal evolution involves the interplay of three universal properties of matter: energy, entropy and interaction. These three properties exert effects on the evolution of living entities across all scales – from molecules through ecosystems. From this perspective, no single entity is “the unit” of evolutionary selection. Survival-of-the-fitted is a mechanism that makes no distinctions between any of the entities susceptible to evolution; life is the expression of its energy-dependant interactions, and all interactions must pass the tests of entropy. We would agree with positions that argue that the whole spectrum of life’s entities takes part in evolution. But we see particular entities—from cells to ecosystems—as secondary manifestations of the primary evolution of networks of interactions. If one wishes to maintain the concept of a unit of evolutionary selection, than the unit is not a particular entity like a gene or individual organism, but rather is the dynamic process of interaction itself. Indeed, the phenotype of a gene is formed by the interactions that generate gene expression, translation into proteins, post-translational modifications and all the rest. The organism, too, is a dynamic product of all its internal and external interactions. We may perceive entities, but all are snapshots of dynamic interactions. The progenitor of evolution is interaction.\n\n\nTheories of evolution influence human behavior\n\nWhether or not Natural Selection is a “law of nature” is controversial among biologists157,158; nevertheless, extreme segments of Western Culture have usurped survival-of-the-fittest to justify an ideology of conquest and dominance. Lamentably, Natural Selection, at one time or another, has been invoked to justify colonialism, racial purity, eugenics, genetic cleansing, dictatorships, unfettered capitalism, unequal rights, and other harsh dissonances (see, for example, 159,160). Obviously, survival-of-the-fittest is a scientific theory and not a benchmark for human behavior; nevertheless, Natural Selection has been hijacked by agendas of exploitation.\n\nUpdating the mechanism of evolution, as we have done here, to highlight the roles of cooperation, interaction and diversity throughout biology might serve to encourage rational human behavior in the realms of governance, economics and social justice.\n\nThe discovery that all multi-cellular organisms are holobionts raises a paradox; at the biological level, there are no individuals; living systems, nevertheless, do manifest group individuality. Yet, the psychological core of each human is his or her feeling of an exclusive existence expressing private thoughts, feelings, and a specific history of being and relationship. Our internal perception of uniqueness is confirmed independently by our individual social and legal responsibilities. Can we resolve the seeming contradiction between our realities as both biological groups and functioning individuals?\n\nLook at it this way: our psychological and institutional individuality, like our body, is created by group interactions and relationships, not merely with microbes, but with the people and the world in which we reside; our identity is the outcome of our history of bonding with others. Echoing Levins and Lewontin34, we construct our niche by the nature of our interactions—we and our worlds co-evolve. We achieve a self by constructing a shared, interactive environment. Thy neighbor is part of thy evolving self.\n\n\nReconciling survival-of-the-fitted with Darwinian survival-of-(only)-the-fittest\n\nPhysicists point out that life, like all the rest of the material universe, must accommodate the Second Law of Thermodynamics and the phenomenon of entropy25,29. Here, we have reasoned that the evolution of life, a dynamic expression of life itself, must also accommodate entropy. Consequently, stable outcomes of the evolutionary process must provide some solution to the spontaneous dissolution of order; entropy, in one way or another, must act as a selective factor in evolution – it would not be reasonable to conclude otherwise. What about the idea of Natural Selection, a cornerstone of modern biology?\n\nIt is conceivable that different manifestations of evolution might be assigned to a combination of fitness (Natural Selection) and fittedness (Entropic Selection). Entropy would seem to account for the more universal adaptations of evolution we have discussed here. In contrast, Natural Selection might drive more local, species-restricted adaptations. Certainly, the quantitative assessment of each mechanism would be different: the advance of evolution by individual fitness would require measurement of the birth rates of generations of descendants of particular test individuals – a difficult, if not impossible task; fittedness based on the strength of ongoing cooperative interactions could be measured, at least at the molecular level, by the amount of energy needed to disrupt the interaction: For example, the strength of the interactions that hold together dsDNA strands is reflected in the amount of heat that has to be applied to disrupt the interaction, as in the PCR reaction161. In any case, a fresh look at the mechanisms of evolution is in order.\n\n\nAnalysis and modeling\n\nA theory can be invoked to explain any phenomenon; a theory is scientific when it proposes a platform for continued rational analysis and when it can support testable predictions. How may we proceed to analyze survival-of-the-fitted?\n\nSurvival-of-the-fitted fits generally into the realm of systems biology, including dynamic complex systems and their attractor states. The perspective and the methods of systems biology go beyond a linear chain of discrete causes and effects; advancing information technologies have extended the scope of biology research, enabling analyses of complex interactions that generate dynamic self-organizing systems. “Big data” are collected, defined and analyzed for associations involving networks, modules, emergent properties and other organizational structures and principles. Systems’ understanding emerges from simulations and mathematical models162,163. The experiments of evolution have been and are being done by nature, analyses of the results need human computation. A system’s biology of evolution is in the offing.\n\nHere, we propose some experiments and predictions, not all of which are currently feasible, to test survival-of-the-fitted:\n\n1. Germ-free mice or rats are raised behind barriers and deprived of gut microbiomes, but they can be reconstituted by feeding them with defined clones of bacteria; one group could be fed with a single monoclonal gut bacterium and other groups could be fed with combinations of diverse bacterial clones. The survival-of-the fittest paradigm would predict that the microbiomes developing in the groups of mice fed with diverse clones of bacteria would evolve over time into microbiomes populated with only a single clone of the fittest bacterium. The microbiomes in the animals fed with a single clone, in the absence of competition, would not evolve.\n\nIn contrast, the survival-of-the-fitted paradigm would predict that the microbiomes of the animals fed only with a single monoclonal species of bacteria would in time respond to entropy to evolve into a diversity of bacteria expressing different genes. The animals fed a diversity of bacteria would continue to express a diverse microbiome. Thus, the contending fitness and fittedness theories of evolution would each lead to contradicting predictions.\n\n2. Evolution will take place in the absence of competition and in environments with unlimited resources; contrary to Darwinian teachings, a struggle for fitness is not required because entropy prevails in the best of worlds. Diversification can be tested under controlled experiments in vitro164. Monoclonal, initially invariant yeast, bacteria or C. elegans could be grown under conditions of “optimal” nutrition and density in monitored cultures and observed for the evolution of genetic and phenotypic diversification in the absence of struggle.\n\n3. Systems that foster diversity and individuality will thrive better than systems limited to an “optimal” archetype; cooperation and symbiosis will emerge in diversified collectives. This prediction can be tested today in a variety of systems at various scales, from single cells to societies and cultures.\n\n4. Sexual reproduction (or an equivalent system of reproducing, programmed diversification) will evolve in multi-cellular living systems. Testing this might require the development of novel in vitro culture systems.\n\n5. Complexity will increase automatically, even in an in vitro culture of initially uniform populations. Observations of new interactions could be made using long term culture systems.\n\n6. Life on other planets will be seen to have evolved, provided that entropy holds sway there.\n\n7. All living systems anywhere will evolve programmed renewal.\n\nPredictions # 6 and # 7 will require the development of suitable probes of habitable planets distant from earth. Perhaps extraterrestrial life may one day be studied at a distance by telemetric analysis of metabolism or other manifestations of life.\n\nFinally, we would predict that the well-being of the biosphere will be aided by realizing intelligent interactive cooperation and shunning selfish struggle. This prediction is now being tested by observing our changing world, obviously without a control group. Let us each work to fulfill this prediction.\n\n\nData availability\n\nNo data is associated with this work.", "appendix": "Acknowledgements\n\nThe authors greatly appreciate the discussions, comments and suggestions by many colleagues from various fields and backgrounds: biology—developmental, molecular, micro-, systems, and evolutionary; bioinformatics; physics; chemistry; computer science; cognitive sciences; philosophy of science; and medicine.\n\n\nReferences\n\nDarwin C: On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. Annals and Magazine of Natural History. John Murray, 1860; 5(26): 132–143. Publisher Full Text\n\nStearns SC: Principles of Evolution, Ecology and Behavior. Open Yale Course EEB 122. 2009. Reference Source\n\nMayr E: Cause and effect in biology. Science. 1961; 134(3489): 1501–1506. PubMed Abstract \| Publisher Full Text\n\nKoonin EV: The Logic of Chance: the Nature and Origin of Biological Evolution. FT press, 2011. Reference Source\n\nMethé BA, Nelson KE, Pop M, et al.: A framework for human microbiome research. Nature. 2012; 486(7402): 215–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZilber-Rosenberg I, Rosenberg E: Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol Rev. 2008; 32(5): 723–735. PubMed Abstract \| Publisher Full Text\n\nRosenberg E, Zilber-Rosenberg I: The hologenome concept of evolution after 10 years. Microbiome. 2018; 6(1): 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenberg E, Zilber-Rosenberg I: The Hologenome Concept: Human, Animal and Plant Microbiota. Springer, 2014. Reference Source\n\nBhattarai Y, Kashyap PC: Germ-free mice model for studying host–microbial interactions. In Mouse Models for Drug Discovery. Springer, 2016; 123–135. Publisher Full Text\n\nMoeller AH, Suzuki TA, Phifer-Rixey M, et al.: Transmission modes of the mammalian gut microbiota. Science. 2018; 362(6413): 453–457. PubMed Abstract \| Publisher Full Text\n\nStencel A, Wloch-Salamon DM: Some Theoretical Insights Into the Hologenome Theory of Evolution and the Role of Microbes in Speciation. Theory Biosci. 2018; 137(2): 197–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott-Phillips TC, Laland KN, Shuker DM, et al.: The Niche Construction Perspective: A Critical Appraisal. Evolution. 2014; 68(5): 1231–1243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaddington CH: Paradigm for an evolutionary process. Towards a theoretical biology. 1969; 2: 106–128. Publisher Full Text\n\nGould SJ: Darwinism and the Expansion of Evolutionary Theory. Science. 1982; 216(4544): 380–387. PubMed Abstract \| Publisher Full Text\n\nLaland KN, Uller T, Feldman MW, et al.: The Extended Evolutionary Synthesis: Its Structure, Assumptions and Predictions. Proc Biol Sci. 2015; 282(1813): 20151019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaland K, Uller T, Feldman M, et al.: Does Evolutionary Theory Need a Rethink? Nature. 2014; 514(7521): 161–4. PubMed Abstract \| Publisher Full Text\n\nCohen IR: Tending Adam’s Garden: evolving the cognitive immune self. Elsevier, 2000. Publisher Full Text\n\nCohen IR: Informational Landscapes in Art, Science, and Evolution. Bull Math Biol. 2006; 68(5): 1213–1229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen IR: Updating Darwin: Information and Entropy Drive the Evolution of Life. F1000Res. 2016; 5: 2808. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoige CA, Day T: A typology of undergraduate textbook definitions of heat across science disciplines. Int J Sci Educ. 2012; 34(5): 677–700. Publisher Full Text\n\nMerriam-Webster Dictionary: On-line dictionary. 2019; (accessed 6/2019). Entry for “Energy”.\n\nWehrl A: General properties of entropy. Rev Mod Phys. 1978; 50(2): 221. Publisher Full Text\n\nOxford English Dictionary (OED): Online Dictionary. 2019; (accessed 6/2019). Entry for “Entropy”.\n\nMartin JS, Smith NA, Francis CD: Removing the entropy from the definition of entropy: clarifying the relationship between evolution, entropy, and the second law of thermodynamics. Evol Educ Outreach. 2013; 6(1): 30. Publisher Full Text\n\nSchrödinger E: What is life? The physical aspect of the living cell and mind. Cambridge University Press Cambridge, 1944. Reference Source\n\nFeynman R: The Feynman Lectures. 1963; Accessed May 2020. Lecture 1: Atoms in Motion. Reference Source\n\nCohen IR, Harel D: Explaining a complex living system: dynamics, multi-scaling and emergence. J R Soc Interface. 2007; 4(13): 175–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoltzmann L: The second law of thermodynamics. In: Theoretical Physics and Philosophical Problems: Selected writings. Springer Science & Business Media, 2012.\n\nAtkins PW: The second law. Scientific American Library, 1984.\n\nBerry S: Entropy, irreversibility and evolution. J Theor Biol. 1995; 175(2): 197–202. PubMed Abstract \| Publisher Full Text\n\nStyer DF: Entropy and evolution. Am J Phys. 2008; 6(11): 1031–1033. Publisher Full Text\n\nDemetrius LA: Boltzmann, Darwin and directionality theory. Phys Rep. 2013; 530(1): 1–85. Publisher Full Text\n\nElena SF, Lenski RE: Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nat Rev Genet. 2003; 4(6): 457–69. PubMed Abstract \| Publisher Full Text\n\nLevins R, Lewontin RC: The Dialectical Biologist. Harvard University Press, 1985. Reference Source\n\nGleick J: Genius: The life and science of Richard Feynman. Vintage, 1993. In Prologue. Reference Source\n\nRovelli C: Carlo rovelli — all reality is interaction. 2017; Accessed May 2020. WNYC Radio Program. Reference Source\n\nDawkins R: The Selfish Gene. Oxford university press, 1976. Reference Source\n\nDawkins R: The Extended Phenotype: The Long Reach of the Gene. Oxford University Press, 1982. Reference Source\n\nHilbe C, Wu B, Traulsen A, et al.: Evolutionary performance of zero-determinant strategies in multiplayer games. J Theor Biol. 2015; 374: 115–124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMargulis L: Symbiosis in Cell Evolution: Life and its Environment on the Early Earth. W. H. Freeman, 1981. Reference Source\n\nEmbley TM, Martin W: Eukaryotic evolution, changes and challenges. Nature. 2006; 440(7084): 623–30. PubMed Abstract \| Publisher Full Text\n\nMbeunkui F, Johann DJ Jr: Cancer and the tumor microenvironment: a review of an essential relationship. Cancer Chemother Pharmacol. 2009; 63(4): 571–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaser MJ: The microbiome revolution. J Clin Invest. 2014; 124(10): 4162–4165. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaser MJ, Webb GF: Host demise as a beneficial function of indigenous microbiota in human hosts. mBio. 2014; 5(6): e02262–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinross JM, Darzi AW, Nicholson JK: Gut microbiome-host interactions in health and disease. Genome Med. 2011; 3(3): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeigh EG Jr: The group selection controversy. J Evol Biol. 2010; 23(1): 6–19. PubMed Abstract \| Publisher Full Text\n\nOnaka T, Takayanagi Y, Yoshida M: Roles of oxytocin neurones in the control of stress, energy metabolism, and social behaviour. J Neuroendocrinol. 2012; 24(4): 587–598. PubMed Abstract \| Publisher Full Text\n\nGruber CW: Physiology of invertebrate oxytocin and vasopressin neuropeptides. Exp Physiol. 2014; 99(1): 55–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarian BJ, Poutahidis T, DiBenedictis BT, et al.: Microbial lysate upregulates host oxytocin. Brain Behav Immun. 2017; 61: 36–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRizzolatti G, Craighero L: The mirror-neuron system. Annu Rev Neurosci. 2004; 27: 169–192. PubMed Abstract \| Publisher Full Text\n\nBaird AD, Scheffer IE, Wilson SJ: Mirror neuron system involvement in empathy: a critical look at the evidence. Soc Neurosci. 2011; 6(4): 327–335. PubMed Abstract \| Publisher Full Text\n\nZentall TR: Imitation by animals: How do they do it? Curr Dir Psychol Sci. 2003; 12(3): 91–95. Publisher Full Text\n\nMacDonald KB: Separation and its discontents: Toward an evolutionary theory of anti-Semitism. Praeger Publishers/Greenwood Publishing Group, 1998. Reference Source\n\nNagasawa M, Mitsui S, En S, et al.: Oxytocin-gaze positive loop and the coevolution of human-dog bonds. Science. 2015; 348(6232): 333–336. Publisher Full Text\n\nMi S, Lee X, Li X, et al.: Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature. 2000; 403(6771): 785–9. PubMed Abstract \| Publisher Full Text\n\nRaymond J, Blankenship RE: Horizontal gene transfer in eukaryotic algal evolution. Proc Natl Acad Sci U S A. 2003; 100(13): 7419–7420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoucy SM, Huang J, Gogarten JP: Horizontal gene transfer: building the web of life. Nat Rev Genet. 2015; 16(8): 472–82. PubMed Abstract \| Publisher Full Text\n\nNowak MA: Evolving cooperation. J Theor Biol. 2012; 299(0): 1–8. PubMed Abstract \| Publisher Full Text\n\nSmith JM: Evolution and the theory of games: In situations characterized by conflict of interest, the best strategy to adopt depends on what others are doing. Am Sci. 1976; 64(1): 41–45. ISSN 00030996. Reference Source\n\nSmith JM, Price GR: The logic of animal conflict. Nature. 1973; 246(5427): 15–18. Reference Source\n\nAxelrod R, Hamilton WD: The evolution of cooperation. Science. 1981; 211(4489): 1390–1396. PubMed Abstract \| Publisher Full Text\n\nPal A, Levy Y: Structure, stability and specificity of the binding of ssDNA and ssRNA with proteins. PLoS Comput Biol. 2019; 15(4): e1006768. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClausen-Schaumann H, Rief M, Tolks-dorf C, et al.: Mechanical stability of single DNA Molecules. Biophys J. 2000; 78(4): 1997–2007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHofreiter M, Serre D, Poinar HN, et al.: Ancient DNA. Nat Rev Genet. 2001; 2(5): 353–9. PubMed Abstract \| Publisher Full Text\n\nMyers AH, Robinson EG, Van Natta ML, et al.: Hip fractures among the elderly: factors associated with in-hospital mortality. Am J Epidemiol. 1991; 134(10): 1128–1137. PubMed Abstract \| Publisher Full Text\n\nZisberg A, Shadmi E, Sinoff G, et al.: Low mobility during hospitalization and functional decline in older adults. J Am Geriat Soc. 2011; 59(2): 266–273. PubMed Abstract \| Publisher Full Text\n\nDave D, Rashad I, Spasojevic J: The effects of retirement on physical and mental health outcomes. Technical report, National Bureau of Economic Research, 2006. Publisher Full Text\n\nSmith JM: Ireland’s Magdalen Laundries and the Nation’s Architecture of Containment. Manchester University Press, 2008. Publisher Full Text\n\nFernhall Bo: Long-term aerobic exercise maintains peak VO2, improves quality of life, and reduces hospitalisations and mortality in patients with heart failure. J Physiother. 2013; 59(1): 56–56. PubMed Abstract \| Publisher Full Text\n\nHoman A: Rhythms of nature in the Barycz Valley. 2010; accessed 12/2019. Reference Source\n\nMilo DS: Good Enough: The Tolerance for Mediocrity in Nature and Society. Harvard University Press, 2019. Reference Source\n\nO’brochta D: Easi-CRISPR: Long ssDNA donors greatly increase efficiency of HR. 2017. Reference Source\n\nFierer N, Jackson RB: The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci U S A. 2006; 103(3): 626–631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwiatczak B, Cohen IR: Gut feelings of safety: tolerance to the microbiota mediated by innate immune receptors. Microbiol Immunol. 2015; 59(10): 573–585. PubMed Abstract \| Publisher Full Text\n\nLewis NS, Nocera DG: Powering the planet: Chemical challenges in solar energy utilization. Proc Natl Acad Sci U S A. 2006; 103(43): 15729–15735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdwards KJ, Becker K, Colwell F: The deep, dark energy biosphere: intraterrestrial life on earth. Ann Rev Earth Plan sci. 2012; 40(1): 551–568. Publisher Full Text\n\nBrown JH, Gillooly JF, Allen AP, et al.: Toward a metabolic theory of ecology. Ecology. 2004; 85(7): 1771–1789. Publisher Full Text\n\nShiraki N, Shiraki Y, Tsuyama T, et al.: Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells. Cell Metab. 2014; 19(5): 780–794. PubMed Abstract \| Publisher Full Text\n\nPalmer CS, Ostrowski M, Balderson B, et al.: Glucose metabolism regulates T cell activation, differentiation, and functions. Front Immunol. 2015; 6: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa C, Han M, Heinrich B, et al.: Gut microbiome–mediated bile acid metabolism regulates liver cancer via NKT cells. Science. 2018; 360(6391): eaan5931. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore S, Evans LDB, Andersson T, et al.: App metabolism regulates tau proteostasis in human cerebral cortex neurons. Cell Rep. 2015; 11(5): 689–696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGinestier C, Monville F, Wicinski J, et al.: Mevalonate metabolism regulates basal breast cancer stem cells and is a potential therapeutic target. Stem Cells. 2012; 30(7): 1327–1337. PubMed Abstract \| Publisher Full Text\n\nBerg JM, Tymoczko JL, Stryer L: Biochemistry. W H Freeman, Section 1.3, Chemical Bonds in Biochemistry. 2002. Reference Source\n\nZahavi A: Mate selection—a selection for a handicap. J Theor Biol. 1975; 53(1): 205–214. PubMed Abstract \| Publisher Full Text\n\nPenn DJ, Számadó S: The handicap principle: how an erroneous hypothesis became a scientific principle. Biological Reviews. 2019; 95(1): 267–290. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMetcalfe NB, Monaghan P: Growth versus lifes-pan: perspectives from evolutionary ecology. Exp Gerontol. 2003; 38(9): 935–940. PubMed Abstract \| Publisher Full Text\n\nMangel M, Kindsvater HK, Bon-sall MB: Evolutionary analysis of life span, competition, and adaptive radiation, motivated by the pacific rockfishes (sebastes). Evolution. 2007; 61(5): 1208–1224. PubMed Abstract \| Publisher Full Text\n\nDiamantis A, Magiorkinis E, Sakorafas GH, et al.: A brief history of apoptosis: from ancient to modern times. Onkologie. 2008; 31(12): 702–706. PubMed Abstract \| Publisher Full Text\n\nKerr JFR: A histochemical study of hypertrophy and ischaemic injury of rat liver with special reference to changes in lysosomes. J Pathol Bacteriol. 1965; 90(2): 419–435. PubMed Abstract \| Publisher Full Text\n\nSulston JE, Horvitz HR: Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol. 1977; 56(1): 110–156. PubMed Abstract \| Publisher Full Text\n\nSandow BA, West NB, Norman RL, et al.: Hormonal control of apoptosis in hamster uterine luminal epithelium. Am J Anat. 1979; 156(1): 15–35. PubMed Abstract \| Publisher Full Text\n\nKoonin EV, Aravind L: Origin and evolution of eukaryotic apoptosis: the bacterial connection. Cell Death Differ. 2002; 9(4): 394–404. PubMed Abstract \| Publisher Full Text\n\nHughes T, Rusten TE: Origin and evolution of self-consumption: autophagy. Adv Exp Med Biol. In: Eukaryotic Membranes and Cytoskeleton. Springer, 2007; 111–118. PubMed Abstract \| Publisher Full Text\n\nAhmed MS, Ikram S, Bibi N, et al.: Hutchinson-gilford progeria syndrome: a premature aging disease. Mol Neurobiol. 2018; 55(5): 4417–4427. PubMed Abstract \| Publisher Full Text\n\nSpalding KL, Arner E, Westermark PO, et al.: Dynamics of fat cell turnover in humans. Nature. 2008; 453(7196): 783–7. PubMed Abstract \| Publisher Full Text\n\nNguyen LV, Vanner R, Dirks P, et al.: Cancer stem cells: an evolving concept. Nat Rev Cancer. 2012; 12(2): 133–43. PubMed Abstract \| Publisher Full Text\n\n1000 Genomes Project ConsortiumAuton A, Brooks LD, et al.: A global reference for human genetic variation. Nature. 2015; 526(7571): 68–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwanton C: Intratumor heterogeneity: evolution through space and time. Cancer Res. 2012; 72(19): 4875–4882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBunzl M: Causal overdetermination. J Philos. 1979; 76(3): 134–150. Publisher Full Text\n\nDoolittle WF, Booth A: It’s the song, not the singer: an exploration of holobiosis and evolutionary theory. Biol Philos. 2017; 32(1): 5–24. Publisher Full Text\n\nFreund J, Brandmaier AM, Lewejohann L, et al.: Emergence of individuality in genetically identical mice. Science. 2013; 340(6133): 756–759. PubMed Abstract \| Publisher Full Text\n\nStern S, Kirst C, Bargmann CI: Neuromodulatory control of long-term behavioral patterns and individuality across development. Cell. 2017; 171(7): 1649–1662.e10. PubMed Abstract \| Publisher Full Text\n\nMilnor J: On the concept of attractor. In: The theory of chaotic attractors. Springer, 1985; 243–264. Publisher Full Text\n\nPao W, Girard N: New driver mutations in non-small-cell lung cancer. Lancet Oncol. 2011; 12(2): 175–180. PubMed Abstract \| Publisher Full Text\n\nGoldschmidt R: The material basis of evolution. Yale University Press, 1940. ISBN 0-300-02823-7. Reference Source\n\nBell G: The masterpiece of nature: the evolution and genetics of sexuality. Routledge, 2019. Reference Source\n\nBurke NW, Bonduriansky R: Sexual conflict, facultative asexuality, and the true paradox of sex. Trends Ecol Evol. 2017; 32(9): 646–652. PubMed Abstract \| Publisher Full Text\n\nMonk JD, Giglio E, Kamath A, et al.: An alternative hypothesis for the evolution of same-sex sexual behaviour in animals. Nat Ecol Evol. 2019; 3(12): 1622–1631. PubMed Abstract \| Publisher Full Text\n\nLivnat A, Papadimitriou CH: Sex as an algorithm: the theory of evolution under the lens of computation. Commun ACM. 2016; 59(11): 84–93. Publisher Full Text\n\nPrum RO: The Evolution of Beauty: How Darwin’s Forgotten Theory of Mate Choice Shapes the Animal World and Us. Doubleday, 2017. Reference Source\n\nSanttila P, Sandnabba NK, Harlaar N, et al.: Potential for homosexual response is prevalent and genetic. Biol Psychol. 2008; 77(1): 102–105. PubMed Abstract \| Publisher Full Text\n\nKimura M: Evolutionary rate at the molecular level. Nature. 1968; 217(5129): 624–626. PubMed Abstract \| Publisher Full Text\n\nKimura M: The neutral theory of molecular evolution. Cambridge University Press, 1983.\n\nJensen JD, Payseur BA, Stephan W, et al.: The importance of the neutral theory in 1968 and 50 years on: A response to Kern and Hahn 2018. Evolution. 2019; 73(1): 111–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKern AD, Hahn MW: The neutral theory in light of natural selection. Mol Biol Evol. 2018; 35(6): 1366–1371. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWikipedia: Entry for “Corporation”. 2019. (accessed 6/2019).\n\nMayr E: What is a Species, and What is Not? Philos Sci. 1996; 63(2): 262–277. Reference Source\n\nGevers D, Cohan FM, Lawrence JG, et al.: Re-evaluating prokaryotic species. Nat Rev Microbiol. 2005; 3(9): 733–9. PubMed Abstract \| Publisher Full Text\n\nBarnes AD, Jochum M, Lefcheck JS, et al.: Energy flux: The link between multitrophic biodiversity and ecosystem functioning. Trends Ecol Evol. 2018; 33(3): 186–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith CR, Glover AG, Treude T, et al.: Whale-fall ecosystems: recent insights into ecology, paleoecology, and evolution. Ann Rev Mar Sci. 2015; 7: 571–596. PubMed Abstract \| Publisher Full Text\n\nLyons TW, Reinhard CT, Planavsky NJ: The rise of oxygen in earth’s early ocean and atmosphere. Nature. 2014; 506(7488): 307–15. PubMed Abstract \| Publisher Full Text\n\nJäättelä M: Heat shock proteins as cellular lifeguards. Ann Med. 1999; 31(4): 261–271. PubMed Abstract \| Publisher Full Text\n\nWood RD, Mitchell M, Sgouros J, et al.: Human DNA repair genes. Science. 2001; 291(5507): 1284–1289. PubMed Abstract \| Publisher Full Text\n\nRimer J, Cohen IR, Friedman N: Do all creatures possess an acquired immune system of some sort? Bioessays. 2014; 36(3): 273–281. PubMed Abstract \| Publisher Full Text\n\nVandermeer JH: Niche theory. Annu Rev Ecol Syst. 1972; 3(1): 107–132. Reference Source\n\nEstrela S, Libby E, Van Cleve J, et al.: Environmentally mediated social dilemmas. Trends Ecol Evol. 2019; 34(1): 6–18. PubMed Abstract \| Publisher Full Text\n\nPrüfer K, Munch K, Hellmann I, et al.: The bonobo genome compared with the chimpanzee and human genomes. Nature. 2012; 486(7404): 527–531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOdling-Smee FJ, Laland KN, Feldman MW: Niche construction: the neglected process in evolution (MPB-37). 2013; 61: Princeton University Press, Reference Source\n\nDay RL, Laland KN, Odling-Smee FJ: Rethinking adaptation: the niche-construction perspective. Perspect Biol Med. 2003; 46(1): 80–95. PubMed Abstract \| Publisher Full Text\n\nHasson O: Pursuit-deterrent signals: communication between prey and predator. Trends Ecol Evol. 1991; 6(10): 325–329. PubMed Abstract \| Publisher Full Text\n\nRitchie EG, Johnson CN: Predator interactions, mesopredator release and biodiversity conservation. Ecol lett. 2009; 12(9): 982–998. PubMed Abstract \| Publisher Full Text\n\nMcNally L, Brown SP: Building the microbiome in health and disease: niche construction and social conflict in bacteria. Philos Trans R Soc Lond B Biol Sci. 2015; 370(1675): 20140298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaser MJ, Kirschner D: The equilibria that allow bacterial persistence in human hosts. Nature. 2007; 449(7164): 843–849. PubMed Abstract \| Publisher Full Text\n\nCohen IR: Rain and Resurrection How the Talmud and Science Read the World. CRC Press, 2010. Publisher Full Text\n\nTung J, Barreiro LB, Burns MB, et al.: Social networks predict gut microbiome composition in wild baboons. eLife. 2015; 4: e05224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaser MJ: Missing Microbes: How the Overuse of Antibiotics Is Fueling Our Modern Plagues. Henry Holt and Company, New York, 2014. Reference Source\n\nDe Laubenfels MW: Dinosaur extinction: one more hypothesis. J Paleontol. 1956; 30(1): 207–212. Reference Source\n\nGokhman D, Lavi E, Prüfe K, et al.: Reconstructing the DNA methylation maps of the Neandertal and the Denisovan. Science. 2014; 344(6183): 523–527. PubMed Abstract \| Publisher Full Text\n\nThompson D: What in the world is causing the retail meltdown of 2017? The Atlantic. 2017; 10(04). Reference Source\n\nLyons SK, Smith FA, Brown JH: Of mice, mastodons and men: human-mediated extinctions on four continents. Evol Ecol Res. 2004; 6(3): 339–358. Reference Source\n\nBar-On YM, Phillips R, Milo R: The biomass distribution on earth. Proceedings of the National Academy of Sciences. 2018; 115(25): 6506–6511. Publisher Full Text\n\nMcShea DW: Complexity and evolution: what everybody knows. Biol Philos. 1991; 6(3): 303–324. Reference Source\n\nWolf YI, Koonin EV: Genome reduction as the dominant mode of evolution. Bioessays. 2013; 35(9): 829–837. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYahalomi D, Atkinson SD, Neuhof M, et al.: A cnidarian parasite of salmon (myxozoa: Henneguya) lacks a mitochondrial genome. Proc Natl Acad Sci. 2020; 117(10): 5358–5363. Publisher Full Text\n\nBarnosky AD, Matzke N, Tomiya S, et al.: Has the earth’s sixth mass extinction already arrived? Nature. 2011; 471(7336): 51–7. PubMed Abstract \| Publisher Full Text\n\nDeLong EF, Pace NR: Environmental diversity of bacteria and archaea. Syst Biol. 2011; 50(4): 470–478. PubMed Abstract \| Publisher Full Text\n\nBronstein JL, Armbruster WS, Thompson JN: Understanding evolution and the complexity of species interactions using orchids as a model system. New Phytol. 2014; 202(2): 373–375. PubMed Abstract \| Publisher Full Text\n\nQuintana FJ, Cohen IR: The HSP60 immune system network. Trends Immunol. 2011; 32(2): 89–95. PubMed Abstract \| Publisher Full Text\n\nGould SJ, Vrba ES: Exaptation—a missing term in the science of form. Paleobiology. 1982; 8(1): 4–15. Publisher Full Text\n\nHodgkin J: What does a worm want with 20,000 genes? Genome Biol. 2001; 2(11): comment2008.1–comment2008.4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEzkurdia I, Juan D, Rodriguez JM: Multiple evidence strands suggest that there may be as few as 19 000 human protein-coding genes. Hum Mol Genet. 2014; 23(22): 5866–5878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavidson EH, Erwin DH: Gene regulatory networks and the evolution of animal body plans. Science. 2006; 311(5762): 796–800. PubMed Abstract \| Publisher Full Text\n\nOlson MV, Varki A: Sequencing the chimpanzee genome: insights into human evolution and disease. Nat Rev Genet. 2003; 4(1): 20–28. PubMed Abstract \| Publisher Full Text\n\nLewontin RC: The units of selection. Annu Rev Ecol Syst. 1970; 1(1): 1–18. Reference Source\n\nElisabeth Lloyd: Units and levels of selection. 2005. Reference Source\n\nRoughgarden J, Gilbert SF, Rosenberg E, et al.: Holobionts as units of selection and a model of their population dynamics and evolution. Biological Theory. 2018; 13(1): 44–65. Publisher Full Text\n\nReed ES: The lawfulness of natural selection. Am Nat. 1981; 118(1): 61–71. Publisher Full Text\n\nByerly HC: Natural selection as a law: Principles and processes. Am Nat. 1983; 121(5): 739–745. Publisher Full Text\n\nPaul DB: Darwin, social darwinism and eugenics. The Cambridge Companion to Darwin. 2003; 214. Publisher Full Text\n\nBergman J: The Darwin Effect: It’s influence on Nazism, Eugenics, Racism, Communism, Capitalism & Sexism. New Leaf Publishing Group. 2014. Reference Source\n\nMullis KB: The unusual origin of the polymerase chain reaction. Sci Am. 1990; 262(4): 56–65. PubMed Abstract \| Publisher Full Text\n\nEfroni S, Harel D, Cohen IR: Toward rigorous comprehension of biological complexity: modeling, execution, and visualization of thymic T-cell maturation. Genome Res. 2003; 13(11): 2485–2497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Shahrour F, Minguez P, Tárraga J, et al.: Babelomics: a systems biology perspective in the functional annotation of genome-scale experiments. Nucleic Acids Res. 2006; 34(Web Server issue): W472–W476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPesce D, Lehman N, de Visser JAGM: Sex in a test tube: testing the benefits of in vitro recombination. Philos Trans R Soc B Biol Sci. 2016; 371(1706): 20150529. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "65121", "date": "23 Jun 2020", "name": "Eugene Rosenberg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCohen and Marron offer a highly original concept of evolution, a subject fundamental to biology and philosophy. They begin by providing a clear and well-written description of neo-Darwinism, recent extended theories of evolution, which take into account epigenetics and microbiomes, and entropy. They then posit their main point- that the evolution of complexity is driven by cooperation, which is a mechanism to avoid entropic destruction. They argue that this cooperation is required at all biological levels, from molecules to ecosystems. For example, they point out the cooperation of individual strands of DNA to form double-stranded DNA helps avoid entropic destruction.\n\nLike most revolutionary ideas in science, the evolution of universal adaptations of life driven by the properties of matter: energy, entropy, and interactions, will be difficult for evolutionary biologists to accept. However, this article should be read and seriously considered by biologists. The authors end the article by suggesting some interesting experiments to test their concept of evolution.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5754", "date": "30 Jul 2020", "name": "Assaf Marron", "role": "Author Response", "response": "We are gratified to see that Eugene Rosenberg judges the ideas presented in the paper to be original and fundamental, and that the proposed experiments are interesting and relevant. We thank Eugene Rosenberg for this expert review. Irun R. Cohen and Assaf Marron" } ] }, { "id": "65123", "date": "13 Jul 2020", "name": "Eugene V. Koonin", "expertise": [ "Reviewer Expertise Evolutionary biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCohen and Marron present a concept of evolution that centers on interactions on different level - molecular, cellular, organismal etc. - which are perceived as a universal anti-entropic force. Entropy is understood as a tendency for increasing disorder and is viewed as the principal selective factor in evolution. The antientropic evolution is postulated to be 'universal', hence no neutral evolutionary processes. The authors, then, forward the principle of 'survival of the fitted' whereby all biological entities that counter entropy with sufficient success to remain in an ordered state survive.\nAlthough not exactly new (indeed, it goes back to Boltzmann who the authors duly cite!), the notion of the fundamental nature of the antientropic tendencies in evolution is worth putting forward. More specifically, the emphasis on interactions permeating the entire biological organization as a universal antientropic factor is worthy and might appeal to many biologists as a fresh perspective eon the general character of evolution..\nHowever, I find several areas within and around these where concepts where I feel compelled to mount criticisms, and not altogether minor ones.\n\nFirst, to me, the 'survival of the fitted' appears to be a non sequitur. Let us say, everything is fine with entropy as a selective factor and interactions, but why not survival of the fittest, that is, those that counteract entropy most efficiently and, as a result, leave more progeny than others?\nSecond, in all their discussion of entropy, the authors refer only to classical,equilibirum thermodynamics. As far as I can see, this discussion cannot be complete without taking into account the concepts of non-equilibrium thermodynamics, in particular, maximization of entropy production and dissipative structures. The literature on these subjects is extensive, starting with Prigogine.\nThird, to me, it is inexplicable how and why, when discussing the pivotal roles of interactions at different levels in evolution, the authors fail to include Maynard Smith-Szathmary's concept of major transitions in evolution (Ref. 11 and references therein) and, especially, the generalized form of this concept that comes uncannily close to the concept of interactions-driven evolution discussed here2.\nFourth, I believe that, along with the key role of interaction, it is essential for developing a general concept of evolution, to consider competition between interactions on different scales and the resulting frustrated states2.\nFinally, more with respect to the presentation of the material. The figures in the current manuscript are over-simplified and, overall, underwhelming. Figure 1 is a trivial illustration of the concept of entropy and is superfluous (readers unfamiliar with the second law will not benefit from reading this article) whereas for Figures 2 and 3, more elaborate schemes are advisable.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "5755", "date": "30 Jul 2020", "name": "Assaf Marron", "role": "Author Response", "response": "We appreciate Eugene Koonin’s positive opening remarks and his expert review and suggestions for improving the paper. The specific recommendations for improvement are five: First, “everything is fine with entropy as a selective factor…but why not survival of the fittest, that is, those that counteract entropy most efficiently and, as a result, leave more progeny than others?” This is an important question and we have addressed it directly in the revision. Briefly, we reason that dealing with entropy does not directly require competition and struggle; one need only to fit in “sufficiently”; moreover, variants that are not “the fittest” readily find other niches, and many advantageous phenotypes are not necessarily associated with reproductive advantage; indeed, increased reproduction is at times more of a problem than a desired outcome. This is, of course, in addition to the distinction the paper makes between universal adaptations driven by entropic selection and local adaptations, which may well be based on reproductive advantage and Darwinian natural selection. See the section titled “Reconciling survival-of-the-fitted with Darwinian survival-of-(only)-the-fittest”. Second, “the authors refer only to classical, equilibrium thermodynamics.” We appreciate the reference to the work of Prigogine and others on the emergence of order in multiple scales from chaotic thermodynamic behavior both in equilibrium and non-equilibrium states. In our revision, we have added the suggested references and others as important elements in quantitative analysis, simulation, and prediction. See the section titled “Analysis and modeling”. We thank Eugene Koonin for calling our attention to these ideas. Third, the first draft of the paper neglected to cite the concepts of Maynard Smith and Szathmary on Major Transitions in Evolution. Our revision now refers to this work which, among others, highlights the pivotal roles of interaction and the relentless increase of complexity. See subsection “Cooperation retards entropy” in section “Interactions”. Fourth, the Reviewer suggested that the paper should “consider competition between interactions on different scales and the resulting frustrated states” as a formative element in cooperative interaction. In the revised paper, we now incorporate this idea and cite the relevant paper by Wolf, Katznelson and Koonin. See subsection “Cooperation retards entropy” in section “Interactions”. We also added references to Maynard Smith & Szathmary and to Wolf, Katznelson and Koonin in the section titled “the emergence of complexity”. Fifth and finally, the Reviewer suggested that the Figures are over-simplified and superfluous (Figure 1) and “for Figures 2 and 3, more elaborate schemes are advisable.” In the revised paper, the captions clarify that the Figures are meant to serve only as simplified conceptual anchors or mnemonics for the principles of the theory. We thank Eugene Koonin for raising these points which we feel have improved the paper considerably. Irun R. Cohen and Assaf Marron" } ] } ]	1	https://f1000research.com/articles/9-626
https://f1000research.com/articles/9-1085/v1	02 Sep 20	{ "type": "Brief Report", "title": "Evaluation of batch fraction, corn silage inclusion level, and mixing duration on long particle distribution of finishing diets for beef cattle", "authors": [ "Elizabeth M. Buckhaus", "Dathan T. Smerchek", "Zachary K. Smith", "Elizabeth M. Buckhaus", "Dathan T. Smerchek" ], "abstract": "Background: Differing fractions of a batch of feed, differing ingredient characteristics, and inadequate mix time can lead to non-uniformity within a mix of feed. Methods: The experiment was designed as a 5 x 2 x 2 factorial arrangement with seven replications per simple treatment mean. Factors included: 1) batch fraction (BF; n = 5); 2) corn silage inclusion level (CSLVL; n = 2) 15% or 30% inclusion (dry matter basis); and 3) mixing duration (DR; n = 2) of 20 or 25 mixer revolutions. Data were analyzed as a completely randomized design using a binomial approach. The Penn State Particle Separator was used to separate fractions of the total mixed ration (TMR). Results: No interactions between BF, CSLVL, and DR were detected (P ≥ 0.31) for any dependent variables. There was an increase (P = 0.01) in retention on the 19 mm sieve from the first BF compared to the last BF. CSLVL altered (P = 0.01) retention on the 19 mm sieve. Increasing DR from 20 to 25 revolutions had no appreciable influence (P = 0.23) on particles greater than 19 mm. CSLVL (P = 0.01) and DR (P = 0.01) altered particle retention on the 8 mm sieve. BF (P = 0.01), CSLVL (P = 0.01), and DR (P = 0.02), influenced particle retention on the 4 mm sieve. CSLVL impacted (P ≤ 0.01) particles remaining in the bottom pan and particles greater than 4 mm. BF (P = 0.01) and CSLVL (P = 0.01) altered particles greater than 8 mm. Conclusions: These data indicate that BF and CSLVL fed alters particle size distribution that in turn could alter dry matter intake, dietary net energy content, and influence daily gain. Mixing DR had no appreciable influence on particle size distribution of the TMR.", "keywords": [ "corn silage", "finishing diet", "mixing duration", "particle size" ], "content": "Introduction\n\nVarying feed ingredient properties such as particle size, shape, density, hygroscopocity, static charge, and adhesiveness can influence how a beef cattle diet mixes prior to feeding. Differing fractions of a batch of feed, differing ingredient characteristics, and mix time can also lead to non-uniformity within a specific mix of feed.\n\nBlom et al. (2020) demonstrated that as the mixer unloads, there is a linear increase in the proportion of long particles fed that results in greater intake, poorer gain, and reduced gain to feed (average daily gain/dry matter intake) in steers during the feedlot receiving phase. Smerchek et al. (2020) demonstrated that as particles greater than 4 mm increase, there is a reduction in average daily gain by approximately 0.02 kg for each percentage point increase in particles greater than 4 mm in the diet.\n\nThe objective of this research was to determine how batch fraction, diet roughage level, and mixing duration influenced particle distribution in finishing diets for beef cattle. The hypothesis was that batch fraction would influence the particles size distribution, greater corn silage inclusion (roughage level) would alter particle size distribution, and mixing duration would have no influence on particle size distribution of the total mixed ration.\n\n\nMethods\n\nNo Institutional Animal Care and Use Committee approval was obtained for this experiment since no animals were used to generate the data used in the present analysis. The study was conducted at the Ruminant Nutrition Center in Brookings, SD, USA.\n\nThe experiment was designed as a 5 × 2 × 2 factorial arrangement with seven replications per simple treatment mean. Factors included: 1) batch fraction (BF; n = 5), where BF 1 was the first 20% of feed unloaded from the mixer and BF 5 was the last 20% of feed unloaded from the mixer; 2) corn silage inclusion level (CSLVL; n = 2) containing (dry matter basis) 15% corn silage or 30% corn silage replacing the corn blend; and 3) mixing duration (DR; n = 2) of 20 or 25 mixer revolutions (5 revolutions·minute-1) prior to unloading. A 2.35 m3 horizontal mixer (Roto-Mix; Dodge City, KS) was used to manufacture all diets. Diets contained corn silage, a 1:1 ratio of dry-rolled corn:high-moisture corn, a liquid supplement (5% dry matter inclusion), and a meal supplement (7% dry matter inclusion). Ingredients were added into the horizontal mixer in the following sequence: high-moisture corn, dry-rolled corn, liquid supplement, dry supplement, and finally corn silage.\n\nThe total mixed ration (TMR) samples were separated using the Penn State Particle Separator (PSPS) using the methods described by (Kononoff et al., 2003). The PSPS had three sieves (19 mm, 8 mm, 4 mm, and pan). The particles retained on the top sieve (19 mm) were considered large, middle sieve (8 mm) were considered medium, and bottom sieve (4 mm) were considered small. Particles less than 4 mm were collected in the pan. Proportions of the TMR on differing sieves was determined on an as-is basis.\n\nData were analyzed as a completely randomized design appropriate for a 5 × 2 × 2 factorial arrangement of treatments using the GLIMMIX procedure of SAS 9.4 (SAS Inst., Inc., Cary, NC) using a binomial approach. There was a total of seven replications for each simple treatment mean (the combination of each BF, CSLVL, and DR). All data are presented as least squares means and the corresponding standard error of the mean. An α of 0.05 was used to determine significance.\n\n\nResults\n\nThe effect of BF, CSLVL, and DR on TMR particle size distribution are presented in Table 1 and full results are available as Underlying data (Smith et al., 2020). Visual representation of the TMR’s fed are shown in Figure 1 (20 DR only). No interactions between BF, DIET, and REV were detected (P ≥ 0.31) for any dependent variables. There was a 53.5% increase (P = 0.01) in retention on the 19 mm sieve from the first BF (first 20% of the TMR unloaded from the mixer) compared to the last BF (last 20% of the TMR unloaded from the mixer). The 15 CSLVL diet had a 71.3% decrease (P = 0.01) in retention on the 19 mm sieve compared to the 30 CSLVL diet. Increasing DR from 20 to 25 revolutions had no appreciable influence (P = 0.23) on particles greater than 19 mm. CSLVL (P = 0.01) and DR (P = 0.01) altered particle retention on the 8 mm sieve. BF (P = 0.01), CSLVL (P = 0.01), and DR (P = 0.02) influenced particle retention on 4 mm sieve. CSLVL impacted (P ≤ 0.01) particles remaining in the bottom pan and particle greater than 4 mm. BF (P = 0.01) and CSLVL (P = 0.01) altered particles greater than 8 mm.\n\n1Determined according to (Kononoff et al., 2003).\n\n2Standard error of the mean.\n\na,bMeans with in a row without a common superscript differ (P ≤ 0.05).\n\n\nConclusions\n\nThese results indicate that BF and CSLVL influences particle size distribution of the TMR fed to feedlot cattle. This potentially could alter dry matter intake, dietary net energy content, and influence animal average daily gain, by altering the actual diet fed from what was formulated to be fed. Mixing DR had no appreciable influence on particle size distribution of the TMR, a shorter mixing duration could have a pronounced impact on the distribution of particles in the TMR, however, a shorter mix DR was not investigated in the present experiment. Future experiments should determine what the shortest possible mix duration could be to effectively manufacture finishing diets fed to feedlot cattle.\n\n\nData availability\n\nFigshare: Evaluation of Batch Fraction, Corn Silage Inclusion Level, and Mixing Duration on Long Particle Distribution of Finishing Diets for Beef Cattle (Smith et al., 2020). https://doi.org/10.6084/m9.figshare.12841469.v1\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors wish to acknowledge Mr. Paul Schlobohm and the Undergraduate employees at the Ruminant Nutrition Center for assistance with the manufacturing of the diets used in this experiment.\n\n\nReferences\n\nBlom EJ, Gentry WW, Pritchard RH, et al.: Evaluation of inclusion of hay, dampened hay, and silage in receiving diets of newly weaned beef calves. Appl Anim Sci. 2020; 36(3): 367–376. Publisher Full Text\n\nKononoff PJ, Heinrichs AJ, Buckmaster DR: Modification of the Penn State forage and total mixed ration particle separator and the effects of moisture content on its measurements. J Dairy Sci. 2003; 86(5): 1858–1863. PubMed Abstract \| Publisher Full Text\n\nSmerchek DT, Buckhaus EM, Miller KD, et al.: Increasing hay inclusion in silage-based receiving diets and its effects on performance and energy utilization in newly weaned beef steers. Transl Anim Sci. 2020; 4(2): txaa026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith Z, Buckhaus E, Smerchek D: Source Data: Evaluation of Batch Fraction, Corn Silage Inclusion Level, and Mixing Duration on Long Particle Distribution of Finishing Diets for Beef Cattle. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12841469.v1" }	[ { "id": "82061", "date": "08 Apr 2021", "name": "Maghsoud Besharati", "expertise": [ "Reviewer Expertise Animal Nutrition", "Ruminant Nutrition", "Dairy cattle", "Beef" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to congratulate authors for the good-quality of the article, the literature reported used to write the paper, and for the clear and appropriate structure. The manuscript is well written, presented and discussed, and understandable to a specialist readership.\n\nIn general, the organization and the structure of the article are satisfactory and in agreement with the journal instructions for authors. The subject is adequate with the overall journal scope.\n\nThe work shows a conscientious study in which a very exhaustive discussion of the literature available has been carried out. The introduction provides sufficient background, and the other sections include results clearly presented and analyzed exhaustively.\n\nHowever, to improve the manuscript, some revisions are recommended such as:\n\nEnglish language needs to be checked; Update the used literature; Revise the paper according to journal's instructions.\n\nThe novelty of the study needs to be highlighted compare to other similar studies.\n\nThe scientific background of the topic is poor. In \"Introduction\" and \"Discussion\", the authors should cite recent references between 2016-2020 from JCR journals (with impact factor) about recent achievements on the subject.\n\nAuthors should state something about Selenium and Vitamin E. At least producer of the additives should stated.\nSo, I recommend the acceptance of the paper after revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6585", "date": "16 Apr 2021", "name": "Zachary Smith", "role": "Author Response", "response": "The authors appreciate the time you took to review our manuscript. Best, Zachary Smith South Dakota State University" } ] }, { "id": "81344", "date": "19 Apr 2021", "name": "Ahmed M. Abd El Tawab", "expertise": [ "Reviewer Expertise I am an associate research professor", "my scope is ruminants nutrition", "animal nutrition", "animal production", "milk production", "dairy and beef cattle", "feed evaluation", "feed technology and rumen simulation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNovelty Study and Clear the aims and Hypotheses: The particle size of feed plays an important role in animal feed strategies and consequence on productive performance and average daily weight gain. The author has studied to determine how batch fraction, diet roughage level, and mixing duration influenced particle distribution in finishing diets for beef cattle.\n\nThe title should be changed to Evaluation of batch fraction, corn silage inclusion level, and mixing duration on particles size distribution of finishing diets for beef cattle.\n\nThe keywords should be included \"beef cattle\".\n\nThe introduction should include information about the digestibility.\n\nThe materials and methods should be added experimental animal paragraph.\n\nTable and figure are good.\n\nResults are good. But the Author should add animal performance table beside the finding table.\n\nConclusion is good.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1085
https://f1000research.com/articles/9-1084/v1	02 Sep 20	{ "type": "Research Article", "title": "High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing", "authors": [ "Devika Ganesamoorthy", "Mengjia Yan", "Valentine Murigneux", "Chenxi Zhou", "Minh Duc Cao", "Tania P. S. Duarte", "Lachlan J. M. Coin", "Mengjia Yan", "Valentine Murigneux", "Chenxi Zhou", "Minh Duc Cao", "Tania P. S. Duarte" ], "abstract": "Background: Tandem repeats (TRs) are highly prone to variation in copy numbers due to their repetitive and unstable nature, which makes them a major source of genomic variation between individuals. However, population variation of TRs has not been widely explored due to the limitations of existing approaches, which are either low-throughput or restricted to a small subset of TRs. Here, we demonstrate a targeted sequencing approach combined with Nanopore sequencing to overcome these limitations. Methods: We selected 142 TR targets and enriched these regions using Agilent SureSelect target enrichment approach with only 200 ng of input DNA. We barcoded the enriched products and sequenced on Oxford Nanopore MinION sequencer. We used VNTRTyper and Tandem-genotypes to genotype TRs from long-read sequencing data. Gold standard PCR sizing analysis was used to validate genotyping results from targeted sequencing data. Results: We achieved an average of 3062-fold target enrichment on a panel of 142 TR loci, generating an average of 97X coverage per sample with 200 ng of input DNA per sample. We successfully genotyped an average of 75% targets and genotyping rate increased to 91% for the highest-coverage sample for targets with length less than 2 kb, and GC content greater than 25%. Alleles estimated from targeted long-read sequencing were concordant with gold standard PCR sizing analysis and highly correlated with alleles estimated from whole genome long-read sequencing. Conclusions: We demonstrate a targeted long-read sequencing approach that enables simultaneous analysis of hundreds of TRs and accuracy is comparable to PCR sizing analysis. Our approach is feasible to scale for more targets and more samples facilitating large-scale analysis of TRs.", "keywords": [ "Tandem repeats", "targeted sequencing", "long-read sequencing" ], "content": "Introduction\n\nRepeated sequences occur in multiple copies throughout the genome; they make up almost half of the human genome1. Repeat sequences can be divided into two categories: interspersed repeats and tandem repeats (TRs). Interspersed repeats are scattered throughout the genome and are remnants of transposons2. TRs consists of repeat units that are located adjacent to each other (i.e. in tandem). There are almost 1 million TRs in the human genome, covering 10.6% of the entire genome3. TRs can be further divided into two types based on the length of the repeat unit; repeats with one to six base pair repeat units are classified as microsatellites or short tandem repeats (STRs) and those with more than six base pair repeat units are known as minisatellites4.\n\nTRs are prone to high rates of copy number variation and mutation due to the repetitive unstable nature, which makes them a major source of genomic variation between individuals. Variation in TRs may explain some of the phenotypic variation observed in complex diseases as it is poorly tagged by single nucleotide variation5,6. Recent studies have shown that 10% to 20% of coding and regulatory regions contain TRs and suggested that variations in TRs could have phenotypic effect7. Although TRs represent a highly variable fraction of the genome, analysis of TRs so far are limited to known pathogenic regions, mainly STRs due to the limitations in analysis techniques.\n\nTraditionally, TR analysis has been carried out via restriction fragment length polymorphism (RFLP) analysis8 or PCR amplification of the target loci followed by fragment length analysis9. These techniques are only applicable to a specific target region and not scalable to high-throughput analysis, which limits the possibility of genome-wide TR analysis. In the recent decade, significant progress has been made in utilising high-throughput short-read sequencing data for genotyping STRs10. Our group and others have also demonstrated targeted sequencing approaches using short-read sequencing for TR analysis11,12. Several computational tools have been developed to improve the accuracy of TR genotyping from short-read sequencing data with varying performance13–19. Yet, most of these tools have focused mainly on the analysis of STRs and analysis of longer TRs remains a hurdle for these approaches. We reported a new approach GtTR in Ganesamoorthy et al.12, which utilizes short-read sequencing data to genotype longer TRs. GtTR reports absolute copy number of the TRs, but it does not report the exact genotype of two alleles due to the use of short-read sequencing data.\n\nSequencing reads that span the entire repeat region are informative to accurately genotype TRs11, and therefore are ideal for genome-wide TR analysis. Long-read sequencing technologies have the potential to span all TRs in human genome, including long TRs. There have been few reports on the use of long-read sequencing for the analysis of specific TRs implicated in diseases20–22. Genotyping tools utilizing long-read sequencing data, such as Nanosatellite21, RepeatHMM23 and Tandem-genotypes24 have been reported in the recent years with varying performance across different length of repeat units and repeat length. We reported VNTRTyper in Ganesamoorthy et al.12 to genotype TRs from long-read whole genome sequencing data. Despite the availability of genotyping tools, long-read sequencing is not widely used for TR analysis, due to the high costs associated with whole genome long-read sequencing. Cost-effective long-read sequencing approaches will be an important and attractive option to genotype TRs in large-scale studies. However, there has been limited progression on targeted long-read sequencing of TRs.\n\nWe have previously demonstrated that targeted sequence capture of repetitive TR sequences are feasible using short-read sequencing technologies12. In this study, we demonstrate the targeted sequence capture of repetitive TRs using Oxford Nanopore long-read sequencing technologies. There have been previous reports on the use of targeted sequencing combined with long read sequencing technologies25; however, enrichment of repeat sequences requires optimization in probe design and probe hybridization approaches. We optimized the protocols and report successful enrichment of repetitive sequences followed by long-read sequencing. We demonstrate the accuracy of genotype estimates from targeted long-read sequencing by comparison with gold-standard PCR sizing analysis. In this study, we predominantly targeted longer TRs (i.e. minisatellites); however, our approach is applicable to all TRs. Our targeted long-read sequencing method presented here provides an accurate and cost-effective approach for large-scale analysis of TRs, which will be useful for researchers to explore the impact of TR variants on diseases and phenotypes.\n\n\nMethods\n\nDNA samples of CEPH/UTAH pedigree 1463 were purchased from Coriell Institute for Medical Research (USA). Seven family members from the pedigree used for sequencing analysis were NA12877, NA12878, NA12879, NA12881, NA12882, NA12889 and NA12890.\n\nThe selection of TRs and design of probes were described in Ganesamoorthy et. al. (2018)12. Briefly, 142 TRs were selected; they range from 112 to 25236 bp in length in the reference human genome (hg19) and the number of repeat units range from 2 to 2300 repeats. TRs used in this study were selected as part of another study to investigate association between TRs and Obesity and these targeted TRs are not disease associated. Agilent SureSelect DNA design (Agilent Technologies) was used to design target probes to capture the targeted regions (including 100-bp flanking regions) and regions flanking the TRs (at least 1000 bp).\n\nAll seven family members from the CEPH pedigree 1463 were used for Nanopore targeted sequencing analysis (NA12877, NA12878, NA12879, NA12881, NA12882, NA12889 and NA1289). Target sequence capture for Nanopore sequencing was performed using Agilent SureSelect XT HS Target Enrichment System (Agilent Technologies) according to the manufacturer’s instructions with slight modifications. Briefly, 200 ng of DNA was fragmented to 3 kb using Covaris Blue miniTUBE (Covaris). Greater than 90% of the targeted TRs are less than 3 kb and SureSelect capture protocol works effectively on fragments less than 4 kb in length; therefore, DNA products were sheared to 3 kb. Fragmented DNA was end-repaired, adapter-ligated and amplified prior to target capture. Extension time for pre-capture amplification was increased to 4 minutes to allow for the amplification of long fragments and 14 cycle amplification was used. Purified pre-capture PCR products were hybridized to the designed capture probes for 2 hours. Streptavidin beads (Thermo Fisher) were used to pull down the DNA fragments bound to the probes. Finally, captured DNA was amplified with long extension time (4 minutes) using Illumina Index adapters provided in the enrichment kit. Post capture PCR products were purified using 0.8X - 1X AMPure XP beads (Beckman Coulter).\n\nNanopore sequencing library preparation was performed using 1D Native barcoding genomic DNA (with EXP-NBD103 and SQK-LSK108) (Oxford Nanopore Technologies) protocol according to the manufacturer’s instructions with minor modifications. Briefly, 100–200ng of post capture PCR products were end repaired and incubated at 20°C for 15 mins and 65°C for 15 mins. End repaired products were ligated with unique native barcodes. Purification steps after end repair and barcode ligation were avoided to minimize the loss of DNA. Barcoded samples were pooled in equimolar concentrations prior to adapter ligation. Adapter ligated samples were purified using 0.4X AMPure XP beads (Beckman Coulter). Samples were split into two sequencing groups: NA12877, NA12878, NA12879 and NA12890 (group 1); and NA12881, NA12882 and NA12889 (group 2). Sequencing was performed using a MinION sequencer (Oxford Nanopore Technologies) using R9.5 flow cell. Both groups were sequenced for 48 hours. Nanopore sequencing data were base called using Albacore (version 2.2.7) and reads were demultiplexed using Albacore (version 2.2.7) based on the barcode sequences.\n\nNanopore WGS data on CEPH Pedigree 1463 sample NA12878 were obtained from the Nanopore WGS consortium (https://github.com/nanopore-wgs-consortium/NA12878/blob/master/nanopore-human-genome/rel_3_4.md)26. PacBio WGS data on NA12878 sample were downloaded from SRA with accession numbers SRX627421 and SRX63831027\n\nSequencing reads were mapped to hg19 reference genome using Minimap2 (version 2.13)28. For Nanopore sequencing ‘-ax map-ont’ and for PacBio WGS ‘-ax map-pb’ parameters were used. VNTRTyper, our in-house tool described by Ganesamoorthy et al.12 was used to genotype TRs from long-read sequencing data. Briefly, VNTRTyper takes advantage of the long-read sequencing to identify the number of repeat units in the TR regions. Firstly, the tool identifies reads that span the repeat region and applies hidden Markov models (HMM) to align the repetitive portion of each read to the repeat unit. Then it estimates the multiplicity of the repeat units in a read using a profile HMM.\n\nRecently, we further improved the accuracy of genotyping estimates by clustering the copy number counts from reads to identify the likely genotypes per target. We used Kmeans clustering and the number of clusters are fixed at two clusters for two alleles. A minimum threshold of two supporting reads per genotype was used to assign genotypes. Furthermore, for heterozygous alleles, both alleles should have at least 10% of reads supporting the allele, if not allele with less than 10% of reads was excluded during the analysis. The updated version of VNTRTyper can be accessed from GitHub Japsa release 1.9–3c and can be deployed using script name jsa.tr.longreads. Details of VNTRTyper analysis are previously reported by Ganesamoorthy et al.12.\n\nWe also used another independent method, Tandem-genotypes, to estimate genotypes from long-read sequencing data. Tandem-genotypes was recently reported for analysis of TR genotypes from long-read sequencing data24 and it can be utilised for both Nanopore and PacBio sequencing technologies.\n\nNanopore and PacBio sequencing data were mapped to the hg19 reference genome using LAST v95929. Calculation of repeat length per sequencing read was performed with Tandem-genotypes as reported by Mitsuhashi et al.24. Copy number changes in reads covering the repeat’s forward and reverse strands were merged and the two alleles with the highest number of supporting reads for each VNTR were extracted. A minimum threshold of two supporting reads per genotype was used to assign genotypes.\n\nA total of 10 targeted VNTR regions which are less than 1 kb in repetitive sequence were validated by PCR sizing analysis in this study (PCR primer sequences provided in Extended data, Supplementary Table 1)30. These ten targets include various repeat unit length and repeat sequence combinations to assess the accuracy of the genotypes determined from sequencing data. The majority of these targets were tested in our previous study12 and the results from the previous PCR analysis were used for these regions. PCRs were performed using HotStar Taq DNA Polymerase (Qiagen) and PCR conditions were optimized for each PCR target. PCR products were purified and subjected to capillary electrophoresis on an ABI3500xL Genetic Analyzer (Applied BioSystems). Fragment sizes were analyzed using GeneMapper 4.0 (Applied BioSystems). Alternatively, STRand or Osiris could be used for fragment size analysis. Capillary electrophoresis plots provided in Extended data, Supplementary Information PCR data30.\n\nLinear regression analysis was used to determine correlation between genotype estimates. All plots were generated using GraphPad Prism (version 7.00 for Windows; GraphPad Software, La Jolla California USA).\n\nWe investigated the effect of GC content, repeat length, repeat period and repeat copy number on target sequencing depth using a multivariate linear regression model. We used ggplot2 (version 3.2.0) to visualize the relationship between these factors and sequencing depth across all seven samples. Thresholds on GC and repeat length were chosen based on this visual analysis. Genotype rate was calculated as the proportion of sample, target pairs which had a predicted genotyped (based on VNTRtyper) amongst all targets which met the GC and repeat length thresholds.\n\n\nResults\n\nWe demonstrate a targeted sequence capture approach combined with Nanopore long-read sequencing to genotype hundreds of TRs.\n\nWe performed sequence enrichment of targeted TRs for 7 samples followed by long-read sequencing using Oxford Nanopore Technologies’ MinION as described in the Methods. Figure 1a shows the read length distribution observed in targeted capture sequencing data. The median read length followed the expected read-length distribution, with the exception of an under-representation of repeats of length >3 kb (Figure 1a). The read length in this study was sufficient to analyse majority of targeted TRs of length less than 2 kb. Sequence coverage varied across targets and samples, on average 97X sequence coverage was achieved, with only 19 targets having less than 1X coverage (Figure 1b) and majority of the low coverage targets (16 of the 19 targets) have less than 25% GC content (Extended data, Supplementary Figure 1)30.\n\nExtended data, Supplementary Table 230 summarises the metrics for targeted sequencing on Nanopore sequencing technologies. Nanopore multiplexing (See Methods) group 1 samples had similar yield between samples; however, Nanopore multiplexing group 2 samples had varying yield per sample. Despite the differences in sequencing yield, we achieved an average of 3062-fold target enrichment and on target capture rate was approximately 50%.\n\n(a) Read length distribution of Nanopore targeted sequencing. Lines indicates the read length distribtuion for each sample and grey bars indicate the length distribution of targeted TRs and (b) Sequence coverage distribution of Nanopore targeted sequencing for all seven samples.\n\nGenotype estimates from targeted long-read sequencing datasets were estimated using our tool VNTRTyper12 with the improvements described in the Methods. We also applied Tandem-genotypes24 to determine the genotypes of TRs from long-read sequencing data. We used a minimum of two reads as read threshold to determine the repeat number for each allele.\n\nPrior to obtaining any sequence data, we generated PCR sizing results as a gold standard on 10 targets for comparison to sequencing analysis. These 10 targets were selected to include various repeat unit length and repeat sequence combinations to assess the accuracy of the genotypes determined from sequencing data. Of these 10 targets, two were excluded for comparison as all seven samples had insufficient number of spanning reads (minimum of two reads required for genotyping) to genotype these targets. Genotype estimates from VNTRtyper on these eight targets correlated well with PCR (Pearson correlation greater than 0.980 for all samples) (Table 1 and Extended data, Supplementary Figure 2)30. Genotype estimates by Tandem-genotypes also correlated well with PCR, with a correlation greater than 0.984 for all samples Extended data, (Supplementary Table 3 and Supplementary Figure 3)30; however, fewer targets had sufficient data to compare with PCR sizing results.\n\nRepeat Number in reference hg19 is provided within brackets for each target\n\nRepeat numbers that do not agree with PCR results are highlighted in red.\n\nND – Sufficient data not available for genotype analysis\n\nGenotype estimates from VNTRTyper and Tandem-genotypes for all 142 targets from Nanopore capture sequencing samples are provided in Extended data, Supplementary Spreadsheet Tables 1 and 230, respectively. Genotype estimates by VNTRtyper and Tandem-genotypes correlate well and the correlation values range from 0.904 to 0.994 for Nanopore targeted sequencing.\n\nWe were able to determine the genotype on average for 60% of the targets (range 48% to 75%) using VNTRTyper and 57% of the targets (range 41% to 74%) using Tandem-genotypes. Both VNTRTyper and Tandem-genotypes failed to genotype targets with low GC sequence content (<25% GC content) and targets which are greater than 2 kb in length, which accounts for approximately 22% of the targets (32 of the 142 targets). Targets with low GC sequence content (<25% GC content) did not have sufficient sequence coverage for analysis due to inefficient sequence enrichment in these regions (Extended data, Supplementary Figure 1)30. Targets which are greater than 2 kb in length did not have sufficient spanning reads for genotyping analysis (See Methods and Figure 1a).\n\nIt was evident that the GC content of the target and size (i.e. repeat length) affected the genotyping efficiency of our targeted capture sequencing approach. Therefore, we assessed the genotyping rate based on the size of the target and GC content of the target (Figure 2). For all 142 targets genotyping rate using VNTRTyper was only 59.8% (Figure 2a); however, the genotyping rate improved to 67% for 125 targets with a size threshold of 2 kb (Figure 2b) and 67.1% for 125 targets with 25% GC threshold (Figure 2c). Furthermore, genotyping rate improved to 75.2% for 110 targets with a combined 2 kb size threshold and 25% GC threshold (Figure 2d). Also, sample with high sequence coverage (NA12889) had the highest genotyping rate of 90.9% for 110 targets (Extended data, Supplementary Figure 4)30. Genotyping rate using Tandem-genotypes also improved to an average of 63.7% for 110 targets (range 43.6% to 85.5%) (Extended data, Supplementary Figure 5)30.\n\nTriangle indicates that greater than 50% of the samples had a genotyping estimate and circle indicates only less than 50% of the samples had a genotyping estimate for the given target. Colours indicate the depth, which is defined as the number of spanning reads detected for the target region. Black thick lines inside the plots indicate the 2-kb size threshold and 25% GC content size threshold. (a) Genotyping rate for all targets, shown as repeat number vs period (i.e. repeat unit). (b) Genotyping rate with 2-kb size threshold. (c) Genotyping rate with 25% GC threshold. (d) Genotyping rate with both 25% GC threshold and 2-kb size threshold.\n\nTo investigate the accuracy of genotype estimates of TRs from targeted sequence capture compared to WGS, we performed genotyping analysis on the targeted regions using VNTRTyper and Tandem-genotypes on whole genome long-read sequencing data. We downloaded whole genome long-read Nanopore and PacBio sequencing data on CEPH Pedigree 1463 NA12878 sample. We have previously reported genotyping estimates by VNTRTyper on PacBio NA12878 WGS data12. Here we use the genotype estimates by VNTRTyper on PacBio NA12878 WGS data to compare genotype estimates by Tandem-genotype and the results of targeted sequencing analysis.\n\nWe compared the accuracy of genotype estimates from WGS data with PCR sizing analysis. Genotype estimates by VNTRTyper and Tandem-genotypes on WGS data were compared with PCR sizing results on 10 targets (Table 2). VNTRTyper and Tandem-genotypes had comparable correlation with PCR sizing analysis for both Nanopore and PacBio WGS. Genotype estimates for all 142 targets from Nanopore and PacBio WGS data determined using VNTRTyper and Tandem-genotypes are provided in Extended data, Supplementary Spreadsheet Table 330.\n\n^NPW_VT – Nanopore WGS VNTRTyper; NPW_TG – Nanopore WGS Tandem-genotypes; PBW_VT – PacBio WGS VNTRTyper; PBW_TG – PacBio WGS Tandem-genotypes; NPC_VT – Nanopore Capture sequencing VNTRTyper; NPC_TG – Nanopore Capture sequencing Tandem-genotypes\n\nRepeat Number in reference hg19 is provided within brackets for each target\n\nRepeat numbers that do not agree with PCR results are highlighted in red.\n\nND – Sufficient data not available for genotype analysis.\n\nWe compared the genotype estimates between WGS data and targeted capture sequencing data (77 targets which had results for both WGS and targeted sequencing). Genotype estimates by VNTRTyper between WGS data and targeted capture sequencing data showed a correlation of 0.9782 (correlation on 154 alleles) (Figure 3a). Genotype estimates by Tandem-genotypes had lower correlation between WGS and targeted capture sequencing data of 0.7694 (correlation on 152 alleles – 76 targets, removing the two outliers improved correlation to 0.9084) (Figure 3b). On the subset of seven targets for which we had generated PCR sizing analysis, Nanopore WGS data correlated with 12/14 genotype estimates on Nanopore capture sequencing using VNTRTyper precisely compared to PCR sizing (Table 2 and Figure 4a). Genotype estimates using Tandem-genotypes on Nanopore WGS data correlated with 11/12 genotype estimates on Nanopore capture sequencing precisely compared to PCR sizing (Figure 4b).\n\nUsing (a) VNTRTyper and (b) Tandem-genotypes for the NA12878 sample.\n\nUsing (a) VNTRTyper and (b) Tandem-genotypes. Red line indicates PCR sizing results. Targets with no genotype estimates are shown as a gap for the corresponding column.\n\nTo assess the extent of variation in repeat numbers between individuals, we compared the genotype estimates to the reported reference (hg19) repeat number. Genotype estimates determined by VNTRTyper on Nanopore capture sequencing on seven members of CEPH pedigree 1463 were used to assess the variation. We found that for a given sample, on average 51% (range 45–60%) of the targets have a genotype which is different to the reference, with more deletions (28%) than duplications (23%) (Figure 5).\n\n\nDiscussion\n\nIn this study, we present a targeted sequencing approach combined with long-read sequencing technology to genotype TRs. To our knowledge, this is the first report on genotyping analysis of hundreds of TRs using targeted long-read sequencing approach. Sequencing reads that span the entire repeat region and flanking region are often useful in providing an accurate estimation of the repeat genotype. Long-read sequencing technologies have the ability to generate reads which can span the entire repeat region and flanking regions. However, whole genome long-read sequencing analysis is still expensive for large-scale population analysis; hence, we developed a targeted long-read sequencing approach for TR analysis.\n\nWe showed that 1) target enrichment of repetitive sequences followed by long-read sequencing is feasible and 2) genotype predictions on targeted TR sequencing are comparable to the accuracy of PCR sizing analysis of repeats. Overall, we achieved an average genotyping rate of 75% for 110 TR loci with repeat length less than 2 kb and GC content greater than 25%. Genotyping rate improved to 91% for the highest-coverage sample, indicating that more sequencing could improve genotyping rate.\n\nTargets with low GC sequence content (<25% GC content) did not have sufficient sequence coverage with targeted sequencing. We have previously performed short-read target capture on these regions12 and observed low sequence coverage in low GC targets. However, both Nanopore and PacBio WGS data do not have any bias in sequence coverage in low GC regions. Hence, the lack of sequence coverage in low GC region for targeted sequencing is likely due to the capture protocol. To overcome the issue of low capture efficiency for low GC regions, it is feasible to increase the number of probes in low GC regions during probe design. This will improve sequence enrichment in low GC targets. Furthermore, use of simulation tools31,32 which can simulate sequencing data from probe sequences designed for capture sequencing can be used to assess the probe design efficiency prior to sequencing. This will allow to improve probe design in regions with low capture efficiency and subsequently improve coverage.\n\nWe also observed targets greater than 2 kb in length could not be genotyped due to the lack of spanning reads for genotyping analysis. This is primarily due to the limitation in sequence read length observed from the capture process. Streptavidin beads used during the capture process has limitations on the size of the fragments it can bind to, which limits the fragment length attainable with this capture protocol. Although there are longer TRs (greater than 2 kb) in the human genome, more than 99% of the TRs reported in human reference genome (hg38) are less than 2 kb in length3. Therefore, our protocol would still be able to successfully genotype most of the TRs in the human genome. TRs greater than 2 kb might need further optimized enrichment protocols.\n\nOur target panel included eight (out of the 142 targets) STRs with longer expansions (>200 number of copies) and seven of these targets failed to genotype. However, three of these had low GC content and one was greater than 4 kb in repeat length. The longer expansions which failed to genotype also had low sequence coverage, however due to the low number of targets we could not conclusively identify the cause for failure for these targets.\n\nWe used VNTRTyper, an in-house genotyping tool described by Ganesamoorthy et al. (2018)12 to determine the repeat number of TRs from long-read sequencing technologies. For comparison, we used Tandem-genotypes24, recently reported genotyping tool for the detection of TR expansions from long-read sequencing. Both genotyping methods were comparable to PCR sizing analysis and genotyping estimates were comparable between the approaches. However, Tandem-genotypes genotyped fewer targets than VNTRTyper. The differences are likely due to the different algorithms used between the methods. Both VNTRTyper and Tandem-genotypes uses reads spanning the repeat region. However, for Tandem-genotypes the flanking length used for analysis is depended on the length of the repeat unit, with a maximum of 100 bp on both sides of the repeat unit. On the other hand, VNTRTyper uses a default 30 bp flanking length for analysis, but it is feasible to change the flanking length. Due to the longer flank length requirement, Tandem-genotypes could have possibly failed to genotype more targets compared to VNTRTyper.\n\nVariations in TRs are a major source of genomic variation between individuals. TRs targeted in this study were initially selected due to the variation observed between case and control samples for obesity analysis12 and these TRs are variable in the population. We show that approximately 50% of the targeted TRs differ from reported reference copy number. However, the major limitation in this analysis is that the sample size is small, and the individuals are related, which introduces a bias in the analysis. Nevertheless, these findings indicate the possibility of variation in TR copy number between individuals and further large-scale studies are required to ascertain the extent of variation.\n\nWe demonstrated that the accuracy of genotype estimates between WGS and targeted capture sequencing were comparable to the accuracy of PCR sizing analysis. However, targeted capture enrichment protocols used in this study have amplification steps, which can introduce errors in TR analysis. This could possibly explain the differences in genotype estimates observed between WGS and targeted capture sequencing for some targets.\n\nAn amplification free targeted analysis with long-read sequencing is an ideal option for accurate genotyping of TRs. Targeted cleavage with Cas9 enzyme followed by Nanopore sequencing33 or PacBio sequencing34 has been recently reported as alternative option for enrichment of regions of interest. This method does not have any amplifications and can be adapted for multiple targets in a single assay. However, currently the DNA input requirements are high and sequencing output are low, which currently restricts wide use of this technique for large-scale analysis.\n\nSelective sequencing approaches utilising Nanopore real-time sequencing capabilities has been reported recently as an alternative approach to enrich regions of interest35,36. Selective sequencing works by mapping a section of the sequence read generated to the regions of interest and if the fragment matches to a region of interest, it will proceed with sequencing the fragment, if not the fragment is ejected from the pore. This approach will be a cost-effective approach to genotype TRs as it removes the need for specific sample preparation for target enrichment; however, the efficiency of this approach for TR analysis is yet to be determined.\n\nThe targeted long-read sequencing approach presented in this study is a cost-effective approach to analyse hundreds of TRs simultaneously. Long-read Nanopore WGS can cost approximately $4000 for 30X coverage of human genome and often with varying coverage across the genome. However, targeted long-read sequencing can be performed for a fraction of cost (less than $300 per sample depending on the multiplexing level) to enrich up to 25 Mb of genomic sequence of interest. The ability to analyse hundreds of TRs for a fraction of cost allows to explore TRs in large-scale studies.\n\nIn summary, we present a targeted approach combined with long-read sequencing to enable cost-effective and accurate approach to genotype TRs using long-read sequencing. Using this method, we have successfully demonstrated the feasibility of targeted capture sequencing of repetitive sequences and genotyping TRs using Nanopore long-read sequencing technology. Our targeted long-read sequencing approach would provide a cost-effective tool for large-scale population analysis of tandem repeats.\n\n\nData availability\n\nNCBI BioProject: Capture Sequencing of Tandem Repeats. Accession number PRJNA422490, https://identifiers.org/ncbi/bioproject:PRJNA422490.\n\nFigshare: Supplementary Information for the \"High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing\" article. https://doi.org/10.6084/m9.figshare.12789278.v130.\n\nThis project contains the following extended data:\n\nSupplementary_Information,pdf, which contains the following:\n\n◦ Supplementary Figure 1. Sequence coverage distribution on targets vs GC%.\n\n◦ Supplementary Figure 2. Correlation of genotype estimates between PCR sizing and VNTRTyper.\n\n◦ Supplementary Figure 3. Correlation of genotype estimates between PCR sizing and Tandem-genotypes.\n\n◦ Supplementary Figure 4. VNTRTyper genotyping rate with 25% GC and 2Kb size threshold.\n\n◦ Supplementary Figure 5. Tandem-genotypes genotyping rate with 25% GC and 2Kb size threshold.\n\n◦ Supplementary Table 1. PCR primer sequences.\n\n◦ Supplementary Table 2. Targeted Sequencing metrics for Nanopore Capture sequencing of tandem repeats.\n\n◦ Supplementary Table 3. Genotype estimates on Nanopore targeted capture sequencing using Tandem-Genotypes.\n\nSupplementary_Spreadsheet_Table1.csv. (Genotype predictions on Nanopore Capture Sequencing data using VNTRtyper.)\n\nSupplementary_Spreadsheet_Table2.csv. (Genotype predictions on Nanopore Capture Sequencing data using Tandem-genotypes.)\n\nSupplementary_Spreadsheet_Table3.csv (Genotype predictions on NA12878 sample Nanopore WGS data and PacBio WGS data using VNTRTyper and Tandem-genotypes.)\n\nSupplementary_Information_PCR_data.pdf. (Capillary electrophoresis results of PCR sizing analysis.)\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Agilent Technologies for their technical assistance with targeted sequencing experiments.\n\n\nReferences\n\nLander ES, Linton LM, Birren B, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract \| Publisher Full Text\n\nJurka J, Kapitonov VV, Kohany O, et al.: Repetitive sequences in complex genomes: structure and evolution. Annu Rev Genomics Hum Genet. 2007; 8: 241–59. PubMed Abstract \| Publisher Full Text\n\nGelfand Y, Rodriguez A, Benson G: TRDB--the Tandem Repeats Database. Nucleic Acids Res. 2007; 35(Database issue): D80–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGemayel R, Cho J, Boeynaems S, et al.: Beyond junk-variable tandem repeats as facilitators of rapid evolution of regulatory and coding sequences. Genes (Basel). 2012; 3(3): 461–480. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArmour JA: Tandemly repeated DNA: why should anyone care? Mutat Res. 2006; 598(1–2): 6–14. PubMed Abstract \| Publisher Full Text\n\nHannan AJ: TRPing up the genome: Tandem repeat polymorphisms as dynamic sources of genetic variability in health and disease. Discov Med. 2010; 10(53): 314–21. PubMed Abstract\n\nGemayel R, Vinces MD, Legendre M, et al.: Variable tandem repeats accelerate evolution of coding and regulatory sequences. Annu Rev Genet. 2010; 44: 445–477. PubMed Abstract \| Publisher Full Text\n\nBidwell JL, Bignon JD: DNA-RFLP methods and interpretation scheme for HLA-DR and DQ typing. Eur J Immunogenet. 1991; 18(1–2): 5–22. PubMed Abstract \| Publisher Full Text\n\nTagliabracci A, Buscemi L, Sassaroli C, et al.: Allele typing of short tandem repeats by capillary electrophoresis. Int J Legal Med. 1999; 113(1): 26–32. PubMed Abstract \| Publisher Full Text\n\nBahlo M, Bennett MF, Degorski P, et al.: Recent advances in the detection of repeat expansions with short-read next-generation sequencing [version 1; peer review: 3 approved]. F1000Res. 2018; 7: F1000 Faculty Rev-736. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuitama J, Zablotskaya A, Gemayel R, et al.: Large-scale analysis of tandem repeat variability in the human genome. Nucleic acids research. 2014; 42(9): 5728–5741. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanesamoorthy D, Cao MD, Duarte T, et al.: GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing. BMC Bioinformatics. 2018; 19(1): 267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGymrek M, Golan D, Rosset S, et al.: lobSTR: A short tandem repeat profiler for personal genomes. Genome Res. 2012; 22(6): 1154–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHighnam G, Franck C, Martin A, et al.: Accurate human microsatellite genotypes from high-throughput resequencing data using informed error profiles. Nucleic Acids Res. 2013; 41(1): e32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao MD, Tasker E, Willadsen K, et al.: Inferring short tandem repeat variation from paired-end short reads. Nucleic Acids Res. 2014; 42(3): e16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWillems T, Zielinski D, Yuan J, et al.: Genome-wide profiling of heritable and de novo STR variations. Nat Methods. 2017; 14(6): 590–592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDashnow H, Lek M, Phipson B, et al.: STRetch: detecting and discovering pathogenic short tandem repeat expansions. Genome Biol. 2018; 19(1): 121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDolzhenko E, van Vugt JJF, Shaw RJ, et al.: Detection of long repeat expansions from PCR-free whole-genome sequence data. Genome Res. 2017; 27(11): 1895–1903. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMousavi N, Shleizer-Burko S, Yanicky R, et al.: Profiling the genome-wide landscape of tandem repeat expansions. Nucleic Acids Res. 2019; 47(15): e90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchule B, McFarland KN, Lee K, et al.: Parkinson's disease associated with pure ATXN10 repeat expansion. NPJ Parkinsons Dis. 2017; 3: 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Roeck A, De Coster W, Bossaerts L, et al.: Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION. Genome Biol. 2019; 20(1): 239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEbbert MTW, Farrugia SL, Sens JP, et al.: Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease. Mol Neurodegener. 2018; 13(1): 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Q, Zhang P, Wang D, et al.: Interrogating the \"unsequenceable\" genomic trinucleotide repeat disorders by long-read sequencing. Genome Med. 2017; 9(1): 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitsuhashi S, Frith MC, Mizuguchi T, et al.: Tandem-genotypes: robust detection of tandem repeat expansions from long DNA reads. Genome Biol. 2019; 20(1): 58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaramitros T, Magiorkinis G: Multiplexed Targeted Sequencing for Oxford Nanopore MinION: A Detailed Library Preparation Procedure. Methods Mol Biol. 2018; 1712: 43–51. PubMed Abstract \| Publisher Full Text\n\nJain M, Koren S, Miga KH, et al.: Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol. 2018; 36(4): 338–345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPendleton M, Sebra R, Pang AWC, et al.: Assembly and diploid architecture of an individual human genome via single-molecule technologies. Nat Methods. 2015; 12(8): 780–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H: Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018; 34(18): 3094–3100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKielbasa SM, Wan R, Sato K, et al.: Adaptive seeds tame genomic sequence comparison. Genome Res. 2011; 21(3): 487–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanesamoorthy D, Yan M, Murigneux V, et al.: Supplementary Information for the \"High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing\" article. figshare . Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12789278.v1\n\nKim S, Jeong K, Bafna V: Wessim: a whole-exome sequencing simulator based on in silico exome capture. Bioinformatics. 2013; 29(8): 1076–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao MD, Ganesamoorthy D, Zhou C, et al.: Simulating the dynamics of targeted capture sequencing with CapSim. Bioinformatics. 2018; 34(5): 873–874. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGilpatrick T, Lee I, Graham JE, et al.: Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol. 2020; 38(4): 433–438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHafford-Tear NJ, Tsai YC, Sadan AN, et al.: CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat. Genet Med. 2019; 21(9): 2092–2102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPayne A, Holmes N, Clarke T, et al.: Nanopore adaptive sequencing for mixed samples, whole exome capture and targeted panels. BioRxiv. 2020; 2020.02.03.926956. Publisher Full Text\n\nKovaka S, Fan Y, Ni B, et al.: Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED. bioRxiv. 2020; 2020.02.03.931923. Reference Source" }	[ { "id": "70683", "date": "17 Sep 2020", "name": "Rick M. Tankard", "expertise": [ "Reviewer Expertise bioinformatics", "statistics", "genomics", "epigenetics", "repeat expansions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important work in sizing tandem repeats from long-read sequencing data in a cost-effective manner.\nSome more STR analysis tools should be cited for \"Several computational tools have been developed to improve the accuracy of TR genotyping from short-read sequencing data with varying performance\", being exSTRa3, ExpansionHunter Denovo2, TREDPARSE4 and TRhist1. The sentence “Yet, most of these tools have focused mainly on the analysis of STRs and analysis of longer TRs remains a hurdle for these approaches.”, it may give the impression that these tools cannot deal with repeat units longer than 6 bp (as defined for STRs in the Introduction), though both GangSTR and ExpansionHunter Denovo deal with up to 20 bp repeat units by default (adjustable with ExpansionHunter Denovo at run time, though there may not be evidence this will give good results).\n\nRegarding reproducibility, the data for comparisons is available. It is not apparent that the software VNTRTyper is available for use, being labelled as an in-house tool. This will make it difficult for others to verify the performance of VNTRTyper on other data. The availability of VNTRTyper would allow other researchers to make use of this work on their own data.\n\nIn Tables 1 and 2, I would appreciate a Root Mean Square Error (RMSE) between the PCR genotypes and the genotypes of other methods to get a sense of the scale of errors. Similarly, for Figure 3, being careful this isn't the RMSE of the linear model.\n\nOverall, I would recommend accepting the article with some small changes.\n\nMinor: Typo in some figure captions: ‘genme’\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "70688", "date": "21 Sep 2020", "name": "Mark T. W. Ebbert", "expertise": [ "Reviewer Expertise Genomics", "long-read sequencing", "statistics", "TRs", "bioinformatics", "computational biology." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGanesamoorthy et al. highlight an important issue and challenge in genotyping large tandem repeats (TR) that cannot be accurately genotyped using short-read sequencing data. This is an important issue because variations in TRs are known to cause many diseases, such as in repeat-expansion and repeat-retraction diseases. Important examples of repeat-expansion diseases include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington’s disease, Fuch’s disease, muscular dystrophy, and many more. A prime example of a repeat-retraction disease is facioscapulohumeral muscular dystrophy (FSHD). Thus, being able to accurately genotype TRs is a critical issue for human health and disease research, as it will help understand disease etiology and how to diagnose and treat diseases.\n\nWhile the questions being asked are important, there are unfortunately many limitations and issues with this particular study. My critique is honestly meant to be helpful to both the authors and the readers of this manuscript, and hope it will be received that way.\n\nThe major issues are as follows:\nWhat may be the biggest issue is the methods used to answer the questions at hand. The authors set out to genotype 142 TRs of various sizes across seven individuals, ranging “from 112 to 25236 bp in length”. This is a fantastic goal and I was excited to see the results. The first methodological issue is that the authors used amplification methods as part of the sequencing. I understand that the authors were seeking to maximize the number of targets they could include in their study based on costs, and to ensure deep coverage, but amplification simply will not work for long TRs (e.g., a TR that is 25236 nucleotides long), or those with high GC content; it should work fine for shorter and less GC-rich TRs, however. Surprisingly, the authors even acknowledge that the Agilent SureSelect protocol they employed “works effectively on fragments less than 4 kb in length”, which clearly indicates that interrogating those ≥4kb will not work.\n\nWhat surprised me most, however, was that the authors then intentionally sheared the amplicons to 3kb, making any attempt to sequence TRs >3kb a nonstarter—and realistically making it difficult to sequence even TRs that approach 3kb, which the authors found in their results. Specifically, the authors found they were most successful sequencing and characterizing TRs <2kb.\nThis alone does not invalidate the utility for all of the authors’ results, however; it simply limits their results to TRs <3kb (maybe <2kb). Thus, these results are still useful, but all downstream results and conclusions should be kept within these bounds.\n\nAdditional minor issues:\nEarly in the introduction, the authors make important points about repetitive sequences in the human genome. Most of the data they present, however, are extremely outdated, including data from the original human genome in 2001 and the number of TRs in the human genome from hg18 (2006). It’s great to cite these early papers, but we now know that they are inaccurate because the reference genome has improved dramatically. Here are some specific points that need to be corrected in the introduction:\nData from the original genome should not be stated as the current estimate for repeated sequences in the human genome, as was stated in the first sentence of the introduction. There are papers that are much more recent and that use a more updated reference genome. The authors make an important point about the number of TRs in the human genome, citing Gelfand et al. The data they present, however, is far outdated. The data from Gelfand et al. is from 2006 using the reference genome hg18. Reference genome hg18 is far too outdated to be used in a paper to be published in 2020. Most researchers are already moving past hg19 (to hg38). The authors need to update these statistics to represent data from hg38, which is actually to their advantage, as it will only further emphasize their point that TRs are plentiful in the human genome. Authors state that “…repeats with one to six basepair repeat units are classified as microsatellites or short tandem repeats (STRs) and those with more than six basepair repeat units are known as minisatellites”, citing Gemayel et al. The paper by Gemayel et al., however, specifically states that microsatellites are between 1 and 10 nucleotides and that minisatellites are >10. After reading a bit more, I see that there is some discrepancy on the exact cutoff for each group, but the authors need to be clearer about this. It is certainly not accurate to state a cutoff of six, citing a paper that clearly states a different cutoff. Authors should clarify and cite additional papers on the matter—perhaps including a more recent publication, though that may not be required.\n\nDevelopment for Albacore ended over 1.5 years ago (last release was January 2019), and the authors used a version that is almost 2 years old (2.2.7 was released in October 2018). Software this old may not normally be an issue, but the technology and algorithms used for Nanopore sequencing have evolved so rapidly over the past two years that it may be important. The authors need to do redo analyses using a current version of Guppy (the current basecaller).\n\nThe authors use two different aligners (Minimap2 and LAST) for genotyping with VNTRTyper and Tandem-genotypes. It appears they did so because authors of Tandem-genotypes recommends LAST for their genotyper. I also assume that VNTRTyper works best with minimap2. It is reasonable to use different aligners if they have been validated for a given pipeline, but the authors should clarify in the methods why they used different aligners rather than simply stating that they did.\n\nAuthors mention optimizing the PCR conditions for each TR, but I do not see specific details for these optimizations. If they are present in supplementary material, they should be clearly referenced in the manuscript.\n\nBased on the methods, it is unclear whether the authors are only using reads that fully span the TRs to genotype (i.e., reads that contain adjacent sequence on both ends of the TR). It would not make sense to include any reads that do not fully span the TR. Authors should also make it clear how they determine whether reads span the TR.\n\nMethods for how authors performed most correlations are unclear. For example, for correlations to PCR, did they perform PCR for each sample across all of the TRs tested? i.e., how many data points are even included?\n\nFigures 3 & 4 misspelled ‘genome’ in the figure title.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1084
https://f1000research.com/articles/9-1080/v1	01 Sep 20	{ "type": "Case Report", "title": "Case Report: Incidentally diagnosed hemangioma of the right atrioventricular groove in an athlete", "authors": [ "Achour Asma", "Mezri Maatouk", "Ahmed Miladi", "Marouane Mahjoub", "Mabrouk Abdelali", "Badii Hmida", "Ahmed Zrig", "Mejdi Ben Massoud", "Walid Mnari", "Mezri Maatouk", "Ahmed Miladi", "Marouane Mahjoub", "Mabrouk Abdelali", "Badii Hmida", "Ahmed Zrig", "Mejdi Ben Massoud", "Walid Mnari" ], "abstract": "The purpose of this article is to illustrate a rare case of a pericardial hemangioma of the right atrioventricular groove of incidental discovery in a tennis player who presented with cough and dyspnea and was treated by surgical excision with a favorable outcome. We also report the role of cardiac magnetic resonance imaging (MRI) in the diagnosis and management of this pericardial tumor.", "keywords": [ "Cavernous hemangioma", "Cardiac tumors", "Pericardium", "Tamponade", "Athletes" ], "content": "Introduction\n\nCardiac hemangioma is a rare benign tumor1 and pericardial localization is extremely rare2–4. It is usually asymptomatic, but it can be serious due to the risk of tamponade. We report the case of a pericardial hemangioma of the right atrioventricular groove in a young athletic patient who presented with cough and dyspnea and was diagnosed incidentally.\n\n\nCase report\n\nA 31-year-old Caucasian female tennis player presented to the emergency department with dyspnea and dry cough for a few days. She had undergone surgery previously for a borderline ovarian tumor eight years ago. There was no history of cardiopulmonary disease, coronary artery disease, or other cardiovascular diseases. No abnormalities were found during the physical examination with no jugular venous distension.\n\nA chest X-ray showed enlargement of the cardiac shadow suggestive of pericardial effusion (Figure 1). Transthoracic echocardiography confirmed a large circumferential pericardial effusion and showed a rounded, well defined pericardial hyperechoic lesion attached to the right atrioventricular groove. There was no right ventricular dysfunction.\n\nA thoracic computed tomography (CT) scan was performed, which showed a large pericardial effusion and confirmed a pericardial mass with homogenous contrast enhancement within the right atrioventricular groove (Figure 2).\n\nCardiac magnetic resonance imaging (MRI) confirmed the large pericardial effusion with a pedunculated ill-defined homogeneous hypointense mass on T1 and a hyperintense mass in the right atrioventricular groove with progressive enhancement after contrast administration on T2 (Figure 3).\n\nCardiac MRI four-chamber view cine steady state free precession (A), black blood T1 weighted without (B) and after gadolinium administration (C): Rounded well defined homogenous hyperintense T2 hypointense T1 mass in right atrioventricular groove with homogenous enhancement after contrast administration (arrow). Note large pericardial effusion (star).\n\nA coronary angiography was performed, which showed tumor blush.\n\nThe patient was referred to a cardiovascular surgery center to be operated on by an experienced cardiac surgeon. General anaesthesia was performed in supine position. Anaesthesia induction was performed by intravenous bolus of propofol (2mg/Kg), tracrium (0.5 mg/Kg) and fentanyl (2 mcg/Kg). Anaesthesia maintenance was performed by isoflurane 1.5% in oxygen and continuous intravenous infusion of tracrium (0.01 mg/Kg/min) and fentanyl (1 mcg/kg/hour). Surgery was initiated by a median sternotomy. Initial examination showed no extension of the mass into the cardiac chamber. A safety total excision of the mass was done using cutting diathermy. Vascular, pericardial and sternal sutures were performed by polypropylene, vicryl and wire, respectively. The anatomopathological examination of the mass revealed conjunctive tumor proliferation, vascular differentiated and concluded with a diagnosis of cavernous hemangioma. Post-procedural medication included antibiotic therapy with cefazolin (1 g intravenously, twice a day) for 48 hours, preventive anticoagulation by low molecular weight heparin (Enoxaparin 0.4 ml subcutaneously, once a day) and analgesic therapy by paracetamol (1 g intravenously, three times a day). Post-operative course was favorable and the patient was discharged after 72 hours.\n\nTwo months after surgery, the patient developed progressive dyspnea vomiting and precordial chest pain. CT scan found loculated left pleural effusion. Chest physiotherapy (one session a day) for two weeks and paracetamol (1 g orally, twice a day) for one week were prescribed with a favorable outcome. The patient remains well after two years of follow-up.\n\n\nDiscussion\n\nCardiac hemangiomas are rare benign vascular tumors and constitute only 2.8% of primary cardiac tumors1. Pericardial localization is extremely rare2–4. Histopathologically, hemangiomas are characterized by benign proliferation of the endothelial cell lining of the blood vessel with increasing vascularization5.\n\nPericardial hemangioma is mostly asymptomatic. Clinical symptoms depend on location, size, and anatomic extension of the tumor5. The most frequents symptoms are dyspnea, cardiac arrhythmia, murmurs, and heart failure. Tamponade due to pericardial effusion can also occur. Imaging is very useful for the diagnosis, localization, and extension of the tumor. CT scans with contrast can show enhancing foci at the arterial phase with diffuse or heterogeneous enhancement at the delayed phase. Small calcifications might be seen also6. Cardiac MRI is a superior tool with a better contrast resolution5. Hemangiomas have an intermediate T1 signal with the same intensity as myocardium and a high T2 signal7. The dynamic postcontrast acquisition shows nodular enhancement with progressive fill-in on delayed images8. Feeding vessel, tumor blush, and flow voids might be seen also1. The tumors are usually ill-defined with no local invasion. Differential diagnoses can be made with solid pericardial masses such as mesothelioma, sarcoma, lymphoma, or paraganglioma4. Surgical total excision is the treatment of choice for resectable tumors9. The use of radiotherapy, corticosteroids, and beta blockers have been reported in some cases1.\n\n\nConclusion\n\nPericardial hemangiomas are extremely rare benign vascular tumors whose prognosis depends on their location and size. Surgical excision constitutes the treatment of choice. Our case demonstrates the importance of cardiovascular MRI as a tool to evaluate the resectability of the tumor.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nLi W, Teng P, Xu H, et al.: Cardiac Hemangioma: A Comprehensive Analysis of 200 Cases. Ann Thorac Surg. 2015; 99(6): 2246–52. PubMed Abstract \| Publisher Full Text\n\nReynen K: Frequency of primary tumors of the heart. Am J Cardiol 1996; 77(1): 107. PubMed Abstract \| Publisher Full Text\n\nKojima S, Sumiyoshi M, Suwa S, et al.: Cardiac hemangioma: a report of two cases and review of the literature. Heart Vessels. 2003; 18(3): 153–6. PubMed Abstract \| Publisher Full Text\n\nGupta N: Intrapericardial hemangioma: a case report. J Clin Diagn Res. 2013; 7(1): 169–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVargis RS, Phansalkar M, Padhi S, et al.: Pericardial Haemangioma: A Common Tumour in an Unusual Location: Case Report and Review of Literature. J Clin Diagn Res. 2017; 11(5): ED15–ED17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRestrepo CS, Vargas D, Ocazionez D, et al.: Primary Pericardial Tumors. RadioGraphics. 2013; 33(6): 1613–30. PubMed Abstract \| Publisher Full Text\n\nHrabak-Paar M, Hübner M, Stern-Padovan R, et al.: Hemangioma of the interatrial septum: CT and MRI features. Cardiovasc Intervent Radiol. 2011; 34 Suppl 2: S90–3. PubMed Abstract \| Publisher Full Text\n\nEdiae J, Lim PS, Addonizio VP, et al.: Pericardial hemangioma taking origin from the posterior wall of the left atrium. Ann Thorac Surg. 2009; 87(6): e54–6. PubMed Abstract \| Publisher Full Text\n\nEsmaeilzadeh M, Jalalian R, Maleki M, et al.: Cardiac cavernous hemangioma. Eur J Echocardiogr. 2007; 8(6): 487–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "70789", "date": "07 Sep 2020", "name": "Jean-Nicolas Dacher", "expertise": [ "Reviewer Expertise Radiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nice case report of a rare condition that shows the importance of pre-operative imaging. I recommend indexing but a minor revision should be made regarding the description of MR findings.\nIn the \"diagnostic assessment\" section (MR part), the authors state that; \"there is progressive enhancement after contrast administration on T2\". This is misleading and should be corrected as usually contrast enhancement is not to be searched on T2-w imaging. I suppose that enhancement was detected either on post contrast T1-w imaging or on first pass perfusion (Saturation Recovery). This section should be re-written as well as the caption of Fig. 3 that is unclear in the present form.\nI suggest the authors to re-use the excellent description from their discussion.\nIn the discussion please change the first sentence of the second chapter; the patient is asymptomatic, not the hemangioma.\nPlease delete the 's' at frequent (second chapter).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5926", "date": "10 Sep 2020", "name": "Asma ACHOUR", "role": "Author Response", "response": "Dear master and colleague,Thank you for the interest you have shown in our topic.I thank you for your relevant comments which will improve the quality of our manuscript." } ] }, { "id": "74537", "date": "13 Jan 2021", "name": "Weikun Hu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reported a rare case of pericardial hemangioma in an athlete, which showed potential clinical significance.\nI recommend indexing after a minor revision.\nThe histological result and image of the tumor should be showed, as this is the important information for the diagnosis of hemangioma.\n\nIn addition, the post-operative cardiac MRI should also be showed, if the authors had done.\n\nThe patient had border-line ovarian tumor eight years ago. Were there any other general physical examinations that had been done before the surgery, such as PET-CT or specific markers of tumor? It is important to distinguish the possibility of tumor recurrence or metastasis.\n\nThe \"diagnostic assessment\" section (MR part) should be corrected as following: a hyperintense mass in the right atrioventricular groove with progressive enhancement after contrast administration on T1 (not T2 as shown in the manuscript).\n\nThe arrow is invisible in the Figure 2 as described in the figure legend.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1080
https://f1000research.com/articles/9-1079/v1	01 Sep 20	{ "type": "Research Article", "title": "Assessment of the phytochemical, antioxidant and antibacterial activities of Heteromorpha arborescens (Spreng.) Cham & Schltdl. leaf extracts", "authors": [ "Taiwo Oluwafunmilola Abifarin", "Gloria Aderonke Otunola", "Anthony Jide Afolayan", "Taiwo Oluwafunmilola Abifarin", "Anthony Jide Afolayan" ], "abstract": "Background: Heteromorpha arborescens (Spreng.) Cham. and Schltdl (Apiaceae) is widely used traditionally for the treatment of a wide range of diseases in Southern and Eastern Africa. Although previous studies have reported the biological activities of hexane, ethyl acetate and methanol extracts of H. arborescens leaves, there is no scientific information on the phytochemical contents, antioxidant and antibacterial activities of acetone, ethanol, aqueous and blanched extracts. This study is therefore aimed to investigate and compare the phytochemical contents, antioxidant and antibacterial activities of acetone, ethanol, aqueous and blanched extracts of H. arborescens leaves. Methods: Phytochemical analysis for the total phenolic, flavonoid, proanthocyanidin, alkaloid and saponin contents of all the fractions were determined by spectroscopic methods, while the free radical scavenging potential of the extracts were evaluated using DPPH, ABTS radical scavenging and total antioxidant capacity assays. Micro dilution method was used to determine the Minimum Inhibitory Concentrations (MIC) of H. arborescens leaf extracts against Bacillus pumilus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. Results: Total phenol content of the extracts ranged between 15.10 mg GAE/g- 42.50 mg GAE/g, proanthocyanidin was 459-8402.1 mg QE/g, and flavonoid content of 109.24-235.79 mg QE/g. In addition, alkaloids (5.59%) and saponins (23.33%) were present in significant amounts. Based on the IC50 values, the ethanol extract exhibited the highest total antioxidant activity (0.013 mg/mL) with highest inhibition against DPPH and ABTS radicals (0.06 and 0.049 mg/mL respectively). Considerable antibacterial activities were observed in the acetone, ethanol and blanched extracts with MIC values ranging from 1.563-12.5 mg/mL; however, the aqueous extract was inactive against all the bacteria strains. Conclusion: The study suggests that H. arborescens leaves could be a valuable source of bioactive compounds. Although the blanching process significantly decreased polyphenolic contents and antioxidant activities of the extracts, it increased the antibacterial compounds.", "keywords": [ "Phytochemicals", "Heteromorpha arborescens", "antioxidant", "antibacterial", "bioactives", "blanching", "traditional", "medicines." ], "content": "Introduction\n\nPlants from the Apiaceae family are generally known to be rich sources of phytochemicals and antioxidants and are commonly used as food, flavoring agents and medicine1. These bioactive constituents may contribute significantly to the protection of humans from a wide range of diseases2–4. Heteromorpha arborescens (Spreng.) Cham. and Schltdl otherwise referred to as parsley tree in English, “wildepitersielie” in Afrikaans and “umbangandlala” in Xhosa is a large shrub or small tree that belongs to the Apiaceae family3.\n\nThe leaves vary from simple to compound and the flowers are small, greenish to yellowish in colour, occurring in compound umbels, with waxy bark which is smooth or glossy in texture5. The species is generally considered as a medicinally important plant throughout Africa since almost all its parts are traditionally used for the treatment of different ailments6. In South Africa, the leaves and roots are used for blood purification, diabetes and shortness of breath7–9. The bark, leaves and roots are also used for respiratory problems in Kenya, Lesotho and Tanzania9. The leaves are consumed as vegetables in Kenya10, while the roots are given to malnourished children in Botswana and Swaziland11. The plant is also used to treat abdominal pains, dysmenorrhea, nervous and mental disorders, as well as a vermicide in children12.\n\nThe healing ability of the plant is attributed to the presence of bioactive compounds, such as tannins, phenols, alkaloids, saponin, flavonoids, and proanthocyanidin. Although very scanty, previous scientific reports exist on the phenolic contents, antioxidant13,14 and antibacterial activities3 of hexane, ethyl acetate and methanol extracts of H. arborescens leaves. To the best of our knowledge no study exists on comparison of polyphenolic contents, antioxidant and antibacterial activities of blanched, aqueous, acetone and ethanol extracts of H. arborescens leaves. The present study was therefore conducted to determine the phytochemical contents, antioxidant and antibacterial activities of blanched, aqueous, acetone and ethanol extracts of H. arborescens leaves.\n\n\nMethods\n\nEthical approval was granted by the University of Fort Hare Animal and Plant Use Research Ethics Committee, South Africa with protocol number OTA011SABI01/19/E.\n\nAll reagents and chemicals including gallic acid, rutin, quercetin, aluminum chloride (AlCl3), ferric chloride (FeCl3.7H20), Folin- Ciocalteu, oxalic acid, trichloroacetic acid (TCA), sulphuric acid (H2SO4), hydrochloric acid (HCl), Na2CO3, ammonium molybdate, potassium ferricyanide (K3Fe(CN)6), catechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylobenzothiazoline-6-sulphonic acid diammonium salt) (ABTS) are products of Sigma-Aldrich, South Africa. Other chemicals such as anhydrous sodium carbonate, sodium nitrite (NaNO2), ethanol, methanol, n-butanol, sodium acetate, butylated hydroxytoluene (BHT), diethyl ether, glacial acetic acid, sulfanilic acid, potassium persulphate, sodium nitroprusside were also purchased from the same company. All the reagents used were of analytical grade. The bacterial strains including; Klebsiella pneumoniae ATCC 13883, Staphylococcus aureus, ATCC 29213, Pseudomonas aeruginosa ATCC 19582, Escherichia coli 25922, Bacillus pumilus ATCC 14884, Staphylococcus epidermis ATCC 12228 were obtained from the Department of Biochemistry and Microbiology, University of Fort Hare, South Africa.\n\nFresh leaves of H. arborescens were obtained in the month of June, 2019 from an area located on latitude 32° 47′ 50.4″ S, 26° 52′ 41.8″ E along Hogsback road, Alice Town, Eastern Cape, South Africa. The plant was authenticated by a taxonomist at the University of Fort Hare and a voucher specimen was preserved in the Giffen Herbarium and assigned number Abif2019/03.\n\nFresh H. arborescens leaves were oven dried at 400C and crushed to coarse powder. 100 g of the powdered plant material was extracted separately in 500 mL acetone, ethanol and water for 24 hours on an orbital shaker. An equal weight of the fresh leaves was immersed into 500 mL of hot water (800C) for 5 min to simulate home cooking and ground with a blender. The extracts were filtered under pressure using a Buchner funnel and Whatman filter paper (150 mm); the acetone and ethanol extracts were concentrated to dryness using a rotary evaporator while the aqueous extract and blanched sample were subjected to freeze drying. The extracts were thereafter stored at 40C until further analysis.\n\nDetermination of total phenolic content. The total phenolic content was determined by spectrometry using Folin-Ciocalteu reagent. Briefly, 0.5 mL of extract (1mg/mL) and gallic acid (0.02 to 0.1mg/mL) were put separately in test tubes. Thereafter, 2.5mL of 10% Folin-Ciocalteu reagent was added into the test tubes, after which 2.5 mL of 7.5% sodium carbonate solution was added to the mixture, stirred and incubated at 400C for 30 min. The absorbance was then measured at 760 nm using a Hewlett Packard VR-2000 spectrophotometer. All samples were analysed in triplicate. Total phenolic content was expressed as milligrams of gallic acid equivalent per gram (mg GAE/g) with the standard curve: y =10.875 x+ 0.1025, R2 = 0.996.\n\nWhere R is the determined coefficient, x is the concentration, and y is the absorbance.\n\nDetermination of flavonoids content. The amount of flavonoids present in the extract was determined using the aluminium chloride (AlCl3) colorimetric assay, as described by Majouli et al.15. A mixture of 0.5 mL of extract was added to an equal volume of 2% AlCl3 solution. The mixture was incubated for 10 min and vigorously shaken, after which the absorbance was measured at 420 nm. Varying concentrations of the standard (quercetin) were also prepared with the same method. All samples were analysed in triplicates. Flavonoid content was expressed as milligrams of quercetin equivalent per gram (mg QE/g) with the standard curve: y = 2.9422x - 0.4438, R2 = 0.913\n\nWhere R is the determined coefficient, x is the concentration, and y is the absorbance.\n\nDetermination of proanthocyanidin (condensed tannins). Proanthocyanidin content was determined as described by Sagbo et al.16. Briefly, a mixture of 3mL of 4% vanillin-methanol solution and 1.5mL of HCl was added to 0.5 mL of each extract. The solution was stirred and incubated at 27∘C for 15 min, after which absorbance was measured at 500 nm. All samples were analysed in triplicate and the amount of proanthocyanidin was expressed as mg/g dry weight of quercetin equivalent (mg QE/g) of the extract with the standard curve: y = 0.0252 x + 0.0482, R2 = 0.9005.\n\nWhere R is the determined coefficient, x is the concentration, and y is the absorbance.\n\nDetermination of alkaloid content. Alkaloid content of H. arborescens leaves was determined as previously described by Abifarin et al.17. 0.5 g of the pulverized leaves was mixed with 200mL of 10% acetic acid in ethanol. The mixture was covered, incubated at room temperature for 4 h, filtered and concentrated to about a quarter of its original volume in a water bath. To the extract, concentrated ammonium hydroxide was added in drops till precipitation was complete. After the solution was allowed to settle, precipitates obtained were washed with dilute ammonium hydroxide and then filtered. The residue was dried in an oven (40°C), weighed and the alkaloid content was determined using the following formula:\n\n% Alkaloid = weight of precipitate / initial weight of sample × 100.\n\nDetermination of saponin content. Saponin content of H. arborescens leaves was determined as previously described by Unuofin et al.18. Briefly, 0.5 g of pulverized H. arborescens leaves was measured into 50 mL of 20% ethanol prepared in distilled water. The mixture was heated in a hot water bath for 4 h at 55°C. The mixture was filtered and the residue extracted again with another 50 mL of 20% ethanol. The two filtrates were combined and reduced to 20 mL over a hot water bath (90°C). The concentrated solution obtained was poured into a 250 mL separating funnel containing 20 mL of diethyl ether. The aqueous layer was collected while the ether layer was discarded. 20 mL of n-butanol was added to the filtrate and then washed thrice with 10 mL of 5% sodium chloride. The mixture was heated in an oven (40°C) to constant weight. The percentage saponin content of the sample was calculated using the following formula:\n\n% Saponins = weight of final filtrate / weight of sample x 100\n\nDPPH radical scavenging activity. DPPH radical scavenging activity for each plant extract was determined as previously described by Ohikhena et al.19. Briefly, a reaction mixture containing 2.5 mL of DPPH solution (0.13 mM) and 2.5 mL of each plant extract or standard (rutin and BHT) dissolved in methanol at varying concentrations (0.005, 0.01, 0.02, 0.04, 0.08 mg/mL), stirred and kept in the dark for 30 min. The absorbance was measured at 517 nm and DPPH radical scavenging activity was calculated as:\n\n% DPPH scavenging activity = [(Abs DPPH − Abs Sample) Abs DPPH)] × 100\n\nWhere Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample extract/standard.\n\nABTS radical scavenging assay. ABTS scavenging activity of the different plant extracts was determined as described by Unuofin et al.20. A mixture was prepared by reacting 7 mM ABTS solution and 2.45 mM K2S2O8 (1:1), which was kept in the dark for 12 hours to produce a bluish green coloration. The solution was adjusted with methanol until an absorbance of 0.700 ± 0.01 at 734 nm was obtained. 1mL of plant extract was allowed to react with 1 mL of the working solution and the absorbance was measured at 734 nm after 7 min. The ABTS scavenging capacity of the extracts were compared with that of BHT and rutin and percentage inhibition was calculated as:\n\nABTS radical scavenging activity (%) = [(Abs control − Abs sample) / (Abs control)] × 100.\n\nWhere Abs control is the absorbance of ABTS radical + methanol; Abs sample is the absorbance of ABTS radical + sample extract/standard.\n\nTotal antioxidant capacity (phosphomolybdenum) assay. The total antioxidant capacity was determined by the method described by Abifarin et al.17. Briefly, 0.3 mL of the extracts and standard (0.025-0.4 mg/mL) were measured into separate test tubes and each was dissolved in 3 mL of reagent solution (0.6 M sulfuric acid, 4 mM ammonium molybdate, and 28 mM sodium phosphate). The test tubes were incubated at 90°C in a water bath for 90 min, allowed to cool to room temperature and the absorbance was measured at 695 nm. Rutin and BHT were used as standards. The percentage inhibition (%TAC) was calculated as:\n\n%TAC = [(Absorbance of sample − Absorbance of control) / (absorbance of sample)] × 100\n\nMicroorganisms and media. The bacteria used in this study were chosen primarily on the basis of their importance as opportunistic microorganisms for humans with diabetes mellitus. The bacterial strains include: Klebsiella pneumoniae ATCC 13883, Staphylococcus aureus, ATCC 29213, Pseudomonas aeruginosa ATCC 19582, Escherichia coli 25922, Bacillus pumilus ATCC 14884, Staphylococcus epidermidis ATCC 12228.\n\nMinimum Inhibitory Concentration (MIC). The MIC of the extracts were evaluated against selected Gram positive (Bacillus pumilus, Staphylococcus aureus and Staphylococcus epidermidis) and Gram negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli) by microdilution method, as described by Mohsenipour et al.21. The bacterial concentration in the inoculum was standardized at 0.5 McFarland turbidity scale, (1 × 108 CFU/ml).\n\nFirstly, sterile round bottom 96-well plates were filled with 100 µl of distilled water. 100 µl stock fractions of 50 mg/mL of plant extract and standard (Erythromycin) were then added into the first row. The fractions were serially diluted to make varying concentrations of the plant extracts (0.78125–12.5 mg/mL) and standard (0.0625–1 µg/mL) and then 50 µl of inoculums was added into the wells. Thereafter, 100 µl sterile nutrient broth culture medium plus 50 µl of the culture of each organism was added into each well and the inoculated micro plates were incubated at 37°C for 24 h. 1% DMSO was used as the negative control while 100 µl of inoculums only was used as the growth hormone. The plates were incubated for 24 h at 37˚C, and subsequently 40 µl of 0.4 mg/mL INT dye was added to each well. The plates were gently agitated and incubated for another 30 min. The MICs were then determined as the lowest concentrations at which there was no indication of colour change to pink (no bacterial growth).\n\nAll data were expressed as mean ± standard deviation (SD) of triplicates and subjected to one-way analysis of variance (ANOVA). Where the data showed significant difference (p < 0.05) among the extracts, a mean separation was done using Fischer’s Least Significant Difference. MINITAB 17 statistical package was used for analysis.\n\n\nResults\n\nThe total phenol, proanthocyanidin and flavonoid contents of H. arborescens leaf extracts are presented in Table 1. The highest phenol content was found in the ethanol extract (42.50 mg GAE/g), followed by the aqueous extract (34.24 mg GAE/g), blanched extract (21.42 mg GAE/g) and acetone extract (15.10 mg GAE/g). Ethanol extract (8402.1 mg QE/g) showed the highest proanthocyanidin content followed by acetone extract (4923 mg QE/g DW), blanched extract (928.6 mg QE/g) and aqueous extract (459 mg QE/g). The concentration of flavonoid was highest in the ethanol extract (235.79 mg QE/g), followed by acetone extract (230.52 mg QE/g), aqueous extract (154.95 mg QE/g) and blanched extract (109.24 mg QE/g). Quantitative determination of the alkaloid and saponin contents of H. arborescens leaves also revealed low alkaloid content (5.59%) with considerable amount of saponin (23.33%).\n\nThe results are expressed as mean ± standard deviation (n=3). Values with different superscripts are significantly different (P < 0.05) across the different extracts. *- not determined.\n\nABTS radical scavenging activity. The ABTS radical scavenging activities of the extracts and standards (BHT and rutin) are presented in Figure 1. The samples exhibited significant ABTS radical scavenging activities, which increased in a concentration dependent manner. Although the standards showed higher ABTS inhibitory potentials than the extracts, the highest inhibitory capacity of the extracts was observed in the ethanol and aqueous extracts. Based on the IC50 (Table 2), ABTS scavenging activity of the samples was in the order: BHT > rutin > ethanol & aqueous > acetone > blanched.\n\nResults are expressed as mean ± standard deviation (n=3). Columns with different letters are significantly different (P < 0.05) across the different samples.\n\nTotal antioxidant capacity. The total antioxidant capacity of the extracts and standards (BHT and rutin) are shown in Figure 2. There was a dose dependent increase in total antioxidant capacity of the samples, with the ethanol extract showing the highest antioxidant capacity when compared with other extracts. Based on the IC50, (Table 2), the values of the total antioxidant capacity of the standards and extracts were in the order: BHT > ethanol > rutin > aqueous > acetone > blanched.\n\nResults are expressed as mean ± standard deviation (n=3). Columns with different letters are significantly different (P < 0.05) across the different samples.\n\nDPPH scavenging activity. There was a dose-dependent increase in percentage inhibitory activity of the extracts and standards against DPPH radical (Figure 3). Ethanol extracts showed the highest DPPH radical inhibitory activity; even though the standards inhibited best when compared with the extracts. With respect to the IC50, (Table 2) the inhibitory capacity of the standards and extracts were in the order: rutin > BHT > ethanol > aqueous > blanched > acetone.\n\nResults are expressed as mean ± standard deviation (n=3). Columns with different letters are significantly different (P < 0.05) across the different samples.\n\nMinimum inhibitory concentration. Antibacterial activity of erythromycin (standard) and extracts of H. arborescens leaves are presented in Table 3. While all Gram-positive and Gram-negative bacteria strains tested were resistant to the aqueous extract at all the concentrations tested, the blanched, acetone, and ethanol extracts showed considerable antibacterial activities with MICs ranging from 1.563-12.5 mg/mL. Ethanol extract showed the best antibacterial activity against B. pumilus, S. epidermidis, S. aureus and P. aeruginosa, exhibiting a low MIC of (1.563 and 3.125 mg/mL). Generally speaking, the Gram-negative bacteria were observed to be more resistant against the extracts tested.\n\nBacillus pumilus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebselia pneumoniae.\n\nBacteria a- Gram positive. Bacteriab- Gram negative. Na- not active at tde highest concentration tested.\n\n\nDiscussion\n\nPlant extracts have been reported to have numerous protective roles due to their phytochemical contents, which contributes to a large extent towards their antioxidant and antimicrobial activities22. Oxidative stress, which leads to the production of free radicals in the body, plays a major role in the development of chronic and degenerative ailments such as cancer, arthritis, aging, autoimmune disorders, cardiovascular and neurodegenerative diseases23. Antioxidants play a vital role in deactivating and scavenging of the free radicals in order to reduce the risk of these chronic diseases24.\n\nIn the present study, the phytochemical, antioxidant and antibacterial activities of the acetone, ethanol, aqueous and blanched extracts of H. arborescens leaves were assessed. Phytochemical constituents varied significantly in all the extracts, which revealed the presence of phenols, flavonoids and proanthocyanidin in considerable amounts. The variation in the phytochemical constituents could be attributed to differences in the extracting capabilities of different solvents. Phenols, a major phytochemical found in plants have been widely studied because of their ability to reduce oxidative stress-related degenerative diseases, including cancer. Flavonoids are a group of hydroxylated phenolics, and their beneficial effects are linked to their antioxidant, antibacterial, anticancer, anti-inflammatory25,26 anti-hypertensive, and cardio protective activities27,28. Proanthocyanidins are a class of compounds belonging to the flavonoid family, with protective effects against tissue damage and cancer, and they also improve blood circulation by strengthening the capillaries, arteries and veins29.\n\nEthanol has been reported to be suitable for extraction of compounds with a wide range of polarity while water is suitable for very polar compounds30. Consequently, a higher quantity of phenolic compounds, proanthocyanidin and flavonoids were observed in the ethanol samples when compared to the acetone, blanched and aqueous extracts; hence, better activity. This is in agreement with previous reports that ethanol is more suitable for the extraction of phenolic compounds in plants30–32. Furthermore, high phenolic compounds were indicated in ethyl acetate and methanol extracts, suggesting the reason for its high antioxidant activity13. Flavonoid content of acetone extract of H. arborescens was comparable to results obtained by Elisha et al.13; however, total phenolic content was much higher as opposed to the current study.\n\nOur results indicate that blanching resulted in a significant decrease in total phenols and flavonoids with significant increase in proanthocyanidin content. This correlates with similar observations by Korus et al33. Jaiswal et al34. and Irondi et al.35. Losses of polyphenolic compounds upon blanching was also observed in some cruciferous vegetables, such as spinach36, kale, broccoli37 and cauliflower38,39. The decrease in polyphenolic contents could be attributed to the loss of water soluble vitamins and nitrogen compounds during the blanching process34. Contrary to the findings of this study, some researchers have claimed that the heating process may alter the cell membrane, causing a release of some membrane-bound phytochemicals which may increase bioavailability as observed by Jimenez-Monreal et al40. Similarly, Ma et al41. observed an increase in polyphenolic contents of Daucus carota L. juice after blanching (at 950 C for 3 min).\n\nSaponins possess medicinal properties such as anti-inflammatory, antibacterial, anticancer and cytotoxic activities42. Alkaloids have also been shown to exhibit antioxidant properties by reducing oxidative damage induced by hydrogen peroxide43. The amount of alkaloids and saponin exhibited by H. arborescens leaves is promising in playing the aforementioned biological roles.\n\nFurther assessment of the antioxidant activities of the extracts revealed the free radical scavenging potential of the extracts, which varied among the methods used. This variation was due to the fact that antioxidants are able to neutralize free radicals by different modes of action such as transition metal chelation, singlet oxygen quenchers, and donation of hydrogen44.\n\nThe total antioxidant capacity and scavenging activity of the extracts against DPPH+ and ABTS+ were compared to the standards (BHT and rutin) and the extracts showed considerable antioxidant activity. Since positive correlation between polyphenolic compounds and antioxidant activities have been reported43,45,46; the highest antioxidant activity exhibited by the ethanol extract could be attributed to its high polyphenolic compounds, as shown in this study. In terms of the relatively higher DPPH and ABTS inhibitory potential of the ethanol extract, these results are in agreement with antioxidant activity of other Apiaceae species such as: Coriandrum sativum47, Ammi majus L.48, Daucus carota L.49, Ferula gummosa50, Seseli libanotis (L.) Koch51, Foeniculum vulgare and Anethum graveolens1. This strengthens the suggestion that the most important bioactive potentials are observed in the plant extracts with high amounts of phenolics and flavonoids. This finding therefore provides some scientific verification for the biological activities of H. arborescens leaves.\n\nIn recent years, more attention has been given to the development of antibacterial drugs from medicinal plants instead of synthetic ones. The antibacterial activities of plant extracts have been confirmed against an array of both Gram-positive and Gram-negative bacteria52. Previous studies have established that the crude, hexane, ethyl acetate, chloroform and butanol extracts reveal antibacterial activities against E. coli, S. aureus and P. aeruginosa. However, P. aeruginosa was more susceptible to three out of the extracts with the butanol fraction showing the highest antibacterial activity53. The resistance of all the bacterial strains to the aqueous extract at all concentrations tested and the significant antibacterial activity of the blanched extract obtained in the present study, could be attributed to poor solubility of the antibacterial compounds in cold water and high extractive power of boiled water. In addition, the higher antibacterial activity observed in the acetone and ethanol extracts could be due to relatively higher flavonoid and phenolic contents present54.\n\nOur findings are in agreement with McGaw et al55. who reported that in the evaluation of antibacterial activities of hexane, ethanol and aqueous leaf extracts of H. arborescens against Bacillus subtilis, S. aureus, E. coli, and K. pneumoniae, only ethanol extract exhibited antibacterial activities. These findings correlate with observation made by Wigmore et al56. that aqueous extracts of plants showed less antibacterial activity when compared with other solvents. Furthermore, in agreement with our findings, the aqueous extract was inactive against all Gram-positive bacteria used. However, contrarily, the aqueous extract was active against S. aureus and S. epidermidis3.\n\n\nConclusion\n\nOur results clearly indicated that the extraction of phenolic compounds and their antioxidant capacity is highly dependent on the solvent of extraction. The study also revealed that H. arborescens aqueous, blanched and ethanol leaf extracts possess various levels and concentrations of phytochemical constituents, which may be essential for human health. A positive correlation between polyphenolic contents, antioxidant and antibacterial activities in extracts of H. arborescens leaves was established, which indicates that certain phenolic compounds may be responsible for high antioxidant activity. It was also observed that the blanching process (at 800 C) significantly decreased polyphenolic content and antioxidant activities but increased antibacterial activity of H. arborescens leaves. This study therefore agrees with its reported use in traditional medicine for the treatment of some bacterial infections and diseases.\n\n\nData availability\n\nFigshare: Absorbance values for antioxidant activity.xlsx, https://doi.org/10.6084/m9.figshare.12654479.v157.\n\nThis project contains the following underlying data:\n\n- Replicate absorbance values for DPPH, TAC and ABTS\n\n- Replicate phytochemical analysis results\n\n- MIC values of antibacterial activity of the extracts\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBagdassarian VLC, Bagdassarian KS, Atanassova MS: Phenolic profile, antioxidant and antimicrobial activities from the Apiaceae family (dry seeds). Mint J Pharm Medi Scie. 2013; 2 (4): 26–31. Reference Source\n\nFotina AA, Fisinin VI, Surai, PF: Recent developments in usage of natural antioxidants to improve chicken meat production and quality. Bulg J Agric Sci. 2013; 19(5): 889–896. Reference Source\n\nNkomo M, Kambizi L: Antimicrobial activity of Gunnera perpensa and Heteromorpha arborescens var. abyssinica. J Med Pl Res. 2009; 3(12): 1051–1055. Reference Source\n\nAbifarin TO, Otunola GA, Afolayan AJ: Cytotoxicity evaluation and anti-inflammatory potentials of Cucumis africanus L.f. leaves. Med Plants-Inter J Phytomed Relat Indus. 2020; 12(1): 48–52. Publisher Full Text\n\nPalgrave MC: Keith Coates Palgrave Trees of Southern Africa. 3rd ed. Cape Town. Struik Publishers (Pty) Ltd. 2002.Reference Source\n\nMaroyi A: Heteromorpha arborescens: a review of its botany, medicinal uses, and pharmacological properties. Asian Journal of Pharmaceutical and Clinical Research. 2018; 11(11): 75–82. Publisher Full Text\n\nMoshi MJ, Mbwambo ZH: Experience of Tanzanian traditional healers in the management of non-insulin dependent diabetes mellitus. Pharm Biol. 2002; 40(7): 552–560. Publisher Full Text\n\nSchmidt E, Lotter M, McCleland W: Trees and Shrubs of Mpumalanga and Kruger National Park. Johannesburg, Jacana; 2002. Reference Source\n\nVan BE Wyk, Van Oudtshoorn N: Medicinal Plants of South Africa. Pretoria: Briza Publications. 2013.\n\nBussmann RW: Ethnobotany of the Samburu of Mt. Nyiru, South Turkana, Kenya. J Ethnobiol Ethnomed. 2006; 2(1): 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSetshogo MP, Mbereki CM: Floristic diversity and uses of medicinal plants sold by street vendors in Gaborone, Botswana. African Journal of Plant Science and Biotechnology. 2011; 5(1): 69–74. Reference Source\n\nNkomo M, Nkeh-Chungag BN, Kambizi L, et al.: Investigation of the antinociceptive and anti-inflammatory properties of Heteromorpha arborescens (Apiaceae). Afr J Tradit Complement Altern Med. 2011; 8(4): 412–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSunil C, Kumar V, Van Staden J: In vitro alpha-glucosidase inhibitory, total phenolic composition, antiradical and antioxidant potential of Heteromorpha arborescens (Spreng.) Cham. & Schltdl. leaf and bark extracts. South African Journal of Botany. 2019; 1(124): 380–386. Publisher Full Text\n\nElisha IL, Dzoyem JP, McGaw LJ, et al.: The anti-arthritic, anti-inflammatory, antioxidant activity and relationships with total phenolics and total flavonoids of nine South African plants used traditionally to treat arthritis. BMC Complement Altern Med. 2016; 16(1): 307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMajouli K, Hamdi A, Hlila MB: Phytochemical analysis and biological activities of Hertia cheirifolia L. roots extracts. Asian Pac J Trop Med. 2017; 10(12): 1134–1139. PubMed Abstract \| Publisher Full Text\n\nSagbo IJ, Afolayan AJ, Bradley G: Antioxidant, antibacterial and phytochemical properties of two medicinal plants against the wound infecting bacteria. Asian Pacific Journal of Tropical Medicine. 2017; 7(9): 817–825. Publisher Full Text\n\nAbifarin TO, Afolayan AJ, Otunola GA: Phytochemical and antioxidant activities of Cucumis africanus L.f: A wild vegetable of South Africa. J Evid Based Integr Med. 2019; 24: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnuofin JO, Otunola GA, Afolayan AJ: Polyphenolic content, antioxidant and antimicrobial activities of Vernonia mespilifolia Less. used in folk medicine in the Eastern Cape Province, South Africa. J Evid Based Integr Med. 2018; 23: 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOhikhena FU, Wintola OA, Afolayan AJ: Quantitative phytochemical constituents and antioxidant activities of the mistletoe, Phragmanthera capitata (Sprengel) Balle extracted with different solvents. Pharmacognosy Res. 2018; 10(1): 16–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnuofin JO, Otunola GA, Afolayan AJ: Phytochemical screening and in vitro evaluation of antioxidant and antimicrobial activities of Kedrostis africana (L.) Cogn. Asian Pacific Journal of Tropical Biomedicine. 2017; 7(10): 901–908. Publisher Full Text\n\nMohsenipour Z, Hassanshahian M: The effects of Allium sativum extracts on biofilm formation and activities of six pathogenic bacteria. Jundishapur J Microbiol. 2015; 8(8): e18971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuspradini H, Wulandari I, Putri AS, et al.: Phytochemical, antioxidant and antimicrobial properties of Litsea angulata extracts [version 2; peer review: 3 approved]. F1000 Res. 2018; 7: 1839. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShinde A, Ganu J, Naik P: Effect of free radicals & antioxidants on oxidative stress: a review. Journal of Dental and Allied Sciences. 2012; 1(2): 63–66. Publisher Full Text\n\nGuleria S, Tiku AK, Singh G, et al.: In vitro antioxidant activity and phenolic contents in methanol extracts from medicinal plants. J Plant Biochem Biotechnol. 2013; 22(1): 9–15. Publisher Full Text\n\nOlajuyigbe OO, Afolayan AJ: Phenolic content and antioxidant property of the bark extracts of Ziziphus mucronata Willd. subsp. mucronata Willd. BMC Complement Altern Med. 2011;11(1): 130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhanam UKS, Oba S, Yanase E, et al.: Phenolic acids, flavonoids and total antioxidant capacity of selected leafy vegetables. J Funct Foods. 2012; 4(4): 979–987. Publisher Full Text\n\nAbushouk AI, Salem AMA, Abdel-Daim MM: Berberis vulgaris for cardiovascular disorders: a scoping literature review. Iran J Basic Med Sci. 2017; 20(5): 503–510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUddin MS, Hossain MS, Al Mamun A, et al.: Phytochemical analysis and antioxidant profile of methanolic extract of seed, pulp and peel of Baccaurea ramiflora Lour. Asian Pac J Trop Med. 2018; 11(7): 443–450. Reference Source\n\nDi Majo D, La Guardia M, Giammanco S, et al.: The antioxidant capacity of red wine in relationship with its polyphenolic constituents. Food Chem. 2008; 111(1): 45–49. Publisher Full Text\n\nSun C, Wu ZS, Wang Z, et al.: Effect of ethanol/water solvents on phenolic profiles and antioxidant properties of Beijing Propolis extracts. Evid Based Complement Alternat Med. 2015; 2015: 595393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWen TN, Prasad KZ, Yang B, et al.: Bioactive substance contents and antioxidant capacity of raw and blanched vegetables. Innovative Food Science & Emerging Technologies. 2010; 11(3): 464–469. Publisher Full Text\n\nJimoh MO, Afolayan AJ, Lewu FB: Antioxidant and phytochemical activities of Amaranthus caudatus L. harvested from different soils at various growth stages. Sci Rep. 2019; 9(1): 12965. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorus A, Lisiewska Z: Effect of preliminary processing and method of preservation on the content of selected antioxidative compounds in kale (Brassica oleracea L. var. acephala) leaves. Food Chem. 2011; 129(1): 149–154. Publisher Full Text\n\nJaiswal AK, Gupta S, Abu-Ghannam N: Kinetic evaluation of colour, texture, polyphenols and antioxidant capacity of Irish York cabbage after blanching treatment. Food Chem. 2012; 131(1): 63–72. Publisher Full Text\n\nIrondi EA, Akintunde JK, Agboola SO, et al.: Blanching influences the phenolics composition, antioxidant activity, and inhibitory effect of Adansonia digitata leaves extract on α-amylase, α-glucosidase, and aldose reductase. Food Science & Nutrition. 2012; 5(2): 233–242. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIsmail A, Lee WY: Influence of cooking practice on antioxidant properties and phenolic content of selected vegetables. Asia Pac J Clin Nutr. 2004; 13: S162–S162. Reference Source\n\nGliszczyńska-Świgło A, Ciska E, Pawlak-Lemańska K, et al.: Changes in the content of health-promoting compounds and antioxidant activity of broccoli after domestic processing. Food Addit Contam. 2006; 23(11): 1088–1098. PubMed Abstract \| Publisher Full Text\n\nZhang D, Hamauzu Y: Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. Food Chem. 2004; 88(4): 503–509. Publisher Full Text\n\nSikora E, Cieślik E, Leszczyńska T, et al.: The antioxidant activity of selected cruciferous vegetables subjected to aquathermal processing. Food Chem. 2008; 107(1): 55–59. Publisher Full Text\n\nJiménez-Monreal AM, García-Diz L, Martínez-Tomé M, et al.: Influence of cooking methods on antioxidant activity of vegetables. J Food Sci. 2009; 74(3): H97–H103. PubMed Abstract \| Publisher Full Text\n\nMa T, Tian C, Luo J, et al.: Influence of technical processing units on polyphenols and antioxidant capacity of carrot (Daucus carrot L.) juice. Food Chem. 2013; 141(3): 1637–44. PubMed Abstract \| Publisher Full Text\n\nAshour AS, El Aziz MMA, Melad ASG, et al.: A review on saponins from medicinal plants: chemistry, isolation, and determination. J Nanomed Res. 2019; 8(1): 6–12. Publisher Full Text\n\nKibiti CM, Afolayan AJ: Preliminary phytochemical screening and biological activities of Bulbine abyssinica used in the folk medicine in the Eastern Cape Province, South Africa. Evid Based Complement Alternat Med. 2015; 2015: 617607. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCioffi G, D’Auria M, Braca A, et al.: Antioxidant and free-radical scavenging activity of constituents of the leaves of Tachigalia paniculata. J Nat Prod. 2002; 65(11): 1526–9. PubMed Abstract \| Publisher Full Text\n\nGomes da Cruz Silva ME, Guimarães AL, Oliveira AP, et al.: HPLC-DAD analysis and antioxidant activity of Hymenaea martiana Hayne (Fabaceae). J Chem Pharm Res. 2012; 4(2): 1160–1166. Reference Source\n\nAdefuye AO, Ndip RN: Phytochemical analysis and antibacterial evaluation of the ethyl acetate extract of the stem bark of Bridelia micrantha. Pharmacogn Mag. 2013; 9(33): 45–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatejić JS, Džamić A, Mihajilov-Krstev T, et al.: Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa. Central European Journal of Biology. 2012; 7(6): 1116–1122. Publisher Full Text\n\nAl-Hadhrami RMS, Hossain MA: Evaluation of antioxidant, antimicrobial and cytotoxic activities of seed crude extracts of Ammi majus grown in Oman. Egyptian Journal of Basic Applied Sciences. 2016; 3(4): 329–334. Publisher Full Text\n\nChatatikun M, Chiabchalard A: Phytochemical screening and free radical scavenging activities of orange baby carrot and carrot (Daucus carota Linn.) root crude extracts. Journal of Chemical and Pharmaceutical Research. 2013; 5(4): 97–102. Reference Source\n\nEbrahimzadeh MA, Nabavi SM, Nabavi SF, et al.: Antioxidant activity of hydroalcholic extract of Ferula gummosa Boiss roots. Eur Rev Med Pharmacol Sci. 2011; 15(6): 658–64. PubMed Abstract\n\nShah MR, Satardeka KV, Barve SS: Screening of phenolic content, antioxidant and in-vitro eye irritation activities from Apiaceae family (dry seeds) for potential cosmetic applications. International Journal of Pharmaceutical Science Research. 2014; 5(10): 4366–4374. Reference Source\n\nBazargani MM, Rohloff J: Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms. Food control. 2016; 61: 156–64. Publisher Full Text\n\nAdamu M, Bagla VP, Eloff JN: Fractionation of Heteromorpha arborescens var abyssinica (Apiaceae) leaf extracts based on polarity leads to a marked change in cytotoxicity that may yield a commercially useful product. South African Journal of Botany. 2016; 103: 36–40. Publisher Full Text\n\nRathee P, Chaudhary H, Rathee S, et al.: Mechanism of action of flavonoids as anti-inflammatory agents: A review. Inflamm Allergy Drug Targets. 2009; 8(3): 229–235. PubMed Abstract \| Publisher Full Text\n\nMcGaw LJ, Jäger AK, Van Staden J, et al.: Antibacterial, anthelmintic and anti-amoebic activity in South African medicinal plants. J Ethnopharmacol. 2000; 72(1–2): 247–263. PubMed Abstract \| Publisher Full Text\n\nWigmore SM, Naiker M, Bean DC: Antimicrobial activity of extracts from native plants of temperate Australia. Pharmacogn Commn. 2016; 6(2): 80–84. Reference Source\n\nAbifarin T, Otunola GA, Afolayan AJ: Absorbance values for antioxidant activity.xlsx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12654479.v1" }	[ { "id": "70661", "date": "17 Sep 2020", "name": "Abimbola Oloye", "expertise": [ "Reviewer Expertise Reproductive toxicology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 prescribed an ethno-medical alternative to treating oxidative stress and perhaps bacterial infections which are largely considered safer, easy to access, and cheaper when compared to most orthodox prescriptions. The wideness of the spread of H. arborescens especially in southern and eastern Africa makes it fit for consideration as an alternative to some chemically compounded drugs.\nThe conclusions drawn about the leaf extracts of this plant by the authors, especially in establishing a relationship between the phenolic content of the extracts and their potencies as antioxidant and antibacterial, were appropriate. However, I wished the authors could give consideration to seasonal variation in concentration statuses of polyphenolics and other bioactive compounds of the plant’s leaves. The month of June during which the study was carried out in the Eastern Cape, South Africa is the winter period, it would be revealing repeating the study in summer (between November and April) and drawing comparisons.\nStudying the effect of blanching was a brilliant one. Indigenes mostly blanch their veggies in the process of making them into edibles or in preparing them as oral medicines for the locals. Blanching, as against chemical extraction, deactivates bioactive compounds hence reduces the therapeutic effects associated with the plant. This has been implied in the study. Interestingly, however, the antibacterial effects of the plant seemed enhanced with blanching. The authors attributed this to enhanced extractive proficiency of water at boiling water temperature. In addition to this, however, I also think the heat-stabile bioactive antibacterial factor should be identified and pinned down.\nThe authors should reconcile the justification for the study enumerated in the abstract with the one stated in the introductory section of the manuscript. The assertion that no literature enumerating the phytochemical contents, antioxidative and antibacterial properties of acetone, ethanol and aqueous exist may not be totally correct. There are few studies on these. (See Citations).1,2 However, they may be correct about the blanched extract. The justification in the introductory section of the main text appears more appropriate.\nThe concentrations of saponin and alkaloids determined in the study were said to be enough to elicit the bioactivities associated with the two compounds. Is there any empirical reference that corroborates this assertion especially considering the fact that the authors considered the alkaloid concentration as low?\nA minor correction: The MIC of Ethanol extract against P. aeruginosa was 12.5 and this did not fall within the range of 1.563- 3.125 stated in the result. This, therefore, revealed an ethanol extract that had a weak antibacterial effect on P. aeruginosa. The authors should take a look at this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "71376", "date": "02 Oct 2020", "name": "Balasubramani Ravindran", "expertise": [ "Reviewer Expertise Phytochemistry and microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: The title reflects sufficiently, the work done and reported by the authors.\n\nAbstract: Adequate\n\nIntroduction: This is adequate with current and relevant citations.\n\nMethods: Methods used were sufficient and reproducible by others.\n\nResults were well presented with Tables and Figures where appropriate. These were also properly captioned and can stand alone.\n\nDiscussion projected the results and made appropriate reference to similar works by other researchers. Replace part of lines 4 and 5 “Oxidative stress which leads to the production of free radicals in the body,” with: Oxidative stress which occurs as a result of imbalance between production and accumulation of free radicals especially reactive oxygen species (ROS),\n\nConclusion is adequate and reflected the findings of the study.\n\nI approve that the article be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1079
https://f1000research.com/articles/8-1683/v1	24 Sep 19	{ "type": "Study Protocol", "title": "Recommendations and Alerting for Delirium Alleviation in Real-Time (RADAR): Protocol for a pilot randomized controlled trial", "authors": [ "Phillip E. Vlisides", "Jacqueline W. Ragheb", "Aleda Leis", "Amanda Schoettinger", "Kim Hickey", "Amy McKinney", "Joseph Brooks", "Mackenzie Zierau", "Alexandra Norcott", "Shirley Yang", "Michael S. Avidan", "Lillian Min", "Jacqueline W. Ragheb", "Aleda Leis", "Amanda Schoettinger", "Kim Hickey", "Amy McKinney", "Joseph Brooks", "Mackenzie Zierau", "Alexandra Norcott", "Shirley Yang", "Michael S. Avidan", "Lillian Min" ], "abstract": "Background: Delirium is a common and serious complication of major surgery for older adults. Postoperative social and behavioral support (e.g., early mobilization, mealtime assistance) may reduce the incidence and impact of delirium, and these efforts are possible with proactive patient-care programs. This pilot trial tests the hypothesis that a multicomponent decision support system, which sends automated alerts and recommendations to patient-care programs and family members for high-risk patients, will improve the postoperative environment for neurocognitive and clinical recovery. Methods: This will be a randomized, controlled, factorial pilot trial at a large academic medical center. High-risk, non-cardiac surgery patients (≥70 years old) will be recruited. Patients will be allocated to a usual care group (n=15), Hospital Elder Life Program (HELP)-based paging system (n=15), family-based paging system (n=15), or combined HELP- and family-based system (n=15). The primary outcome will be the presence of delirium, defined by positive long-form Confusion Assessment Method screening. Secondary outcomes will include additional HELP- and family-based performance metrics along with various neurocognitive and clinical recovery measures. Exploratory outcomes include the incidence of positive family-based delirium assessments post-discharge, 36-item Short Form Survey, PROMIS Cognitive Function Abilities Subset 4a, and 30-day readmission rates. Ethics and dissemination: This trial has received approval by the University of Michigan Medical Institutional Review Board (IRBMED). Dissemination plans include presentation at scientific conferences, publication in medical journals, and distribution via educational and news media. Registration: ClinicalTrials.gov Identifier NCT04007523, registered on 7/3/2019.", "keywords": [ "Clinical Trial Protocol", "Decision Support Systems", "Delirium", "Feasibility Studies", "Perioperative Care" ], "content": "Introduction\n\nDelirium is a distressing and common surgical complication, affecting approximately 20–50% of older surgical patients1,2. Postoperative delirium is associated with increased mortality3 and cognitive and functional decline4–6, and healthcare resource utilization7,8. Of the diverse prevention strategies that have been tested with variable success7,9, one notable proactive patient-care program, the Hospital Elder Life Program (HELP), has been shown to reduce delirium incidence through social and behavioral interventions (e.g., mealtime assistance, support with visual/hearing aids)10. However, substantial resources are needed for program sustainment, and delirium still persists in high-risk patients11–13. In 2018, we found that <50% of surgical patients ≥70 years old at Michigan Medicine were officially enrolled in the program by the end of the second postoperative day. Furthermore, the average length of cumulative therapeutic activity was only 10 minutes across the first three postoperative days. This is pertinent given that the peak incidence of postoperative delirium occurs within the first 48 hours2,14. As such, complementary strategies that improve patient triage and support may lead to earlier identification and therapeutic intervention for high-risk patients.\n\nClinical decision support systems can serve as a candidate strategy for mitigating delirium risk. Such systems provide targeted patient- and disease-specific information, presented in a timely manner, for improving healthcare quality15,16. In the context of delirium, automated pages could be sent to supportive healthcare services, such as HELP, along with family members and caretakers, with alerts and targeted recommendations. An alert page could be sent to HELP program officials on the first postoperative morning requesting early evaluation and enhanced treatment protocols. This may improve high-risk patient triage, early resource allocation, and cumulative therapeutic time spent with patients. A similar paging system could be implemented for family members and caretakers, as family-based interventions may provide additional support for patients at risk for delirium. Feasibility has been demonstrated with family-based protocols for hospitalized medical patients, with therapeutic focus on re-orientation, visual and hearing aid assistance, and conversational stimulation17. Similar protocols could be adapted for surgical patients, as surgery is generally a predictable event (and thus possibly amenable to familial planning), and family support may correlate with overall postoperative recovery18. A recent systematic review also demonstrated that family-performed delirium screens demonstrated improved psychometrics compared to family-informed delirium screens (i.e., those not performed by family members)19. Thus, family members and caretakers could be recruited to actively participate in postoperative recovery by performing family-based delirium assessments20 and implementing therapeutic protocols. An electronic, paging-based alerting system could provide family members with reminders and alerts for conducting such a program.\n\nThe premise of this pilot proposal is thus formed by the above considerations: preliminary evidence that suggests (1) suboptimal delirium prevention resource utilization and (2) the potential role for a clinical decision support system involving HELP and family members. The primary objective of this study is to determine whether pager-based clinical decision support systems enhance HELP- and family-based therapeutic activities. A secondary objective will be to identify facilitators and barriers to delivering therapeutic interventions for both HELP and family members. Overall, this pilot trial will test the hypothesis that a multicomponent decision support system will improve the postoperative environment for neurocognitive and clinical recovery in older, high-risk surgical patients.\n\n\nMethods and analysis\n\nThis is a single-center, randomized, factorial pilot trial at Michigan Medicine (Ann Arbor MI, USA). Approval was obtained from the University of Michigan Medical School Institutional Review Board (HUM00165251), and the trial has been registered at www.clinicaltrials.gov (NCT04007523). This protocol is also compliant with the Consolidated Standards of Reporting Trials (CONSORT) extension for pilot and feasibility trials and the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Guidelines21,22. Lastly, all study team members are certified in Good Clinical Practice.\n\nAfter enrollment, patients (n=60) will be allocated (1:1:1:1), via block-randomization, stratified by gender, to one of four groups: usual care (n=15), HELP-based paging system (n=15), family-based paging system (n=15), or both HELP- and family-based paging system (n=15) (Figure 1). The randomization code will be created by the biostatistician (AL) and concealed from the rest of the research team. On the morning of surgery, allocation assignments will be delivered via sequentially numbered, opaque, sealed envelopes to unblinded research team members who will initiate arm-specific operations. The support systems will consist of automated pager alerts to the HELP program and/or family members and caretakers, depending on group allocation, for providing additional delirium evaluation and therapeutic prevention activities (see Interventions: clinical decision support systems). Family members in the intervention group will also be provided with preoperative education on delirium and training in the Family Confusion Assessment Method (FAM-CAM) instrument20. Although it will not be possible to blind patients and family members to family-based interventions, study team members performing daily assessments will remain blinded to group allocation.\n\nHELP = Hospital Elder Life program; CAM = Confusion Assessment Method, LTAC = Long-Term Acute Care.\n\nParticipants will be screened and recruited at preoperative clinics, preoperative holding areas, and surgical wards (if patients are pre-admitted). Written informed consent will be obtained from all participants and family members (or caretakers) prior to scheduled surgery. Template forms are provided as Extended data23. Supplemental recruitment materials will be distributed in conjunction with the Michigan Institute for Clinical and Health Research, the NIH-funded Clinical and Translational Science Award Institute at the University of Michigan. Specifically, recruitment fliers will be posted throughout preoperative clinics, and informative postcards will be sent to potentially eligible patients preoperatively.\n\nEligibility criteria will reflect the pragmatic nature of the trial balanced with the aim of recruiting patients at high risk for postoperative delirium. Inclusion criteria include the following: age ≥ 70 years of age; major non-cardiac, non-intracranial neurologic, and non-major vascular surgery (as defined by work-related value units suggestive of high surgical complexity)24; anticipated length of hospital stay at least 72 hours; and at least one family member, or caretaker, available on each of the three first postoperative days. Exclusions include emergency surgery, severe cognitive impairment (precluding ability to perform delirium assessments), planned post-operative ICU admission (HELP unavailable in the ICU), and non-English speaking.\n\nThis proposal will build upon previous decision support systems launched by our department for reducing intraoperative awareness and delivering protective lung ventilation strategies25,26. For participants randomized to the HELP-based support system, a single page will be sent to the on-call HELP staff during the first postoperative morning as the team begins ward rounds (Table 1). The page will request an enhanced treatment protocol, which includes HELP visitations three times daily. Therapeutic treatment will be administered during each visit per program protocols, which generally includes cognitive engagement, mealtime assistance, mobility and range of motion exercises, and assistance with visual and hearing aids. During the final evening visit, a sleep protocol will be implemented. For this protocol, HELP officials offer sleep and relaxation exercises, relaxation massages, and warm milk and/or tea.\n\nFAM-CAM = Family Confusion Assessment Method. MRN = Medical Record Number\n\nFor participants randomized to the family-based system, family members (or caretakers) will receive preoperative education on delirium (including an educational video), a folder with an informational flyer and therapeutic activities checklists, and FAM-CAM training. Suggested therapeutic activities include daily assistance with visual and hearing aids, providing assistance with drinking and mealtime assistance, handwashing, re-orientation to time and place, and cognitive stimulation activities. Lastly, family members will also receive a pager, and automated pages will be sent twice daily with reminders to perform these activities (Table 1). Completion of activities will then be logged daily in conjunction with unblinded members of the research team. Study activities for each group are listed in Table 2.\n\nHELP = Hospital Elder Life Program; FAM-CAM = Family Confusion Assessment Method.\n\nDuring this pilot phase, characterizing the success and barriers encountered with trial interventions will be essential for analyzing fidelity. As a separate, but complimentary line of investigation, facilitators and barriers to support system implementation will be characterized for both HELP personnel and family members. The following strategies for characterizing implementation efforts are driven by the Consolidated Framework for Implementation Research (CFIR)27, which described five major domains that shape implementation effectiveness: intervention characteristics, outer setting, inner setting, characteristics of individuals involved, and the process of implementation. Survey-based questions and focus groups described below are guided by these implementation themes.\n\nHELP-based implementation barriers will be elucidated via combination of focus groups and online surveys. This strategy has been previously used for successfully identifying facilitators and barriers to delirium prevention involving multidisciplinary bundles28. Prior to paging system implementation, an anonymous survey will be distributed to HELP staff members. The survey includes Likert-scale29 questions derived from the “Safety Attitudes Questionnaire,” which reports views on teamwork, safety, collaboration, resource availability, and collegiality30. Open-ended questions are then provided for participants to express additional thoughts and insights. These surveys will be sent again 6 and 12 months after system implementation. Within a month after each of these surveys are collected, focus groups will be held with available HELP team members. Focus groups will be tape recorded and common themes will be elicited from transcriptions28,31. All responses will remain anonymous from both groups and surveys. The final objective will be to delineate clear barriers and facilitators to HELP-based triage and therapy implementation strategies.\n\nAll family members will be provided with surveys (available as Extended data)23 on postoperative day three (or discharge, whichever is sooner). These surveys also contain a similar combination of Likert-scale and open-ended questions to identify barriers to completing delirium screening and prevention activities.\n\nLastly, a sensitivity analysis will be performed, which will report daily proportions – and reasons – for missing HELP- and family-based assessments (see Sensitivity Analysis and Missing Data).\n\nThe primary outcome of this pilot trial will be the presence of delirium, defined by a positive long-form Confusion Assessment Method (CAM)32 screening. The following secondary outcomes will also be collected and analyzed: delirium severity (long-form CAM severity scale), new symptoms of depression or anxiety (using the Hospitalized Anxiety and Depression Scale, HADS)33, falls (proportion, %), length of hospital stay (days), discharge disposition (e.g., home, long-term care facility), delayed discharge due to cognitive impairment (proportion, %), incidence of any new non-surgical site infection (%), incidence of new multidrug resistant organism colonization (%), and mortality (%). Exploratory outcomes will include the incidence of positive FAM-CAM assessments (%) 30 days post-discharge, PROMIS Cognitive Function Abilities (Short Form 4a), 36-Item Short Form Survey, and 30-day readmission rates.\n\nProtocol fidelity measures. Lastly, protocol fidelity measures will be reported for both HELP- and family-based interventions. HELP-based measures include the following: total therapeutic time spent with HELP staff during the first three postoperative days, proportion of participants visited and enrolled by HELP (%), and time to initial HELP evaluation. For family-based interventions, the following measures will be reported: cumulative time family members spent with patients, proportion of daily tasks (e.g., assistance with glasses/hearing aids, handwashing), successfully completed, length of time spent on stimulating activity, and overall agreement of the FAM-CAM with interview-rated CAM assessments.\n\nData collection. At Michigan Medicine, HELP data collection is standard throughout surgical and medical wards. The time at which patients are first evaluated, total therapeutic time (minutes) spent with patients, and nature of therapeutic activities (e.g., cognitive stimulation, mealtime assistance) are all collected daily and logged on computer files. Unblinded research team will have access to these logs via secured, shared drive within the Michigan Medicine network. These research personnel will review HELP logs daily and meet with HELP leadership as needed to discuss problems that may arise regarding HELP data collection and logging.\n\nFor delirium assessment, research team members will screen for delirium using the long-form CAM32 twice daily (once in the morning, and again in the afternoon) for the first three postoperative days. These team members will be blinded to group allocation. Our research group has extensive experience with CAM in prior trials2,34,35, and our international group has created a program for training investigators in CAM methodology with a previously high inter-rater reliability (Fleiss kappa=0.88 [95% CI 0.85 to 0.92])35. Our study team members who have previously received this training will lead CAM assessment efforts for this trial. Additionally, the study PI (Vlisides), has received complementary CAM training from the NIH-funded (K07AG041835) Center of Excellence for Delirium in Aging: Research, Training and Educational Enhancement (CEDARTREE). For new team members not previously trained, the PI will lead an on-site training session using online long-form CAM training videos available from the Hospital Elder Life Program. Then, after each team member has successfully scored two non-delirious and two delirious patients identically – in terms of symptom recognition – with a previously trained study team member, the trainees will be considered independently trained for CAM assessment2. For those enrolled in the intervention bundle, family members (or caretakers) will perform the FAM-CAM20 independently of the research team. FAM-CAM assessments will be requested once daily in the afternoon.\n\nDepression and anxiety measures will take place both at preoperative baseline and during postoperative day three (or day of discharge, whichever is sooner). For assessment of falls, study team members will ask about fall occurrences during each study visit, and the medical record will also be reviewed for any falls during the study period. Additional clinical secondary outcomes described will be collected from the electronic medical record. On postoperative day three, for patients not randomized to the family-support groups, the research team will ask family members about cumulative time spent with patients, and any interactive activities performed, during the first three postoperative days for comparison to family-based intervention groups.\n\nFinally, research data recorded on paper will be stored in participant charts that will be located in locked cabinets in the Department of Anesthesiology at Michigan Medicine. Electronic data will be de-identified and stored online using the REDCap electronic research database, which resides on a secured, password-protected network managed by the Michigan Institute of Clinical and Health Research.\n\nSample size and power. Given its fluctuating and recurrent nature, delirium presence will be primarily assessed over time with logistic generalized estimating equation models as we have done previously36. In brief, time and group will serve as fixed factors, and a group by time interaction term will be included. Interaction terms will be removed from models if no significant interaction effect is observed. These models allow for longitudinal data analysis in the setting of incomplete and missing data. Group models will be constructed individually with the control group serving as a reference, and an intention-to-treat approach will be followed. Power calculations were then conducted with generalized estimating equations for the time-averaged difference between two groups (i.e., control group and intervention group, either HELP- or family-based support) in a repeated-measures design with the binary outcome of delirium. Accounting for six equally spaced measurements (twice daily delirium assessments for the first three postoperative days), with an autoregressive correlation structure (baseline correlation 0.3) and linear missing data structure, a total sample size of 60 patients (n=30 in each intervention group) will provide >80% power to detect a difference in proportions of 15% (approximate Cohen’s effect size difference of 0.9) for experiencing an episode of delirium between groups, assuming a baseline proportion of 20% in the control group, with α=0.05. Power analysis was conducted using PASS 16 (PASS 2019 Power Analysis and Sample Size Software [2019]. NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/pass). As an exploratory analysis, the interaction between HELP- and family-based support groups will be assessed using a generalized estimating equations model.\n\nDescriptive statistics will be reported for secondary and exploratory outcomes. Inferential statistics will be deferred given the small sample size and pilot nature of the trial. Rigorous statistical analysis will be deferred for planned, follow-up, large-scale investigation. However, inferential statistics will be reported for fidelity measures described previously (Outcomes – Protocol Fidelity Measures). For continuous data, the Shapiro-Wilk test will be used to assess for normal distribution, and either independent t-tests or the Mann-Whitney U-test will be used, as appropriate. For categorical data, chi-squared or Fisher’s exact test will be used, as appropriate. Cohen’s kappa will be used to assess agreement between research-based CAM delirium assessments and FAM-CAM assessments.\n\nSensitivity analysis and missing data. Missing data are anticipated for multiple outcomes described in this study. For each HELP visit, cumulative therapeutic time is routinely logged, as are reasons for deferred visits. Thus, the proportion of deferred shift visits, compared to all available shifts (excluding shifts missed due to early discharge) will be reported along with associated reasons (Table 3). Nine total visits are anticipated during the first three postoperative days – daily morning, afternoon, and evening sleep visits. Similarly, missing CAM and FAM-CAM data are expected as well. Reasons for missing assessments will be presented in conjunction with barriers that family members and caretakers report for conducting FAM-CAM assessments. Given the relatively small sample size and pilot nature of this trial, imputation will be deferred for missing data.\n\nHELP, Hospital Elder Life Program; FAM-CAM, Family Confusion Assessment Method.\n\nAs described, a complementary line of analysis will focus on facilitators and barriers to implementing therapeutic protocols described, both from HELP- and family-based perspectives. Results will be used to inform therapeutic protocol design for a larger, follow-up trial. Descriptive reporting, based on mixed methods and Likert scale survey methodology29,31, will also be used to report experiences with clinical decision support systems. This sub-study analysis involving HELP staff members has received exemption from the University of Michigan Institutional Review Board (HUM00166883).\n\nThe RADAR Trial interventions were designed with intentions for high generalizability. HELP is now present at more than 200 hospital systems worldwide, and decision-support systems may help triage and organize support operations across such sites, particularly for those limited by personnel and/or resources. Alternatively, for hospitals without HELP, this trial will also assess the feasibility and efficacy of similar interventions administered by family members and caretakers.\n\nTo further study the pragmatic and explanatory elements of RADAR, trial members completed the PRagmatic-Explanatory Continuum Indicator Summary (PRECIS-2) toolkit37. This is an assessment tool that characterizes the pragmatic and explanatory elements of clinical trial design, and the results inform as to where trial design resides on the pragmatic-explanatory continuum. For PRECIS-2 assessment, nine study domains are analyzed: eligibility criteria, recruitment, setting, intervention organization, flexibility of intervention delivery, flexibility of adherence, follow up, primary outcome, and primary analysis. Raters score each domain on a scale of 1 – 5, with lower scores reflecting explanatory trial characteristics, and higher scores suggesting a more pragmatic nature. RADAR Trial members independently scored each domain using the associated instructions, and median scores are illustrated in Figure 2. Each domain received a median score of 4 or 5, reflecting a relatively pragmatic study design. Regarding (1) eligibility criteria, the trial will recruit a heterogeneous, well-rounded group of surgical patients that will receive interventions similar to those administered postoperatively. There will be some exclusions based on family and caretaker availability, cognitive function, and surgical subtype. Recruitment (2) will be conducted at regularly scheduled preoperative clinic appointments, which occur as part of routine, standard care. The setting (3) The setting will be identical to where patients otherwise receive perioperative care. Regarding (4) the expertise and resources needed to deliver the interventions, the HELP-based interventions are closely related to usual care, though they may occur sooner and more thoroughly with the pager alerting system. However, there will be some additional resources and training required, particularly for family-based interventions. For (5) flexibility of intervention delivery, pager alerts can be reliably delivered and modified as needed. There are also many opportunities throughout the day to implement clinical protocols as outlined. However, to demonstrate effectiveness, the proposed interventions likely need to occur consistently and with adherence to the protocol. In terms of flexibility adherence (6), daily pager alerts will be reliably and automatically sent to HELP and family members to enhance protocol fidelity. Checklists will also be made available to family members. Follow-up (7) for most outcomes and operations will occur in the immediate postoperative period, and many outcomes described are obtainable via chart review. However, certain follow-up measures (e.g., CAM, 30-day surveys) require prospective collection from research team members, though raters did not raters generally did not anticipate this to be particularly burdensome or prohibitive. Delirium is the primary outcome (8), which is a common and serious postoperative outcome that is relevant to surgical patients. Lastly, the primary analysis plan (9) follows an intention-to-treat approach with longitudinal modeling that accounts for missing data. Although raters generally scored the trial design as pragmatic, raters were part of the trial team, and thus not independent assessors. This may introduce bias with regards to objectively rating explanatory and pragmatic elements of a trial38.\n\nAfter reviewing training materials, members of the RADAR team independently scored each of the 9 domains included in the PRECIS-2 Toolkit. For each domain, scores range from 1 to 5, with lower scores reflecting an explanatory nature, and higher scores reflecting pragmatic characteristics. Median scores are presented from all team members (n=10) that completed the PRECIS-2 toolkit scoring. The median score for each domain was either a 4 or 5, reflecting a fairly pragmatic study design overall.\n\nAll participants will be monitored throughout the entire perioperative course by both the research team (including direct oversight by the PI) and clinical teams per standard care. The research team will monitor for adverse events, which will be reported per IRB guidelines. Participants will also have phone and pager numbers to the study coordinator and study PIs, and they are encouraged to contact our study team with any concerns that arise. While admitted to the hospital, participants will otherwise undergo routine monitoring and management per standard clinical practice. There will otherwise be no additional data management committee, and no interim analyses or audits are planned for this trial. For data storage, primary source paper documents will be stored in locked files within the Department of Anesthesiology at Michigan Medicine. Electronic data will be de-identified and entered into the online REDCap, database, which is managed by the Michigan Institute of Clinical and Health Research Management Core. Lastly, all protocols and consent forms approved by the University of Michigan Medical School IRB are reviewed yearly.\n\nMultiple strengths of this trial are worth noting. First, trial design is relatively pragmatic, as perioperative care will be minimally altered. The HELP-based activities described already take place at Michigan Medicine; a single page will be sent to HELP to assist with triage and focus therapeutic activity on relatively high-risk patients. The system can conceivably benefit any patient regardless of surgical subspecialty, as supportive protocols outlined could be implemented – or adapted – as part of enhanced recovery protocols irrespective of surgical service. The study also offers an innovative approach to integrating family members and caretakers in the postoperative recovery process. Preliminary data suggest that family involvement is both feasible17 and may improve clinical recovery after major surgery18. Thus, both HELP- and family-based support systems offered might provide an effective, practical approach to mitigating delirium risk while minimizing strain to the healthcare system.\n\nConsiderable limitations are also worth discussion. First, both HELP officials and family members may be subject to the Hawthorne effect39. That is, individuals may modify their behavior under study conditions. Both HELP officials and family members may rigorously perform study protocols knowing that performance is being monitored. As such, protocol effectiveness will likely decrease in non-research settings. For the HELP program, performance measures will be compared to historical controls (2018 HELP records) to assess for this effect. Family members of patients not randomized to family-based support interventions may still elect to spend more time with patients after learning about the trial and proceeding with enrollment. To assess for latent family support in the control groups, the research team will ask family members about time and activities with patients for comparisons to structured family-based support allocation groups. This will be assessed on the afternoon of postoperative day three, at the end of the inpatient study window, to avoid inadvertent introduction of study-related family support interventions. Lastly, only patients with family members and/or caretakers – who will be available for the first three days of hospitalization – will be eligible for the trial. Thus, patients without such social support will be ineligible. These eligibility criteria thus exclude a group of patients who may be particularly vulnerable to delirium (i.e., less social support)40 and reduce trial pragmatism.\n\nEmanuel et al.41 have proposed seven universal requirements, drawn from landmark codes and declarations, for comprehensively incorporating all relevant ethical considerations for clinical research, particularly in the context of aiming to improve health and/or increase understanding of human biology. These considerations are presented in question format, along with responses for this trial, in Table 4.\n\n\nDissemination\n\nThe trial will be presented at academic conferences, presentations, and medical journals. As mentioned, the trial was registered on www.clinicaltrials.gov (NCT04007523), and any protocol changes will be made publicly available on this registry. This manuscript currently reflects the 2nd version of the protocol (September 15, 2019).\n\nIf the results demonstrate improved HELP evaluation and therapeutic practices, the paging-based support intervention will be tested in a large-scale trial to assess effectiveness for reducing delirium incidence and related consequences. Family-based interventions may be included as well depending on success and feasibility with family-led delirium screening and prevention procedures described. The nature of such future interventions may be modified depending on survey results from HELP personnel and family members.\n\n\nConclusions\n\nDelirium remains a pressing public health issue, and associated consequences bear significant morbidity. The proposed clinical decision support system has the potential to improve the environment for neurocognitive and clinical recovery for high-risk patients. The paging support system is also relatively pragmatic, and if successful, could be used across various healthcare systems and tailored accordingly. If encouraging preliminary results are demonstrated, the proposed interventions will be tested in a large-scale trial for clinical effectiveness.\n\n\nData availability\n\nNo data are associated with this article\n\nOpen Science Framework: Recommendations and Alerting for Delirium Alleviation in Real-Time (RADAR): Extended Data. https://doi.org/10.17605/OSF.IO/UYZ9223.\n\nThis project contains the following extended data:\n\nRADAR Family Consent v1.0 7-23-2019.pdf\n\nRADAR Subject Consent v1.0 7-23-2019.pdf\n\nHELP Focus Group Discussion Guide.pdf\n\nRADAR – HELP Facilitators and Barriers Survey.pdf\n\nRADAR Family Survey – Facilitators and Barriers.pdf\n\nThese data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nData collection forms related to the Hospital Elder Life Program (e.g., Confusion Assessment Method, Family Confusion Assessment Methods) are subject to copyright restriction and are available on the Hospital Elder Life Program Website (https://www.hospitalelderlifeprogram.org/). The Hospital Anxiety and Depression Scale is also subject to copyright restriction and can be accessed at the following website: https://www.gl-assessment.co.uk/. The 36-Item Short Form Survey (SF-36) can be found on the Rand Healthcare website (https://www.rand.org/health-care/surveys_tools/mos/36-item-short-form.html), and the PROMIS cognitive function assessments can also be found on the associated website (http://www.healthmeasures.net/explore-measurement-systems/promis).\n\nOpen Science Framework: CONSORT Pilot and SPIRIT checklists, and WHO Trial Registration Data Set for ‘Recommendations and Alerting for Delirium Alleviation in Real-Time (RADAR): Protocol for a pilot randomized controlled trial’. https://doi.org/10.17605/OSF.IO/UYZ9223.\n\nRADAR CONSORT Pilot Checklist.doc\n\nRADAR SPIRIT Checklist.doc\n\nRADAR WHO Trial Registration Data Set.docx\n\nReporting guidelines are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to acknowledge George A. Mashour, MD, PhD, Sharon Inouye, MD, MPH, and Ann Kolanowski, PhD, RN, FAAN, for study design consultation. Additionally, we acknowledge the Michigan Institute for Clinical and Health Research and Michigan Medicine Office of Patient Experience for assistance with designing recruitment materials. We also acknowledge the Hospital Elder Life Program for permissions with the Confusion Assessment Method (Copyright 2003, Hospital Elder Life Program, LLC) and Family Confusion Assessment Method (FAM-CAM), Copyright 1988, 2011. Hospital Elder Life Program. Not to be reproduced without permission.\n\n\nReferences\n\nDasgupta M, Dumbrell AC: Preoperative risk assessment for delirium after noncardiac surgery: a systematic review. J Am Geriatr Soc. 2006; 54(10): 1578–89. PubMed Abstract \| Publisher Full Text\n\nAvidan MS, Maybrier HR, Abdallah AB, et al.: Intraoperative ketamine for prevention of postoperative delirium or pain after major surgery in older adults: an international, multicentre, double-blind, randomised clinical trial. Lancet. 2017; 390(10091): 267–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWitlox J, Eurelings LS, de Jonghe JF, et al.: Delirium in elderly patients and the risk of postdischarge mortality, institutionalization, and dementia: a meta-analysis. JAMA. 2010; 304(4): 443–51. PubMed Abstract \| Publisher Full Text\n\nHshieh TT, Saczynski J, Gou RY, et al.: Trajectory of Functional Recovery After Postoperative Delirium in Elective Surgery. Ann Surg. 2017; 265(4): 647–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInouye SK, Marcantonio ER, Kosar CM, et al.: The short-term and long-term relationship between delirium and cognitive trajectory in older surgical patients. Alzheimers Dement. 2016; 12(7): 766–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaczynski JS, Marcantonio ER, Quach L, et al.: Cognitive trajectories after postoperative delirium. N Engl J Med. 2012; 367(1): 30–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGleason LJ, Schmitt EM, Kosar CM, et al.: Effect of Delirium and Other Major Complications on Outcomes After Elective Surgery in Older Adults. JAMA Surg. 2015; 150(12): 1134–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeslie DL, Marcantonio ER, Zhang Y, et al.: One-year health care costs associated with delirium in the elderly population. Arch Intern Med. 2008; 168(1): 27–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOh ES, Fong TG, Hshieh TT, et al.: Delirium in Older Persons: Advances in Diagnosis and Treatment. JAMA. 2017; 318(12): 1161–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHshieh TT, Yang T, Gartaganis SL, et al.: Hospital Elder Life Program: Systematic Review and Meta-analysis of Effectiveness. Am J Geriatr Psychiatry. 2018; 26(10): 1015–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInouye SK, Bogardus ST Jr, Charpentier PA, et al.: A multicomponent intervention to prevent delirium in hospitalized older patients. N Engl J Med. 1999; 340(9): 669–76. PubMed Abstract \| Publisher Full Text\n\nBradley EH, Schlesinger M, Webster TR, et al.: Translating research into clinical practice: making change happen. J Am Geriatr Soc. 2004; 52(11): 1875–82. PubMed Abstract \| Publisher Full Text\n\nBradley EH, Webster TR, Baker D, et al.: After adoption: sustaining the innovation. A case study of disseminating the hospital elder life program. J Am Geriatr Soc. 2005; 53(9): 1455–61. PubMed Abstract \| Publisher Full Text\n\nMarcantonio ER, Goldman L, Mangione CM, et al.: A clinical prediction rule for delirium after elective noncardiac surgery. JAMA. 1994; 271(2): 134–9. PubMed Abstract \| Publisher Full Text\n\nClinical Decision Support. Office of the National Coordinator for Health Information Technology (ONC), Washington, DC. Content last reviewed April 2018. Reference Source\n\nClinical Decision Support. Agency for Healthcare Research And Quality (AHRQ), Rockville, MD. Content last reviewed June 2019. Reference Source\n\nRosenbloom-Brunton DA, Henneman EA, Inouye SK: Feasibility of family participation in a delirium prevention program for hospitalized older adults. J Gerontol Nurs. 2010; 36(9): 22–33; quiz 34–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCardoso-Moreno MJ, Tomás-Aragones L: The influence of perceived family support on post surgery recovery. Psychol Health Med. 2017; 22(1): 121–8. PubMed Abstract \| Publisher Full Text\n\nRosgen B, Krewulak K, Demiantschuk D, et al.: Validation of Caregiver-Centered Delirium Detection Tools: A Systematic Review. J Am Geriatr Soc. 2018; 66(6): 1218–25. PubMed Abstract \| Publisher Full Text\n\nSteis MR, Evans L, Hirschman KB, et al.: Screening for delirium using family caregivers: convergent validity of the Family Confusion Assessment Method and interviewer-rated Confusion Assessment Method. J Am Geriatr Soc. 2012; 60(11): 2121–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEldridge SM, Chan CL, Campbell MJ, et al.: CONSORT 2010 statement: extension to randomised pilot and feasibility trials. BMJ. 2016; 355: i5239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan AW, Tetzlaff JM, Altman DG, et al.: SPIRIT 2013 statement: defining standard protocol items for clinical trials. Ann Intern Med. 2013; 158(3): 200–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVlisides PE: Recommendations and alerting for delirium alleviation in real-time (RADAR): extended data. 2019. Retrieved from osf.io/uyz92\n\nMin L, Hall K, Finlayson E, et al.: Estimating Risk of Postsurgical General and Geriatric Complications Using the VESPA Preoperative Tool. JAMA Surg. 2017; 152(12): 1126–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMashour GA, Shanks A, Tremper KK, et al.: Prevention of intraoperative awareness with explicit recall in an unselected surgical population: a randomized comparative effectiveness trial. Anesthesiology. 2012; 117(4): 717–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlum JM, Stentz MJ, Maile MD, et al.: Automated alerting and recommendations for the management of patients with preexisting hypoxia and potential acute lung injury: a pilot study. Anesthesiology. 2013; 119(2): 295–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDamschroder LJ, Aron DC, Keith RE, et al.: Fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science. Implement Sci. 2009; 4: 50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalas MC, Burke WJ, Gannon D, et al.: Implementing the awakening and breathing coordination, delirium monitoring/management, and early exercise/mobility bundle into everyday care: opportunities, challenges, and lessons learned for implementing the ICU Pain, Agitation, and Delirium Guidelines. Crit Care Med. 2013; 41(9 Suppl 1): S116–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLikert T: A technique for the measurement of attitudes. Arch Psychology. 1932; 22(140): 55. Reference Source\n\nSexton JB, Helmreich RL, Neilands TB, et al.: The Safety Attitudes Questionnaire: psychometric properties, benchmarking data, and emerging research. BMC Health Serv Res. 2006; 6: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrueger RA, Casey MA: Focus groups: A Practical Guide for Applied Research. 4th ed. London: Sage Publications; 2009. Reference Source\n\nInouye SK, van Dyck CH, Alessi CA, et al.: Clarifying confusion: the confusion assessment method. A new method for detection of delirium. Ann Intern Med. 1990; 113(12): 941–8. PubMed Abstract \| Publisher Full Text\n\nZigmond AS, Snaith RP: The hospital anxiety and depression scale. Acta Psychiatr Scand. 1983; 67(6): 361–70. PubMed Abstract \| Publisher Full Text\n\nVlisides PE, Das AR, Thompson AM, et al.: Home-based Cognitive Prehabilitation in Older Surgical Patients: A Feasibility Study. J Neurosurg Anesthesiol. 2019; 31(2): 212–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaybrier HR, Mickle AM, Escallier KE, et al.: Reliability and accuracy of delirium assessments among investigators at multiple international centres. BMJ Open. 2018; 8(11): e023137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVlisides PE, Thompson A, Kunkler BS, et al.: Perioperative Epidural Use and Risk of Delirium in Surgical Patients: A Secondary Analysis of the PODCAST Trial. Anesth Analg. 2019; 128(5): 944–952. PubMed Abstract \| Publisher Full Text\n\nLoudon K, Treweek S, Sullivan F, et al.: The PRECIS-2 tool: designing trials that are fit for purpose. BMJ. 2015; 350: h2147. PubMed Abstract \| Publisher Full Text\n\nGlasgow RE, Gaglio B, Bennett G, et al.: Applying the PRECIS criteria to describe three effectiveness trials of weight loss in obese patients with comorbid conditions. Health Serv Res. 2012; 47(3 Pt 1): 1051–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCambridge J, Witton J, Elbourne DR: Systematic review of the Hawthorne effect: new concepts are needed to study research participation effects. J Clin Epidemiol. 2014; 67(3): 267–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBassuk SS, Glass TA, Berkman LF: Social disengagement and incident cognitive decline in community-dwelling elderly persons. Ann Intern Med. 1999; 131(3): 165–73. PubMed Abstract \| Publisher Full Text\n\nEmanuel EJ, Wendler D, Grady C: What makes clinical research ethical? JAMA. 2000; 283(20): 2701–11. PubMed Abstract \| Publisher Full Text" }	[ { "id": "56796", "date": "17 Dec 2019", "name": "Balachundhar Subramaniam", "expertise": [ "Reviewer Expertise Postoperative delirium", "Perioperative hemodynamics", "Resilience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 designed an elegant study to maximize their ability to use existing supportive interventions in the prevention of delirium. This study is very much needed and wish the very best for the authors to complete all the elements proposed in this study.\nThe authors note this as a factorial design. In Fig. 1 this is categorized as usual HELP vs. Enhanced HELP system. The study is powered for interventions vs. controls. Enhanced HELP, Family support and HELP plus family support are all intervention groups. Is this powered to 15 vs. 45 (unbalanced)? While they note that n=30 in their power calculations. This is confusing.\n\nGiven 1, is factorial design the best suited for this study?\n\nIn the HELP group, all efforts are taken to make sure the investigators get two assessment right before they become eligible to score CAM. Why aren't the same efforts taken for FAMCAM as well? I suppose there will be logical issues, perhaps scoring with case videos might be one option.\n\nInclusion criteria mentions high-risk group and yet age is the only one chosen to define high risk. Or have I missed other risk factors in the inclusion criteria?\n\nFamily support can do certain things and HELP can do certain things. I presume this will be complimentary. It will be interesting to find out the results of this study.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5845", "date": "01 Sep 2020", "name": "Phillip Vlisides", "role": "Author Response", "response": "The authors have designed an elegant study to maximize their ability to use existing supportive interventions in the prevention of delirium. This study is very much needed and wish the very best for the authors to complete all the elements proposed in this study. The authors note this as a factorial design. In Fig. 1 this is categorized as usual HELP vs. Enhanced HELP system. The study is powered for interventions vs. controls. Enhanced HELP, Family support and HELP plus family support are all intervention groups. Is this powered to 15 vs. 45 (unbalanced)? While they note that n=30 in their power calculations. This is confusing. RESPONSE: We would like to thank the reviewer for the thorough and thoughtful review. We can certainly understand the confusion regarding power/sample size calculations and will attempt to clarify. The power calculations pool two intervention groups together for each calculation. For example, for detecting effects specific to the HELP intervention, the HELP arm (n=15) and combined arm (n=15) were pooled together and compared to the control arm (n=15) and family-support only arm (n=15). This strategy was also used for the family-specific intervention calculations. This is a conventional strategy for factorial trials described by Montgomery et al. (BMC Med Res Methodol. 2003; 3:26). We have added this language to the Statistical Analysis section of the manuscript. We acknowledge that his powering strategy has shortcomings. For example, this strategy crucially assumes there is no interaction between the interventions (e.g., HELP- and family-based support), which may not be the case in this trial. However, in the context of a pilot clinical trial for generating effect size data, testing feasibility, and informing future trial protocols, we surmised that this would be a reasonable initial strategy. We have added this limitation (and additional explanation) to the protocol manuscript. Given 1, is factorial design the best suited for this study? RESPONSE: As discussed above, there are indeed methodological limitations with this approach. The main objective of this pilot study, however, is to determine both efficacy and feasibility with these decision-support systems for both HELP- and family-based support. The factorial design allows for separate (and combined) analyses with respect to each intervention arm. This is particularly salient given that many hospital systems do not have HELP, and such family-based support systems might be alternative supportive care options for such hospital systems. In fact, the results from this study might inform future design for separate trials (one specific to hospitals with HELP, and a separate trial for hospitals without HELP). The factorial design allows us to (1) efficiently collect initial data on these interventions simultaneously and (2) analyze effects both separately and jointly via exploratory interaction analysis. In the HELP group, all efforts are taken to make sure the investigators get two assessment right before they become eligible to score CAM. Why aren't the same efforts taken for FAMCAM as well? I suppose there will be logical issues, perhaps scoring with case videos might be one option. RESPONSE: The reviewer raises important points about CAM and FAM-CAM methodology. Logistical issues are indeed a concern. Family members will only be scoring their own family members, and there will be a limited number of assessments (i.e., three days) prior to discharge. Thus, training opportunities would be limited. This is nonetheless an important methodological issue. We did not incorporate case videos for family members, but this could be considered for future protocols. We have added this as a study limitation in the protocol manuscript. Inclusion criteria mentions high-risk group and yet age is the only one chosen to define high risk. Or have I missed other risk factors in the inclusion criteria? RESPONSE: The high-risk label is derived from one of our group’s prior studies, in which patients at least 70 years of age, presenting for surgery with work-related value units suggestive of high surgical complexity were among the highest risk of perioperative complications (including delirium) regardless of comorbidity burden (JAMA Surg. 2017 152(12): 1126-1133). Patients 70 years of age presenting for major surgery were assigned nine points in the screening tool published (JAMA Surg. 2017 152(12): 1126-1133), and a score ³9 points was proposed as the optimal cutoff for predicting major perioperative complications (e.g., infection, delirium, falls). We have clarified this language in the updated protocol manuscript. Family support can do certain things and HELP can do certain things. I presume this will be complimentary. It will be interesting to find out the results of this study. RESPONSE: The reviewer is correct – the HELP- and family-based activities will be complementary. In the future, these interventions may work in a combined, complementary manner, or they each may be suitable as an independent set of interventions. Family-based support protocols might, for example, be helpful for hospitals without HELP availability. We otherwise thank the reviewer for the review and analysis of the protocol." } ] }, { "id": "69809", "date": "20 Aug 2020", "name": "Nicolai Goettel", "expertise": [ "Reviewer Expertise Delirium", "postoperative neurocognitive disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this work.\n\nVlisides and coworkers present the protocol for a pilot randomized controlled trial (RCT) investigating the effect of a multicomponent decision support system, which sends automated alerts and recommendations to patient-care programs and family members for high-risk patients, on the incidence of postoperative delirium.\n\nHigh-risk, non-cardiac surgery patients (≥70 years old) are allocated to 4 groups: (1) usual care group (n=15), (2) Hospital Elder Life Program (HELP)-based paging system (n=15), (3) family-based paging system (n=15), and (4) combined HELP- and family-based system (n=15).\n\nPrimary outcome measure is the incidence of postoperative delirium assessed by Confusion Assessment Method (CAM) screening. Secondary outcome measures are diverse performance metrics for postoperative neurocognitive disorders and clinical recovery.\n\nThe study protocol is methodologically sound and closely follows the CONSORT and SPIRIT guidelines.\n\nEven though patients are randomly allocated to treatment groups, there is a risk of apprehension bias and the Hawthorne effect. Study participants, especially those in groups 2–4, may respond differently due to being observed, or individuals may modify an aspect of their behavior in response to their awareness of being observed.\n\nThere is also a considerable risk of compliance bias. Preventive interventions for postoperative neurocognitive disorders tend to be rather complex and time-consuming for all persons involved. Participants compliant with the intervention may differ in some way from those not compliant, which can systematically affect the outcome of interest. In this context, one could argue that higher-functioning patients are more compliant with the intervention; hence, they may present less postoperative delirium.\n\nFigure 1 (CONSORT flow chart): There seems to be a two-step group allocation rather than the 1:1:1:1 group allocation mentioned in the text. What is the difference between “usual HELP care” and “enhanced HELP system”?\n\nOne eligibility criterion for recruitment is a high risk for postoperative delirium. However, the inclusion criteria only partially reflect a high-risk surgical population. In other words, being 70 years of age and older and undergoing major non-cardiac, non-intracranial, and non-major vascular surgery might not be sufficient to reach a high-risk level for postoperative delirium. I would suggest to extend the inclusion criteria to participants with an even higher risk of postoperative neurocognitive disorders, such as patients with pre-existing cognitive impairment.\nGiven the above, the overall incidence of postoperative delirium (or other postoperative neurocognitive disorders) could potentially be quite low in the studied population, or at least not attain the 20% in the control group assumed by the authors. This would subsequently lead to statistically weak results (underpowered study).\n\nIn summary, this research is hypothesis-driven and the study is well-designed. I comment the authors for making the effort to include most (if not all) state-of-the-art nonpharmacological interventions to prevent postoperative neurocognitive disorders in a single RCT. I wish the investigators best of luck with the ongoing trial.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5846", "date": "01 Sep 2020", "name": "Phillip Vlisides", "role": "Author Response", "response": "Thank you for the opportunity to review this work. Vlisides and coworkers present the protocol for a pilot randomized controlled trial (RCT) investigating the effect of a multicomponent decision support system, which sends automated alerts and recommendations to patient-care programs and family members for high-risk patients, on the incidence of postoperative delirium. High-risk, non-cardiac surgery patients (≥70 years old) are allocated to 4 groups: (1) usual care group (n=15), (2) Hospital Elder Life Program (HELP)-based paging system (n=15), (3) family-based paging system (n=15), and (4) combined HELP- and family-based system (n=15). Primary outcome measure is the incidence of postoperative delirium assessed by Confusion Assessment Method (CAM) screening. Secondary outcome measures are diverse performance metrics for postoperative neurocognitive disorders and clinical recovery. The study protocol is methodologically sound and closely follows the CONSORT and SPIRIT guidelines. Even though patients are randomly allocated to treatment groups, there is a risk of apprehension bias and the Hawthorne effect. Study participants, especially those in groups 2–4, may respond differently due to being observed, or individuals may modify an aspect of their behavior in response to their awareness of being observed. RESPONSE: We would like to thank the reviewer for the thorough, thoughtful review. We agree that apprehension bias, and the Hawthorne effect, are real possibilities with this trial. We have attempted to mitigate this bias with the following strategies (as outlined in the Strength and Limitations section): First, we will compare HELP metrics (specifically, time to initial evaluation and cumulative time spent with patients) with this trial compared to historical controls (2018 records). If performance measures are improved in the control group compared to the 2018 historical controls, this suggests that the Hawthorne effect may be present. If performance is similar, this would weight against the presence of the Hawthorne effect. Second, we will assess cumulative time family members spent with patients, along with any potential therapeutic activities performed, even for patients/families not randomized to family-based support interventions. We will wait to collect these data until postoperative day three, so as to not introduce the idea of providing enhanced family support during the first three postoperative days. There is also a considerable risk of compliance bias. Preventive interventions for postoperative neurocognitive disorders tend to be rather complex and time-consuming for all persons involved. Participants compliant with the intervention may differ in some way from those not compliant, which can systematically affect the outcome of interest. In this context, one could argue that higher-functioning patients are more compliant with the intervention; hence, they may present less postoperative delirium. RESPONSE: We agree that this limitation is present. Indeed, patients who are able – and motivated – to engage with HELP- and family-based support may inherently be less prone to postoperative delirium and related neurocognitive disorders. We have added this limitation to the protocol manuscript. Figure 1 (CONSORT flow chart): There seems to be a two-step group allocation rather than the 1:1:1:1 group allocation mentioned in the text. What is the difference between “usual HELP care” and “enhanced HELP system”? RESPONSE: The reviewer is correct. This two-step allocation helps to facilitate our randomization procedures, but the final 1:1:1:1 allocation remains the same. At our institution, with usual HELP care, the team manually reviews the census of surgical wards daily and then sees various patients throughout the day based on volunteer availability and visitation patterns. There is presently no structured triage system for prioritizing high risk patients, as outlined in the manuscript. As a result, some high risk patients may not be seen until the second or third postoperative day, as discussed in the Introduction section. This information has been included in the Interventions section of the protocol manuscript. One eligibility criterion for recruitment is a high risk for postoperative delirium. However, the inclusion criteria only partially reflect a high-risk surgical population. In other words, being 70 years of age and older and undergoing major non-cardiac, non-intracranial, and non-major vascular surgery might not be sufficient to reach a high-risk level for postoperative delirium. I would suggest to extend the inclusion criteria to participants with an even higher risk of postoperative neurocognitive disorders, such as patients with pre-existing cognitive impairment. RESPONSE: We agree with the reviewer that there exists a higher-risk tier, which includes patients with pre-existing cognitive impairment (along with other risk factors). Data from a frailty prediction model for surgical patients ≥70 years old, developed by our institution, suggest that high surgical complexity alone (i.e., procedure requiring considerable perioperative resources with inpatient admission) was associated with increased risk of geriatric complications, including delirium and related outcomes (AOR 12.1, 95% CI: 6.4 – 22.7; p<0.001) (Min et al., JAMA Surg. 2017 Dec; 152(12): 1126–1133). As such, for a feasibility/pilot trial, we surmised that this population would be appropriate for testing both efficacy and feasibility with such candidate interventions. Focusing on patients with prior neurocognitive impairment may be more challenging logistically, specifically with regards to determining basic barriers to implementation. However, we will certainly consider this population in the future as we continue to determine the impact and barriers with respect to such decision-support systems. Given the above, the overall incidence of postoperative delirium (or other postoperative neurocognitive disorders) could potentially be quite low in the studied population, or at least not attain the 20% in the control group assumed by the authors. This would subsequently lead to statistically weak results (underpowered study). RESPONSE: Indeed, this is an important consideration with postoperative delirium. Prior trials at our institution have revealed an incidence of approximately 20% with such non-cardiac surgery populations (Avidan et al., Lancet 2017; Vlisides et al., JNA 2019). In fact, the inclusion criteria for these prior trials included patients >60 years of age. As such, we anticipate that this 20% estimate will be accurate. Nonetheless, if the incidence differs significantly in this pilot trial, these results will better inform future power calculations for subsequent trials. In summary, this research is hypothesis-driven and the study is well-designed. I comment the authors for making the effort to include most (if not all) state-of-the-art nonpharmacological interventions to prevent postoperative neurocognitive disorders in a single RCT. I wish the investigators best of luck with the ongoing trial. RESPONSE: We would like to thank the reviewer for the constructive, encouraging feedback." } ] } ]	1	https://f1000research.com/articles/8-1683
https://f1000research.com/articles/9-49/v1	27 Jan 20	{ "type": "Research Article", "title": "In silico study on RNA structures of intronic mutations of beta-globin gene", "authors": [ "Nur Imaniati Sumantri", "Kenny Lischer", "Dian Rachma Wijayanti", "Tomy Abuzairi", "Nur Imaniati Sumantri", "Kenny Lischer", "Dian Rachma Wijayanti" ], "abstract": "Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and RNA Composer, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.", "keywords": [ "Intervening sequence", "IVS1nt5", "IVS1nt1", "beta globin gene", "RNA structure", "beta thalassemia major" ], "content": "Introduction\n\nThalassemia is a hereditary blood disorder that induces the production of an abnormal form or inadequate amount of hemoglobin in the body. Hemoglobinopathy has been identified in approximately 71% of all countries around the world, and more than 50% of cases of β-thalassemia occur in Southeast Asia1. β-thalassemia is one of the most prevalent blood disorders worldwide. It can result in various traits and the coinheritance of thalassemia minor, intermedia, or major depends on many factors. People with β-thalassemia exhibit reduced hemoglobin production, and low levels of hemoglobin results in a lack of oxygen supply throughout the body2.\n\nβ-thalassemia is caused by mutations in the human beta-globin (HBB) gene, which is responsible for producing β-globin, a subunit of hemoglobin3. Most mutations associated with β-thalassemia are caused by the substitution of one or a limited number of nucleotides in HBB. These mutations affect the functions of the gene including transcription, RNA processing or translation of β-globin mRNA. β zero (β0)-thalassemia is caused by mutations in HBB that stop the production of beta-globin. Conversely, other mutations only reduce the amount of beta-globin protein produced, a condition termed β plus (β+)-thalassemia4.\n\nPeople with β0- and β+-thalassemia have been diagnosed with thalassemia major and thalassemia intermedia, respectively. Thalassemia major has a more severe phenotype than thalassemia intermedia. Most patients with thalassemia major die at a young age5. Unfortunately, as many as 23,239 babies are born with inherited β-thalassemia major every year1.\n\nConformational changes in regulatory RNA induce specific human diseases. More than 95% of mutations result in only small local changes in the conformation of RNA. The same phenotype (disease) can be caused by different mutations with varying degrees of effects on the overall RNA structure6.\n\nNumerous variants of non-deletional β-thalassemia are caused by mutations that interfere with processing of the primary mRNA transcript7. These mutations affect AG or GT dinucleotides at the exon-intron splice junction. These mutations eventually ablate regular splicing and induce β0-thalassemia, resulting in a β-thalassemia major phenotype4. Mutations in intervening sequence 1 (IVS1) at nucleotides 143 (change from guanine to thymine, IVS1nt1G>T) and 147 (change from guanine to cytosine, IVS1nt5G>C) are common mutations in Southeast Asia that result in β-thalassemia minor and major, respectively8.\n\nA study on mutations that interfere with mRNA transcription may explain the mechanism by which the mRNA structure is changed by mutations in HBB. Specific in silico RNA analysis and visualization tools to study these structural changes have been developed, such as ViennaRNA Web Services, RNA Composer, and UCSF Chimera9–12. ViennaRNA Web Services and RNA Composer are web interface packages that offer structure modeling for RNA. This study evaluated the effects of the intronic HBB mutations IVS1nt1 and IVS1nt5 on the RNA structure using specific in silico tools.\n\n\nMethods\n\nThe complete genome sequence of HBB was obtained from Ensembl for transcriptional regulation detail by selecting transcript HBB-201 ENST00000335295.413. Concerning mutations in IVS1, introns 1–2 were mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The sequences of wild-type and mutant HBB were aligned using BioEdit version 7.214. The complete wild-type and mutant sequences were transcribed into mRNA using Nucleic Acid Converter (https://skaminsky115.github.io/nac/).\n\nWe used the RNAfold Server from ViennaRNA Web Services to predict the RNA secondary structures9. By selecting this applet, thermodynamic parameters, including centroids, partition function, and minimum free energy (MFE), were automatically calculated by the software as considered for structure folding. To create the RNA best structure tree plot, the wild-type and mutants sequences and structural elucidations were forwarded to the Barriers server, which is part of the ViennaRNA Web Services package15,16. The tools were utilized with their respective default parameters.\n\nFree energy minimization and base pairing probabilities are the most used methods in predicting single-sequence structure using the following equations17. When a structure i is at equilibrium with the unpaired state, the equilibrium is described by the equilibrium constant Ki as presented in Equation 1:\n\n\n\nThe Gibbs free energy change for structure i, namely ΔGi, quantifies the favorability of a given secondary structure compared with the unpaired state. Similarly, the equilibrium between two structures i and j is described in Equation 2 as follows:\n\n\n\nThus, the Gibbs free energy change quantifies the favorability of a structure at a given temperature. The structure with the lowest Gibbs free energy change will be the most prevalent in solution at equilibrium.\n\nSupplementing a free energy minimization calculation with a partition function calculation can identify the pairs that are more likely to be correct. The partition function Q is defined as the sum of the equilibrium constants Ki for all possible structures.\n\n\n\nThus, the probability of a particular structure i being found in solution can be calculated according to Equation 4 as follows:\n\n\n\nFurthermore, the probability of a base pair i–j can be calculated by summing the equilibrium constants for structures that contain that pair and dividing by the partition function.\n\n\n\nIn Equation 5, the sum of the index k is taken over all structures that have the base pair i–j.\n\nRNA Composer was used to construct the tertiary structures. As many as 353 nucleotides from introns 1–2 to exon 2 (ENSE00001057381) of wild-type and mutant HBB were entered to generate 3D structures10. The files were downloaded in the .pdb format. The interactive model was visualized using the University of California at San Francisco (UCSF) Chimera version 1.13.118.\n\n\nResults and Discussion\n\nIntronic mutations of HBB can lead to β-thalassemia major. These mutations have spread widely around the world19. In this study, we observed the effects of intronic mutations on HBB mRNA structure. HBB is encoded by 3931 nucleotides located on chromosome 11 with its various regulatory genes. This gene contains 1608 bp and consists of three exons and two introns. Introns 1–2 cover nucleotides 143–272. This region is known as IVS1 (Figure 1a). In these introns, we highlighted two mutations, namely IVS1nt5G>C and IVS1nt1G>T, located at nucleotides 143 and 147, respectively (Figure 1b).\n\n(A) Details of HBB showing the target mutations. (B) Sequence alignment of wild-type and mutant HBB was performed using BioEdit version 7.2.\n\nOur observation revealed differences in the MFE and other thermodynamic characteristics between wild-type and mutant HBB. We found that different point mutations occurred in the same IVS region, reflecting unique features. MFE, centroid, and partition function properties are depicted in a mountain plot in Figure 2.\n\n(A) Centroid, (B) minimum free energy, and (C) partition function.\n\nThe MFE has important roles for secondary structure prediction. Lower values are associated with better structures. Conformation of MFE secondary structure by RNA folding is irreversible because the sequence space sets up the shape space20. Centroid-based analysis is an alternative method for predicting the secondary structure. This calculation yields 30% less prediction errors than the MFE21. The stability of RNA molecules fluctuates in different low energy structure. Therefore, we need to analyze the equilibrium ensemble of RNA structures via the partition function22. The partition function measures the quality of secondary structure by determining the confidence in base pairs predicted by free energy minimization23.\n\nBased on the mountain plot result, the mutations of IVS1nt1G>T and IVS1nt5G>C changes the mRNA structure of HBB. However, IVS1nt5G>C leads to a much larger free energy change versus the wild-type. A previous study found that IVS1nt1G>T and IVS1nt5G>C resulted in splice junction disturbances24. Therefore, we believe that this disturbance may affect the mRNA structure.\n\nAlthough introns are known as non-coding regions, their alteration influences mRNA expression, which can lead to several diseases. Using the RNAfold applet from the ViennaRNA package, we analyzed the thermodynamic characteristics and structural changes of wild-type and mutant HBB (Figure 3 and Figure 4)7,9.\n\n(A) Wild-type, (B) IVS1nt1G>T, and (C) IVS1nt5G>C. Structural changes of RNA are highlighted by blue boxes.\n\n(A) Wild-type, (B) IVS1nt1G>T, and (C) IVS1nt5G>C.\n\nThe secondary structure of mRNA is beneficial for tracing the refolding pathways of an RNA molecule. The next question that arises is how the secondary structure of mRNA is affected by mutation of HBB. To obtain the mRNA structure, the RNAfold system was used (see Methods). The prediction of the mRNA secondary structure is based on the centroid-based pair probability (Figure 3). Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>T mRNA structures compared with the wild-type structure. In the secondary structure, the higher the number of the larger loops, the more unstable the structure. This folding makes the MFE lower than that of the wild-type. To further clarify the effect of folding, barrier tree plot analysis was performed (Figure 4). The result revealed that changes in the population occur earlier in IVS1nt5G>C carriers than in people carrying wild-type or IVS1nt1G>T HBB. The earlier changes in the population result in different populations toward the end of the barrier tree plot.\n\nThese intronic HBB mutations may lead to critical folding changes that could drastically affect the tertiary structure of the beta-globin chain as well as the quaternary structure. To further clarify the effects of the mutations, 3D models of wild-type and mutant and HBB were visualized using UCSF Chimera software (Figure 5). In general, the tertiary structure of the IVS1nt1G>T mutant was similar to that of wild-type HBB, excluding a small change as presented in the lower left part of Figure 5, whereas IVS1nt5G>C exhibited significant alteration of its folding. Previous research reported the predicted pathogenic effects of these intronic mutations using the HSF server. IVS1nt5G>C was predicted to be a silencer motif new site, intronic identifying elements and exonic splicing silencer (ESS) site were broken. ESS provides binding sites for proteins, promoting exon exclusion, and helps the spliceosome to decoy splice sites25. In addition, Seo et al. (2013) stated that most of the mutations affecting splicing disrupted the highly conserved donor and acceptor sites (GT/AG) at the exon–intron junctions and polypyrimidine tract, and the branch-point sequence may be disrupted by mutations affecting the splicing sites26.\n\nThis prediction shows complex structural folding considering the base pairing probability.\n\nRNA structure stability depends on metal or ligand binding, the alteration of which can affect the binding site activity and structure stability. The properties of RNA structures related to multiple ligand binding, ligand binding-induced conformational changes, and the ion-stabilized catalytic core have been explored. The study by Miao et al. stated that the positive charge of metal cations plays compensates for the negative charge of the RNA phosphate backbone27. In addition to that function, metal ions also bind to extremely specific locations on RNA 3D structures27. All of these findings reveal that IVS1nt5G>C significantly changes the mRNA structure. However, the protein structure of HBB still needs to be elucidated to understand the effects of the mutations on protein features after translation.\n\nRNAs start to form after the transcription process is completed. Once done, the structure of RNA is critical for its activities. This study examined the effects of mutations in terms of the minimum energy and the impact on the secondary and tertiary structures of HBB mRNA. Changes in the RNA formation may lead to disturbances of the protein structure. Protein expression can be influenced by mRNA folding, other local and global sequence features, and modulation of mRNA stability by codon usage28. Regarding the length of introns 1–2, the mutations occur in small RNA-affected regions where analysis is important for detection, annotation, quantification, and de novo discovery of putative small RNA molecules29. However, it will be interesting to clarify the effects of splicing, debranching, and trimming on the mRNA structure in future research.\n\n\nConclusion\n\nOur research revealed the impact of intronic mutations on the secondary and tertiary structure of mRNA. Different point mutations occurring in the same IVS region were associated with unique characteristics. We have expanded our understanding of the thermodynamic aspects of these mutants. The limitations of this study included our inability to clarify the interaction of secondary structures due to the lack of an RNA analysis platform function for intronic mutations. RNA molecular simulation and wet lab experiments are required to elucidate the detailed features of the intronic mutants and their interactions with ions or ligands to further assess their functional failure, especially in genetic disease.\n\n\nData availability\n\nFigshare: DNA sequence of wild-type and intronic mutation human beta-globin gene, https://doi.org/10.6084/m9.figshare.11565726.v230.\n\nFigshare: RNA sequence of wild-type and intronic mutation human beta-globin gene, https://doi.org/10.6084/m9.figshare.11566596.v131.\n\nFigshare: Mountain plot of wild-type and intronic mutation human beta-globin gene, https://doi.org/10.6084/m9.figshare.11556675.v332.\n\nFigshare: Centroid base pair probability of wild-type and intronic mutation human beta-globin gene, https://doi.org/10.6084/m9.figshare.11565711.v233.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank Enago (www.enago.com) for the English language review.\n\n\nReferences\n\nModell B, Darlison M: Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008; 86(6): 480–487. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusharraf SG, Iqbal A, Ansari SH, et al.: β-Thalassemia Patients Revealed a Significant Change of Untargeted Metabolites in Comparison to Healthy Individuals. Sci Rep. 2017; 7: 42249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNgo DA, Steinberg MH: Genomic approaches to identifying targets for treating β hemoglobinopathies. BMC Med Genomics. 2015; 8: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThein SL: The molecular basis of β-thalassemia. Cold Spring Harb Perspect Med. 2013; 3(5): a011700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFucharoen S, Winichagoon P: Haemoglobinopathies in southeast Asia. Indian J Med Res. 2011; 134: 498–506. PubMed Abstract \| Free Full Text\n\nHalvorsen M, Martin JS, Broadaway S, et al.: Disease-associated mutations that alter the RNA structural ensemble. PLoS Genet. 2010; 6(8): e1001074. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThein SL: Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis. 2018; 70: 54–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi CK: New trend in the epidemiology of thalassaemia. Best Pract Res Clin Obstet Gynaecol. 2017; 39: 16–26. PubMed Abstract \| Publisher Full Text\n\nGruber AR, Lorenz R, Bernhart SH, et al.: The Vienna RNA websuite. Nucleic Acids Res. 2008; 36(Web Server issue): W70–W74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPopenda M, Szachniuk M, Antczak M, et al.: Automated 3D structure composition for large RNAs. Nucleic Acids Res. 2012; 40(14): e112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorenz R, Wolfinger MT, Tanzer A, et al.: Predicting RNA secondary structures from sequence and probing data. Methods. 2016; 103: 86–98. PubMed Abstract \| Publisher Full Text\n\nFallmann J, Will S, Engelhardt J, et al.: Recent advances in RNA folding. J Biotechnol. 2017; 261: 97–104. PubMed Abstract \| Publisher Full Text\n\nHunt SE, McLaren W, Gil L, et al.: Ensembl variation resources. Database (Oxford). 2018; 2018: bay119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser. 1999; 41: 95–98. Reference Source\n\nFlamm C, Hofacker IL, Stadler PF, et al.: Barrier trees of degenerate landscapes. Z Phys Chem. 2002; 216(2): 155. Publisher Full Text\n\nWolfinger MT, Svrcek-Seiler WA, Flamm C, et al.: Efficient computation of RNA folding dynamics. J Phys A Math Gen. 2004; 37: 4731–4741. Publisher Full Text\n\nSeetin MG, Mathews DH: RNA Structure Prediction: An Overview of Methods. In: Keiler K. (eds) Bacterial Regulatory RNA. Methods Mol Biol. Humana Press, Totowa, NJ. 2012; 37: 99–122. PubMed Abstract \| Publisher Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–1612. PubMed Abstract \| Publisher Full Text\n\nDe Sanctis V, Kattamis C, Canatan D, et al.: β-Thalassemia Distribution in the Old World: an Ancient Disease Seen from a Historical Standpoint. Mediterr J Hematol Infect Dis. 2017; 9(1): e2017018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorenz R, Bernhart SH, Höner Zu Siederdissen C, et al.: ViennaRNA Package 2.0. Algorithms Mol Biol. 2011; 6: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchuster P: Prediction of RNA secondary structures: from theory to models and real molecules. Rep Prog Phys. 2006; 69(5). Publisher Full Text\n\nDing Y, Chan CY, Lawrence CE: RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble. RNA. 2005; 11(8): 1157–1166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHofacker IL, Losrenz R: Predicting RNA structure: advances and limitations. In: Waldsich C, editor. RNA folding methods and protocols. Humana Press. New York. 2014; 1086: 1–19. PubMed Abstract \| Publisher Full Text\n\nMathews DH, Moss WN, Turner DH: Folding and finding RNA secondary structure. Cold Spring Harb Perspect Biol. 2010; 2(12): a003665. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahdieh N, Rabbani B: Beta thalassemia in 31,734 cases with HBB gene mutations: Pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East. Blood Rev. 2016; 30(6): 493–508. PubMed Abstract \| Publisher Full Text\n\nSeo S, Takayama K, Uno K, et al.: Functional analysis of deep intronic SNP rs13438494 in intron 24 of PCLO gene. PLoS One. 2013; 8(10): e76960. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiao E, Westhof Z: RNA Structure: Advances and Assessment of 3D Structure Prediction. Annu Rev Biophys. 2017; 46: 483–503. PubMed Abstract \| Publisher Full Text\n\nBoel G, Letso R, Neely H, et al.: Codon influence on protein expression in E. coli correlates with mRNA levels. Nature. 2016; 529(7586): 358–363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPantano L, Pantano F, Marti E, et al.: Visualization of the small RNA transcriptome using seqclusterViz [version 2; peer review: 2 approved]. F1000Res. 2019; 8(ISCB Comm J): 232. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbuzairi T: DNA sequence of wild-type and intronic mutation human beta-globin gene. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11565726.v2\n\nAbuzairi T: RNA sequence of wild-type and intronic mutation human beta-globin gene. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11566596.v1\n\nAbuzairi T: Mountain plot of wild-type and intronic mutation human beta-globin gene. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.11556675.v3\n\nAbuzairi T: Centroid base pair probability of wild-type and intronic mutation human beta-globin gene. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.11565711.v2" }	[ { "id": "59162", "date": "12 Feb 2020", "name": "Zhichao Miao", "expertise": [ "Reviewer Expertise RNA structure prediction", "single cell omics" ], "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\nNur and colleagues report a computational study of the beta-globin gene mRNA structure based on secondary and tertiary structure prediction. However, the prediction is not reliable enough to fully support the conclusion. Therefore, I suggest to keep the paper in record but not use it as a conclusion. Major and minor comments are listed here:\nMajor comments:\nThe paper is fully based on computational prediction. However, both the prediction approaches are not yet accurate enough to draw a conclusion without experimental validation.\n\nFor secondary structure prediction, MFE (RNAfold) only consider the cis-Watson-Crick base pair situation without pseudoknots. However, different sequences that can be aligned together are expected to have the same secondary structure. Using the sequence co-evolution information may further improve secondary structure prediction accuracy. That is why ViennaRNA has RNAalifold to predict 2D structure from sequence alignment. Considering the modeling only includes point mutations, one cannot rule out the possibility that both wild type and mutants use the same structure but the mutation point use a base pair conformation different from cis-Watson-Crick (e.g., WC-sugar). In this case, the main topology of the structure should stay the same. The authors show the mutation in Figure 1, but do not show the positions paired with the mutational position.\n\nAccording to the 2D structures predicted in Figure 3, the proposed structures are not likely to fold in 3D. To improve, I suggest: 1) Predict the existence of Pseudoknots; 2) Check if the mutants can use the same 2D structure according to the base pair; 3) Use multiple sequence alignment (MSA) to predict secondary structure and show the co-evolution.\n\nFor 3D structure prediction, it is difficult for an automatic method (e.g., RNAComposer) to handle a structure of more than 150nt without a good secondary structure assignment. This paper tries to use RNAComposer to predict a structure of >350nt. This is beyond the current capability of automatic prediction. From Figure 5, we can find severe atomic clashes, which are not expected in 3D structures. To improve, I suggest to focus on the mutational region, use the MSA-based 2D prediction result to predict 3D structure, and use energy minimization (e.g., Rosetta) to optimize the 3D structure. However, the only information from 3D structure prediction is the feasibility of the 2D structure rather than the functional inferences expected from this paper.\n\nMinor comments:\nThe free energy calculation is not part of this paper. The authors should just cite the related publications rather introducing the ideas.\n\nThe 3D structure prediction method should be spelt as \"RNAComposer \".\n\nFigure 1B should include the base pair information (2D structure) of the alignment.\n\nThe y-axis of Figure 2 needs more explanation.\n\nFigure 4 needs more explanation. Its meaning is not clear.\n\nIt could be good to share the predicted structure information to increase the reproducibility.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "60477", "date": "11 Mar 2020", "name": "Kholis Abdurachim Audah", "expertise": [ "Reviewer Expertise My area of research is in protein engineering", "protein-protein interactions/ biomolecules interactions/ structure-function relationships which include but not limited to DNA manipulation", "DNA mutagenesis", "cloning and expression", "protein purification (various chromatography methods)", "protein characterization employing different biophysical methods (CD", "MALDI-MS", "FLuorescence Spectroscopy)", "Enzyme kinetics. My research interest is in the area of infectious diseases and drug discovery." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very helpful if the authors included the full structure of the wild type and compare it with the two constructed mutants' structure.\n\nFigure 2 showed a subtle difference in MFE and other thermodynamics characteristics, is there any value that can be presented as a result of statistical analysis or based on best fitting model?\n\nFigure 3 seems to have significant importance in terms of the structure stability (clinical consequence, for example). Any supporting literature?\n\nHowever, the author did not elaborate more on this. For example, IVS1nt5G>T mRNA structures that showed a significant difference (blue-circled). This needs to be discussed more.\n\nAgain, the value needs to be shown here: \"In the secondary structure, the higher the number of the larger loops, the more unstable the structure\".\n\nFigure 5 seems to differ significantly between WT and the mutants, but the author stated otherwise? Except this statement: \"excluding a small change as presented in the lower left part\". This needs to be clarified and explain/discuss any structure-function relationship alteration.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5496", "date": "20 Jul 2020", "name": "Tomy Abuzairi", "role": "Author Response", "response": "It would be very helpful if the authors included the full structure of the wild type and compare it with the two constructed mutants’ structure. Response: We thank the reviewer for their interest in the study and their comments. We agree with this comment. The secondary structure prediction (Figure 3) already displayed the full structure of wildtype and mutants. Meanwhile, we have revised Figure 5 as full sequence RNA tertiary structure that was predicted using 3dRNA from http://biophy.hust.edu.cn/services.html Figure 2 showed a subtle difference in MFE and other thermodynamics characteristics, is there any value that can be presented as a result of statistical analysis or based on best fitting model Response: We show all the value of thermodynamic characteristics in the mountain plot to show the different on the every position paired. By using plot, we can easily compare the structures. For example, we can easily compare wild-type, IVS1nt1G>T, and IVS1nt5G>C for the MFE as shown in Figure 2(b) that shows IVS1nt5G>C has the lowest value compare with wild-type and IVS1nt1G>T. Therefore, the plot is sufficient to show subtle difference in thermodynamic characteristics. Figure 3 seems to have significant importance in terms of the structure stability (clinical consequence, for example). Any supporting literature? Response: We agree with this and have incorporated your suggestion throughout the manuscript. “The principle of this methods is to create the secondary structure with minimal base pair distance to all other secondary structures in the Boltzmann ensemble9.” “Both IVS1nt5G>C and IVS1nt1G>T give implications to the clinical condition due to protein instability. There are some disruptions in producing beta globin chains by these mutations, hence the beta thalassemia major (BTM) patients produce beta globin chain in a limited number or even not at all. This mechanism results in unbalanced alpha and beta globin chains in haemoglobin that expedite the haemolysis and ineffective erythropoiesis contributing to reduced production of mature red blood cells28. Thus, the BTM patients requires lifelong blood transfusions and commonly suffer complications, such as endocrine complications and cardiac complications29” However, the author did not elaborate more on this. For example, IVS1nt5G>C mRNA structures that showed a significant difference (blue-circled). This needs to be discussed more. Response: We have updated our discussion for general alteration mainly happens in IVS1nt5G>C. “The blue box in Figure 4 highlighted the main alteration formed within the three sequences. However, the structure of IVS1nt5G>C seems to be the best predicted according to the red color in the base-pairing probability.” Again, the value needs to be shown here: \"In the secondary structure, the higher the number of the larger loops, the more unstable the structure\". Response: The prediction tool does not provide loop counting of the secondary structure, hence we omit the sentence and create additional sentences to support our results. “In general, the tertiary structure of each sequence was unique. The point mutation gave tremendous folding changes in the 3D model. Once the base shifted, base pairing mechanism was influenced massively.” Figure 5 seems to differ significantly between WT and the mutants, but the author stated otherwise? Except this statement: \"excluding a small change as presented in the lower left part\". This needs to be clarified and explain/discuss any structure-function relationship alteration. Response: This figure has been revised totally into full sequence 3D structure, while the structure-function relation already discussed, e.g. “IVS1nt5G>C was predicted to be a silencer motif new site, intronic identifying elements and exonic splicing silencer (ESS) site were broken”." } ] } ]	1	https://f1000research.com/articles/9-49
https://f1000research.com/articles/8-33/v1	09 Jan 19	{ "type": "Research Note", "title": "Pangenome guided pharmacophore modelling of enterohemorrhagic Escherichia coli sdiA", "authors": [ "DJ Darwin Bandoy" ], "abstract": "Enterohemorrhagic Escherichia coli (EHEC) continues to be a significant public health risk. With the onset of next generation sequencing, whole genome sequences are a potential resource for predictive modelling of the different regulatory mechanism of pathogens, particularly quorum sensing. We used a pangenome approach to determine EHEC genome clustering, determine the synonymous and nonsynonymous mutations across the EHEC sdiA and modelled the associated amino acid changes. Across the EHEC population, nonsynonymous variants are notably absent in ligand binding site for quorum sensing, indicating that population wide conservation of sdiA ligand site can be targeted for potential prophylactic purposes. Applying pathotype-wide pangenomics as a guide for determining evolution of pharmacophore sites is a potential approach in drug discovery.", "keywords": [ "pangenome", "pharmacophore", "EHEC", "Escherichia coli" ], "content": "Introduction\n\nOne of the more prominent strain of Escherichia coli is the enterohemorrhagic (EHEC) pathotype associated with global outbreaks of bloody diarrhea and hemolytic uremic syndrome1. EHEC is one of the classical of examples of one health disease as the interface of animal health spills into human health. Within the cattle reservoir, sdiA gene is required by E. coli to survive the acidic rumen environment. SdiA is used by E. coli to sense acyl homoserine in a quorum sensing system2. However, it is considered as an orphan as the cognate acyl homoserine synthase is absent, and hence sdiA is considered an environmental sensor to sense the nearby microbial community. SdiA is stabilized by acyl homoserine lactone and acts as transcription factor glutamate decarboxylase needed for survival in the acidic environment. Hence blocking the ability of EHEC to survive the acidic ruminal environment is a proposed mechanism to control shedding in the cattle reservoir.\n\nWhole genome sequencing of bacterial pathogens, particularly EHEC, is quickly transforming the workflows of epidemiological investigations. However, most bioinformatic pipelines used in clinical investigation use data reduction of genomes and artificially reducing diversity due to comparison of a limited number of housekeeping genes3. While wgMLST attempts to increase the number of genes, the assignment of a single reference genome appears to be inadequate in light of the pangenome. Various studies have shown that a significant number of genes that are present to the entire universe of genes within a species is missed for variant calling if only a single reference gene is used4. In this study we applied multi-scale approach to generate genome wide clustering using pangenome phylogeny using genome wide gene presence absence variation (PAV). We used this genome wide clusters as guide in generating the pharmacophore of sdiA as potential design strategy to control shedding by reducing the acid survival in the rumen.\n\nWe applied the concept of the pangenome, which represents the entirety of the genes that are present within a species, to a pathotype level. The EHEC pangenome represents the combination of genes seen in the EHEC pathotype. While the pangenome of E. coli was published in 2008 contained 8 genomes, we generated an updated EHEC pangenome with 153 genomes. The pangenome enables clustering of isolates using gene presence and absence. We used the genome wide comparison to generate clusters and genomic clade specific pharmacophore model of sdiA. This strategy enables to capture the pangenome wide variation of sdiA and ensures all conserved variants are targeted by the drug discovery pipeline enabling a pangenome to pharmacophore approach.\n\n\nMethods\n\nWhole genome sequences with the associated EHEC metadata was downloaded from Patric Database 3.5.28 using the keyword search for E. coli as organism and EHEC as the pathotype within the E. coli species5,6. This resulted to 196 results and 152 of which are closed genome and draft genomes while the remaining sequences are EHEC associated plasmids (Underlying data: Metadata from Patric Database of EHEC E. coli pangenome7).\n\nThe genomes were annotated with Prokka 1.13.3 as per published protocol8. Gff files were extracted as input for the pangenome pipeline Roary 3.11.2 using the following parameters for not splitting paralogs (roary -s -p 32 *.gff) and the resulting presence absence matrix (Underlying data: EHEC E. coli pangenome presence absence matrix7) together with the accessory genome phylogeny visualized in Phandango 1.3.0 and is represented as Figure 1B9,10. Each blue bar represents an individual gene and solid blue blocks represent gene clusters. The accessory genome-based phylogeny newick file was visualized using iTol 4.311–13.\n\nSnippy variant calling pipeline 4.3.5 was used to determine the synonymous and nonsynonymous protein mutations using sdiA of Escherichia coli O157:H7 str. Sakai as reference. The –contigs option was added to the standard commandline (snippy –outdir –ref sdiA_sakai.gbk). The resulting individual variants of sdiA was merged into EHEC E. coli sdiA variant calling data (Underlying data7).\n\nSdiA genes were extracted from the pangenome output of Roary and protein in silico modelling performed using SWISS-MODEL14–18. SdiA protein sequences were used as targets to search for protein templates within the SWISS-MODEL library. Model selection was based on the template with the highest quality prediction by the target-template alignment.\n\n\nResults and discussion\n\nThree main genomic clades were generated using the pangenome PAV of genes (Figure 1A). Clade I includes the genomes isolated from the earliest reported outbreak in 1982 as well as the more recent cases. Clade II includes genomes from European (predominantly Czech and German) enterohemorrhagic E. coli isolates (EHEC) belonging to the O26 serotype, while Clade III includes cluster of cases associated with acute renal failure in children. Clade III is associated with hybrid pathotype of serogroup O80 has aside from Shiga toxin, an extra-intestinal virulence plasmid (pS88), is currently emerging in France. Specific gene clusters are found in cluster 1 and noticeably absent in cluster III, particularly prophages and associated genes (Underlying data: Table 2, Figure 1B). This indicates that specific genome wide gene presence and absence can be used for functional cluster instructive of the underlying epidemiological dynamics. The acquisition of genomic islands unique to individual isolates are well defined in the pangenome gene presence absence matrix (Figure 1B). The core genome is 2145 (Table 1) and total gene count within the EHEC pangenome is 17152, which is not that far off the total E. coli pangenome 22,00019. This enormous difference between the core gene and total gene highlights the variation between the different isolates, which can be strain specific and individual isolate specific as indicated by the pangenome data.\n\nAll of the examined genomes contain sdiA gene, which is contained within the 2145 core genome. Using sdiA of Escherichia coli O157:H7 str. Sakai for variant calling, we identified the nonsynonymous mutations that are widely distributed across the EHEC isolates (Figure 3). 45.0% (49/109) of the nonsynonymous mutation is due to conversion of arginine to lysine at position 189 of sdiA (Figure 2A). This amino acid is located with the α-6 domain, adjacent to the amino acid clusters associated with sdiA dimerization. Previous protein modelling determined role of the guanidinium group of arginine which enables interactions in three different directions enabling a more complex electrostatic interaction versus lysine as well as the higher pKa value in arginine that can yield a more stable ionic interaction compared to lysine20. The arginine to lysine mutations are found mostly in clade III isolates. The second ranked nonsynonymous mutation asparagine to serine at amino acid position 101 with 17.4% (19/109) which is located adjacent to η-4 phenylalanine which is associated with the ligand and distributed among the different clades (Figure 2B). β-5 domain alanine to threonine change at amino acid position 140 is the third ranked nonsynonymous mutation with 9.2 % (10/109) (Figure 2C). The fourth ranked nonsynonymous mutation is at amino acid position 138 with the conversion of cysteine to serine (Figure 2D) also within the β-5 domain. Both nonsynonymous variants of amino acid position 140 and 138 are found within clade II and III. None of the highly ranked nonsynonymous mutations impact the ligand interaction, indicating the conservation of the sdiA motif across the population in geographic and temporal distribution, which suggests the possibility of targeting sdiA for quorum sensing inhibition.\n\n\nConclusion\n\nWhile EHEC pangenome is remarkably diverse, the allelic variants of sdiA, particularly nonsynonymous mutants, indicate the conservation of quorum sensing domain, indicating that targeting this structure can be effective across the different lineages of EHEC pathotype.\n\n\nData availability\n\nAll underlying and extended data available from Open Science Framework: Supplemental Data for Pangenome guided pharmacophore modelling of enterohemorrhagic Escherichia coli sdiA, https://doi.org/10.17605/OSF.IO/BNZ857\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nTable 1 Metadata from Patric Database of EHEC E. coli pangenome\n\nTable 2 EHEC E. coli pangenome presence absence matrix\n\nTable 3 EHEC E. coli sdiA variant calling data\n\nSWISS-MODEL Homology Modelling Report available at osf.io/bnz85.", "appendix": "Grant information\n\nThis research was funded by the University of the Philippines Enhanced Creative Work and Research Grant.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nRohde H, Qin J, Cui Y, et al.: Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4. N Engl J Med. 2011; 365(8): 718–724. PubMed Abstract \| Publisher Full Text\n\nSperandio V: SdiA sensing of acyl-homoserine lactones by enterohemorrhagic E. coli (EHEC) serotype O157:H7 in the bovine rumen. Gut Microbes. 2010; 1(6): 432–435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaiden MC, Bygraves JA, Feil E, et al.: Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A. 1998; 95(6): 3140–3145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMéric G, Yahara K, Mageiros L, et al.: A reference pan-genome approach to comparative bacterial genomics: identification of novel epidemiological markers in pathogenic Campylobacter. PLoS One. 2014; 9(3): e92798. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntonopoulos DA, Assaf R, Aziz RK, et al.: PATRIC as a unique resource for studying antimicrobial resistance. Brief Bioinform. 2017; bbx083. PubMed Abstract \| Publisher Full Text\n\nWattam AR, Davis JJ, Assaf R, et al.: Improvements to PATRIC, the all-bacterial Bioinformatics Database and Analysis Resource Center. Nucleic Acids Res. 2017; 45(D1): D535–D542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandoy D: Supplemental Data for Pangenome Guided Pharmacophore Modelling of Enterohemorrhagic Escherichia Coli sdiA. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/BNZ85\n\nSeemann T: Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014; 30(14): 2068–2069. PubMed Abstract \| Publisher Full Text\n\nHadfield J, Croucher NJ, Goater RJ, et al.: Phandango: an interactive viewer for bacterial population genomics. Bioinformatics. 2018; 34(2): 292–293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage AJ, Cummins CA, Hunt M, et al.: Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015; 31(22): 3691–3693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLetunic I, Bork P: Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016; 44(W1): W242–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLetunic I, Bork P: Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res. 2011; 39(Web Server issue): W475–478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLetunic I, Bork P: Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics. 2007; 23(1): 127–128. PubMed Abstract \| Publisher Full Text\n\nWaterhouse A, Bertoni M, Bienert S, et al.: SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46(W1): W296–W303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBienert S, Waterhouse A, de Beer TA, et al.: The SWISS-MODEL Repository-new features and functionality. Nucleic Acids Res. 2017; 45(D1): D313–D319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBertoni M, Kiefer F, Biasini M, et al.: Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Sci Rep. 2017; 7(1): 10480. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenkert P, Biasini M, Schwede T: Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics. 2011; 27(3): 343–350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuex N, Peitsch MC, Schwede T: Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis. 2009; 30 Suppl 1: S162–173. PubMed Abstract \| Publisher Full Text\n\nRobins-Browne RM, Holt KE, Ingle DJ, et al.: Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing? Front Cell Infect Microbiol. 2016; 6: 141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSokalingam S, Raghunathan G, Soundrarajan N, et al.: A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS One. 2012; 7(7): e40410. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "47169", "date": "24 Apr 2019", "name": "Kerry K. Cooper", "expertise": [ "Reviewer Expertise I am an expert in foodborne bacterial genomics", "epidemiology and pathogenesis", "particularly E. coli", "Salmonella", "Campylobacter", "and Listeria." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would state the biggest issue with the manuscript is the work is not technically sound, because upon examining the metadata file from Patric, numerous strains included in the EHEC pangenome were in fact not EHEC strains. Many of the isolates are UPEC, STEC or EPEC strains that should not be included in the analysis and are resulting in potential errors in the results and conclusions of the manuscript. All O55 strains are EPECs, O104 from the Germany outbreak is a STEC not EHEC, O127 is an EPEC, and CFT073 is an UPEC strain, just as some examples.\nFurthermore, the analysis also includes O157:H7 strains that are wild type and mutant strains, and the mutants should not be included in the analysis. Additionally, the authors mention three clades include O157 (Clade I), O26 (Clade II) and O80 (Clade III), however these clades are formed because the vast majority of the strains used in the analysis come from those three serotypes. The analysis is missing three serotypes from the \"big 6\" serotypes, including O45, O121 and O145, and only include one or two representatives of two of the other serotypes O111, O103. There are numerous genomes available for each of these serotypes through NCBI that should be included in this analysis. Particularly as the \"big 6\" serotypes represent >50% of the infections, and in the United States represent adulterants in ground beef or other meat products. Therefore, they are a vital aspect for the development of pharmacophore modelling of sdiA to prevent colonization in cattle.\nAdditionally, several genomic studies by Ogura et al (2009)1 and Cooper et al (2014)2 have shown that many of these \"big 6\" serotypes arise along different evolutionary pathways or split from O157 at different time points thus acquire different genes. It would vital to include these in analysis to see if these different pathways impacted the conservation of sdiA. The author should also provide a much cleaner version of the metadata as a separate tab in the spreadsheet that includes only those strains that were included in the analysis. Unfortunately, the above-mentioned issue means that all of the results in the manuscript are potential erroneous and need to be completely re-done with the elimination of non-EHEC strains and the inclusion of additional \"big 6\" genomes to provide a scientifically sound analysis.\nIt would also be helpful to include in the methods section the date of the search, as the database is constantly changing making reproduction a little bit easier by other researchers. Upon the new analysis it would be helpful to include a brief table or statement of the serotype breakdown included in the EHEC pangenome that would also eliminate some of the above-mentioned issues and make it easier for readers to get a sense of those serotypes included in the analysis.\n\nThere are a number of grammatical errors or poor phrasing in the manuscript that should be reviewed and corrected. Such as there is only one author and no indication of other researchers on the manuscript, yet the manuscript keeps stating we instead of I.\n\nFinally, there are several points made in the introduction and discussion that do not have references. For example, in the introduction the author mentions \"pangenome of E. coli was published in 2008 contained 8 genomes\" but does not reference the paper. Additionally, the author mentions \"serogroup O80 has aside from Shiga toxin, an extra-intestinal virulence plasmid (pS88), is currently emerging in France\" but do not reference anything indicating the emergence in France. I have also provided the citations for Ogura et al.1 and Cooper et al.2 for the author to review and potentially cite in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4650", "date": "16 May 2019", "name": "DJ Darwin Bandoy", "role": "Author Response", "response": "I appreciate the work of the reviewer for going through the metadata. The inclusion of non-EHEC is necessary as outgroup comparison group and does not debunk the validity of the analysis pipeline. In light of this clarification, since this is the sole reason for the non-approval, I appeal for an approval with reservations as the analysis is technically and scientifically sound. I am in the process of including the big 6 serotypes in the analysis based on the reviewer's comments, as well as the additional references which are very constructive additions to the paper. Again, as the reviewer sees the value in redoing a more inclusive analysis of EHEC serotypes, this is another justification to approve with reservation the paper submitted.I beg to disagree with the comment of using \"inclusive we\" in place of I as a grammatical error. The use of royal or inclusive we in lieu of I is a matter of preference. This is the only part of the review I do not agree with." } ] }, { "id": "46991", "date": "15 May 2019", "name": "Olivier Tenaillon", "expertise": [ "Reviewer Expertise Microbial genomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present manuscript presents a state-of-the-art pan genome analysis of EHEC strains and a subsequent analysis of the variation in sdiA.\nThe analysis of sdiA could have been completed with simple KA/Ks analysis and compared to that of the core genome. For now, there is no connection between the two analysis, the authors could have simply performed the analysis of sdiA.\nSome mutants have frame shifts in the gene, this is not discussed.\nI think the author could try to connect more the two parts of the analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4651", "date": "16 May 2019", "name": "DJ Darwin Bandoy", "role": "Author Response", "response": "I thank the reviewer for the effort in doing the review. I accept all the suggestions and will add the population genetic analysis in the next version of the paper." } ] } ]	1	https://f1000research.com/articles/8-33
https://f1000research.com/articles/9-339/v1	07 May 20	{ "type": "Brief Report", "title": "Intraspecific mitochondrial gene variation can be as low as that of nuclear rRNA", "authors": [ "Tshifhiwa G. Matumba", "Jody Oliver", "Nigel P. Barker", "Christopher D. McQuaid", "Peter R. Teske", "Tshifhiwa G. Matumba", "Jody Oliver", "Nigel P. Barker", "Christopher D. McQuaid" ], "abstract": "Background: Mitochondrial DNA (mtDNA) has long been used to date historical demographic events. The idea that it is useful for molecular dating rests on the premise that its evolution is neutral. Even though this idea has long been challenged, the evidence against clock-like evolution of mtDNA is often ignored. Here, we present a particularly clear and simple example to illustrate the implications of violations of the assumption of selective neutrality. Methods: DNA sequences were generated for the mtDNA COI gene and the nuclear 28S rRNA of two closely related rocky shore snails, and species-level variation was compared. To our knowledge, this is the first study to use nuclear rRNA at this taxonomic level, presumably because this marker is assumed to evolve so slowly that it is only suitable for phylogenetics.\n\nResults: Even though high inter-specific divergence reflected the faster evolutionary rate of COI, intraspecific genetic variation was similar for both markers. As a result, estimates of population expansion times based on mismatch distributions differed between the two markers by millions of years. Conclusions: Assuming that 28S evolves effectively clock-like, these findings can be explained by variation-reducing purifying selection in mtDNA at the species level, and an elevated divergence rate caused by diversifying selection between the two species. Although these two selective forces together make mtDNA suitable as a marker for species identifications by means of DNA barcoding because they create a ‘barcoding gap’, estimates of demographic change based on this marker can be expected to be highly unreliable. Our study contributes to the growing evidence that the utility of mtDNA sequence data beyond DNA barcoding is limited.", "keywords": [ "Purifying selection", "diversifying selection", "mismatch distribution", "molecular dating", "demographic history", "population expansion" ], "content": "Introduction\n\nMitochondrial DNA (mtDNA) has long been a marker of choice for investigating concepts as diverse as estimating genetic diversity and effective population sizes, reconstructing species’ evolutionary histories, exploring spatial genetic subdivisions, and identifying cryptic species. All these methods assume that mtDNA variation conforms to the neutral model of molecular evolution1, but violations of this premise have long been recognised2. Over the past decades, much evidence has accumulated that mtDNA can be strongly affected by selective sweeps and background selection3–6. As a result, the usefulness of the marker in assessing genetic diversity7 and exploring spatial genetic structure in continuously distributed populations8 has been questioned, and corrections of the mitochondrial molecular clock that account for selection have been proposed9,10.\n\nThe implications of reduced genetic diversity at the species or population levels due to purifying selection has so far received little attention. When mutations in mitochondrial genes occur at fewer sites than expected under the neutral model11, molecular dating of historical demographic events by means of evolutionary rate estimates that are typically based on inter-specific divergence12,13 will result in considerable underestimates. This is particularly likely because divergence between species can be strongly affected by diversifying selection that is driven by different environmental conditions14,15, resulting in a faster accumulation of mutations characterising each species than is expected under the neutral model.\n\nHere, we explore this issue using mitochondrial and nuclear DNA sequence data from two common southern African snails of the genus Afrolittorina. The finding that data from two genetic markers whose mutation rates are assumed to differ by at least an order of magnitude have similar levels of intraspecific variation challenges the usefulness of mitochondrial DNA sequences for studying historical demographic changes.\n\n\nMethods\n\nSpecimens of the snails Afrolittorina africana and A. knysnaensis were collected at 34 sites throughout South Africa (Table 1). DNA was extracted using the CTAB protocol16, amplified with universal COI primers17 and 28S primers LSU518 and LSU160019 following Williams et al.19, and sequenced on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) using Big Dye Terminator v3.1 chemistry. Sequences were edited using MEGA720, and 28S sequences were phased in PHASE v2.1.121 using default settings. Genealogical relationships between COI haplotypes and 28S alleles were reconstructed using the median-joining algorithm22 in popArt v1.723. To explore the effect of using interspecific evolutionary rates to estimate species-level population size changes, we calculated population expansion time24 using Arlequin v3.525 using each marker’s slowest and fastest published rates for marine gastropods (Table 2).\n\n34 sites along the South African coastline were sampled, and these are arranged from west to east.\n\nThe moment estimator τ is equal to 2ut, where u equals 2 µk (μ is the mutation rate and k is the length of the sequence), and t is the time of expansion in million years (my).\n\n1Malaquias & Reid 2009; 2Williams & Reid 2004. A generation time of one year was used.\n\n\nResults\n\nSpecies-specific genetic clusters reconstructed from COI sequences were highly distinct (Figure 1a), with a minimum number of 44 nucleotide differences between the two species’ most closely related haplotypes. In contrast, differentiation between 28S sequences (Figure 1b) was an order of magnitude smaller (4 differences).\n\nMedian-joining haplotype networks constructed from a) COI sequences and b) 28S rRNA sequences of Afrolittorina knysnaensis (grey) and A. africana (white). Low intra-specific variation and high inter-specific variation of COI potentially illustrate purifying and diversifying selection, respectively. The size of circles is proportional to the frequency of each haplotype, cross-bars represent nucleotide differences, and black dots are missing haplotypes not found in the samples.\n\nIn contrast to the high inter-specific differentiation between COI haplotypes, intra-specific genetic differentiation was comparatively low for this marker, and similar to that of 28S. In A. knysnaensis, six COI haplotypes and seven 28S haplotypes were found, while the maximum differentiation between the COI haplotypes was only two nucleotide differences, but 10 for 28S. The number of haplotypes was greater for A. africana, where 14 were found for COI and 10 for 28S. Maximum nucleotide differences for this species were seven in the COI network and five for 28S.\n\nThe practical implications of two markers with very different evolutionary rates based on inter-specific divergence having similar levels of intraspecific variation are illustrated in Table 2. Using published rates, estimates of population expansion times were more than an order of magnitude greater for the 28S data than for the COI data.\n\n\nDiscussion\n\nThe usefulness of the mtDNA COI gene to uncover overlooked biodiversity is undisputed because of the marker’s tendency to have a well-defined barcoding gap, as was found here. The two study species’ COI sequences were much more strongly differentiated than their 28S sequences, potentially reflecting diversifying selection as a result of adaption to different thermal environments. In contrast, there was comparatively little genetic variation at the intraspecific level for either marker, which is likely due to the commonly reported strong purifying selection acting upon the COI gene6,9.\n\nMany researchers explore their mtDNA sequence data for additional information, but the selective forces that together create the barcoding gap26 make its utility for other applications questionable7,8. In the present study, we have highlighted a largely unexplored problem that likely arises from selection effects in mtDNA data: the fact that demographic events using gene regions under variation-reducing purifying selection are dated using molecular clock calibrations affected by variation-increasing diversifying selection. The finding that intraspecific mtDNA variation can be as low as that of nuclear rRNA cautions against the continued use of mtDNA for exploring demographic trends by means of mismatch distributions or Bayesian skyline plots27, a practice that continues to dominate the recent literature28–32.\n\nIn our opinion, it is time to discontinue the use of fixed mtDNA rates based on divergence dating of closely related taxa, such as the closure of the Central American Seaway to date phylogenies of marine species12,13 or the 2% rule in birds33. The very large datasets generated using next-generation sequencing have considerable potential to facilitate more accurate dating by identifying nuclear markers that conform to the assumptions of the molecular clock but curiously, fixed rates based on mtDNA data are still being used to calibrate such datasets when no suitable fossil calibration points exist34. A possible solution may involve the identification of a suite of neutral markers that can be used to assess divergence between the species used in the original molecular dating studies, and 28S rRNA may be a suitable candidate.\n\n\nData availability\n\nDNA sequences generated in this study were submitted to GenBank (COI accession numbers: MT331645–MT331814; 28S rRNA accession numbers: MT329760–MT330099).", "appendix": "Acknowledgements\n\nA previous version of this article is available on bioRxiv: https://doi.org/10.1101/2020.03.31.017764.\n\n\nReferences\n\nKimura M: The neutral theory of molecular evolution. Cambridge: Cambridge University Press; 1983. Reference Source\n\nBallard JW, Kreitman M: Is mitochondrial DNA a strictly neutral marker? Trends Ecol Evol. 1995; 10(12): 485–8. PubMed Abstract \| Publisher Full Text\n\nBlier PU, Dufresne F, Burton RS: Natural selection and the evolution of mtDNA-encoded peptides: evidence for intergenomic co-adaptation. Trends Genet. 2001; 17(7): 400–6. PubMed Abstract \| Publisher Full Text\n\nBallard JWO, Rand DM: The population biology of mitochondrial DNA and its phylogenetic implications. Annu Rev Ecol Evol Syst. 2005; 36(1): 621–42. Publisher Full Text\n\nMeiklejohn CD, Montooth KL, Rand DM: Positive and negative selection on the mitochondrial genome. Trends Genet. 2007; 23(6): 259–63. PubMed Abstract \| Publisher Full Text\n\nStewart JB, Freyer C, Elson JL, et al.: Purifying selection of mtDNA and its implications for understanding evolution and mitochondrial disease. Nat Rev Genet. 2008; 9(9): 657–62. PubMed Abstract \| Publisher Full Text\n\nBazin E, Glémin S, Galtier N: Population size does not influence mitochondrial genetic diversity in animals. Science. 2006; 312(5773): 570–2. PubMed Abstract \| Publisher Full Text\n\nTeske PR, Golla TR, Sandoval-Castillo J, et al.: Mitochondrial DNA is unsuitable to test for isolation by distance. Sci Rep. 2018; 8(1): 8448. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoares P, Ermini L, Thomson N, et al.: Correcting for purifying selection: an improved human mitochondrial molecular clock. Am J Hum Genet. 2009; 84(6): 740–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo SY, Lanfear R, Bromham L, et al.: Time-dependent rates of molecular evolution. Mol Ecol. 2011; 20(15): 3087–101. PubMed Abstract \| Publisher Full Text\n\nLawrie DS, Messer PW, Hershberg R, et al.: Strong purifying selection at synonymous sites in D. melanogaster. PLoS Genet. 2013; 9(5): e1003527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchubart CD, Diesel R, Hedges B: Rapid evolution to terrestrial life in Jamaican crabs. Nature. 1998; 393: 363–5. Publisher Full Text\n\nKnowlton N, Weigt LA: New dates and new rates for divergence across the Isthmus of Panama. Proc Biol Sci. 1998; 265(1412): 2257. Publisher Full Text \| Free Full Text\n\nLamb AM, Gan HM, Greening C, et al.: Climate-driven mitochondrial selection: a test in Australian songbirds. Mol Ecol. 2018; 27(4): 898–918. PubMed Abstract \| Publisher Full Text\n\nSun JT, Jin PY, Hoffmann AA, et al.: Evolutionary divergence of mitochondrial genomes in two Tetranychus species distributed across different climates. Insect Mol Biol. 2018; 27(6): 698–709. PubMed Abstract \| Publisher Full Text\n\nDoyle J: DNA protocols for plants. In: Molecular techniques in taxonomy. 1991; 283–93. Publisher Full Text\n\nFolmer O, Black M, Hoeh W, et al.: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994; 3(5): 294–9. PubMed Abstract\n\nLittlewood DT, Curini-Galletti M, Herniou EA: The interrelationships of Proseriata (Platyhelminthes: Seriata) tested with molecules and morphology. Mol Phylogenet Evol. 2000; 16(3): 449–66. PubMed Abstract \| Publisher Full Text\n\nWilliams ST, Reid DG, Littlewood DT: A molecular phylogeny of the Littorininae (Gastropoda: Littorinidae): unequal evolutionary rates, morphological parallelism, and biogeography of the Southern Ocean. Mol Phylogenet Evol. 2003; 28(1): 60–86. PubMed Abstract \| Publisher Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7): 1870–4. PubMed Abstract \| Publisher Full Text\n\nStephens M, Smith NJ, Donnelly P: A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001; 68(4): 978–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandelt HJ, Forster P, Röhl A: Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999; 16(1): 37–48. PubMed Abstract \| Publisher Full Text\n\nLeigh JW, Bryant D: popart: full-feature software for haplotype network construction. Methods Ecol Evol. 2015; 6(9): 1110–6. Publisher Full Text\n\nRogers AR, Harpending H: Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol. 1992; 9(3): 552–69. PubMed Abstract \| Publisher Full Text\n\nExcoffier L, Lischer HE: Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 2010; 10(3): 564–7. PubMed Abstract \| Publisher Full Text\n\nStoeckle MY, Thaler DS: DNA barcoding works in practice but not in (neutral) theory. PLoS One. 2014; 9(7): e100755. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrummond AJ, Rambaut A, Shapiro B, et al.: Bayesian coalescent inference of past population dynamics from molecular sequences. Mol Biol Evol. 2005; 22(5): 1185–92. PubMed Abstract \| Publisher Full Text\n\nLow VL, Norma-Rashid Y, Yusoff A, et al.: Pleistocene demographic expansion and high gene flow in the globe skimmer dragonfly Pantala flavescens Fabricius (Odonata: Libellulidae) in Peninsular Malaysia. Zool Anz. 2017; 266: 23–7. Publisher Full Text\n\nGao TX, Yang TY, Yanagimoto T, et al.: Levels and patterns of genetic variation in Japanese whiting (Sillago japonica) based on mitochondrial DNA control region. Mitochondrial DNA A DNA Mapp Seq Anal. 2019; 30(1): 172–83. PubMed Abstract \| Publisher Full Text\n\nIván Pérez-Quiñonez C, Quiñonez-Velázquez C, García-Rodríguez FJ: Genetic homogeneity of the Pacific thread herring (Opisthonema libertate) (Günther, 1867) in the Eastern Pacific, inferred from mtDNA sequences. Mitochondrial DNA A DNA Mapp Seq Anal. 2019; 30(3): 517–24. PubMed Abstract \| Publisher Full Text\n\nDiringer B, Pretell K, Avellan R, et al.: Genetic structure, phylogeography, and demography of Anadara tuberculosa (Bivalvia) from East Pacific as revealed by mtDNA: implications to conservation. Ecol Evol. 2019; 9(8): 4392–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrant WS: Problems and cautions with sequence mismatch analysis and Bayesian skyline plots to infer historical demography. J Hered. 2015; 106(4): 333–46. PubMed Abstract \| Publisher Full Text\n\nShields GF, Wilson AC: Calibration of mitochondrial DNA evolution in geese. J Mol Evol. 1987; 24(3): 212–7. PubMed Abstract \| Publisher Full Text\n\nTrucchi E, Gratton P, Whittington JD, et al.: King penguin demography since the last glaciation inferred from genome-wide data. Proc Biol Sci. 2014; 281(1787): 20140528. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "66088", "date": "20 Jul 2020", "name": "Andrew G. Briscoe", "expertise": [ "Reviewer Expertise Molecular biology", "mitogenomics", "phylogenetics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting manuscript addressing the methodological issues with the continued trend of using mitochondrial DNA sequence data to infer genetic variation and population structure. The results are based on a very limited data set, however, they are only intended to further illustrate the issues highlighted by the authors. Apart from that, the study does not require much improvement. It is well written and structured and the results support the conclusions and therefore suggest indexing the article once the following issues have been considered and adequately resolved;\n\nThe authors claim that this is the first study to use 28S ribosomal RNA at this taxonomic level. Do they mean in snails, as this gene has been used extensively for phylogenetics and some population level analysis? More info is needed.\n\nCould the authors include a reference when stating that there is an order of magnitude of difference between the mutation rates of the two markers used in the study?\n\nReference 29 is cited as an example of the continued use of mtDNA for exploring demography, however, that study uses the mitochondrial control region rather than sequence data Protein Coding Genes (PCGs). Is there evidence that the mutation rates of this mitochondrial locus is under the same kind of diversifying selection as PCGs? If not, then the authors should consider changing or removing this reference.\n\nIn the discussion the authors refer to the different thermal environments of the two snail species used in the study. It would be useful if the authors could elaborate on this as very little background about the species being studied is provided.\n\nThe authors state that they are assuming 28S evolves in a clock-like manner. Can they provide a references/evidence for this as it is quite a bold statement, which has implications on some of the conclusions being drawn.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5835", "date": "28 Aug 2020", "name": "Peter Teske", "role": "Author Response", "response": "This is an interesting manuscript addressing the methodological issues with the continued trend of using mitochondrial DNA sequence data to infer genetic variation and population structure. The results are based on a very limited data set, however, they are only intended to further illustrate the issues highlighted by the authors. Apart from that, the study does not require much improvement. It is well written and structured and the results support the conclusions and therefore suggest indexing the article once the following issues have been considered and adequately resolved; COMMENT: The authors claim that this is the first study to use 28S ribosomal RNA at this taxonomic level. Do they mean in snails, as this gene has been used extensively for phylogenetics and some population level analysis? More info is needed.RESPONSE: We have conducted an extensive literature review on this issue, and have not found any comparable example. Even though intraspecific variation in 18S or 28S rRNA has been reported in various invertebrate taxa, upon closer inspection it becomes evident that this variation is either located in the more rapidly evolving ITS regions rather than in the rRNA, or the species studied have geographically distinct populations that may constitute cryptic species. However, as we cannot rule out the possibility that comparable examples exist, we have changed the sentence to: “Nuclear rRNA is not usually used to study intraspecific variation in species that are not spatially structured, presumably because this marker is assumed to evolve so slowly that it is more suitable for phylogenetics.” COMMENT: Could the authors include a reference when stating that there is an order of magnitude of difference between the mutation rates of the two markers used in the study? RESPONSE: The paper already includes several references that have estimated mutation rates specifically for marine snails. Please see Table 2, where the 28S rates range from 0.01-0.05 %/myr, whereas the COI rates range from 0.26-0.50 %/myr. We now cite both references in the last paragraph of the Introduction. COMMENT: Reference 29 is cited as an example of the continued use of mtDNA for exploring demography, however, that study uses the mitochondrial control region rather than sequence data Protein Coding Genes (PCGs). Is there evidence that the mutation rates of this mitochondrial locus is under the same kind of diversifying selection as PCGs? If not, then the authors should consider changing or removing this reference.RESPONSE: This is a valid point, and while a signature of diversifying selection in the control region is likely because this marker is linked to the PCGs by virtue of being part of the same genome, it is clearly not under selection itself. For that reason, we have decided to remove this reference. COMMENT: In the discussion the authors refer to the different thermal environments of the two snail species used in the study. It would be useful if the authors could elaborate on this as very little background about the species being studied is provided. RESPONSE: To comply with word count requirements for Brief Reports, we have removed more detailed descriptions of the species’ distribution ranges, morphology and physiology. In response to similar queries from the other reviewers, we now cite the PhD thesis on which the present study is based. This thesis not only provides information on the temperature-defined marine biogeographical provinces in which these species occur, but it also shows evidence for adaptive differences assessed using physiological experiments. COMMENT: The authors state that they are assuming 28S evolves in a clock-like manner. Can they provide a references/evidence for this as it is quite a bold statement, which has implications on some of the conclusions being drawn.RESPONSE: We have found no specific information on this, as the issue of selection on the molecular clock has primarily received attention in the context of mtDNA. However, we believe that the considerable difference in terms of genetic differentiation between species justifies such as statement. There is now strong evidence that the barcoding gap in COI is a result of selective forces, and the comparatively low level of genetic divergence based on 28S implies that similar selective forces are not acting on this marker. We have, however, made the statement in the Abstract less bold and have changed it to: “Assuming that 28S evolution is more clock-like…”." } ] }, { "id": "68386", "date": "12 Aug 2020", "name": "Abigail Hui En Chan", "expertise": [ "Reviewer Expertise Molecular systematics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 aimed to explore the genetic variation of two common southern African snails of the genus Afrolittorina. The main finding of this study is that intraspecific variations of nuclear 28S rDNA sequences are higher than such value from the mitochondrial gene in the snail populations. However, many points need to concern before acceptance. I listed the major points for revising the manuscript.\nIntroduction part, the introduction part seems to focus on the selection process in the population to increase/decrease the genetic variation accumulation in the mt genome. The authors did not emphasize on the research questions; for example, Why did you want to study in two snails of the genus Afrolittorina? Why did you use the 28S rDNA as another genetic marker? What is the research question or hypothesis of this study? What was the reason behind deciding to study on A. aficana and A. knysnaensis? Finally, what is the main objective of this work? Whether you aimed to study the genetic differentiation between species of the snail based on different kinds of markers or study on population genetic, the content of this study could not support both objectives. For example, if the authors want to present on comparing the genetic variations that came out from the COI gene and 28S rDNA gene, the analysis used in this study was not suitable, the haplotype network analysis may not fit enough for the genetic variations you want to get. If the author wants to analyze the population genetically, the weak point is the numbers of the snails collected? And why the authors decided to collect from the various sites? What is the hypothesis behind your sampling?\n\nI don't understand why the authors have to analyze the molecular clock by comparing different originated markers like COI from one of the protein-coding genes in the mitochondrial genome and LSU (RNA-specifying gene) from the nuclear genome. Of course, the rate of evolution acting on these two genomes is different.\n\nIf the authors considered in Figure 1, you would realize that 28S rDNA is not good enough for species discrimination between A. aficana and A. knysnaensis comparing with the COI gene. Only 2 nucleotide differences between those two snail species, while the nucleotide variation in the intra-specific level of A. knysnaensis is higher than that.\n\nThe authors didn't discuss the evidence in snails with regards to variation, which is the point that showed in the result.\n\nThere is no analysis to estimate either positive or negative selection, but the authors discussed it as the condition forced on the snail populations. It becomes over speculation.\n\nThe title of this study should mention snail species because the genetic variation patterns are various depending on the groups of organisms.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5836", "date": "28 Aug 2020", "name": "Peter Teske", "role": "Author Response", "response": "Reviewer Report: Abigail Hui En Chan and Urusa Thaenkham, Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, ThailandAPPROVED WITH RESERVATIONSCOMMENT: This study aimed to explore the genetic variation of two common southern African snails of the genus Afrolittorina. The main finding of this study is that intraspecific variations of nuclear 28S rDNA sequences are higher than such value from the mitochondrial gene in the snail populations.RESPONSE: The statement that “intraspecific variations of nuclear 28S rDNA sequences are higher than such value from the mitochondrial gene” is not entirely correct, as in A. africana, COI has more haplotypes, whereas in A. knysnaensis, 28S has more haplotypes. We specifically state that “intra-specific genetic differentiation was comparatively low for this marker [i.e., COI], and similar to that of 28S.”COMMENT: However, many points need to concern before acceptance. I listed the major points for revising the manuscript. Introduction part, the introduction part seems to focus on the selection process in the population to increase/decrease the genetic variation accumulation in the mt genome. The authors did not emphasize on the research questions; for example, Why did you want to study in two snails of the genus Afrolittorina? Why did you use the 28S rDNA as another genetic marker? What is the research question or hypothesis of this study? What was the reason behind deciding to study on A. aficana and A. knysnaensis? Finally, what is the main objective of this work?RESPONSE: This article is a Brief Report, which has very strict word limits. For that reason, it is not possible to provide lengthy explanations for the issues raised. Briefly, the original purpose of the research was to determine whether each species is divided into regional populations (which was not found). The purpose was not to compare intraspecific variation between two markers with very different evolutionary rates, as that is an unexpected finding, and essentially a novel discovery that is reported in this paper. To answer the additional questions, we would like to refer the reviewers to the original PhD thesis on which this paper is based, which is now included among the references, and which is accessible via the following link: http://vital.seals.ac.za:8080/vital/access/manager/Repository/vital:5588?site_name=GlobalView COMMENT: Whether you aimed to study the genetic differentiation between species of the snail based on different kinds of markers or study on population genetic, the content of this study could not support both objectives. For example, if the authors want to present on comparing the genetic variations that came out from the COI gene and 28S rDNA gene, the analysis used in this study was not suitable, the haplotype network analysis may not fit enough for the genetic variations you want to get. If the author wants to analyze the population genetically, the weak point is the numbers of the snails collected?RESPONSE: The aim of the study was not to analyse each population genetically. We assume here that the reviewers equate ‘populations’ with sampling sites, please see the following paper for an explanation why this is problematic: Waples & Gaggiotti (2006) Molecular Ecology 15:1419-1439. As neither species shows regional sub-structure (see also the PhD thesis mentioned earlier) and each species thus comprises a single ‘population’, it is possible to pool data from all sites for intraspecific genetic analysis. The number of samples collected (93 and 82) is actually large compared to other papers of this nature. For example, in their highly cited paper on the barcoding gap (whose detection depends on both intra- and inter-specific variation), Paulay & Meyer (2005) used an average number of 7.7 samples per cowrie species. The fact that neither species of Afrolittorina is genetically subdivided throughout its range is clearly important, and was removed from an earlier version of this article so as not to exceed the word count for Brief Reports. We have decided to briefly mention it in the last paragraph of the Introduction: “Here, we explore this issue using mitochondrial and nuclear DNA sequence data from two common southern African snails of the genus Afrolittorina that show no spatial genetic structure throughout their ranges.” We now also cite the PhD thesis for anyone interested in the biology of these snails. COMMENT: And why the authors decided to collect from the various sites? What is the hypothesis behind your sampling?RESPONSE: The original purpose of collecting samples from multiple sites that span much of each species’ range was to determine whether or not these species are genetically subdivided into regional populations, which was not found. However, this is clearly not relevant to the present study, which focuses on intraspecific variation, not genetic structure. Obtaining large numbers of samples from a few sites would be problematic as one cannot rule out that additional, genetically distinct ‘populations’ exist that were not sampled. Please note that Table 1 is not particularly important to understand this paper, and for that reason should not be the focus of a lengthy discussion related to sampling. We originally had it in an Extended Data section, but the editor requested us to move it to the main text. COMMENT: I don't understand why the authors have to analyze the molecular clock by comparing different originated markers like COI from one of the protein-coding genes in the mitochondrial genome and LSU (RNA-specifying gene) from the nuclear genome. Of course, the rate of evolution acting on these two genomes is different.RESPONSE: The difference in evolutionary rates between these two markers is crucial to highlight the potential role of variation-reducing selection in COI. Without understanding this, it is not possible to understand the take-home message of this paper. We essentially point out that the known rate differences between these two markers manifest at the inter-specific level. The novel finding of this study is that intra-specific levels of variation are surprisingly similar. To clarify, the statement “Of course, the rate of evolution acting on these two genomes is different” is only true between species, not within species. COMMENT: If the authors considered in Figure 1, you would realize that 28S rDNA is not good enough for species discrimination between A. aficana and A. knysnaensis comparing with the COI gene. Only 2 nucleotide differences between those two snail species, while the nucleotide variation in the intra-specific level of A. knysnaensis is higher than that.RESPONSE: The purpose of this paper is not to suggest that 28S is a marker that is suitable for species discrimination, particularly when compared to COI. As we state in the first sentence of the Discussion, “The usefulness of the mtDNA COI gene to uncover overlooked biodiversity is undisputed because of the marker’s tendency to have a well-defined barcoding gap, as was also found here.” It is not clear what the reviewers mean by “not good enough”, the two genetic clusters are clearly distinct, although inter-specific divergence is clearly much lower. The likely reason for this is that 28S evolves in a more clock-like fashion while COI is strongly affected by selective forces, and this is another important point to understand, as a marker that does not evolve in a clock-like fashion should not be used for intraspecific molecular dating. Please see the second paragraph in the Discussion: “In the present study, we have highlighted a largely unexplored problem that likely arises from selection effects in mtDNA data: the fact that demographic events using gene regions under purifying selection are dated using molecular clock calibrations affected by variation-increasing inter-specific divergence.” Given the slow evolutionary rate of 28S, minimal differentiation between species is expected. What is unexpected are the similar levels of intraspecific variation (see title). COMMENT: The authors didn't discuss the evidence in snails with regards to variation, which is the point that showed in the result.RESPONSE: The results report the simplest and most intuitive means of describing instraspecific variation: the number of haplotypes, and the maximum number of mutations between them. Several sentences in the Discussion deal with this, e.g. first paragraph: “In contrast, there was comparatively little genetic variation at the intraspecific level for both markers, which is likely due to the commonly reported strong purifying selection acting upon the COI gene.” COMMENT: There is no analysis to estimate either positive or negative selection, but the authors discussed it as the condition forced on the snail populations. It becomes over speculation.RESPONSE: The paper originally included a test for selection, but as it was not informative and exceeded the word limit for Brief Reports, it was removed. We used a McDonald-Kreitman test (McDonald & Kreitman 1991) in MKT (Egea et al. 2008) to test for selection in COI. Out of 58 mutations for the whole dataset, only one was non-synonymous. Although the test had the highest possible proportion of adaptive substitutions (alpha) of 1.0, it was non-significant (P = 0.57), supposedly because of the lack of non-synonymous polymorphism.Further, recent genomic evidence indicates that tests comparing synonymous and non-synonymous mutations are not actually conclusive about whether or not selection has taken place, because synonymous sites may be affected by strong selection even though no amino acid changes have taken place (Lawrie DS et al. (2013) Strong Purifying Selection at Synonymous Sites in D. melanogaster. PLoS Genet 9(5): e1003527). Again, a detailed discussion of these issues would far exceed the word limit, and would not add much to the study. The simple dataset used here is clearly not sufficiently informative to conclude that purifying selection is present, and for that reason we have changed the earlier title “Purifying selection can reduce intraspecific mitochondrial gene variation to that of nuclear rRNA” (https://www.biorxiv.org/content/10.1101/2020.03.31.017764v1) to the present one, which merely focuses on intraspecific variation rather than selection.We nonetheless do not think that mentioning selection is this context is over-speculative. There is strong evidence that a finding of barcoding gaps in hundreds of studies is the result of selective forces, and we cite several important papers in this context, including two at the end of the following sentence in the Discussion: “In contrast, there was comparatively little genetic variation at the intraspecific level for both markers, which is likely due to the commonly reported strong purifying selection acting upon the COI gene.” In terms of finding a COI barcoding gap, our study is merely an additional example, but the finding that intraspecific variation-reducing selection can be so significant that COI shows levels of variation similar to a marker that evolves much more slowly is novel. COMMENT: The title of this study should mention snail species because the genetic variation patterns are various depending on the groups of organisms.RESPONSE: We respectfully disagree – the practice of including taxonomic information in the title is standard procedure in taxonomy journals, but it is undesirable in a multidisciplinary journal such as F1000Research. In general, it is well known that shorter titles that report results are more likely to get accessed (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351256/). In the present case, the snail species, their geographic structure, their biology etc. are far less important than are the two genetic markers, and anyone who does not work on littorinid snails may consider the paper irrelevant to their own work if species names are included in the title. However, the finding of this study is likely applicable to a wide range of organisms given the mounting evidence for variation-reducing selection in mtDNA, and giving the impression that it is unique to two rocky shore snails would be an obstacle to further investigation into this issue. To provide an example, had Stoeckle & Thaler 2014 mentioned in the title of their article “DNA barcoding works in practice but not in (neutral) theory” that these findings are based entirely on birds, we would probably not have cited their paper, even though it is highly relevant. For that reason, we believe that it should be sufficient to mention “rocky shore snails” in the Abstract." } ] } ]	1	https://f1000research.com/articles/9-339
https://f1000research.com/articles/9-1060/v1	28 Aug 20	{ "type": "Research Article", "title": "Independent accretion of TIM22 complex subunits in the animal and fungal lineages", "authors": [ "Sergio A. Muñoz-Gómez", "Shannon N. Snyder", "Samantha J. Montoya", "Jeremy G. Wideman", "Sergio A. Muñoz-Gómez", "Shannon N. Snyder", "Samantha J. Montoya" ], "abstract": "Background: The mitochondrial protein import complexes arose early in eukaryogenesis. Most of the components of the protein import pathways predate the last eukaryotic common ancestor. For example, the carrier-insertase TIM22 complex comprises the widely conserved Tim22 channel core. However, the auxiliary components of fungal and animal TIM22 complexes are exceptions to this ancient conservation. Methods: Using comparative genomics and phylogenetic approaches, we identified precisely when each TIM22 accretion occurred. Results: In animals, we demonstrate that Tim29 and Tim10b arose early in the holozoan lineage. Tim29 predates the metazoan lineage being present in the animal sister lineages, choanoflagellate and filastereans, whereas the erroneously named Tim10b arose from a duplication of Tim9 at the base of metazoans. In fungi, we show that Tim54 has representatives present in every holomycotan lineage including microsporidians and fonticulids, whereas Tim18 and Tim12 appeared much later in fungal evolution. Specifically, Tim18 and Tim12 arose from duplications of Sdh3 and Tim10, respectively, early in the Saccharomycotina. Surprisingly, we show that Tim54 is distantly related to AGK suggesting that AGK and Tim54 are extremely divergent orthologues and the origin of AGK/Tim54 interaction with Tim22 predates the divergence of animals and fungi. Conclusions: We argue that the evolutionary history of the TIM22 complex is best understood as the neutral structural divergence of an otherwise strongly functionally conserved protein complex. This view suggests that many of the differences in structure/subunit composition of multi-protein complexes are non-adaptive. Instead, most of the phylogenetic variation of functionally conserved molecular machines, which have been under stable selective pressures for vast phylogenetic spans, such as the TIM22 complex, is most likely the outcome of the interplay of random genetic drift and mutation pressure.", "keywords": [ "TIM22 complex", "Mitochondrial protein import", "Mitochondrial evolution", "Neutral evolution" ], "content": "Introduction\n\nMitochondria evolved from an ancient alphaproteobacterial endosymbiont (Martijn et al., 2018; Roger et al., 2017). The integration of the symbiont into host cell processes required the evolution of dedicated machinery for the import of host-encoded proteins (Roger et al., 2017). The establishment of symbiont protein import allowed the transfer of many genes from the symbiont to the host genome as well as domestication of symbiont metabolic processes (e.g., via the evolution of mitochondrial carrier family proteins MCFs) (Cavalier-Smith, 2006). Understanding how symbionts become organelles and integrate into host cell processes requires an understanding of how protein import machineries originate and diversify.\n\nProteins imported into the mitochondria require several dedicated protein complexes to ensure proper sorting and assembly into mitochondrial subcompartments, including the mitochondrial outer membrane (MOM), the mitochondrial inner membrane (MIM), the intermembrane space (IMS) and the matrix (Wiedemann & Pfanner, 2017). The translocase of the mitochondrial outer membrane complex (TOM) facilitates the transport of all mitochondrial proteins (with the exception of some MOM proteins (Becker et al., 2008; Becker et al., 2011; Dimmer et al., 2012; Doan et al., 2020)). The sorting and assembly machinery complex (SAM) is required for the import and assembly of MOM β-barrel proteins like Tom40, Sam50, Mdm10, and Porin. Two translocase of the mitochondrial inner membrane (TIM) complexes facilitate MIM protein import. The TIM23 complex is required for the membrane translocation of proteins with presequences that are directed into the matrix as well as single-pass transmembrane domain (TMD) proteins (Mokranjac & Neupert, 2010). The TIM22 complex is responsible for inserting and assembling multi-pass TMD proteins like MCFs into the MIM (Horten et al., 2020).\n\nAll of these protein complexes are inferred to have been present in the last eukaryotic common ancestor (LECA) and the general phylogenetic profiles of their components have been recently reported (Fukasawa et al., 2017; Mani et al., 2015). Investigations have so far focused on the broad distribution of subunits across eukaryotes, leaving some details unexplored, like the evolution of TIM22 complex components.\n\nThe TIM22 complex functions to assemble multi-pass TMD proteins like MCFs into the MIM (Kerscher et al., 1997). In both human and yeast cells, TIM22 substrates first cross the MOM via TOM and are delivered to the small tim complexes (Tim9-10 and Tim8-13 complexes; Tim12 is additionally present in S. cerevisiae, whereas Tim10a and Tim10b, as well as Tim8a and Tim8b are present in human) which shuttle proteins through the IMS to TIM22 (Beverly et al., 2008; Davis et al., 2007; Gentle et al., 2007; Hoppins & Nargang, 2004; Koehler et al., 1998; Sirrenberg et al., 1998).\n\nIn S. cerevisiae, the TIM22 complex comprises Tim22, Tim54, Tim12, Tim18, and Sdh3, whereas in human, the complex contains Tim22, Tim29, AGK (acyglycerol kinase), and a subset of small Tims (Tim9, Tim10a, and Tim10b); as the small Tims are soluble proteins that shuttle hydrophobic substrates, they might not constitute stable components of the TIM22 complex. In S. cerevisiae, Tim54 plays a role in TIM22 complex stability as well as assembly of the Yme1 complex (Hwang et al., 2007; Kerscher et al., 1997). Sdh3 (a component of succinate dehydrogenase [SDH] – Complex II of the electron transport chain) and Tim18 (a paralogue of Sdh4) facilitate the integration of Tim54 into the TIM22 complex (Gebert et al., 2011; Kerscher et al., 2000; Koehler et al., 2000). A recent cryo-EM structure has been solved for the human TIM22 complex, showing how the components interact within the MIM (Qi et al., 2019). In human mitochondria, Tim29 stabilizes the TIM22 complex and links it to TOM (Callegari et al., 2016; Kang et al., 2016). AGK plays a role in the function of TIM22 as well as lipid metabolism (Kang et al., 2017; Vukotic et al., 2017).\n\nBecause animal and fungal TIM22 complexes are best characterized, both structurally and functionally, these lineages offer an ideal case to dissect the fine-grained evolutionary history of multi-protein complexes. Apart from the central Tim22 subunit (Fukasawa et al., 2017; Žárský & Doležal, 2016), the origin and evolution of the TIM22 complex components in animals and fungi has not been extensively investigated. In this paper, we explore the evolutionary history of the TIM22 complex in animals and fungi, using homology searching and phylogenetic methods. We found that each lineage’s TIM22 complex accreted subunits independently. We place our findings in a larger theoretical framework recently developed by Lynch (Lynch, 2020; Lynch & Trickovic, 2020). We argue that most of the structural variation seen in the functionally conserved TIM22 complex across the Holozoa and the Holomycota is non-adaptive. The evolutionary history of the TIM22 complex probably represents an example of effectively neutral divergence from an optimal mean phenotype which has primarily been governed by the joint forces of drift and mutation.\n\n\nResults and discussion\n\nRecent investigations have identified complex components beyond Tim22 in human cells, namely Tim29 and AGK (Callegari et al., 2016; Kang et al., 2016; Kang et al., 2017; Mårtensson & Becker 2017; Qi et al., 2019; Vukotic et al., 2017). Tim29 was originally identified as a complex component primarily involved in complex assembly and stability (Kang et al., 2016) but was subsequently also shown to be important for protein import (Callegari et al., 2016). The Sengers syndrome-associated AGK was originally shown to catalyze the phosphorylation of acylglycerols to lysophosphatidic and phosphatidic acid in the MIM (Bektas et al., 2005), but has recently been identified as a TIM22 complex member in human cells (Kang et al., 2017; Vukotic et al., 2017). A cryo-EM structure that includes Tim22, Tim29, AGK, and the small Tims (Tim9, Tim10a, and Tim10b) has recently been reported (Qi et al., 2019). Since Tim29, AGK, and Tim10b are not present in fungi (Fukasawa et al., 2017), we sought to determine when in the holozoan lineage these proteins were gained.\n\nUsing the reciprocal best BLAST search method, we identified orthologues of Tim29 in the genomes of most animal species and unicellular eukaryotes (i.e., protists) most closely related to animals (Figure 1). This means Tim29 originated prior to the origin of animals. We could not identify Tim29 in Gallus gallus; however, orthologous sequences were recovered from other birds and reptiles, suggesting loss of Tim29 is limited to chickens. We used our set of Tim29 sequences to search for orthologues across eukaryotes using the HMMer server (Finn et al., 2011) at EBI. When restricting our taxon searching to exclude holozoans, we retrieved no hits below our 0.01 e-value significance cut off, strongly suggesting that no homologues of Tim29 exist outside Holozoa (Extended data, Supplemental Text File 1; Wideman et al., 2020).\n\nThe Coulson plot was generated using the Coulson Plot Generator (Field et al., 2013). Abbreviations: Vertebrates: Hsap, Homo sapiens; Mdom, Monodelphis domesticus; Drer, Danio rerio; Xtro, Xenopus tropicalis; Ggal, Gallus gallus; Mmus, Mus musculus; Invertebrates: Dmel, Drosophila melanogaster; Cele, Caenorhabditis elegans; ; Tric, Trichoplax sp.; Acro, Acropora sp.; Aque, Amphimedon queenslandica; Mlei, Mnemiopsis leidyi; Nvec, Nematostella vectensis; Bran, Branchiostoma sp.; Unicellular Holozoa: Mbre, Monosiga brevicollis; Cowc, Capsaspora owczarzaki; Sarc, Sphaeroforma arctica; Sros, Salpingoeca rosetta; Fungi: Spom, Schizosaccharomyces pombe; Scer, Saccharomyces cerevisiae; Ncra, Neurospora crassa; Cneo, Cryptococcus neoformans; Umay, Ustilago maydis; Lsta, Lipomyces starkeyi; Ylip, Yarrowia lipolytica, Bden, Batrachochytrium dendrobatidis; Mdap, Mitosporidium daphnia; Ecun, Encephalitozoon cuniculi; Piro, Piromyces sp.; Spun, Spizellomyces punctatus; Rirr, Rhizophagus irregularis; Crev, Coemansia reversa; Ccor, Conidiobolus coronatus; Cang, Catenaria anguillulae; Rall, Rozella allomyces; Fonticulids: Fonticula alba; Apusozoa: Ttra, Thecamonas trahens.\n\nTo determine when in the holozoan lineages the TIM22 subunits AGK, Tim10b, and Tim8b first appeared, we collected sequences related to AGK and the small Tims from diverse holozoan genomes. We aligned sequences using MUSCLE (Edgar, 2004) and performed phylogenetic reconstructions using RAxML (Stamatakis, 2014) and MrBayes (Ronquist et al., 2012).\n\nThe phylogenetic reconstruction of AGK and related sequences clearly distinguish putative clades of holozoan AGK, ceramide kinase, and sphingosine kinase indicative of a pre-metazoan ancestry of these enzymes (Figure 2A). We did not include representatives from an outgroup as the best BLAST hits of AGK outside the holozoan lineage were cyanobacteria, oomycetes, and plants, suggesting that a detailed analysis of this gene family is required to understand its origin and evolutionary history in eukaryotes.\n\n(A) Acylgylcerol Kinase (AGK) was present in the ancestral holozoan. (B) Tim10b arose from a duplication of Tim9 at the base of metazoans. (C) Tim8a and Tim8b arose from a duplication of Tim8 at the base of chordates. Homologues of AGK and small tims were collected from a subset of holozoan taxa using BLASTp. Sequences were aligned and trimmed resulting in 296, 75, and 73 sites for AGK, Tim9/10, and Tim8/13, respectively. The asterisks indicate true Tim8a and Tim8b orthologues that are not supported in our tree but are confirmed by best BLAST analysis. The resulting alignments were subjected to phylogenetic analysis using MrBayes (Ronquist et al., 2012) for posterior probability and RAxML (Stamatakis, 2014) for maximum likelihood frameworks.\n\nFor the small Tims, the reconstructed phylogenies (Figures 2B and 2C) include well-supported Tim10a and Tim13 clades. This suggests that Tim10b was the result of a duplication of Tim9 at the base of animals (Figure 2B), whereas Tim8a and Tim8b are the result of a duplication at the base of chordates (Figure 2C). Although we did not recover Tim8a and Tim8b sequences from Branchiostoma floridae within the vertebrate clades of Tim8a and Tim8b (Figure 2C, asterisks), their best BLAST hits are clearly Tim8a and Tim8b from vertebrates, respectively. We therefore conclude that Tim8a and Tim8b arose prior to the divergence of chordates from the rest of animals. We were unable to recover small Tim sequences from the Choanoflagellate Monosiga brevicollis, but this is likely due to an incomplete database as Tim9 and Tim10 are probably essential in holozoans.\n\nThese results demonstrate that TIM22 complex subunits were accreted very early in the holozoa. Tim29 and AGK (but see below) appear to be gained shortly after the holozoan lineage diverged from the holomycotan lineage, Tim10b originated shortly after the origin of animals, and Tim8b originated after the origin of chordates. This means that Tim29 and AGK predate the origin of animals and have persisted in this lineage for about a billion years and Tim10b arose shortly thereafter.\n\nComponents of the TIM22 complex were identified in fungi much earlier than the recent discoveries in animals (Kerscher et al., 1997; Kerscher et al., 2000; Koehler et al., 2000). Tim54 appears to be involved in complex stability as well as integration of MIM proteases (Hwang et al., 2007; Kerscher et al., 1997), but its mechanism of action is unclear. In yeast, Tim18 is related to Sdh4 of the SDH complex (Kerscher et al., 2000; Koehler et al., 2000). Another SDH complex member, Sdh3 interacts with Tim18 as a TIM22 complex module that is integrated into the MIM by the OXA complex (Stiller et al., 2016). Sdh3 and Tim18 are involved in the biogenesis and assembly of Tim22 and Tim54 into a functional TIM22 complex (Gebert et al., 2011). Finally, Tim12 is a small Tim that is not found outside the yeast lineage.\n\nUsing the reciprocal best BLAST method, we were able to identify Tim54 in representatives from every major fungal lineage as well as Fonticula alba, but no other eukaryotic lineage (Figure 1). We were unable to identify Tim54 in Rozella allomycis or microsporidians except Mitosporidium daphniae, a short-branching microsporidian with canonical mitochondria (Haag et al., 2014). We used our set of Tim54 sequences to search for orthologues across eukaryotes using the HMMer (Finn et al., 2011) server at EBI. When restricting our taxon searching to exclude fungi and Fonticula, we surprisingly retrieved AGK sequences from animals (141 hits above threshold) and Capsaspora as top hits (Extended data, Supplemental Text File 2; Wideman et al., 2020). These results indicate that Tim54 and AGK likely share a common ancestor; however, the diacylglycerol kinase (DAGK) domain is now virtually undetectable in fungal sequences.\n\nTo determine when Tim18 and Tim12 originated, sequences related to Tim18, Sdh4, Tim10, and Tim12 were collected from all Saccharomycotina in the Mycocosm database (Grigoriev et al., 2013). We trimmed long sequences and removed any spurious hits (as determined by reciprocal BLAST into the S. cerevisiae S288c genome). Sequences were aligned using MUSCLE (Edgar, 2004), manually trimmed, and phylogenies reconstruction using RAxML (Stamatakis, 2014) and MrBayes (Ronquist et al., 2012) for likelihood and posterior probability calculations, respectively. The phylogenetic reconstruction of Tim18 indicates that a duplication occurred after the divergence of early-branching Saccharomycotina (e.g. Lipomyces and Yarrowia), but before the divergence of a major clade that includes Wickerhamomyces and Saccharomyces (Figure 3A). An additional Sdh4 paralogue Shh4 is the result of the more recent Saccharomyces lineage whole genome duplication/hybridization event (Kellis et al., 2004; Marcet-Houben & Gabaldón, 2015; Wolfe & Shields, 1997). The phylogenetic reconstruction of Tim12 suggests that a duplication of the Tim10 protein occurred even earlier in Saccharomycotina (Figure 3B) as only the earliest-branching species lack clear Tim12 representatives (e.g., Lipomyces starkeyi).\n\n(A) Tim18 arose from a duplication of Sdh4 deep within the yeast lineage. (B) Tim12 arose from a duplication of Tim10 near the base of Saccharomycotina. Homologues of Tim18/Sdh4 and Tim10/12 were collected from sequenced Saccharomycotina on Mycocosm (Grigoriev et al., 2013) using BLASTp. Sequences were aligned and trimmed resulting in 128 and 82 sites for Tim18 and small tims, respectively. The resulting alignments were subjected to phylogenetic analysis using MrBayes (Ronquist et al.,2012) for posterior probability and RAxML (Stamatakis, 2014) for maximum likelihood frameworks.\n\nIn contrast to the animal TIM22 complex, which accreted subunits early in the evolution of animals, we demonstrate that a gradual accretion of TIM22 complex subunits occurred in the lineage leading to S. cerevisiae. Tim54 is likely a divergent fungal AGK which lost the DAGK domain after the divergence of holomycota from holozoa (Figure 4). Tim18 and Tim12 are respectively derived from duplications of Sdh4 and Tim10 deep within the Saccharomycotina. It is unknown if other fungal lineages have undergone similar expansions of the TIM22 complex.\n\nThe ancestral TIM22 complex comprises a single subunit, Tim22, which likely interacted with the small Tims, Tim9 and Tim10. The ancestral opisthokont TIM22 complex contained an additional subunit, AGK, which retains a diacylglycerol kinase (DAGK) domain in holozoans. In the holomycotan lineage, the DAGK domain was lost or diverged beyond recognition, but stabilized into Tim54, which is well-conserved across Holomycota. Early in the evolution of Saccharomycotina, Tim12 was gained via a duplication of Tim10, and Tim18 was gained via a duplication of Sdh4. Shortly after the divergence of Holozoa from Holomycota, Tim29 was gained as a subunit of the TIM22 complex. Tim10b was gained after the divergence of Metazoa from the unicellular holozoans from a duplication of Tim9. Tim8a and Tim8b result from a duplication of Tim8 in the lineage leading to chordates (not shown).\n\n\nConclusions\n\nThe first elements of a larger theoretical framework to quantitatively understand the macroevolution of cells, their organelles, and molecular machines have been developed (Lynch, 2020; Lynch & Trickovic, 2020). Our findings are consistent with the predictions made by the theory of effectively neutral divergence of mean phenotypes across major phylogenetic lineages (Lynch, 2020). This theory assumes that the selective pressures on many molecular machines have remained relatively constant for long stretches of macroevolutionary time. This is most likely the case for many multi-protein complexes whose functions are strongly conserved across vast phylogenetic spans. Mitochondrial protein import complexes, such as TIM22, are good examples of such strongly functionally conserved systems. The TIM22 complex has to physically interact with dozens or even hundreds of substrates for their proper insertion into the MIM. This implies that its functional divergence is constrained (and/or buffered) by its many physical interactions. However, slight deviations from an optimal (functional) phenotypic mean could be expected as a consequence of a permissive population-genetic environment, i.e., stronger drift due to historical bottlenecks or smaller effective population sizes. As selective pressures are assumed to remain constant for many conserved multi-protein complexes in stable cellular environments, most phenotypic divergence would be dictated by the combined action of random genetic drift and mutation pressure. Most divergence seen in multi-protein complexes or molecular machines, therefore, would primarily be non-functional and non-adaptive but mostly structural (e.g., subunit composition) in character.\n\nMany other observations appear to be compatible with this view. The common recruitment of paralogous subunits, as well as the presence of highly derived and lineage-specific subunits in multi-protein complexes provide general examples. More specifically, kinetoplastids offer another example in a protein-import complex. Trypanosoma brucei lacks the TIM23 complex and instead contains a bifunctional Tim22 protein that acts both as a presequence and a carrier translocase complex (Harsman et al., 2016; Schneider, 2020; Singha et al., 2008). We have recently suggested that the bifunctional Tim22 complex of kinetoplastids evolved neutrally via homologue replacement of ancestral components followed by loss of TIM23 (von Känel et al., 2020). This massive divergence from the ancestral state probably added no obvious benefit to the organism. As long as the whole still performs the same function, it doesn’t matter what parts are used. This is also the sentiment behind neutral evolution of cellular structures (Wideman et al., 2019; Zhang, 2018). Evolutionary divergence primarily dictated by drift and mutation (under stable selective pressure in constant cellular environments (Lynch, 2020)); would certainly allow for the constructive, ratchet-like, structural evolution of multi-protein complex ((Gray et al., 2010; Stoltzfus, 1999; Stoltzfus, 2012; Wideman et al., 2019); a.k.a., constructive neutral evolution). Further comparative investigations at the bench are required to determine what functional differences exist between fungal and animal TIM22 complexes, and whether other eukaryotic lineages have accreted subunits in a similar way to opisthokonts (Figure 4).\n\n\nMethods\n\nTim22 orthologues were identified previously (Žárský & Doležal, 2016). We used the reciprocal best hit method to identify Tim54 and Tim29 orthologues in fungi and holozoans, respectively. Briefly, S. cerevisiae Tim54 and Homo sapiens Tim29 were used as BLASTp (Altschul et al., 1997) queries into opisthokont predicted proteomes (see Figure 1 for organism list) using the NCBI BLAST server or Mycocosm database (Grigoriev et al., 2013). The top hit was retrieved and used as a BLASTp query into S. cerevisiae (for Tim54) or H. sapiens (for Tim29) proteome. If the top hit was the original BLASTp query, then the retrieved protein was considered orthologous. Using HMMer (Finn et al., 2011) at EBI we used our collected Tim54 and Tim29 sequences to build Hidden Markov Model profiles to identify orthologues across eukaryotes. Orthologue distribution across opisthokonts was visualized using the Coulson Plot Generator (Field et al., 2013).\n\nHomologues of AGK, and small Tims were collected from metazoan predicted proteomes using BLASTp. Homologues of Tim18, Sdh4, Tim10, and Tim12 were collected from all sequenced Saccharomycotina from the Mycocosm database using BLASTp (Grigoriev et al., 2013). Pertinent homologues were aligned with MUSCLE (Edgar, 2004) and manually trimmed using Mesquite v.2.75. Phylogenetic tree reconstructions were performed using MrBayes v.3.2.6 for Bayesian analysis (Ronquist et al., 2012). MrBayes analyses were run with the following parameters: prset aamodelpr = fixed (WAG); mcmcngen = 2,000,000; samplefreq = 1000; nchains = 4; startingtree = random; sumt burnin = 250. Split frequencies were checked to ensure convergence. Maximum-likelihood bootstrap values (100 pseudoreplicates) were obtained using RAxML v.8.2.10 (Stamatakis, 2014) under the LG model (Le & Gascuel, 2008).\n\n\nData availability\n\nSequence accession are provided in Extended data, Supplemental Table 1 (Wideman et al., 2020). Sequences are from the NCBI database, the Mycocosm database at the Joint Genome Institute, or from the Mnemiopsis leydii genome database.\n\nFigshare: Extended Data: Independent accretion of TIM22 complex subunits in the animal and fungal lineages. https://doi.org/10.6084/m9.figshare.12818786.v1 (Wideman et al., 2020).\n\nThis project contains the following extended data:\n\nSupplemental Table 1. TIM22 complex subunit accessions collected in this investigation.\n\nSupplemental Text File 1. Tim29 HMMer results .txt file.\n\nSupplemental Text File 2. Tim54 HMMer results .txt file.", "appendix": "References\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker T, Pfannschmidt S, Guiard B, et al.: Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors. J Biol Chem. 2008; 283(1): 120–7. PubMed Abstract \| Publisher Full Text\n\nBecker T, Wenz LS, Krüger V, et al.: The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins. J Cell Biol. 2011; 194(3): 387–395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBektas M, Payne SG, Liu H, et al.: A novel acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in prostate cancer cells. J Cell Biol. 2005; 169(5): 801–811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeverly KN, Sawaya MR, Schmid E, et al.: The Tim8-Tim13 Complex Has Multiple Substrate Binding Sites and Binds Cooperatively to Tim23. J Mol Biol . 2008; 382(5): 1144–1156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCallegari S, Richter F, Chojnacka K, et al.: TIM29 is a subunit of the human carrier translocase required for protein transport. FEBS Lett. 2016; 590(23): 4147–4158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCavalier-Smith T: Origin of mitochondria by intracellular enslavement of a photosynthetic purple bacterium. Proc Biol Sci. 2006; 273(1596): 1943–1952. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavis AJ, Alder NN, Jensen RE, et al.: The Tim9p/10p and Tim8p/13p complexes bind to specific sites on Tim23p during mitochondrial protein import. Mol Biol Cell. 2007; 18(2): 475–486. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDimmer KS, Papić D, Schumann B, et al.: A crucial role for Mim2 in the biogenesis of mitochondrial outer membrane proteins. J Cell Sci. 2012; 125(Pt 14): 3464–3473. PubMed Abstract \| Publisher Full Text\n\nDoan KN, Grevel A, Mårtensson CU, et al.: The Mitochondrial Import Complex MIM Functions as Main Translocase for α-Helical Outer Membrane Proteins. Cell Rep. 2020; 31(4): 107567. PubMed Abstract \| Publisher Full Text\n\nEdgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004; 5: 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nField HI, Coulson RMR, Field MC: An automated graphics tool for comparative genomics: the Coulson plot generator. BMC bioinformatics. 2013; 14(1): 141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinn RD, Clements J, Eddy SR: HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011; 39(Web Server issue): W29–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukasawa Y, Oda T, Tomii K, et al.: Origin and Evolutionary Alteration of the Mitochondrial Import System in Eukaryotic Lineages. Mol Biol Evol. 2017; 34(7): 1574–1586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGebert N, Gebert M, Oeljeklaus S, et al.: Dual function of Sdh3 in the respiratory Chain and TIM22 protein translocase of the mitochondrial inner membrane. Mol Cell. 2011; 44(5): 811–818. PubMed Abstract \| Publisher Full Text\n\nGentle IE, Perry AJ, Alcock FH, et al.: Conserved motifs reveal details of ancestry and structure in the small TIM chaperones of the mitochondrial intermembrane space. Mol Biol Evol. 2007; 24(5): 1149–60. PubMed Abstract \| Publisher Full Text\n\nGray MW, Lukes J, Archibald JM, et al.: Cell biology. Irremediable complexity? Science. 2010; 330(6006): 920–921. PubMed Abstract \| Publisher Full Text\n\nGrigoriev IV, Nikitin R, Haridas S, et al.: MycoCosm portal: gearing up for 1000 fungal genomes. Nucleic Acids Res. 2013; 42(Database issue): D699–D704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaag KL, James TY, Pombert JF, et al.: Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites. Proc Natl Acad Sci U S A. 2014; 111(43): 15480–15485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarsman A, Oeljeklaus S, Wenger C, et al.: The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins. Nat Commun. 2016; 7: 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoppins SC, Nargang FE: The Tim8-Tim13 Complex of Neurospora crassa Functions in the Assembly of Proteins into Both Mitochondrial Membranes. J Biol Chem. 2004; 279(13): 12396–12405. PubMed Abstract \| Publisher Full Text\n\nHorten P, Colina-Tenorio L, Rampelt H: Biogenesis of mitochondrial metabolite carriers. Biomolecules. 2020; 10(7): 1008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang DK, Claypool SM, Leuenberger D, et al.: Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion. J Cell Biol. 2007; 178(7): 1161–1175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang Y, Baker MJ, Liem M, et al.: Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability. Elife. 2016; 5: e17463. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang Y, Stroud DA, Baker MJ, et al.: Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex. Mol Cell. 2017; 67(3): 457–470.e5. PubMed Abstract \| Publisher Full Text\n\nKellis M, Birren BW, Lander ES: Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature. 2004; 428(6983): 617–624. PubMed Abstract \| Publisher Full Text\n\nKerscher O, Holder J, Srinivasan M, et al.: The Tim54p-Tim22p complex mediates insertion of proteins into the mitochondrial inner membrane. J Cell Biol. 1997; 139(7): 1663–1675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerscher O, Sepuri NB, Jensen RE: Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. Mol Biol Cell. 2000; 11(1): 103–116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoehler CM, Merchant S, Oppliger W, et al.: Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins. EMBO J. 1998; 17(22): 6477–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoehler CM, Murphy MP, Bally NA, et al.: Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane. Mol Cell Biol. 2000; 20(4): 1187–1193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe SQ, Gascuel O: An improved general amino acid replacement matrix. Mol Biol Evol. 2008; 25(7): 1307–20. PubMed Abstract \| Publisher Full Text\n\nLynch M: The evolutionary scaling of cellular traits imposed by the drift barrier. Proc Natl Acad Sci U S A. 2020; 117(19): 10435–10444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynch M, Trickovic B: A Theoretical Framework for Evolutionary Cell Biology. J Mol Biol. 2020; 432(7): 1861–1879. PubMed Abstract \| Publisher Full Text\n\nMani J, Meisinger C, Schneider A: Peeping at TOMs - Diverse entry gates to mitochondria provide insights into the evolution of eukaryotes. Mol Biol Evol. 2015; 33(2): 337–351. PubMed Abstract \| Publisher Full Text\n\nMarcet-Houben M, Gabaldón T: Beyond the whole-genome duplication: Phylogenetic evidence for an ancient interspecies hybridization in the baker’s yeast lineage. PLoS Biol. 2015; 13(8): e1002220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMårtensson CU, Becker T: Acylglycerol Kinase: Mitochondrial Protein Transport Meets Lipid Biosynthesis. Trends Cell Biol. 2017; 27(10): 700–702. PubMed Abstract \| Publisher Full Text\n\nMartijn J, Vosseberg J, Guy L, et al.: Deep mitochondrial origin outside the sampled alphaproteobacteria. Nature. 2018; 557(7703): 101–105. PubMed Abstract \| Publisher Full Text\n\nMokranjac D, Neupert W: The many faces of the mitochondrial TIM23 complex. Biochim Biophys Acta. 2010; 1797(6–7): 1045–1054. PubMed Abstract \| Publisher Full Text\n\nQi L, Wang Q, Guan Z, et al.: Cryo-EM Structure of the Human Mitochondrial Translocase TIM22 Complex. bioRxiv. 2019; 869289. Publisher Full Text\n\nRoger AJ, Muñoz-Gómez SA, Kamikawa R: The Origin and Diversification of Mitochondria. Curr Biol. 2017; 27(21): R1177–R1192. PubMed Abstract \| Publisher Full Text\n\nRonquist F, Teslenko M, van der Mark P, et al.: MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61(3): 539–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider A: Evolution of mitochondrial protein import - lessons from trypanosomes. Biol Chem. 2020; 401(6-7): 663–676. PubMed Abstract \| Publisher Full Text\n\nSingha UK, Peprah E, Williams S, et al.: Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei. Mol Biochem Parasitol. 2008; 159(1): 30–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSirrenberg C, Endres M, Folsch H, et al.: Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5. Nature. 1998; 391(6670): 912–915. PubMed Abstract \| Publisher Full Text\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStiller SB, Höpker J, Oeljeklaus S, et al.: Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins. Cell Metab. 2016; 23(5): 901–908. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoltzfus A: On the possibility of constructive neutral evolution. J Mol Evol. 1999; 49(2): 169–81. PubMed Abstract \| Publisher Full Text\n\nStoltzfus A: Constructive neutral evolution: exploring evolutionary theory’s curious disconnect. Biol Direct. 2012; 7: 35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Känel C, Muñoz-Gómez SA, Oeljeklaus S, et al.: Homologue replacement in the import motor of the mitochondrial inner membrane of trypanosomes. Elife. 2020; 9: e52560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVukotic M, Nolte H, König T, et al.: Acylglycerol Kinase Mutated in Sengers Syndrome Is a Subunit of the TIM22 Protein Translocase in Mitochondria. Mol Cell. 2017; 67(3): 471–483.e7. PubMed Abstract \| Publisher Full Text\n\nWideman JG, Novick A, Muñoz-Gómez SA, et al.: Neutral evolution of cellular phenotypes. Curr Opin Genet Dev. 2019; 58-59: 87–94. PubMed Abstract \| Publisher Full Text\n\nWideman J, Munoz-Gomez S, Montoya S, et al.: Extended Data: Independent accretion of TIM22 complex subunits in the animal and fungal lineages. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12818786.v1\n\nWiedemann N, Pfanner N: Mitochondrial Machineries for Protein Import and Assembly. Annu Rev Biochem. 2017; 86: 685–714. PubMed Abstract \| Publisher Full Text\n\nWolfe KH, Shields DC: Molecular evidence for an ancient duplication of the entire yeast genome. Nature. 1997; 387(6634): 708–13. PubMed Abstract \| Publisher Full Text\n\nŽárský V, Doležal P: Evolution of the Tim17 protein family. Biol Direct. 2016; 11(1): 54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J: Neutral Theory and Phenotypic Evolution. Mol Biol Evol. 2018; 35(6): 1327–1331. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71396", "date": "24 Sep 2020", "name": "Kevin R. Lynch", "expertise": [ "Reviewer Expertise Sphingolipid biochemistry", "specifically sphingosine-1-phosphate synthesis", "transport", "degradation and receptor binding. Thus", "as stated in my comments", "my focus on AGK", "which is a member of the sphingolipid kinase family." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe well-studied S. cerevisiae and human Tim22 complexes both function in the transport/insertion of integral membrane proteins into the inner mitochondrial membrane, yet the only conserved protein of the complexes is Tim22 itself. Through a variety of protein sequence comparison techniques, the authors document the appearance of various Tim22 complex proteins in holozoan and holomycotan lineages. Surprisingly, the authors found that the animal Tim22 complex protein, AGK, is distantly related to the fungal Tim22 complex protein, Tim54. I do not have particular expertise in Molecular Evolution, rather my interest in the study is primarily through the narrower lens of an ongoing interest in the biochemistry of AGK.\n\nThe authors do not state what regions of AGK and Tim54 were found to be related, and this information is of interest, because even when comparing AGK with the closely related sphingolipid kinases, the only similarity (detected by BLASTP searches) is in the amino terminal, ATP-binding region (identified by inference from human SPHK1 crystal structure). The authors report that AGK orthologs are readily identifiable when searching metazoan databases and can likewise be identified in a few unicellular eukaryotes that are closely related to animals (e.g. choanoflagelattes, Capsaspora). This is readily verified with simple BLASTP searches using human AGK as a query sequence. However, after reading the paper twice, I remain confused as to whether AGK orthologs have been identified in other protist databases. Specifically, how do the authors distinguish between AGK orthologs and the paralogous DAG kinases?\n\nWhile it is tangential to the subject of this paper, I note that AGK-focused publications have a somewhat tortuous history (of which the authors are, understandably, probably unaware). As documented in their Figure 2A, AGK is most closely related to sphingolipid kinases. That group in turn is more distantly related to the diacylglycerol kinase (DAGK) family and the NAD+ kinases. The earliest AGK literature focused on its putative kinase activity. The authors reference the 2005 Bektas et al. paper in support of the contention that AGK is an acylglycerol kinase as is the common practice in AGK papers since 2005. However, there is little in the Bektas paper or elsewhere to validate this claim. Indeed, AGK appeared in the literature originally as a multi-substrate lipid kinase (PMID: 152520461) and later as a possible ceramide kinase in Drosophila (PMID: 220694802). Another group reported a failure to detect any kinase activity, i.e. AGK as an “orphan” kinase (PMID: 162698263). I recommend that the authors consider citing all of these mutually contradictory reports since all save one have been neglected in the subsequent AGK literature – for no apparent reason except ‘everybody else does it’. The more recent AGK literature includes the discovery that humans born deficient in the protein suffer from Sengers syndrome, which is characterized by mitochondrial insufficiency. Subsequent to that discovery, AGK was found to be a component of the Tim22 complex in human cells. Interestingly, a single amino change (G126E) in human AGK that would eliminate enzyme activity (by inference from the better characterized sphingosine kinases) does not inhibit the import function of the Tim22 complex in cultured cells.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "70541", "date": "28 Sep 2020", "name": "Thomas Becker", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe TIM22 complex promotes import of carrier proteins into the inner mitochondrial membrane. Recent studies showed the composition of the TIM22 complex in human mitochondria, which displayed remarkable differences to the yeast homolog. Munoz-Gomez and colleagues investigates the phylogenetic origin of the TIM22 subunits. The authors found that yeast Tim54 and human AGK are distantly related. The furthermore showed that yeast Tim12 and Tim18 arose from gene duplication of Tim10 and Sdh3, respectively. Tim29 is specific for holozoan, while Tim12, Sdh3, Tim18 are specific for holomycota. Overall, the findings are interesting and well presented. I have only minor recommendations.\n\nThe authors should describe in more detail the substrates of the carrier pathway, including substrates with an uneven number of transmembrane spans that have been reported recently (Gomkale et al., 20201; Rampelt et al., 20202).\n\nThe authors should name all import pathways into mitochondria in the introduction. The current version is rather incomplete and lacks the MIM-dependent import pathway into the outer membrane. Furthermore, appropriate citations for TOM and SAM complexes should be added.\n\nThe section about the TIM22 components should be modified. The authors should describe that Tim18/Sdh3 are important for the stability of the TIM22 complex. Tim54 could serve as a docking site for small TIM chaperones of the intermembrane space.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1060
https://f1000research.com/articles/9-486/v1	01 Jun 20	{ "type": "Systematic Review", "title": "The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis", "authors": [ "Faizal Andri", "Asri Nurul Huda", "Marjuki Marjuki", "Faizal Andri", "Asri Nurul Huda" ], "abstract": "Background: Due to their antimicrobial properties and safety, essential oils are currently proposed as a sustainable option for antibiotic alternatives in the livestock sector. This current systematic review and meta-analysis investigated the effects of dietary essential oil supplements on growth response of small ruminants. Methods: A total of 12 studies (338 small ruminants) were included in this meta-analysis. The overall effect size was quantified using Hedges’ g with 95% confidence interval (CI) using a fixed-effect model. Publication bias was inspected using Begg’s and Egger’s tests, followed by trim and fill method to detect the number of potential missing studies. Results: Insignificant heterogeneity among studies was detected both on dry matter intake (DMI; P of Q = 0.810; I-square = 0.00%), average daily gain (ADG; P of Q = 0.286; I-square = 17.61%), and feed conversion ratio (FCR; P of Q = 0.650; I-square = 0.00%). The overall effect size showed that essential oils supplementation had no significant impact on DMI (Hedges’ g = -0.12; 95% CI = -0.50 to 0.26; P = 0.429) and FCR (Hedges’ g = -0.17; 95% CI = -0.55 to 0.22; P = 0.284), but had a significant positive impact on ADG (Hedges’ g = 0.44; 95% CI = 0.12 to 0.76; P = 0.002). The result of publication bias analysis showed that DMI, ADG, and FCR did not present any significant biases (P > 0.10), and no potential missing studies detected. Conclusions: Dietary essential oil could improve ADG of small ruminants, without any alteration on DMI and FCR. Further research in this topic is still required to provide stronger evidence of the potency of essential oil as a growth promoter for small ruminants.", "keywords": [ "Antibiotics alternative", "Average daily gain", "Goats", "Natural feed additives", "Protozoa", "Secondary metabolites", "Sheep." ], "content": "Introduction\n\nIn animal nutrition, antibiotics become the first choice of feed additive due to their substantial benefit toward health and productivity. However, the routine use of this chemical additive yields residues in livestock products, and is also responsible for the development of microbial antibiotic resistance1,2. These factors represent a dangerous risk to human health, which has led to the global drive to reduce antibiotic use in the livestock sector. As a result, several natural products have been proposed to be used as antibiotic alternatives3,4.\n\nAmong natural feed additives, essential oils have a unique mechanism of action in livestock production. They can manipulate rumen fermentation characteristics5,6 and subsequently improve growth rate7,8. However, other findings showed no meaningful effect of this feed additive on productive performance9,10, while another study showed a negative impact11. The inconsistent results among studies requires an appropriate tool to quantify the overall effect. Therefore, this study was conducted to measure the quantitative effects of dietary essential oil supplementation on the growth response of small ruminants using a systematic review and meta-analysis approach.\n\n\nMethods\n\nThe systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline12. A completed PRISMA checklist is available in Reporting guidelines13.\n\nThe inclusion and exclusion of the study were based on participants, interventions, comparisons, outcomes, and study design (PICOS) criteria as indicated in Table 1. Additionally, only publications written in English which was included in this study. All dates up until the date last searched were included.\n\nDMI: dry matter intake; ADG: average daily gain; FCR: feed conversion ratio.\n\nThe literature search was carried out using the following electronic databases: Scopus, PubMed, and SciELO. The search was last performed on 30 April 2020. Table 2 shows the full electronic search strategy.\n\nResults from the search were firstly checked for duplicates. After duplicate studies were removed, F.A. and A.N.H. screened titles and abstracts independently using the eligibility criteria (Table 1). Full texts of the selected studies were then further examined to find eligible studies. In the case of any disagreement, this was resolved by adjudication from M.M. The authors of the included studies were not contacted for further clarification.\n\nData extraction was performed independently by F.A. and A.N.H. The senior investigator (M.M.) solved any disagreements by discussion. Data extracted included the following items: 1) authors; 2) animal species; 3) number of animals; 4) essential oil source; and 5) growth response variables. Growth response variables consisted of dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR). Standard error or standard error of means were converted into standard deviation14. The data was pooled when a study used more than one dose of essential oils or tested both sexes of experimental animals15.\n\nThe overall effect size was quantified using Hedges’ g16 using a fixed-effect model. This model was chosen due to the insignificant heterogeneity among studies after checked using Cochran’s Q16 and I-square17.\n\nPublication bias was inspected using Begg’s18 and Egger’s tests19, with P <0.10 set to determine the existence of publication bias. The trim and fill method20 was employed to detect the number of potential missing studies and to adjust the overall effect size. All meta-analysis procedures were performed using Meta-Essentials version 1.421.\n\n\nResults\n\nFigure 1 shows the PRISMA flow diagram. A total of 137 records were identified through database searching. Of these, 12 studies were eligible for the current meta-analysis. The essential oil sources included oregano11,22–24, thyme25,26, chavil27, juniper7,28, and mixed product9,29. Unfortunately, one study did not define the source of essential oil8. The main characteristics of the included studies are shown in Table 3. Extracted data of outcome measures is available as Extended data30.\n\nn: number of experimental animals; EO: essential oil; NI: no information; Mix A: a mixture of thymol, carvacrol, eugenol, limonene, and cinnamaldehyde EO; Mix B: a mixture of thyme leaf, daphne leaf, sage tea leaf, fennel seed, orange cortes, and myrtle leaf EO; DMI: dry matter intake; ADG: average daily gain; FCR: feed conversion ratio.\n\nData of ADG from two studies11,25 were considered as outliers because their standardized residual was >\|3\| and thus were excluded from effect size quantification. Insignificant heterogeneity among studies was detected both for DMI (P of Q = 0.810; I-square = 0.00%), ADG (P of Q = 0.286; I-square = 17.61%), and FCR (P of Q = 0.650; I-square = 0.00%). As can be seen in Figure 2, the overall effect size showed that essential oil supplementation had no significant impact on DMI (P = 0.429) and FCR (P = 0.284), but had a significant positive impact on ADG (P = 0.002). The result of publication bias analysis showed that DMI, ADG, and FCR did not present any significant biases (P >0.10) (Table 4). The trim and fill method also did not detect any potential missing studies for all parameters.\n\nDMI: dry matter intake; ADG: average daily gain; FCR: feed conversion ratio.\n\nDMI: dry matter intake; ADG: average daily gain; FCR: feed conversion ratio.\n\n\nDiscussion\n\nThe current meta-analysis showed that dietary essential oils significantly increased ADG of small ruminants. This finding probably related to the antimicrobial activity of essential oils, which could reduce ruminal protozoa population31,32. Protozoa population may represent up to 50% of the total biomass of rumen microbes33. They have a negative impact on nitrogen utilization by ruminants because they engulf and digest bacteria, thus reducing microbial protein flow to abomasum34. Additionally, the presence of protozoa is also associated with methane production, which is responsible for the loss of up to 12% of gross energy intake by ruminants35. Thereby, the reduction of the ruminal protozoa population by essential oil could increase microbial protein, as well as energy supply, which ultimately could improve the growth rate of small ruminants.\n\nThis study provides insight of the potency of essential oil as a growth promoter for small ruminants. However, the current findings should be interpreted with caution due to the limited data available. Moreover, the literature search only covers published literature, which could lead to publication bias. For that reason, further research in this topic is highly encouraged to provide stronger evidence.\n\n\nConclusions\n\nThe current meta-analysis reveals that dietary essential oil could improve average daily gain of small ruminants, without any alteration on dry matter intake and feed conversion ratio. However, further research in this topic is still highly recommended to provide more robust evidence.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Extended data for ‘The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis’. https://doi.org/10.6084/m9.figshare.12298913.v330.\n\nThis project contains extracted data of outcome measures (dry matter intake, average daily gain, and feed conversion ratio).\n\nFigshare: PRISMA checklist for ‘The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis’. https://doi.org/10.6084/m9.figshare.12298034.v213.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBen Y, Fu C, Hu M, et al.: Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: A review. Environ Res. 2019; 169: 483–493. PubMed Abstract \| Publisher Full Text\n\nHe Y, Yuan Q, Mathieu J, et al.: Antibiotic resistance genes from livestock waste: occurrence, dissemination, and treatment. npj Clean Water. 2020; 3(1): 4. Publisher Full Text\n\nOh J, Hristov AN: Effects of plant-derived bio-active compounds on rumen fermentation, nutrient utilization, immune response, and productivity of ruminant animals. ACS Symp Ser. 2016; 1218: 167–186. Publisher Full Text\n\nDhanasekaran DK, Dias-Silva TP, Filho ALA, et al.: Plants extract and bioactive compounds on rumen methanogenesis. Agrofor Syst. 2019; 6. Publisher Full Text\n\nLin B, Lu Y, Salem AZM, et al.: Effects of essential oil combinations on sheep ruminal fermentation and digestibility of a diet with fumarate included. Anim Feed Sci Technol. 2013; 184(1–4): 24–32. Publisher Full Text\n\nPoudel P, Froehlich K, Casper DP, et al.: Feeding essential oils to neonatal holstein dairy calves results in increased ruminal prevotellaceae abundance and propionate concentrations. Microorganisms. 2019; 7(5): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaves AV, Stanford K, Dugan MER, et al.: Effects of cinnamaldehyde, garlic and juniper berry essential oils on rumen fermentation, blood metabolites, growth performance, and carcass characteristics of growing lambs. Livest Sci. 2008; 117(2–3): 215–224. Publisher Full Text\n\nLei Z, Zhang K, Li C, et al.: Dietary supplementation with Essential-oils-cobalt for improving growth performance, meat quality and skin cell capacity of goats. Sci Rep. 2018; 8(1): 11634. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalekkhahi M, Tahmasbi AM, Naserian AA, et al.: Effects of essential oils, yeast culture and malate on rumen fermentation, blood metabolites, growth performance and nutrient digestibility of Baluchi lambs fed high-concentrate diets. J Anim Physiol Anim Nutr (Berl). 2015; 99(2): 221–229. PubMed Abstract \| Publisher Full Text\n\nPukrop JR, Campbell BT, Schoonmaker JP: Effect of essential oils on performance, liver abscesses, carcass characteristics and meat quality in feedlot steers. Anim Feed Sci Technol. 2019; 257: 114296. Publisher Full Text\n\nCanbolat O, Filya I, Kamalak A: Effect of oregano oil on growth performance, rumen fermentation parameters and blood metabolites of growing lambs. Livest Res Rural Dev. 2018; 30(4): 59. Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndri F, Huda AN, Marjuki M: PRISMA checklist for ‘The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis’. 2020. http://www.doi.org/10.6084/m9.figshare.12298034.v2\n\nGreig JD, Waddell L, Wilhelm B, et al.: The efficacy of interventions applied during primary processing on contamination of beef carcasses with Escherichia coli: A systematic review-meta-analysis of the published research. Food Control. 2012; 27(2): 385–397. Publisher Full Text\n\nHiggins JPT, Li T, Deeks JJ: Choosing effect measures and computing estimates of effect. In: Cochrane Handbook for Systematic Reviews of Interventions. 2019; 143–176. Publisher Full Text\n\nHedges LV, Olkin I: Statistical Methods for Meta-Analysis. San Diego, CA USA: Academic Press; 1985. Reference Source\n\nHiggins JPT, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med. 2002; 21(11): 1539–1558. PubMed Abstract \| Publisher Full Text\n\nBegg CB, Mazumdar M: Operating characteristics of a rank correlation test for publication bias. Biometrics. 1994; 50(4): 1088–1101. PubMed Abstract \| Publisher Full Text\n\nEgger M, Smith GD, Schneider M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997; 315(7109): 629–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuval S, Tweedie R: A nonparametric “trim and fill” method of accounting for publication bias in meta-analysis. J Am Stat Assoc. 2000; 95(449): 89–98. Publisher Full Text\n\nSuurmond R, van Rhee H, Hak T: Introduction, comparison, and validation of Meta-Essentials: A free and simple tool for meta-analysis. Res Synth Methods. 2017; 8(4): 537–553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanbolat Ö, Karabulut A: Effect of urea and oregano oil supplementation on growth performance and carcass characteristics of lamb fed diets containing different amounts of energy and protein. Turk J Vet Anim Sci. 2010; 34(2): 119–128. Publisher Full Text\n\nGümüş R, Erol HS, İmik H, et al.: The effects of the supplementation of lamb rations with oregano essential oil on the performance, some blood parameters and antioxidant metabolism in meat and liver tissues. Kafkas Univ Vet Fak Derg. 2017; 23(3): 395–401. Publisher Full Text\n\nAydin ÖD, Merhan O, Yildiz G, et al.: The effect of oregano oil (Origanum vulgare) on the fattening performance and blood oxidant-antioxidant balance in post-weaned tuj lambs. Kafkas Univ Vet Fak Derg. 2020; 26(1): 59–64. Publisher Full Text\n\nBaytok E, Kara K, Aksu T, et al.: The effect of Mediterranean thyme (Thymbra spicata L. var. spicata) essential oil on fattening performance and ruminal parameters in lamb. J Anim Feed Sci. 2017; 26(4): 319–325. Publisher Full Text\n\nRibeiro ADB, Ferraz Junior MVC, Polizel DM, et al.: Thyme essential oil for sheep: Effect on rumen fermentation, nutrient digestibility, nitrogen metabolism, and growth. Arq Bras Med Vet e Zootec. 2019; 71(6): 2065–2074. Publisher Full Text\n\nParvar R, Ghoorchi T, Kashfi H, et al.: Effect of Ferulago angulata (Chavil) essential oil supplementation on lamb growth performance and meat quality characteristics. Small Rumin Res. 2018; 167: 48–54. Publisher Full Text\n\nYesilbag D, Biricik H, Cetin I, et al.: Effects of juniper essential oil on growth performance, some rumen protozoa, rumen fermentation and antioxidant blood enzyme parameters of growing Saanen kids. J Anim Physiol Anim Nutr (Berl). 2017; 101(5): e67–76. Publisher Full Text\n\nÖzdoǧan M, Önenç SS, Önenç A: Fattening performance, blood parameters and slaughter traits of Karya lambs consuming blend of essential oil compounds. African J Biotechnol. 2011; 10(34): 6663–6669. Publisher Full Text\n\nAndri F, Huda AN, Marjuki M: Extended data for ‘The use of essential oils as a growth promoter for small ruminants: a systematic review and meta-analysis’. 2020. http://www.doi.org/10.6084/m9.figshare.12298913.v3\n\nPatra AK, Yu Z: Effects of essential oils on methane production and fermentation by, and abundance and, diversity of rumen microbial populations. Appl Environ Microbiol. 2012; 78(12): 4271–4280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoltan YA, Natel AS, Araujo RC, et al.: Progressive adaptation of sheep to a microencapsulated blend of essential oils: Ruminal fermentation, methane emission, nutrient digestibility, and microbial protein synthesis. Anim Feed Sci Technol. 2018; 237: 8–18. Publisher Full Text\n\nSylvester JT, Karnati SKR, Yu Z, et al.: Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen. J Dairy Sci. 2005; 88(6): 2083–2095. Publisher Full Text\n\nNewbold CJ, De la Fuente G, Belanche A, et al.: The role of ciliate protozoa in the rumen. Front Microbiol. 2015; 6: 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWanapat M, Cherdthong A, Phesatcha K, et al.: Dietary sources and their effects on animal production and environmental sustainability. Anim Nutr. 2015; 1(3): 96–103. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "64169", "date": "17 Jun 2020", "name": "C.J. Linde du Toit", "expertise": [ "Reviewer Expertise Small ruminant nutrition and livestock GHG emissions" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article the researchers conducted a meta analysis on the effects of essential oils on production parameters of small ruminants.\n\nThis is a well written article. The objective and methods employed are suitable and clearly defined within the text. The authors could have included the experimental design in the selection criteria as well s the type of rations used in the various studies. The researchers could have broadened the criteria to include more studies in the Meta analysis. Less than 20 studies were included but the authors did discuss this and the need for further research in their discussion. The quality of the article is acceptable for indexing.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5842", "date": "28 Aug 2020", "name": "Faizal Andri", "role": "Author Response", "response": "We are grateful to the reviewer for his valuable comments and suggestions. Please see our responses and changes as detailed below. 1. The authors could have included the experimental design in the selection criteria as well as the type of rations used in the various studies. The use of experimental design and type of ration as selection criteria will left only a small number of eligible studies for synthesis, therefore we do not consider these elements as selection criteria. However, we showed these information as additional study characteristics (see Table 3). 2. The researchers could have broadened the criteria to include more studies in the Meta-analysis. Less than 20 studies were included but the authors did discuss this and the need for further research in their discussion. The studies in this aspect is currently still very limited and we have addressed this issue (see Discussion section)." } ] }, { "id": "64168", "date": "01 Jul 2020", "name": "Liang Chou Hsia", "expertise": [ "Reviewer Expertise Agricultural production", "Aquaculture production", "Ecologic system", "Conservation", "Rural education and extension", "Reproduction of animal (ex AI for animals)", "Nutrition on poultry", "pigs", "cattle", "sheep", "goat", "dog and cat", "etc.", "Management for animal production", "Animal house design and arrangement", "Animal behavior and welfare", "Feed processing", "Animal waste management", "Extension education." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 purpose of this paper tries to investigate whether essential oils have any positive or negative effects on growth performance, such as dry matter intake (DMI), average daily gain (ADG), and feed conversion ratio (FCR). The results shown that the essential oils provided for sheep and goats in 12 studies. Most experiments shown the essential oils can improve the above performance. The present study used the Begg's and Egger's tests which were meta-analysis procedures. Please indicate the original words and then can put abbreviations. It is not necessary to point out the authors' duties in study selection and data extraction. The authors need to make clarification from the literature on why the essential oils can be used as a growth promoter for small ruminants.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5843", "date": "28 Aug 2020", "name": "Faizal Andri", "role": "Author Response", "response": "We would like to thank the reviewer for his helpful comments and suggestions. Please see our responses and changes as detailed below. 1. Please indicate the original words and then can put abbreviations. We have made sure that the original words have indicated before using abbreviations. 2. It is not necessary to point out the authors' duties in study selection and data extraction. We have deleted the statement regarding the authors’ duties (see Study Selection and Data Extraction sections). 3. The authors need to make clarification from the literature on why the essential oils can be used as a growth promoter for small ruminants. We have included the clarification about why essential oils can be used as a growth promoter for small ruminants (see Discussion section)." } ] } ]	1	https://f1000research.com/articles/9-486
https://f1000research.com/articles/9-446/v1	26 May 20	{ "type": "Research Article", "title": "Association between categorization of emotionally-charged and neutral visual scenes and parameters of event-related potentials in carriers of COMT, HTR2A, BDNF gene polymorphisms", "authors": [ "Elena V. Vorobyeva", "Pavel N. Ermakov", "Evgenij F. Borokhovski", "Ekaterina M. Kovsh", "Alexander S. Stoletniy", "Pavel N. Ermakov", "Evgenij F. Borokhovski", "Ekaterina M. Kovsh" ], "abstract": "Background: This study aimed to discover the association between parameters of event-related potentials (ERPs) and categorization of images of visual scenes, both emotionally-charged and neutral, in carriers of polymorphisms of the COMT, HTR2A, BDNF genes. Methods: Electroencephalogram (EEG) and ERPs were recorded at 128 leads, with two ear referents. Images of different visual scenes were presented to the study participants sequentially on a monitor screen. The participants’ task was to examine these images and indicate what emotions (negative, neutral or positive) they elicit. Comparison of event-related potentials was carried out using unpaired Student t-test in EEGLAB toolbox. Results: COMT. A stronger reaction, as reflected in the amplitude of the ERPs, in participants with the recessive homozygous Met/Met genotype was observed on latency around 200 ms to the stimuli, assessed as positive. Carriers of dominant homozygous Val/Val genotype had higher amplitude of 200 ms peak when assessed scene images as either neutral or negative in comparison to other genotypes. Participant with the Val/Met heterozygous genotype had higher amplitude of ERP that Met/Met group on same latency when assessed stimuli as negative. HTR2A. Significant increase in negativity in the parietal-occipital regions revealed in the range of 350-420 ms in participants with the recessive homozygous A/A genotype when choosing any type of assessment, compared to carriers of the heterozygous genotype A/G and the dominant homozygous G/G genotype. BDNF. Participants with Val/Val genotype categorized the visual images more thoroughly, as reflected in greater activation of the parietal-occipital zones and higher amplitude on ERP peak on 190 ms (negative assessment) and 160 ms (neutral assessment) then Val/Met carriers. Conclusions: The COMT, HTR2A, BDNF gene polymorphisms are associated with the process of categorizing emotionally charged and neutral visual scenes, and this relationship is reflected in the ERP parameters.", "keywords": [ "Categorization of emotionally-charged and neutral visual scenes", "parameters of event-related potentials", "COMT", "HTR2A", "BDNF genes polymorphisms." ], "content": "Introduction\n\nStudying hereditary factors that determine specifics of the categorization of visual images are very important for opening up new opportunities for individualizing educational experiences, i.e. those that could take into account individual visual characteristics of students. Specifically, emotions that actualize these visual images can play an essential role in the process of categorizing visual scenes. As it was shown in our previous work, particular features of image categorization may vary dependent on genetic differences among carriers at the level of genes that control the functioning of brain neurotransmitter systems (Ermakov et al., 2017; Kovsh et al., 2018) .\n\nFrom an evolutionary standpoint, emotions arose to serve as quick adaptive responses to the environment to increase chances of survival (Nesse, 1990). Modern psychophysiology entertains the idea that genetic changes that affect neurotransmitter pathways can also influence the manifestations of emotions (Alfimova et al., 2019).\n\nIn this work, we studied specific parameters of event-related potentials (ERPs) and categorization of images of visual scenes, both emotionally-charged and neutral, in carriers of polymorphisms of COMT, HTR2A, BDNF genes.\n\nThe catechol-O-methyltransferase (COMT) gene encodes the production of the catechol-O-methyltransferase enzyme that regulates the inactivation of catecholamines (dopamine, norepinephrine, adrenaline). The COMT enzyme is involved in the metabolic degradation of the dopamine neurotransmitter in brain regions such as the prefrontal cortex, amygdala and striatum. Research have shown that the COMT gene is associated with the activity of the brain stem, amygdala, basal ganglia and prefrontal regions, and also is involved in the modulation of neural substrate structures responsible for processing negative and positive emotions (Williams et al., 2010), motivation (Åberg et al., 2011), recognition of negative emotions (Gohier et al., 2014), and is associated with aggressiveness (van Dongen et al., 2018). There is a connection between COMT gene and the thickness of the cerebral cortex in associative areas of the brain (prefrontal, parietal, and cingulate), in which dopamine content is typically high (Williams et al., 2010). As a result, carriers of the Met allele have a thicker cortical layer in these particular areas (Miranda et al., 2019).\n\nOur work also included genotyping of study participants on the serotonin 5-HTR2A receptor gene and the brain neurotrophic factor gene (BDNF). The genotypes for these genes (as well as for the COMT gene) could be represented in a homozygous dominant form, homozygous recessive and heterozygous forms. These genotypes are associated with different sensitivity of neuron receptors to the presence of neurotransmitters in the synaptic cleft; in the case of the HTR2A gene, the level of survival of dopaminergic neurons, while in the case of the BDNF gene, the nutrition of serotonergic neurons (Ermakov et al., 2017).\n\nThe serotonin receptor gene HTR2A is located in the 13ql4 – q21 region of the thirteenth chromosome that has two introns and three exons. It is the main excitatory G-protein-coupled serotonin receptor, and may also have an inhibitory effect on the visual and orbitofrontal cortex. Serotonin receptors encoded by the 5HTR2A gene are found in large numbers in the hippocampus and in the anterior cerebral cortex, i.e. in structures closely related to emotional processes. In the 5HTR2A gene, a 1438A/G polymorphism located in the promoter region is possible, which causes a change in the functional activity of the receptor depending on the presence of A or G alleles. The serotonin system is actively involved in the processes of cognitive and emotional control. It was also shown that the HTR2A serotonin receptor gene is involved in the emotional assessment of visual stimuli (Mills et al., 2016).\n\nBDNF is seen as a common neurotrophin in the brain that has an activating effect on neuronal differentiation and synaptic neuroplasticity, and affects neuron survival in adulthood (Chen et al., 2004; Pearson-Fuhrhop et al., 2009). Neurotrophins are combinations of growth factors in the brain that play a key role in neuronal plasticity. The BDNF gene is located on the short arm of chromosome 11 and contains Val66Met single nucleotide polymorphism (SNP), in which guanine can be replaced by adenine (G196A). Such a substitution causes the amino acid valine to be replaced by methionine in codon 66, and the alleles of this gene are called Val and Met. The Met allele interferes with the intracellular traffic of BDNF, reducing the secretion of neurotrophic brain factor, which is associated with the transition from plasticity to stability in neural networks (Egan et al., 2003). The Met allele affects the transport of neuropeptides within the cell and reduces depolarization-dependent secretion of BDNF (Alfimova et al., 2009).\n\nOur previous work confirmed the hypothesis that there is specific induced electrical activity in the brain associated with the analyses of emotiogenic images in individuals with reduced (Val/Met genotype of the brain neurotrophic factor BDNF) and high (Val/Val genotype of the brain neurotrophic factor BDNF gene) cortical plasticity, as well as in individuals with different genotypes for the HTR2A serotonin receptor gene (Ermakov et al., 2017).\n\nThe aim of this work is to study parameters of ERPs while categorizing images of neutral and emotionally-charged visual scenes in carriers of polymorphisms of COMT, HTR2A, BDNF genes.\n\n\nMethods\n\nThe use of experimental subjects is in accordance with ethical guidelines as outlined in the Declaration of Helsinki. In addition, the design of the experiment, the methodological approach, the conditions of confidentiality and use of oral consent for the subjects was performed according to the Code of Ethics of Southern Federal University (SFU; Rostov-on-Don, Russia) and approved ethically by the Academic Council of the Academy of Psychology and Pedagogy of SFU, on 22 February, 2017. Before the start, each participant was informed about the goal of the research, procedure, experimental conditions, and safety of their personal data. Oral consent was obtained from each participant before continuing with the study.\n\nThis study involved 73 participants, of both sexes, aged between 18 and 28 years. The study was conducted from March 24, 2017 to May 25, 2017 at the Laboratory of Psychophysiology and Experimental Psychology, Department of Psychophysiology and Clinical Psychology, Academy of Psychology and Education Sciences, Southern Federal University (Rostov-on-Don, Russia). The participants were students of Rostov universities, including the Southern Federal University (SFU). SFU students received bonus points in their academic disciplines for their participation in the study.\n\nDNA analysis was carried out as follows: after two hours of dry hunger, buccal epithelium was collected from the inner surface of the participant’s cheeks by two sterile cotton probes. Then, probes with biological material were immersed in a transport medium and sent to the «Биологические решения и технологии» (Moscow, Russia), where DNA was extracted from clinical material using the AmpliPrim DNA-sorb-AM reagent kit (ООО “НекстБио”, Russia). Real-time polymerase chain reaction (PCR) genotyping with the detection of various alleles by TaqMan type fluorescent probes was performed using the «Терцик» programmable thermostat (ДНК-Технология, Russia) using SNP-express reagents (Литех, Russia). GelDoc UV-transilluminator (Bio-Rad, USA) was used to visualize the results. For analysis, we used genomic DNA preparations obtained from biological samples using DNA extraction kits with the removal of PCR inhibitors and the presence of at least 100 genomic copies in 1 μl. During the genetic examination, the following DNA sections were analyzed:\n\n– COMT catechol-O-methyltransferase gene (Genebank sequence AY341246, mutation 23753G> A, Val158Met, rs4680 code). Possible genotypes: Val/Val, Val/Met, Met/Met;\n\n– BDNF brain neurotrophic factor gene (GenBank sequence NG_011794, mutation 68690G> A Val66Met, rs6265 code). Possible genotypes: Val/Val, Val/Met, Met/Met;\n\n– HTR2A serotonin receptor gene (GenBank sequence, NG_013011, mutation 4692G> A, rs6311 (Tr2)). Possible genotypes: G/G, G/A, A/A.\n\nGenetic analysis showed that carriers of dominant (GG), heterozygous (GA), and minor (AA) genotypes of Tr2 polymorphism of the second type of serotonin receptor gene have similar genotypes for Tr3 polymorphism (CC, TC, TT, respectively). From now on, we will use the designation of genotypes by Tr2 polymorphism (A/G), implying the presence of a carrier of a similar genotype by Tr3 polymorphism.\n\nThe genotyping resulted in the following sample composition: COMT gene, 25 participants were carriers of Val/Val genotype, 41 Val/Met and 7 Met/Met genotypes; HTR2A gene, 19 participants were carriers of A/A genotype, 29 A/G and 25 G/G; BDNF gene, 46 participants were carriers of Val/Val genotype, 26 Val/Met and 1 Met/Met.\n\nImages of different visual scenes were presented to the study participants sequentially on a monitor screen. The participants’ task was to examine these images and indicate what emotions they elicit – by pressing the corresponding key on the keyboard: “1”– negative, “2” – neutral, and “3” – positive emotions. The distance to the monitor was 100 cm. The stimulus database (Stoletniy et al., 2020b) of the experiment, contained 445 images, has already been used in our previous works (Ermakov et al., 2016; Vorobyeva et al., 2016). All stimuli were aligned by their luminance characteristics and had a resolution of 1024×768 pixels. The sequence of presentation of the images was random with the interstimulus interval ranging from 500 to 1500 ms.\n\nElectroencephalogram (EEG) recording was carried out using Neurovision-136 equipment manufactured by «МКС» (Russia), with a discretization frequency of 1000 Hz. ERPs were recorded at 128 leads, using «5–10» system, and with two ear referents.\n\nThe frequencies of emotionally different responses were processed using the REdaS Package for R, version 0.9.3., in order to calculate confidence intervals for averaged response rate (α = 0.05) (Maier, 2015). The ERPs were processed using the EEGLAB version 4.11. – an interactive Matlab/Octave toolbox for processing electrophysiological data (Delorme & Makeig, 2004). Before averaging, the recording was filtered to eliminate epochs of ERP with artifacts. For the analysis were taken pre-stimulus interval 100 ms before the response key was pressed, and the analysis era was 500 ms from the moment the response key was pressed. ERPs were averaged and analyzed according to type of responses – negative, neutral and positive. An intragroup comparison of ERPs was carried out using unpaired Student t-test (p <0.05). ERPs to emotionally different responses were compared separately for samples of carriers of different genotypes for the COMT, BDNF, and HTR2A genes, e.g. in COMT group ERP compared as Val/Val vs Val/Met vs Met/Met carriers when they chose negative, neutral or positive answer, etc. All visualization was conducted in EEGLAB.\n\n\nResults\n\nBehavioral results. The calculation of confidence intervals for assessing relative frequencies of differential responses to emotiogenic stimuli resulted in the following outcomes for participants with COMT genotypes. Participants with different genotypes of the COMT gene selected negative responses to the stimuli with approximately equal average frequency, as illustrated in Figure 1. However, the distribution of neutral and positive responses is of greater interest.\n\nParticipants with the Met/Met genotype reliably responded with the neutral reaction 10% more often than participants with Val/Met and 15% more often than Val/Val genotype carriers. Also, the same Met/Met subgroup of participants gave on average 10% and 13% fewer positive answers, respectively.\n\nERP data analysis. The analysis of ERPs in response to images of different emotional modality showed the following. When respondents viewed the presented image as emotionally negative, the amplitude of the ERP in the parietal and occipital regions of the brain (range 150 – 250 ms) in the subgroup with the Met/Met genotype was significantly lower (p <0.03, t = -2.40) than in the Val/Met subgroup (Figure 2). Comparison of ERP for the Met/Met and Val/Val subgroups when the respondents choose to evaluate presented images as emotionally negative showed a significantly higher amplitude (p <0.001; t = -0.40) for the Val/Val subgroup in the parietal-occipital leads, in the range of 180 – 220 ms. The amplitude of the ERPs in response to the negative images was also significantly higher (p <0.04; t = -0.13) for the Val/Val subgroup compared to the Val/Met subgroup in the same latency.\n\nIn addition, the topography of the potentials distribution also shows that the main differences in brain activity when evaluating a stimulus as being emotionally negative are observed mainly in the parietal-occipital regions of the cortex (Figure 3). Examples of amplitudes values of the ERPs of the Pz lead are presented in Table 1.\n\nWhen the respondents evaluated emotiogenic stimuli as neutral, the following electrophysiological picture was observed. In participants with the Met/Met genotype, the amplitude of the ERP in the parietal-occipital areas within the 150 – 230 ms timeframe was significantly lower than in the Val/Met (p <0.035, t = -0.18) and Val/Val (p <0.001 t = -3.59) subgroups of participants (Figure 4). Also, in the Val/Val genotype subgroup, the amplitude of ERPs in the same time range and region was significantly higher (p <0.07; t = -2.77) than for the Val/Met subgroup (Figure 4). Examples of the amplitude for the Pz lead are presented in Table 1.\n\nThe topography of the potentials distribution also shows that the main changes in brain activity associated with the selection of neutral responses appear in the parieto-occipital regions of the cortex, as is the case with the selection of negative responses (Figure 5). Nevertheless, the amplitude and spatial differences in the latter case show that the activity of these regions in the Met/Met and Val/Met subgroups was lower, while in the Val/Val subgroup the activity was slightly higher.\n\nWhen emotiogenic stimuli were evaluated to be positive, our analyses of the ERPs showed the following. In respondents with the Met/Met genotype of the COMT gene, the amplitude of ERP wave in the range from 199 to 210 ms was significantly higher than in the Val/Met subgroup (p <0.05, t = -0.09) and Val/Val (p <0.05 t = -0.28 ) subgroup (Figure 6). In respondents with the Val/Met genotype, the ERP amplitude was significantly higher than in respondents with Val/Val (p <0.469; t = -0.74) in the same time range and region (Figure 6). It is worth noting that in this case, the highest amplitude of the ERPs was observed in the Met/Met subgroup, and the lowest in the Val/Val subgroup, in contrast with the other two (negative and neutral) response options. Examples of the amplitude values for the POz lead are presented in Table 1.\n\nThe topography of the distribution of cortical potentials (Figure 7) demonstrates higher activity at a latency of 200 ms in the parieto-occipital leads for all subgroups as the trend shown in Figure 6 reflects. Nevertheless, in comparison selecting negative and neutral options, it is apparent that the choice of a positive response required less involvement of these brain areas.\n\nPresented below are the amplitudes of ERPs at the Pz and Poz leads that exemplify selection of different response options when evaluating stimuli by respondents with different genotypes of the COMT gene. Table 1 illustrates the amplitude values of the ERPs with respect to the zero reference line.\n\nBehavioral results. The calculation of confidence intervals for the average frequency by selection of the response option showed the following for participants with HTR2A geneotypes. Confidence intervals overlap in all figures, which indicates the absence of any significant differences between the means (Figure 8). This fact indicates that the participants in this sample selected responses of different emotional modality with approximately the same frequency, regardless of which genotype of HTR2A gene they carried.\n\nERP data analysis. Analyses of ERPs in responses to images of different emotional modality by participants with different genotypes of the HTR2A gene showed the following. When a stimulus was evaluated as negatively emotionally charged, the amplitude of ERPs in the latency from 350 to 420 ms in participants with the A/A genotype showed a significantly more powerful increase in negativity than in participants with the A/G (p <0.007, t = -2.88) and G/G (p <0.022 t = -2.42) genotypes. At the same time, the ERP in subgroups of participants with the A/G and G/G genotypes, when compared with each other, did not have any significant differences (p <0.57, t = 0.56), and their amplitudes differed very slightly (Figure 9). Examples of the amplitude values at the Oz lead are presented in Table 1.\n\nThe topography of the ERPs for the latency of 400 ms demonstrates a high and homogeneously distributed positivity in the parieto-occipital and posterior parietal regions of the brain in subgroups with the A/G and G/G genotypes compared to the A/A subgroup (Figure 10).\n\nWhen a neutral response to an emotiogenic stimulus was selected in the time range from 390 to 410 ms, negativity of responses significantly increased in participants with the A/A genotype compared to the A/G (p <0.014, t = -2.58) and G/G (p < 0.008 t = -2.81) subgroups. The ERP of the A/G and G/G subgroups remained positive, and when comparing between these two subgroups, the corresponding potentials did not show significant differences (p <0.62, t = - 0.49) (Figure 11). Examples of the amplitude values at the Oz lead are presented in Table 1.\n\nThe topography of the distribution of ERPs for the latency of 400 ms associated with a neutral response showed a picture similar to the situation of choosing a negative response, namely spatially uniform and higher positivity in the parietal-occipital and posterior temporal regions in subgroups with the genotypes A/G and G/G, compared with the increasing negativity in the A/A subgroup in the same brain regions (Figure 12).\n\nWhen the visual stimuli were evaluated as emotionally positive, the amplitude of the corresponding ERPs in the latency from 370 to 405 ms in participants with the A/A genotype showed increasing negativity, significantly different from that in subgroups with the A/G (p <0.004, t = -3 , 03) and G/G (p <0.04, t = -2.11) genotypes. Comparison between the A/G and G/G subgroups revealed no significant differences (p <0.75, t = 0.31), in terms of the amplitude of the indicated latency (Figure 13). It is useful to note ERPs of the A/G and G/G subgroups with the latency of 400 ms remained positive, but also expressed the tendency for further increase in negativity, similar to situations when participants selected responses of a different emotional valence (Figure 9 and Figure 11). Examples of the amplitude values at the Oz lead are presented in Table 1.\n\nThe topography of the distribution of ERP for the latency of 400 ms associated with positive responses showed a picture similar to the two previous situations. The parieto-occipital and posterior temporal regions showed higher positive amplitude of event-related potentials in subgroups with the A/G and G/G genotypes compared to the A/A subgroup, which had the increased negativity in these brain regions (Figure 14).\n\nPresented below are the amplitudes of ERPs at the Oz lead that exemplify selection of different response options when evaluating stimuli by respondents with different genotypes of the HTR2A gene. Table 1 illustrates the amplitude values of the ERPS with respect to the zero reference line.\n\nThe calculation of confidence intervals for assessing the frequency of responses different in their emotional valence was carried out only for the Val/Val and Val/Met subgroups of the BDND gene as only these subgroups were sufficiently represented in the sample. Participants with the indicated genotypes on average selected negative and positive responses to the presented stimuli with an approximately equal frequency, as illustrated in Figure 15A and C. At the same time, the Val/Val subgroup chose to judge presented visual stimuli as neutral rating significantly more often (57%) than the Val/Met subgroup did (Figure 15B).\n\nAnalyses of brain ERPs for the BDNF subgroup was also performed only for participants with the Val/Val and Val/Met genotypes. When the presented images were evaluated negatively emotionally charged, the amplitude of the ERPs in the parietal-occipital areas within in the 180-195 ms latency for the Val/Val subgroup was significantly higher (p <0.04, T = -2.05) than for the Val/Met subgroup (Figure 16). Table 1 contains examples of the amplitude values for the Oz lead with the latency of 190 ms.\n\nThe topography of the distribution of ERPs demonstrates a higher amplitude in the parieto-occipital regions for the Val/Val subgroup of participants. It should also be noted that in the right hemisphere, the levels of activity are slightly higher than in the left, and this is true for the participants of both subgroups (Figure 17).\n\nThe analysis of the ERPs when participants evaluated visual stimuli as emotionally neutral showed significant differences in the latency of 130 – 180 ms from the stimulus onset (Figure 18). The amplitude of the ERPs in the subgroup with the Val/Val genotype of the BDNF gene in this case was statistically significantly greater (p <0.005, T = –2.89) than for the subgroup with the Val/Met genotype (the amplitudes for the Oz lead are shown in Table 1). The range of significant differences is somewhat shifted towards its earlier latency in comparison with the situations when participants were selecting a negative response.\n\nThe topography of the distribution of cortical potentials clearly indicates their significantly higher amplitude in the parieto-occipital regions of the cortex for the subgroup with the Val/Val genotype than the subgroup with the Val/Met genotype of the BDNF gene at the latency of 160 ms (Figure 19).\n\nComparison of the ERPs in response to stimuli perceived as emotionally positive by participants with different genotypes of the BDNF gene did not reveal any statistically significant differences (p> 0.05, T = -1.15) (Figure 20). Examples of the amplitude values at the Oz lead with the latency of 200 ms are presented in Table 1.\n\nThe topography of the ERPs distribution in response to the stimuli evaluated to be emotionally positive shows an even activity of the parieto-occipital regions for 200 ms from the stimuli onset, regardless of the genotype of the BDNF gene (Figure 21).\n\nPresented below are the amplitudes of ERPs at the Oz lead that exemplify selection of different response options when evaluating stimuli by respondents with different genotypes of the BDNF gene. Table 1 illustrates the amplitude values of the ERPs with respect to the zero reference line.\n\n\nDiscussion\n\nA functional polymorphism of the catechol-O-methyltransferase gene (COMT), Val158Met, is involved in the catecholamine system associated with the perception of emotions. Dopamine in areas, such as the prefrontal cortex, tonsil and striatum, modulates brain activity in response to aversive stimuli and regulates the processing of emotions. For example, it was found that the COMT Val158Met gene genotype is associated with the success of the detection of emotions, and subsequent perception of the emotions’ valence (Tamm et al., 2016).\n\nOur work demonstrates that, when asked to assess emotional valence of visual stimuli presented to them, participants with the Met/Met genotype of the COMT gene more often perceived these stimuli as neutral. At the same time, participants with the Val/Val genotype of the same gene evaluated these stimuli as emotionally positive with a higher frequency. Yet, we found no statistically significant difference between the subgroups with different genotypes of the COMT gene in their evaluation of the presented stimuli as emotionally negative.\n\nThere is a possibility that the assessment of the stimuli as negative by the study participants may be accompanied by a fear-type reaction. It is known that individual differences in dopaminergic genotypes of the COMT gene can affect the initial fear reactions (Panitz et al., 2018). Another study also found lower rates in respondents with the Val/Val expression of Val158Met COMT gene compared with carriers of the Met allele in fear recognition (Heller et al., 2018).\n\nBy means of the analysis of the ERPs recorded during the evaluation of the emotional valence of visually presented stimuli, it was found that there are statistically significant differences between carriers of different genotypes of the COMT gene for components of the ERPs in the parietal and occipital regions in the range from 150 to 250 ms. These results could be indicative of the presence of more intense processing of visual stimuli by the carriers of the Val/Val and Val/Met genotypes of the COMT gene in response to the stimuli perceived as neutral or negative, and by the Met/Met genotype carriers of the COMT gene in response to emotionally positive stimuli.\n\n(Storozheva et al., 2019), during studying auditory ERPs, found that the responses with the highest amplitude were given by people with the Val/Val genotype of the COMT gene, which is largely consistent with our data.\n\nMoreover, in this work, we did not obtain statistically reliable data on the differences between the genotype subgroups of the COMT gene for the late components of ERPs. However, such data do exist in the periodicals. For instance, (McLoughlin et al., 2018) found that people with the Met/Met genotype of the COMT gene demonstrated the best task performance during the registration of event-related brain potentials, and the homozygous Met/Met genotype of the COMT gene was associated with a smaller frontal source of P3. The latter indicates that, although having more dopamine in the frontal areas of the cortex has advantages in some tasks, it can also jeopardize the reactive inhibition function.\n\nAlso, (Shalev et al., 2019) showed the presence of significant relationships between the COMT genotype and the effective threshold for visual perception, and found that insufficient or excessive catecholaminergic activity can have equally harmful effects on ability to maintain stable attention focus.\n\nThe receptor for 5-hydroxytripamine (serotonin) 2A (5-HTR2A) is a key receptor involved in monoaminergic regulation of the basic biological functions of the body and of human behavior. A polymorphic version of the HTR2A rs6313 gene (102 T> C), potentially associated with the distortion of the effectiveness of post-transcriptional processes, is considered in the medical literature to be a risk factor for cognitive pathologies. A polymorphism rs6311, located in close proximity to the promoter region of the gene, is associated with changes in the expression of the HTR2A gene (Zabotina et al., 2018). It is assumed that anomalies in emotional regulation may result from dysfunctional serotonergic regulation of the limbic and prefrontal areas, especially of the amygdala, anterior cingulate gyrus, and the prefrontal cortex. Serotonergic genes are involved in controlling recognition of emotions (Piel et al., 2018).\n\nThe MRI data obtained in the work of (Matsunaga et al., 2017), indicates that the polymorphisms of the HTR2A gene are associated with individual differences in the manifestation of empathy and in experiencing happiness. Brain structures responsible for providing a mental model of empathizing with other people, include the medial prefrontal cortex, as well as the temporoparietal region. It was also found that participants with the dominant homozygous and heterozygous GG genotypes of the HTR2A gene experienced more pronounced feelings of happiness and greater activation of a part of the mentalization network, than people with the minor homozygous AA genotype.\n\n(Gonzalez et al., 2019) showed that the genotype of the HTR2A gene correlates with the manifestation of emotions of anger in a sample of adult males.\n\nIn our present work, when participants evaluated emotional valence presented to them via visual stimuli, there was no statistically significant difference in the frequency of assessing these stimuli as neutral, positive or negative in those with different genotypes of the HTR2A gene (A/A, A/G and G/G).\n\nAnalysis of the ERPs recorded while assessing emotional valence of presented visual stimuli found that in participants with the A/A genotype of the HTR2A gene, there was a statistically significant increase in negativity in the parieto-occipital regions (the time range of 350–420 ms) in comparison with participants who carried genotypes A/G and G/G of the HTR2A gene. This latter finding can be interpreted in support of the fact that decision-making in evaluating visual stimuli in participants with the A/A genotype of the HTR2A gene occurs more intensively (compared with subjects with the A/G and G/G genotypes), regardless of what response regarding emotional valence of a particular stimulus was selected by the participants.\n\nExisting studies that feature data from the post-mortem analysis of the brain of mentally healthy people found that carriers of different alleles of the T102C polymorphic marker differ in the amount of 5HTR2A mRNA and synthesized on its basis protein product, where the carriers of the A/A genotype showed a lower level of the gene expression (Polesskaya & Sokolov, 2002).\n\nThe interactions between genes and the environment lead to changes in the brain, depending on emotions- and behavior-transforming experiences. A positive interaction with the environment through new experiences and physical activity can engage the neuroplasticity mechanisms to improve brain functioning. Adult neurogenesis and brain neurotrophic factor (BDNF) serve as mediators of neuroplasticity (Rogers et al., 2019).\n\nIn our work, when the study participants evaluated emotional valence of visually presented stimuli, those with the Val/Val genotype of the BDNF gene choose the neutral response significantly more often than the participants with the Val/Met genotype did.\n\nOur analysis of the ERPs, recorded while the participants were assessing the emotional valence of visually presented stimuli, found that when negative responses were given, the amplitude of the ERPs in the parieto-occipital regions of the Val/Val subgroup was significantly higher than that of the Val/Met subgroup in the latency of 180–195 ms. Similarly, when the participants evaluated stimuli as being emotionally neutral, the amplitude of the ERPs in the range of 130–180 ms in the parieto-occipital regions was statistically significantly higher in the subgroup with the Val/Val genotype of the BDNF gene than it was in the subgroup with the Val/Met genotype. No statistically significant differences between these genotypes of the BDNF gene were detected in response to the stimuli perceived by the participants as emotionally positive.\n\nThe obtained data can be interpreted as indicating that individuals with high cortical plasticity (i.e., with the Val/Val genotype of the BDNF gene), more carefully process the details of the visual stimuli, which manifests itself as an increase in activation of the parietal-occipital zones (with a statistically significantly higher peak amplitude at 190 ms of the induced activity), as it is reflected in their responses to the images estimated to be emotionally negative or neutral.\n\nOther authors using the method of recording event-related brain potentials showed that the carriers of the Met allele of the BDNF gene have lower electrophysiological indicators of attention compared to the carriers of the Val allele, which is manifested in a decrease in the amplitude and increase in the latent period of the P300 component (Getzmann et al., 2013; Schofield et al., 2009).\n\nThe Met allele of the Val66Met polymorphism of the BDNF gene is associated with the reduced levels of functioning of the amygdala and hippocampus. It also is implicated in manifestations of depression and post-traumatic stress disorder, with episodic memory deficit. There is a study that analyzed ERPs in response to recognition of emotionally negative, neutral and positive words. In it, a reduced late component P300 was observed in Met carriers compared with the carriers of the Val homozygotes in response to recognizing negatively- and positively-colored words, which could indicate that conscious experience of emotional recollection may differ depending on the BDNF Val66Met genotype (Jones et al., 2019).\n\n\nConclusions\n\nAs this experimental study shows, when assessing emotional valence of visually presented stimuli, participants with the recessive homozygous Met/Met genotype of the COMT gene at a behavioral level more often perceived presented images as emotionally neutral. In contrast, participants with the dominant Val/Val homozygous genotype of the COMT gene were more likely to evaluate the visual stimuli as emotionally positive. No statistically significant differences between carriers of different genotypes of the COMT gene were observed in response to the stimuli perceived as emotionally negative.\n\nA stronger reaction, as reflected in the amplitude of the ERPs, in participants with the recessive homozygous Met/Met genotype of the COMT gene was observed to the stimuli, assessed as positive (in the parietal and occipital regions in the time range from 150 to 250 ms). In those with the dominant homozygous Val/Val genotype of the COMT gene, a higher amplitude of the ERPs was observed for the stimuli assessed as either neutral or negative (in the parietal and occipital regions in the time range from 150 to 250 ms). The same tendency, but to a lesser extent, was detected for the study participants with the Val/Met heterozygous genotype of the COMT gene.\n\nThere were no statistically significant differences in the frequency of evaluating visually presented stimuli as either emotionally neutral, positive, or negative, as reflected in responses of the participants with different genotypes of the HTR2A gene (A/A, A/G, and G/G).\n\nAnalysis of the ERPs associated with participants’ evaluation of presented visual stimuli as emotionally negative, neutral, or positive revealed a statistically significant increase in negativity in the parietal-occipital regions in the range of 350–420 ms in participants with the recessive homozygous A/A genotype of the HTR2A gene, compared to participants with the heterozygous genotype A/G and the dominant homozygous G/G genotype of the HTR2A gene.\n\nWhen the study participants evaluated emotional valence of visually presented stimuli, those with high cortical plasticity (carriers of the Val/Val genotype of the BDND gene) chose neutral responses significantly more often than participants with reduced cortical plasticity (carriers of the Val/Met genotype).\n\nParticipants with high cortical plasticity (Val/Val genotype of the BDNF gene), processed the visual images more thoroughly, as reflected in greater activation of the parietal-occipital zones (with a statistically significantly higher peak amplitude at 190 ms of the ERP), which is shown to be typical for people with the homozygous Val/Val genotype when they evaluate emotional valence of visual information.\n\n\nData availability\n\nOpen Science Framework: EEGLAB datasets for study of event-related potentials during categorization of emotionally-charged and neutral visual scenes in carriers of polymorphisms of the COMT, HTR2A, BDNF genes, https://doi.org/10.17605/OSF.IO/84BV6 (Stoletniy et al., 2020a).\n\nOpen Science Framework: Stimul database for studying emotional assessment of emotionally-charged images, https://doi.org/10.17605/OSF.IO/ZYG54 (Stoletniy et al., 2020b).\n\nData is made available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nÅberg E, Fandiño-Losada A, Sjöholm LK, et al.: The functional Val158Met polymorphism in catechol-O-methyltransferase (COMT) is associated with depression and motivation in men from a Swedish population-based study. J Affect Disord. 2011; 129(1–3): 158–166. PubMed Abstract \| Publisher Full Text\n\nAlfimova MV, Golimbet VE, Barkhatova AN, et al.: [The role of genotype-environment interactions in the development of symptoms of anxiety and depression related to the disease burden for family]. Zh Nevrol Psikhiatr Im S S Korsakova. 2009; 109(12): 50–54. PubMed Abstract\n\nAlfimova MV, Golimbet VE, Korovaitseva GI, et al.: The Role of the Interaction between the NMDA and Dopamine Receptor Genes in Impaired Recognition of Emotional Expression in Schizophrenia. Neuroscience and Behavioral Physiology. 2019; 49(1): 153–158. Publisher Full Text\n\nChen ZY, Patel PD, Sant G, et al.: Variant Brain-Derived Neurotrophic Factor (BDNF) (Met66) Alters the Intracellular Trafficking and Activity-Dependent Secretion of Wild-Type BDNF in Neurosecretory Cells and Cortical Neurons. J Neurosci. 2004; 24(18): 4401–4411. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelorme A, Makeig S: EEGLAB: An open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods. 2004; 134(1): 9–21. PubMed Abstract \| Publisher Full Text\n\nEgan MF, Kojima M, Callicott JH, et al.: The BDNF val66met Polymorphism Affects Activity-Dependent Secretion of BDNF and Human Memory and Hippocampal Function. Cell. 2003; 112(2): 257–269. PubMed Abstract \| Publisher Full Text\n\nErmakov P, Kovsh E, Vorobyeva E: Features of induced brain activity in female carriers of various genotypes using the maoa-uVNTR marker when evaluating emotional scenes [Osobennosti vyzvannoj aktivnosti mozga devushek-nositel’nic razlichnyh genotipov po markeru MAOA -uVNTR pri ocenke jemocional’no okrashennyh scen]. Russian Psychological Journal. 2016; 13(4): 232–253. Publisher Full Text\n\nErmakov P, Vorobyeva E, Kovsh E, et al.: Features of evoked brain activity during recognition of emotional images in carriers of polymorphic variants of the BDNF and HTR2A genes [Osobennosti vyzvannoj aktivnosti mozga pri analize izobrazhenij jemociogennogo haraktera u nositelej polimorfnyh variantov genov BDNF i HTR2A]. Experimental Psychology. 2017; 10(3): 65–85. Publisher Full Text\n\nGetzmann S, Gajewski PD, Hengstler JG, et al.: BDNF Val66Met polymorphism and goal-directed behavior in healthy elderly - Evidence from auditory distraction. NeuroImage. 2013; 64: 290–298. PubMed Abstract \| Publisher Full Text\n\nGohier B, Senior C, Radua J, et al.: Genetic modulation of the response bias towards facial displays of anger and happiness. Eur Psychiatry. 2014; 29(4): 197–202. PubMed Abstract \| Publisher Full Text\n\nGonzalez I, Polvillo R, Ruiz‐Galdon M, et al.: Dysmorphic contribution of neurotransmitter and neuroendocrine system polymorphisms to subtherapeutic mood states. Brain Behav. 2019; 9(2): e01140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeller J, Mirzazade S, Romanzetti S, et al.: Impact of gender and genetics on emotion processing in Parkinson’ s disease—A multimodal study. Neuroimage Clin. 2018; 18: 305–314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones R, Craig G, Bhattacharya J: Brain-Derived Neurotrophic Factor Val66Met Polymorphism Is Associated With a Reduced ERP Component Indexing Emotional Recollection. Front Psychol. 2019; 10: 1922. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKovsh EM, Vorobeva EV, Ermakov PN: Psychological features of Russian woman with different diplotypes of HTR2A, COMT, BDNF genes. Behavior Genetics. 2018; 48(6): 484.\n\nMaier MJ: Companion package to the book R: Einführung durch angewandte Statistik R Package Version 0.9 3. 2015. Reference Source\n\nMatsunaga M, Kawamichi H, Umemura T, et al.: Neural and Genetic Correlates of the Social Sharing of Happiness. Front Neurosci. 2017; 11: 718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLoughlin G, Palmer J, Makeig S, et al.: EEG Source Imaging Indices of Cognitive Control Show Associations with Dopamine System Genes. Brain Topogr. 2018; 31(3): 392–406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMills M, Wieda O, Stoltenberg SF, et al.: Emotion moderates the association between HTR2A (rs6313) genotype and antisaccade latency. Exp Brain Res. 2016; 234(9): 2653–2665. PubMed Abstract \| Publisher Full Text\n\nMiranda M, Morici JF, Zanoni MB, et al.: Brain-Derived Neurotrophic Factor: A Key Molecule for Memory in the Healthy and the Pathological Brain. Front Cell Neurosci. 2019; 13: 363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNesse RM: Evolutionary explanations of emotions. Hum Nat. 1990; 1(3): 261–289. PubMed Abstract \| Publisher Full Text\n\nPanitz C, Sperl MFJ, Hennig J, et al.: Fearfulness, neuroticism/anxiety, and COMT Val158Met in long-term fear conditioning and extinction. Neurobiol Learn Mem. 2018; 155: 7–20. PubMed Abstract \| Publisher Full Text\n\nPearson-Fuhrhop KM, Kleim JA, Cramer SC: Brain Plasticity and Genetic Factors. Top Stroke Rehabil. 2009; 16(4): 282–299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiel JH, Lett TA, Wackerhagen C, et al.: The effect of 5-HTTLPR and a serotonergic multi-marker score on amygdala, prefrontal and anterior cingulate cortex reactivity and habituation in a large, healthy fMRI cohort. Eur Neuropsychopharmacol. 2018; 28(3): 415–427. PubMed Abstract \| Publisher Full Text\n\nPolesskaya OO, Sokolov BP: Differential expression of the “C” and “T” alleles of the 5-HT2A receptor gene in the temporal cortex of normal individuals and schizophrenics. J Neurosci Res. 2002; 67(6): 812–822. PubMed Abstract \| Publisher Full Text\n\nRogers J, Renoir T, Hannan AJ: Gene-environment interactions informing therapeutic approaches to cognitive and affective disorders. Neuropharmacology. 2019; 145(Pt A): 37–48. PubMed Abstract \| Publisher Full Text\n\nSchofield PR, Williams LM, Paul RH, et al.: Disturbances in selective information processing associated with the BDNF Val66Met polymorphism: Evidence from cognition, the P300 and fronto-hippocampal systems. Biol Psychol. 2009; 80(2): 176–188. PubMed Abstract \| Publisher Full Text\n\nShalev N, Vangkilde S, Neville MJ, et al.: Dissociable Catecholaminergic Modulation of Visual Attention: Differential Effects of Catechol-O-Methyltransferase and Dopamine Beta-Hydroxylase Genes on Visual Attention. Neuroscience. 2019; 412: 175–189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoletniy A, Kovsh E, Vorobyeva E: EEGLAB datasets for study of event-related potentials during categorization of emotionally-charged and neutral visual scenes in carriers of polymorphisms of the COMT, HTR2A, BDNF genes. 2020a. http://www.doi.org/10.17605/OSF.IO/84BV6\n\nStoletniy A, Kovsh E, Yavna D: Stimul database for studying emotional assessment of emotionally-charged images. 2020b. http://www.doi.org/10.17605/OSF.IO/ZYG54\n\nStorozheva ZI, Kirenskaya AV, Bochkarev VK, et al.: Effects of the Val158Met Polymorphism of the Catechol-O-Methyltransferase Gene on Measures of Sensory Gating in Health and Schizophrenia. Neuroscience and Behavioral Physiology. 2019; 49(5): 595–602. Publisher Full Text\n\nTamm G, Kreegipuu K, Harro J: Perception of emotion in facial stimuli: The interaction of ADRA2A and COMT genotypes, and sex. Prog Neuropsychopharmacol Biol Psychiatry. 2016; 64: 87–95. PubMed Abstract \| Publisher Full Text\n\nvan Dongen JDM, van Schaik RHN, van Fessem M, et al.: Association between the COMT Val158Met polymorphism and aggression in psychosis: Test of a moderated mediation model in a forensic inpatient sample. Psychology of Violence. 2018; 8(2): 269–276. Publisher Full Text\n\nVorobyeva E, Kovsh E, Yavna D: Visual evoked potentials elicited by culturally-specific images in women with different levels of hostility. International Journal of Psychophysiology. 2016; 108: 96. Publisher Full Text\n\nWilliams LM, Gatt JM, Grieve SM, et al.: COMT Val(108/158)Met polymorphism effects on emotional brain function and negativity bias . NeuroImage. 2010; 53(3): 918–925. PubMed Abstract \| Publisher Full Text\n\nZabotina AM, Belinskaya MA, Zhuravlev AS, et al.: The Influence of Rs6311 and Rs6313 Polymorphisms of Serotonin 2a Receptor Gene (HTR2A) on Its mRNA and Protein Levels in Peripheral Blood Leukocytes in Treatment with Antipsychotics. Cell and Tissue Biology. 2018; 12(5): 382–390. Publisher Full Text" }	[ { "id": "63932", "date": "17 Jun 2020", "name": "Sergey B. Malykh", "expertise": [ "Reviewer Expertise behavioral genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present article is devoted to the analysis of differences in ERP parameters depending on genetic differences (COMT, BDNF, and HTR2A gene polymorphisms) during facial recognition. The idea is interesting, however, some issues occur:\nIn the Introduction (P. 3, left and the right column) it is stated “… in carriers of polymorphisms of COMT, HTR2A, BDNF genes.” From a genetics point of view, this sentence is incorrect, since everyone has genetic polymorphism, the authors should rephrase this sentence.\n\nIn the Introduction (P. 3, left column) it is stated “As a result, carriers of the Met allele have a thicker cortical layer in these particular areas.” Although it is indicated later, what SNP the authors selected for the analysis, its designation (Val158Met), and functional significance have to be reported in the Introduction section. In addition, it is well documented that Val158Met alleles are codominant. Please, indicate this information also.\n\nThere is a mistake in the Introduction (P. 3, right column) “In the 5HTR2A gene, a 1438A/G polymorphism located in the promoter region is possible…”. Please report “-1438A/G”, since this SNP is located in the promoter gene region. Although, mention the data regarding on association of HTR2A allele with reduced/enhanced expression.\n\nIn Materials and Methods (P.4, left column) the authors have to transliterate or to present unique designations for the Russian phrases ≪Биологические решения и технологии≫,ООО “НекстБио”, ≪Терцик≫, ДНК-Технология, Литех, МКС.\n\nIn Materials and Methods it is indicated that the authors conducted real-time PCR using “Tertsik” DNA-analyzer; however, this PCR machine is not used for Real-time PCR. The authors should report the correct DNA Analyzer and the appropriate determination of genotyping. It remains unclear, why the authors did used GelDoc UV-transilluminator for TaqMan-based fluorescent PCR. They have to use Real-time DNA Analyzer.\n\nIn Materials and Methods Section please provide information regarding to % of women, mean age, since sex differences may modulate recognition of emotions.\n\nIn Materials and Methods Section (P. 4, left column) the authors use the term “mutation” for the studied SNPs instead of polymorphism or SNP.\n\nIn Materials and Methods Section (P. 4, left column) the authors should indicate genotype frequency of the observed genotypes.\n\nStatistical analysis should be based on the appropriate statistical criterion. Since the authors deal with quantitative data (the amplitude of the ERP), the use of unpaired Student t-test requires the testing for the normality distribution and the assumption of the homogeneity of variance. Assuming small sample size (N = 73), the normality of distribution is unlikely. Please, provide required normality of distribution testing or in the case of its failure, use non-parametric statistical tests. In addition, operations with genetic data (allele and genotype frequencies) require the check for the Hardy-Weinberg disequilibrium. Please, report this information.\n\nI suggest to include BDNF Met/Met-genotype together with Met/Val-genotype into one group (Met-allele carriers) for the statistical analysis.\n\nThe study sample is rather small (N = 73), therefore I suggest to indicate a preliminary character of conducted study and to mention this issue in Limitations.\n\nThe text has to be checked for the sequence of tenses.\nThe article can be indexed after all corrections (including appropriate statistical analysis).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5818", "date": "28 Aug 2020", "name": "Alexander Stoletniy", "role": "Author Response", "response": "Thank you for your report! Question 1-8: Corrections made in text of the article according to report. Question 9: Hardy-Weinberg equilibrium verification results were added in the \"Procedure\" section. There are table 1 with frequency distribution and text with description for it. Concerning about ERP statistics and normality distribution. Usually in articles with ERP method,such a question is not raised, since the technique itself, due to the number of trials (recomended more >25) and a subsuqent averaging, assumes a normal distribution of event-related potentials. For example, in our case the quantity of numeriacal data is very lagre (number of participants per number of electrodes per number of trials - 73 * 128 * 450.) Therefore, researchers are fairly free to use parametric methods without checking data for normality. This is why a T-test was used. However, a table of Shapiro Wilk normality test results for latencies at which differences were found are attached. Type of response Shapiro-Wilk test statistics and p-value on latency (ms) 200 300 400 Negative W = 0,97 W = 0,96 W = 0,98 p = 0,16 p = 0,07 p = 0,41 Neutral W = 0,98 W = 0,97 W = 0,97 p = 0,36 p = 0,17 p = 0,26 Positive W = 0,97 W = 0,99 W = 0,99 p = 0,11 p = 0.95 p = 0,98 As seen in example - all data in this latency for 73 participants and 3 responses type are normaly distributed (p>0.05) Question 10: Since there was only one Met\\Met genotype carriers, his\\here results were excluded from research. There is a note about this in the text, in the begging of section \"Results / BDNF\". Question 11: \"Limitations\" section was added in end of the article. Two majors limitations were described in the section, and futher direction of research was added. Question 12: Corrections made in text of the article." } ] }, { "id": "65487", "date": "06 Jul 2020", "name": "Vladimir Barabanshikov", "expertise": [ "Reviewer Expertise Psychophysiology", "experimental psychology", "general psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is devoted to the actual problem of reflection of evoked potentials in the parameters of the process of evaluating visual scenes as causing positive, negative or neutral emotions in respondents. The analysis was carried out taking into account the representation of different genotypes in the study participants for the COMT, BDNF and HTR2A genes.\nComments:\nDescribe in detail the characteristics of the study sample (average age, gender distribution).\n\nIt is desirable to describe the database of stimulus images (visual scenes) that was used in the work – what types of visual images were used. The work is characterized by novelty, relevance and from a scientific point of view is quite interesting.\n\nI recommend indexing it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5817", "date": "28 Aug 2020", "name": "Alexander Stoletniy", "role": "Author Response", "response": "Thank you for your report! 1. Correction was made in the text (see \"Participant\") 2. Correction was made in the text (see \"Procedure\" section)" } ] } ]	1	https://f1000research.com/articles/9-446
https://f1000research.com/articles/9-1059/v1	28 Aug 20	{ "type": "Research Article", "title": "Predictors of presumptive treatment of uncomplicated malaria among children in private retail outlets in Kenya: mixed effects logistic regression modelling", "authors": [ "Diana Omache", "Nelson Owuor", "Beatrice Machini", "Nelson Owuor", "Beatrice Machini" ], "abstract": "Background: The health seeking behavior in Kenya raises concerns in malaria case management at the private sector. Adherence to the national guidelines for the diagnosis, treatment and prevention of malaria is key in management of the disease. Presumptive treatment remains a major challenge in Kenya, especially in the private sector, with major gaps in literature identified on predictors of this treatment. Mixed-effects regression modelling considers county clustering, is more accurate in prediction and is more efficient and flexible. Methods: The study design was a cross-sectional, nationally representative, retail outlet survey secondary data analysis. The study populations included the health care providers in the retail outlets sampled randomly in both the rural and urban settings in Kenya. The primary outcome of interest was the proportion of health care providers who treated patients presumptively. Multivariable analysis was conducted for the significant variables, adjusting for clustering at the county level to determine the predictors of presumptive treatment. The best fitting model was examined using the Akaike Information Criterion (AIC). Results: Out of the 333 health care providers who treated patients, 190 (57%) treated patients presumptively. From the mixed effects logistic regression model, the predictors of presumptive treatment of uncomplicated malaria were case management training (AOR = 0.44; 95% CI = (0.18 – 1.09)), asked signs or symptoms (AOR = 0.19; 95% CI = (0.10 - 0.37)) and results presented (AOR = 0.08 95% CI = (0.03 - 0.19)). Conclusions: Presumptive treatment of uncomplicated malaria remains a challenge in the private retail sector. Malaria case management training and health care providers asking of signs and symptoms and results presented predicts presumptive treatment. To address the issue of presumptive treatment of Malaria, strengthening of malaria case management training is key for health care providers in the private sector.", "keywords": [ "Presumptive Treatment", "Uncomplicated Malaria among Children", "Private Sector", "Mixed Effects Models" ], "content": "Introduction\n\nMalaria is a health problem across the globe; the World Health Organization (WHO) estimates that nearly one-half (3.4 billion) of the population in the world is at risk of the disease1. Africa continues to have a disproportionately large share of the global malaria burden. According to the 2018 WHO Malaria Report, in 2017 the region reported 92% and 93% of malaria morbidity and mortality, respectively2. In 2010, Kenya adopted the Test and Treat case management policy by WHO, which is in line with the malaria treatment guidelines in Kenya3. Although the burden is high in Sub-Saharan Africa, Kenya has made major efforts to reduce and eliminate malaria. The Ministry of Health through the Division of National Malaria Programme has implemented comprehensive evidence-based strategies and policies to fight this disease. The key interventions used include the provision of mosquito nets and indoor residual spraying of households for vector control, case-management interventions & surveillance and monitoring and evaluation. Case management focuses on prompt diagnosis, effective treatment, capacity building among health workers and provision of quality diagnostics and effective medicines in all health facilities4.\n\nTo ensure universal health coverage of these interventions, recognition of the private sector’s role in malaria is clearly stated as one of the strategies of case management for the Division of National Malaria Programme. This includes ensuring access to affordable malaria medicines, training of health workers among others4. Though medical care is majorly sought in the public sector by the population, up to 25% of the population medical care is sought from the private sector5. However, there were challenges in access in the private sector primarily due to price6.\n\nThe Affordable Medicine Facility for malaria (AMFm) project, was implemented in nine countries as a pilot activity including Kenya between mid-2010 and 2012. The four major objectives of the AMFm was to increase Artemisinin-based combined therapies (ACTs) availability, affordability and use7. The AMFm went through a transition of two years, finally adapting to the private sector co-pay where grant funds were dedicated at initio to procure subsidized medicines and implement supporting interventions. The AMFm strategy has proved to be effective in the countries piloted7. Though ACTs and rapid diagnostic tests (RDTs) have been made available in the private sector in Kenya, adherence to the national guidelines for malaria treatment is key by the health care providers. Several studies have shown that children are treated presumptively especially when they have fever cases8–12.\n\nLogistic regression is a statistical method of analysis that is used when the dependent (outcome) variable is binary (dichotomous)13. In this study, the outcome variable is presumptive treatment (Yes/No), which is a binary outcome variable. Mixed effects models, on the other hand, means that the predictor variables includes both the fixed and random effects whereby the random effects adjust for cluster correlations with multilevel data14. The health care providers in this study are nested in the retail outlets which are nested in the counties. The predictor variables included the county as the random effect to adjust for inter-county correlations. This study used the application of mixed effects logistic regression modeling to predict treatment of children with uncomplicated malaria presumptively in the retail outlets.\n\nThe health seeking behavior in Kenya raises concerns in improvement in provision of health services in the private retail sector. About 25% of the population in Kenya seek medical care from the private retails sector5. The AMFm has made ACTs available in the private retail sector through co-payment done at the manufacturer level to enable public and private buyers in approved countries purchase high-quality ACTs at a fraction of the market price. Though there is access to medicines at affordable prices as well as health worker capacity building on management of malaria, adherence to the national guidelines for the treatment of malaria is a key factor to be considered by the health care providers.\n\n“Inappropriate” treatment practices (presumptive treatment) of uncomplicated malaria among children could be caused several factors. This study examined these factors using a mixed effects logistic regression model. This study will inform strengthening of interventions in malaria management in the private sector. The study will also give insight for further research on malaria in the private retail sector. This study focused on application of mixed effects logistic regression modelling in establishing the predictors of treatment of children with uncomplicated malaria presumptively in the private retail sector in Kenya.\n\n\nMethods\n\nThis study design was a secondary data analysis from a cross-sectional, nationally representative, retail outlet survey that was measuring levels in key indicators on availability of ACTs and RDTs, and dispensing practices of ACTs in accordance with the diagnosis, treatment and prevention national treatment guidelines for malaria in Kenya.\n\nThis secondary analysis proposed in this study explored the predictors of ‘inappropriate ‘treatment practices (presumptive treatment) of health care providers in the private retail outlets in Kenya.\n\nThe study populations included the health care providers who treated the patients in the retail outlets during the survey (September 2018), sampled randomly in both the rural and urban settings in Kenya.\n\nThe study included health care providers who treated patients in privately owned and predominantly dispensing retail outlets while health care providers from Public facilities, faith-based outlets and NGOs, non-functional outlets and pharmacies within a private facility were not included in this study.\n\nThis study adopted the sample size in the primary study that was determined based on the Cochran’s formula15:\n\n\n\nWhere:\n\nn = the desired sample size\n\nZ = standard normal deviate at required 95% confidence level =1.96\n\np = Variability in the proportion of patients treated in the private sector assumed to be 50%16\n\nq = 1-p\n\nd = margin of error = 0.05\n\nTo cater for non-response the sample was raised by 5%, hence the sample size was 405.\n\nThis study used the data for the health care providers who treated the patients during the primary study. In the primary study, a multistage random sampling method was used to obtain data in the retail outlets with each outlet having one health care provider surveyed. Specifically, all the 47 counties were included in the survey, but the sampling was done in two categories. In the first category, the 43 counties with less than 10 sub-counties had two sub-counties randomly sampled while in the second category, four counties with more than 10 sub-counties (namely Nairobi, Kiambu, Kakamega and Nakuru) had four sub-counties randomly sampled.\n\nIn the first stage for the first category, two sub-counties were randomly sampled. In the second stage, a rural and an urban area were randomly sampled from each sub-county. In the third stage, each of the rural/urban setting with two retail outlets was sampled: one registered and one unregistered. However, two registered retail outlets in the sub-county were selected. (If two or fewer, all were selected; if more than two, the researcher randomly selected two.) Two unregistered retail outlets in the Sub-county were selected to a maximum of two. (If two or fewer, all were sampled; if more than two, the researcher randomly sampled two.) A total of eight outlets were sampled in each of the counties for this category.\n\nIn the first stage for the second category, four sub-counties were randomly sampled. In the second stage, a rural and an urban area were randomly sampled from each sub-county (with the exception of Nairobi, where researchers went to one that is a relatively formal settlement and one that is a relatively informal settlement (slum). In the third stage, each of the rural/urban setting had two retail outlets sampled; one registered and one unregistered. However, two registered retail outlets in the sub-county were selected. (If two or fewer, all were sampled; if more than two, the researcher randomly sampled two.) Two unregistered retail outlets in the Sub-county were selected to a maximum of 2 (If 2 or less, all were sampled; if more than two, researcher randomly sampled two). A total of 16 retail outlets were sampled in each of the counties for this category.\n\nThe list of unregistered retail outlets was obtained from the county pharmacist, the drug inspectors or the public health officer. Registered retail outlets were more likely to be around the municipal areas, unlike the unregistered ones. The registered retail outlets were selected from a list from the pharmacy and Poisons Board (PPB).\n\nThis study drew the data from the primary study that was conducted in September 2018 in the sampled private retail outlets in Kenya. The primary study accessed the availability of ACTs and RDTs and the dispensing practices of the health care providers in the private retail outlets. The data was valuable in this study since presumptive treatment was identified from the primary study report and the variables to investigate the predictors of this treatment were available for this study. The data was requested from the Division of National Malaria Programme (DNMP) for secondary data analysis in this study. The variables not of interest from the primary data were dropped, leaving only the variables of interest to be used in this study. After the study conclusions for the primary study, the primary researcher had planned to store the data for a period of 5 years.\n\nDuring the primary study, each survey team consisting of two persons visited a number of facilities as assigned. For the mystery shopper to yield positive results, only one of the data collectors obtained consent and administered the survey questionnaire while the other posed as the mystery shopper. One member of the team introduced him/herself to the outlet staff and seek audience with the superintendent. The superintendent of the retail outlets was told that the DNMP was monitoring national availability of case management for malaria commodities and malaria case management practices as part of the DNMP’s Monitoring and Evaluation activities. He/she was advised on the confidentiality of the results. The data collectors were given a letter from the MoH specifying the nature and purpose of the survey.\n\nOne method was through administration of a standardized retail outlets questionnaire where each retail outlet was assessed to determine the availability of non-expired, recommended and non-recommended ACTs and other anti-malarial drugs on the day of the survey. The presence of functional malaria microscopy service and availability of RDTs was assessed on the day of the survey and retrospectively over the last three months. Finally, the availability of weighing scales, ACT guidelines, and health workers’ exposure to malaria case-management training was also established. The retail outlets assessment data was collected using a combination of methods including direct observations of antimalarials in stock and interviewing superintendent of the retail outlets.\n\nSecondly, the mystery shopper presented themselves to the retail outlets and requested for medicine based on a case scenario. After the encounter the information was recorded on a standardized tool, designed to answer key counseling and dispensing of drugs tasks performed during this retail outlets visit.\n\nThirdly, the retail outlets superintendent and assistants who attended to the patients were interviewed on key aspects of in-service training, guidelines accessibility, supervision and practice at the retail outlets. The team thanked the outlet staff and departed.\n\nThe potential predictor variables accessed comprised of: zone, malaria risk, availability of rapid diagnostic tests, availability of artemisinin-based combination therapies, artemisinin-based combination therapies (acts) price, health care provider cadre, client asked signs/symptoms, health care provider case management training, client presented results, any supervision and access to national malaria guidelines. The outcome variable was presumptive treatment of uncomplicated malaria among children. Table 1 shows the measurement methods and study variables pertinent to this article assessed using the questionnaire, while Figure 1 shows the relationship between the predictor and outcome variables.\n\nData cleaning, coding and analysis was done using STATA version 15 and R version 3.5.3 software. Exploratory data analysis was done to detect missing data, check for assumptions, and determine relationships between explanatory and outcome variables.\n\nDescriptive statistics formed the basis of analysis for the variables of interest using frequencies. Specifically, the descriptive statistics determined were proportion of health care providers who treat patients presumptively, cadre, asked about any signs/symptoms, had gone through the case management training, had any supervision, had a case management supervision and had access to the national malaria guidelines. In addition, the proportion of the private retail outlets with RDTs and ACTs available was determined as well as the zone and the malaria risk. Finally, the median for the price of ACTs was since the data was skewed.\n\nBivariate analysis was performed using a mixed effects logistic regression at 95% CI to examine the association between the outcome and predictor variables which are categorical (zone, malaria risk, RDTs availability, ACTs availability, cadre, signs/symptoms asked, case management training, results presented, any supervision, case management supervision and access to national malaria guidelines) adjusting for county effects. Bivariate analysis is the analysis of two variables or an analysis that attributes to a two-way classification.\n\nMultivariable logistic regression modelling was conducted to obtain the adjusted odds ratio, p-values, 95% CI and standard errors. The random effects were included to adjust for clustering at the county level to obtain adjusted odds ratios. The accuracy of the model was examined using the Akaike Information Criterion and the predictor variables for the best fitting model was used to predict the outcome of interest. Data were presented in tables clearly indicating the odds ratios, p-values, confidence intervals and standard errors.\n\nEthical approval for this study was obtained from the Kenyatta National Hospital/University of Nairobi – Ethics and Research Committee (KNH/UON/ ERC) – P457/06/2019. Permission to use the data for this study was obtained from the Division of National Malaria Programme (DNMP). During the primary study, an informed written consent was obtained from the respondents before they participated in the study.\n\n\nResults\n\nThe proportion of health care providers who treated patients presumptively was the first objective of this study. Out of the 333 health care providers who were interviewed, slightly more than a half, 190 (57%) treated patients presumptively (Table 2). Less than one-third, 70 (21%) and 44 (13%) had access to malaria guidelines and attended case management training, respectively. A majority (163, 47%) of the health care providers were pharmaceutical technologists. During treatment, 170 (51%) of the health care providers asked the signs and symptoms of the patients while 112 (34%) of the patients presented results.\n\nIn terms of the retail outlet characteristics, out of the 333 outlets where the health care providers were interviewed, almost all, 322 (97%) had availability of ACTs. Only 77 (23%) of the retail outlets had availability of RDTs.\n\nThe distribution of the retail outlet zone was almost equal, where 159 (48%) and 174 (52%) were located in the rural and urban areas, respectively. Finally, slightly more than one-third, 106 (32%) of the health workers accessed were from the high malaria risk area.\n\nThe de-identified raw dataset is available as Underlying data17,18.\n\nThe outcome variable of interest was presumptive treatment (Yes/No). The potential predictor variables were zone, RDT availability, ACT availability, drug price, cadre, health care provider asked signs or symptoms, case management training, results presented, any supervision, access to national malaria guidelines and malaria risk.\n\nOut of the 10 variables, a health care provider who asked signs or symptoms, results were presented and had access to the national malaria case management guidelines were significantly associated with presumptive treatment of malaria at 95% CI adjusting for county effects.\n\nSpecifically, health care providers who asked signs or symptoms from the patient were negatively associated with presumptive treatment compared to those who did not (OR = 0.24; P<0.001, 95% CI = (0.13 - 0.45). In addition, health care providers who were presented with results from the patients were negatively associated with presumptive treatment compared to those who were not presented with results (OR = 0.08; P<0.001, 95% CI = (0.03 - 0.20). Finally, health care providers who had access to national malaria case management guidelines were negatively associated with presumptive treatment compared to those who have not (OR = 0.49; P-value = 0.038, 95% CI = (0.25 - 0.96).\n\nThere was not sufficient statistical evidence to indicate that there was a significant association between the other potential predictor variable and presumptive treatment of uncomplicated malaria among the health care providers. Table 3 shows the results of the factors associated with presumptive treatment of uncomplicated malaria among children in the retail outlets.\n\np<0.05.\n\nA mixed effects logistic regression analyses was conducted and a liberal p-value of <0.2 was used to select the potential variables that were used to predict presumptive treatment of uncomplicated malaria19. The mixed effects logistic regression found that a health care provider who asked patients signs or symptoms and results were presented were predictors that were statistically significant at 95% CI. The county variance was 0.99, which indicated that there was evidence for variance of the different counties. A health care provider who asked the patient any signs or symptoms was 80% less likely to treat the patient presumptively to those who did not adjusting for the variability in the counties (AOR = 0.20; P<0.001, 95% CI = (0.10 - 0.39). In addition, health care providers who had results presented by the patient were 91% less likely to treat a patient presumptively compared to one the one whom he results were not presented adjusting for county effects (AOR = 0.09; P-value = <0.001, 95% CI = (0.04 - 0.20). Table 4 shows the results for the mixed effects logistics regression analysis.\n\np<0.05.\n\nThe model with the best fit based on the Akaike Information Criterion (AIC) was the one with health care provider who has undergone a malaria case management training, who asked the patient for the signs and symptoms and for whom results were presented (Table 5). The built model will therefore be:\n\nThe smaller the AIC value, the better the model.\n\nAIC, Akaike Information Criterion.\n\nLogit [pj] = 2.24 – 1.67 X1 - 0.83X2 - 2.49X3 + u j; where u j ~N (0, δg 2)\n\nThe probability of treating a patient presumptively = logit -1 (β0 + β1 X1 + β2X2 + β3X3+ u j)\n\nWhere u j = 1.96δg\n\nTherefore, the probability (Treating a patient presumptively) of any hypothetical county = logit -1 (2.24 - 1.67X1- 0.83 X2 - 2.49 X3 + 1.96 (1.013)\n\nConsidering the different value of the predictor variables i.e. Yes = 1 or No = 0, Table 6 below illustrates the different probabilities. The mixed effects logistic regression for the best-fitted model was conducted (Table 6).\n\nFinally, the probability of a health care provider treating a patient presumptively if the patient presented results, were asked about signs and symptoms and the health care provider has been trained was 0.32. However, when the health care provider has not gone for a case management training but the results were presented and the health care provider asked for the signs and symptoms, the probability of treating a patient presumptively is 0.85 (Table 7).\n\n\nDiscussion\n\nThe proportion of health care providers who treat uncomplicated malaria presumptively was 57% out of the 333 health care providers. A study conducted in Tanzania found that 51% of the patients testing negative were treated presumptively for uncomplicated malaria20. Another study conducted in the Coastal area of Kenya found that presumptive treatment of patients ranged from 0 to 13.9% in the private sectors21. In Nigeria a study conducted found that presumptive treatment among health care providers was higher in the private sector (95%) than the public sector (23%)22. These studies have shown that presumptive treatment in the private sector is being practiced. The study conducted in Tanzania had a percentage of health care providers who treat patients presumptively almost equal (51%) to that in this study (57%).\n\nThe potential factors associated with presumptive treatment were as follows: zone, RDT availability, ACT availability, drug price, cadre, health care provider asked signs or symptoms, case management training, results presented, any supervision, access to national malaria guidelines and malaria risk. However, the factors that were associated with presumptive treatment of uncomplicated malaria considering the county effect were zone, signs/symptoms asked, case management training, results presented and access to malaria case management guidelines. Several other studies have investigated the factors associated with presumptive treatment of uncomplicated malaria.\n\nA study that was conducted in Kenya assessed the association between presumptive treatment and case management training among health care providers in the private retail sector. The study showed that malaria case management training was significantly associated with presumptive treatment23. These results are in line with that of this study as malaria case management training was significantly associated with presumptive treatment taking into account the county effects. A cluster randomized trail conducted in Ghana found that there was a significant association between availability of RDTs and presumptive treatment of uncomplicated malaria24. The results contradict those of this study since the data did not provide enough evidence to indicate the association between availability of RDTs and presumptive treatment of uncomplicated malaria taking into account the county effect.\n\nAvailability of antimalarial drugs was significantly associated with presumptive treatment of uncomplicated malaria in a study that was conducted in Nigeria22. However, in this study, the data did not provide sufficient evidence to indicate that there was an association.\n\nBoth the test for association and model building for this study was done using the mixed effects logistic regression models. The random effect which was the county was taken into account due to the county clustering. This method has been proved to be more accurate for prediction compared to the ordinary logistic regression models25. This study applied the mixed effects logistic regression model with the best fitted model used as the AIC. Generally, comparing the AIC value, the mixed effects logistic regression model had a better fit compared to the ordinary logistic regression model. The mixed effects models have also been proven to be more efficient and flexible26.\n\nBased on the AIC values for the different candidate models to predict presumptive treatment of uncomplicated malaria among children in the private retail outlets, if the test was presented, if the health care provider asked the signs and symptoms and had undergone case management training were the predictor variables that fitted the best model. The model further predicted the probability of a health care provider to treat a patient presumptively based on the three factors. For a health care provider who has undergone a malaria case management training, who asked the patient for the signs and symptoms and for whom results were presented, the probability is 0.32. For one who has gone through a malaria case management training, for whom results were presented but did not ask for signs or symptoms of malaria the probability is 0.71. For a health care provider who had no results presented, the probability was 0.52. If the test results were presented, and the health care provider asked for the signs and symptoms but had not undergone a case management training, the probability of treating a patient presumptively was 0.85. These results show that all the three predictors are crucial for a health care provider. However, malaria case management training plays a key role in treatment of malaria at the private retail sector.\n\nIn conclusion, the health care providers practices and knowledge in case management of malaria is key in diagnosis, treatment and prevention of malaria in Kenya. Presumptive treatment is due to misdiagnosis of a patient, which may lead to drug resistance.\n\n\nConclusion\n\nHealth seeking behavior in the private retail sector raises concerns in the provision of health care services in the private sector. Much has been done in the public sector but there is a need to also strengthen interventions in the private sector in realization of Universal Health Care in Kenya to reach the Sustainable Development Goals (SDGs). Presumptive treatment of uncomplicated malaria among children in Kenya has been an issue which depends on the health care providers as well as the retail outlet factors. Adherence to the national malaria case management guidelines in key in ensuring quality of treatment to the malaria patients. Interventions in place to curb presumptive treatment include provision of malaria case management guidelines, case management training, supervision and availability of RDTs to ensure testing before treatment. On the other hand, availability of ACTs due to the AMFm subsidies could possibly influence presumptive treatment of uncomplicated malaria.\n\nThe results from this study show that case management training, asking about signs/symptoms and having results presented predict presumptive treatment of uncomplicated malaria. A mixed effects logistic regression model was used to adjust for the county clustering effect. This model provides more accurate prediction compared to an ordinary logistic regression model measured by the AIC values. Apart from asking the signs and symptoms or results presented, case management training for the health care providers play a key role in management of malaria by health care providers in the private retail outlets.\n\nMalaria case management training touches on testing and asking of patients for signs/symptoms before diagnosis and treatment of a patient, However, this study gives and insight for policy makers to emphasize on testing and asking signs/symptoms when training health care providers on malaria case management.\n\nBased on the discussion and conclusion drawn, this study recommends:\n\n1. The Division of National Malaria Programme (DNMP) and other partners should focus on malaria case management in the private sector to improve the quality of treatment.\n\n2. Further research to look into the factors that may predict presumptive treatment since the data in this study did not provide evidence to indicate the other potential factors predictability.\n\n3. There is need to strengthen interventions in the private sector in realization of Universal Health Care in Kenya.\n\n\nData availability\n\nFigshare: PPTUM_Kenya.xlsx. https://doi.org/10.6084/m9.figshare.12783128.v217.\n\nThis file contains the de-identified dataset used in the present study.\n\nFigshare: PPTUMK_DD. https://doi.org/10.6084/m9.figshare.12783188.v218.\n\nThis file contains the data dictionary for the de-identified dataset.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nI would like to acknowledge the following people who have mentored and supported me throughout this project. I would also like to thank Dr. Marshal Mweu (University of Nairobi) for his continuous mentorship and advice. The University of Nairobi Institute of Tropical and Infectious Diseases (UNITID) for their support. The Division of National Malaria Programme (DNMP) in collaboration with the Global Fund for their support during this project.\n\n\nReferences\n\nWorld Health Organization: World Malaria Report 2015. World Health, 2015. Reference Source\n\nWHO: WORLD MALARIA REPORT 2018. 2018. Reference Source\n\nWorld Health Organization: World malaria Report 2012. World Health Organization, 2012. Reference Source\n\nNational Malaria Control Program [Ministry of Health]: Kenya Malaria Strategy 2009 - 2018 (Revised 2014). 2014; 76. Reference Source\n\nKenya National Bureau of Statistics: Malaria Indicator Survey 2015. Minist. Heal. Kenya, 2016; 2. Reference Source\n\nO’Connell KA, Gatakaa H, Poyer S, et al.: Got ACTs? Availability, price, market share and provider knowledge of anti-malarial medicines in public and private sector outlets in six malaria-endemic countries. Malar J. 2011. 10: 326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAMFm: Independent Evaluation of Phase 1 of the Affordable Medicines Facility - malaria (AMFm), Multi-Country Independent Evaluation Report:Final Report. Calverton, Maryl. London ICF Int. London Sch. Hyg. Trop. Med., 2012. Reference Source\n\nJuma E, Zurovac D: Changes in health workers’ malaria diagnosis and treatment practices in Kenya. Malar J. 2011; 10: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMosha JF, Conteh L, Tediosi F, et al.: Cost implications of improving malaria diagnosis: Findings from North-Eastern Tanzania. PLoS One. 2010; 5(1): e8707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nD’Alessandro U, Ubben D, Hamed K, et al.: Malaria in infants aged less than six months - Is it an area of unmet medical need? Malar J. 2012; 11: 400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBilal JA, Gasim GI, Abdien MT, et al.: Poor adherence to the malaria management protocol among health workers attending under-five year old febrile children at Omdurman Hospital, Sudan. Malar J. 2015; 14: 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnsumana R, Jacobsen KH, Gbakima AA, et al.: Presumptive self-diagnosis of malaria and other febrile illnesses in Sierra Leone. Pan Afr Med J. 2013; 15: 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott AJ, Hosmer DW, Lemeshow S: Applied Logistic Regression. Biometrics. 2006.\n\nMaxwell SE, Maxwell SE: Mixed-Effects Models. In: Designing Experiments and Analyzing Data.2017. Reference Source\n\nYuan M, Lin Y: Model selection and estimation in regression with grouped variables. J R Stat Soc Ser B Stat Methodol. 2006; 68(1): 49–67. Publisher Full Text\n\nMugenda O, Mugenda AG: Research Methods – Quantitative & Qualitative Approaches. African Cent Technol Stud Nairobi Kenya. 2003. Reference Source\n\nKemunto D: PPTUM_Kenya.xlsx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12783128.v2\n\nKemunto D: PPTUMK_DD. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12783188.v2\n\nDohoo IR, Martin SW, Stryhn H: Methods in epidemiologic research . 2012. Reference Source\n\nReyburn H, Mbakilwa H, Mwangi R, et al.: Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: Randomised trial. BMJ. 2007; 334(7590): 403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoyer S, Musuva A, Njoki N, et al.: Fever case management at private health facilities and private pharmacies on the Kenyan coast: Analysis of data from two rounds of client exit interviews and mystery client visits. Malar J. 2018; 17(1): 112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBamiselu OF, Ajayi I, Fawole O, et al.: Adherence to malaria diagnosis and treatment guidelines among healthcare workers in Ogun State, Nigeria. BMC Public Health. 2016; 16(1): 828. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKangwana BP, Kedenge SV, Noor AM, et al.: The effect of an anti-malarial subsidy programme on the quality of service provision of artemisinin-based combination therapy in Kenya: A cluster-randomized, controlled trial. Malar J. 2013; 12: 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnsah EK, Narh-Bana S, Affran-Bonful H, et al.: The impact of providing rapid diagnostic malaria tests on fever management in the private retail sector in Ghana: A cluster randomized trial. BMJ. 2015; 350: h1019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalazsi L, Matyas L, Wansbeek T: Fixed effects models. In: Advanced Studies in Theoretical and Applied Econometrics.2017; 50: 1–34. Publisher Full Text\n\nBagiella E, Sloan RP, Heitjan DF: Mixed-effects models in psychophysiology. Psychophysiology. 2000; 37(1): 13–20. PubMed Abstract \| Publisher Full Text" }	[ { "id": "82322", "date": "08 Apr 2021", "name": "Moses Ocan", "expertise": [ "Reviewer Expertise Community medicine use", "Antimicrobial resistance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have enjoyed reading the work and here below are the summary of my comments:\nClearly define/provide the age range for the children considered in the study.\n\nProvide key elements of the Kenya malaria treatment policy related to this study and how it is implemented in the country.\n\nAbstract, last sentence of the background should be deleted. Add the aim of the study to the background.\n\nThe methods section; what provide retail outlets where included in this study, give details.\n\nProvide details of the healthcare cadres targeted for this study.\n\nLevel of significance in the analysis of the data.\n\nHow many retail outlets were visited or considered in this study.\n\nInclude results for univariate and Bivariate analysis in the abstract\n\nIntroduction section\nUse the most recent world malaria report (WHO malaria report, 2020).\n\nFourth sentence ...In 2010, Kenya adopted......., this sentence is not clear and should be re-written.\n\nSecond paragraph sentence, .....Though medical care is........, is this referring to general healthcare or specifically malaria treatment?\n\nThird paragraph should include specific information on how AMFm has impacted malaria treatment in Kenya.\n\nParagraph four should be removed from the introduction section of the manuscript and transferred to the methods section.\n\nThe sentence, .....The health seeking behavior in Kenya....., is not clear, please clarify with some detail.\n\nMethods section\nClearly define what you mean by retail outlet.\n\nProvide the professions of the healthcare cadres targeted in this study.\n\nHow many retail outlets were included in this study and how was the number arrived at?\n\nHow was the data to be included in this study selected from the large dataset?\n\nWhere is the dataset of the original study kept? Provide details and when the data was accessed by the study team.\n\nHow was confidentiality of the study participants ensured while accessing the dataset?\n\nRevise the description of the sampling procedure. Focus on how you selected the data that you included in your analysis from the large dataset of the primary study.\n\nProvide details of ethical approval of the primary study as well.\n\nDescribe how you collected data for your study. Please describe how the data points you analyzed were extracted from study units obtained from the original study. Rewrite the section on data collection.\n\nWhat is the study unit for your study?\n\nHow was quality control ensured in this study?\n\nResults\nWhat patients are you referring to, please be specific.\n\nStratify the results by the type of healthcare cadre and retail outlet type.\n\nWhat were the high malaria risk areas and how many study participants were obtained from this areas.\n\nProvide details of the ACTs used in the retail outlets (type, formulation etc).\n\nHow was malaria diagnosed among the study participants?\n\nWho were your study participants, healthcare cadres or children?\n\nPlease clarify what you mean by healthcare providers who were presented with results by the patients. Your target population for this study children, please clarify.\n\nWhen was the malaria case management training conducted for the healthcare cadres?\n\nDiscussion\nWhat is the prevalence of malaria in children in Kenya and what proportion of these is treated presumptively from previous studies?\n\nIs the extent of presumptive treatment of malaria among children similar to that in adults?\n\nWhy would asking for signs and symptoms of disease as found in your study reduce the risk of presumptive treatment. Please explain.\n\nThe meaning of the key results of the study has not been clearly described in the discussion section.\n\nConclusion and recommendations\nSummarize the conclusion and should come from the main finding of the study.\n\nSummarize the recommendation, who should take action and what action should they take.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "89369", "date": "17 Aug 2021", "name": "Luca Nelli", "expertise": [ "Reviewer Expertise Quantitative Ecology and Epidemiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI’ve read the manuscript entitled “Predictors of presumptive treatment of uncomplicated malaria among children in private retail outlets in Kenya: mixed effects logistic regression modelling” with great interest. This is a very interesting and scientifically sound work, presenting an original investigation of the possible drivers of “inappropriate” malaria treatment in private health facilities in Kenya. The methods used are sound and the conclusion reflects the main findings.\nI only have some minor comments with respect to the general presentation. In addition, most of the manuscript is well written, but there are some typos that should be checked for. Please also check punctuation errors.\nPlease see detailed comments:\nABSTRACT:\nThe summary is interesting and concise. However, instead of reporting only those 3 coefficients (and also, the first one is not significant, are you sure it’s one of the main findings you want to put in the abstract?), I was wondering if you could provide some more results. If you don’t have space, I guess you can easily reduce the method subsection (which I think contains too many details for an abstract).\nINTRODUCTION:\nI feel like some parts of the introduction could be removed (or at least reduced). For example, you have a whole paragraph with a general description of what a logistic regression is, how the response variable is distributed, and what a mixed-effect model is. I think all of this is irrelevant for the introduction, and you have already mentioned this in the methods section. I would suggest removing the paragraph.\nOn the contrary, I would recommend expanding a little bit on the (plausible) drivers of presumptive treatment. You only mention that “Inappropriate treatment practice […] could be caused by several factors”, but it would be good to see some reference as to what these factors could be. In other words, how do you justify the choice of your variables in the model? If there’s no prior knowledge on this, that’s fine but you should flag this as an important knowledge gap (this would also strengthen your whole work).\nI don’t agree with the final sentence: “This study focused on application of mixed effect model […]”. While it is true that you used mixed-effect models to investigate, I think you have done much more than that (i.e. not “just” applying a method – which is well established and you haven’t established anything new from a methodological point of view – so I would put more emphasis on the main aim of the study (understanding which factors are influential on inappropriate practices). If, on the contrary, your main aim was to investigate if mixed-effect models can be applied to these data, I think the whole paper (including title, introduction, and discussion) should definitely have a different angle.\nMETHODS:\nThe whole method section is very long and full of unnecessary (just my opinion, of course) details and repetitions. For example:\nUnclear who/what the “mystery shopper” is. Please define this.\n\nI really enjoyed the description of the Data Collection Procedure. It’s like reading a novel :-)\n\nYou have plenty of repetition in the description of your covariates (e.g. page 5 in the left column, page 5 in the right column, Table 1, Figure 1). Surely you can reduce this?\n\nYou mention microscopy availability in the Data Collection, but you haven’t used that as a predictor in your models. Why?\n\nAkaike Information Criterion (first paragraph of page 6) doesn’t assess model accuracy. It’s a method to compare different models (as you rightly do in Table 5), but to validate the model I would suggest calculating the percentage of correct classification and values of area under the ROC curve.\n\nRESULTS:\nAgain, plenty of repetitions here. For example, you don’t need to write again what response variable and predictors you used in the models. You already told us in the method.\nI don’t think that the formulas on page 9 (and in Table 7) add anything to the interpretation of the results (you already report the coefficients). But at the same time, I like that you present the 4 different scenarios (Table 7) with all the possible combinations. Can you find a way to present this as a figure/plot instead? This would give a clearer picture of the five different scenarios (and it would make your manuscript more balanced – as it definitely has too many tables now).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1059
https://f1000research.com/articles/9-1054/v1	27 Aug 20	{ "type": "Research Article", "title": "Diagnosis of Helicobacter pylori infection using immunochromatography among patients attending Tamboul Hospital in Gezira State, Sudan: a cross-sectional study", "authors": [ "Rawda Mohmmed Elhassan Ali Noor", "Wafaa Mohammed Abdalla", "Ahmed Bakheet Abd Alla", "Ahmed Ibrahim Hashim", "Rawda Mohmmed Elhassan Ali Noor", "Wafaa Mohammed Abdalla", "Ahmed Ibrahim Hashim" ], "abstract": "Background: Helicobacter pylori causes a major health problem worldwide; more than half of the world’s population are infected with this pathogen. The diagnosis of the infection was initially made through invasive methods, but now non-invasive methods have been developed to make diagnosis easier. This study aimed to screen the presence of H.pylori antibodies and antigen among symptomatic and asymptomatic patients at Tamboul City in Gezira State. Methods: A cross-sectional study was conducted in Tamboul city, Gezira State, Sudan between March 2016 and December 2019 to compare between antigen and antibody tests results used for diagnosis of H. pylori infection among symptomatic and asymptomatic Sudanese patients. Stool and blood samples were collected and analyzed for presence of antigen and antibodies to H. pylori using immunochromatography (ICT) cards. Results: Serum and stool samples were collected from 100 patients; 50 were symptomatic and 50 were asymptomatic. In symptomatic patients, 18/50 (36%) were men (32; 64%, women) with mean age of 16.7±24.6 years. In this group, 35/50 (70%) showed positive results for stool antigen, while 30/50 (60%) were positive for serum antibodies. In asymptomatic patients, 19/50 (38%) were men (31; 62%, women) with mean age of 16.7±20.4 years. In this group, 18/50 (36%) were positive for stool antigen and 25/50 (50%) for serum antibodies. There was a significant association between antigen results and patient group (P=0.001), but there was an insignificant association between antibodies results and patient group (P=0.317). Age group, history of infected persons in the family, blood group, and previous treatment were all not associated with H. pylori infection (P≥0.05). Conclusion: The frequency of H. pylori antigen was higher than antibodies in symptomatic patients, while the frequency of H. pylori antibodies was higher than antigen in asymptomatic patients.", "keywords": [ "H.pylori", "Tamboul", "ICT", "Gazira state." ], "content": "Introduction\n\nHelicobacter pylori is a Gram-negative, microaerophilic, spiral-shaped bacteria that inhabits the mucous layer of the antral gastric (Basher et al., 2011). Humans are living things infected (Abdallah et al., 2014). H. pylori is typically symptomless and has no clinical manifestations. Signs and symptoms associated with the disease are mainly due to gastric or peptic ulcer disease or duodenal inflammation. Other symptoms, such as nausea, vomiting, and stomach pain, can be related to other gastrointestinal diseases (Gold et al., 2014). The prevalence of H. pylori is still high in most countries, with approximately one-third of adults infected in northern Europe and North America, while the prevalence in southern and eastern Europe, South America and Asia is higher than 50% (Eusebi et al., 2014). Up to 93.6% of adults in developing countries are infected with H. pylori (Olokoba et al., 2013) and the prevalence of infection in Sudan was estimated to be between 65.8% and 80% (Abdallah et al., 2014).\n\nThe attachment of H. pylori to the gastric mucosa is mediated by the Lewis (Le6) antigen. The availability of these receptors may be reduced in blood groups A and B compared to blood group O. H antigen is also an important gastroduodenal mucosal cell receptor for H. pylori attachment (Alcout et al., 2000).\n\nLaboratory testing for H. pylori infection is very important part of the diagnosis process for gastric and duodenal inflammatory disease. A number of different diagnostic test methods, both invasive and non- invasive, are available. Invasive methods, such as antibody (Ab) detection is cheap and easy to perform, but may give false positive results (Quartero et al., 2000). The stool antigen (Ag) test has recently become more acceptable as it is non-invasive, convenient for patients and can be performed easily even in small laboratories. Other non-invasive tests may also include detection of H. pylori antigens in saliva (logan & walker, 2001; Malfertheiner et al., 2007).\n\nThis study aimed to screen the presence of H.pylori antibodies and antigen among symptomatic and asymptomatic patients at Tamboul City in Gezira State.\n\n\nMethods\n\nThis descriptive, cross-sectional, hospital-based study was conducted at Tamboul Hospital in Gezira State, Sudan, between March 2016 and December 2019.\n\nEthical approval was obtained from the Scientific Research Committee of the College of Medical Laboratory Sciences, Sudan University of Science and Technology (Ethical No SRC-MLS-01-19). Written informed consent was initially obtained from participants for participation in the study and publication of data; however, the researchers noticed that participants preferred to agree verbally. After requesting permission from the Scientific Research Committee to change the consent process, verbal informed consent was obtained from participants for participation in the study and publication of data.\n\nPatients were selected by non-probability, convenience sampling and were recruited by physicians.\n\nPatients with and other without symptoms of H. pylori infection with different age and gender were included in this research.\n\nExclusion criteria: symptomatic patients undergoing treatment of H. pylori infection and patients with other diseases.\n\nA structured questionnaire containing demographic data and medical history was used to collect information from participants.\n\nSample size was determined according to the formula below.\n\nn=t2p (1-p)/µ2\n\nt=1.96 (t2=3.8416)\n\np=prevalence (0.80) (80%)\n\nµ=0.05 (µ2=0.0025).\n\n308410.8(0.92)/0.0025=113\n\nDue to high cost, the sample size was kept at 100 sera and stool samples; 50 symptomatic patients and 50 asymptomatic patients.\n\nSerum sample. Five ml blood sample were collected from each patient in a plain blood container. Serum was then separated by centrifugation (3000 rpm for five minutes). H. pylori antibodies (IgG and IgM) were detected in the serum using ALL TEST (Hangzho Biotics, China; REF IHP-302). 2 drops of sample (about 100µl) were added into the sample well (S) of the test device and then 1 drop buffer was added to this. As the test began to work, a purple color moving across the window to the center of the device.\n\nThe presence of two-color bands as test band (T) and control band (C) within the results window, no matter which band appeared first, indicated a positive result. It should be noted that some invalid results are because the directions have been followed incorrectly or the test may have deteriorated beyond the expiration date (Ansorg et al., 1991).\n\nStool sample. Patients were asked to provide a stool sample at the hospital. Fresh stool samples were collected into spoon-cover and outer-labeled stool container for antigen detection. Using a wood stick, a small portion of the stool sample was transferred into buffer, incubated for 2 minutes and then two to three drops of the mixture were poured in the hole of the ICT of H. pylori stool antigen detection (ALLTEST; Hangzho Biotics, China; catalog number 30226). The color migrated from the well containing the tested sample in the ICT device (Ansorg et al., 1991). The presence of two bands (test band (T) and control band (C)) within the result window, no matter which band appeared first, indicated a positive result.\n\nStatistical Package for Social Science program (SPSS) version 20 was used for analysis. Frequencies were presented as tables and figures. Chi-square test was used to compare between variables and level of significance was set as P ≥ 0.05.\n\n\nResults\n\nA total of 100 stool and 100 sera specimens were collected from 50 asymptomatic and 50 symptomatic patients, with different gender, age and blood groups. Age was classified as following: <15 years, 15-50 years, and >50 years old.\n\nIn asymptomatic patients, the mean age was 16.7±24.6 years. The majority of the patients in this group were women (32; 64%), In symptomatic patients, the mean age was 16.7± 20.4 years, and the majority of patients were women (31; 62%).\n\nFor both groups of patients, those who had previous treatment for H. pylori had low frequency of infection compared to those with no previous treatment. For both groups, 60% had family members who had also been diagnosed with H. pylori infection (Table 1).\n\nThere was a higher number of symptomatic patients with a positive antigen result (35/50; 70%) compared with asymptomatic patients (18/50; 36%). There was a significant association between antigen result and patient group (P=0.001; Table 2).\n\nSimilarly, there was a higher number of symptomatic patients with a positive antibody result (30/50; 60%) compared to asymptomatic patients (25/50; 40%). There was no significant association between antibody results and patient group (P=0.317; Table 3).\n\nPositive results were those with the presence of both IgM and IgG; presence of either was considered a negative result.\n\nSymptomatic patients in the 15–50 years age group had the highest number of positive results for antigen and antibodies (antigen, 27 (54%); antibody, 24 (48%)). In asymptomatic patients for this age group, a positive antigen and antibody result was seen in 17 (34%) and 23 (46%) patients, respectively. However, there was no association between age group and positive result for antigen and antibodies to H. pylori in either group of patients.\n\nWomen showed higher positive results than men for both antigen and antibody in both asymptomatic and symptomatic groups (asymptomatic women, Ag 12 (24%), Ab 14 (28%); asymptomatic men, Ag 9 (18%), Ab 11 (22%); symptomatic women, Ag 21 (42%), Ab 19 (38%); symptomatic men, Ag 9 (18%), Ab 11 (22%)). This association between gender and antigen/antibody presence was not significant in either group of patients.\n\nPatients with O positive blood group showed higher positive results than other blood groups for both antigen and antibody in both asymptomatic and symptomatic groups (asymptomatic O positive, Ab 14 (28%), Ag 17 (34%); symptomatic O positive, Ab 10 (20%), Ag 7 (14%)). The association between blood group and antigen/antibody presence was not significant in either group of patients.\n\nPatients in both groups with family members who also had H. pylori infection showed higher positive antigen results than those without family members who had the infection (symptomatic patients with family infection, 21 (42%), without family infection 14 (48%); asymptomatic patients with family infection, 12 (21%), without family infection 6 (12%). There was no significant association between antigen results and family member infection.\n\nAsymptomatic patients who had no previous treatment for H. pylori showed higher antigen positivity (12/50; 24%) than those with previous treatment. This was similar in symptomatic patients (no previous treatment Ag, 25/50; 50%). There was no significant association between previous treatment and antigen result in asymptomatic and symptomatic patients.\n\n\nDiscussion\n\nH. pylori infection is a co-factor in the development of three important upper gastrointestinal diseases: duodenal or gastric ulcers, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (McColl, 2010).\n\nIn the present study, H. pylori antibodies were present in 25/50 (50%) asymptomatic patients and 30/50 (60%) symptomatic patients. These results were similar to a study carried by Naji et al. (2014) in Yemen, which found antibodies in 72%. In contrast, our results are higher than those found by Douraghi et al. (2013) in Iran, who found antibodies in 4.6% of patients, and lower than those found by Alfadil (2016) in Sudan who found antibodies in 48% of patients.\n\nIn our asymptomatic patients, H. pylori antigen was detected in 18/50 (36%) patients, and 35/50 (70%) symptomatic patients. These results are similar to a study carried out in Nigeria by Omosor et al. (2018), where stool antigen was found in 56% of patients. Our results for antigen were higher than those reported by Douraghi et al. (2013) in Iran (12.4%) and lower than Naji et al. (2014) in Yemen (49%). The variation may be due to difference in socioeconomic status of the patients.\n\nOur study results did not find a significant association between age groups and H. pylori infection (as diagnosed with antigen/antibody testing). This is similar to study done in Iran by Alvali et al. (2010), but it differed from Kabir (2007) in Sweden who reported that the percentage of infection increases with age. In our study, the age group 15–50 years showed the highest rate of infection in both symptomatic and asymptomatic patients for antigen and antibodies. That may be because the vast majority of individuals acquire H. pylori infection during childhood, and the infection associated with poor hygiene and overcrowding.\n\nWe also found that women were more affected than men both in the symptomatic group (62% vs 38%.) and asymptomatic group (64% vs 36%). This was a similar result to that found in Turkey by Kanbay et al. (2005) (60.6% vs 42.9%), but a dissimilar results to those found in Iran by Metanant et al. (2015) (32.8% vs 36.4%). The differences between studies could be due to random selection of patients regardless of their gender in our study.\n\nIn the current study, patients with blood group O positive had the highest frequency of positive H. pylori infection, both for symptomatic (48%) and asymptomatic (38%) patients. These results were the same as another study done in Gezira State, Sudan by Mohammed et al. (2015), who found that 58.1% of blood group O positive patients were positive for H. pylori infection. This is in contrast to a study done in Turkey by Kanbay et al. (2005) who was reported that patients with blood group A positive had a higher frequency of H. pylori infection than other blood groups at 38.2%. This dissimilarity may be due differences in ethnic background, and the fact that blood group O positive is the most frequent blood group in Sudan (Hassan, 2010).\n\nIn this study, 12/50 (24%) of asymptomatic patients who had family members with H. pylori infection were positive for antigen and/or antibody. These results are similar to Gravina et al. (2016) in Italy, who found H. pylori infection in 28.4% household contacts. However, this study was higher than Mabeku et al. (2018) in Cameroon (80.39%). In symptomatic patients, the frequency of antigen and antibodies with family member infection were 21(42%) and 20(40%) correspondingly, that were similar to result of Gravina et al. (2016) in Italy who found H. pylori infection in 43.2% household contacts. This finding was higher than Manfredi et al. (2016) in Italy (81.7%), and variation may be due to personal hygiene.\n\nIn the current study, the frequency of antigen in asymptomatic patients who had previous treatment for H. pylori were 12% (6/50) while in symptomatic patients this was 20% (10/50). This agreed with a study carried out by Gisbert et al. (2005), but was higher than the results reported by Nanivadekar et al. (1990) in India (2.4%), and lower than the results found by Bapat et al. (2000) in India (60%). This discrepancy may be due to resistance of standard triple therapy. In this study, the association between previous treatment and H. pylori reinfection was not statistically significant.\n\n\nConclusions\n\nThe frequency of H. pylori antigen was higher than antibodies in symptomatic patients, while the frequency of H. pylori antibodies was higher than antigen in asymptomatic patients.\n\n\nData availability\n\nFighshare: Diagnosis of H. pylori in Tamboul Hospital in Gazira State, https://doi.org/10.6084/m9.figshare.12488432.v2 (Abd Alla & Alhassan, 2020).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAbd Alla A, Alhassan R: Diagnosis of H. pylori in Tamboul Hospital in Gazira State. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12488432.v2\n\nAbdallah TM, Mohammed HB, Ali AA: Sero-prevalence and factors associated with Helicobacter pylori infection in Eastern Sudan. Asian Pac J Trop Dis. 2014; 4(2): 115–119. Publisher Full Text \| Free Full Text\n\nAlcout A, Blackwell C, Weir D: Increased inflammatory responses of persons of blood group O to Helicobacter pylori. J infect Dis. 2000; 181(4): 1364–1569. PubMed Abstract \| Publisher Full Text\n\nAlfadil A: Compression between antigen and antibodies detection test in diagnosis of Helicobacter pylori infection (MSc thesis). Sudan University for Science and Technology. 2016; 24.\n\nAlvali SM, Adel SH, Raja AR: Seroprevalence study of Helicobacter pylori infection among visitors of cardiac patients in Razi Hosbital in Ahvaz, Iran. Jundishapur J Microbiol. 2010; 3(1): 28–31. Reference Source\n\nAnsorg R, Von Recklinghausen G, Pomarius R, et al.: Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter pylori. J Clin Microbiol. 1991; 29(1): 51–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBapat M, Abraham P, Bhandarkar P, et al.: Acquisition of Helicobacter pylori infection and reinfection after its eradication are uncommon in Indian adults. Indian J Gasteroentrol. 2020; 19(4): 172–4. PubMed Abstract\n\nBashir AH, Yousif SM, Mahmoud MO: Clinicoepidemiological study in Sudanese patients: prevalence and effect of eradicative triple therapy on extra digestive Helicobacter pylori skin manifestations, EdHpSm. Clinical reviews and opinions. 2011; 3(2): 14–9. Reference Source\n\nDouraghi M, Nateghi M, Goudarzi H, et al.: Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children. Microbiol Immunol. 2013; 57(11): 772–7. PubMed Abstract \| Publisher Full Text\n\nEusebi LH, Zagari RM, Bazzoli F: Epidemiology of Helicobacter pylori infection. Helicobacter. 2014; 19 Suppl 1: 1–5. PubMed Abstract \| Publisher Full Text\n\nGold B, Gilger M, Czinn S: New diagnostic strategies for detection of Helicobacter pylori infection in pediatric patients. Gastroenterol Hepatol (N Y). 2014; 10(12 Suppl 7): 1–19. PubMed Abstract \| Free Full Text\n\nGisbert J, Trapero M, Pajares J: Evaluation of 3 different tests for the detection of stool antigens to confirm Helicobacter pylori eradication after treatment. A pilot study. Gastroenterología y Hepatología. 2005; 28(10): 615–618. Publisher Full Text\n\nGravina AG, Federico A, Dallio M, et al.: Intrafamilial Spread of Helicobacter pylori Infection. J Gatroenterol Hepatol Endosc. 2016; 1(1): 1003. Reference Source\n\nHassan MF: Frequency of ABO, subgroup ABO and Rh(D) Blood Groups in Major Sudanese Ethnic Groups. Pak J Med Pes. 2010; 49(1): 21. Reference Source\n\nKabir S: The current status of Helicobacter pylori vaccines: a review. Helicobacter. 2007; 12(2): 89–202. PubMed Abstract \| Publisher Full Text\n\nKanbay M, Gür G, Arslan H, et al.: The relationship of ABO blood group, age, gender, smoking, and Helicobacter pylori infection. Dig Dis Sci. 2005; 50(7): 1214–7. PubMed Abstract \| Publisher Full Text\n\nLogan RP, Walker MM: Epidemiology and Diagnosis of Helicobacterpylori infection. BMJ. 2001; 323(7318): 920–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMabeku LBK, Ngamga MLN, Leundji H: Potential risk factors and prevalence of Helicobacter pylori infection among adult patients with dyspepsia symptoms in Cameroon. BMC infect Dis. 2018; 18(1): 278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalfertheiner P, Megrand E, Morain C, et al.: Current concepts in the management of Helicobacter pylori Infection. Gatroenterol Hepatol. 2012; 28(16): 167–180.\n\nManfredi M, Iuliano S, Gismondi P, et al.: Helicobacter pylori infection: We Should Always Verfiy the Interfamilial Transmission. Biol Med. 2016; 9: 1. Reference Source\n\nMcColl KEL: Clinical practice. Helicobacter pylori infection. N Engl J Med. 2010; 362(17): 1597–1604. PubMed Abstract \| Publisher Full Text\n\nMetanant M, Sharifi B, Izadi S: Prevalence of Helicobacter pyloriinfection in healthcare workers. TurK J Med Sci. 2010; 40(6): 965–969. Reference Source\n\nMohammed M, Suliman O, Khalfalla O: Association between Helicobacter pylori infection, ABO Blood Groups and Rhesus factor in Peptic Ulcer Disease patients, in Gezira, Central Sudan. BJMM. 2015; 7(1): 11–16. Publisher Full Text\n\nNaji SA, Ameri AG, Alkadasi NM, et al.: Comparison of stool antigen and blood antibodies test methods for detection of Helicobacter pylori infection and risk factors. Int J Microbiol App Sci. 2014; 3(12): 118–127. Reference Source\n\nNanivadekar S, Sawant P, Patel H, et al.: Association or peptic ulcer with Helicobacter pylori and the recurrence rate. A three year follow up study. J Assoc Physician India. 1990; 38 Suppl 1: 703–6. PubMed Abstract\n\nOmosor KI, Omosor OH, Adejumo BG, et al.: Comparative evaluation of stool antigen and blood antibodiestest methods for the screening of Helicobacter pylori infection in asymptomatic adult population in Delta State, Nigeria. J Mol Microbiol. 2018; 2(1): 3. Reference Source\n\nOlokoba AB, Gashau W, Bwala S, Adamu A, Salawu FK: Helicobacter pylori Infection in Nigerians with Dyspepsia. Ghana Med J. 2013; 47(2): 79–81. PubMed Abstract \| Free Full Text\n\nQuartero A, Numans M, de Melker R, et al.: In-practice evaluation of whole-blood Helicobacter pylori test: its usefulness in detecting peptic ulcer disease. Br J Gen pract. 2000; 50(450): 13–16. PubMed Abstract \| Free Full Text" }	[ { "id": "75335", "date": "14 Dec 2020", "name": "Milagrosa Montes", "expertise": [ "Reviewer Expertise Graduated in Pharmacy (1988) and Doctor in Pharmacy from the Universidad de Navarra (1996). Specialist in Microbiology and Clinical Parasitology (1991). I currently work in the Microbiology Service of University Hospital Donostia. I have participated in more than 15 scientific projects", "and in more than 95 scientific publications related to the molecular mechanisms of resistance to antimicrobials", "diagnosis and epidemiology of Communicable Infections", "especially those that are preventable by vaccination", "in infection by Streptococcus pyogenes and Helicobacter pylori." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe two ICT methods to diagnose Helicobacter infection in two population groups: symptomatic and asymptomatic. I see two major errors. The first one, to use the detection of antibodies (Ab) to diagnose Helicobacter infection. Serology should only be used for epidemiological studies as it does not differentiate between current and past infections. The second failure that I observe is that speaking of non-invasive diagnostic methods to detect Helicobacter infection, they do not name the urea breath test (UBT), which, to date, is the non-invasive test with the highest sensitivity and specificity, and the one recommended by all Helicobacter infection diagnostic guidelines. My suggestion to the authors would be that they write another paper with the stool antigen data, saying that it is a good technique to detect Helicobacter infection when UBT is not available, it is logical that positivity is higher in symptomatic subjects (70 % vs 36%), and to use the serology data to give the seroprevalence in Gezira State; 55 patients have Ab (30 in the symptomatic group and 25 in the asymptomatic group) a figure lower than the 65-80% reported for Sudan by Abdallah et al. They should further describe in detail the test used to detect Helicobacter Ag in feces, whether it uses monoclonal or polyclonal Ab. It would be good to give the data of each test in previously treated patients, it would give an idea of the treatment failures. The heading of Table 1, \"Demographic variables for patients with Helicobacter pylori in patients from Gezira State, Sudan\" is not correct, since the 100 patients of the study are included, those with and those without infection, it should say \"Demographic variables of the 100 patients from Gezira State (Sudan) included in the study\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "97083", "date": "23 Nov 2021", "name": "Vikram Kate", "expertise": [ "Reviewer Expertise Gastroenterology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 demonstrated the use of immunochromatography in this cross-sectional, hospital-based study conducted at Tamboul Hospital in Gezira State, Sudan, between March 2016 and December 2019. In the introduction the authors could have mentioned some studies which used immunochromatography for the diagnosis of H. pylori. This is an upcoming non-invasive method used for the diagnosis of H. pylori infection.\nThe test was carried out for both antigen and antibody. Sera and stool samples were used. The H. pylori status was compared with different parameters in this study. However, it would have been desirable to compare the test results with a gold standard which was not carried out in this study. The gold standard could have been to stool antigen test against which the serology could have been compared, as done in a study by Obaid et al. (2021)1. The statistical analysis could have determined the sensitivity and specificity of this test with the gold standard. That would have given the efficacy of this new test with the existing standards. The authors could have given the details of the data tables which would clarify the results better. This helps the readers to interpret the results better.\nHowever, this study shows that non-invasive methods can be used to test H. pylori for antigen as well as antibodies. That’s a message taken from this study. The authors concluded that the frequency of H. pylori antigen was higher in symptomatic patients, while the frequency of H. pylori antibodies was higher in asymptomatic patients when compared to the antibodies. This indicates that in symptomatic patients it may be more prudent to use antigen related tests for the diagnosis of H. pylori and for screening antibody test can be recommended using immunochromatography.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1054
https://f1000research.com/articles/9-1052/v1	27 Aug 20	{ "type": "Research Article", "title": "Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2", "authors": [ "Timothy P. Levine" ], "abstract": "Background: FAM161A is a microtubule-associated protein conserved widely across eukaryotes, which is mutated in the inherited blinding disease Retinitis Pigmentosa-28. FAM161A is also a centrosomal protein, being a core component of a complex that forms an internal skeleton of centrioles. Despite these observations about the importance of FAM161A, current techniques used to examine its sequence reveal no homologies to other proteins. Methods: Sequence profiles derived from multiple sequence alignments of FAM161A homologues were constructed by PSI-BLAST and HHblits, and then used by the profile-profile search tool HHsearch, implemented online as HHpred, to identify homologues. These in turn were used to create profiles for reverse searches and pair-wise searches. Multiple sequence alignments were also used to identify amino acid usage in functional elements. Results: FAM161A has a single homologue: the targeting protein for Xenopus kinesin-like protein-2 (Tpx2), which is a strong hit across more than 200 residues. Tpx2 is also a microtubule-associated protein, and it has been shown previously by a cryo-EM molecular structure to nucleate microtubules through two small elements: an extended loop and a short helix. The homology between FAM161A and Tpx2 includes these elements, as FAM161A has three copies of the loop, and one helix that has many, but not all, properties of the one in Tpx2. Conclusions: FAM161A and its homologues are predicted to be a previously unknown variant of Tpx2, and hence bind microtubules in the same way. This prediction allows precise, testable molecular models to be made of FAM161A-microtubule complexes.", "keywords": [ "Structural Bioinformatics", "Microtubule Associated Protein", "Centrosome", "Primary Cilium", "Retinitis Pigmentosa", "Nucleation Factor", "Tpx2", "FAM161A" ], "content": "Introduction\n\nInherited eye disease affects approximately 1 in 1500 people in Western societies. The largest grouping within these disorders is Retinitis Pigmentosa (RP) (Daiger et al., 2013). RP is itself a diverse array of conditions that share a final common pathway of loss of photoreceptor function, usually with initial loss of rod photoreceptors diminishing peripheral and night vision, followed by critical central vision defects in both rods and cones.\n\nMutations in at least 56 genes cause inherited RP syndromes (Daiger et al., 2013). Some of the proteins coded by genes mutated in RP, such as rhodopsin, are obviously linked to photoreceptor function (Athanasiou et al., 2018). Other RP proteins have general functions in many cells, for example in the mRNA spliceosome, and studies of these forms of RP have revealed how specific house-keeping pathways are critical for photoreceptor function (Ruzickova & Stanek, 2017). A small, third group of RP proteins have remained mysterious because no molecular function can be assigned. The lack of information stems from these proteins having no domains with recognisable functional activities. The failure of routine methods to identify domains exists not only for RP proteins, but also across the human proteome. Even the most highly annotated proteome, the budding yeast Saccharomyces cerevisiae, has a substantial minority of proteins (20%) without useful functional information (Wood et al., 2019).\n\nAmong 56 proteins linked to RP, functional information is missing in several, including FAM161A, truncations of which cause recessive RP28 (Bandah-Rozenfeld et al., 2010; Langmann et al., 2010). FAM161A binds to microtubules (Zach et al., 2012), and localises to the primary cilium (Di Gioia et al., 2012; Estrada-Cuzcano et al., 2012; Nishimura et al., 2010; Zach et al., 2012), which is vital in photoreceptor function through its specialisation as the outer segment. FAM161A is also a centrosomal protein (Di Gioia et al., 2015), and is a key component of a complex with three other proteins that together form an internal framework for centriolar microtubules (Le Guennec et al., 2020).\n\nFAM161A has retained its systematic name in part because no function has been detectable from its sequence. The region of greatest conservation in FAM161A has been annotated as UPF0564, one of >10,000 domains of unknown function (DUFs) and uncharacterized protein families (UPFs) (Bateman et al., 2010). It has also been named PF10595 by the protein families (PFAM) project (Finn et al., 2016). While analysis of UPF0564/PF10595 shows that FAM161 proteins are distributed across eukaryote evolution, the lack of any homologues of known function, means that the function of the entire protein family remains unknown.\n\nThe question of proteins of unknown function would be addressed in large measure by determining their structure, which narrows the range of possible functions (Lobb & Doxey, 2016; Zhang et al., 2014). In the absence of solved structure, an interim measure is to predict domain structures using structural bioinformatics, one major branch of which is remote homology sensing of distant homologies between DUFs and domains of known function (Bateman et al., 2010). Following the widespread application of sequence-sequence comparison tools such as BLAST (Altschul et al., 1990), search tools were made more sensitive by adding iterations where the output of one search is submitted as the query for the next. This creates a protein alignment that represents multiple homologues in a profile that carries information about sites of conservation and variation, including where deletions and insertions are tolerated. Iterative tools such as PSI-BLAST perform profile-sequence searches, which find homologues with greater sensitivity (Altschul et al., 1997). A further step-change increase is to use profiles on both sides (i.e. both as query and as target), which is described as a profile-profile search, which requires the preparation in advance of large libraries of profiles (for example: all human proteins, or all PFAM entries) (Yona & Levitt, 2002).\n\nThis article describes sequence-sequence, profile-sequence, and profile-profile searches that strongly predict FAM161A is a homologue of Targeting Protein for Xklp2 (TPX2). The implication is that FAM161 homologues across evolution act as microtubule nucleation factors.\n\n\nMethods\n\nSearches were carried out at the Tuebingen Toolkit, unless otherwise stated (Zimmermann et al., 2018). FAM161A isoform 2 (660 aa, accession number Q3B820) was used in searches, and numbering is for this isoform. Likewise, the Tpx2 sequence used is isoform X1 (747 aa, Q9ULW0).\n\nPSI-BLAST was carried out querying the nr30 or nr50 databases (sequence redundancy reduced so that no sequence shares more than 30% and 50% identity with any other respectively), with inclusion threshold e-value =0.001 for forwarding to subsequent rounds. PSI-BLAST with low complexity filter was carried out at NCBI using the Refseq database (nr100, but 40% smaller than the database called “nr”). HHblits was used iteratively in the same way, except all queries used the nr30 database. Structural predictions were made by PSI-PRED 3.0 within HHpred (Alva et al., 2016; Zimmermann et al., 2018). HHpred was used with default settings, in particular with the Maximum Accuracy realignment off, unless otherwise stated, in which case the default threshold of 0.3 was chosen. Note that all search results exclude hits with pSS <10%, so these weak hits cannot be reported. Simple alignment of two sequences was carried out in NCBI BLASTP with standard settings. To align two proteins with maximal sensitivity, pairwise HHpred profile-profile searches were carried out by replacing the target library with a single converged target profile. To visualise alignments of small regions, multiple sequence alignments were pruned in Jalview by hand to reduce redundancy and fragmentary sequences. Logos were drawn with Weblogo (Crooks et al., 2004). Sequences not already aligned by PSI-BLAST, HHblits or HHpred were aligned with MAFFT.\n\n\nResults\n\nFAM161A is homologous to the microtubule nucleation factor Tpx2\n\nHumans have three members of the FAM161 family (FAM161A/B/C, 569-660 residues), which overlap to a varying extent with the UPF0564/PF10595 domain (Figure 1). FAM161A and FAM161B, are vertebrate-only paralogues, the human proteins being 31% identical (+16% homologous) across the region of 403 residues that matches 90% of UPF0564/PF10595 (Bandah-Rozenfeld et al., 2010). The remainder of this study will focus on FAM161A, with description of FAM161B largely omitted because it is so similar (data not shown). FAM161C (also called testis-specific protein 10-interacting protein, TSGA10IP) in only found in a few mammals. It contains ~40% of the C-terminus of UPF0564/PF10595, and is 29% identical (+24% homologous) to FAM161A across 227 residues in six blocks. This makes it an open question of whether FAM161C has a different function.\n\nAlignments include small gaps (up to 60 residues) that are not indicated. No protein contains 100% of UPR0564/PF10595. Regions of proteins that align with UPR0564/PF10595 are indicated by gray shading, with limits of alignment indicated by wavy lines. Note that alignment between proteins outside the identified domain is not shown.\n\nDatabases created with profile-search tools report UPF0564/PF10595 homologues to be universal in animals, and also to be present in a minority of fungi, protists and plants, indicating that UPF0564/PF10595 has a conserved, fundamental function beyond photoreceptors. Profiles of FAM161A were created to prepare for profile-profile searches. A PSI-BLAST search identified known algal homologues on the first iteration. On the second iteration not only were known fungal homologues added, but also several large proteins annotated as titin (Table 1). These positive alignments were based on large numbers of charged residues dispersed across the whole of FAM161A (Extended data: Supplementary Figure 1A). This makes it very likely the hits represent non-specific false positives, which can occur with PSI-BLAST (Schaffer et al., 2001). In the third iteration, most new hits were titins and further iterations produced massive increases in hits, which were mostly long and repetitive proteins (Table 1). This shows that a profile of FAM161A and its homologues cannot be built using PSI-BLAST with standard settings.\n\nThe numbers of hits with significant homologies (cut-off e-value ≤0.001) are shown for each iteration using four different strategies. 1/2: PSI-BLAST without a filter searching non-redundant databases of different sizes (nr50 and nr30 for #1 and #2 respectively); 3: PSI-BLAST with low complexity filter searching a comprehensive database (nr100); 4. HHblits searching a non-redundant database (nr30). Also showing the number of hits annotated as either titin for #1 and #2 or Tpx2 for #3 and #4 also showing e-value of hit. Red shading indicates multiple sequence alignment was too large for more iterations. Blue shading indicates convergence, with the next iteration identical.\n\nTwo different approaches were used to circumvent this problem. The first was to implement PSI-BLAST with a filter for regions of low complexity, which excluded 9 blocks in FAM161A (Extended data: Supplementary Figure S1B). Multiple sequence alignments of this search barely grew after the second iteration and converged after the sixth iteration (Table 1). The second approach was to use HHblits, which uses hidden Markov models (HMMs) to build the profile (Remmert et al., 2012). While traditional profile tools apply fixed rules on gap opening and deletions, HMMs develop alignment-specific rules. Using HHblits, the profile for FAM161A almost reached its final size at the third iteration, and converged at the ninth iteration.\n\nThe FAM161A HHblits profile was submitted to the profile-profile search tool HHsearch, which is implemented online at the Tuebingen Toolkit as HHpred (Soding, 2005; Soding et al., 2005; Zimmermann et al., 2018). HHpred was set to examine homologues in three databases: solved protein structures (PDB), PFAM, and the human proteome. There was a single significant hit: Tpx2, which stands for targeting protein for Xenopus kinesin-like protein-2 (Xklp2) (Figure 2A). The hit was strong, with predicted shared structure (pSS) ≥95%, and 225 residues were matched (detail in Extended data: Supplementary Figure 2A). This is highly significant, as there were no false positives with hits of this strength in a benchmarking study (Fidler et al., 2016). A similar hit (pSS=98%) was obtained submitting the non-converged profile from the second iteration of PSI-BLAST (data not shown). The fully converged PSI-BLAST profile was too large to submit. Reverse searches, seeded with Tpx2 or its internal domain “Tpx2_importin” (PF12214), showed the same homologies to FAM161A/UPF0564 (Figure 2A), which is important as profile-profile searches can sometimes produce asymmetric results (Levine, 2019).\n\n(A) Results of pair-wise HHpred searches between FAM161A, Tpx2 and the “Tpx2_importin” domain (PF12214, which covers residues 362-498 of Tpx2- see part B). The probability of shared structure (pSS, as %) and the number of columns (in brackets below) are reported for all pairwise searches. Results in PDB are identical to the human proteome, because Tpx2 is present in PDB as 6BJC_T with no additional DSSP secondary structural information. NB: self-searches (long diagonal) have pSS=100% and include all columns. (B) Alignment of FAM161A with Tpx2. 320 residues of FAM161A (229–548) align with 228 residues of Tpx2 (474-701), which spans repeats 5 to 8. The probability of shared structure (pSS) =96%. Two adjacent repeats in FAM161A (79 and 88 residues, 239-317 and 318-395, both within the portion homologous to UPF0564) also align (pSS=96%, shaded semi-circle). Tpx2 repeats are coloured in a rainbow from violet to red. The region included in a cryo-EM structure with tubulin (residues 300-341) is shown by the dotted purple box. The region included in PF12214 is shown by the grey transparent box from repeat 3 to 6. (C) pSS of pairwise comparisons of each repeat in FAM161A and Tpx2, where repeat boundaries are 25 residues down-stream of those in B (arrow). Shading of each cell represents the average pSS of two searches on a grey scale with pSS from 10-100% repesented by grey 10-100%, with FAM161A as query (bottom left); and with Tpx2 as query (top right). The structures labelled R and W in repeat 2 of Tpx2 are the ridge and wedge that bind tubulin (see Figure 3).\n\nThe aligned region of FAM161A was residues 229–548; the region of Tpx2 that matched it was 474–701 (Figure 2B). Tpx2 has previously been shown to contain nine repeats of length 45–60 residues occupying its final two thirds (residues 222–747) (Sanchez-Pulido et al., 2016). The alignment covered from the C-terminal half of repeat 5 to the start of repeat 9. FAM161A also produced out-of-register hits with itself, indicating the presence of two repeats of ~70 residues (237–312 and 320–385, Figure 2B). In addition, a partial third repeat was found ~60 residues downstream (447-463) (Figure 2B, and see below). Using a non-default setting designed to increase accuracy of alignments, the Maximum Accuracy algorithm (MAC), repeats 1 and 2 only (FAM161A 236-361) showed homology to four regions of Tpx2, the strongest focussed on repeat 6, and others shifted either one or two whole repeats further on, or two repeats earlier (Extended data: Supplementary Figure S2B).\n\nSince repeats can occasionally align out of register, reducing the specificity for individual repeats, all repeats from FAM161A and Tpx2 were compared against each other. This showed that repeat 1 of FAM161A is most like Tpx2 (particularly its repeats 4/6/7), followed by repeat 2 of FAM161A and then repeat 3 (Figure 2C). In addition, the presence of non-conserved unstructured regions just prior to each repeat in FAM161A and in the centres of each repeat in Tpx2 suggest that the boundaries of Tpx2 repeats should be drawn in a different register that matches FAM161A, approximately 25 residues further toward the C-terminus than previously (Sanchez-Pulido et al., 2016). This shortens repeat 9, which shows no homologies to FAM161A, as might be expected given its well-defined function to bind to kinesin (Eckerdt et al., 2008). Homologies detected between individual repeats might be revealing. Among the Tpx2 repeats, repeat 2 is an outlier. While all the others are homologous to 3 others, it shows homologies only to itself (data not shown). However, it does show weak homology to a single region of FAM161A in repeat 3 (Figure 2C).\n\nFinally, a re-examination of the profile-sequence searches supported the homology between FAM161A and Tpx2 homology: PSI-BLAST and HHblits both produced occasional significant hits to Tpx2 (Table 1, rows 3 and 4); also a large proportion of the weak hits marginally outside the inclusion cut-off are Tpx2 (data not shown).\n\nOverall, these results strongly predict that FAM161A is a homologue of Tpx2.\n\nFAM161A has three repeats of the extended loop in Tpx2 that nucleates microtubules\n\nTpx2 is a microtubule-associated protein (MAP) (Wittmann et al., 1998), as is FAM161A (Zach et al., 2012). While the interaction with microtubules by FAM161A is not understood in detail, the tubulin–Tpx2 interaction has been studied by cryo-EM in ultrastructural detail at 3.3 Angstrom resolution (Zhang et al., 2017). Two short structural elements in Tpx2 in a region of 42 residues starting in repeat 2 (300-341, purple box in Figure 2B) contact four tubulins, showing how Tpx2 can stimulate microtubule nucleation (Figure 3A, left) (Zhang et al., 2017). This region coincides with the redrawn boundaries of repeat 2 (Figure 2C). Further analysis is required to determine if the homology uncovered by HHpred between this repeat and repeat 3 of FAM161A is consistent with FAM161A having elements that crosslink tubulin.\n\nThe first microtubule nucleating element in repeat 2 of Tpx2 is the extended loop 300GCTIVKPFNLSQ311, which forms a “ridge” that runs parallel to protofilaments to crosslink α- and β-Tubulin. The conserved residue F307 binds in a hydrophobic pocket formed by both tubulins (Figure 3A, right) (Zhang et al., 2017). Profile-profile searches showed that five other Tpx2 repeats (1/4/6/7/8) contain ridge-like sequences, which are characterised by Pro-Phe-charged/polar-hydrophobic (PF±Ø) motifs (Figure 3B, left). The motif is missing from repeats 3 and 5, although other aspects of these repeats are preserved (Figure 3B, right).\n\n(A) Left: scheme of binding of one copy of Tpx2 residues 300-341 (rainbow blue→red) binding to four tubulin monomers. Right: close up of the surfaces of the tubulins and Tpx2, shown as a ribbon, with the ridge (300-311) and wedge (323-341), but residues 312-322 modelled as a straight rod (length 2.4 nm) as they not seen in the cryo-EM structure. Conserved side-chains in Tpx2 (F307 F334: black, H335: blue) bind into pockets formed at tubulin interfaces. Image derived from PDB 6BJC chains A-D and P (Zhang et al., 2017). The asterisk indicates space at the C-terminus of the wedge (see Discussion). (B) FAM161A and Tpx2 contain repeated ridge-like motifs. Left: Ungapped alignment of extended loops from repeats 1–3 of FAM161A and repeats 1–8 of Tpx2, indicating PF±Ø motifs. Pink box indicates the ridge sequence in repeat 2. Sequence directly after repeat 2 is also included, showing the linker (green letters) and wedge (red letters, helix in blue box). Centre: yellow boxes indicate charged predicted helices (average length 25 aa, numbers of missing residues shown). Right: Extended loops following the helices, containing FKA±PxP motifs. The analogous sequences in repeat 3 of both proteins do not form motifs, but are included for comparison (grey letters). Shading is according to the Clustal scheme. Numbers on the right indicate omitted residues between repeats. (C) Comparison of the wedge helix in Tpx2 with the amphipathic helix in FAM161A. Both the wedge of Tpx2 (blue box, left) and the predicted amphipathic helix of FAM161A (green box, right) are accompanied by logos made from 40 diverse sequences, and by a consensus in which bold lettering indicates a strong conservation, Ø is hydrophobic (black), ± is charged or polar, and other colouring is: D/E-red, KRH-blue, Q-purple, A-black.\n\nFAM161A repeats 1 and 2 both contain ridge-like sequences with PF±Ø. In addition, a third ridge-like sequence was found in FAM161A ~80 residues beyond C-terminus of repeat 2, and this was therefore designated as a partial third repeat. This region of FAM161A is homologous to FAM161C (Extended data: Supplementary Figure S2B), and although FAM161C lacks the key phenylalanine (336PQKL339), a variant PF±Ø motif may lie upstream (318PWDL321).\n\nRidge-like sequences in Tpx2 repeats 4–8 run directly into helices (Alfaro-Aco et al., 2017). The helices are on average 25 aa, and they are highly enriched for charged residues. The charged helices are followed by unstructured loops that contain characteristic FKA±PxP motifs (Figure 3B, centre and right), which have been noted previously (Alfaro-Aco et al., 2017). Significantly, FAM161A repeats 1 and 2 both have the same loop-charged helix-loop form (Figure 3B). Therefore, overall FAM161A contains three copies of sequences that align without gaps to the ridge in Tpx2, which is itself repeated six times. Two of the FAM161A repeats and four of the Tpx2 repeats have the same arrangement, namely loop (ridge-like, PF±Ø)–charged helix–loop (FKA±PxP).\n\nFAM161A contains an amphipathic helix that resembles the “wedge” of Tpx2\n\nA second element in Tpx2 that nucleates microtubules is a short helix (10 residues) that has been described as a “wedge”. This fits into a deep pocket that forms between four tubulin monomers between side-by-side protofilaments (Extended data: Supplementary Figure S3) (Zhang et al., 2017). The helix is amphipathic, having hydrophobic residues in positions 1/5/8 (i.e. all on the same face), with charges or large polar residues at most other positions (Figure 3C). Two conserved residues on the helix (334FH335) contribute to microtubule nucleation by binding into a hydrophobic pocket formed by two tubulin monomers (Figure 3A). The wedge is separated from the ridge by an 11 aa linker, which is long enough to reach across one β Tubulin molecule between the two different interfaces (Figure 3A). Unlike the ridge which is repeated six times, Tpx2 only contains one short amphipathic helix, as indicated by a higher hydrophobic moment compared to the charged helices in the other repeats (Extended data: Supplementary Figure S4) (Gautier et al., 2008).\n\nWhile one of the five helices found in repeats with ridge-like sequences is amphipathic, neither of the two helices in FAM161A repeats are amphipathic (Extended data: Supplementary Figure S4). However, another location in FAM161A, immediately before the third ridge-like sequence, does contain a predicted amphipathic helix (Extended data: Supplementary Figure S2). This is the only region of FAM161A that shows specific homology to repeat 2 of Tpx2 (Figure 2C). While this helix is 14 residues, making it one turn longer than the helix in Tpx2, it is otherwise similar, with regularly spaced hydrophobic residues, including a phenylalanine at the 8th position, as found in Tpx2 (Figure 3C). This region is widely conserved in FAM161A homologues across evolution (Extended data: Supplementary Figure S5), though there are some exceptions: in mouse the FAM161A helix is shorter, and in trypanosomes the key aromatic side chain is missing. FAM161B and FAM161C contain variations of this amphipathic helix (375YEGLYKAFQRRAAK388 and 321LEKLHRQLQRDL332 respectively). In FAM161C this overlaps the PF±Ø-like motif identified above, so binding of the helix and the ridge would be mutually exclusive.\n\nThus, FAM161A has a sequence that has many properties of the wedge in Tpx2, except for making one additional helical turn.\n\n\nDiscussion\n\nHere, FAM161A is shown to contain repeats similar to those in Tpx2. Since the way Tpx2 interacts with microtubules is now understood in great detail (Zhang et al., 2017), it has been possible to show that the sequence of FAM161A shares most of the requirements to bind microtubules too, in particular having three ridge-like extended loops, as well as a more variant wedge-like helix.\n\nThe residues of both the ridge and wedge of Tpx2 fit precisely into the surface of tubulin. This suggests that ridges and wedges may not tolerate gaps or insertions, and is consistent with the strongly conserved pattern found in ungapped alignments of FAM161A (Extended data: Supplementary Figure S5A). The homology might be missed if gaps are tolerated during alignment (Sanchez-Pulido et al., 2016). The application of HMMs, which adjust these parameters for each search, might underlie the considerable gain in sensitivity obtained here.\n\nThe main outstanding question is whether the FAM161A helix is functionally like the wedge in Tpx2. Given the requirement to bind onto the surface of tubulin, which is highly conserved, the additional full turn in the only predicted amphipathic helix in FAM161A might be expected to prevent it fitting into the deep pocket in tubulin, despite it sharing many biochemical properties with the wedge of Tpx2 (Figure 3C). However, the wedge in some Tpx2 homologues is also predicted to be extended by one turn, as they lack the helix-breaking proline (Extended data: Supplementary Figure S6). On this point, the tubulin structure leaves unfilled space room beyond the C-terminus of the Tpx2 wedge (asterisk in Figure 3A and Extended data: Supplementary Figure S3B). Another piece of evidence is that repeat 2 in Tpx2 shows homology only to repeat 3 of FAM161A. Although this alignment is based solely on the ridge (Figure 2C and data not shown), it suggests that the nearby amphipathic helices may share a function. One way forward to examine this question would be to study protein docking in silico. As for the finding that the proposed ridges and wedge in FAM161A are not arranged in the same relationship as found in Tpx2, such differences exist among Tpx2 homologues. For example, in algae the wedge precedes all ridge repeats (data not shown). If it were determined that the FAM161A helix cannot act as a wedge, it is worth noting that Tpx2 homologues in some species do not have any wedge: for example, none of the three short Tpx2 homologues in flies have one (data not shown) (Sanchez-Pulido et al., 2016).\n\nOne of the first molecular studies of FAM161A described it as a MAP (Zach et al., 2012). At endogenous levels, it localises to the basal body at the base of the primary cilium and in the inner segment which contains a mixture of organelles (Di Gioia et al., 2012). When over-expressed it localises to microtubules. Its 140 interactors detected by yeast two-hybrid are enriched for residents of the cilium (Di Gioia et al., 2012) and also residents of the Golgi, centrosome and microtubules (Di Gioia et al., 2015). Two centrosomal interactors are well described: transforming acidic coiled-coil-3 (TACC3) (Gomez-Baldo et al., 2010), and proteome of the centriole-1B (POC1B, also called WDR51B) (Di Gioia et al., 2015). The centriole-specific function of FAM161A is now understood in ultrastructural detail, and involves the interaction with POC1B, as well as POC5 and centrin form a complex that multimerises into a cylindrical scaffold adhering to the inside of centrioles (Le Guennec et al., 2020). This scaffold is different from the centriole’s internal SAS-6 cartwheel that projects radially from the central axis (Gonczy, 2012). FAM161A plays a central role, binding not only the three other components, but also tubulin. The molecular basis for that binding has previously been unknown, but the findings here provide models for this interaction. Given that another cylindrical layer containing WDR90 is proposed to lie between the FAM161A layer and microtubules (Steib et al., 2020), FAM161A may only be able bind microtubules through gaps in the WDR90 layer. This might explain how evolution has produced small microtubule-binding domains of short loops and helices (Figure 4).\n\nSequence and structural motifs as shown in key: prism = loop similar to ridge with PF±Ø; ovoid = loop with FKA±PxP; half cylinder = amphipathic helix: wedge–blue, FAM161A–green; yellow spheres: NLS; zig-zag = all charged helix. Repeat boundaries shown as in Figure 2C, with two known binding sites in Tpx2: aurora kinase A at N-terminus (black) and kinesin at C-terminus (red).\n\nThe precise function of FAM161A can be interpreted by comparing its overall form with Tpx2 (Figure 4). Tpx2 has three separate microtubule binding sites: (1) the N-terminal 240 residues, which includes the extreme N-terminal Aurora A kinase binding site (Brunet et al., 2004); (2) residues 236-352 (repeats 1&2, including ridge and wedge) (Trieselmann et al., 2003); (3) residues 547-579 (repeat 6) and an undefined amount of flanking sequence (i.e. ridge repeats, possibly more than one) (Trieselmann et al., 2003). A truncation of six repeats (2–7, residues 274-659) can nucleate microtubules, albeit sub-optimally (Roostalu et al., 2015). Maybe the three repeats in FAM161A can act in the same way. Tpx2 is regulated by RanGTP (Gruss et al., 2001), but this is not likely to occur in FAM161A. The NLS in Tpx2 overlaps the ridge, so that binding to importin and tubulin are mutually exclusive (Giesecke & Stewart, 2010). In contrast, the predicted NLS in FAM161A (224-230) is not near the tubulin binding sites (Figure 4). The second extended loop found after the charged helix in most repeats of both Tpx2 and FAM161A has yet to be studied, but its conserved motif suggests a specific interaction (Alfaro-Aco et al., 2017), which might be with tubulin, other copies of Tpx2 or another MAP in stoichiometric complexes.\n\nIn summary, the homologies discovered here (Figure 2) strongly predict that FAM161A is a previously unknown homologue of Tpx2. This could underlie its binding to tubulins in centrioles, and also allow the pool of FAM161A in other parts of the cell, possibly the Golgi apparatus (Di Gioia et al., 2012), to be a microtubule nucleation factor. The homologies produce testable models to study the function of FAM161A by directed mutagenesis of residues that align with Tpx2.\n\n\nData availability\n\nUniProtKB - Q3B820 (F161A_HUMAN), Accession number Q3B820: https://www.uniprot.org/uniprot/Q3B820\n\nUniProtKB - Q9ULW0 (TPX2_HUMAN), Accession number Q9ULW0: https://www.uniprot.org/uniprot/Q9ULW0\n\nHarvard Dataverse: Extended data, https://doi.org/10.7910/DVN/EVAGZU (Levine, 2020).\n\nThis project contains the file ‘Supplementary figures.pdf’, which contains the following extended data:\n\nSupplementary Figure S1: A. Hit to titin in the second iteration of PSI-BLAST into the nr50 database; B. Low complexity regions in FAM161A.\n\nSupplementary Figure S2: A. HHpred result of symmetrical pairwise alignment of Fam161A and Tpx2; B. HHpred search with alignment realigned with Maximum Accuracy algorithm\n\nSupplementary Figure S3: The wedge helix of Tpx2 is buried deeply in the pocket formed by four tubulin monomers\n\nSupplementary Figure S4: Properties of helices following ridge sequences in Tpx2 and FAM161A\n\nSupplementary Figure S5: Sequences in amphipathic helices in the FAM161 family\n\nSupplementary Figure S6: Variation of Tpx2 ridge and wedge sequences across species.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nI thank Dr Matt Hayes for discussions.\n\n\nReferences\n\nAlfaro-Aco R, Thawani A, Petry S: Structural analysis of the role of TPX2 in branching microtubule nucleation. J Cell Biol. 2017; 216(4): 983–997. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J Mol Biol. 1990; 215(3): 403–410. PubMed Abstract \| Publisher Full Text\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlva V, Nam SZ, Söding J, et al.: The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res. 2016; 44(W1): W410–415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAthanasiou D, Aguila M, Bellingham J, et al.: The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy. Prog Retin Eye Res. 2018; 62: 1–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBandah-Rozenfeld D, Mizrahi-Meissonnier L, Farhy C, et al.: Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010; 87(3): 382–391. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBateman A, Coggill P, Finn RD: DUFs: families in search of function. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010; 66(Pt 10): 1148–1152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunet, S, Sardon T, Zimmerman T, et al.: Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts. Mol Biol Cell. 2004; 15(12): 5318–5328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrooks GE, Hon G, Chandonia JM, et al.: WebLogo: a sequence logo generator. Genome Res. 2004; 14(6): 1188–1190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaiger SP, Sullivan LS, Bowne SJ: Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013; 84(2): 132–141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Gioia SA, Farinelli P, Letteboer SJ, et al.: Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network. Hum Mol Genet. 2015; 24(12): 3359–3371. PubMed Abstract \| Publisher Full Text\n\nDi Gioia SA, Letteboer SJ, Kostic C, et al.: FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies. Hum Mol Genet. 2012; 21(23): 5174–5184. PubMed Abstract \| Publisher Full Text\n\nEckerdt F, Eyers PA, Lewellyn AL, et al.: Spindle pole regulation by a discrete Eg5-interacting domain in TPX2. Curr Biol. 2008; 18(7): 519–525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEstrada-Cuzcano A, Neveling K, Kohl S, et al.: Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement. Am J Hum Genet. 2012; 90(1): 102–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFidler DR, Murphy SE, Courtis K, et al.: Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains. Traffic. 2016; 17(11): 1214–1226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinn RD, Coggill P, Eberhardt RY, et al.: The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016; 44(D1): D279–285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGautier R, Douguet D, Antonny B, et al.: HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics. 2008; 24(18): 2101–2102. PubMed Abstract \| Publisher Full Text\n\nGiesecke A, Stewart M: Novel binding of the mitotic regulator TPX2 (target protein for Xenopus kinesin-like protein 2) to importin-alpha. J Biol Chem. 2010; 285(23): 17628–17635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomez-Baldo L, Schmidt S, Maxwell CA, et al.: TACC3-TSC2 maintains nuclear envelope structure and controls cell division. Cell Cycle. 2010; 9(6): 1143–1155. PubMed Abstract \| Publisher Full Text\n\nGönczy P: Towards a molecular architecture of centriole assembly. Nat Rev Mol Cell Biol. 2012; 13(7): 425–435. PubMed Abstract \| Publisher Full Text\n\nGruss OJ, Carazo-Salas RE, Schatz CA, et al.: Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell. 2001; 104(1): 83–93. PubMed Abstract \| Publisher Full Text\n\nLangmann T, Di Gioia SA, Rau I, et al.: Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa. Am J Hum Genet. 2010; 87(3): 376–381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe Guennec M, Klena N, Gambarotto D, et al.: A helical inner scaffold provides a structural basis for centriole cohesion. Sci Adv. 2020; 6(7): eaaz4137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevine TP: Remote homology searches identify bacterial homologues of eukaryotic lipid transfer proteins, including Chorein-N domains in TamB and AsmA and Mdm31p. BMC Mol Cell Biol. 2019; 20(1): 43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevine TP: EXTENDED DATA: for Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/EVAGZU\n\nLobb B, Doxey AC: Novel function discovery through sequence and structural data mining. Curr Opin Struct Biol. 2016; 38: 53–61. PubMed Abstract \| Publisher Full Text\n\nNishimura DY, Baye LM, Perveen R, et al.: Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71. Am J Hum Genet. 2010; 86(5): 686–695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRemmert M, Biegert A, Hauser A, et al.: HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods. 2012; 9(2): 173–175. PubMed Abstract \| Publisher Full Text\n\nRoostalu J, Cade NI, Surrey T: Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module. Nat Cell Biol. 2015; 17(11): 1422–1434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRůžičková Š, Staněk D: Mutations in spliceosomal proteins and retina degeneration. RNA Biol. 2017; 14(5): 544–552. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanchez-Pulido L, Perez L, Kuhn S, et al.: The C-terminal domain of TPX2 is made of alpha-helical tandem repeats. BMC Struct Biol. 2016; 16(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchaffer AA, Aravind L, Madden TL, et al.: Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 2001; 29(14): 2994–3005. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSöding J: Protein homology detection by HMM-HMM comparison. Bioinformatics. 2005; 21(7): 951–960. PubMed Abstract \| Publisher Full Text\n\nSöding J, Biegert A, Lupas AN: The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005; 33(Web Server issue): W244–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteib E, Gambarotto D, Laporte M, et al.: WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity. BioRxiv. 2020; Publisher Full Text\n\nTrieselmann N, Armstrong S, Rauw J, et al.: Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation. J Cell Sci. 2003; 116(Pt 23): 4791–4798. PubMed Abstract \| Publisher Full Text\n\nWittmann T, Boleti H, Antony C, et al.: Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein. J Cell Biol. 1998; 143(3): 673–685. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWood V, Lock A, Harris MA, et al.: Hidden in plain sight: what remains to be discovered in the eukaryotic proteome? Open Biol. 2019; 9(2): 180241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYona G, Levitt M: Within the twilight zone: a sensitive profile-profile comparison tool based on information theory. J Mol Biol. 2002; 315(5): 1257–1275. PubMed Abstract \| Publisher Full Text\n\nZach F, Grassmann F, Langmann T, et al.: The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule association. Hum Mol Genet. 2012; 21(21): 4573–4586. PubMed Abstract \| Publisher Full Text\n\nZhang D, Iyer LM, Burroughs AM, et al.: Resilience of biochemical activity in protein domains in the face of structural divergence. Curr Opin Struct Biol. 2014; 26: 92–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang R, Roostalu J, Surrey T, et al.: Structural insight into TPX2-stimulated microtubule assembly. eLife. 2017; 6: e30959. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmermann L, Stephens A, Nam SZ, et al.: A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. J Mol Biol. 2018; 430(15): 2237–2243. PubMed Abstract \| Publisher Full Text" }	[ { "id": "70563", "date": "01 Oct 2020", "name": "Vikram Alva", "expertise": [ "Reviewer Expertise Protein bioinformatics and evolution" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article by Timothy Levine substantiates a homologous relationship between the microtubule-associated ciliary protein FAM161A and the microtubule-associated spindle assembly factor Tpx2. While the interaction between Tpx2 and microtubules has been characterized structurally using cryo-EM, the interaction between FAM161A and microtubules remains poorly understood. Based on the conservation of microtubule-binding elements of Tpx2 in FAM161A, the author proposes that both these proteins bind microtubules in a similar fashion.\nThe homologous connection between FAM161A and Tpx2, although distant, is well substantiated. The manuscript is very well written, and I have only some minor comments.\nPage 3, Methods section: \"PSI-BLAST with low complexity filter was carried out at NCBI using the Refseq database (nr100, but 40% smaller than the database called “nr”)\".\nRefseq is not the same as nr100! https://www.ncbi.nlm.nih.gov/books/NBK50679/#RefSeqFAQ.what_is_a_reference_sequence_r\n\nPage 3, Methods section: \"HHblits was used iteratively in the same way, except all queries used the nr30 database\".\nI believe the HHblits searches reported in this study were carried out against the Unclust30 database and not the nr30 database; this also needs to be corrected in Table 1.\n\nThe author refers to the probability values yielded by HHpred as the probability of shared structure (pSS). This is, however, incorrect. While the sequence similarity of proteins reflects homology, structural similarity alone may often be analogous because only a limited number of folded conformations are available to the polypeptide chain. Consequently, proteins unrelated in sequence may exhibit substantially similar structures. However, in such cases, HHpred typically does not report high probability values. The probability value reported by HHpred is an estimate for a match to be a true positive. It allows inferring if an obtained match is homologous to the query or is just a chance hit.\n\nPage 4, paragraph 1: \"FAM161A and FAM161B, are vertebrate-only paralogues, the human proteins being 31% identical (+16% homologous) across the region of 403 residues that matches 90% of UPF0564/PF10595\".\nIt is unclear what the author means by 16% homologous. Sequence identity can be quantified, but homology is a qualitative description of the relationship between proteins and cannot be quantified.\n\nFAM161A and Tpx2 appear to contain multiple coiled-coil segments. It would be useful to the readers if the author could comment on this in the text.\n\nAre the microtubule-binding elements observed in Tpx2 and FAM161A also present in FAM161B and FAM161C? The author could consider including a few sentences on this in the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "70565", "date": "12 Oct 2020", "name": "Paul Guichard", "expertise": [ "Reviewer Expertise Cell Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTimothy Levine's research paper is a bioinformatic analysis of the FAM161a protein. This study identified regions within FAM161A homologous to the Tpx2 protein. FAM161a is a microtubule-associated protein that is localized at the centrioles/basal bodies, Golgi apparatus, and at the connecting cilium of the photoreceptors. Truncations of this protein cause retinitis pigmentosa, a disease leading to photoreceptor degeneration and subsequent blindness. Since this protein is poorly studied, this bioinformatic analysis is important because it defines potential functional domains for this protein. This analysis can be used as a basis for future in-depth biochemistry and structural biology studies.\n\nComputer databases indicate that a domain called UPR0564/PF10595 exists in this protein, as well as in its homologs FAM161A and FAM161C. However, no function has been attributed to this domain. From that point on, T. Levine used PSI-Blast and hidden Markov models (HMMs) approaches to find homologs. Importantly, HMMs identified TPX2, a well-characterized microtubule-binding protein that contains nine repeats of length 45–60 residues, as a putative homolog. In addition, T. Levine identified two to three repeats in the sequence of FAM161A of about 60-70 residues that showed homology to four regions of TPX2. Further analysis, using the known structural interaction of TPX2 with microtubules, leads to propose that FAM161A homologous sequence contains three ridge-like extended loops, as well as a more variant wedge-like helix, structural domains that interact with microtubules and therefore are potentially crucial for FAM161A function.\n\nThis bioinformatics analysis is very interesting and reveals a surprising homology with TPX2. Thanks to this work, T. Levine defines potential functional domains of this protein. This work is a crucial starting point to study the structure and function of FAM161A in order to better understand the mechanisms that leads to retinitis pigmentosa.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1052
https://f1000research.com/articles/9-415/v1	21 May 20	{ "type": "Research Article", "title": "Inflated citations and metrics of journals discontinued from Scopus for publication concerns: the GhoS(t)copus Project", "authors": [ "Andrea Cortegiani", "Mariachiara Ippolito", "Giulia Ingoglia", "Andrea Manca", "Lucia Cugusi", "Anna Severin", "Michaela Strinzel", "Vera Panzarella", "Giuseppina Campisi", "Lalu Manoj", "Cesare Gregoretti", "Sharon Einav", "David Moher", "Antonino Giarratano", "Mariachiara Ippolito", "Giulia Ingoglia", "Andrea Manca", "Lucia Cugusi", "Anna Severin", "Michaela Strinzel", "Vera Panzarella", "Giuseppina Campisi", "Lalu Manoj", "Cesare Gregoretti", "Sharon Einav", "David Moher", "Antonino Giarratano" ], "abstract": "Background: Scopus is a leading bibliometric database. It contains the largest number of articles cited in peer-reviewed publications. The journals included in Scopus are periodically re-evaluated to ensure they meet indexing criteria and some journals might be discontinued for publication concerns. These journals remain indexed and can be cited. Their metrics have yet to be studied. This study aimed to evaluate the main features and metrics of journals discontinued from Scopus for publication concerns, before and after their discontinuation, and to determine the extent of predatory journals among the discontinued journals. Methods: We surveyed the list of discontinued journals from Scopus (July 2019). Data regarding metrics, citations and indexing were extracted from Scopus or other scientific databases, for the journals discontinued for publication concerns. Results: A total of 317 journals were evaluated. Ninety-three percent of the journals (294/318) declared they published using an Open Access model. The subject areas with the greatest number of discontinued journals were Medicine (52/317; 16%), Agriculture and Biological Science (34/317; 11%), and Pharmacology, Toxicology and Pharmaceutics (31/317; 10%). The mean number of citations per year after discontinuation was significantly higher than before (median of difference 64 citations, p<0.0001), and so was the number of citations per document (median of difference 0.4 citations, p<0.0001). Twenty-two percent (72/317) were included in the Cabell’s blacklist. The DOAJ currently included only 9 journals while 61 were previously included and discontinued, most for 'suspected editorial misconduct by the publisher'. Conclusions: The citation count of journals discontinued for publication concerns increases despite discontinuation and predatory behaviors seemed common. This paradoxical trend can inflate scholars’ metrics prompting artificial career advancements, bonus systems and promotion. Countermeasures should be taken urgently to ensure the reliability of Scopus metrics both at the journal- and author-level for the purpose of scientific assessment of scholarly publishing.", "keywords": [ "predatory", "journal", "Scopus", "metrics", "indexing", "citation count" ], "content": "Introduction\n\nScopus is a leading bibliometric database launched in 2004 by the publishing and analytics company Elsevier. It was developed by research institutions, researchers and librarians, and contains the largest number of abstracts and articles cited in peer reviewed academic journal articles that cover scientific, technical, medical, and social science fields1.\n\nScopus provides bibliometric indicators that many institutions use to rank journals to evaluate the track record of scholars who seek hiring or promotion. These metrics are also used to allocate financial bonuses or to evaluate funding applications2–4. Ensuring the quality of the content of the Scopus database is, therefore, of great importance.\n\nTo be indexed in Scopus, journals are evaluated and periodically reviewed by an independent and international Content Selection and Advisory Board (CSAB), which is a group of scientists, researchers and librarians, comprised of 17 Subject Chairs, each representing a specific subject field, and by a computerized algorithm1. At any time after a journal inclusion, concerns regarding its quality may be raised by a formal complaint, thereby flagging the journal for re-evaluation by the CSAB. Should the CSAB panel determine that the journal no longer meets Scopus standards, new articles from that journal are no longer be indexed1. One of the most common reasons for discontinuation is ‘publication concerns’, which refers to the quality of editorial practices or other issues that have an impact on its suitability for continued coverage5. The list of the discontinued sources is publicly available and is updated approximately every six months6. However, publications from no longer indexed journals may not be removed retrospectively from Scopus. Hence, articles indexed prior to the date of discontinuation could remain part of the database7.\n\nIt has been claimed that a number of journals discontinued from Scopus for publication concerns might be so-called ‘predatory’ journals6–8. Predatory journals “prioritize self-interest at the expense of scholarship and are characterized by false or misleading information, deviation from best editorial and publication practices, a lack of transparency, and/or the use of aggressive and indiscriminate solicitation practices”9. Since researchers are pressured to publish in indexed journals, predatory journals are constantly trying to be indexed in the Scopus database, thereby boosting their attractiveness to researchers2,7,8,10. Having articles from predatory journals indexed in Scopus poses a threat to the credibility of science and might cause harm particularly in fields where practitioners rely on empirical evidence in the form of indexed journal articles10,11.\n\nWe hypothesize that, even though Scopus coverage is halted for discontinued journals, still they can get citations, as all their documents already indexed remain available to users. To date, the metrics of those journals discontinued for publication concerns have not been studied yet. Therefore, by the present analysis we set out to (1) evaluate the main scientific features and citation metrics of journals discontinued from Scopus for publication concerns, before and after discontinuation, and (2) determine the extent of predatory journals included in the discontinued journals.\n\n\nMethods\n\nThe freely accessible and regularly updated Elsevier list12 of journals discontinued from the Scopus database (version July 2019)13 was accessed on 24th January 2020 (See Underlying data12). We restricted our analysis to journals discontinued for “publication concerns”. Journals were checked for relevant data (described below), then independently collected by eight of the authors in pairs (MI, GI, AM, LC, AS, MS, VP, AC) using a standardized data extraction form (Underlying data Table 1). A second check was performed by other four authors (LM, CG, SE, AG) to confirm the data and resolve discrepancies. Data collection was initiated on 24th January and completed by the end of February 2020. Confirmed data were registered on an Excel datasheet (Underlying data, Table 112).\n\nData were extracted either from the Scopus database13 or by searching other sources, such as SCImago Journal & Country Rank (SJCR)14, Journal Citation Reports, Centre for Science and Technology Studies (CWTS) Journal Indicators15, Beall’s updated List16, Directory of Open Access Journals (DOAJ)17, PubMed18 and Web of Science19. Open Access policy was checked on journals websites. A standardized data extraction form, independently applied by eight authors (MI, GI, AM, LC, AS, MS, VP, AC), was used to collect the following data: journal title, name and country of the publisher, the number of years of Scopus coverage, year of Scopus discontinuation, subject areas and sub-subject areas, Impact Factor (IF), CiteScore, SCImago Journal Rank (SJR), Source Normalized Impact per Paper (SNIP), best SCImago quartile, inclusion in PubMed, Web Of Science (WOS) and DOAJ (for open access journals) databases, presence in the updated Beall’s List, total number of published documents and total number of citations. All the metrics were checked on the year of Scopus discontinuation. In cases of discrepancies between Scopus data and other sources, the Scopus database was the preferential source.\n\nWe defined the ‘before discontinuation’ time frame as the period comprised within the first year of journal coverage by Scopus and the year of discontinuation, which was not included in our calculations. By ‘after discontinuation’ time frame, we referred to the period comprised within the year of Scopus discontinuation and the year of our data collection. In cases of multiple discontinuations, we considered the last one, according to the date of the last document displayed in the Scopus database. Citations ‘before’ and ‘after’ the date of discontinuation were manually counted based on either the Scopus journal overview or the downloadable tables made available by Scopus upon request (see Source data). When evaluating the journal inclusion in PubMed, WOS and DOAJ, year 2019 was considered as the reference year, preventing disadvantages for journals with time gaps for publication.\n\nFinally, one author (AS) checked whether discontinued journals were present in Cabell’s whitelist or blacklist20 or the DOAJ’s list of discontinued journals21. As some of the journals included in the blacklist lack ISSNs or other unique identifiers, the comparison of the three lists with Scopus’s discontinued journals was based on matching the journals’ names by similarity using the Jaro-Winkler algorithm in RStudio Desktop 1.2.5033 and RecordLinkage 0.4–11.2 following the approach developed by Strinzel et al. (2019)22,23. The Jaro-Winkler metric, scaled between 0 (no similarity) and 1 (exact match), was calculated for all possible journals’ pairings24. We manually inspected all pairs with a Jaro-Winkler metric smaller than one in order to include cases where, due to the orthographical differences between the lists, no exact match was found. For each matched pair, we compared the journals’ publishers and, where possible, ISSNs, to exclude any cases where two journals had the same or a similar name but were edited by different publishers.\n\nFull definitions and descriptions of the sources and metrics are reported in the Extended Data Appendix 125.\n\nAll data management and calculations were performed using Microsoft Excel (version 2013, Microsoft Corporation®, USA) and GraphPad Prism (version 8.3.1, 322, GraphPad software®, San Diego California). The normality of the distribution was assessed with the D’Agostino-Pearson test. Means and standard deviations (SDs) for variables with normal distribution or medians, interquartile ranges (IQRs, 25th–75th) and ranges (minimum value - maximum value) for non-normally distributed data were reported. Categorical data were expressed as proportions and percentages.\n\nThe paired sample t test or the Wilcoxon matched-pairs signed ranked test were used to compare journals’ data before and after Scopus discontinuation, as appropriate.\n\n\nResults\n\nData could be retrieved regarding 317 of the 348 journals listed as discontinued (91.1%).\n\nAmong the 135 publishers identified, the publishers with the largest number of discontinued journals were: Academic Journals Inc. (39/317; 12.3%), Asian Network for Scientific Information (19/317; 6.0%), and OMICS Publishing Group (18/317; 5.7%). Table 1 reports the distribution of Scopus discontinued journals by publisher. United States (76/317, 24%), India (63/317, 20%) and Pakistan (49/317, 15%) were the most common countries where publishers declared they were headquartered (Figure 1 and Table 2).\n\nData are reported as percentages and fractions. Publishers with less than four Scopus discontinued journals were grouped as ‘Others’.\n\nThe map chart shows the different frequencies of distribution by country with different colors.\n\nData were retrieved from Scimago Journal & Country Rank and are reported as percentages and fractions. Countries with less than four Scopus discontinued journals were grouped as ‘Others’\n\nThe subject areas with the greatest number of discontinued journals were Medicine (52/317; 16%), Agriculture and Biological Science (34/317; 11%), and Pharmacology, Toxicology and Pharmaceutics (31/317; 10%) Table 3 and Extended data Table 126 report the distribution of discontinued journals by subject area and sub-area in full. Of these journals, 93% (294/318) declared they published using an Open Access model.\n\nData were retrieved from Scopus and are reported as percentages and fraction\n\nTable 4 shows the characteristics and metrics of journals at the time of discontinuation. The median time of Scopus coverage prior to discontinuation of the journals was 8 years (IQR 6–10, range 1–54). In total, 299 journals had been assigned to a SCImago quartile (Q); 39 of them (13%) listed in Q1 or Q2, and 260 in Q3 or Q4 (87%). Only ten of the discontinued journals had an Impact Factor at the year of discontinuation, with a median value of 0.84 (IQR 0.37–2.29, range 0.28–4).\n\nData are reported as medians, interquartile ranges [IQRs] and ranges (minimum value – maximum value) or as percentages and fractions.\n\n* No missing data. The analyses were conducted on all the 317 Scopus discontinued journals.\n\n† Data were available and calculated for 10 journals.\n\n‡ Data were available and calculated for 304 journals.\n\n§ Data were available and calculated for 299 journals.\n\n° Data were available and calculated for 82 journals.\n\nSjR: SCImago Journal & Country Rank; SNIP: Source Normalized Impact per Paper; IF: Impact Factor\n\nTable 5 shows the total number of documents and citations, the total number of documents per journal and the citations count before and after Scopus discontinuation. The total number of citations received after discontinuation was 607,261, with a median of 713 citations (IQR 254-2,056, range 0-19,468) per journal.\n\nData are reported as medians, interquartile ranges [IQRs] and ranges (minimum value – maximum value), if not otherwise specified.\n\n* No missing data. The analyses were conducted on all the 317 Scopus discontinued journals.\n\nPaired t-tests revealed that the mean number of citations per year after discontinuation was significantly higher than before (median of difference 64 citations, p<0.0001). Likewise, the number of citations per document proved significantly higher after discontinuation (median of difference 0.4 citations, p<0.0001) (Table 2).\n\nOf the discontinued journal, 22% (72/317) were included in the Cabell’s blacklist, while 29 (9%) were currently under review for inclusion. Only five journals (2%) were in included in the Cabell’s whitelist. In 243 cases (243/317), either the journal’s publisher was included in the updated Beall’s list of predatory publishers or the journal was included in the corresponding list of standalone journals (76.6%). The DOAJ currently includes only 9 journals. In total, 61 journals were previously included and discontinued by DOAJ; in 36 cases the reason was ‘suspected editorial misconduct by the publisher’ while in 23 instances it was ‘journal not adhering to best practice’ and in one case ‘no open access or license info’.\n\nTable 6 shows the indexing in PubMed, Web of Science, updated Beall’s list, Cabell’s white- and blacklist, and DOAJ (both included and discontinued).\n\nData are reported as percentages and fractions.\n\nDOAJ: Directory of Open Access Journals\n\n\nDiscussion\n\nThe present study was aimed at scrutinizing the main features of journals whose coverage was discontinued by Scopus due to publication concerns. To do so, (a) we counted and compared citation metrics per journal and per document obtained before and after discontinuation, and (b) accessed well-known and established blacklists and whitelists dealing with the issue of predatory publishing, i.e. Cabell’s and updated Beall’s list, as well as the DOAJ.\n\nOur main finding was that articles published in these journals before discontinuation, remain available to users and continue to receive a relevant number of citations after discontinuation, more than before. Moreover, a large number of the discontinued journals are likely to be predatory.\n\nAlthough Scopus applies a rigorous control of content quality and warns users when a journal is discontinued in its source details, the average users tend not to access journal’s details but articles’ contents. By doing so, they remain unaware that the article they have accessed was issued by a journal discontinued for publication concerns. Therefore, articles issued by journals whose scientific reputation is currently deemed questionable, continue to be displayed and to get cited as contents from legitimate, up-to-standard journals. When quantifying how coverage discontinuation affected the likelihood of these journals to be cited, data indicate that their articles received significantly more citations after discontinuation than before.\n\nBeyond the dangerous exposure of scholars, clinicians and even patients to potentially dubious or low quality contents, the considerable number of citations received after discontinuation by “ghost journals” can be a serious threat to scientific quality assessment by institutions and academia. In fact, these citations contribute to the calculation of the authors’ metrics by Scopus, including the Hirsch index (H-index)27, which is still among the main descriptors of productivity and scientific impact, based on career advancements are determined2–4. The fact that “ghost journals” can help to move up in academia is a relevant issue, and has inspired the allegorical vignette depicted in Figure 2: ghost journals can inflate authors’ metrics lifting them unnaturally and effortlessly.\n\nOf greatest concern is our finding that many of the discontinued journals display predatory behaviors in claiming to be open access. Exploitation of the open-access publishing model has been shown to go hand in hand with deviation from best editorial and publication practices for self-interest9. Such journals are not only associated with poor editorial quality, but are also deceptive and misleading by nature, i.e. they prioritize self-interest at the expense of scholars, and lack transparent and independent peer review9,28. Young researchers from low-income and middle-income countries are probably most susceptible to the false promises and detrimental practices of predatory journals. However, “predatory scholars” also seem to exist, possibly sharing a common interest with deceptive journals and publishers and knowingly using them to achieve their own ends7,29,30.\n\nThe policy underlying the decision to keep publications prior to discontinuation of indexing is clear. Some of these publications may actually fulfill publishing criteria (e.g. International Committee of Medical Journal Editors, Committee on Publication Ethics). It would be unfair to punish researchers for an eventual deterioration in journal performance; changes in the standards employed by the journal may change over time and the researchers may be unaware of quality issues. On the other hand, as the integrity of the editorial process cannot be vouched for, it is ethically untenable to keep such data available without clearer warnings.\n\nWe believe that Scopus should evaluate deleting the discontinued journals from the database contents or, at least, stop tracking their citations. In alternative, we propose that the CSAB could apply these measures case-by-case, after evaluating the severity of the potential misconducts. At the author-level, an alternative may be the provision of two metrics: one with and one without citations from publications in discontinued journals.\n\nThis analysis is not free of limitations. First, we included the year of discontinuation in the “after discontinuation” period, starting from January 1th. This decision may have led to some overestimation in the number of citations received after discontinuation. Second, we included only those journals discontinued from Scopus for “publication concerns” but were not able to retrieve details regarding the specific concern raised. Finally, we did not evaluate the impact of the citations received after discontinuation on author-level metrics.\n\n\nConclusions\n\nThe citation count of journals whose coverage in Scopus has been halted for publication concerns, increases despite discontinuation. This paradoxical trend can inflate scholars’ metrics prompting career advancements and promotions. Countermeasures should be taken to ensure the validity and reliability of Scopus metrics both at journals- and author-level for the purpose of scientific assessment of scholarly publishing. Creative thinking is required to resolve this issue without punishing authors who have inadvertently published good quality papers in a failing or predatory journal.\n\n\nData availability\n\nDiscontinued sources from Scopus are available from the following link: https://www.elsevier.com/__data/assets/excel_doc/0005/877523/Discontinued-sources-from-Scopus.xlsx\n\nAll the relevant data are freely retrievable from Scopus ‘journal overview’ or can be requested to Scopus through https://www.scopus.com/sources.\n\nFigshare: Underlying data Table 1.xlsx. https://doi.org/10.6084/m9.figshare.12231083.v212\n\nThis project contains the following underlying data:\n\n- Underlying data Table 1.xlsx (Standardized data extraction form with data collected)\n\nFigshare: Extended data Appendix 1.\n\nhttps://doi.org/10.6084/m9.figshare.12231110.v225\n\nThis project contains the following extended data:\n\n- Extended data Appendix 1.docx (Definitions of sources and metrics used in the manuscript of the GhoS(t)copus Project)\n\nFigshare: Extended data Table 1. https://doi.org/10.6084/m9.figshare.12233171.v226\n\nThis project contains the following extended data:\n\n- Extended data table.docx (Distribution of Scopus discontinued journals by subject sub-areas)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to thank Dr. Antonio Corrado (“Korrado 20”) for creating and providing the Figure 2.\n\n\nReferences\n\nElsevier: How Scopus works. [accessed 28 February 2020]. Reference Source\n\nCortegiani A, Manca A, Lalu M, et al.: Inclusion of predatory journals in Scopus is inflating scholars’ metrics and advancing careers. Int J Public Health. 2020; 65: 3–4. Publisher Full Text\n\nBagues M, Sylos-Labini M, Zinovyeva N: A walk on the wild side: “Predatory” journals and information asymmetries in scientific evaluations. Research Policy. 2019; 48(2): 462–477. Publisher Full Text\n\nHedding DW: Payouts push professors towards predatory journals. Nature. 2019; 565(7739): 267. Publisher Full Text \| Free Full Text\n\nHolland K, Brimblecombe P, Meester W, et al.: The importance of high-quality content: curation and re-evaluation in Scopus. [accessed 28 February 2020]. Reference Source\n\nElsevier: Content Policy and Selection. [accessed 28 February 2020]. Reference Source\n\nCortegiani A, Manca A, Giarratano A: Predatory journals and conferences: why fake counts. Curr Opin Anaesthesiol. 2020; 33(2): 192–197. PubMed Abstract \| Publisher Full Text\n\nCortegiani A, Misseri G, Gregoretti C, et al.: The challenge of the predatory open-access publishing outbreak. Eur J Anaesthesiol. 2019; 36(11): 810–813. PubMed Abstract \| Publisher Full Text\n\nGrudniewicz A, Moher D, Cobey KD, et al.: Predatory journals: no definition, no defence. Nature. 2019; 576(7786): 210–212. PubMed Abstract \| Publisher Full Text\n\nCortegiani A, Longhini F, Sanfilippo F, et al.: Predatory Open-Access Publishing in Anesthesiology. Anesth Analg. 2019; 128(1): 182–187. PubMed Abstract \| Publisher Full Text\n\nSeverin A, Low N: Readers beware! Predatory journals are infiltrating citation databases. Int J Public Health. 2019; 64(8): 1123–1124. PubMed Abstract \| Publisher Full Text\n\nCortegiani A, Ippolito M, Ingoglia G, et al.: Underlying data Table 1: Standardized data extraction form with data collected. 2020. http://www.doi.org/10.6084/m9.figshare.12231083\n\nElsevier: Scopus®, registered trademark of Elsevier B.V. [accessed 28 February 2020]. Reference Source\n\nScimago Lab, Copyright 2007-2020. [accessed 28 February 2020]. Reference Source\n\nCentre for Science and Technology Studies: About CWTS. Leiden University, The Netherlands. [accessed 28 February 2020]. Reference Source\n\nBeall’s list of predatory journals and publishers. [accessed 28 February 2020]. Reference Source\n\nDirectory of Open Access Journals. Licensed under CC BY-SA. [accessed 28 February 2020]. Reference Source\n\nPubMed Help. Bethesda (MD): National Center for Biotechnology Information (US); 2005; PubMed Help. [Updated 2019 Jul 25]. [accessed 28 February 2020]. Reference Source\n\nClarivate Analytics Company. [accessed 28 February 2020]. Reference Source\n\nCabell’s Scholarly Analytics. [accessed 28 February 2020]. Reference Source\n\nDirectory of Open Access Journals: DOAJ publishes lists of journals removed and added. Directory of Open Access Journals Blog. [accessed 28 February 2020]. Reference Source\n\nStrinzel M, Severin A, Milzow K, et al.: Blacklists and Whitelists To Tackle Predatory Publishing: a Cross-Sectional Comparison and Thematic Analysis. Wolf JM, editor. mBio. 2019; 10(3): pii: e00411-19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWinkler WE: String Comparator Metrics and Enhanced Decision Rules in the Fellegi-Sunter Model of Record Linkage. ERIC. 1990. Reference Source\n\nPorter EH, Winkler WE: Approximate string comparison and its effect on an advanced record linkage system. Citeseer. 1997. Reference Source\n\nCortegiani A, Ippolito M, Ingoglia G, et al.: Extended data Appendix 1. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12231110.v2\n\nCortegiani A, Cugusi L, Ippolito M, et al.: Extended data Table 1. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12233171.v2\n\nHirsch JE: An index to quantify an individual’s scientific research output. Proc Natl Acad Sci U S A. 2015; 102(46): 16569–16572. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButterworth JF 4th, Vetter TR: Predatory Journals Undermine Peer Review and Cheapen Scholarship. Anesth Analg. 2019; 128(1): 11–12. PubMed Abstract \| Publisher Full Text\n\nCobey KD, Grudniewicz A, Lalu MM, et al.: Knowledge and motivations of researchers publishing in presumed predatory journals: a survey. BMJ Open. 2019; 9(3): e026516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPond BB, Brown SD, Stewart DW, et al.: Faculty Applicants' Attempt to Inflate CVs Using Predatory Journals. Am J Pharm Educ. 2019; 83(1): 7210. PubMed Abstract \| Free Full Text" }	[ { "id": "63789", "date": "16 Jun 2020", "name": "Johann Mouton", "expertise": [ "Reviewer Expertise Bibliometrics", "scholarly publishing", "science policy", "sociology of science" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very relevant study in the growing scholarship around predatory publishing. It is one of the first studies that look at how predatory or at least questionable journals that have been delisted from citation database continue to have a presence in academia. More specifically, the paper asks the very important question why databases like Scopus (and others) continue to track the citations of journals that have been removed. This creates a distortion at many levels, including at the individual publication profile level.\nI am happy to recommend indexing of this paper as it is (some minor grammatical editing is required).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5813", "date": "26 Aug 2020", "name": "Andrea Cortegiani", "role": "Author Response", "response": "Dear reviewer, We are glad to submit a revised version of our manuscript, previously entitled ‘Inflated citations and metrics of journals discontinued from Scopus for publication concerns: the GhoS(t)copus Project’. The comment you provided was helpful in revising and improving the manuscript. We hereby provide our reply. Best regards, Andrea Cortegiani, MD On behalf of co-authors _______________________________________________________________________ Response to Reviewer 1 (Johann Mouton, Centre for Research on Evaluation, Science and Technology (CREST), DST/NRF Centre of Excellence in Scientometrics and Science, Technology and Innovation Policy, Stellenbosch University, Stellenbosch, South Africa) Comment: This is a very relevant study in the growing scholarship around predatory publishing. It is one of the first studies that look at how predatory or at least questionable journals that have been delisted from citation database continue to have a presence in academia. More specifically, the paper asks the very important question why databases like Scopus (and others) continue to track the citations of journals that have been removed. This creates a distortion at many levels, including at the individual publication profile level. I am happy to recommend indexing of this paper as it is (some minor grammatical editing is required). Reply: We thank the reviewer for the comment. We have now edited the manuscript and corrected errors and typos." } ] }, { "id": "64881", "date": "31 Jul 2020", "name": "Pablo Iriarte", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving us the opportunity to read this article in which the authors describe the characteristics, citations and metrics of journals that have been indexed in the Scopus database, at some point, and have afterwards been “discontinued” within Scopus for different reasons, summarised as “publication concerns”. Since the articles that have been published before the journal’s indexation was discontinued remain in the database, and can still be found, they may still be cited. The authors find this to be particularly problematic since they believe that these journals may be what are often called “predatory journals”, and therefore may threaten the credibility of science, by polluting the database with “weak research”. Therefore, the authors aimed to compare the number of citations per year, and per journal, and per document, before and after the journal was delisted. They conclude that the number of citations was actually higher after the journal was discontinued from Scopus. Although we understand the problem these authors try to highlight, we have some major concerns regarding some aspects of this study (mainly related to baseline assumptions and lack of clear definition) and also some minor ones.\nMajor concerns:\nBaseline assumption: In this article, the authors suggest that if a journal is being indexed, even for a long period of time (half of them have been indexed for 8 to 54 years), and is encountering “publication concern”, then all the previously published articles should become suspicious of bad science. We are not sure this should be considered straight forward, for the reason developed under our second major concern.\n“Predatory journals”: the problem of the lack of a clear definition of what a predatory journal is, remains. The authors use different sources to try to identify journals as “predatory” and we can only realise that the sources do not seem to agree. Although authors auto cite their own “consensus definition” of predatory journals and publishers “(..) entities that prioritize self-interest at the expense of scholarship and are characterized by false or misleading information, deviation from best editorial and publication practices, a lack of transparency, and/or the use of aggressive and indiscriminate solicitation practices.” they fail to underline that not everybody agrees with this definition. Also, the recent COVID-19 debacle of very low-quality scientific publications, published in usually highly regarded journals, suggests that bad peer-review and misleading articles may not be a characteristic of any journal. Also, it remains unclear to us how a journal may be indexed for 8 to 10 years, and all of a sudden become “predatory”. Or was it predatory in the first place, but was only uncovered after such a long time? If this is what the authors suggest, then what should we think about “recently” indexed journals? They may all be predatory as well, and will only be uncovered in 5 to 10 years?\n“Publication concerns”: This term needs to be better defined in order to really understand what lies behind it. It remains unclear why these journals have been excluded from the Scopus database at some point. Interestingly, half of these journals have been deemed good enough to figure in the database for more than 8 years… that’s a lot! And all of a sudden, they are not judged acceptable anymore and are discontinued from Scopus. Ok, why not. It may take some time before someone alerts Scopus of the misbehaviour of a given journal, although more than 10 years for 25% of them seems a lot. Or could it be a problem behind the vague concept of “publication concerns”? Could it be that the publication has stopped? Or the journal has changed its name? Or has merged with another one? Or has changed in quality over time? Illustrating some of the reason for discontinuation would help the reader understand the context.\nAccording to Scopus’ document cited in the article (ref. 5), there are 3 causes prompting Scopus to launch a journal re-evaluation: Under performance – metrics ; Outlier performance – radar ; Publication concerns. It might have been interesting to analyse the journals removed using the criteria “outlier performance – radar” as well as, according to Scopus document (ref. 5 cited by the authors) it is “particularly effective in flagging potential predatory journals.” Scopus describes it as ”an algorithm that flags journals based on approximately 40 outlier predictors, including sudden change in output volume, sudden change in publishing country and/or affiliations, and high journal/author self-citation rates.”\nIncrease in citations: The authors are worried that the citations of these journal have increased after the journal’s indexation was discontinued in the Scopus database. The problem here is that they do not seems to consider the fact that this may be the case for all journals (those indexed and those discontinued) which is probably due to the rapid increase in the number of publications over time. Unfortunately, this study lacks a “control group” (journals whose coverage has NOT been discontinued in the Scopus database) which could have help the reader understand whether the increase in citation of these journals was similar, was higher, or was lower than that of “legitimate journals”.\nUnderlying discourse: The term \"inflated\" used in the title, in Figure 2 and conclusion suggests manipulation or distortion of citations and an artificial advantage for authors of articles published in predatory journals before they are removed from Scopus. This is not demonstrated by the reasoning and data used in the article as a basis for comparison is missing.\nMethods and reproducibility: While the authors have provided data alongside the article, we have not been able to reproduce some of their results, such as “citations per year” presented in table 5. Data presented in “underlying data table 1” would benefit from better variable descriptions, such as where exactly was the information collected from, and the date of its collection. Some variable names and analysis are misleading, such as “Actual Pubmed”, described in methods section as “inclusion in PubMed” and in table 6 as “main database indexing”. It does not reflect whether the journal is currently indexed in PubMed, but may in some cases only indicate that a single article is present in PubMed or selected citations, due to their deposit in PMC (eg. “Advanced Materials Letters“). Some data seem a bit bizarre… and information provided by the authors like “Citation before and after the date of discontinuation were manually counted based on either the Scopus journal overview or the downloadable tables made available by Scopus upon request (see source data)” (p.3) did not allow us to double check some numbers that were weirdly extreme, and potential typos. Some counts of the number of citations seem erroneous, leading to an aberrant number of citations per document for journals like \"Mental Health in Family Medicine\" (80 citations per document before discontinuation) or \"Pharmacognosy Reviews\" (170 after). Other example of bizarre data: according to “underlying data table 1” the journal “Advanced material research” has been indexed for 10 years (from 2004 to 2014) and has received during this period only 3 citations. However, after having been delisted from the Scopus database, during a 6 year period (2014 to 2019), it has received 13875 citations. Any thoughts on how/why this could have happened?\n\nMinor concerns:\nAbstract:\nBackground: “contains the largest number of abstract and articles…” -> \"One of the largest\" could be better, some databases are bigger than Scopus (Google Scholar and Dimensions, see https://doi.org/10.1016/j.joi.2018.03.0061 or https://arxiv.org/abs/2004.14329)\n\nBackground: The term “publication concerns” may not be clear to everyone.\n\nBackground: “These journals remain indexed and can be cited.” This sentence is confusing. The articles published before the exclusion remains indexed and their citations continue to be taken into account but new issues of the journals are not indexed any more.\n\nMethods: The use of the term “discontinued” both for DOAJ (Results) and for journal publication (Background) is confusing. Should we say “excluded” or “delisted”?\n\nResults: “317 journals were evaluated” but next sentence states ninety-three percent of the journals (294/318)” -> typo for 318?\n\nResults: “the mean number of citations per year after discontinuation was significantly higher than before, and so was the number of citation per document”. Unclear whether the median difference of 64 is per journal, or cumulative across all “discontinued” journals? What are “documents”? Do you mean “articles”? or are there any other types of publication?\n\nConclusions: it’s unclear how the conclusion regarding “predatory journals” is drawn. Also, we don’t think the career advancement are “artificial”, they are real! Although maybe “undue”?\nIntroduction:\n\n“publications from no longer indexed journals may not be removed retrospectively … hence articles … could remain part of the database7” p.3 -> this conditional statement seems to contradict abstract which categorically states “These journals remains indexed” as well as the author’s conclusion “we propose that CSAB could apply these measure case-by-case”. Reference 7, linked with statement, was not helpful to clarify.\n\nMethods:\n“Independently collected by eight of the authors in pairs”: not very clear: two by two, or checked by two different people independently?\n\n“the year of our data collection”: more precision maybe?\nResults:\nWhy were data from 31 journals not retrieved? What was the problem?\n\nTable 1: Interestingly, none of these journals (discontinued from Scopus for publication concerns) have been published by Elsevier. Could there possibly be a selection bias? An interesting option could be to check if Elsevier’s journals that are still in Scopus might have been discontinued from others sources (DOAJ, WOS), and on which grounds?\n\nTable 1: Maybe the table could be enhanced to provide information about whether other journals by that publisher are still included in Scopus or not? Eg. 39 journals discontinued from Scopus were published by Academic journal Inc. Are there any other journals by this publisher still in Scopus?\n\nTable 3: don’t need 2 decimal precision in %.\n\nTable 3: Subject area are repetitive in Scopus, and a journal can have more than one, while this table and underlying data mention only 1 per journal, presumably the first appearing in Scopus ? If so, probably worth indicating and better to remove the percentages in table 3 falsely suggesting mutually exclusive categories?\n\nTable 5 and results (text): unclear where the median difference of 64 comes from? Or the 0.4?\n\nTable 5: total number of citation does not match number of citation before and after discontinuation. Any thoughts on how/why this could have happened? A note of explanation about that would be useful.\n\nCitations by year in table 5: The number of years before and after the journal is removed from Scopus is very different, the average is more than 9 years before and 4 years after (median of 8 and 4 respectively) which makes the comparison in Table 5 not relevant. Indeed, the number of citations per year is higher during the 2 or 3 years following the publication of the article and decreases sharply with time (DOI:10.1371/journal.pone.01537302) so that the ratio of citations per year also decreases if a larger number of years is used.\n\nDistribution of articles: of the 317 journals analysed, 5 contain more than half of the articles concerned by this question. This very inhomogeneous distribution means that the statistical analyses and the percentages per journal do not take this kind of distribution into account.\n\nPage 5, first paragraph: table 2 should probably read table 5?\n\nPage 5, 2nd paragraph: In 243 case (243/317)… is useless here. Maybe the authors meant 76.6% (243/217)\n\nTable 6: maybe a good idea to separate the “positive” facts (being indexed in Pubmed, WoS or Cabell’s whitelist) from the negative ones (Beall’s list, Cabell’s blacklist DOAJ discontinued.)\nDiscussion:\np.7 Unclear why the term “ghost journal” suddenly appear and how it is defined.\n\np. 7-8 “Of greatest concern is our finding that many of the discontinued journals display predatory behaviors in claiming to be open access” -> do you mean “displaying… are claiming”? to our understanding the article does not say that open access systematically means predatory. According to ref 9 et 22, the large majority of DOAJ indexed journals were not found in Beall’s list or Cabell’s Blacklist.\n\np. 8: “Such journals” unclear: predatory journals or OA journals?\n\nThe authors highlight that a limitation of their methodology is that they have included the year of discontinuation in the period “after discontinuation”, which could have led to overestimations. Then why not present the 2 analyses with the year of discontinuation included in the period BEFORE and in the period AFTER discontinuation, so that the reader can check for him-herself what bias this has induced?\n\nA mention of or comparison with other databases’ practices with regards to removing journals for indexing could be interesting. Do their approaches differ from Scopus’?\nConclusions:\nProposals are missing to solve the problem addressed and to avoid the stigmatization of the authors of the \"suspect\" articles. For example, a new open peer-review for articles published within X months before the journal's exclusion would be a possibility.\n\nMaybe another idea would be to flag published articles that have been published in journals that are not indexed anymore “NB: this article was been published in YEAR, in a journal that has encountered publication concerns in YEAR”\nReferences:\nReference 5 URL should be https://www.elsevier.com/__data/assets/pdf_file/0004/891058/ACAD_LIB_SC_ART_Importance-of-high-quality-content_WEB.pdf\n\nLink in reference 12 does not work properly due to a superfluous space in the middle.\n\nReference style is not harmonized (cf. 1st § of methods section makes 4 references to the same underlying data table 1 (ref number 12 in the reference list, 2 of whom are not correct and should refer to source data, the rest having various citation style).\n\nThere might be some mix up in references: ex. §4 on page 3 lists ref 6-8. Is it possible it should read 5, 7-8 instead?\n\nThe literature review would benefit from additional references to complete or contrast with the author’s findings: ex. doi:10.3390/publications80200173 that concludes that “articles published in predatory journals have little scientific impact.”\n\nAuto-citations: There are different ways to increase a researchers’ number of citations or H-index. Publishing in a “predatory journal” may be one of them, but auto-citation is also one. Of the 30 references cited at the end of this paper, 11 (37%) are auto-citations (citation of a reference including at least on author of the present paper), 7 (23%) are articles from others, and the remaining 12 were websites.\nTypo/language\nIn Underlying data table 1: last column title - DOAJ instead of DOAH ?\n\np.3 §3 “journal are no longer be indexed” -> “journal are no longer indexed”\n\np.3 §5 “still they can get” -> “they can still get”", "responses": [ { "c_id": "5814", "date": "26 Aug 2020", "name": "Andrea Cortegiani", "role": "Author Response", "response": "Dear reviewers, We are glad to submit a revised version of our manuscript, previously entitled ‘Inflated citations and metrics of journals discontinued from Scopus for publication concerns: the GhoS(t)copus Project’. The comments you provided were more than helpful in revising and improving the manuscript. We hereby provide a point-by-point reply to the comments. Best regards, Andrea Cortegiani, MD On behalf of co-authors _______________________________________________________________________ Response to Reviewers team 2 (Pablo Iriarte, Library of the University of Geneva, Geneva, Switzerland; Floriane Muller, Library of the University of Geneva, Geneva, Switzerland; Nadia Elia, Division of Anaesthesiology, Department of Acute Medicine, Geneva University Hospitals, Institute of Global Health, Faculty of Medicine, University of Geneva, Geneva, Switzerland) Comment 1: Thank you for giving us the opportunity to read this article in which the authors describe the characteristics, citations and metrics of journals that have been indexed in the Scopus database, at some point, and have afterwards been “discontinued” within Scopus for different reasons, summarised as “publication concerns”. Since the articles that have been published before the journal’s indexation was discontinued remain in the database, and can still be found, they may still be cited. The authors find this to be particularly problematic since they believe that these journals may be what are often called “predatory journals”, and therefore may threaten the credibility of science, by polluting the database with “weak research”. Therefore, the authors aimed to compare the number of citations per year, and per journal, and per document, before and after the journal was delisted. They conclude that the number of citations was actually higher after the journal was discontinued from Scopus. Although we understand the problem these authors try to highlight, we have some major concerns regarding some aspects of this study (mainly related to baseline assumptions and lack of clear definition) and also some minor ones. Reply: We are very grateful for the insights of the reviewers which have led us to improve our manuscript. We have now submitted a revised version of the manuscript and herein is our point-by-point reply to the comments. English form was also revised. Major comments: Comment 2: Baseline assumption: In this article, the authors suggest that if a journal is being indexed, even for a long period of time (half of them have been indexed for 8 to 54 years), and is encountering “publication concern”, then all the previously published articles should become suspicious of bad science. We are not sure this should be considered straight forward, for the reason developed under our second major concern. Reply: Thank you for the opportunity to clarify this. We made no such claim in the paper and, in fact, our key message was different. We clearly stated in the discussion that “It would be unfair to punish researchers for an eventual deterioration in journal performance; changes in the standards employed by the journal may change over time and the researchers may be unaware of quality issues”. We aimed to provide an analysis and describe the main scientific features and citation metrics of journals discontinued from Scopus for publication concerns as we strongly believe that this phenomenon merits discussion. We fully agree with the reviewer that further evaluation is required before the best solution for all aspects of this complex issue is determined. In fact, this study is the first to provide some of the information required to answer this question, albeit not all. We also agree with the reviewer regardless of the solution that is decided upon in the future, it should ensure that researchers are not unfairly punished. This is clearly stated. However, as we also point out, this issue can no longer be ignored; it involves a large number of journals and published documents. Comment 3: “Predatory journals”: the problem of the lack of a clear definition of what a predatory journal is, remains. The authors use different sources to try to identify journals as “predatory” and we can only realise that the sources do not seem to agree. Although authors auto cite their own “consensus definition” of predatory journals and publishers “(..) entities that prioritize self-interest at the expense of scholarship and are characterized by false or misleading information, deviation from best editorial and publication practices, a lack of transparency, and/or the use of aggressive and indiscriminate solicitation practices.” they fail to underline that not everybody agrees with this definition. Also, the recent COVID-19 debacle of very low-quality scientific publications, published in usually highly regarded journals, suggests that bad peer-review and misleading articles may not be a characteristic of any journal. Reply: First, we would like to highlight that the definition we reported for “predatory” is not the authors’ own. It was taken from an international collaboration of 35 authors who extensively studied the topic. Although we agree that no definition is perfect, this is most certainly not something we decided on ourselves and a consensus process was involved in its determination. If the reviewer wishes to argue with the definition provided, this should ideally be taken up with those involved in the consensus process. We surveyed recognized lists (i.e. Cabell, updated Beall, DOAJ) to evaluate the extent of predatory journals among the discontinued journals. With regards to the comment regarding the quality of COVID-19 research: Very true. We too have been following this topic with great interest. However, two wrongs do not make a right. In fact, this precise issue makes the discussion of journal metrics and our responsibilities towards them even more pertinent. Our research highlights some of the issues that arose with monitoring of the publication process from a different angle. It also promotes the need to continue to increase awareness within the scientific community itself regarding the damage that could potentially be caused by low-quality papers. Comment 3: Also, it remains unclear to us how a journal may be indexed for 8 to 10 years, and all of a sudden become “predatory”. Or was it predatory in the first place, but was only uncovered after such a long time? If this is what the authors suggest, then what should we think about “recently” indexed journals? They may all be predatory as well, and will only be uncovered in 5 to 10 years? Reply: It is our impression that the process may occur in two manners: (1) Some of the more recently indexed journals may indeed turn out to be predatory. So indeed perhaps newly indexed journals need to undergo more rigorous monitoring than well established journals. Whether our impression is correct and, if so, how this should be done, are questions far beyond the scope of our research; (2) Some of the discontinued older journals probably did deteriorate slowly. Our impression was that this process is typically a “slippery slope” and does not have an abrupt cutoff. As our analysis was not intended to study this question, we prefer not to speculate on the ideal timing for journal discontinuation. More data and expert input is needed on how to identify this process in the future. Comment 4: “Publication concerns”: This term needs to be better defined in order to really understand what lies behind it. It remains unclear why these journals have been excluded from the Scopus database at some point. Interestingly, half of these journals have been deemed good enough to figure in the database for more than 8 years… that’s a lot! And all of a sudden, they are not judged acceptable anymore and are discontinued from Scopus. Ok, why not. It may take some time before someone alerts Scopus of the misbehaviour of a given journal, although more than 10 years for 25% of them seems a lot. Or could it be a problem behind the vague concept of “publication concerns”? Could it be that the publication has stopped? Or the journal has changed its name? Or has merged with another one? Or has changed in quality over time? Illustrating some of the reason for discontinuation would help the reader understand the context. Reply: The term ‘publication concerns’ is not one which spontaneously decided upon. It is the term defined and used by Scopus. Indeed, we report in the manuscript all the available definitions and details provided by Scopus. Unfortunately, no additional details are publicly available regarding the criteria used to discontinue a journal because of ‘publication concerns’. We too would be delighted to receive more details as they may be important. Having said this, we honestly doubt that merging with another paper or changing a journal name is cause for publication concern. With regards to the reviewers’ rumination on the time gap for discontinuation: As noted above, it is indeed possible that some journals have changed quality over time or that they were evaluated only several years after indexing. This information would most certainly be interesting if it were publicly available, but it is not. Furthermore, as also stated above, this is not within the scope of our project. Comment 5: According to Scopus’ document cited in the article (ref. 5), there are 3 causes prompting Scopus to launch a journal re-evaluation: Under performance – metrics ; Outlier performance – radar ; Publication concerns. It might have been interesting to analyse the journals removed using the criteria “outlier performance – radar” as well as, according to Scopus document (ref. 5 cited by the authors) it is “particularly effective in flagging potential predatory journals.” Scopus describes it as ”an algorithm that flags journals based on approximately 40 outlier predictors, including sudden change in output volume, sudden change in publishing country and/or affiliations, and high journal/author self-citation rates. Reply: We thank the reviewer for this insightful comment which suggests some new research directions for the future. We have added this suggestion to the concluding paragraph of our paper. Comment 6: Increase in citations: The authors are worried that the citations of these journal have increased after the journal’s indexation was discontinued in the Scopus database. The problem here is that they do not seems to consider the fact that this may be the case for all journals (those indexed and those discontinued) which is probably due to the rapid increase in the number of publications over time. Unfortunately, this study lacks a “control group” (journals whose coverage has NOT been discontinued in the Scopus database) which could have help the reader understand whether the increase in citation of these journals was similar, was higher, or was lower than that of “legitimate journals”. Reply: Please see below our response to this and the next comment together. Comment 7: Underlying discourse: The term \"inflated\" used in the title, in Figure 2 and conclusion suggests manipulation or distortion of citations and an artificial advantage for authors of articles published in predatory journals before they are removed from Scopus. This is not demonstrated by the reasoning and data used in the article as a basis for comparison is missing. Reply: Indeed, the lack of a control group is a study limitation. We now point this out in the discussion section (see page 13). However, the authors have no interest vested in presenting an “underlying discourse” we have taken this comment very seriously. We have now removed the term “inflated” from both the title and the conclusions. We also modified the caption of Figure 2, substituting ‘can’ with ‘may’. Comment 8.1: Methods and reproducibility: While the authors have provided data alongside the article, we have not been able to reproduce some of their results, such as “citations per year” presented in table 5. Data presented in “underlying data table 1” would benefit from better variable descriptions, such as where exactly was the information collected from, and the date of its collection. Reply: We salute the reviewer for being so thorough as to attempt to reproduce our results. Our decision to submit the full database for publication and to select an Open Research publishing platform stems from precisely this reason – we would be delighted if this study was repeated and expanded on in the future. We calculated “citations per year” as the ratio between the total number of citations (before discontinuation plus after discontinuation) and the number of Scopus years. In the revised version of underlying data table 1 we have now added a box with a more detailed description to enable the readers to repeat our analysis. However, we must point out that online data changes daily. Therefore, in order to reproduce the data to perfection, one would need to know for which one of the 317 journals that we studied - on which day through the duration of the study period we downloaded the data. The overall process took about a month as described in the paper. This issue may render the data not reproducible to the dot. However, at any time of examination, the overall trends should remain the same. Comment 8.2: Some variable names and analysis are misleading, such as “Actual Pubmed”, described in methods section as “inclusion in PubMed” and in table 6 as “main database indexing”. It does not reflect whether the journal is currently indexed in PubMed, but may in some cases only indicate that a single article is present in PubMed or selected citations, due to their deposit in PMC (eg. “Advanced Materials Letters“). Reply: Thank you for pointing out this omission. We have now revised both the manuscript and underlying data table 1 to specify that we collected data on the inclusion of articles in PubMed. We also changed the title of Table 6 as follows: “Table 6. Discontinued journals’ current Open Access policy and the indexing of their articles in major databases”. Comment 8.3: Some data seem a bit bizarre… and information provided by the authors like “Citation before and after the date of discontinuation were manually counted based on either the Scopus journal overview or the downloadable tables made available by Scopus upon request (see source data)” (p.3) did not allow us to double check some numbers that were weirdly extreme, and potential typos. Some counts of the number of citations seem erroneous, leading to an aberrant number of citations per document for journals like \"Mental Health in Family Medicine\" (80 citations per document before discontinuation) or \"Pharmacognosy Reviews\" (170 after). Other example of bizarre data: according to “underlying data table 1” the journal “Advanced material research” has been indexed for 10 years (from 2004 to 2014) and has received during this period only 3 citations. However, after having been delisted from the Scopus database, during a 6 year period (2014 to 2019), it has received 13875 citations. Any thoughts on how/why this could have happened?” Reply: Bizarre or not, the data is what it is. Here we describe how the data was collected in detail: We searched the name of each journal using the Scopus database “Sources” page (https://www.scopus.com/sources.uri?zone=TopNavBar&origin=searchbasic), then we opened the page of the journal and selected “view all documents”. At the following page, we checked the box “All” to select all the documents and then clicked “View citation overview”. At this stage, two options may appear: a) a page with a line chart reporting the number of citations per year, or b) a flag inviting a request for a citation overview download due to the large size of the overview which does not allow on-site display as a line chart. In the a) situation, we manually summed the citations displayed. In the b) situation, we inserted in the request form an email address and Scopus sent us back a .csv file for each request, containing the requested data. As this detailed description add little to the main text of the paper we have not added it to the description of the methods. We wish to reiterate the point made in our previous response – that data collection was not completed in a single day. The data for this study required accessing several sources for each paper, documentation of different variables from each source and performance of individual calculations. For this reason, we report the time frame during which the data was collected (24th January - end of February 2020). Citations are continuously updated as they grow and new journals are indexed in the database, so at any time other than the exact time we downloaded the data, the number of citations would be slightly different than ours. Specifically, with regards to the examples given for “bizarre” data, we have re-checked these items and our replies are as follows: Our table 1 did not and does not state “80 citations per document before discontinuation” for Mental Health in Family Medicine. We report a total of 175 documents, with 213 citations before discontinuation and 14123 after discontinuation. Thus, 1.22 citations per document before discontinuation and 80 after discontinuation. Regarding Pharmacognosy Reviews - We are grateful to the reviewer for picking up on this typo - In the table, the number of citations before discontinuation was erroneously written as ‘43451’ rather than ‘4345’. This led to the number of 170 citations per year for the period before discontinuation. We have corrected the resultant calculations. We have also re-checked the database for additional typos (none were found). The main findings of the manuscript did not change after this correction. Nonetheless thank you for pointing out the mistake. Regarding Advanced Material Research - we have re-checked the data and confirm that it is correct. We do not have an explanation for the huge difference between the period preceding and succeeding discontinuation. Minor comments: Comment 1: Abstract: Background: “contains the largest number of abstract and articles…” -> \"One of the largest\" could be better, some databases are bigger than Scopus (Google Scholar and Dimensions, see https://doi.org/10.1016/j.joi.2018.03.0061 or https://arxiv.org/abs/2004.14329) Reply: Modified as suggested. Comment 2: Background: The term “publication concerns” may not be clear to everyone. Reply: Thank you for this comment. As this is a Scopus term, we cannot modify it. We have added quotation marks to make this clear to the readers. Comment 3: Background: “These journals remain indexed and can be cited.” This sentence is confusing. The articles published before the exclusion remains indexed and their citations continue to be taken into account but new issues of the journals are not indexed any more. Reply: Thank you for pointing out this misnomer. We have substituted ‘journals’ for ‘previously published articles’. Comment 4: Methods: The use of the term “discontinued” both for DOAJ (Results) and for journal publication (Background) is confusing. Should we say “excluded” or “delisted”? Reply: The term ‘discontinued’ is that used in the Scopus database. The label ‘coverage discontinued in Scopus’ is also displayed on the discontinued journals’ page. The downloadable list of journals whose coverage has been discontinued is also named by Scopus as ‘Discontinued sources from Scopus’. As it is important that the labels used in the manuscript remain consistent with official labels and definitions, we felt we could not change the term ‘discontinued’. Comment 5: Results: “317 journals were evaluated” but next sentence states ninety-three percent of the journals (294/318)” -> typo for 318? Reply: Thank you. It was indeed a typo which has been corrected. Once again, thank you for the scrupulous review which has improved our paper. Comment 6: Results: “the mean number of citations per year after discontinuation was significantly higher than before, and so was the number of citation per document”. Unclear whether the median difference of 64 is per journal, or cumulative across all “discontinued” journals? What are “documents”? Do you mean “articles”? or are there any other types of publication? Reply: We calculated the median number of cumulative citations across all discontinued journals per year of coverage and defined it as ‘Citations per year’. We calculated the median number of cumulative citations across all discontinued journals per document (‘Citations per document’). We did not give a subjective definition of ‘document’ but included all the indexed documents provided by the Scopus database. We have added this detailed description in the methods section of the paper, and we also specified the calculations performed in underlying table 1. Comment 7: Conclusions: it’s unclear how the conclusion regarding “predatory journals” is drawn. Also, we don’t think the career advancement are “artificial”, they are real! Although maybe “undue”? Reply: As a result of this comment we have modified the conclusions to state as follows: “Journals whose coverage in Scopus has been halted for publication concerns continue to be cited. This paradox may influence scholar metrics, potentially prompting career advancements and promotions. Further studies are needed, also investigating the journals discontinued from Scopus using the criteria “outlier performance – radar”, particularly effective in flagging potential predatory journals. Countermeasures should be taken to ensure the validity and reliability of Scopus metrics for both journals and authors due to their importance for scientific assessment of scholarly publishing. Creative thinking is required to resolve this issue without punishing authors who have inadvertently published good quality papers in a failing or predatory discontinued journal.” Comment 8: Introduction: “publications from no longer indexed journals may not be removed retrospectively … hence articles … could remain part of the database7” p.3 -> this conditional statement seems to contradict abstract which categorically states “These journals remains indexed” as well as the author’s conclusion “we propose that CSAB could apply these measure case-by-case”. Reference 7, linked with statement, was not helpful to clarify. Reply: Thank you for pointing out that the language in this sentence requires improvement. We have revised this to read more succinctly: “The list of the discontinued sources is publicly available and is updated approximately every six months 6. However, articles published in journals that were discontinued and are no longer indexed, are probably not removed from the Scopus database. It has been claimed…” Comment 9: Methods: “Independently collected by eight of the authors in pairs”: not very clear: two by two, or checked by two different people independently? Reply: Agree. We specified that four pairs of authors independently collected the data (i.e. two people independently collected the same quarter of the data. The entire database is the result of eight people collecting the data). Comment 10: “the year of our data collection”: more precision maybe? Reply: Agree. We changed “the year of our data collection” with “2020”. Comment 11: Results: Why were data from 31 journals not retrieved? What was the problem? Reply: The journals were not found on Scopus database using the search tool. This is now also stated in the paper. Comment 12: Table 1: Interestingly, none of these journals (discontinued from Scopus for publication concerns) have been published by Elsevier. Could there possibly be a selection bias? An interesting option could be to check if Elsevier’s journals that are still in Scopus might have been discontinued from others sources (DOAJ, WOS), and on which grounds? Reply: We agree that this would be an interesting finding. In this study, we focused on journals discontinued by Scopus for ‘publication concerns’, and we afterwards checked whether they had also been delisted from DOAJ. Should the data mentioned by the reviewers regarding WOS be available online, it may worth further investigation. Thank you for this important insight. Comment 13: Table 1: Maybe the table could be enhanced to provide information about whether other journals by that publisher are still included in Scopus or not? Eg. 39 journals discontinued from Scopus were published by Academic journal Inc. Are there any other journals by this publisher still in Scopus? Reply: The relation between the publisher and de-indexing of articles in Scopus after discontinuation is an important question that should be addressed in further research. We aimed to provide a snapshot of the effect of ongoing article availability, rather than explore publisher and/or Scopus policies associated with journal discontinuation. Comment 14: Table 3: don’t need 2 decimal precision in %. Reply: This has been changed in accordance with the reviewer’s request. Comment 15: Table 3: Subject area are repetitive in Scopus, and a journal can have more than one, while this table and underlying data mention only 1 per journal, presumably the first appearing in Scopus? If so, probably worth indicating and better to remove the percentages in table 3 falsely suggesting mutually exclusive categories? Reply: Indeed the reviewer is correct. The table caption now reads “First subject area as displayed in Scopus. Note: a journal may have more than one subject area”. Comment 16: Table 5 and results (text): unclear where the median difference of 64 comes from? Or the 0.4? Reply: Thank you for asking. These are the results of the Wilcoxon matched-pairs signed rank test. We removed the reference to the table to avoid misunderstandings. The results differ slightly after corrections made following previous reviewers’ comments. The raw data are available in underlying data table 1 and the analyses can be easily recreated by the readers. Comment 17: Table 5: total number of citations does not match number of citation before and after discontinuation. Any thoughts on how/why this could have happened? A note of explanation about that would be useful. Reply: Again- we are grateful for the reviewers’ sharp eye. We rechecked the data and found a typo: The number of citations after discontinuation was reported as 607621 when it should have been 607261 (this can be seen in our underlying data table 1 and in the main text). We have corrected this in the new version of the manuscript. Comment 18: Citations by year in table 5: The number of years before and after the journal is removed from Scopus is very different, the average is more than 9 years before and 4 years after (median of 8 and 4 respectively) which makes the comparison in Table 5 not relevant. Indeed, the number of citations per year is higher during the 2 or 3 years following the publication of the article and decreases sharply with time (DOI:10.1371/journal.pone.01537302) so that the ratio of citations per year also decreases if a larger number of years is used. Reply: This is probably true. Although some papers may undergo resurgence this is probably not common. However, we found no better way of coping with the issue of the different number of Scopus coverage years across journals. And furthermore, this “decay” is likely to be fairly consistent across all journals both before and after discontinuation. As we also added to the study limitations the lack of a control group of non-discontinued journals, we have also inserted a word of caution regarding our results. Comment 19: Distribution of articles: of the 317 journals analysed, 5 contain more than half of the articles concerned by this question. This very inhomogeneous distribution means that the statistical analyses and the percentages per journal do not take this kind of distribution into account. Reply: We used non-parametric tests to reduce the effect of non normal distribution of data on our findings precisely for this reason. We also present IQRs and ranges to be more informative. Comment 20: Page 5, first paragraph: table 2 should probably read table 5? Reply: We have removed this reference to table 2 in order to accommodate a previous reviewer’s comment (Comment 16). Comment 21: Page 5, 2nd paragraph: In 243 case (243/317)… is useless here. Maybe the authors meant 76.6% (243/217) Reply: This has been amended. Thank you. Comment 22: Table 6: maybe a good idea to separate the “positive” facts (being indexed in Pubmed, WoS or Cabell’s whitelist) from the negative ones (Beall’s list, Cabell’s blacklist DOAJ discontinued.) Reply: This was a good idea. We have changed the order of the items in Table 6 accordingly. Comment 23: Discussion: p.7 Unclear why the term “ghost journal” suddenly appear and how it is defined. Reply: We modified the phrase; the text now reads “The fact that discontinued journals might help to move up in academia is a relevant issue, and has inspired the vignette depicted in Figure 2: “Discontinued journals may inflate authors’ metrics lifting them unnaturally and effortlessly.” We decide not to remove the term ‘inflate’ from Figure 2 caption as the figure is an allegorical and so intentionally exaggerated representation of the phenomenon. Comment 24: p. 7-8 “Of greatest concern is our finding that many of the discontinued journals display predatory behaviors in claiming to be open access” -> do you mean “displaying… are claiming”? to our understanding the article does not say that open access systematically means predatory. According to ref 9 et 22, the large majority of DOAJ indexed journals were not found in Beall’s list or Cabell’s Blacklist. Reply: We have changed the phrase “Of greatest concern is our finding that many of the discontinued journals display predatory behaviours in claiming to be open access” to “Of greatest concern is our finding that many of the discontinued journals display predatory behaviours in claiming to be open access without actually being indexed in DOAJ.” Comment 25: p. 8: “Such journals” unclear: predatory journals or OA journals? Reply: ‘Predatory’. This is now written. Comment 26: The authors highlight that a limitation of their methodology is that they have included the year of discontinuation in the period “after discontinuation”, which could have led to overestimations. Then why not present the 2 analyses with the year of discontinuation included in the period BEFORE and in the period AFTER discontinuation, so that the reader can check for him-herself what bias this has induced? Reply: We mentioned the possibility of overestimation in order to be entirely honest. However, our impression, just from eyeballing the data during collection, was that this would not lead to much of a change. More importantly, in the early stage, we planned no such analysis and therefore did not collect the data that would be required to do this analysis. At this stage performing such an analysis practically requires that the data be recollected in near entirety again which is no simple task. Comment 27: A mention of or comparison with other databases’ practices with regards to removing journals for indexing could be interesting. Do their approaches differ from Scopus’? Reply: This question again is one of policy and therefore not in the scope of our study. The reviewers’ comments indeed present much food for thought in terms of future research. Comment 28: Conclusions: Proposals are missing to solve the problem addressed and to avoid the stigmatization of the authors of the \"suspect\" articles. For example, a new open peer-review for articles published within X months before the journal's exclusion would be a possibility. Maybe another idea would be to flag published articles that have been published in journals that are not indexed anymore “NB: this article was been published in YEAR, in a journal that has encountered publication concerns in YEAR” Reply: We appreciate the reviewers’ proposals and have now added them to the discussion. Solutions should also address metrics and citations deriving from these journals. We have therefore added that while this may be an immediately implementable temporising measure, additional thought should be dedicated to address of these aspects as well while maintaining fairness. Comment 29: References: Reference 5 URL should be https://www.elsevier.com/__data/assets/pdf_file/0004/891058/ACAD_LIB_SC_ART_Importance-of-high-quality-content_WEB.pdf Reply: Corrected. Comment 30: Link in reference 12 does not work properly due to a superfluous space in the middle. Reply: Corrected. Comment 31: Reference style is not harmonized (cf. 1st § of methods section makes 4 references to the same underlying data table 1 (ref number 12 in the reference list, 2 of whom are not correct and should refer to source data, the rest having various citation style). Reply: Now harmonised. Comment 32: There might be some mix up in references: ex. §4 on page 3 lists ref 6-8. Is it possible it should read 5, 7-8 instead? Reply: We again salute the work done by the reviewer. An additional critical eye is always helpful. We have re-reviewed the references to ensure that no additional issues exist, but are of course willing to make any additional corrections if such are identified. Comment 33: The literature review would benefit from additional references to complete or contrast with the author’s findings: ex. doi:10.3390/publications80200173 that concludes that “articles published in predatory journals have little scientific impact.” Reply: Thank you. We have now added this reference and discussed it. Comment 34: Auto-citations: There are different ways to increase a researchers’ number of citations or H-index. Publishing in a “predatory journal” may be one of them, but auto-citation is also one. Of the 30 references cited at the end of this paper, 11 (37%) are auto-citations (citation of a reference including at least on author of the present paper), 7 (23%) are articles from others, and the remaining 12 were websites. Reply: Auto-citations are indeed an issue in the literature. However, in this case, we wish to highlight several points that make this comment moot: The reviewer included in their count three references: Underlying data Table 1: Standardized data extraction form with data collected [previously Ref. n° 12], Extended data Appendix 1 [previously Ref. n° 25] and Extended data Table 1. [previously Ref. n°26], that are study materials. F1000research policy explicitly requires adding supplementary study materials and all the underlying data (e.g. databases) in the reference list. Consequently, the real percentage of self quotations is actually lower. This manuscript has a long list of authors, many of which have published (1) together (2) on this topic. Hence the probability of citing previous research by these authors is high. Is the reviewer suggesting that researchers never base their newest work on their previous work? Experts that consistently research any field of research are likely to work together and to cite their own work - each step supports the next. Despite the nature of this comment and our concern that it may detract from the quality of our paper, we have removed some of the references. The authors of this paper have no need of self citations – for promotion or any other purpose. Comment 35: Typo/language In Underlying data table 1: last column title - DOAJ instead of DOAH ? Reply: Corrected. Comment 36: p.3 §3 “journal are no longer be indexed” -> “journal are no longer indexed” Reply: Corrected. Comment 37: p.3 §5 “still they can get” -> “they can still get” Reply: Corrected." } ] } ]	1	https://f1000research.com/articles/9-415
https://f1000research.com/articles/9-516/v1	04 Jun 20	{ "type": "Research Article", "title": "Analgesic efficacy of intravenous nefopam after spine surgery: a randomized, double-blind, placebo-controlled trial", "authors": [ "Jatuporn Eiamcharoenwit", "Haruthai Chotisukarat", "Kanjana Tainil", "Nalinrat Attanath", "Phuping Akavipat", "Jatuporn Eiamcharoenwit", "Haruthai Chotisukarat", "Kanjana Tainil", "Nalinrat Attanath" ], "abstract": "Background: The incidence of moderate to severe pain is high among patients undergoing spinal surgery. Nefopam can be used as an adjuvant analgesic postoperatively after spine surgery. The study aimed to assess the analgesic efficacy and side effects of nefopam on 24-hour postoperative morphine consumption after spine surgery. Methods: The study is a randomized, double-blinded, placebo-controlled trial. A total of 96 patients were randomized into 4 treatment groups, 24 each. In group 1, patients received normal saline before surgical incision and before the end of surgery. In group 2, patients received 30 mg nefopam before surgical incision and normal saline before the end of surgery. In group 3, patients received normal saline before surgical incision and 30 mg of nefopam before the end of surgery. In group 4, patients received 30 mg of nefopam in both timings. Patient-controlled analgesia morphine was used for the postoperative period. Outcomes were to determine 24-hour morphine consumption and incidence of side effects. Results: Of 96 patients enrolled, 21 in placebo-placebo, 22 in nefopam-placebo, 22 in placebo-nefopam and 21 in nefopam-nefopam groups completed the study. Analysis of the Kruskal-Wallis test on the intention-to-treat basis shows no significant difference in 24-hour postoperative morphine consumption between four groups, which were 18 [IQR 13.5-29], 20 [IQR 11-28.3], 17 [IQR 11.5-28.5], 13 [IQR 8.5-18.5] mg., respectively (p = 0.223). Incidence of side effects, including tachycardia, sedation, sweating and nausea/ vomiting, did not differ. Conclusions: Adding perioperative nefopam to opioid analgesic does not improve analgesic efficacy in patients who underwent spine surgery. Registration: Thai Clinical Trials Registry ID TCTR20171115001; registered on 15 November 2017.", "keywords": [ "Non-opioid analgesics", "Postoperative pain", "Morphine consumption" ], "content": "Introduction\n\nThe incidence of moderate to severe pain after spine surgery is 30–64%, especially in the first 3 days after surgery1,2. Currently, opioids are primarily considered for postoperative pain control. However, a high dose of opioids may cause side effects such as nausea, vomiting, drowsiness and respiratory depression3,4. As a result, patients recover slowly after surgery1,5.\n\nIn addition to opioids, adjuvant drugs are also used for postoperatively after spine surgery to reduce the amount and side effects of opioids, including non-steroidal anti-inflammatory, drugs: NSAIDs, gabapentinoids (such as pregabalin gabapentin) and paracetamol6–9. NSAIDs work by inhibiting the production of prostaglandins both in the central nervous system and peripheral nervous system through the inhibition of cyclooxygenase (COX) isoenzymes. The results of these effects reduce inflammation and pain after surgery. Although NSAIDs have an opioid-sparing effect, there are adverse effects; traditional NSAIDs inhibit the aggregation of platelets and may cause abnormal bleeding during surgery, increasing the risk of bleeding from ulcers in the gastrointestinal tract. In patients receiving COX-2 inhibitors, the risk of thrombosis increases, especially the coronary arteries. Also, elderly patients receiving NSAIDs may exhibit impaired kidney function, potentially leading to acute renal failure10.\n\nNefopam is a non-opioid analgesic drug used to treat postoperative pain. Mechanism of action is inhibiting the re-uptake of serotonin and norepinephrine11. It also reduces glutamate release via modulating sodium and calcium channels12. In previous studies, multiple timings of systemic nefopam were administered during the perioperative period. Nefopam was administered either before surgical incision, defined as preemptive analgesia13–16, or at the end of surgery17–23 for post-operative pain control; however, the correct timing is not known. Therefore, the objectives of this study were to determine the analgesic efficacy and side effects of nefopam that administered before surgical incision, or before the end of the surgery, or both timings compared with placebo on postoperative morphine consumption.\n\n\nMethods\n\nThe study was a randomized, double-blinded, placebo-controlled trial. It was approved by the Institutional Review Board of Prasat Neurological Institute (IRBPNI) [Bangkok] and informed consent was obtained from all patients. The patients enrolled in the study comprised all patients undergoing spine surgery at Prasat Neurological Institute, February 2018 to March 2019.\n\nInclusion criteria were patient with age >18 years, who were undergoing lumbosacral spine surgery under general anesthesia; elective case; not more than three-level spinal surgery; ASA physical status I-III; expected length of operation of 4–6 hours; and no history of nefopam or morphine allergy. Exclusion criteria were: patients with ischemic heart disease or arrhythmia; epilepsy; liver disease; creatinine clearance <30 ml/min; receiving nefopam within 24 hours or five elimination half-lives of nefopam; received strong opioids for more than 2 weeks or received monoamine oxidase inhibitor within 2 weeks before surgery; and who are unable to use a patient-controlled analgesia (PCA) device.\n\nPatients were allocated into four treatment groups by the computer-generated random sequence and their allocation placed in a sealed envelope. A total of 96 patients were randomized into 4 treatment groups: 24 in placebo-placebo, 24 in nefopam-placebo, 24 in placebo-nefopam and 24 in nefopam-nefopam groups. The envelopes were opened only after the enrolled participants by the nurse anesthetists who was not involved in the study. All participants and researchers were blinded to the group allocation. At enrolment, patients were explained on a 0 to 10 numerical rating scale (NRS): 0 corresponds to no pain and 10 to the worst imaginable pain for postoperative pain assessment, and how to use the patient-controlled analgesia device on a day before surgery. Patients received premedication with 7.5 mg of midazolam within 30 – 60 minutes before anesthesia. No patients received NSAIDs, serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressant, gabapentinoid or opioids on the morning of surgery.\n\nWhen the patients arrived at the operating theatre, each group of patients received two treatment timings: period A, 30 minutes before surgical incision; or period B, 30 minutes before the end of surgery or both timings compared with matching placebo. Nurse anesthetists not involved in the research project opened sealed envelopes containing the allocation to group in the order of patients. Study medications were in identical appearance bottles; 100 ml of transparent, colorless solution, containing either 30 mg of nefopam (Acupan® BIOCODEX) or placebo, which was prepared in sealed envelopes by the nurse anesthetist who was not involved in the study. Study medications were given intravenously within 20 minutes each time. The treatment team and the data collector will not know which group of participants is in the study group. The patients were withdrawn and labels were opened if they exhibited a heart rate >150/min, arrhythmia, development of extreme unexpected events (such as acute ischemic heart disease, pulmonary embolism), and if the patient stopped using the PCA device before 24 hours after surgery.\n\nIn the operating theatre, Patients were monitored routinely with an electrocardiogram, noninvasive blood pressure, and pulse oximetry. Additionally, a bispectral index (BIS) monitor was utilized to assess the depth of anesthesia. Pre-oxygenation with high-flow oxygen through a facemask was given for 3 to 5 minutes. Anesthesia was then induced with intravenous propofol (1.5–2 mg/kg), and intravenous cisatracurium (0.15–0.2 mg/kg) was administered to facilitate the endotracheal intubation. Anesthesia was maintained at 1 MAC of desflurane with oxygen and nitrous oxide. Anesthesiologists provided cisatracurium and morphine by adjusting the depth of anesthesia to the Bispectral index (BIS) of 40–60. All patients received local wound infiltration with 20 ml of 0.5% bupivacaine at the end of the operation. At the recovery room, the patients were asked pain scores every 15 minutes using a numerical rating scale. If the pain score is greater than or equal to 4 points, the patients were injected with 2 mg of morphine every 10 minutes until the patients reported pain scores of less than 4. Then, the patients started to use the PCA device in the post-operative period. The PCA devices used in this study were IVAC® PCAM® Syringe Pumps (Alaris, United Kingdom). The protocol PCA setting of morphine 1 mg/ml; no basal rate, bolus dose of 1 mg, lockout interval of 5 minutes, 4-hour limit of 40 mg. If the patients required more than 40 mg of morphine within 4 hours, the cause of pain was reevaluated and neuropathic pain was ruled out by using the Thai-language Neuropathic Pain Diagnostic Questionnaire (Thai DN4)24. Any other analgesics were not permitted during the study period.\n\nDemographic data, American Society of Anesthesiologist (ASA) Physical status, comorbid disease, average pain score in a 24-hour period before surgery, operation, anesthetic time, duration of surgery, vital signs every 2.5 minutes during nefopam administration, intraoperative and 24 hours postoperative morphine consumption, first time to rescue morphine, pain score during postoperative 24 hours and side effects were recorded.\n\nThe primary outcome of the study was to determine the analgesic efficacy. The primary efficacy was defined as the percentage of a reduction in 24-hour morphine consumption during each time of nefopam administration. Secondary outcome comparisons were 24-hour postoperative pain score, and incidence of side effects such as tachycardia, sedation, sweating and nausea/ vomiting. Sedation was defined as a Pasero Opioid-induced Sedation Scale (POSS) score that was greater than or equal to 325. Clinically important postoperative nausea and vomiting (PONV) were defined as PONV intensity scale of ≥7526.\n\nPrevious studies have shown that when nefopam was given before the end of surgery, reductions in postoperatively 24-hour morphine consumption were 19.2–51%17,18. The sample size was determined from an average percentage of reduction in postoperatively 24-hour morphine consumption equaled to 35% before the end of the surgery group. Neither the percentage of a reduction in 24-hour morphine consumption in the group receiving nefopam before surgical incision nor the group receiving nefopam before surgical incision plus at the end of surgery compared with placebo was reported in these previous studies. Therefore, this sample size estimated the level of reduction in 24-hour morphine consumption in both groups. The sample size gives the trial a power of 80%, sets a two-tailed α at 0.05. The calculation resulted in 21 patients per group. To compensate for 10% attrition rate, we included 24 patients per group. The total sample size is 96 patients.\n\nCriteria for interim analysis and early termination of the study were as follows: 1) a heart rate of more than 150 beats per minute; 2) arrhythmias; and 3) patients developed extreme unexpected events such as acute ischemic heart disease, pulmonary embolism. Analysis of the analgesic efficacy measures was performed by the Kruskal-Wallis test. The main analysis was analyzed by intention-to-treat (ITT). Descriptive statistical analyses were performed and expressed in median (IQR) for continuous variables and number (percent) for categorical variables as appropriate. The software program SPSS version 16 was used. Safety data analysis was performed on ITT and a statistical chi-square test was used to present the results. Statistically significance was considered if p-value < 0.05.\n\n\nResults\n\nA total of 112 patients were eligible; 12 patients did not meet inclusion criteria and 4 patients declined to participate. As such, 96 patients were randomly assigned to the four groups: 21 in placebo-placebo, 22 in nefopam-placebo, 22 in placebo-nefopam and 21 in nefopam-nefopam groups. There were three patients in the placebo-placebo group, two patients in nefopam-placebo group, two patients in placebo-nefopam group and three patients in nefopam-nefopam group excluded because 10 patients had an operative time of less than 4 hours. The total number of patients assessed was 86 (Figure 1).\n\nDemographic characteristics of patients, preoperative pain score, number of surgical levels, surgery time and anesthetic time are shown in Table 1. The four groups are comparable in age, sex, body mass index, ASA Classification, type of operation, number of levels of spinal surgery, surgery time and anesthetic time. The demographic characteristics and baseline clinical characteristics of patients are similar.\n\nData given as n (%) or median [IQR].\n\nASA Classification: American Society of Anesthesiologists Physical Status Classification System;\n\nTLIF: transforaminal lumbar interbody fusion; TPS: transpedicular screw.\n\nThe postoperative 24-hour morphine consumption (median [IQR]) for the placebo-placebo, nefopam-placebo, placebo-nefopam and nefopam-nefopam groups were similar, at 18 [13.5–29], 20 [11–28.3], 17 [11.5–28.5], 13 [8.5–18.5] mg, respectively (p = 0.223). Time to first dose of morphine were similar, at 30 [7.5–85], 16.5 [10–41], 30 [12.5–67.5], 30 [12.5–91.5] min, respectively (p = 0.710). Pain score and total intraoperative morphine for four treatment groups were also similar (Table 2). All raw data are available from Figshare27.\n\nThe incidence of sedation, nausea and vomiting, sweating and intraoperative arrhythmia did not differ significantly between the groups (Table 3). No patients in four groups developed tachycardia. There was no statistically significant difference in heart rate between the four groups (Figure 2). No patients discontinued the treatment due to adverse events.\n\nPeriod A: before surgical incision; Period B: before the end of surgery.\n\nGroup 1: Placebo - Placebo; Group 2: Nefopam - Placebo; Group 3: Placebo - Nefopam; Group 4: Nefopam - Nefopam. There was no statistically significant difference in heart rate between groups.\n\n\nDiscussion\n\nThe main results of this study showed no difference in 24 hours postoperatively morphine consumption between nefopam group and placebo. Additionally, the analgesic efficacy of nefopam that administered before surgical incision, or before the end of surgery, or both timings compared with placebo were similar. There were no significant differences in early or late side effects between the four groups. The result of this study was similar to previous studies. Merle et al.19 and Remérand et al.20 reported that nefopam was given at the end of surgery and continuous infusion for 24–48 hours postoperatively, did not reduce morphine consumption. Similarly, Richebé et al.13 showed that nefopam given in three periods—at the induction of anesthesia, at the end of surgery and continuous infusion 48 hours postoperatively—did not significant differences in 48 hours morphine consumption. Additionally, Cuvillon et al. showed that continuous intravenous 120 mg nefopam and placebo effects were not different at 48 hours after major abdominal surgery23. These previous studies reported the efficacy of nefopam did not significantly differ to placebo, which might be because most of the previous studies studied the usual dose of nefopam 20 mg per dose by intravenous injection. Although intravenous administration of nefopam was given in a single dose of 20 mg, this gave an analgesic efficacy equivalent to 6–12 mg morphine17,28. On the other hand, many previous studies reported that nefopam had analgesic efficacy to reduce the 24-hours postoperative morphine consumption by up to 19.2–51%17,18. However, those studies studied patients who underwent minor or moderate surgery, such as laparoscopic or breast surgery, which caused mild to moderate post-operative pain (except for two studies that assessed hip arthroplasty and hepatic resection)17,18. The recommended dose of nefopam is 20 mg per dose by the manufacturer. The maximum dose of nefopam is no more than 120 mg per day. This study studied in patients who underwent spinal surgery that could cause moderate to severe pain. So, we used the dose of nefopam 30 mg per dose by intravenous injection which the dose injected could be close to the median effective dose (ED50) of studies by Delage et al. and Beloeil et al. (28 and 27.3 mg), respectively29,30. The side effects of the ED50 doses were not different from the control group. Another study reported that the ED50 of nefopam for postoperative analgesia in patients who have undergone laparoscopic cholecystectomy was 62.1 mg (95% CI, 52.9–72.9 mg). However, there were 27.6% and 20.7% of the patient developed pain upon injection and phlebitis, respectively31. Nevertheless, the present study assessed a 30-mg dose of nefopam; the main result showed no difference in 24 hours postoperatively morphine consumption between nefopam group and placebo. This study reported negative outcomes, that may be a significant change in research result if 1) extension to the study duration of postoperative nefopam, 2) titration of nefopam dosage achieves an optimal dose and 3) the study design is conducted in patients undergoing minor to moderate surgery.\n\nOur study had some limitations. Firstly, we studied a single dose of nefopam; no continuous intravenous infusion or around-the-clock dosing. However, the half-life of a single dose of nefopam administered intravenously is 3–5 hours, so the analgesic effect of one or two- doses of nefopam did not extend to 24 hours postoperatively. Secondly, almost all patients only experienced preoperative mild to moderate pain. We suggested that perioperative nefopam administration has little analgesic effect, especially when given without opioids. The recommendation for clinical usage is used in combination or adjuvant therapy with the other types of analgesics or multimodal analgesia approach.\n\n\nConclusion\n\nAdding perioperative nefopam to opioid analgesic does not improve analgesic efficacy in patients who underwent spine surgery.\n\n\nData availability\n\nFigshare: Raw Data: Analgesic Efficacy of Intravenous Nefopam after Spine Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial. https://doi.org/10.6084/m9.figshare.12029256.v5.27\n\nThis project contains the individual-level data for each participant.\n\nFigshare: Information of abbreviation data set Title: Analgesic Efficacy of Intravenous Nefopam after Spine Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial Untitled Item. https://doi.org/10.6084/m9.figshare.12090753.v132.\n\nThis project contains definitions used in the above dataset27.\n\nFigshare: CONSORT checklist for ‘Analgesic efficacy of intravenous nefopam after spine surgery: a randomized, double-blind, placebo-controlled trial’. https://doi.org/10.6084/m9.figshare.12033693.v433.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBajwa SJ, Haldar R: Pain management following spinal surgeries: An appraisal of the available options. J Craniovertebr Junction Spine. 2015; 6(3): 105–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSommer M, de Rijke JM, van Kleef M, et al.: The prevalence of postoperative pain in a sample of 1490 surgical inpatients. Eur J Anaesthesiol. 2008; 25(4): 267–74. PubMed Abstract \| Publisher Full Text\n\nTobias JD: A review of intrathecal and epidural analgesia after spinal surgery in children. Anesth Analg. 2004; 98(4): 956–65. PubMed Abstract \| Publisher Full Text\n\nFineberg SJ, Nandyala SV, Kurd MF, et al.: Incidence and risk factors for postoperative Ileus following anterior, posterior, and circumferential lumbar fusion. Spine J. 2014; 14(8): 1680–5. PubMed Abstract \| Publisher Full Text\n\nBuvanendran A, Thillainathan V: Preoperative and postoperative anesthetic and analgesic techniques for minimally invasive surgery of the spine. Spine (Phila Pa 1976). 2010; 35(26 Suppl): S274–80. PubMed Abstract \| Publisher Full Text\n\nKehlet H: Postoperative opioid sparing to hasten recovery: what are the issues? Anesthesiology. 2005; 102(6): 1083–5. PubMed Abstract \| Publisher Full Text\n\nWhite PF, Kehlet H: Improving postoperative pain management: what are the unresolved issues? Anesthesiology. 2010; 112(1): 220–5. PubMed Abstract \| Publisher Full Text\n\nPuvanesarajah V, Liauw JA, Lo SF, et al.: Analgesic therapy for major spine surgery. Neurosurg Rev. 2015; 38(3): 407–18; discussion 419. PubMed Abstract \| Publisher Full Text\n\nDevin CJ, McGirt MJ: Best evidence in multimodal pain management in spine surgery and means of assessing postoperative pain and functional outcomes. J Clin Neurosci. 2015; 22(6): 930–8. PubMed Abstract \| Publisher Full Text\n\nLanas A, Benito P, Alonso J, et al.: Safe prescription recommendations for non steroidal anti-inflammatory drugs: consensus document ellaborated by nominated experts of three scientific associations (SER-SEC-AEG). Reumatol Clin. 2014; 10(2): 68–84. PubMed Abstract \| Publisher Full Text\n\nEvans MS, Lysakowski C, Tramèr MR: Nefopam for the prevention of postoperative pain: quantitative systematic review. Br J Anaesth. 2008; 101(5): 610–7. PubMed Abstract \| Publisher Full Text\n\nVerleye M, André N, Heulard I, et al.: Nefopam blocks voltage-sensitive sodium channels and modulates glutamatergic transmission in rodents. Brain Res. 2004; 1013(2): 249–55. PubMed Abstract \| Publisher Full Text\n\nRichebé P, Picard W, Rivat C, et al.: Effects of nefopam on early postoperative Hyperalgesia after cardiac surgery. J Cardiothorac Vasc Anesth. 2013; 27(3): 427–35. PubMed Abstract \| Publisher Full Text\n\nYoo JY, Lim BG, Kim H, et al.: The analgesic effect of nefopam combined with low dose remifentanil in patients undergoing middle ear surgery under desflurane anesthesia: a randomized controlled trial. Korean J Anesthesiol. 2015; 68(1): 43–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNa HS, Oh AY, Koo BW, et al.: Preventive Analgesic Efficacy of Nefopam in Acute and Chronic Pain After Breast Cancer Surgery: A Prospective, Double-Blind, and Randomized Trial. Medicine (Baltimore). 2016; 95(20): e3705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi SK, Yoon MH, Choi JI, et al.: Comparison of effects of intraoperative nefopam and ketamine infusion on managing postoperative pain after laparoscopic cholecystectomy administered remifentanil. Korean J Anesthesiol. 2016; 69(5): 480–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu Manoir B, Aubrun F, Langlois M, et al.: Randomized Prospective study of the Analgesic effect of nefopam after orthopaedic surgery. Br J Anaesth. 2003; 91(6): 836–41. PubMed Abstract \| Publisher Full Text\n\nMimoz O, Incagnoli P, Josse C, et al.: Analgesic efficacy and safety of nefopam vs. propacetamol following hepatic resection. Anaesthesia. 2001; 56(6): 520–5. PubMed Abstract \| Publisher Full Text\n\nMerle JC, Vandroux D, Odin I, et al.: [Analgesic effect of continuous intravenous nefopam after urological surgery]. Ann Fr Anesth Reanim. 2005; 24(1): 13–8. PubMed Abstract \| Publisher Full Text\n\nRemérand F, Le Tendre C, Rosset P, et al.: Nefopam after total hip arthroplasty: role in multimodal analgesia. Orthop Traumatol Surg Res. 2013; 99(2): 169–74. PubMed Abstract \| Publisher Full Text\n\nLee JH, Kim JH, Cheong YK: The analgesic effect of nefopam with fentanyl at the end of laparoscopic cholecystectomy. Korean J Pain. 2013; 26(4): 361–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark HJ, Park JU, Yoo W, et al.: Analgesic effects of nefopam in patients undergoing bimaxillary osteotomy: A double-blind, randomized, placebo-controlled study. J Craniomaxillofac Surg. 2016; 44(2): 210–4. PubMed Abstract \| Publisher Full Text\n\nCuvillon P, Zoric L, Demattei C, et al.: Opioid-sparing effect of nefopam in combination with paracetamol after major abdominal surgery: a randomized double-blind study. Minerva Anestesiol. 2017; 83(9): 914–20. PubMed Abstract \| Publisher Full Text\n\nChaudakshetrin P, Prateepavanich P, Chira-Adisai W, et al.: Cross-cultural adaptation to the Thai language of the neuropathic pain diagnostic questionnaire (DN4). J Med Assoc Thai. 2007; 90(9): 1860–5. PubMed Abstract\n\nPasero C: Assessment of sedation during opioid administration for pain management. J Perianesth Nurs. 2009; 24(3): 186–90. PubMed Abstract \| Publisher Full Text\n\nWengritzky R, Mettho T, Myles PS, et al.: Development and validation of a postoperative nausea and vomiting intensity scale. Br J Anaesth. 2010; 104(2): 158–66. PubMed Abstract \| Publisher Full Text\n\nEiamcharoenwit J: Raw Data: Analgesic Efficacy of Intravenous Nefopam after Spine Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial. figshare. 2020; Dataset. http://www.doi.org/10.6084/m9.figshare.12029256.v5\n\nMcLintock TT, Kenny GN, Howie JC, et al.: Assessment of the analgesic efficacy of nefopam hydrochloride after upper abdominal surgery: a study using patient controlled analgesia. Br J Surg. 1988; 75(8): 779–81. PubMed Abstract \| Publisher Full Text\n\nDelage N, Maaliki H, Beloeil H, et al.: Median effective dose (ED50) of nefopam and ketoprofen in postoperative patients: a study of interaction using sequential analysis and isobolographic analysis. Anesthesiology. 2005; 102(6): 1211–6. PubMed Abstract \| Publisher Full Text\n\nBeloeil H, Eurin M, Thévenin A, et al.: Effective dose of nefopam in 80% of patients (ED80): a study using the continual reassessment method. Br J Clin Pharmacol. 2007; 64(5): 686–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim H, Lee DK, Lee MK, et al.: Median effective dose of nefopam to treat Postoperative Pain in patients who have undergone laparoscopic cholecystectomy. J Int Med Res. 2018; 46(9): 3684–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEiamcharoenwit J: Information of abbreviation data set Title: Analgesic Efficacy of Intravenous Nefopam after Spine Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial Untitled Item. figshare. 2020; Dataset. http://www.doi.org/10.6084/m9.figshare.12090753.v1\n\nEiamcharoenwit J: CONSORT checklist - Analgesic Efficacy of Intravenous Nefopam after Spine Surgery: A Randomized, Double-Blind, Placebo-Controlled Trial. figshare. 2020; Dataset. http://www.doi.org/10.6084/m9.figshare.12033693.v4" }	[ { "id": "65114", "date": "07 Jul 2020", "name": "Rattaphol Seangrung", "expertise": [ "Reviewer Expertise pain management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is a randomized, double-blinded, placebo-controlled trial that used nefopam 30 mg perioperative period for assessing its efficacy in spine surgery. The primary efficacy was defined as the percentage of a reduction in 24-hour morphine consumption during each time of nefopam administration (every four hours). However, one question I have is why the results of morphine consumption (table 2) were showed in the only milligram of morphine consumption at each time point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5777", "date": "29 Jul 2020", "name": "Jatuporn Eiamcharoenwit", "role": "Author Response", "response": "Thank you for your review and comment. Because 24 hours postoperatively total opioid consumption in the placebo group is less than in nefopam groups, and the result shows no significant difference in 24-hour postoperative morphine consumption between four groups. So, the primary outcome was to determine the milligram of 24-hour morphine consumption." }, { "c_id": "5812", "date": "26 Aug 2020", "name": "Jatuporn Eiamcharoenwit", "role": "Author Response", "response": "Dear Dr. Rattaphol Seangrung,Thank you for giving a chance to improve our research article.We changed the primary efficacy to “the primary efficacy was defined as the cumulative morphine dose received 24 hours postoperatively by Patient Controlled Analgesia (PCA) during each time of nefopam administration”We already edited as new version. Hopefully, you would appreciate our revised version. Regards,Jatuporn Eiamcharoenwit" } ] }, { "id": "65113", "date": "30 Jul 2020", "name": "Francis Remérand", "expertise": [ "Reviewer Expertise anesthesiology and critical care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reports no morphine consumption reduction in the 24 first hours after spine surgery in the 4 tested groups: placebo-placebo, single preincisional nefopam bolus – placebo, placebo-end surgery single nefopam bolus, preincisional and end surgery nefopam injections.\nDespite a well design study (randomized monocentric double blind controlled study), major points have to be clarified, before interpreting the results.\nThe sample size calculation is unclear:\n\nIn the methods chapter, it is calculated using a “percentage of reduction\" of morphine consumption during the first 24 postoperative hours. The mean consumption to calculate the sample size is not given. By using the shared data of this study, the overall morphine consumption (N=96) is 22 mg +/- 20 (mean +/- standard deviation). With a power of 80%, an alpha at 0.05, 99 patients per group are required to detect the chosen 35% reduction in a two tailed test (https://biostatgv.sentiweb.fr), while the authors' calculation resulted in 21 patients per group. This study seems therefore largely underpowered.\n\nThe perioperative analgesic use should be more accurately described:\nWas intraoperative analgesia obtained using morphine? (Methods, paragraph 6). If yes, the total dose should be mentioned in Table 2 (I suppose the 10-15mg intraoperative morphine mentioned in table 2 is for analgesia, not for a 6 hours anesthesia). Morphine was injected before the PACU in some patients (table 2 and data file): can you describe how the physician decided to inject or not morphine at the end of the surgery?\n\nMethods, end of paragraph 4: no patient received preoperative gabapentinoid or opiod or NSAID. In the shared data file, 39/96 patients had gabapentinoid, 11 had NSAID and several had tramadol. Please explain these contradictory data.\n\nData analysis:\nResults, first paragraph: 10 of 96 patients were excluded because operative time < 4 hours - Why did you choose such a limit? This is quite in opposition with the “intention to treat analysis” mentioned in the last paragraph of methods. Please explain why we find 39 patients (and not 10) with an operative time < 240 min in the shared data file. Please clearly identify in the shared file which patients have been excluded.\n\nHow were the lacking data seen in the shared data file managed? (“NA”).\n\nResults:\nPlease replace in the third paragraph “similar” by “not different” (2 occurrences).\n\nTable 3: are we dealing with nb of patients, nb of side effect occurrence? Can you mention cumulative data for patients (nb of patients having at least one occurrence of the side effect during the first 24 hours)?\n\nI don’t understand Figure 2: can you explain what each “time 0” is?\n\nThe detailed comments on the discussion have to be performed after the above points have been clarified. The global comment on discussion is that the main limitation of this study is it is largely underpowered. So conclusions about negative results have to be very cautious (more than in the present version of the paper). Nevertheless, these data have to be published, since they are fitted for a potential future meta-analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5811", "date": "26 Aug 2020", "name": "Jatuporn Eiamcharoenwit", "role": "Author Response", "response": "Dear Dr. Francis Remérand,Thank you for giving a chance to improve our research article. Hopefully, you would appreciate our revised version. Regards,Jatuporn Eiamcharoenwit Response to reviewer: This study reports no morphine consumption reduction in the 24 first hours after spine surgery in the 4 tested groups: placebo-placebo, single preincisional nefopam bolus – placebo, placebo-end surgery single nefopam bolus, preincisional and end surgery nefopam injections.Despite a well designed study (randomized monocentric double-blind controlled study), major points have to be clarified, before interpreting the results. Comments:The sample size calculation is unclear:In the methods chapter, it is calculated using a “percentage of reduction\" of morphine consumption during the first 24 postoperative hours. The mean consumption to calculate the sample size is not given. By using the shared data of this study, the overall morphine consumption (N=96) is 22 mg +/- 20 (mean +/- standard deviation). With a power of 80%, an alpha at 0.05, 99 patients per group are required to detect the chosen 35% reduction in a two tailed test (https://biostatgv.sentiweb.fr), while the authors' calculation resulted in 21 patients per group. This study seems therefore largely underpowered.Response: We changed the primary efficacy to “the primary efficacy was defined as the cumulative morphine dose received 24 hours postoperatively by Patient Controlled Analgesia (PCA) during each time of nefopam administration” (Outcome, paragraph 2). Response: According to previous study, PCA-administered morphine over 24 hours was significantly less for the nefopam group than control group 21.2 (15.3) and 27.3 (19.2) mg respectively; P=0.02). In nefopam group, patients received every 4-hour i.v. nefopam 20 mg. The first dose was infused in the operating room at the onset of deep-wound closure1. Reference Du Manoir B, Aubrun F, Langlois M, et al.: Randomized Prospective study of the Analgesic effect of nefopam after orthopaedic surgery. Br J Anaesth. 2003; 91:836-41. We reanalyzed the sample size calculation. We will calculate sample size using One Way Analysis of Variance (ANOVA).Table 1: The cumulative morphine dose received 24 hours postoperatively in 4 group.Groups Cumulative morphine dose received 24 hours postoperatively, mg (SD)Placebo - Placebo 27.3 (19.2) using previous data1Nefopam - Placebo 24 estimated from ascendingPlacebo - Nefopam 21.2 (15.3) using previous data1Nefopam - Nefopam 18 estimated from descendingVariance of means, V 11.792Table 2: Power and Sample Size for One Way Analysis of Variance (ANOVA)Test Significance Level, α 0.05Number of Groups, G 4Variance of Means, V 11.792Common Standard Deviation, σ 9.120Effect Size, Δ² = V/σ² 0.142Power (%) 80Sample Size per Group, n 21Previous studies have shown that when nefopam was given before the end of surgery, postoperatively 24-hour morphine consumption was 21.2 (15.3). When the sample size in each of the 4 groups is 21, a one-way analysis of variance will have 80% power to detect at the 5% level a difference in means characterized by a variance of means, V, of 11.792, assuming that the common standard deviation is 9.12. Response: We revised information for the sample size calculation and statistical analysis chapter, paragraph 1. “Previous studies have shown that when nefopam was given before the end of surgery, postoperatively 24-hour morphine consumption was 21.2 (15.3) mg. Morphine dose received 24 hours postoperatively in control group was 27.3 (19.2) mg1. The sample size was determined from total postoperatively 24-hour morphine consumption. Neither the mean of 24-hour morphine consumption in the group receiving nefopam before surgical incision nor the group receiving nefopam before surgical incision plus at the end of surgery compared with placebo was reported in these previous studies. Therefore, this sample size estimated the level of reduction in 24-hour morphine consumption in both groups. The sample size gives the trial a power of 80%, sets a two-tailed α at 0.05 in means characterized by a variance of means of 11.792, assuming that the common standard deviation is 9.12. The calculation resulted in 21 patients per group. To compensate for 10% attrition rate, we included 24 patients per group. The total sample size is 96 patients” (Sample size calculation and statistical analysis, paragraph 2).The perioperative analgesic use should be more accurately described:Was intraoperative analgesia obtained using morphine? (Methods, paragraph 6). If yes, the total dose should be mentioned in Table 2 (I suppose the 10-15mg intraoperative morphine mentioned in table 2 is for analgesia, not for 6 hours anesthesia). Morphine was injected before the PACU in some patients (table 2 and data file): can you describe how the physician decided to inject or not morphine at the end of the surgery?Response: (Methods, paragraph 6) Intraoperative morphine 10 – 15 mg was not for post operate analgesia. It was obtained for an average of 6 hours of anesthesia. So, we will move intraoperative morphine (mg) in table 2 to table 1.Response: (table 2 and data file) The wording of MO_dose_beforePCA in the shared data file is a loading dose before the administration of Patient Controlled Analgesia (PCA) at PACU. It was not administered morphine before admitting the PACU.At the PACU, the patients were asked pain scores every 15 minutes using a numerical rating scale. If the pain score is greater than or equal to 4 points, the patients were injected with 2 mg of morphine every 10 minutes until the patients reported pain scores of less than 4. Then, the patients started to use the PCA device in the post-operative period. Methods, end of paragraph 4: no patient received preoperative gabapentinoid or opiod or NSAID. In the shared data file, 39/96 patients had gabapentinoid, 11 had NSAID and several had tramadol. Please explain these contradictory data. Response: We wrote unclear. In the shared data file, the wording of Preop_Med is mean that current medications of patients. They were not premedication before day surgery. No patients received NSAIDs, gabapentinoid, or opioids on the morning of surgery. We change the word to “Current_Med” in the shared data file. Data analysis:Results, first paragraph: 10 of 96 patients were excluded because operative time < 4 hours - Why did you choose such a limit? This is quite in opposition with the “intention to treat analysis” mentioned in the last paragraph of methods. Please explain why we find 39 patients (and not 10) with an operative time < 240 min in the shared data file. Please clearly identify in the shared file which patients have been excluded.Response: We have incorrectly written. Ten patients were unable to use patient-controlled analgesia (PCA) device postoperatively that cause patients to drop out of clinical trials. We change the sentence to “There were three patients in the placebo-placebo group, two patients in nefopam-placebo group, two patients in placebo-nefopam group and three patients in nefopam-nefopam group were excluded from analysis. Because 10 patients were unable to use patient-controlled analgesia device postoperatively” (Results, paragraph 1). The sample size was anticipated by 10% attrition rates. Patients have been excluded who were number 31, 62 and 83 in placebo-placebo group, number 24 and 75 in nefopam-placebo group, number 69 and 73 in placebo-nefopam group, number 41, 66 and 72 in nefopam-nefopam group. Ten patients in the shared data file are highlighted in yellow (Shared data file). We were correcting code for each group (Shared data file, Group).Response: The sentence “The main analysis was analyzed by intention-to-treat (ITT)” was deleted. And, we change the sentence to “Safety data analysis was analyzed by a statistical chi-square test” (Sample size calculation and statistical analysis, paragraph 2).Response: We change the sentence to “Analysis of the Kruskal-Wallis test shows no significant difference in 24-hour postoperative morphine consumption between four groups, which were 18 [IQR 13.5-29], 20 [IQR 11-28.3], 17 [IQR 11.5-28.5], 13 [IQR 8.5-18.5] mg., respectively (p = 0.223)” (The results section in an abstract) How were the lacking data seen in the shared data file managed? (“NA”).Response: The lacking data (“NA”) were 10 patients who were unable to use patient-controlled analgesia (PCA) device postoperatively. Ten patients who dropped out of the study (Shared data file, highlighted in yellow). Results:Please replace in the third paragraph “similar” by “not different” (2 occurrences).Response: We replace the word to “not different” in the third paragraph of results. Table 3: are we dealing with nb of patients, nb of side effect occurrence? Can you mention cumulative data for patients (nb of patients having at least one occurrence of the side effect during the first 24 hours)?Response: The data were rechecked. We intended to report the occurrence of side effects. We change the word to “Variables, number of occurrences” in Table 3. We mentioned the number of patients having at least one occurrence of the side effect during the first 24 hours in the last row of table 3 (Table 3). I don’t understand Figure 2: can you explain what each “time 0” is?Response: Figure 2 – Heart rate at time 0 is heart rate before starting the study drug before surgical incision (Figure 2, left).Heart rate at time 0 is heart rate at the end of surgery (Figure 2, right)." } ] } ]	1	https://f1000research.com/articles/9-516
https://f1000research.com/articles/9-668/v1	02 Jul 20	{ "type": "Case Report", "title": "Case Report: Cutaneous chilblains-like lesions (CCLL) versus COVID-19 toes during the pandemic. Confocal findings and proposal for new acronym, CCLL – is there a diagnostic window?", "authors": [ "Joanna Ludzik", "Alexander Witkowski", "Donna E. Hansel", "Philipp W. Raess", "Kevin White", "Sancy Leachman", "Joanna Ludzik", "Donna E. Hansel", "Philipp W. Raess", "Kevin White", "Sancy Leachman" ], "abstract": "The COVID-19 outbreak caused by the novel coronavirus, SARS-CoV-2, typically presents with symptoms including fever, cough, headache, myalgia, asthenia, anosmia, diarrhea, and sometimes pneumonia, which can be fatal. Recently, new dermatologic findings have been described in association with the disease that can potentially be a distinguishing feature of infection. One such feature resembles chilblains and this case report represents a unique presentation of this feature.", "keywords": [ "coronavirus", "Covid-19", "pernio", "chilblains", "dermoscopy", "confocal", "diagnostic window" ], "content": "Introduction\n\nThe COVID-19 outbreak caused by the novel coronavirus, SARS-CoV-2, typically presents with symptoms including fever, cough, headache, myalgia, asthenia, anosmia, diarrhea, and sometimes pneumonia, which can be fatal. Recently, new dermatologic findings have been described in association with the disease that can potentially be a distinguishing feature of infection. One such feature resembles chilblains1. Cutaneous chilblains-like lesions (CCLL), described in multiple case reports from around the globe, have demonstrated either an erythematous-edematous or blistering skin lesion that mostly affects the toes and soles. Over the course of one to two weeks, lesions become more purpuric and flatten, finally resolving spontaneously without any treatment. The majority of patients with CCLL are generally in good health, without significant coronavirus symptoms, may have a recent history of mild upper respiratory symptoms, but no prior history of similar cutaneous lesions2. By contrast, non-COVID-19-related chilblains are associated with exposure to low temperatures and may be associated with autoimmune disorders (chilblain lupus), hematologic disorders and rarely viral infections. During recent months, CCLL has been reported in association with COVID-19, although the timing of these lesions relative to active infection appears to vary. In a cohort of patients reported from Italy, some patients developed skin findings only during the initial course of disease, while others reported onset at later stages3.\n\nThe ill-defined timing of presentation of CCLL in confirmed COVID-19 positive patients may be associated with onset, progression or resolution of the disease. Due to potentially unreliable and subjective patient reporting, lack of awareness that these lesions may be a symptom of COVID-19 disease, and limited coronavirus testing availability, the diagnosis of COVID-19 and/or CCLL may be missed or unreported. Based on our experience with the COVID-19 population in Oregon, we hypothesize that there may be a limited and possibly non-overlapping diagnostic window for COVID-19 infection and CCLL. If true, this could result in classic CCLL features in the absence of a positive COVID-19 diagnostic test. Here, we present the case of a 48-year-old healthy woman, who presented with CCLL on her toes and tested as negative for COVID-19.\n\n\nCase report\n\nA 48-year-old healthy female patient presented to our hospital with CCLL on her toes. She had no other underlying diseases and denied recent exposure to cold temperatures. She is a healthcare provider with potential exposure to SARS-CoV-2. The patient reported upper respiratory symptoms four weeks prior to development of cutaneous lesions, which included mild sore throat, non-productive cough, and chest pain. A viral test was unavailable at that time. Her highest temperature was 99.4°F. Concurrent with the onset of cutaneous manifestations, she and her husband both experienced intermittent diarrhea over a period of 3 days without any recurrent respiratory symptoms or fever. Two 9-year old children in the home were asymptomatic. The patient began developing blister-like lesions on both her feet, starting with a single, isolated lesion on the bottom of one toe. Additional lesions developed on the lateral aspects and top of 6 toes, primarily at the distal aspect. The cutaneous lesions slowly progressed from pink macules and papules to violaceous lesions with surrounding pink erythema (Figure 1).\n\n(A) Clinical images taken of the patient’s left foot, showing multiple well circumscribed violaceous discolorations with surrounding pink erythema on the distal toes. (B) Closeup of the cutaneous chilblains-like lesions. (C) Dermoscopy image of the fourth toe showing multiple red globules. (D) Patient-submitted clinical photos of her indoor pet cat with multiple red-purple well-circumscribed slightly raised spots bilaterally that also resolved spontaneously. Additionally, ocular discharge was present.\n\nA 3mm punch biopsy was taken on the patient’s third left lateral toe in the most prominent area of CCLL findings approximately 2 weeks after the onset of lesions and during early stages of lesion resolution. Pathologic findings included parakeratosis, overlying vacuolar alteration of the basal layer with dyskeratosis, fibrinoid degeneration and edema of the papillary dermis (Figure 2). No thrombi were seen. Superficial and deep perivascular lymphocytic infiltrate with extravasated red cells were also present with a final pathologic diagnosis of chilblains/perniosis, consistent with those seen in COVID-associated chilblains-like lesions (“COVID-toes”). Immunohistochemical analysis demonstrated the perivascular lymphocytic infiltrate to be composed predominantly of CD3+ T cells, with very rare CD20+ B cells. The T cells demonstrated an unremarkable CD4:CD8 ratio. Rare CD163+ histiocytes were scattered throughout.\n\n(A) Reflectance confocal microscopy (RCM) image (1mm x 1mm) of the biopsied toe showing an enlarged keratinocytes and presence of dendritic cells (activated Langerhans cells in the circle). (B) RCM image (1mm x 1mm) of the epidermis and dermal epidermal junction showing bright white dots representing inflammatory cells (↑) and an enlarged, bright and reflective swollen dermal papillae (↓). (C) Histopathology H&E image at 10x magnification. (D) Histopathology H&E image at 10x magnification stained for CD8, which shows the vast majority of lymphocytes to be T cells with a relatively unremarkable CD4:CD8 ratio, present mostly in a perivascular distribution and in the superficial dermis.\n\nAn in vivo virtual biopsy with reflectance confocal microscopy (RCM; Vivascope 3000, Caliber I.D., Rochester, NY, USA) was performed on the same toe that was examined histologically. Findings included an irregular epidermis with broadened honeycombed pattern and absence of pagetoid cells. In the dermal-epidermal junction, enlarged and swollen highly reflective papillae as well as dendritic cells (activated Langerhans cells) and bright dots representing inflammatory infiltrate were present. Within two days of the biopsy and virtual biopsy, the patient was tested for SARS-COV-2 (PCR) and antibodies, neither of which yielded a positive result. The timing of the tests or failure of many to mount an antibody-mediated response make it difficult to determine whether this is a COVID-related case or not, since the specificity of available testing is low. No therapeutic intervention was made and upon follow-up the patients symptoms resolved.\n\nOf interesting note, the patient reported that at the same time she and her husband developed the gastrointestinal symptoms, her elderly pet cat also developed an upper respiratory infection with a three-day history of lethargy, loss of appetite and increased sneezing and coughing, which spontaneously resolved. Additionally, the interior distal aspect of the cat’s ears developed multiple red-purple, well-circumscribed, slightly raised spots bilaterally that resembled the patient’s cutaneous findings. These lesions resolved within 3 weeks of onset.\n\n\nDiscussion\n\nCutaneous lesions associated with coronavirus-induced vasculitis has been previously reported in a cat with feline infectious peritonitis and concurrent feline immunodeficiency virus infection4. In this case, the animal presented with a similar two-week history of pyrexia, loss of appetite and weight loss, sneezing, bilateral nasal and ocular discharge to our patient’s cat. The cat described in this previous case report presented with multiple well-circumscribed slightly raised, red nodules and positive FCoV4. Recently at the Bronx Zoo in New York, eight big cats (5 tigers and 3 lions) have tested positive for the COVID-19 virus and are believed to have been infected by an asymptomatic zookeeper. The cats started showing symptoms including a dry cough, with one tiger who did not show any symptoms for coronavirus5. A recent publication also suggests that cats may be intermediate hosts of the SARS-Cov-26,7.\n\nDue to the potential for additional publications about cutaneous findings in COVID-19 patients and the possibility of confusion between the previously reported acronym for chilblains-like lesions (CLL) and chronic lymphocytic leukemia (CLL), we propose to precisely distinguish the nomenclature of these new dermatologic findings with the acronym of cutaneous chilblains-like lesions (CCLL). In addition, this is the first report of reflectance confocal microscopy findings in CCLL and the large number of dendritic antigen-presenting cells may be a distinguishing feature of CCLL. Further studies are needed to determine if these RCM findings are exclusive to CCLL or also found in traditional chilblains. It is not possible to conclude that our patient had COVID-19-associated CCLL because she did not have a positive viral or antibody test and may be a limitation to our findings. However, she did have a pattern of development that is highly suspicious for infection-induced CCLL, including a history of respiratory and gastrointestinal symptoms, aged below 50 years, no comorbidities, a previous state of good health, lack of prior history of skin findings consistent with chilblains, lack of cold temperatures in the region, latency between mild systemic symptoms and the morphology of CCLL and the development of similar finding in her pet cat.\n\nWhile these findings in combination may be coincidental, we hypothesize that there may be a delayed immune-mediated reaction to SARS-COV-2 in genetically predisposed patients who may test negative during a certain diagnostic window. Further studies are needed to determine the accurate diagnostic window for COVID-19 diagnosis and possible cutaneous manifestations.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAlramthan A, Aldaraji W: A case of COVID-19 presenting in clinical picture resembling chilblains disease. First report from the Middle East. Clin Exp Dermatol. 2020. Publisher Full Text\n\nNyssen A, Benhadou F, Magnée M, et al.: Chilblains. Vasa. 2020; 49(2): 133–140. PubMed Abstract \| Publisher Full Text\n\nPiccolo V, Neri I, Filippeschi C, et al.: Chilblain-like lesions during COVID-19 epidemic: a preliminary study on 63 patients. J Eur Acad Dermatol Venereol. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCannon MJ, Silkstone MA, Kipar AM: Cutaneous lesions associated with coronavirus-induced vasculitis in a cat with feline infectious peritonitis and concurrent feline immunodeficiency virus infection. J Feline Med Surg. 2005; 7(4): 233–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttps://www.nationalgeographic.com/animals/2020/04/tiger-coronavirus-covid19-positive-test-bronx-zoo/\n\nHalfmann PJ, Hatta M, Chiba S, et al.: Transmission of SARS-CoV-2 in Domestic Cats. N Engl J Med. 2020. PubMed Abstract \| Publisher Full Text\n\nGollakner R, Capua I: Is COVID-19 the first pandemic that evolves into a panzootic? Vet Ital. 2020; 56(1): 7–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66284", "date": "17 Jul 2020", "name": "Marco Ardigo", "expertise": [ "Reviewer Expertise Rare skin diseases and non invasive techniques for skin analysis" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a report of a case of chilblains-like lesions on a COVID-19+ patient. Even if the case could be potentially interesting (several cases have been already reported in the literature) and it has been well presented, the use of confocal microscopy in this specific case does not add any new information with no distinctive features.\nFew points:\nDermal features (important for diagnosis) cannot be seen on confocal microscopy.\n\nWhy the authors underline the absence of pagetoid cells in a non suspected melanocytic lesion?\n\nHow the authors define activated Langerhans cells based on confocal microscopy observation alone?\n\nIn my view, this case report does not merit to be considered for indexing as it is presented.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "66287", "date": "27 Jul 2020", "name": "Adam Reich", "expertise": [ "Reviewer Expertise general dermatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 demonstrated a case of chilblain-like lesions in a 48-year old woman. This case report is interesting, however, I have several comments, which can possibly be of relevance. Recently, \"A COVID toe\" has been described as a manifestation of COVID-19 infection. However, most reports are focused on single patients or demonstrate only a case series. Today, it is a matter of debate, whether indeed this is a specific cutaneous manifestation of COVID-19 infection or whether it may also result from other viral infections. In a recent study by Hubiche et al. (Hubiche T et al. Negative SARS-CoV-2 PCR in patients with chilblain-like lesions [published online ahead of print, 2020 Jun 18]. Lancet Infect Dis. 2020;S1473-3099(20)30518-11) in only 30% of patients, COVID-19 infection (current or past) could be proofed. It is also important to exclude other causes of chilblains, mainly lupus erythematosus, which is not documented in current report.\n\nThe authors also propose a new acronym for chilblains-like lesions with an additional word cutaneous (CCLL) to distinguish it from CLL - chronic lymphocytic leukemia. However, I disagree with such a proposal as adding a word \"cutaneous\" implicates that there are also extracutaneous manifestations of chilblains-like lesions, which is not the case in the current situation.\n\nSome minor comments: The authors should avoid to use the term \"unique\" in the abstract It is unclear, what the authors meant under the term \"blister-like lesions\"\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5809", "date": "26 Aug 2020", "name": "Alexander Witkowski", "role": "Author Response", "response": "To Reviewer Prof. Adam Reich: All comments addressed, the term \"unique\" in the abstract has been removed ; \"blister-like lesions\" have been changed to blisters as this was the actual presentation ; CCLL has been removed and it remains Chilblains-like lesions ; new citation added (number 8) and added in the discussion. Thank you for your comments and suggestions" } ] }, { "id": "66288", "date": "30 Jul 2020", "name": "Magdalena Żychowska", "expertise": [ "Reviewer Expertise Dermoscopy", "non-invasive skin imaging techniques" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented an interesting case of chilblains-like lesions in a 48-year-old woman. The case report is well-organized and written in a clear fashion. Furthermore, it includes dermoscopic and confocal findings of chilblains-like lesions. However, I have several comments and suggestions:\nthe abstract could be longer and should include more details regarding the presented case and findings\n\nif available, please include lab findings (antinuclear antibodies? serology for Mycoplasma/Chlamydia infection? C-reactive protein?)\n\nI'm not a big supporter of the acronym introduced by the authors just to separate the name of the dermatological lesions from chronic lymphocytic leukemia (CLL).\nThe dermoscopic and confocal findings presented by the authors may be of some value for future diagnostic process of COVID-19-related lesions, but it should be taken into consideration that the infection was not confirmed in the presented patient and the authors only hypothesize that there may be a delayed immune-mediated reaction. Undoubtedly, further studies, comparing the dermoscopic and confocal features of COVID-19 toes and chilblains-like lesions resulting from other conditions are needed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5810", "date": "26 Aug 2020", "name": "Alexander Witkowski", "role": "Author Response", "response": "To Reviewer Dr. Magdalena Żychowska: All comments addressed, the abstract has been made longer including details regarding the presented case and findings; lab findings unavailable ; CCLL has been removed and it remains Chilblains-like lesions ; new citation added (number 8) and added in the discussion. Thank you for your comments and suggestions" } ] } ]	1	https://f1000research.com/articles/9-668
https://f1000research.com/articles/9-609/v1	15 Jun 20	{ "type": "Research Article", "title": "Prediction of repurposed drugs for treating lung injury in COVID-19", "authors": [ "Bing He", "Lana Garmire", "Bing He" ], "abstract": "Background: Coronavirus disease (COVID-19) is an infectious disease discovered in 2019 and currently in outbreak across the world. Lung injury with severe respiratory failure is the leading cause of death in COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there still lacks efficient treatment for COVID-19 induced lung injury and acute respiratory failure. Methods: Inhibition of angiotensin-converting enzyme 2 (ACE2) caused by the spike protein of SARS-CoV-2 is the most plausible mechanism of lung injury in COVID-19. We performed drug repositioning analysis to identify drug candidates that reverse gene expression pattern in L1000 lung cell line HCC515 treated with ACE2 inhibitor. We confirmed these drug candidates by similar bioinformatics analysis using lung tissues from patients deceased from COVID-19. We further investigated deregulated genes and pathways related to lung injury, as well as the gene-pathway-drug candidate relationships. Results: We propose two candidate drugs, COL-3 (a chemically modified tetracycline) and CGP-60474 (a cyclin-dependent kinase inhibitor), for treating lung injuries in COVID-19. Further bioinformatics analysis shows that 12 significantly enriched pathways (P-value <0.05) overlap between HCC515 cells treated with ACE2 inhibitor and human COVID-19 patient lung tissues. These include signaling pathways known to be associated with lung injury such as TNF signaling, MAPK signaling and chemokine signaling pathways. All 12 pathways are targeted in COL-3 treated HCC515 cells, in which genes such as RHOA, RAC2, FAS, CDC42 have reduced expression. CGP-60474 shares 11 of 12 pathways with COL-3 and common target genes such as RHOA. It also uniquely targets other genes related to lung injury, such as CALR and MMP14. Conclusions: This study shows that ACE2 inhibition is likely part of the mechanisms leading to lung injury in COVID-19, and that compounds such as COL-3 and CGP-60474 have potential as repurposed drugs for its treatment.", "keywords": [ "COVID-19", "SARS-CoV-2", "lung injury", "ACE2", "COL-3", "CGP-60474" ], "content": "Abbreviations\n\nCOVID-19: coronavirus disease 2019, SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, ACE2: angiotensin-converting enzyme 2, AGER: advanced glycosylation end-product specific receptor, LBP: lipopolysaccharide binding protein, SCGB1A1: secretoglobin family 1A member, SFTPD: surfactant protein D, RAS: renin–angiotensin system, Ang II: angiotensin II, Ang-(1-7): angiotensin (1-7), ARDS: acute respiratory distress syndrome, ACE2i: inhibition of ACE2, NS: not significant, NA: not available.\n\n\nIntroduction\n\nCoronavirus disease 2019 (COVID-19) is an infectious disease discovered in 2019 and currently in outbreak across the world, resulting in more than 4.3 million infections and over 291,354 deaths as of 12th May. It is causing tens of thousands of new infections and thousands of mortalities every day. Patients with COVID-19 present with respiratory symptoms. Severe viral pneumonia related lung injury with acute respiratory failure is the main reason for COVID-19 related death1. However, there still lacks efficient treatment for COVID-19 induced lung injury and acute respiratory failure.\n\nCoronaviruses (CoVs), are a large family of enveloped, positive-sense, single-stranded RNA viruses, which can be found in many vertebrates, such as birds, pigs and humans, and cause various diseases. A novel CoV, termed severe acute respiratory syndrome (SARS)-CoV-2, is the cause of COVID-19. Lung injury with acute respiratory failure was also the main reason for death in patients with SARS2. The spike protein of SARS-CoV-2 shares 79.5% sequence identity with the SARS-CoV virus3–5, which caused the SARS pandemic in 2002, resulting in 774 deaths in 8096 confirmed patients in 29 countries6. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the entry receptor and cellular serine protease TMPRSS2 for S protein priming to allow fusion of viral and cellular membranes7, similar to SARS-CoV8,9. Since in SARS-CoV infection, the spike protein of SARS-CoV inhibits ACE2 to cause severe lung injury and acute respiratory failure10,11, it is highly likely that SARS-CoV-2 uses the same mechanism. Inhibition of ACE2 may be part of the pathogenic mechanism in SARS-CoV-2 induced lung injury and acute respiratory failure. Therefore, a drug repurposing pipeline aiming to reverse the gene expression pattern due to ACE2 inhibition may be a candidate for treating lung injury in COVID-19.\n\nTowards this goal, we performed drug repositioning analysis to identify drugs and compounds for treating SARS-CoV-2 induced lung injury. To explore the mechanisms of proposed drug treatment, we further investigated deregulated genes and pathways in both human lung cells treated with ACE2 inhibitor and human lung tissues from patients deceased from COVID-19. Our results revealed that lung injury related molecular mechanisms are shared between ACE2 inhibition and SARS-CoV-2 infection. Moreover, our proposed drugs can target key genes in these mechanisms, and therefore may prevent lung injury in COVID-19.\n\n\nMethods\n\nRNA-Seq data from human lung tissues from two COVID-19 deceased patients and age-matched healthy lung tissues, as well as human lung A549 cells with or without H1N1 infection, were downloaded from Gene Expression Omnibus (GEO) database (accession number: GSE147507), as reported by Melo et al.12. Level 5 LINCS L1000 data, a collection of gene expression profiles for thousands of perturbagens at a variety of time points, doses, and cell lines, were downloaded from the GEO database (accession numbers: GSE70138 and GSE92742). Gene expression profiles in lung cells were extracted from the downloaded L1000 dataset using R scripts (code is available on GitHub)13. The extracted data include 37,366 treatments of 12,707 drugs in 13 lung cell lines at different time points and doses. Two lung cell lines, A549 and HCC515, were treated with 10 µM moexipril, a homologue of ACE2 that inhibits ACE2 and ACE. Gene expression profiles were collected from A549 and HCC515 cells at six and 24 hours after treatment. Upon moexipril treatment, ACE2 level decreased with time in HCC515 as expected; however, levels increased in A549. This prompted us to focus the analysis on the HCC515 line, which showed the inhibition effect of moexipril. Differential expression of genes was measured by z-score14.\n\nThe RNA-Seq data were analyzed using DESeq215 (version: 1.26.0). Differential gene expressions were identified by comparing cases and controls (e.g. COVID-19 lung tissue vs. the healthy lung tissue, or cells with H1N1 infection vs. those without H1N1 infection). The top 1000 differential expressed genes were selected by the absolute z-score value. These genes were then used for pathway enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.816. Significant pathways (P-value <0.05) were compared between HCC515 cells with ACE2 inhibitor inhibition and lung tissues from COVID-19 deceased patients. A gene is called “consistent”, if it shows changes in the same direction (increase or decrease) with ACE2 inhibitor treatment and SARS-CoV-2 infection. The importance of pathways was ranked using the following score:\n\n\n\nPvalueACE2i is the P-value from pathway enrichment analysis for the top 1000 differentially expressed genes in HCC515 cells treated with ACE2 inhibitor. PvalueCOVID19 is the P-value from pathway enrichment analysis for the top 1000 differentially expressed genes in human lung tissue infected by SARS-CoV-2. nconsistent is the number of consistent genes in that pathway.\n\nThe importance of genes was ranked by the following score:\n\nZscoreACE2i is the z-score of the gene in HCC515 cells treated with ACE2 inhibitor. ZscoreCOVID19 is the z-score of the gene in human lung tissue infected by SARS-CoV-2. npathway is the number of significant pathways this gene is involved.\n\nThe differential gene expression list was transformed into a gene rank list. An effective drug treatment is one that reverts the aberrant gene expression in disease back to the normal level in health. DrInsight Package17 (version: 0.1.1) was used for this purpose, and the outlier-sum (OS) statistic was retrieved, which models the overall disease-drug connectivity by aggregating disease transcriptome changes with drug perturbation. The Kolmogorov–Smirnov (K-S) test was then applied to the OS statistic, to show the significance level of one drug treatment relative to the background of all other drugs and compounds in the reference drug dataset. The reference drug dataset contains gene rank lists from 12,707 drug treatments in the LINCS L1000 data, as mentioned above. The false discovery rate (FDR) was used to adjust P-values from the K-S test to avoid false significance due to multiple comparisons. FDR<0.05 was used as the threshold to select significant drug candidates for the disease.\n\nFigure 1 and Figure 5 were generated in Microsoft PowerPoint 2016. Figure 2 and Figure 3 were generated in R (version: 3.6.3) with ggplot2 package (version: 3.3.0)18. Figure 4 was generated in Cystoscope (version: 3.7.2)19.\n\nInput data include gene expression in A549 cells with H1N1 infection, HCC515 cells with ACE2 inhibitor (ACE2i), human lung tissues from COVID-19 deceased patients and cells with drug treatment. Reversing analysis is conducted to search for drugs that can reverse the gene expression changes upon treatment. The candidate drug to is compared to all other drugs and compounds, in order to estimate its significance level at treating the disease. Candidate drugs for H1N1 are used for validation of the computational pipeline. Candidate drugs identified in both HCC515 cells treated with ACE2 inhibitor and in human lung tissues from COVID-19 deceased patients are used for downstream mechanism analysis.\n\nZ-score: z score of differential expression of genes in the sample; ACE2i: HCC515 cells with ACE2 inhibitor inhibition; SARS-CoV-2: human lung tissues from COVID-19 patients deceased from SARS-CoV-2 induced lung complications; COL-3: HCC515 cells treated with COL-3; CGP-60474: HCC515 cells treated with CGP-60474.\n\nX-axis and Y-axis show -log10 transformed P-values in human COVID-19 patient lung tissues (SARS-CoV-2) and HCC515 cells with ACE2 inhibitor inhibition (ACE2i), respectively. Size of the bubble shows the average value of -log10 transformed P-value in SARS-CoV-2 and ACE2i.\n\nAll pathways were significant enriched in both human COVID-19 patient lung tissues and HCC515 cells with ACE2 inhibitor inhibition. The abnormal gene expression patterns in these pathways were reversed by COL-3 and/or CGP-60474. Blue diamond: down-regulated gene in disease; orange diamond: up-regulated gene in disease; hexagon: pathway; blue line: drug decreases gene expression; orange line: drug increases gene expression; blue/orange line width corresponds to the ability to change gene expression; dark green line: interaction between gene and pathway; diamond size: importance of gene in the disease; hexagon size: importance of pathway in the disease.\n\n\nResults\n\nOur drug repositioning is based on the assumption that if a drug can reverse the abnormality of gene expression pattern in the disease, the drug should be able to treat the disease20,21. Towards this we have implemented the computational framework as shown in Figure 1. We collected differential gene expression patterns in the disease and in cells with drug treatment. Then we searched reversible genes whose expression changes in drug treatment are opposite to those in disease to estimate the effect of a drug for the disease. We further compared effect of every drug to all other candidates to estimate the significance of a drug for treating the disease.\n\nAs COVID-19 is an emerging disease with much unknown, we first demonstrate the feasibility of the drug repositioning pipeline using H1N1 virus infection, where much more research has been done and multiple drugs are approved by the United States Food and Drug Administration. We computed the differentially expressed genes from RNA-Seq data of A549 lung cells with or without H1N1 virus infection. We then identified the best candidates that could reverse the expression pattern of these differentially expressed genes, by analyzing 12,707 drugs and compounds from LINCS L1000 pharmacogenomics data14. The results show that CGP-60474 (FDR= 2.514×10-4), sirolimus (FDR= 3.040×10-4), COL-3 (FDR= 9.452×10-4), PIK-75 (FDR= 0.002), and wortmannin (FDR= 0.046) could significantly (FDR<0.05) reverse the gene expression in H1N1 infection in A549 lung cells (Table 1). Sirolimus, the second-best candidate by FDR, also known as rapamycin, is a potent immunosuppressant that acts by selectively blocking the transcriptional activation of cytokines, thereby inhibiting cytokine production. It was previously shown clinically effective in H1N1 infected patients with severe pneumonia and acute respiratory failure22 as adjuvant treatment with steroids. In summary, our drug repositioning pipeline has shown merit in discovering effective drugs through the example of H1N1 infection.\n\nNS, not significant; NA, not available; ACE2i, inhibition of ACE2; FDR, false discovery rate.\n\nTo repurpose drugs for inhibition of ACE2, we conducted differential gene expression analysis in HCC515 and A549 lung cells with the inhibition of ACE2 by moexipril, from the LINCS L1000 project14 using a similar approach as for H1N1 infection described above. Upon examination of ACE2 expression at different time points (six and 24 hours), we opted to focus on HCC515 cells, which have reduced ACE2 expression upon treatment with moexipril, an ACE2 inhibitor. At six hours after treatment with moexipril, narciclasine (FDR=0.006) and geldanamycin (FDR=0.006) could significantly reverse the gene expression changes due to the ACE2 inhibitor (Table 1). At 24 hours post treatment of moexipril, the effect of CGP-60474 (FDR=1.337×10-7), panobinostat (FDR=2.443×10-05), trichostatin-a (FDR=3.546×10-03) and COL-3 (FDR= 0.002) became significant (Table 1).\n\nTo further confirm if these effects shown in cell lines are physiologically relevant for human lung injury due to COVID-19, we analyzed the RNA-Seq data of human lung tissues from two COVID-19 deceased patients with age-matched normal lung tissues, as reported by Melo et al.12 Gene expression of individual markers for lung injury, advanced glycosylation end-product specific receptor (AGER), lipopolysaccharide binding protein (LBP) and secretoglobin family 1A member (SCGB1A1)23 is up-regulated in the HCC515 cell line treated with ACE2 inhibitor and human COVID-19 patient lung tissue (Figure 2), whereas expression of surfactant protein D (SFTPD), a gene encoding a protein involved in the innate immune response to protect the lungs against inhaled microorganisms and chemicals, is decreased. This indicates the similarity between ACE2 inhibition by moexipril in the cell line and lung injury from COVID-19. Next we extracted the differentially expressed genes in COVID-19 lung tissues vs. normal lungs and used them as target genes to be reversed by the same drugs and compounds in the drug repositioning framework as shown in Figure 1. The results show that sirolimus (FDR=0.003), COL-3 (FDR=0.003), CGP-60474 (FDR=0.003), staurosporine (FDR=0.003) and mitoxantrone (FDR=0.003) are significant in reversing the target genes’ expression in the human lung tissues due to COVID-19 mentioned earlier (Table 1). Thus, together COL-3 and CGP-60474 show consistent effects for reversing gene expression changes in both the HCC515 cell line treated with ACE2 inhibitor and human COVID-19 patient lung tissue (Table 1). Moreover, COL-3 and CGP-60474 both can reversely decrease the expression of marker genes for lung injury, AGER, LBP, SCGB1A1, and reversely increase SFTPD expression in HCC515 cell line pre-treated with ACE2 inhibitor moexipril. CGP-60474 (0.12 µM) appears to be more potent than COL-3 (2.5 µM). In conclusion, COL-3 and CGP-60474 show promise as potential purposeful drugs to treat lung injury in COVID-19.\n\nWe performed pathway enrichment analysis with the top 1000 deregulated genes in HCC515 cells with ACE2 inhibitor inhibition and human COVID-19 patient lung tissues. It was found that 12 significantly enriched pathways (P-value <0.05) overlap between HCC515 cells with ACE2 inhibitor inhibition and human COVID-19 patient lung tissues (Figure 3, Table 2). As expected, multiple pathways involved in virus infection are enriched. Various signaling pathways, such as the TNF signaling pathway, MAPK signaling pathway and chemokine signaling pathway, with well-known associations with lung injury, are also enriched24–26. Moreover, other pathways related to cancers (e.g. ‘viral carcinogenesis’ and ‘proteoglycans in cancer’), or cardiovascular diseases (e.g. ‘viral myocarditis’) also show up significantly enriched in the results (Figure 3, Table 2). A total of 66 genes in these overlapped pathways show consistent changes between the ACE2 inhibited lung cell line and SARS-CoV-2 lung tissues (Table 2).\n\nACE2i, inhibition of ACE2.\n\nWe further analyzed the genes and pathways associated with the two drugs COL-3 and CGP-60474, which show coherent effects in reversing the gene expression patterns in HCC515 cells with ACE2 inhibitor inhibition and human COVID-19 patient lung tissues (Figure 4). For COL-3, from the molecular point of view, it leads to decreased expression of many genes including RHOA, RAC2, FAS and CDC42 in lung cells, as part of the mechanisms to protect lung from injury (Figure 4). These genes are important players in pathways such as the chemokine signaling pathway (for CCL2, ADCY7, GNG11, PXN, CDC42, RAC2, RHOA, WAS), TNF signaling pathway (for CCL2, MMP3, JUN, BCL3, FAS, MAP2K6) and MAPK signaling pathway (for HSPA1A, CDC42, RAC2, PAK2, FAS, MAP2K6, JUN, GADD45B, GADD45A). All 12 significantly enriched pathways in Figure 3 are also observed in COL-3 treatment. CGP-60474 shares 13 gene targets with COL-3, including RHOA, WAS, HSPA1A, SNX2, RAB8A, IL2RG, MMP3, BCL2L1, JUN, HIST1H2BK, GNG11, IQGAP1 and MYL12B. It also has a unique set of target genes related to lung injury, such as CALR and MMP14 (Figure 4). It decreases the expression of CALR, a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum27. It also increases the expression of MMP14, a member of the matrix metalloproteinase (MMP) family with anti-inflammatory properties. CGP-60474 treatment affects 11 out of 12 significantly enriched pathways in COL-3, but not the viral myocarditis pathway. More details on the molecular mechanisms of the target genes and pathways of these two drug candidates are discussed below.\n\n\nDiscussion\n\nThe inhibition of ACE2 promotes lung injury via the renin–angiotensin system (RAS)28. In pulmonary RAS, ACE2 converts angiotensin II (Ang II), an octapeptide hormone, to Ang-(1-7), an heptapeptide hormone (Figure 5). Ang II triggers pulmonary inflammation and activates the TNF signaling pathway and MAPK signaling pathway to promote lung injury29,30. On the other hand, Ang-(1–7) inhibits inflammation and protects lungs from injury31 by inhibiting the MAPK signaling pathway32, lowering cytokine release33 and downregulating the RHOA/ROCK pathway34. Thus, inhibition of ACE2 will increase Ang II levels, decrease Ang-(1–7), and deregulate various downstream pathways, such as TNF and MAPK signaling pathways to promote lung injury (Figure 5). Our pathway analysis on the HCC515 lung cell line confirmed that inhibition of ACE2 by moexipril can deregulate TNF signaling, MAPK signaling and cytokine signaling pathways. We further showed that these pathways are also deregulated in human lung tissues from COVID-19 deceased patients (Table 2). Moreover, inhibition of ACE2 induced similar expression patterns of lung injury markers to that in human lung tissues from COVID-19 deceased patients (Figure 2). This evidence suggests that inhibition of ACE2 may indeed be part of the molecular mechanisms of lung injury in COVID-19. Moreover, other pathways related to cancers (e.g. ‘viral carcinogenesis’ and ‘proteoglycans in cancer’), or cardiovascular diseases (e.g. viral myocarditis) also show up significantly enriched in the results (Table 2). These results may help to explain the increased risks of fatality among COVID-19 patients with underlying conditions (cancers, heart diseases)35,36. Additionally, myocarditis has been clinically observed in a patient with COVID-1937, showing a direct link between the two conditions.\n\nOur drug repositioning analysis suggested five possible drugs based on RNA-Seq data from patients deceased from COVID-19. Among them, clinical trial has started for treating patients with COVID-19 pneumonia with sirolimus (NCT04341675). Two other drugs (or compounds), COL-3 and CGP-60474, also have additional evidence of effectiveness from the L1000 data of the lung HCC515 cell line treated with ACE2 inhibitor moexipril. Moreover, both COL-3 and CGP-60474 could reverse the expression patterns of lung injury markers in HCC515 cells with ACE2 inhibitor inhibition and human COVID-19 patient lung tissues (Figure 2). This phenotypic evidence suggests that COL-3 and CGP-60474 may be effective in treating lung injury in COVID-19 (Figure 5). Therefore, we further analyzed the target genes and pathways of these two drugs in treating lung injury in COVID-19.\n\nCOL-3, also known as incyclinide or CMT-3, is a chemically modified tetracycline. It reversed the expression patterns of many lung injury related genes and pathways, such as RHOA, RAC2 and FAS in the chemokine signaling pathway, TNF signaling pathway and MAPK signaling pathway (Figure 4). RHOA, also known as ras homolog family member A, is a member of the Rho family of small GTPases. The activation of RHOA is crucial for lung injury38. Inhibition of RHOA is a promising approach to acute lung injury treatment39. RAC2, also known as Ras-related C3 botulinum toxin substrate 2, is a member of the Ras superfamily of small guanosine triphosphate (GTP)-metabolizing proteins. Rac2 plays an important role in inflammation-mediated lung injury40,41. FAS, also known as Fas cell surface death receptor, is a member of the TNF-receptor superfamily. FAS activation is essential in inducing acute lung injury42. Small interfering RNA targeting Fas reduced lung injury in mice43. Previous results from many pre-clinical animal models have supported the role of COL-3 in reducing lung injury and improves survival of experimented animals. For example, COL-3 prevented lung injury and acute respiratory distress syndrome (ARDS) in a clinically applicable porcine model44–50. It also improved acute respiratory distress syndrome (ARDS) survival in an ovine model51. Given all the evidence, COL-3 may be an attractive candidate for a clinical trial treating severe viral pneumonia related lung injury with respiratory failure in COVID-19 (Figure 5).\n\nCGP-60474, on the other hand, is an inhibitor of cyclin-dependent kinase (Figure 5). CGP-60474 not only shared target genes with COL-3, such as RHOA, WAS, HSPA1A, SNX2, RAB8A, IL2RG, MMP3, BCL2L1, JUN, HIST1H2BK, GNG11, IQGAP1 and MYL12B, but also has unique target genes that related to lung injury, like CALR and MMP14 (Figure 4). Blocking CALR activity attenuated murine acute lung injury by inducing polarization of M2 subtype macrophages, which are anti-inflammatory52. MMP14 was shown to trigger the anti-inflammatory proteolytic cascade to prevent lung injury in mice53. Interestingly, so far only a few studies have reported some biological functions of CGP-6047454–56. One drug reposition study using L1000 data also pointed to CGP-60474 as the most potent drug based on anti-inflammatory effects56. The authors then experimentally showed that CGP-60474 alleviated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in activated macrophages, downregulated the NF-κB activity, and reduced the mortality rate in lipopolysaccharide induced endotoxemia mice. Another in silico drug prediction study suggested that CGP-60474 could target multiple cancers, though no experiments were conducted54. Although cyclin-dependent kinase inhibition by another drug, seliciclib, reduced lung damage in a mouse model of ventilator-induced lung injury55, further in vivo investigation of CGP-60474 is needed to test its role in treating lung injury.\n\nIn summary, we propose two candidate drugs, COL-3 and CGP-60474, which can reverse the gene expression patterns in COVID-19 lung injury and a lung cell line with ACE2 being inhibited. We further analyzed potential molecular and biological mechanisms of lung injury in COVID-19. The work will hopefully gain the interest of the biomedical and clinical community for further validations in vivo for both candidate drugs, and even possibly clinical trials on COL-3 to save lives from severe respiratory failure in COVID-19.\n\n\nData availability\n\nRNA-Seq data from Gene Expression Omnibus, Accession number GSE147507: https://identifiers.org/geo:GSE147507\n\nPhase I LINCS L1000 data from Gene Expression Omnibus, Accession number GSE92742: https://identifiers.org/geo:GSE92742\n\nPhase II LINCS L1000 data from Gene Expression Omnibus, Accession number GSE70138: https://identifiers.org/geo:GSE70138\n\nZenodo: lanagarmire/COVID19-Drugs-LungInjury: Prediction of repurposed drugs for treating lung injury in COVID-19. https://doi.org/10.5281/zenodo.382327757\n\nThis project contains the following underlying data:\n\n- HCC515_6_data_for_drug.csv (Differential expression of genes in HCC515 cell at 6 h after treatment of ACE2 inhibitor)\n\n- HCC515_24_data_for_drug.csv (Differential expression of genes in HCC515 cell at 24 h after treatment of ACE2 inhibitor)\n\n- COVID19-Lung_data_for_drug.csv (Differential expression of genes in lung tissues with COVID-19)\n\n- HCC515_6_drug.csv (Drugs for HCC515 cell at 6 h after transfection of ACE2 inhibitor)\n\n- HCC515_24_drug.csv (Drugs for HCC515 cell at 24 h after transfection of ACE2 inhibitor)\n\n- COVID19-Lung_drug.csv (Drugs for lung tissuse from COVID-19 patients)\n\n- COL-3_single_treatment_response_data.csv (Differential expression of genes in HCC515 cell at 24h after treatment of COL-3)\n\n- CGP-60474_single_treatment_response_data.csv (Differential expression of genes in HCC515 cell at 24h after treatment of CGP-60474)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nSource code available from: https://github.com/lanagarmire/COVID19-Drugs-LungInjury\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.382292313\n\nLicense: GNU General Public License v3.0", "appendix": "References\n\nZhou F, Yu T, Du R, et al.: Clinical Course and Risk Factors for Mortality of Adult Inpatients With COVID-19 in Wuhan, China: A Retrospective Cohort Study. Lancet. 2020; 395(10229): 1054–1062. PubMed Abstract \| Publisher Full Text\n\nLew TW, Kwek TK, Tai D, et al.: Acute Respiratory Distress Syndrome in Critically Ill Patients With Severe Acute Respiratory Syndrome. JAMA. 2003; 290(3): 374–80. PubMed Abstract \| Publisher Full Text\n\nLu R, Zhao X, Li J, et al.: Genomic Characterisation and Epidemiology of 2019 Novel Coronavirus: Implications for Virus Origins and Receptor Binding. Lancet. 2020; 395(10224): 565–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F, Li W, Farzan M, et al.: Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed With Receptor. Science. 2005; 309(5742): 1864–8. PubMed Abstract \| Publisher Full Text\n\nOu X, Liu Y, Lei X, et al.: Characterization of Spike Glycoprotein of SARS-CoV-2 on Virus Entry and Its Immune Cross-Reactivity With SARS-CoV. Nat Commun. 2020; 11(1): 1620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Wit E, van Doremalen N, Falzarano D, et al.: SARS and MERS: Recent Insights Into Emerging Coronaviruses. Nat Rev Microbiol. 2016; 14(8): 523–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann M, Kleine-Weber H, Schroeder S, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2): 271–280.e8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShulla A, Heald-Sargent T, Subramanya G, et al.: A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry. J Virol. 2011; 85(2): 873–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi W, Moore MJ, Vasilieva N, et al.: Angiotensin-converting Enzyme 2 Is a Functional Receptor for the SARS Coronavirus. Nature. 2003; 426(6965): 450–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuba K, Imai Y, Rao S, et al.: A Crucial Role of Angiotensin Converting Enzyme 2 (ACE2) in SARS Coronavirus-Induced Lung Injury. Nat Med. 2005; 11(8): 875–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaga S, Yamamoto N, Nakai-Murakami C, et al.: Modulation of TNF-alpha-converting Enzyme by the Spike Protein of SARS-CoV and ACE2 Induces TNF-alpha Production and Facilitates Viral Entry. Proc Natl Acad Sci U S A. 2008; 105(22): 7809–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanco-Melo D, Nilsson-Payant BE, Liu W-C, et al.SARS-CoV-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems. bioRxiv. 2020. Publisher Full Text\n\nGarmire group: lanagarmire/COVID19-Drugs-LungInjury: Prediction of repurposed drugs for treating lung injury in COVID-19 (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3822923\n\nSubramanian A, Narayan R, Corsello SM, et al.: A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles. Cell. 2017; 171(6): 1437–52 e17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Huber W, Anders S: Moderated Estimation of Fold Change and Dispersion for RNA-seq Data With DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang DW, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1): 44–57. PubMed Abstract \| Publisher Full Text\n\nChan J, Wang X, Turner JA, et al.: Breaking the Paradigm: Dr Insight Empowers Signature-Free, Enhanced Drug Repurposing. Bioinformatics. 2019; 35(16): 2818–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. 2016. Reference Source\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 2003; 13(11): 2498–504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHodos RA, Kidd BA, Shameer K, et al.: In Silico Methods for Drug Repurposing and Pharmacology. Wiley Interdiscip Rev Syst Biol Med. 2016; 8(3): 186–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSirota M, Dudley JT, Kim J, et al.: Discovery and Preclinical Validation of Drug Indications Using Compendia of Public Gene Expression Data. Sci Transl Med. 2011; 3(96): 96ra77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang CH, Chung FT, Lin SM, et al.: Adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe H1N1 pneumonia and acute respiratory failure. Crit Care Med. 2014; 42(2): 313–21. PubMed Abstract \| Publisher Full Text\n\nMokra D, Kosutova P: Biomarkers in acute lung injury. Respir Physiol Neurobiol. 2015; 209: 52–8. PubMed Abstract \| Publisher Full Text\n\nPatel BV, Wilson MR, O'Dea KP, et al.: TNF-induced death signaling triggers alveolar epithelial dysfunction in acute lung injury. J Immunol. 2013; 190(8): 4274–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMukhopadhyay S, Mukherjee S, Stone WL, et al.: Role of MAPK/AP-1 signaling pathway in the protection of CEES-induced lung injury by antioxidant liposome. Toxicology. 2009; 261(3): 143–51. PubMed Abstract \| Publisher Full Text\n\nWard PA: Role of complement, chemokines, and regulatory cytokines in acute lung injury. Ann N Y Acad Sci. 1996; 796: 104–12. PubMed Abstract \| Publisher Full Text\n\nGelebart P, Opas M, Michalak M: Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum. Int J Biochem Cell Biol. 2005; 37(2): 260–6. PubMed Abstract \| Publisher Full Text\n\nKuba K, Imai Y, Penninger JM: Angiotensin-converting enzyme 2 in lung diseases. Curr Opin Pharmacol. 2006; 6(3): 271–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarshall RP, Gohlke P, Chambers RC, et al.: Angiotensin II and the fibroproliferative response to acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2004; 286(1): L156–64. PubMed Abstract \| Publisher Full Text\n\nTao W, Li PS, Xu G, et al.: Soluble Epoxide Hydrolase Plays a Vital Role in Angiotensin II-Induced Lung Injury in Mice. Shock. 2018; 50(5): 589–94. PubMed Abstract \| Publisher Full Text\n\nLu W, Kang J, Hu K, et al.: Angiotensin-(1-7) inhibits inflammation and oxidative stress to relieve lung injury induced by chronic intermittent hypoxia in rats. Braz J Med Biol Res. 2016; 49(10): e5431. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeng Y, Yu CH, Li W, et al.: Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis protects against lung fibrosis by inhibiting the MAPK/NF-kappaB pathway. Am J Respir Cell Mol Biol. 2014; 50(4): 723–36. PubMed Abstract \| Publisher Full Text\n\nRodrigues Prestes TR, Rocha NP, Miranda AS, et al.: The Anti-Inflammatory Potential of ACE2/Angiotensin-(1-7)/Mas Receptor Axis: Evidence from Basic and Clinical Research. Curr Drug Targets. 2017; 18(11): 1301–13. PubMed Abstract \| Publisher Full Text\n\nMeng Y, Li T, Zhou GS, et al.: The angiotensin-converting enzyme 2/angiotensin (1-7)/Mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the NOX4-derived ROS-mediated RhoA/Rho kinase pathway. Antioxid Redox Signal. 2015; 22(3): 241–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNovel Coronavirus Pneumonia Emergency Response Epidemiology T: The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China. Zhonghua Liu Xing Bing Xue Za Zhi. 2020; 41(2): 145–51. PubMed Abstract \| Publisher Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInciardi RM, Lupi L, Zaccone G, et al.: Cardiac Involvement in a Patient With Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2020. PubMed Abstract \| Publisher Full Text\n\nRafikov R, Dimitropoulou C, Aggarwal S, et al.: Lipopolysaccharide-induced lung injury involves the nitration-mediated activation of RhoA. J Biol Chem. 2014; 289(8): 4710–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbedi F, Hayes AW, Reiter R, et al.: Acute lung injury: The therapeutic role of Rho kinase inhibitors. Pharmacol Res. 2020; 155: 104736. PubMed Abstract \| Publisher Full Text\n\nDooley JL, Abdel-Latif D, St Laurent CD, et al.: Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2009; 297(6): L1091–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArizmendi N, Puttagunta L, Chung KL, et al.: Rac2 is involved in bleomycin-induced lung inflammation leading to pulmonary fibrosis. Respir Res. 2014; 15(1): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatute-Bello G, Lee JS, Liles WC, et al.: Fas-mediated acute lung injury requires fas expression on nonmyeloid cells of the lung. J Immunol. 2005; 175(6): 4069–75. PubMed Abstract \| Publisher Full Text\n\nDel Sorbo L, Costamagna A, Muraca G, et al.: Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury. Crit Care Med. 2016; 44(8): e604–13. PubMed Abstract \| Publisher Full Text\n\nSadowsky D, Nieman G, Barclay D, et al.: Impact of chemically-modified tetracycline 3 on intertwined physiological, biochemical, and inflammatory networks in porcine sepsis/ARDS. Int J Burns Trauma. 2015; 5(1): 22–35. PubMed Abstract \| Free Full Text\n\nSteinberg J, Halter J, Schiller H, et al.: Chemically modified tetracycline prevents the development of septic shock and acute respiratory distress syndrome in a clinically applicable porcine model. Shock. 2005; 24(4): 348–56. PubMed Abstract \| Publisher Full Text\n\nRoy SK, Kubiak BD, Albert SP, et al.: Chemically modified tetracycline 3 prevents acute respiratory distress syndrome in a porcine model of sepsis + ischemia/reperfusion-induced lung injury. Shock. 2012; 37(4): 424–32. PubMed Abstract \| Publisher Full Text\n\nRoy SK, Kendrick D, Sadowitz BD, et al.: Jack of all trades: pleiotropy and the application of chemically modified tetracycline-3 in sepsis and the acute respiratory distress syndrome (ARDS). Pharmacol Res. 2011; 64(6): 580–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim JH, Suk MH, Yoon DW, et al.: Inhibition of matrix metalloproteinase-9 prevents neutrophilic inflammation in ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol. 2006; 291(4): L580–7. PubMed Abstract \| Publisher Full Text\n\nCarney DE, McCann UG, Schiller HJ, et al.: Metalloproteinase inhibition prevents acute respiratory distress syndrome. J Surg Res. 2001; 99(2): 245–52. PubMed Abstract \| Publisher Full Text\n\nCarney DE, Lutz CJ, Picone AL, et al.: Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass. Circulation. 1999; 100(4): 400–6. PubMed Abstract \| Publisher Full Text\n\nZhou X, Wang D, Ballard-Croft CK, et al.: A tetracycline analog improves acute respiratory distress syndrome survival in an ovine model. Ann Thorac Surg. 2010; 90(2): 419–26. PubMed Abstract \| Publisher Full Text\n\nJiang Z, Chen Z, Hu L, et al.: Calreticulin Blockade Attenuates Murine Acute Lung Injury by Inducing Polarization of M2 Subtype Macrophages. Front Immunol. 2020; 11: 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAguirre A, Blazquez-Prieto J, Amado-Rodriguez L, et al.: Matrix metalloproteinase-14 Triggers an Anti-Inflammatory Proteolytic Cascade in Endotoxemia. J Mol Med (Berl). 2017; 95(5): 487–97. PubMed Abstract \| Publisher Full Text\n\nTaguchi YH: Drug Candidate Identification Based on Gene Expression of Treated Cells Using Tensor Decomposition-Based Unsupervised Feature Extraction for Large-Scale Data. BMC Bioinformatics. 2019; 19(Suppl 13): 388. PubMed Abstract \| Publisher Full Text\n\nHoogendijk AJ, Kuipers MT, van der Poll T, et al.: Cyclin-dependent Kinase Inhibition Reduces Lung Damage in a Mouse Model of Ventilator-Induced Lung Injury. Shock. 2012; 38(4): 375–80. PubMed Abstract \| Publisher Full Text\n\nHan HW, Hahn S, Jeong HY, et al.: LINCS L1000 Dataset-Based Repositioning of CGP-60474 as a Highly Potent Anti-Endotoxemic Agent. Sci Rep. 2018; 8(1): 14969. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe B, Garmire L: Prediction of repurposed drugs for treating lung injury in COVID-19 [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3823277" }	[ { "id": "64847", "date": "30 Jun 2020", "name": "Xia Yang", "expertise": [ "Reviewer Expertise Bioinformatics", "systems biology", "complex diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, He and Garmire utilized publicly available gene expression datasets on COVID-19 lungs and drug treatment studies in lung cell lines to conduct drug repositioning analysis. They found that the genes and pathways altered by an AEC2 inhibitor in the HCC515 lung cell line mimic those of COVID-19 lungs, supporting that ACE2 inhibition likely underlines the lung injury induced by the SARS-CoV2 virus. They used the gene profiles from these datasets to identify drugs in the L1000 database that can reverse the genes and pathways affected by ACE2 inhibition and in COVID-19 lungs and prioritized two drugs, COL-3 and CGP-60474, that have such properties and may have the potential to treat COVID-19. The study was well-designed and executed, the paper was well-written and easy to follow, and the findings are interesting and promising. The genes, pathways, and drugs uncovered offer insights into COVID-19. Overall a very timely study to address a critical health issue. There are some technical aspects to be addressed, as detailed below.\nMajor:\nSome of the statistical analyses need justification. The arbitrary use of top 1000 genes instead of any statistical cutoff is unconventional. Pathway analysis used raw p values instead of FDRs, which is also unconventional. Please justify.\n\nUnclear what type of genes are considered in n_consistent in the pathway score calculation. Among top 1000 genes in both ACE2i and COVID19? If so, state it clearly.\n\nAs many curated pathways that are closely related (e.g., inflammation pathways) share overlapping genes, they tend to show coordinated over- or under-enrichment in pathway analysis. The use of n_pathway in the gene score calculation can be misleading due to such overlaps in non-independent pathways.\n\nWhich method was used in the FDR calculation for drug repositioning analysis? Please clarify.\n\nDatasets related to H1N1 was used as a positive control to test the drug repositioning pipeline since numerous FDA-approved drugs are available. This is a good design but only one of the drugs predicted was discussed. What about the other top ranked drugs? Any evidence for H1N1 treatment effect for the other top ranked drugs? What about the FDR approved drugs for H1N1? Were they retrieved by the analysis? These are unclear based on the results descriptions. If not, the conclusion \"our drug repositioning pipeline has shown merit in discovering effective drugs through the example of H1N1 infection\" is too strong.\n\nFor genes in Table 2, not sure if the direction of changes is consistent. Please clarify.\n\nMinor:\nAdd year to the date \"12th May\".\n\nIt is interesting that the ACE2 inhibitor only inhibited ACE2 in HCC515 cell line but not the A549 cell line. Any explanation on why the drug did not inhibit ACE2 in A549?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5826", "date": "26 Aug 2020", "name": "Bing He", "role": "Author Response", "response": "In this study, He and Garmire utilized publicly available gene expression datasets on COVID-19 lungs and drug treatment studies in lung cell lines to conduct drug repositioning analysis. They found that the genes and pathways altered by an AEC2 inhibitor in the HCC515 lung cell line mimic those of COVID-19 lungs, supporting that ACE2 inhibition likely underlines the lung injury induced by the SARS-CoV2 virus. They used the gene profiles from these datasets to identify drugs in the L1000 database that can reverse the genes and pathways affected by ACE2 inhibition and in COVID-19 lungs and prioritized two drugs, COL-3 and CGP-60474, that have such properties and may have the potential to treat COVID-19. The study was well-designed and executed, the paper was well-written and easy to follow, and the findings are interesting and promising. The genes, pathways, and drugs uncovered offer insights into COVID-19. Overall a very timely study to address a critical health issue. There are some technical aspects to be addressed, as detailed below. Major: Some of the statistical analyses need justification. The arbitrary use of top 1000 genes instead of any statistical cutoff is unconventional. Pathway analysis used raw p values instead of FDRs, which is also unconventional. Please justify. Response: Thank you for your comments. We got gene expression data in response to AEC2 inhibitor in the HCC515 lung cell line from LINC1000 project. It only provides differential gene expression levels measured in Z-score. So we can only select differential genes by using top n genes. To be consistent, in this study, we also transformed all differential expressions in Z-score and used top n to select differentially expressed genes. Top 1000 differentially expressed genes in HCC515 cell with AEC2 inhibitor treatment and COVID-19 lungs mapped to 43 and 54 kegg pathways, respectively. The FDR for the most significantly pathway in HCC515 cell with AEC2 inhibitor treatment is 0.21. If we use FDR instead of p value in pathway analysis, then no further analysis could be performed in this study. Unclear what type of genes are considered in n_consistent in the pathway score calculation. Among top 1000 genes in both ACE2i and COVID19? If so, state it clearly. Response: Thank you for the comment. We have revised the manuscript accordingly. “n_consistent is the number of consistent genes in that pathway among top 1000 differential expressed genes, for both HCC515 cells with ACE2 inhibitor treatment and lung tissues of deceased COVID-19 patients”. As many curated pathways that are closely related (e.g., inflammation pathways) share overlapping genes, they tend to show coordinated over- or under-enrichment in pathway analysis. The use of n_pathway in the gene score calculation can be misleading due to such overlaps in non-independent pathways. Response: Thank you for your comment. We removed n_pathway from gene score calculation according to your suggestion, and updated figure 4 according to new gene score. Which method was used in the FDR calculation for drug repositioning analysis? Please clarify. Response: The FDR calculation used Benjamini-Hochberg (BH) procedure. We have clarified this in the manuscript according to your comment. Datasets related to H1N1 was used as a positive control to test the drug repositioning pipeline since numerous FDA-approved drugs are available. This is a good design but only one of the drugs predicted was discussed. What about the other top ranked drugs? Any evidence for H1N1 treatment effect for the other top ranked drugs? What about the FDR approved drugs for H1N1? Were they retrieved by the analysis? These are unclear based on the results descriptions. If not, the conclusion \"our drug repositioning pipeline has shown merit in discovering effective drugs through the example of H1N1 infection\" is too strong. Response: Thank you for your comments. Our drug response data in lung cells are collected from LINC1000 project, which unfortunately do not include treatment using current four FDA approved drugs (peramivir, zanamivir, oseltamivir phosphate, and baloxavir marboxil) for H1N1. We have toned down the statement to “our drug repositioning pipeline has shown promise through the example of H1N1 infection\". We also added discussion of other top ranked drugs and revised the conclusion according to your suggestions. For genes in Table 2, not sure if the direction of changes is consistent. Please clarify. Response: Yes, all genes listed in table 2 have same direction in both HCC515 cell with AEC2 inhibitor treatment and COVID-19 lungs. We have added the annotation to the table 2. Minor: Add year to the date \"12th May\". Response: We added the year according to your suggestion. It is interesting that the ACE2 inhibitor only inhibited ACE2 in HCC515 cell line but not the A549 cell line. Any explanation on why the drug did not inhibit ACE2 in A549? Response: The ACE2 inhibitor inhibited ACE2 in both HCC515 cell and A549 cell. But in A549 cell, Z-score of ACE2 at 24h after treatment is higher than that at 6h, rather than being lower as expected (if ACE2 inhibitor is effective). This implies the inhibitor is not effective in A549 cell for some reason. Due to the concern of data quality, we didn’t use data from A549 cell." } ] }, { "id": "65750", "date": "10 Jul 2020", "name": "Leng Han", "expertise": [ "Reviewer Expertise Computational biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHe et al. implemented a computational framework of drug repositioning to identify drug candidates for COVID-19 lung injury treatment. The authors first proved feasibility of drug repositioning pipeline in H1N1 infection data. They extracted genes that showed differential expression in ACE2 inhibitor-treated cell line and COVID-19 patients. The authors next screened drugs that could reverse expression of these differential genes, wherein COL-3 and CGP-60474 showed the most potent to treat lung injury in COVID-19.\nIn the moexipril-treated HCC515 cell line, the effects of narciclasine and geldanamycin disappeared at 24 hours post treatment, which could significantly reverse the gene expression changes at six hours after treatment. Please provide more discussion on this.\n\nHow many genes (key genes) were deregulated in the same direction in both ACE2 inhibition-treated HCC515 cell line and COVID-19 patient lung tissue?\n\nFor referring drug candidates, did you use all the differentially expressed genes in COVID-19 lung tissues vs. normal lungs, or only part of differential genes?\n\nIn Table 1, column \"SARS-CoV-2 infection Human lung tissue\", the P values of effective drugs are the same, please provide more details to explain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5827", "date": "26 Aug 2020", "name": "Bing He", "role": "Author Response", "response": "He et al. implemented a computational framework of drug repositioning to identify drug candidates for COVID-19 lung injury treatment. The authors first proved feasibility of drug repositioning pipeline in H1N1 infection data. They extracted genes that showed differential expression in ACE2 inhibitor-treated cell line and COVID-19 patients. The authors next screened drugs that could reverse expression of these differential genes, wherein COL-3 and CGP-60474 showed the most potent to treat lung injury in COVID-19. In the moexipril-treated HCC515 cell line, the effects of narciclasine and geldanamycin disappeared at 24 hours post treatment, which could significantly reverse the gene expression changes at six hours after treatment. Please provide more discussion on this. Response: Thank you for your comment. We have added more discussion on this according to your suggestion.\"Among these predicted drugs, narciclasine and geldanamycin are significant in HCC515 cells at 6h after treatment but no longer significant in cells at 24h after treatment. Both narciclasine and geldanamycin have anti-inflammatory effects and can reduce lung injury caused by other diseases in animal model. On the other hand, in HCC515 cells treated with moexipril, the ACE2 level at 24h is lower than that at 6h, suggesting that ACE2 inhibition is enhanced over time. Thus drugs such as narciclasine and geldanamycin that are effective in early treatment may not be suitable for sustained administration.” How many genes (key genes) were deregulated in the same direction in both ACE2 inhibition-treated HCC515 cell line and COVID-19 patient lung tissue? Response: There are 5390 genes deregulated in the same direction in both ACE2 inhibition-treated HCC515 cell line and COVID-19 patient lung tissue. Among them, 797 genes are in top 1000 differentially expressed genes in either ACE2 inhibition-treated HCC515 cell line or COVID-19 patient lung tissue, and 119 genes are in significantly enriched pathways. We have added these statistics into the manuscript according to your comment. For referring drug candidates, did you use all the differentially expressed genes in COVID-19 lung tissues vs. normal lungs, or only part of differential genes? Response: We used all differentially expressed genes in drug repurposing analysis. In Table 1, column \"SARS-CoV-2 infection Human lung tissue\", the P values of effective drugs are the same, please provide more details to explain. Response: Good question. Table 1 shows BH FDR adjusted P value for every drug. The raw p value for the top 5 drugs are 5.11e-7,6.38e-7,9.02e-7,1.21e-6 and 1.34e-6. There are 12706 drugs. Since FDR=(Number of drugs x P.value)/Drug rank , FDR for the top 5 drugs are 0.00649, 0.00405,0.00382,0.00385 and 0.00340. According to BH procedure, if FDR of a drug is bigger than the FDR of the next lower ranked drug, the FDR of that drug is reset to the FDR of next lower ranked drug. So the FDR for the top 4 drugs are set to the FDR of the fifth drug, which is 0.00340. This is a conservative measure. That’s why FDR of all top 5 drugs are 0.00340, which is rounded to 0.003 in table 1." } ] } ]	1	https://f1000research.com/articles/9-609
https://f1000research.com/articles/9-1048/v1	26 Aug 20	{ "type": "Method Article", "title": "Measurement, modeling and QALYs", "authors": [ "Paul C. Langley", "Stephen P. McKenna", "Stephen P. McKenna" ], "abstract": "Over the past 30 years, a mainstay of health technology assessment has been the creation of modeled incremental cost-per-quality adjusted life year (QALY) claims. These are intended to inform resource allocation decisions. Unfortunately, the reliance on the construction of QALYs from generic utility scales is misplaced. Those advocating QALY-based lifetime modeled claims fail to appreciate the limitations placed on these constructs by the axioms of fundamental measurement. Utility scales, such as those created by the EQ-5D-3L instrument, are nothing more than multidimensional, ordinal scales. Such scales cannot support basic arithmetic operations. Interval scales can support addition and subtraction; ratio scales the further operations of multiplication and division. Those who advocate the construction of QALYs fail to appreciate that such an operation is only possible if the utility scale is unidimensional and has ratio properties with a true zero. The utility measures available do not meet these requirements. As we cannot produce meaningful utility values, the QALY is an invalid construct. Consequently, cost-per-incremental QALY claims are impossible to sustain and the application of cost-per QALY thresholds meaningless. As utility is a latent, unidimensional variable, the best a measure of utility could achieve would be unidimensionality and interval scaling properties. Where such measures are available, they could support claims for response to therapy. Consequently, there would be no need to continue constructing imaginary lifetime value assessment frameworks. Admitting that the QALY is a fatally flawed construct means rejecting 30 years of cost-per-QALY models.", "keywords": [ "Imaginary QALY", "ordinal scores", "impossible models" ], "content": "Introduction\n\nThe value framework advocated by the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) is quite clear: “Leaders in the field of economic evaluation in health care have long recommended that analysts seeking to inform resource allocation decisions approximate the value of interventions in terms of incremental cost per Quality Adjusted Life Year (QALY) gained”1. The application of this value framework is probably best exemplified in the reference case technology assessment guidelines put in place by groups such as the National Institute for Health and Care Excellence (NICE) in the UK, the Canadian Agency for Drugs and Technologies in Health (CADTH) and the Institute for Clinical and Economic Evaluation (ICER) in the US. In each case pharmaceutical manufacturers and others (including the ICER itself) are asked to make a case for comparative cost effectiveness. This is done by constructing an imaginary (yet apparently believably ‘realistic’) simulation model extending, in the default case, for the lifetime of persons with a chronic disease. The costs and benefits of comparator interventions for the defined hypothetical population are then calculated. Benefits are expressed in terms of incremental cost-per-QALY claims. There is no intention that the resulting claims should meet the standards of normal science for credibility, evaluation and replication2. The model is not about the discovery of new facts; it is purely speculative. This is made clear in the latest version of the Canadian guidelines where it states: “Economic evaluations are designed to inform decisions. As such they are distinct from conventional research activities, which are designed to test hypotheses”3. By rejecting the construction of empirically verifiable theories and hypotheses, the imaginary simulated worlds of economic evaluations fail the demarcation test; they are pseudoscience not science4.\n\n\nCreating QALYs\n\nThere is no ‘gold standard’ measure that can be used to generate QALYs. Several generic multiattribute instruments have been developed for this purpose. These differ considerably and produce markedly dissimilar scores for the same health states. The most used measures are the EQ-5D-3L and EQ-5D-5L, the HUI Mk2 and Mk3 and the SF-6D. These are designed to generate utility or value metrics on a scale from 0 = death to 1 = perfect health. Unfortunately, in the case of the EQ-5D-3L, the most widely used instrument, the algorithms applied to create utility scores can generate negative utility. The same argument, the production of negative utilities, applies to the other instruments. With the EQ-5D-3L utilities are allowed to range from −0.59 to 1.0. The negative utilities generated are considered to indicate states ‘worse than death’. The zero value in each measure is arbitrary, and it is not clear whether a utility of zero or lower makes any sense. The utility value is then applied to the simulated time spent in various hypothetical disease states over the course of a disease and a value adjusted time spent measure created: the QALY. QALYs are then aggregated (and discounted) over the simulated course of the disease to generate lifetime QALYs. Given estimated lifetime costs, the analyst can then produce lifetime cost-per-QALY, and eventually a simulated incremental cost-per-QALY claim.\n\nFor the utility value to support these operations it has to meet the axioms of fundamental measurement5. Four main types of measurement scale are recognized: nominal, ordinal, interval and ratio. Each satisfies one or more of the properties of: (i) identity - where each value has a unique meaning; (ii) magnitude where each value has an ordered relationship to other values; (iii) interval where the distances between scale units are equal to one another; and (iv) ratio where there is a ‘true zero’ below which no value exists. Nominal scales are purely descriptive and have no inherent numerical value in terms of magnitude. Ordinal scales have both identity and magnitude in an ordered relation but the distance between the ranks can differ considerably, generating only medians and modes (e.g., EQ-5D scales). The interval scale has identity, magnitude and equal intervals. It supports mathematical operations of addition and subtraction. A ratio scale satisfies all properties, supporting the additional operations of multiplication and division.\n\nThe question that must be addressed for those supporting QALYs is whether the utility value has ratio measurement properties. If we consider the EQ-5D-3L, there is no evidence that it measures at an interval level, let alone that it has ratio measurement properties.5 Quite the opposite. It can generate negative utilities and then negative QALYs. Put simply, it does not have a true zero. As the EQ-5D-3L is based on symptoms defined by ordinal response levels, the resulting EQ-5D-3L score can only have ordinal properties, not ratio properties. The same argument applies to the other instruments. There is no evidence to suggest that the question of fundamental measurement was considered in its development. The principal objective was a simple, functionally based capture of five symptoms with three ordinal response levels. Across any disease state, patients respond to the same five symptoms. Community preference weights are then applied and an algorithmic value is produced. The result is an ordinal score. Multiplying this score by time spent in a disease state is mathematically impossible.\n\nUnless it can be demonstrated that the EQ-5D-3L (or any other value scale) has ratio properties for any target patient population, the concept of a generic utility QALY collapses; it defies measurement. The implications are interesting: the reference case incremental cost-per-QALY value framework is unintelligible, the claims for simulated QALY based cost-effectiveness claims with willingness to pay thresholds is redundant and some 30 years of advocating the construction of simulated imaginary worlds irrelevant. Rather than seeking real-world evidence, we are locked into a paradigm for imaginary world evidence.\n\n\nAbandoning the QALY\n\nCan the QALY be rescued; or, more to the point, do we want to put in the effort to rescue it? Certainly, it could be possible to start from scratch and develop a new measure from first principles employing modern rather than classical test theory measurement. This recognizes the application of Rasch measurement theory (RMT) in its application of conjoint simultaneous measurement (CSM). However, even with the application of RMT, we are unable to develop a scale with ratio properties unless there is a clear specification equation guiding its content6. At best we might develop a value set with interval properties, but this would preclude relating health status to time spent in a disease state (a multiplicative function) to create a QALY.\n\nDo we need a QALY? Is there really a need to talk in terms of incremental cost-per-QALY claims? If we are concerned with quality of life and not the more narrowly defined health-related quality of life that characterizes almost all patient-reported outcome measures (PROMs), then we should consider disease-specific measurements. This is overdue; for we can say unequivocally that PROMs that were developed utilizing classical test theory, will not meet Rasch measurement standards. Quite simply, they were not designed to reflect an underlying latent construct with items selected to conform to Rasch measurement requirements. In some cases, it is possible, ex post facto, to ‘rescue’ an instrument through item assessment and possible removal of misfitting items7,8. A more positive approach would be to go back to first principles, as put forward by Rasch some 60 years ago, and meet fundamental CSM in the development of instruments9.\n\nA further obstacle to rescuing the QALY is the fact that the utility manifest score can take negative values. This has been shown across many disease states for both the EQ-5D-3L and EQ-5D-5L10,11. In the former, the lowest possible manifest score, as noted above, is −0.59; in the latter the lowest score is −0.29. These negative scores, assuming we ignore the standards of fundamental measurement, lead to the intriguing possibility of negative QALYs. In other words, over a hypothetical lifetime, patients can conceivably hop into and out of negative QALY disease stages. With aggregate lifetime QALYs the sum of the time spent in these positive and negative QALY states could cancel each other out. It is not clear how we would interpret this ordinal score construction of negative time? Particularly where the lifetime summation of QALYs by disease stage is negative: cost per negative QALY?\n\n\nNeed fulfillment and Rasch\n\nIt is a puzzle why those developing PROMs that are focused on functional status and symptom response should ignore the interests of the patient and, often, caregivers. After all, there is no reason why a physician’s view of response to therapy should necessarily be concordant with that of the patient or caregiver. If quality of life has any meaning it should focus on the patient as the principal ‘beneficiary’ of therapy interventions. A patient-centric approach, where life maintains its quality if patient needs are fulfilled, is not a new concept. It was first proposed in the early 1990s and has been the driving force in disease-specific instrument development within the Rasch measurement framework12,13.\n\n\nThe Rasch model\n\nMeasurement is critical for the advancement of science. The focus, as in the physical sciences, should be on the development of unidimensional indices rather than profiles. We need to focus on one attribute at a time (e.g., temperature14 or pain), not confusing several attributes into a meaningless single score. Despite this, fundamental measurement scales are rare in medicine. If they are to advance beyond ordinal raw scores, they must meet the axioms of invariance and sufficiency15. Where the object to be measured is a latent construct, such as quality of life, we require a framework for identifying, if they exist, inherent measurement structures with interval properties. This is provided in the application of the axioms of conjoint simultaneous measurement developed independently by Rasch, and Luce and Tukey in the early 1960s16,17. To reflect an underlying unidimensional latent construct such as need-based quality of life, the CSM model argues that two requirements must be met by any outcome measure: (i) item difficulty (the easier the item in a questionnaire, the more likely it is to be affirmed), and (ii) respondent ability (the more able the respondent, the more likely are they to affirm the item).\n\nIf we consider quality of life measures, where the latent construct is need fulfillment, the items are generated by qualitative patient interviews in a specific disease state. Where data generated by the measure fit the Rasch model, a single index with interval properties is produced that captures response to therapy. QALYs and imaginary lifetime models are irrelevant. In other words, a patient-centric quality of life measure is generated, not a multi-attribute outcome such as the EQ-5D-3L that confuses a clinically based set of symptoms and responses to produce a meaningless outcome.\n\nThis is not to say that the Rasch model has been ignored. There are now several need-based disease-specific quality of life instruments available for clinical trials and for evaluating the impact of competing interventions on quality of life18.\n\n\nNext steps\n\nScience can only make significant advances if measures are developed that have the required measurement properties; unidimensionality and ratio level measurement. Utility measures produce composite scores, as they add together several different types of outcome, for example, pain, emotional distress and physical mobility. Composite measurement cannot replace unidimensional measurement.\n\nWe have known how to develop unidimensional measures for the last 60 years, through the application of RMT. However, this also requires the development of theoretical models that explain the nature of the outcome that is to be measured and generating relevant content from people who are the true experts (patients in the case of quality of life). Such measurement is rare. Fitting measure data to the Rasch model is also a challenge, because of its strict requirements. For this reason, researchers continue to use dated methodologies and look for measurement models that are less demanding. Unfortunately, the consequences of failing to meet the requirements for fundamental measurement implies that the cost-per-QALY construct is an analytical dead end and much of the utility modeling conducted in the past 30 years has been profitless.\n\nAbandoning the QALY would be, to say the least, embarrassing. A centerpiece of health technology assessment would be shown to have no discernible value. It is not just a question of pointing to the shortcomings of QALYs, but making it clear that the QALY, as exemplified in incremental cost-per-QALY modeled claims, is an impossible construct. Claims for pricing and access for pharmaceutical products and devices must be rejected; they are not realistic.\n\nThis article is intended to demonstrate that, in failing to appreciate the axioms of fundamental measurement, the utility values included in QALY analyses are an analytical dead end. If we are to assess the impact on patients of emerging therapies accurately, we need a disease-specific framework that provides a coherent assessment of the comparative benefits to patients and caregivers. We cannot include approximate information as an element in the evidence (real or imaginary) presented to formulary committees. Just as claims based on phase 3 clinical trials are recognized as robust, so should claims for quality of life and utility meet the same standards. This would free us to return to normal science and hypothesis testing.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nNeumann PJ, Willke RJ, Garrison LP: A Health Economics Approach to US Value Assessment Frameworks – Introduction: An ISPOR Special Task Force Report (1). Value Health. 2018; 21(2): 119–123. PubMed Abstract \| Publisher Full Text\n\nLangley P: Nonsense on Stilts – Part 1: The ICER 2020-2023 value assessment framework for constructing imaginary worlds. Innov Pharm. 2020; 11(1). Publisher Full Text\n\nCADTH: Guidelines for the economic evaluation of health technologies. Ottawa. 2017Reference Source\n\nPiglucci M: Nonsense on Stilts: How to tell science from bunk. Chicago; University of Chicago Press. 2010. Reference Source\n\nMerbitz C, Morris J, Grip JC: Ordinal scales and foundations of misinference. Arch Phys Med Rehabil. 1989; 70(4): 308–12. PubMed Abstract\n\nStenner AJ: Measuring reading comprehension with the lexile framework. Paper presented at: Fourth North American Conference on Adolescent/Adult Literacy; Washington, DC. 1996. Reference Source\n\nPallant JF, Tennant A: An introduction to the Rasch measurement model: an example using the Hospital Anxiety and Depression Scale (HADS). Br J Clin Psychol. 2007; 46(Pt 1): 1–18. PubMed Abstract \| Publisher Full Text\n\nHeaney A, McKenna SP, Hagell P, et al.: Improving scoring precision and internal construct validity of the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) using Rasch Measurement Theory. J Rheumatol. 2019; jrheum.180943. Publisher Full Text\n\nBond T, Cox C: Applying the Rasch Model. (3rd Ed.). New York: Routledge. 2015Reference Source\n\nParkin D, Devlin N, Feng Y: What determines the shape of an EQ-5D index distribution. Med Decis Making. 2016; 36(8): 941–51. PubMed Abstract \| Publisher Full Text\n\nFeng Y, Devlin N, Bateman A, et al.: Distribution of the EQ-5D-5L profiles and values in three patient groups. Value Health. 2019; 22(3): 355–61. Publisher Full Text\n\nMcKenna SP, Heaney A, Wilburn J, et al.: Measurement of patient-reported outcomes. 1: The search for the Holy Grail. J Med Econ. 2019; 22(6): 516–22. PubMed Abstract \| Publisher Full Text\n\nMcKenna SP, Heaney A, Wilburn J, et al.: Measurement of Patient-Reported Outcomes. 2: Are Current Measures Failing Us? J Med Econ. 2019; 22(6): 523-30. PubMed Abstract \| Publisher Full Text\n\nChang H: Inventing Temperature: Measurement and Scientific Progress. New York: Oxford: University Press. 2007. Reference Source\n\nGrimby G, Tennant A, Tesio L: The Use of Raw Scores From Ordinal Scales: Time to End Malpractice? J Rehabil Med. 2012; 44(2): 97–98. PubMed Abstract \| Publisher Full Text\n\nRasch G: Probabilistic models for some intelligence and attainment tests. Copenhagen: Danmarks Paedagogiske. Institut. 1960. Reference Source\n\nLuce RD, Tukey JW: Simultaneous conjoint measurement. A new type of fundamental measurement. J Math Psychol. 1964; 1(1): 1–27. Publisher Full Text\n\nRouse M, Twiss J, McKenna SP: Co-calibrating Quality-Of-Life Scores From Three Pulmonary Disorders: Implications for Comparative-Effectiveness Research. J Med Econ. 2016; 19(6): 596–603. PubMed Abstract \| Publisher Full Text" }	[ { "id": "70294", "date": "07 Sep 2020", "name": "Ariel Beresniak", "expertise": [ "Reviewer Expertise Methodology in health decision making" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 (Measurement, modeling and QALYs) is a methodological review paper about the limitations of the QALY indicator used in health decision making. Compared to the high number of publications presenting economic models in many therapeutic areas expressed in incremental costs per QALY, few papers have been published explaining the criticisms of QALY calculation, confirming the interest of this paper in the scientific littérature. This article presents clearly the basic metric properties necessary to allow calculations and why the QALY indicator does not fulfill the conditions to be used in economic assessments. Some explanations are missing for such methodological review. Firstly the multiplicative formula should be presented for calculating QALY. Secondly, the assumptions underlying the QALY approach should be discussed to better understand the controversy. A reference to the ECHOUTCOME European project specifically testing the validity of the QALY assumptions and concluding that the QALY approach is flawed should be mentioned as the most extensive research about the dangers of using QALY in health decision making, and how this new paper confirms or complete the findings.1\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "72004", "date": "02 Oct 2020", "name": "Jonathan Belsey", "expertise": [ "Reviewer Expertise Health economics and biostatistics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 considers the mathematical underpinnings of current methods used to quantify utility and considers the implications the shortcomings that he highlights may have for the interpretation of QALYs and consequently the results of traditional cost utility analyses.\nThe authors highlight the fact that, in order to support the manipulations inherent to a cost utility model, the tool used to generate utility must satisfy a number of requirements (unidimensionality, ratio properties, true zero), without which it is not meaningful to carry out the arithmetic manipulations inherent in a cost utility approach.\nUsing the EQ-5D-3L instrument as an example, the authors demonstrate that these requirements are signally not met, and in consequence, the conclusions of analyses based on this approach are fatally undermined.\nI cannot fault the authors' chain of logic and in consequence, must agree with their conclusions. This is not a qualitative surprise - anyone who works in health economics is well aware that the QALY is a flawed measure - but I had never really thought through the mathematical inconsistencies before, in the detailed and logical way that the authors highlight.\nOne would like to think that we could behave as scientists and begin to look at how we can modify our approach to the economic assessment of healthcare interventions to address these shortcomings. Unfortunately, it seems likely that there is too much political and intellectual capital invested in the QALY to anticipate its downfall any time soon. Unfortunately, there is a long and undistinguished history of misused metrics remaining embalmed in our approach to data analysis on the basis that: \"It may be flawed, but at least it is equally flawed for everyone\". I fear that the QALY will continue to dictate healthcare spending for a good many years yet, but I nonetheless congratulate the authors for their incisive critique of its derivation.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1048
https://f1000research.com/articles/9-1044/v1	25 Aug 20	{ "type": "Case Report", "title": "Case Report: Primary pure clear cell gastric carcinoma", "authors": [ "Imen Ben Ismail", "Hakim Zenaidi", "Raja Jouini", "Saber Rebii", "Ayoub Zoghlami", "Hakim Zenaidi", "Raja Jouini", "Saber Rebii", "Ayoub Zoghlami" ], "abstract": "Clear cell carcinoma has been described in numerous anatomic sites, but the renal location is the most frequent. Its occurrence in the stomach is exceptional. Here, we report the case of a 51-year-old woman who presented with epigastric pain of four months. The upper gastrointestinal endoscopic examination revealed a polypoid tumor of the greater curvature of the stomach. Biopsies showed a poorly differentiated carcinoma with a signet-ring cell component. The CT scan revealed a polypoid mass of the vertical part of the greater gastric curvature. There was no renal lesion. A distal subtotal gastrectomy was performed, and the post-operative course was uneventful. The gross exam showed a 6.5 cm, polypoid ulcerated tumor of the antrum. Histological analysis showed a clear cell gastric carcinoma. The immunohistochemical study, performed to rule out a metastasis from renal carcinoma, showed that tumor cells didn’t express CD10 and vimentin. We therefore retained the diagnosis of a primary gastric clear cell carcinoma. Pure primary clear cell carcinomas of the stomach are exceedingly rare and are associated with a poor prognosis. Immunohistochemistry is the cornerstone of the diagnosis of these tumors to rule out metastases from a renal clear cell carcinoma.", "keywords": [ "Clear cells", "gastric cancer", "gastrectomy" ], "content": "Introduction\n\nClear cell carcinoma (CCC) generally develops in organs originating from the Mullerian system, such as the lower urinary and female genital tracts1. Its occurrence in the gastrointestinal tract is uncommon, and cases occurring in the stomach are exceedingly rare2. Clear cytoplasm, and hence clear cells, are the result of intracellular accumulation of glycogen, lipid, water, or mucin3. Due to its rarity, the clinicopathological and biological behaviors of this entity remain unclear.\n\nUntil now, only a few cases of gastric CCC have been reported in the English literature. These reports generally included gastric carcinoma with focal clear cell changes. We report herein the third case of a pure gastric CCC.\n\n\nCase report\n\nWe report the case of a 51-year-old north African woman, who presented with a history of epigastric pain of four months. The abdominal examination found mild epigastric tenderness but no palpable mass. The upper gastrointestinal endoscopic examination revealed a polypoid tumor of the greater curvature of the stomach measuring 6×4 cm (Figure 1). Biopsies were performed using digestive endoscopic biopsy forceps. The anatomopathological examination showed a poorly differentiated carcinoma with a signet-ring cell component. No immunohistochemical examination was performed.\n\nA CT scan of the thorax and abdomen, performed as part of the extension assessment, showed a pedicled budding mass with endoluminal development of the vertical part of the greater gastric curvature measuring 6 × 4 × 5cm (Figure 2). Otherwise, it did not show evidence of any renal tumor or hepatic or pulmonary localization.\n\nIn order to reduce tumor volume and improve the R0 resection rate, the patient received four courses (one course per two weeks) of perioperative chemotherapy with 5-fluorouracil (2600mg/m2), folinic acid (350mg), oxaliplatin (85mg/m2) and docetaxel (50mg/m2) according to the FLOT regimen, administrated through a totally implantable venous access port via the internal jugular left vein. Then the patient underwent distal subtotal gastrectomy with a manual Roux-en-Y esophagojejunostomy, via a midline incision. This was the procedure of choice of the clinical lecturer who performed the intervention in case of proximal gastric cancer.\n\nGross examination revealed an ulcerated polypoid tumor with endoluminal development. The histological exam showed an invasive tumor arranged in lobules, clusters, and nests within a highly vascularized stroma (Figure 3a). There were necrotic changes. Tumor cells had abundant clear cytoplasm and well-defined cytoplasmic borders. The nuclei had marked atypia and prominent eosinophilic nucleoli (Figure 3b). The tumor infiltrates to the subserosa without serosal invasion. Moreover, we noted the absence of vascular emboli, perineural tumor invasion and lymph node metastasis. Periodic acid-Schiff and alcian blue stains were negative. An immunohistochemical study was performed to rule out a renal origin. The tumor cells were negative for CD10 and vimentin. They were positive for cytokeratin with diffuse cytoplasmic and membranous staining. The diagnosis of primary gastric CCC in its pure form was made.\n\n(a) Clear cell gastric carcinoma (top right: normal gastric mucosa; bottom left: gastric carcinoma; He x 100). (b) Tumor cells are clear with marked nuclear atypia and numerous mitotic figures.\n\nThe postoperative course was uneventful and the patient was discharged on the fifth postoperative day on analgesic treatment and low-molecular-weight heparin for thirty days. The patient received four courses of adjuvant chemotherapy (FLOT regimen). The CT scan done after six months showed no local or distant recurrence.\n\n\nDiscussion\n\nA CCC can develop in various organs, and the most common sites are the kidneys and the female genital tract1. Its occurrence in the gastrointestinal tract is uncommon. Only few case reports or small series have been described in the colon4, pancreas5, and the biliary system6.\n\nEven though the presence of clear cell changes in gastric carcinoma has been reported in 8.5 % of cases7, the pure form of CCC of the stomach is an extremely rare oncologic entity. There is no specific reference to CCC in the latest WHO classification of gastric carcinoma. This entity has not been well documented, with only limited literature available on the topic.\n\nRegarding the clinical characteristics, Kim et al.7 have demonstrated, in a large cohort study, that gastric carcinomas with clear cell changes were associated with younger age and tended to be located in the gastric antrum. However, Ghotli et al.8 showed that gastric CCC had a predilection for the gastroesophageal junction. Moreover, these tumors are polypoid and histologically characterized by a tubulo-papillary pattern. These features are consistent with the characteristics of the tumor in our case, which was polypoid and located in the vertical part of the greater gastric curvature.\n\nWhat makes this case remarkable is that the present tumor is made of 100% clear cells. This situation is different from previously reported case series, in which only a portion of the adenocarcinoma showed clear cell cytoplasm. Ghotli et al.8 reported 12 cases and defined the CCC as adenocarcinoma composed of more than 10% of clear cells. Kim et al.7 reported 65 cases and defined CCC as a carcinoma composed of more than 5% of clear cells. To the best of our knowledge, only Terada9 and Yamada et al.10 have reported the pure form.\n\nIt has been shown that the presence of clear cell changes is an independent indicator of poor prognosis7 since it is associated with advanced depth of invasion, presence of lymphovascular tumor emboli, and lymph node metastases, compared to gastric adenocarcinoma without clear cell changes.\n\nAdvances made in techniques used for pathological examinations and immunohistochemistry made the diagnosis of gastric CCC easier. Immunophenotypically, it has been demonstrated that CCC carcinoma shows overexpression of cyclin D18. It has also been noted that clear cell tumors of the stomach may produce alpha-fetoprotein (AFP) in the serum and within the tumor11. In our case, AFP was not measured.\n\nRecently, hepatocyte nuclear factor-1b (HNF-1b) has been accepted as a unique biomarker of CCC for tumors of the female genital tract, bladder, and pancreas5,12,13. In the stomach, carcinomas with clear cell changes also show increased positive immunostaining of HNF-1b as it has a role in cellular glycogen synthesis7. Nevertheless, until now, there has been no reports about the role of HNF-1b in gastric adenocarcinomas.\n\nDue to its rarity, there are no therapeutic guidelines for CCC. It is managed like conventional gastric carcinomas, and its surgical treatment depends on its localization. In our case, the tumor was located in the vertical part of the great gastric curvature and necessitated total gastrectomy.\n\n\nConclusions\n\nPure primary CCC of the stomach are exceedingly rare and are associated with a poor prognosis. Immunohistochemistry is the cornerstone of the diagnosis of these tumors to rule out metastases from a renal CCC.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nEvans H, Yates WA, Palmer WE, et al.: Clear cell carcinoma of the sigmoid mesocolon: a tumor of the secondary mullerian system. Am J Obstet Gynecol. 1990; 162(1): 161–163. PubMed Abstract \| Publisher Full Text\n\nGovender D, Ramdial PK, Clarke B, et al.: Clear cell (glycogenrich) gastric adenocarcinoma. Ann Diagn Pathol. 2004; 8(2): 69–73. PubMed Abstract \| Publisher Full Text\n\nBittinger A, Altekrüger I, Barth P: Clear cell carcinoma of the gallbladder: A histological and immunohistochemical study. Pathol Res Pract. 1995; 191(12): 1259–1265. PubMed Abstract \| Publisher Full Text\n\nJewell LD, Barr JR, McCaughey WTE, et al.: Clear-cell epithelial neoplasms of the large intestine. Arch Pathol Lab Med. 1988; 112(2): 197–199. PubMed Abstract\n\nKim L, Liao J, Zhang M, et al.: Clear cell carcinoma of the pancreas: histopathologic features and a unique biomarker: hepatocyte nuclear factor-1b. Mod Pathol. 2008; 21(9): 1075–1083. PubMed Abstract \| Publisher Full Text\n\nVardaman C, Albores-Saavedra J: Clear cell carcinoma of the gallbladder and extrahepatic bile ducts. Am J Surg Pathol. 1995; 19(1): 91–99. PubMed Abstract \| Publisher Full Text\n\nKim JY, Park DY, Kim GHa, et al.: Does clear cell carcinoma of stomach exist ? Clinicopathological and prognostic significance of clear cell changes in gastric adenocarcinomas. Histopathology. 2014; 65(1): 90–99. PubMed Abstract \| Publisher Full Text\n\nGhotli ZA, Serra S, Chetty R: Clear cell (glycogen-rich) gastric adenocarcinoma: a distinct tubulo-papillary variant with a predirection for the cardia/gastroesophageal region. Pathology. 2007; 39(5): 466–9. PubMed Abstract \| Publisher Full Text\n\nTerada T: Pure Glycogen-Rich Clear Cell Adenocarcinomaof the Stomach. J Gastrointest Cancer. 2014; 45(3): 372–6. PubMed Abstract \| Publisher Full Text\n\nYamada R, Horiguchi S, Onishi T, et al.: Early Gastric Cancer with Purely Enteroblastic Differentiation and No Conventional Adenocarcinoma Component. Case Rep Pathol. 2018; 28: 3620293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsunou H, Konishi F, Jalal RE, et al.: Alphafetoprotein- producing gastric carcinoma with enteroblastic differentiation. Cancer. 1994; 73(3): 534–40. PubMed Abstract \| Publisher Full Text\n\nTsuchiya A, Sakamoto M, Yasuda J, et al.: Expression profiling in ovarian clear cell carcinoma: identification of hepatocyte nuclear factor-1 beta as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma. Am J Pathol. 2003; 163(6): 2503–2512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrimo F, Herawi M, Sharma R, et al.: Hepatocyte nuclear factor-1b expression in clear cell adenocarcinomas of the bladder and urethra: diagnostic utility and implications for histogenesis. Hum Pathol. 2011; 42(11): 1613–1619. PubMed Abstract \| Publisher Full Text" }	[ { "id": "88574", "date": "06 Jul 2021", "name": "Bassam Musa Sadik Al-Musawi", "expertise": [ "Reviewer Expertise Medical human genetics", "clinical genetics", "cytogenetics", "molecular diagnosis", "biochemical genetics", "birth defects and teratology", "and genetic counseling." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is concise, nicely written and presented with clear concept for an important topic.\nHowever, due to its rarity, some more details may be required, both clinically and histopathologically.\nBeing an exceedingly rare tumor, all available clinical and demographic data are required; e.g. origin, ethnicity, martial status of the patient, history of dyspepsia or indigestion, nausea or vomiting, loss of appetite or weight, smoking, drug histories, etc.\n\nHave the authors considered that neoadjuvant chemotherapy might have altered the immunohistochemical reactivity of this tumor?\n\nThe authors relied on minimal immunohistochemical markers as a diagnostic tool to exclude renal origin. They should have exhaust all possible markers to exclude a renal and other less common origins and to prove a gastric origin: Please, see below: ((The commonly used immunomarkers can differentiate and distinguish most of the clear-cell tumors. Clear cell carcinoma (CCC) of female genital tract (FGT) are CD15, Napsin A, and CA125 positive and estrogen receptor (ER), Wilms tumor 1 (WT1),carcinoembryonic antigen(CEA), inhibin, and alpha-fetoprotein (AFP) negative; clear cell renal cell carcinoma (CCRCC) are CD10, epithelial membrane antigen (EMA), vimentin, Pax 2, and CK20 negative; variable CK7 andmelanomas are HMB45 and Melan A positive; skin CCCs are CK and HMWK positive; sarcomas are positive with vimentin and variably positive for desmin, smooth muscle actin, S-100, and CD68. In the central nervous system (CNS), immunomarkers such as glial fibrillary acidic protein (GFAP), neuron-specific-enolase (NSE), S-100, vimentin, and EMA are used for differentiation of entities. By combining morphology with immunohistochemistry (IHC).)) Even Perivascular Epithelioid Cell (PEComa) must be excluded. This is a major drawback on the report.\n\nThey also did not specify which cytokeratin was used (pancytokeratin, CK7 or CK20?\n\nAlso H&E images are insufficient. They should provide more photos of H&E as well as for all immunohistochemical markers used to convince the reader of a primary pure CCC of stomach.\n\nTumor stage was not precisely determined. It must be clearly mentioned.\n\nClear cell carcinomas are usually aggressive and carry a poor prognosis. Have the authors followed the case up and for how long?\n\nBeing an aggressive tumor, why have you tried the conventional treatment for adenocarcinoma? would a more aggressive therapy been more appropriate?\n\nThe authors have stated that ((Then the patient underwent distal subtotal gastrectomy with a manual Roux-en-Y esophagojejunostomy, via a midline incision)) in case report section, but later in discussion they state that ((In our case, the tumor was located in the vertical part of the great gastric curvature and necessitated total gastrectomy)). Would that be considered a controversy?\n\nA statement that all authors have approved the submitted manuscript and all subsequent revisions is required and must be added.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "127229", "date": "28 Mar 2022", "name": "Sung-Jig Lim", "expertise": [ "Reviewer Expertise pathology", "immunooncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is interesting and clinically challenging. And there might be some concerns to be addressed.\nClear cell carcinoma of stomach is not included in the subtype of gastric cancer in WHO classification and diagnostic criteria is not unsettled. Then the authors need to describe the details of microscopic findings: tumor cell growth architectures and surrounding vascular patterns, tumor cell morphology, and percentage of clear cells etc.\n\nWith the microscopic findings, figures with clear focus should be added.\n\nVarious markers for differential diagnosis with figures are necessary to rule out metastatic tumor, or other types of carcinoma.\n\nThey should add the diagnostic criteria of clear cell carcinoma in other organs and the reason for the presence of clear cell gastric carcinoma as a diagnostic entity in the discussion.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1044
https://f1000research.com/articles/9-1043/v1	25 Aug 20	{ "type": "Review", "title": "Open science approaches to COVID-19", "authors": [ "Edwin G. Tse", "Dana M. Klug", "Matthew H. Todd", "Edwin G. Tse", "Dana M. Klug" ], "abstract": "In only a matter of months, the coronavirus disease of 2019 (COVID-19) has spread around the world. The global impact of the disease has caused significant and repeated calls for quick action towards new medicines and vaccines. In response, researchers have adopted open science methods to begin to combat this disease via global collaborative efforts. We summarise here some of those initiatives, and have created an updateable list to which others may be added. Though open science has previously been shown as an accelerator of biomedical research, the COVID-19 crisis has made openness seem the logical choice. Will openness persist in the discovery of new medicines, after the crisis has receded?", "keywords": [ "Sars-CoV-2", "COVID-19", "open science", "open data", "open access", "open source" ], "content": "Introduction\n\nIn late 2019, reports began to emerge from Wuhan, China concerning cases of pneumonia of an unknown origin. Shortly thereafter, Chinese authorities identified this to be a novel type of coronavirus disease (now known as coronavirus disease 2019; COVID-19) caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), with the outbreak being declared by the World Health Organization (WHO) as a public health emergency of international concern. Over the next two months, increasing numbers of COVID-19 cases were reported in countries outside China at an alarming rate, prompting the WHO to declare COVID-19 a global pandemic in March 20201. To date, according to the WHO COVID-19 Situation Dashboard2, there have been around 18 million cases worldwide, in over 200 countries, resulting in around 690,000 deaths. As COVID-19 is caused by a novel coronavirus, there are no established methods for its treatment, and measures such as social distancing and self-isolation have become crucial to prevent further spread. The urgency to overcome this pandemic is furthered by the devastating effect on the world economy that has been seen as a result of the prolonged implementation of these measures3.\n\nThe enormous impact of this disease has resulted in significant activity towards new therapeutics, particularly for the development of a new vaccine. Much of this work is taking place in the private sector, alongside the usual requirement in that sector for secrecy. However, there has in parallel been a significant push for a more open approach because it is understood that openness leads to research acceleration. Broadly speaking, these initiatives can be grouped together as three types: 1) open access (the availability of research publications that are free to access and, often, re-use), 2) open data (the same, but with data) and 3) open source (in which broader community participation is allowed via liberal licence terms). There are many well-known and frequently-described advantages of openness (e.g. reduction of duplication of effort, faster communication of important outcomes) that nevertheless compete with a need for secrecy for many researchers, arising from the need for protected intellectual property or a perceived competitive advantage. These motivations for secrecy seem, in a time of crisis, to be lessened, and the increased prevalence of open initiatives relating to COVID-19 research has been striking.\n\nOpen science in biomedical research has gained increased traction over the past decade4,5 from screening projects (e.g., CO-ADD6) and the sharing of physical samples (e.g., SGC probes7, MMV Boxes8) through to fully-fledged drug discovery (e.g., Open Source Malaria9, MycetOS10) and development (e.g., M4K Pharma11) campaigns. For COVID-19, data are being generated and shared (e.g. protein target structures, fragment hits), and initiatives have been created to identify and fast-track candidate compounds into clinical use. Even the lengthy process of drug approval is thought to be something that can be shortened: the urgency of the current situation and the use of open science has opened the possibility of reducing the timeline significantly to as little as 1.5 years12 (though the fastest vaccine to be developed, for the 2014–2016 Ebola virus epidemic, took 5 years)13,14.\n\nThis article collates the key open science resources and initiatives currently available for COVID-19 research (Figure 1). The three previously mentioned categories will be used to group the resources and a brief description of each will be given. This article forms the basis of a “living” collection of open science resources for COVID-19. As more resources become available, anyone may update the repository and discuss those additions.\n\nRepresentative examples from the development timeline include the COVID Moonshot project, the ICR canSAR tool, the Diamond XChem fragment screen and preprint servers.\n\n\nOpen access\n\nAn obvious example of open access is the preprint server, such as bioRxiv15 and medRxiv16. To date, there have been more than 7000 COVID-19 focussed articles submitted to these servers17. While this is a valuable resource, the articles are preliminary reports and are yet to be peer-reviewed: the main purpose of preprints is to allow researchers to quickly disseminate their results before official publication. It is noted that this surge in COVID-related studies has led to preprint servers like bioRxiv to implement stricter quality control of submitted articles, resulting in purely computational-based papers no longer being accepted18.\n\nUnlike preprint servers, open access journals publish peer-reviewed articles that are freely available for anyone to view without payment. Examples of open access journals featuring COVID-19 collections include the Public Library of Science journals (e.g. PLoS One, PLoS Medicine, etc.)19, Nature Communications20 and Wellcome Open Research21. Other journals which are not fully open access, such as JAMA and The BMJ, have made their COVID collections free and open22,23.\n\n\nOpen data\n\nThe Diamond Light Source is a synchrotron facility located in the UK that is utilised for a range of scientific areas including the investigation of protein structures and properties. During the coronavirus pandemic, Diamond scientists have used their facility to generate data on protein targets and fragments, all of which have been made publicly available24. Notably, by solving a high-resolution structure (1.39 Å) of the SARS-CoV-2 main protease25, it was possible to perform a screen of multiple fragment libraries to identify the most promising hits for fragment-based drug discovery. This screen resulted in 74 high interest hits and the full results of this screen have been made available to researchers and initiatives such as the PostEra COVID Moonshot project (vide infra). This main protease structure has also been used to screen for covalent probes from a library of electrophile fragments26.\n\nThe Protein Data Bank (PDB) is an open database containing the 3D structural data of proteins and nucleic acids deposited by researchers from around the world. This database is an important resource for scientific research and many scientific journals now require authors to submit their structural data to the PDB. The PDB are maintaining a collection of a wide range of SARS-CoV-2 structures including the main protease and spike protein/receptors27.\n\nThe National Center for Advancing Translational Sciences (NCATS) has focussed its efforts on drug repurposing for COVID-19 by generating datasets created from the screening of SARS-CoV-2-related assays against FDA-approved drugs and anti-infectious agents. Multiple compound collections are actively being screened in eight assays (with more in development) that focus on various stages of the SARS-Cov-2 life cycle in both human and viral targets. The results of the screen, as well as all assay protocols, have been made available online28.\n\nThe Institute of Cancer Research (ICR) have developed a tool named canSAR which is a knowledgebase that collates multidisciplinary data and applies machine learning approaches to provide useful predictions for cancer drug discovery. The ICR have repurposed this tool for the current research efforts against coronavirus, allowing people to freely search for information including the druggable interactome, ongoing and completed clinical trials, and lists of active compounds, probes and targets under investigation29.\n\nA number of platforms have been created with the purpose of aggregating and curating openly available data that has been generated during COVID-19 research. The European Commission is working with a number of partners, including the EMBL-EBI, to create a platform that aggregates data ranging from sequencing and expression data to protein structures, drug targets and compounds30. The CORD-19 dataset is a large, machine-readable database intended to facilitate machine learning and data mining approaches to COVID-19 research31,32. The COVID-19 Molecular Structure and Therapeutics Hub maintains a repository of input files and analysis scripts for molecular simulation and dynamics studies related to COVID-1933.\n\n\nOpen source\n\nPostEra AI is a for-profit startup company that specialises in integrating molecular design with chemical synthesis. As a result of the coronavirus pandemic, and stemming from the data produced by the Diamond Light Source (vide supra), PostEra have collaborated with academic institutions and industry around the world, while adopting open science principles, to design new inhibitors of the SARS-CoV-2 main protease34. This global collaboration effort allows anyone to suggest new inhibitors based on the initial Diamond fragment hits. Following this, the most attractive compounds will be identified using machine learning algorithms, synthesised by a contract synthesis company and evaluated in inhibition assays (fluorescence and RapidFire mass spectrometry) against the SARS-CoV-2 main protease in labs around the world. Importantly, all stages of this process will be made publicly available. To date, there have been over 12000 unique compounds designed by the community. Over 1300 compounds have been ordered commercially, around 950 compounds have been synthesised, and over 900 compounds have been assayed against SARS-CoV-2.\n\nThe Joint European Disruptive Initiative (JEDI) is a search for breakthrough technologies in the European Union. The GrandChallenge is a three stage campaign for the development of lead compounds against multiple SARS-CoV-2 targets35. Stage 1 is an open competition that focuses on in silico screening of compounds against high-resolution protein structures. Teams use simulation approaches (e.g. molecular dynamics, deep learning, docking, etc.) to score libraries of compounds against a chosen target. By comparing multiple different approaches errors can be averaged out and the best compounds for each protein target chosen for progression to the next stage. Stage 2 is an in vitro screening stage focussing on identifying the compounds from Stage 1 that provide 99% viral suppression. Teams must provide experimental evidence for this through either selective testing, high-throughput screening or smart combinatorial methods. Stage 3 is the in vivo screening stage aimed at finding novel drug combinations. This stage is run independently from the first two stages but lead compounds from those stages may be incorporated in this stage (provided they have been FDA-approved). Following the conclusion of each stage, the top-ranked team will be awarded a cash prize (€250,000 in both Stage 1 and 2, and up to €1,000,000 in Stage 3).\n\nThe Medicines for Malaria Venture (MMV) is a not-for-profit organisation that brings together the public and private sectors for the discovery and development of new antimalarial medicines. MMV have previously created, and freely distributed, collections of promising candidate compounds in well plates to researchers around the world to enable a more efficient starting point for new drugs (see Malaria Box36, Pathogen Box37 & Pandemic Box38). MMV have now created, and made available on request, the COVID Box, which contains 80 compounds of both marked drugs and compounds in development that possess known or predicted activity against SARS-CoV-239. A stipulation of this open research project is that the resulting data generated by researchers using the COVID Box must be shared in the public domain within 2 years of its generation.\n\nThe COVID-19 Protein Portal is a UK-based initiative led by Wellcome and UKRI, that provides SARS-CoV-2-related protein reagents for UK scientists to use, free of charge40. These include viral proteins, human proteins and antibodies, all of which are searchable in their online database. All results generated from the use of these reagents will be made publicly available.\n\nNextstrain is an open source, interactive data visualisation platform that provides “real-time tracking of pathogen evolution” via the analysis of sequencing data41. The tools used to achieve this are freely available to use and modify, and have already been used to track the evolution of a range of pathogens including the seasonal flu, Zika virus, and the West Nile virus. In response to the current pandemic, Nextstrain is maintaining a SARS-CoV-2 phylogenetic tree based on the analysis of contributed sequencing data.\n\nFolding@home (F@H) is a distributed computing project involving multiple research labs and citizen scientists from around the world that focuses on simulating protein dynamics. F@H provides software that enables users to donate unused computing power towards the computational analysis of protein folding42. Thus far, F@H’s COVID-19 projects have focused on simulating the interactions between the SARS-CoV-2 spike protein and the human ACE2 receptor to which it binds. All input files are available through GitHub, which is also where the research outputs will be made openly available43.\n\nThe Open Source COVID-19 (OSC19) Drug Discovery program is utilising computer science and biochemistry to enable the rapid screening of existing drug molecules for use against COVID-1944. Scientists from a range of fields are encouraged to participate, including synthetic chemists to make drug candidates, biochemists and virologists to run assays and donors and volunteers to aid in publicity and fundraising. As an open source project, all research results will be freely available with no intellectual property claims for any discoveries made.\n\n\nOther resources\n\nIn a similar manner to this article, a number of additional resources have been created to help spread word of the growing list of open science efforts for COVID-19 research. Examples include Initiatives such as Joinup EU45 and SPARC46, which have created hubs of open source research projects and resources. Funding agencies like the UKRI and UKCDR have provided lists of funded COVID-19 research projects to help researchers identify and fill funding gaps47,48. To further advocate the use of open science, the Open COVID Pledge has aimed to encourage researchers and businesses to make their COVID-related intellectual property freely available by providing the Open COVID Licence49. Finally, the Virus Outbreak Data Network (VODAN) is working to ensure that data related to the COVID outbreak is findable, accessible, interoperable, and reusable50.\n\n\nConclusion\n\nA significant amount of effort has been made to progress COVID-19 research since the beginning of the pandemic. With so many scientific minds working on this problem together, it is clear that conducting scientific research in an open manner can accelerate the research process. The resources and initiatives highlighted in this article demonstrate the benefits of open science approaches and its potential to accelerate research timelines. This article itself will form the basis of an open science “living” resource hosted on a public repository. It is understandable that a global public health crisis causes us to adopt innovations in how we work in the search for an effective solution. After the COVID-19 crisis has faded there will remain many other crises that we face in the search for an effective therapy to alleviate suffering, whether the affected population is a billion people or a single individual in search of a cure. It is hoped that open science will be seen as a “new normal” approach in those crises too.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nWHO Director-General’s Open Remarks at the Media Briefing on COVID-19 – 11 March 2020. 2020; Accessed 9 June, 2020. Reference Source\n\nWHO Coronavirus Disease (COVID-19) Dashboard. 2020; Accessed 9 June, 2020. Reference Source\n\nGopinath G: The Great Lockdown: Worst Economic Downturn Since the Great Depression. 2020; Accessed 24 June, 2020. Reference Source\n\nMcKiernan EC, Bourne PE, Brown CT, et al.: How Open Science Helps Researchers Succeed. eLife. 2016; 5: e16800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaw DL: Is Open Science the Future of Drug Development? Yale J Biol Med. 2017; 90(1): 147–151. PubMed Abstract \| Free Full Text\n\nZuegg J, Hansford KA, Elliott AG, et al.: How to Stimulate and Facilitate Early Stage Antibiotic Discovery. ACS Infect Dis. 2020; 6(6): 1302–1304. PubMed Abstract \| Publisher Full Text\n\nMüller S, Ackloo S, Arrowsmith CH, et al.: Donated Chemical Probes for Open Science. eLife. 2018; 7: e34311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Voorhis WC, Adams JH, Adelfio R, et al.: Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond. PLoS Pathog. 2016; 12(7): e1005763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliamson AE, Ylioja PM, Robertson MN, et al.: Open Source Drug Discovery: Highly Potent Antimalarial Compounds Derived from the Tres Cantos Arylpyrroles. ACS Cent Sci. 2016; 2(10): 687–701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim W, Melse Y, Konings M, et al.: Addressing the Most Neglected Diseases Through an Open Research Model: The Discovery of Fenarimols as Novel Drug Candidates for Eumycetoma. PLoS Negl Trop Dis. 2018; 12(4): e0006437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorgan MR, Roberts OG, Edwards AM: Ideation and Implementation of an Open Science Drug Discovery Business Model − M4K Pharma [version 1; peer review: 2 approved, 1 approved with reservations]. Wellcome Open Res. 2018; 3: 154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLurie N, Saville M, Hatchett R, et al.: Developing Covid-19 Vaccines at Pandemic Speed. N Engl J Med. 2020; 382(21): 1969–1973. PubMed Abstract \| Publisher Full Text\n\nWHO Prequalifies Ebola Vaccine, Paving the Way for its use in High-Risk Countries. 2019; Accessed 9 June, 2020. Reference Source\n\nHerder M, Graham JE, Gold R: From Discovery to Delivery: Public Sector Development of the rVSV - ZEBOV Ebola Vaccine. J Law Biosci. 2020; lsz019. Publisher Full Text\n\nbioRxiv - The Preprint Server for Biology. 2020; Accessed 8 June, 2020. Reference Source\n\nmedRxiv - The Preprint Server for Health Sciences. 2020; Accessed 8 June, 2020. Reference Source\n\nCOVID-19 SARS-CoV-2 Preprints from medRxiv and bioRxiv. 2020; Accessed 8 June, 2020. Reference Source\n\nKwon D: How Swamped Preprint Servers are Blocking Bad Coronavirus Research. Nature. 2020; 581(7807): 130–131. PubMed Abstract \| Publisher Full Text\n\nPLoS COVID-19 Updates. 2020; Accessed 23 June, 2020. Reference Source\n\nSARS-CoV-2 - Latest Research and News. 2020; Accessed 23 June, 2020. Reference Source\n\nArticles from collection Coronavirus (COVID-19). 2020; Accessed 23 June, 2020. Reference Source\n\nCoronavirus (COVID19). 2020; Accessed 29 June, 2020. Reference Source\n\nCoronavirus (covid-19): Latest News and Resources. 2020; Accessed 29 June, 2020. Reference Source\n\nMain Protease Structure and XChem Fragment Screen. 2020; Accessed 8 June, 2020. Reference Source\n\nOwen CD, Lukacik P, Strain-Damerell CM, et al.: SARS-CoV-2 main protease with unliganded active site (2019-nCoV, coronavirus disease 2019, COVID-19). wwPDB.2020. Publisher Full Text\n\nResnick E, Bradley A, Gan J, et al.: Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening. J Am Chem Soc. 2019; 141(22): 8951–8968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCOVID-19/SARS-CoV-2 Resources. 2020; Accessed 30 June, 2020. Reference Source\n\nOpenData \| COVID-19. 2020; Accessed 8 June, 2020. Reference Source\n\ncanSAR Coronavirus Research Tool. 2020; Accessed 16 June, 2020. Reference Source\n\nThe COVID-19 Data Portal. 2020; Accessed 23 June, 2020. Reference Source\n\nCOVID-19 Open Research Dataset Challenge (CORD-19). 2020; Accessed 24 June, 2020. Reference Source\n\nCORD-19 COVID-19 Open Research Dataset. 2020; Accessed 24 June, 2020. Reference Source\n\nCOVID-19 Molecular Structure and Therapeutics Hub. 2020; Accessed 24 June, 2020. Reference Source\n\nCOVID Moonshot. 2020; Accessed 8 June, 2020. Reference Source\n\nJEDI Grand Challenge. 2020; Accessed 8 June, 2020. Reference Source\n\nThe Malaria Box. 2020; Accessed 16 June, 2020. Reference Source\n\nThe Pathogen Box. 2020; Accessed 16 June, 2020. Reference Source\n\nThe Pandemic Response Box. 2020; Accessed 16 June, 2020. Reference Source\n\nThe COVID Box. 2020; Accessed 16 June, 2020. Reference Source\n\nCOVID-19 Protein Portal. 2020; Accessed 23 July, 2020. Reference Source\n\nReal-Time Tracking of Pathogen Evolution. 2020; Accessed 24 June, 2020. Reference Source\n\nCOVID-19. 2020; Accessed 24 June, 2020. Reference Source\n\nFolding@home COVID-19 Efforts. 2020; Accessed 24 June, 2020. Reference Source\n\nOpen Source COVID-19 Research Consortium. 2020; Accessed 16 July, 2020. Reference Source\n\nDigital Response to COVID-19 Open Source Solutions. 2020; Accessed 24 June, 2020. Reference Source\n\nThe Coronavirus and Open Science: Our Reads and Open Use Cases. 2020; Accessed 24 June, 2020. Reference Source\n\nCOVID-19 Research and Innovation Supported by UKRI. 2020; Accessed 24 June, 2020. Reference Source\n\nCOVID-19 Research Project Tracker by UKCDR & GloPID-R. 2020; Accessed 24 June, 2020. Reference Source\n\nOpen COVID Pledge. 2020; Accessed 24 June, 2020. Reference Source\n\nVirus Outbreak Data Network (VODAN). 2020; Accessed 24 June, 2020. Reference Source" }	[ { "id": "70313", "date": "14 Sep 2020", "name": "Matthew Hall", "expertise": [ "Reviewer Expertise Open data sharing", "Translational science", "Assay development", "Experimental therapeutics", "Drug discovery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments to the Author\n\nThe authors have worked to summarize the open science efforts made in the wake of the COVID-19 pandemic. They have collected COVID-19-related open research resources and categorized them as open access, open data, or open source. In summarizing these approaches, the authors seek to highlight the benefits of open science and the promise of accelerated therapeutic development in this unprecedented COVID era.\n\nThe review as a whole is succinct, well-written and easy-to-understand, and presents a clear summary of the accumulated open science resources. The curation work the authors have undertaken is commendable, and given the number of COVID-related initiatives becoming public, it could prove valuable to researchers looking to find an appropriate resource.\n\nIt’s clear the review was meant to summarize the state of open science initiatives for COVID-19 research without offering commentary or analysis on the approaches taken, but this reviewer can’t help but wonder what lessons the scientific community will learn from this first big foray (for many) into open science. E.g. who were the main users of these open science resources? Are there (or should there be) minimum requirements to be considered an “open science” resource? Is it enough to say that data/results will be made public in the future?\n\nRegarding the list being compiled by the authors, this reviewer understands why Github is a logical home for a living, open source resource, but it may inadvertently limit the audience reached (or require a bit of exploration for users new to the platform). Github will likely be familiar to those in the informatics or developer communities, but to those in basic/bench research, the sight of a Github page may signal that the resource is not meant for them. The authors might consider mirroring the resource somewhere in a format more familiar to bench researchers or perhaps work to direct users to the wiki rather than the code tab, where the sight of a classic README.md file/structure might scare away potential readers who otherwise recognize that as the sign of a wrong turn taken.\n\nTo improve the utility of their lists, the authors might also consider categorizing the resources by subject matter (e.g. structural data vs small molecule screening/activity vs prediction platforms) in addition to classifying by type of open science resource. It’s likely that users may be in search of a particular type of COVID resource/dataset matched to their area of expertise, more so than a particular class of open science platform (open data vs open source). Those in search of the former will need to read through a handful of separate resource write-ups before being able to decide which, if any, are relevant to them.\n\nThe value of this type of resource is highly dependent on how frequently it is updated and curated, so the authors should be commended on their undertaking of this work.\n\nMinor edit to text: The NCATS OpenData Portal (cited as #28) also has a preprint which goes into greater detail about the mission and design of the site: https://www.biorxiv.org/content/10.1101/2020.06.04.135046v11\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "70312", "date": "26 Oct 2020", "name": "Alexander Tropsha", "expertise": [ "Reviewer Expertise Cheminformatics and Computer Aided Drug Discoevry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 timely, excellent manuscript that both highlights and supports open drug discovery research especially for COVID-19 and possible future pandemics. The authors provide a nice overview of different types of open research activities related to the research response to COVID-19.\nThere has been indeed an unprecedented level of sharing data and resources since the beginning of the pandemic and it is difficult to cover all of these developments in a short review. I would nevertheless suggest a few additions. Thus, I recommend adding a statement on the availability of computational resources besides Folding@home such as COVID-19 HPC consortium (https://www.xsede.org/covid19-hpc-consortium).\n\nI would also suggest a broader acknowledgment of multiple journals and publishers that agreed to provide open access to all papers on the topic of COVID-19. A possibly complete list is available from the Wellcome Trust (https://wellcome.org/press-release/publishers-make-coronavirus-covid-19-content-freely-available-and-reusable).\n\nFinally, I would recommend that the authors expand on the important issue of the quality of research publications. The authors mention that even preprint servers stopped accepting manuscripts with countless purely computationally repurposed drugs against the disease with no experimental confirmation. It would be prudent to cite a recent publication in Nature Biotechnology on the need for automated peer review of the publications tsunami (https://www.nature.com/articles/s41587-020-0686-x)1 and perhaps, emphasize the importance of rigor, experimental support, and reproducibility of research findings reported in both preprint servers and peer-reviewed publications.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1043
https://f1000research.com/articles/9-242/v1	07 Apr 20	{ "type": "Systematic Review", "title": "Post-stroke fatigue: a scoping review", "authors": [ "Ghazaleh Aali", "Avril Drummond", "Roshan das Nair", "Farhad Shokraneh", "Avril Drummond", "Roshan das Nair", "Farhad Shokraneh" ], "abstract": "Background: Post-stroke fatigue (PSF) is one of the most common and frustrating outcomes of stroke. It has a high prevalence and it can persist for many years after stroke. PSF itself contributes to a wider range of undesirable outcomes that affect all aspects of daily life. The aim of this review was to identify and summarise the most recent research on PSF, in order to update the evidence base. Methods: We updated an existing review (Hinkle et al. 2017) systematically searching CINAHL, MEDLINE, PsycINFO, and PubMed to cover new research studies between 1st March 2016 and the search date (19th January 2020). We included interventional and observational research, and clinical practice guidelines that were not covered in the original review. After duplicate removal in EndNote, two reviewers screened the search results in Rayyan, and data from eligible full texts were extracted onto an Excel spreadsheet. Finally, we used RobotReviewer and a human reviewer to assess the risk of bias of randomised trials for this scoping review. Results: We identified 45 records for 30 studies (14 observational, 10 interventional studies, and 6 guidelines). Apart from one, the interventional studies were single-centred, had high risk of bias and small sample size (median 50). They investigated exercise, pharmacotherapy, psychotherapy, education, and light therapy. Observational studies mainly reported the factors related to PSF including co-morbidities, depression and anxiety, quality of life, activities of daily living, stroke severity, medication use and polypharmacy, polymorphism, pain, apathy, limb heaviness, neuroticism, mobility, and thyroid-stimulating hormone. Guidelines either did not report on PSF or, when reported, their recommendations were supported by little or low level of evidence. Conclusion: Although we identified a number of recent studies which have added to our current knowledge on PSF, none are robust enough to change current clinical practice.", "keywords": [ "Post-Stroke Fatigue", "Scoping Review" ], "content": "Introduction\n\nPost-stroke fatigue (PSF) has been defined as ‘overwhelming feeling of exhaustion or tiredness’, which is unrelated to exertion, and does not typically improve with rest1. It is one of the most common outcomes of stroke and its prevalence varies between 25% and 85%; however, it is generally accepted that it affects 50% of people after stroke2. PSF is linked to undesirable stroke outcomes and affects patients’ participation in studies, adherence to medication, and effectiveness of rehabilitation3. This has a negative impact on patients’ quality of life and daily life activities4–7, and also contributes to the burden on family members and carers8.\n\nAlthough researchers have attempted to explain PSF mechanisms9, its aetiology still remains unclear. This is partly because there are many contributing factors to PSF8,10–32, and each research team may focus only on some of the factors to find a route for preventing, treating or managing PSF. Any endeavour to find the most effective intervention in the research literature leads to a collection of heterogeneous interventions from physiotherapy33 and exercise34–38 to psychotherapy, pharmacotherapy, and recently laser therapy39–41.\n\nAs a systematic effort to review these scattered interventions, a Cochrane review42,43 compared all the tested PSF treatments to a control group, to standard care, or to each other, through reviewing randomised controlled trials (RCTs). This review concluded that there was insufficient evidence of the efficacy of the tested interventions in trials, and more robust research with adequate sample sizes was required42,43. Since then, more recent systematic reviews until 2019 have attempted to summarise the evidence of effectiveness of modafinil, mindfulness training, a traditional Chinese medicine, and smart technologies, but still came toa similar conclusion tothat of the Cochrane review in 201544–47.\n\nAs a result of such uncertainty, current clinical practice guidelines rely on low levels of evidence, such as expert consensus, to make recommendations for PSF48,49. However, the efforts to design and test treatments continue, which makes it necessary to keep up-to-date with new research and practice literature.\n\nThe objective of this review was to identify and summarise the most recent research literature related to PSF in order to update the evidence base. As there was an existing review covering the literature up until 201650, we only updated the literature not covered in this review.\n\n\nMethods\n\nIn 2017, Hinkle et al.50 published a review covering emerging evidence relating to the management of PSF, up to and including February 2016. Because of the comprehensiveness of this review, we only searched for literature published after 1st March 2016. As the search methods of the Hinkle et al. review were not reproducible, and the search strategies and results were not available, we contacted the corresponding author and their librarian on 15th October 2019. Since we did not receive a reply, we designed the search methods for the reported databases in order to capture the majority of the literature included in Hinkle et al.’s review.\n\nWe followed Arksey and O’Malley framework51 for conducting this scoping review. We also used Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Extension for Scoping Reviews(PRISMA-ScR) for reporting52. The relevant PRSIMA-ScR checklist is available as Extended data and the flow diagram is reported in the Results section (Figure 1).\n\nWe ran a search to include literature between 1st March 2016 and 19th January 2020 (search date) in CINAHL via EBSCOhost, MEDLINE via Ovid SP, PubMed (excluding MEDLINE), and PsycINFO via Ovid. There were no limitations to language, document type (e.g., thesis), study completion status (e.g., ongoing), and publication status (e.g., unpublished) at the search stage. We report the search strategies for all databases in Extended data.\n\nWe imported the search results into EndNote X6 and de-duplicated them based on title, and additionally double-checked the automatically identified duplicates manually. Two reviewers (GA and FS) screened the results independently against the eligibility criteria using Rayyan, which is a recommended screening system53. Discrepancies were resolved through discussions or asking a third reviewer (AD).\n\nTwo reviewers (GA and FS) also investigated the full texts of relevant search results against the same criteria involving a third reviewer (AD) in case of disagreement. At full text screening stage, we also investigated the reference lists of the relevant studies to identify additional relevant studies. Since one study may have multiple reports or publications, we kept a record and cited all the reports of a single study to provide a better overview of the new research evidence.\n\nWe included the following studies:\n\n- Studies of adult humans with PSF – any definition of PSF – at any stage of the stroke care continuum;\n\n- Any interventional (clinical trial) or observational (cohort, case-control, and cross-sectional) studies, and clinical practice guidelines;\n\n- Studies reporting findings that had not been included in the previous review;\n\n- Studies included in relevant systematic reviews.\n\nWe excluded the following studies:\n\n- Studies with case reports, case series, and qualitative design;\n\n- Studies included in Hinkle et al. or results which repeated the summarised knowledge in that review;\n\n- Studies of pre-clinical nature;\n\n- Clinical studies where fatigue was reported only as a side effect of the treatment;\n\n- Studies focusing on single muscle fatigue or muscle fatigue in general;\n\n- Studies not focusing on fatigue and/or stroke or focusing on heat stroke, athletes’ fatigue or carers’ fatigue;\n\n- Systematic or narrative or review papers;\n\n- Ongoing studies or protocols with no results (listed and cited in this paper for further follow-up);\n\n- Tool validation studies without reporting new findings on PSF.\n\nOne reviewer (GA) extracted and entered the data in Excel 2007 and the second reviewer (FS) checked the extracted and entered data against the full text and, if appropriate, corrected or amended the data.\n\nFor interventional studies, we extracted PICOS (participants, intervention, comparison, outcomes, and study design) and other data points:\n\n- Study name and year;\n\n- Clinical trial registration number (for further check on selective reporting bias);\n\n- Country of origin;\n\n- Number of centres;\n\n- Patients: Number of patients, type of stroke, time passed after stroke;\n\n- Intervention and controls: name of intervention and duration;\n\n- Primary and secondary outcomes measures in general and fatigue measures in particular, outcome endpoints, and main findings related to PSF;\n\n- Study design (single-arm clinical trial (CT), controlled clinical trial (CCT), or RCT);\n\nFor observational studies, we extracted:\n\n- Study name and year;\n\n- Clinical trial registration number (for further check on selective reporting bias);\n\n- Country of origin;\n\n- Number of centres;\n\n- Patients: Number of patients, type of stroke, time passed after stroke;\n\n- Primary and secondary outcomes measures in general and fatigue measures in particular, outcome endpoints, and main findings related to PSF;\n\n- Study design (cohort, case-control, or cross-sectional).\n\nFor clinical practice guidelines, we extracted the following data:\n\n- Study name and year;\n\n- Country and organisation who produced the guideline;\n\n- Recommendations on PSF;\n\n- Evidence base reporting the level of evidence or study designs related to the level of evidence.\n\nWe used RobotReviewer for assessing the risk of bias in the four categories of the Cochrane Risk of Bias tool54 for included RCTs. Although this automation system is reliable for checking the risk of bias for certain bias categories55,56, one of the reviewers (GA) also double-checked and revised RobotReviewer’s assessment and corrected the data where necessary. We also added a ‘selective reporting of outcomes’ category to the list of biases to cover the main biases in Cochrane Risk of Bias tool.\n\nWe summarised the data from the new relevant literature in tables. We did not proceed to a meta-analysis for fatigue outcomes due to the heterogeneity of studies. We checked if any of the interventional studies considered following the CONSORT57 for reporting RCTs or TIDieR checklist58 to report the components of new interventions.\n\n\nResults\n\nThe search identified 1021 results. After screening, we included 45 relevant records related to 24 studies and 6 guidelines (Figure 1).\n\nThe characteristics of included interventional studies have been charted in Table 1. The table shows eight RCTs some with multiple reports and one with a follow-up study59–73, one CCT74, and two single-arm trials75,76. All studies were based on single centre studies, except for West et al. (2019) which had two centres71. In studies that reported the intervention delivery details, the psychological interventions were delivered individually and face-to-face – rather than online – bypsychologists. We also assessed the risk of bias for RCTs and reported the categories of risk in Table 2 with supporting statements in Extended data.\n\n* Psychologists with doctoral qualifications in clinical neuropsychology.\n\nRCT: Randomised Controlled Trial; CCT: CONTROLLED CLINICAL TRIAL; CT: Clinical Trial; NR: Not Reported; NA: Not Applicable; CHF: Congestive Heart Failure; TIA: Transient Ischaemic Attack; D: Day; W: Week; M: Month; S: Session\n\nQuestion marks in red cells indicate unclear or high risk of bias and plus signs in green cells show low risk of bias.\n\nMost of the interventional studies have a medium to high risk of bias. Table 2 shows only two studies in green (indicating low risk of bias) but both have small sample size consisting of 34 (MIDAS study63–68) and 64 randomised patients61 respectively.\n\nWe identified 14 observational studies of which half had a prospective cohort design77–90 and the other half were cross-sectional surveys91–97. Three cross-sectional surveys were embedded within cohort studies91,94,97. Only one of the studies (NotFAST) had a follow-up report81–85. Details of all studies are reported in Table 3 as well as the Extended data.\n\nNR: Not Reported; Y: Year; M: Month; W: Week; D: Day\n\nFor cohort studies, the left number shows the number of participants who finished follow-up, and the right number is the number of participants who started and took part in the study; for cross-sectional studies within cohort studies, the left number shows the number of participants in cross-sectional study and the right number is the number of participants in cohort study.\n\nTable 4 summarises the main finding of each interventional study all of which either have high risk of bias or small sample size. Such limitations make it hard to transfer the research findings to practice.\n\nFAS: Fatigue Assessment Scale; VAS: Visual Analogue Scale; BFI: Brief Fatigue Index; MFI: Multidimensional Fatigue Inventory; FSS: Fatigue Severity Scale; FACIT: Functional Assessment of Chronic Illness Therapy; W: Weeks; D: Day; M: Month; S: Session; TAU: Treatment As Usual; CBT: Cognitive Behavioural Therapy.\n\n Grey cells contain findings from low risk studies;however they have small sample size.\n\nThe majority of observational studies investigated factors related to PSF including co-morbidities, physical and mental outcomes, illness characteristics, characteristics of interventions, and biomarkers (Table 5).\n\nFSS: Fatigue Severity Scale; FIS: Fatigue Impact Scale; FACIT: Functional Assessment of Chronic Illness Therapy; FAS: Fatigue Assessment Scale; FAI: Fatigue Assessment Inventory; PSD: Post-Stroke Depression; CFS: Chalder Fatigue Scale; CLCE: Checklist for Cognitive and Emotional consequences following stroke\n\nWe identified six recent guidelines from three English-speaking countries including the UK49 and two North American countries (one from Canada48 and four from the USA98–101). Among these, the Canadian guideline was the most recent and the only one with comprehensive recommendations on PSF. The UK guideline will be updated in 2021. Half of these guidelines, that is, all those from USA, have not provided specific recommendations on PSF, as reported in Table 6. In almost all the guidelines, the reliance on ‘experts’ consensus’ is apparent because of the limited evidence base for PSF (Table 6).\n\nAHA: American Heart Association; ASA: American Stroke Association; VA/DoD: Department of Veterans Affairs/Department of Defense; NICE: National Institute for Health and Care Excellence; CSBPR: Canadian Stroke Best Practice Recommendations by Management of Mood, Cognition and Fatigue Following Stroke Best Practice Writing Group/Heart & Stroke Canadian Stroke Best Practices and Quality Advisory Committee/Canadian Stroke Consortium; NR: Not Reported; RCT: Randomised Controlled Trial; CCT: Controlled Clinical Trial; CT: Clinical Trial; SR: Systematic Review.\n\n\nDiscussion\n\nWe conducted this review to identify and summarise the most recent research studies on PSF since Hinkle et al.’s review (2017)50. We therefore documented the interventional and observational research and clinical practice guidelines since March 2016. However, there were some key contributors to the weak evidence base: (i) recording and reporting only some contributing factors to PSF in observational studies, (ii) the heterogeneity of designed interventions, (iii) high risk of bias, (iv) small sample size in interventional studies, and (v) variety of outcome measures in both observational and clinical studies. This, in turn, is reflected in the quality of the clinical recommendations for PSF.\n\nDespite the high prevalence of PSF2 and its obvious effects on treatment adherence102, in practice, only half of recent stroke guidelines have clinical recommendations on PSF. Of those that do, two guidelines provide only brief recommendations, and only one provides comprehensive recommendations, but these are based on low levels of evidence48. The weak evidence base and the need to rely on expert consensus is likely to be the main reason that PSF is generally not covered in the guidelines.\n\nThe dominance of single-centred interventional studies with small sample sizes and interventions delivered within a 12-week period may be the reasons for absence of follow-up studies. MIDAS (interventional)63–65 and NotFAST (observational)81–85 are the only recent studies with novel and potentially long-term findings with larger sample size (in case of MIDAS 2)103 or with the intention to design an intervention (NotFAST2)104.\n\nWhile the observational studies reported the type of stroke, the interventional studies did not include this important data, which makes it difficult to summarise studies. Most of participants entered the interventional studies three-months after stroke. This is likely to be due to a number of reasons; for example, fatigue is not recognised immediately after a stroke, some studies want to ensure that participants have a stabile fatigue, and there is competition for recruitment in the early stages to more acute trials.\n\nThe variety of the interventions tested in studies and trials underlines the complexity of PSF and is an indication to researchers that probably the most effective interventions need to target multiple aspects of fatigue. While current reporting practice of interventions in RCTs included in our review is of concern (none followed TIDieR and two followed CONSORT), future studies should consider following reporting guidelines such as CONSORT and TIDieR for interventional studies, STROBE for observational studies, and RIGHT105, AGREE106, or CheckUP107 for clinical practice guidelines.\n\nAmong the observational studies, the populations-based study from the stroke register in New Zealand91 and Sweden77 provides valuable insights about the link between co-morbidities and increased PSF in long-term (4–7 years). This, and other similar register-based studies, represent the added value of having high-quality data in health system databases for long-term observational and register-based studies108.\n\nPsychologists delivered the psychotherapies in RCTs to individual patients and there was no intervention using online platforms as the media of delivery. This may be due to a number of reasons: it is usual to test the efficacy of an intervention face to face before moving to another medium; participants with stroke may have other problems which mean it is more difficult to deliver treatments online, e.g. communication issue and cognitive problems.\n\nFatigue Severity Scale (FSS) was the main outcome measure for PSF in observational studies, whereas Fatigue Assessment Scale (FAS)was used more frequently than other measures in interventional studies. The main reason that the FSS has been used is probably because it is now seen as a way to compare different studies: in simple terms, researchers use it because other researchers have used it. It is also relatively straight forward to complete.\n\nOnly one of observational studies and half of the interventional studies were registered in clinical trial registers, with the remaining unregistered trials potentially introducing bias in selective reporting of outcomes109,110. One of the interventional studies was registered retrospectively with potential for the same bias69,70.\n\nIt is possible that we overlooked studies which did not report PSF in the searchable part of the paper or if the report was not indexed in the searched databases. In such cases, we invite the audience of this review paper to contact us or comment on the paper online.\n\nThe current trend of research on PSF shows the continued importance of this topic globally. Our review identified a weak evidence base that highlights the need for more research that could have the following characteristics: I) studies to design and test multi-component interventions for PSF; and II) Robust RCTs with adequate sample sizes to produce the evidence for recommendations in guidelines. From our current knowledge on PSF, none of the recent studies are robust enough to change current clinical practice.\n\n\nData availability\n\nOpen Science Framework: Post-Stroke Fatigue: A Scoping Review, https://doi.org/10.17605/OSF.IO/XJKCS111.\n\nRegistration DOI: https://doi.org/10.17605/OSF.IO/XJKCS\n\nThis project contains the following underlying data:\n\n- Extracted data from included studies\n\nOpen Science Framework: Post-Stroke Fatigue: A Scoping Review, https://doi.org/10.17605/OSF.IO/XJKCS111.\n\nThis project contains the following extended data:\n\n- Full search strategies\n\n- Risk of bias assessment\n\nUnedited draft of risk of bias reported by RobotReviewer: https://robotreviewer.vortext.systems/#report/_gk5JV8oLUAd9lhmsx71G\n\nOpen Science Framework: PRISMA-ScR checklist for ‘Post-stroke fatigue: a scoping review’, https://doi.org/10.17605/OSF.IO/XJKCS111.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nDe Groot MH, Phillips SJ, Eskes GA: Fatigue associated with stroke and other neurologic conditions: Implications for stroke rehabilitation. Arch Phys Med Rehabil. 2003; 84(11): 1714–20. PubMed Abstract \| Publisher Full Text\n\nCumming TB, Packer M, Kramer SF, et al.: The prevalence of fatigue after stroke: A systematic review and meta-analysis. Int J Stroke. 2016; 11(9): 968–77. PubMed Abstract \| Publisher Full Text\n\nACTRN12619000826145: Too tired to recover: evaluation of a post-stroke fatigue management guideline. 2019. Reference Source\n\nLerdal A, Gay CL: Acute-Phase Fatigue Predicts Limitations with Activities of Daily Living 18 Months after First-Ever Stroke. J Stroke Cerebrovasc Dis. 2017; 26(3): 523–31. PubMed Abstract \| Publisher Full Text\n\nRamírez-Moreno JM, Munoz-Vega P, Alberca SB, et al.: Health-Related Quality of Life and Fatigue After Transient Ischemic Attack and Minor Stroke. J Stroke Cerebrovasc Dis. 2019; 28(2): 276–84. PubMed Abstract \| Publisher Full Text\n\nToy G, Singer J, Balucani C, et al.: Post Stroke Fatigue and Depression Predict Lower Health Related Quality of Life in Stroke Patients. Stroke (00392499). 2017; 48: ATP153–ATP. Reference Source\n\nBroussy S, Saillour-Glenisson F, Garcia-Lorenzo B, et al.: Sequelae and Quality of Life in Patients Living at Home 1 Year After a Stroke Managed in Stroke Units. Front Neurol. 2019; 10: 907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMandliya A, Das A, Unnikrishnan JP, et al.: Post-stroke Fatigue is an Independent Predictor of Post-stroke Disability and Burden of Care: A Path analysis Study. Top Stroke Rehabil. 2016; 23(1): 1–7. PubMed Abstract \| Publisher Full Text\n\nDe Doncker W, Dantzer R, Ormstad H, et al.: Mechanisms of poststroke fatigue. J Neurol Neurosurg Psychiatry. 2018; 89(3): 287–93. PubMed Abstract \| Publisher Full Text\n\nPaciaroni M, Acciarresi M: Poststroke Fatigue. Stroke. 2019; 50(7): 1927–33. PubMed Abstract \| Publisher Full Text\n\nAarnes R, Stubberud J, Lerdal A: A literature review of factors associated with fatigue after stroke and a proposal for a framework for clinical utility. Neuropsychol Rehabil. 2019; 1–28. PubMed Abstract \| Publisher Full Text\n\nCumming TB, Yeo AB, Marquez J, et al.: Investigating post-stroke fatigue: An individual participant data meta-analysis. J Psychosom Res. 2018; 113: 107–12. PubMed Abstract \| Publisher Full Text\n\nDelva II, Delva MY, Poltorapavlov VA: Clinical factors related to post-stroke fatigue within the first 3 month after acute cerebrovascular events. Wiad Lek. 2017; 70(3 pt 2): 581–5. PubMed Abstract\n\nDelva II, Lytvynenko NV, Delva MY: Post-stroke fatigue and its dimensions within first 3 months after stroke. Wiad Lek. 2017; 70(1): 43–6. PubMed Abstract\n\nDelva I: Factors associated with post-stroke fatigue dimensions over the second year after acute cerebrovascular events. Bulletin of Problems Biology and Medicine. 2018; 2(147): 139–42. Publisher Full Text\n\nDelva I, Lytvynenko N, Delva M: Factors Associated with Post-Stroke Fatigue within the First 3 Month after Stroke. Georgian Med News. 2017; 6(267): 38–42. PubMed Abstract\n\nDelva M, Lytvynenko N, Delva I: Factors Associated with Post-Stroke Fatigue during the Second Half Year after Stroke. Georgian Med News. 2017; 11(272): 59–64. PubMed Abstract\n\nDelva M, Lytvynenko N, Delva I: Factors Associated with the Time-Based Phenomenology of Post-Stroke Fatigue over the First Year after Stroke Occurrence. Georgian Med News. 2018; 6(279): 92–7. PubMed Abstract\n\nDelva MY, Delva II, Lytvynenko NV: Post-stroke fatigue and its dimensions over the second half year after stroke. Wiad Lek. 2018; 71(2 pt 2): 314–7. PubMed Abstract\n\nGalligan NG, Hevey D, Coen RF, et al.: Clarifying the associations between anxiety, depression and fatigue following stroke. J Health Psychol. 2016; 21(12): 2863–71. PubMed Abstract \| Publisher Full Text\n\nGoh HT, Stewart JC: Poststroke Fatigue Is Related to Motor and Cognitive Performance: A Secondary Analysis. J Neurol Phys Ther. 2019; 43(4): 233–9. PubMed Abstract \| Publisher Full Text\n\nGraber M, Garnier L, Duloquin G, et al.: Association Between Fatigue and Cognitive Impairment at 6 Months in Patients With Ischemic Stroke Treated With Acute Revascularization Therapy. Front Neurol. 2019; 10: 931. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolmqvist A, Berthold Lindstedt M, Möller MC: Relationship between fatigue after acquired brain injury and depression, injury localization and aetiology: an explorative study in a rehabilitation setting. J Rehabil Med. 2018; 50(8): 725–31. PubMed Abstract \| Publisher Full Text\n\nKhan A, Delargy M: Post Stroke Fatigue; Point Prevalence, Characterization, Associations and Radiological Correlation in a Rehabilitation Hospital. Ir Med J. 2017; 110(10): 650. PubMed Abstract\n\nLagogianni C, Thomas S, Lincoln N: Examining the relationship between fatigue and cognition after stroke: A systematic review. Neuropsychol Rehabil. 2018; 28(1): 57–116. PubMed Abstract \| Publisher Full Text\n\nLiu X: Fatigue and depression in Chinese stroke patients: A 1-year longitudinal study. Dissertation Abstracts International: Section B: The Sciences and Engineering. 2018; 79(1).\n\nMurphy S, Durand M, Negro F, et al.: The Relationship Between Blood Flow and Motor Unit Firing Rates in Response to Fatiguing Exercise Post-stroke. Front Physiol. 2019; 10: 545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutai H, Furukawa T, Houri A, et al.: Factors associated with multidimensional aspect of post-stroke fatigue in acute stroke period. Asian J Psychiatr. 2017; 26: 1–5. PubMed Abstract \| Publisher Full Text\n\nSarfo FS, Berchie P, Singh A, et al.: Prevalence, Trajectory, and Predictors of Poststroke Fatigue among Ghanaians. J Stroke Cerebrovasc Dis. 2019; 28(5): 1353–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchow T, Teasdale TW, Quas KJ, et al.: Problems with balance and binocular visual dysfunction are associated with post-stroke fatigue. Top Stroke Rehabil. 2017; 24(1): 41–9. PubMed Abstract \| Publisher Full Text\n\nShepherd AI, Pulsford R, Poltawski L, et al.: Physical activity, sleep, and fatigue in community dwelling Stroke Survivors. Sci Rep. 2018; 8(1): 7900. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSyla D, Sweenie A, Lendrem D, et al.: A scoping exercise to gauge the incidence of early post-stroke fatigue for patients at Newcastle community stroke services. Int J Ther Rehabil. 2017; 24(2): 52. Publisher Full Text\n\nAlahmari W, Alhowimel A, Alotaibi M, et al.: Effectiveness of physiotherapy interventions for post stroke fatigue (PSF): a systematic review. Physiotherapy. 2019; 105(Suppl 1): e172. Publisher Full Text\n\nGbiri CAO, Olawale OA, Ogunmola OT: Task-oriented circuit training for post-stroke fatigue, sleeppattern, functional performance and societal re-integration in stroke survivors with post-stroke fatigue: a clinical controlled study. International journal of stroke Conference: 11th world stroke congress, WSC 2018 Canada. 2018; 13(2 Supplement 1): 86. Publisher Full Text\n\nDurand MJ, Boerger TF, Nguyen JN, et al.: Two weeks of ischemic conditioning improves walking speed and reduces neuromuscular fatigability in chronic stroke survivors. J Appl Physiol (1985). 2019; 126(3): 755–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurand MJ, Murphy SA, Schmit BD, et al.: Two Weeks of Ischemic Preconditioning Training on the Paretic Leg Improves Leg Strength and Delays Muscle Fatigue in Chronic Stroke. Stroke. 2017; 48: ATP150–ATP. Reference Source\n\nShahar N, Schwartz I, Portnoy S: Differences in muscle activity and fatigue of the upper limb between Task-Specific training and robot assisted training among individuals post stroke. J Biomech. 2019; 89: 28–33. PubMed Abstract \| Publisher Full Text\n\nVecchio M, Gracies JM, Panza F, et al.: Change in Coefficient of Fatigability Following Rapid, Repetitive Movement Training in Post-Stroke Spastic Paresis: a Prospective Open-Label Observational Study. J Stroke Cerebrovasc Dis. 2017; 26(11): 2536–40. PubMed Abstract \| Publisher Full Text\n\nda Silva BP, Souza GADS, Filho AADN, et al.: Analysis of the effects of low-level laser therapy on muscle fatigue of the biceps brachii muscle of healthy individuals and spastic individuals: Study protocol for a single-center, randomized, double-blind, and controlled clinical trial. Medicine (Baltimore). 2019; 98(39): e17166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ndas Neves M, Reis M, Andrade E, et al.: Effects of low-level laser therapy (LLLT 808 nm) on lower limb spastic muscle activity in chronic stroke patients. Lasers Med Sci. 2016; 31(7): 1293–300. PubMed Abstract \| Publisher Full Text\n\ndas Neves MF, Aleixo DC, Mendes IS, et al.: Long-term analyses of spastic muscle behavior in chronic poststroke patients after near-infrared low-level laser therapy (808 nm): a double-blinded placebo-controlled clinical trial. Lasers Med Sci. 2019. PubMed Abstract \| Publisher Full Text\n\nKennedy C, Kidd L: Interventions for post-stroke fatigue: A Cochrane review summary. Int J Nurs Stud. 2018; 85: 136–137. PubMed Abstract \| Publisher Full Text\n\nWu S, Kutlubaev MA, Chun HY, et al.: Interventions for post-stroke fatigue. Cochrane Database Syst Rev. 2015; (7): CD007030. PubMed Abstract \| Publisher Full Text\n\nXu L, Xu XY, Hou XQ, et al.: Adjuvant therapy with Astragalus membranaceus for post-stroke fatigue: a systematic review. Metab Brain Dis. 2020; 35(1): 83–93. PubMed Abstract \| Publisher Full Text\n\nKettlewell J, das Nair R, Radford K: A systematic review of personal smart technologies used to improve outcomes in adults with acquired brain injuries. Clin Rehabil. 2019; 33(11): 1705–1712. PubMed Abstract \| Publisher Full Text\n\nUlrichsen KM, Kaufmann T, Dorum ES, et al.: Clinical Utility of Mindfulness Training in the Treatment of Fatigue After Stroke, Traumatic Brain Injury and Multiple Sclerosis: A Systematic Literature Review and Meta-analysis. Front Psychol. 2016; 7: 912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPacheco RL, Latorraca COC, da Silva LDGM, et al.: Modafinil for poststroke patients: A systematic review. Int J Clin Pract. 2019; 73(2): e13295. PubMed Abstract \| Publisher Full Text\n\nLanctot KL, Lindsay MP, Smith EE, et al.: Canadian Stroke Best Practice Recommendations: Mood, Cognition and Fatigue following Stroke, 6th edition update 2019. Int J Stroke. 2019; InPress:InPress. PubMed Abstract \| Publisher Full Text\n\nNational Institute for Health and Care Excellence. Stroke and TIA. 2017. Reference Source\n\nHinkle JL, Becker KJ, Kim JS, et al.: Poststroke Fatigue: Emerging Evidence and Approaches to Management: A Scientific Statement for Healthcare Professionals From the American Heart Association. Stroke. 2017; 48(7): e159–e170. PubMed Abstract \| Publisher Full Text\n\nArksey H, O'Malley L: Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018; 169(7): 467–73. PubMed Abstract \| Publisher Full Text\n\nHarrison H, Griffin SJ, Kuhn I, et al.: Software tools to support title and abstract screening for systematic reviews in healthcare: an evaluation. BMC Med Res Methodol. 2020; 20(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Altman DG: Assessing Risk of Bias in Included Studies.2008; 187–241. Publisher Full Text\n\nMarshall IJ, Kuiper J, Wallace BC: RobotReviewer: evaluation of a system for automatically assessing bias in clinical trials. J Am Med Inform Assoc. 2016; 23(1): 193–201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGates A, Vandermeer B, Hartling L: Technology-assisted risk of bias assessment in systematic reviews: a prospective cross-sectional evaluation of the RobotReviewer machine learning tool. J Clin Epidemiol. 2018; 96: 54–62. PubMed Abstract \| Publisher Full Text\n\nSchulz KF, Altman DG, Moher D, et al.: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann TC, Glasziou PP, Boutron I, et al.: Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014; 348: g1687. PubMed Abstract \| Publisher Full Text\n\nChen PC, Liaw MY, Wang LY, et al.: Inspiratory muscle training in stroke patients with congestive heart failure: A CONSORT-compliant prospective randomized single-blind controlled trial. Medicine. 2016; 95(37): e4856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CH, Hung KS, Chung YC, et al.: Mind-body interactive qigong improves physical and mental aspects of quality of life in inpatients with stroke: A randomized control study. Eur J Cardiovasc Nurs. 2019; 18(8): 658–66. PubMed Abstract \| Publisher Full Text\n\nLiu CH, Tsai CH, Li TC, et al.: Effects of the traditional Chinese herb Astragalus membranaceus in patients with poststroke fatigue: A double-blind, randomized, controlled preliminary study. J Ethnopharmacol. 2016; 194: 954–962. PubMed Abstract \| Publisher Full Text\n\nLiu F, Li Y, Xu Y: The intervention effect of wuling capsule on inflammatory reaction inpatients with post-stroke fatigue. Biomedical Research (India). 2018; 29(6): 1070–2. Publisher Full Text\n\nBivard A, Lillicrap PT, Krishnamurthy V, et al.: MIDAS (Modafinil in Debilitating Fatigue After Stroke): A Randomized, Double-Blind, Placebo-Controlled, Cross-Over Trial. Stroke. 2017; 48(5): 1293–1298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBivard A, Lillicrap T, Krishnamurthy V, et al.: Modafinil In Debilitating fatigue After Stroke (MIDAS): a randomized, doubleblind, placebo-controlled, cross-over trial. Eur Stroke J. 2017; 2(Suppl 1): 31. Publisher Full Text\n\nLillicrap T, Krishnamurthy V, Attia J, et al.: Modafinil In Debilitating fatigue After Stroke (MIDAS): study protocol for a randomised, double-blinded, placebo-controlled, crossover trial. Trials. 2016; 17(1): 410. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLillicrap TP, Levi CR, Holliday E, et al.: Short- and Long-Term Efficacy of Modafinil at Improving Quality of Life in Stroke Survivors: A Post Hoc Sub Study of the Modafinil in Debilitating Fatigue After Stroke Trial. Front Neurol. 2018; 9: 269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVisser MM, Goodin P, Parsons MW, et al.: Modafinil treatment modulates functional connectivity in stroke survivors with severe fatigue. Sci Rep. 2019; 9(1): 9660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVisser MM, Maréchal B, Goodin P, et al.: Predicting Modafinil-Treatment Response in Poststroke Fatigue Using Brain Morphometry and Functional Connectivity. Stroke. 2019; 50(3): 602–609. PubMed Abstract \| Publisher Full Text\n\nACTRN12616001211459: Effectiveness of cognitive behavior therapy for post-stroke fatigue and sleep disturbance: a pilot randomised controlled trial. 2016. Reference Source\n\nNguyen S, Wong D, McKay A, et al.: Cognitive behavioural therapy for post-stroke fatigue and sleep disturbance: a pilot randomised controlled trial with blind assessment. Neuropsychol Rehabil. 2019; 29(5): 723–738. PubMed Abstract \| Publisher Full Text\n\nWest A, Simonsen SA, Jennum P, et al.: An exploratory investigation of the effect of naturalistic light on fatigue and subjective sleep quality in stroke patients admitted for rehabilitation: A randomized controlled trial. NeuroRehabilitation. 2019; 45(2): 187–200. PubMed Abstract \| Publisher Full Text\n\nWest A, Simonsen SA, Jennum PJ, et al.: The effects of circadian light on fatigue and subjective sleep quality in stroke patients admitted for rehabilitation. European Stroke Journal. 2017; 2(Suppl 1): 175.\n\nWest A, Simonsen SA, Sennels H, et al.: The effect of naturalistic light on depressive mood, fatigue, subjective sleep quality and melatonin and cortisol blood levels in stroke patients admitted for rehabilitation. Neuropsychobiology. 2016; 74(4): 255.\n\nDelva II: Effectiveness of acetylsalicylic acid in correction of post-stroke fatigue during acute cerebrovascular events. Medical and Ecological Problems. 2019; 23(1–2): 8–12. Publisher Full Text\n\nVan Heest KNL, Mogush AR, Mathiowetz VG: Effects of a One-to-One Fatigue Management Course for People With Chronic Conditions and Fatigue. Am J Occup Ther. 2017; 71(4): 7104100020p1–7104100020p9. PubMed Abstract \| Publisher Full Text\n\nWu S, Chalder T, Anderson KE, et al.: Development of a psychological intervention for fatigue after stroke. PLoS One. 2017; 12(8): e0183286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlomgren C, Samuelsson H, Blomstrand C, et al.: Long-term performance of instrumental activities of daily living in young and middle-aged stroke survivors-Impact of cognitive dysfunction, emotional problems and fatigue. PLoS One. 2019; 14(5): e0216822. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen K, Marsh EB: Chronic post-stroke fatigue: It may no longer be about the stroke itself. Clin Neurol Neurosurg. 2018; 174: 192–197. PubMed Abstract \| Publisher Full Text\n\nChoi-Kwon S, Ko M, Jun SE, et al.: Post-Stroke Fatigue May Be Associated with the Promoter Region of a Monoamine Oxidase A Gene Polymorphism. Cerebrovasc Dis. 2017; 43(1–2): 54–58. PubMed Abstract \| Publisher Full Text\n\nDouven E, Köhler S, Schievink SHJ, et al.: Temporal Associations between Fatigue, Depression, and Apathy after Stroke: Results of the Cognition and Affect after Stroke, a Prospective Evaluation of Risks Study. Cerebrovasc Dis. 2017; 44(5–6): 330–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrummond A, Hawkins L, Sprigg N, et al.: The Nottingham Fatigue after Stroke (NotFAST) study: Factors Associated With Severity of Fatigue in Stroke Patients Without Depression. Clin Rehabil. 2017; 31(10): 1406–15. PubMed Abstract \| Publisher Full Text\n\nHawkins L, Drummond A: Post stroke fatigue: a priority for stroke nursing? Br J Neurosci Nurs. 2017; 13(Sup2): S6. Publisher Full Text\n\nHawkins L, Drummond A, Birks E, et al.: The Nottingham Fatigue After Stroke study The frequency of fatigue and associated factors in stroke patients without depression...The 35th Scientific Meeting of the Physiotherapy Research Society 16 April 16, versity of Leicester, UK. International Journal of Therapy & Rehabilitation. 2016; 23(6): S270–1.\n\nHawkins L, Lincoln NB, Sprigg N, et al.: The Nottingham Fatigue After Stroke (NotFAST) Study: Results From Follow-Up Six Months After Stroke. Top Stroke Rehabil. 2017; 24(8): 592–6. PubMed Abstract \| Publisher Full Text\n\nWorthington E, Hawkins L, Lincoln N, et al.: The day-to-day experiences of people with fatigue after stroke: Results from the Nottingham Fatigue After Stroke study. Int J Therapy & Rehabili. 2017; 24(10): 449–55. Publisher Full Text\n\nPonchel A, Labreuche J, Bombois S, et al.: Influence of Medication on Fatigue Six Months after Stroke. Stroke Res Treat. 2016; 2016: 2410921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBunevicius A: Reader Response: Depressed TSH Level as a Predictor of Poststroke Fatigue in Patients With Acute Ischemic Stroke. Neurology. 2019; 93(12): 563–4. PubMed Abstract \| Publisher Full Text\n\nGanesh A, Galetta S: Editors' note: Depressed TSH level as a predictor of poststroke fatigue in patients with acute ischemic stroke. Neurology. 2019; 93(12): 563. Publisher Full Text\n\nWang J, Gu M, Sun W, et al.: Author response: Depressed TSH level as a predictor of poststroke fatigue in patients with acute ischemic stroke. Neurology. 2020. Reference Source\n\nWang J, Li F, Xiao L, et al.: Depressed TSH level as a predictor of poststroke fatigue in patients with acute ischemic stroke. Neurology. 2018; 91(21): e1971–8. Publisher Full Text\n\nMahon S, Theadom A, Barker-Collo S, et al.: The Contribution of Vascular Risk Factors in Prevalence of Fatigue Four Years Following Stroke: Results from a Population-Based Study. J Stroke Cerebrovasc Dis. 2018; 27(8): 2192–9. PubMed Abstract \| Publisher Full Text\n\nChoi-Kwon S, Choi SH, Suh M, et al.: Musculoskeletal and central pain at 1 year post-stroke: associated factors and impact on quality of life. Acta Neurol Scand. 2017; 135(4): 419–25. PubMed Abstract \| Publisher Full Text\n\nKuppuswamy A, Clark E, Rothwell J, et al.: Limb Heaviness: A Perceptual Phenomenon Associated With Poststroke Fatigue? Neurorehabil Neural Repair. 2016; 30(4): 360–2. PubMed Abstract \| Publisher Full Text\n\nElf M, Eriksson G, Johansson S, et al.: Self-Reported Fatigue and Associated Factors Six Years after Stroke. PLoS One. 2016; 11(8): e0161942. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLau CG, Tang WK, Liu XX, et al.: Neuroticism and Fatigue 3 Months After Ischemic Stroke: A Cross-Sectional Study. Arch Phys Med Rehabil. 2017; 98(4): 716–21. PubMed Abstract \| Publisher Full Text\n\nMacIntosh BJ, Edwards JD, Kang M, et al.: Post-stroke Fatigue and Depressive Symptoms Are Differentially Related to Mobility and Cognitive Performance. Front Aging Neurosci. 2017; 9: 343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Rijsbergen MWA, Mark RE, Kop WJ, et al.: Psychological factors and subjective cognitive complaints after stroke: Beyond depression and anxiety. Neuropsychol Rehabil. 2019; 29(10): 1671–84. PubMed Abstract \| Publisher Full Text\n\nBraun LT, Grady KL, Kutner JS, et al.: Palliative Care and Cardiovascular Disease and Stroke: A Policy Statement From the American Heart Association/American Stroke Association. Circulation. 2016; 134(11): e198–225. PubMed Abstract \| Publisher Full Text\n\nPeberdy MA, Gluck JA, Ornato JP, et al.: Cardiopulmonary Resuscitation in Adults and Children With Mechanical Circulatory Support: A Scientific Statement From the American Heart Association. Circulation. 2017; 135(24): e1115–34. PubMed Abstract \| Publisher Full Text\n\nManagement of Stroke Rehabilitation Work Group, Office of Quality Safety and Value, Office of Evidence Based Practice: VA/DoD Clinical Practice Guideline for the Management of Stroke Rehabilitation: Department of Veterans Affairs and Department of Defense. 2019. Reference Source\n\nWinstein CJ, Stein J, Arena R, et al.: Guidelines for Adult Stroke Rehabilitation and Recovery: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2016; 47(6): e98–e169. PubMed Abstract \| Publisher Full Text\n\nZhang J, Gong Y, Zhao Y, et al.: Post-stroke medication adherence and persistence rates: a meta-analysis of observational studies. J Neurol. 2019. PubMed Abstract \| Publisher Full Text\n\nACTRN12618000602224: Modafinil In Debilitating Fatigue After Stroke 2 (MIDAS 2). 2018. Reference Source\n\nDrummond A: NotFAST2 - Nottingham Fatigue study: developing a fatigue intervention. Stroke Association and University of Nottingham. 2019. Reference Source\n\nChen Y, Yang K, Marušic A, et al.: A Reporting Tool for Practice Guidelines in Health Care: The RIGHT Statement. Ann Intern Med. 2017; 166(2): 128–32. PubMed Abstract \| Publisher Full Text\n\nBrouwers MC, Kerkvliet K, Spithoff K, et al.: The AGREE Reporting Checklist: a tool to improve reporting of clinical practice guidelines. BMJ. 2016; 352: i1152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVernooij RW, Alonso-Coello P, Brouwers M, et al.: Reporting Items for Updated Clinical Guidelines: Checklist for the Reporting of Updated Guidelines (CheckUp). PLoS Med. 2017; 14(1): e1002207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbildstrom SZ, Torp-Pedersen C, Madsen M: Register-based studies of cardiovascular disease. Scand J Public Health. 2011; 39(7 Suppl): 165–9. PubMed Abstract \| Publisher Full Text\n\nJones CW, Keil LG, Holland WC, et al.: Comparison of registered and published outcomes in randomized controlled trials: a systematic review. BMC Med. 2015; 13: 282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathieu S, Boutron I, Moher D, et al.: Comparison of registered and published primary outcomes in randomized controlled trials. JAMA. 2009; 302(9): 977–84. PubMed Abstract \| Publisher Full Text\n\nShokraneh F, Aali G: Post-Stroke Fatigue: A Scoping Review. 2020. http://www.doi.org/10.17605/OSF.IO/XJKCS" }	[ { "id": "62111", "date": "22 Apr 2020", "name": "Carina U. Persson", "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 found this scoping review very exciting to read since it addresses a very important issue that needs to be managed daily in post-stroke rehabilitation. I believe that the interest in post-stroke fatigue is great from both researchers and clinically active colleagues. I am positive to and welcome this type of “game-changer” by a transparent scientific publishing.\nThe title identifies the manuscript as a scoping review and reflects the population (individuals post-stroke) and the construct (post-stroke fatigue). The abstract is presented with a clear structure, including background, methods, results and conclusion, of which all parts relate to the aim of the study. The rational for the review is described; there is insufficient evidence of the efficacy of the interventions related to post-stroke fatigue in conducted trials and more robust research with adequate sample sizes is required. In addition, it was time to make an update. The objective is well described, to identify and summarise the most recent research related to post-stroke fatigue in order to update the evidence base. The update covers new research studies between 1st March 2016 and 19th January 2020. The search strategies for the databases are reported in a separate link (Extended data). In the results, data related to the objective of the study is presented with a clear structure in six tables. These tables include characteristics of, and summary of the findings from, the included interventional and observational studies, risk of bias and a summary of included guidelines. Due to the heterogeneity of studies, no meta-analysis for fatigue outcomes was performed. The interventional studies were limited by either having high risk of bias or small sample size. Consequently, transferring the research findings to practice was hard.\n\nThe current manuscript is well written and easy to follow. It is clear that you have put in a lot of effort and made a great job. Even so, I have following minor comments/suggestions that could possibly improve your article:\n\nMethod:\n\nAs written, I am uncertain about limitations to language. According to the manuscript, there were no limitations to language. However, in the Extended data, English language seems to be a limitation in the search strategies. Please, clarify so there is no doubt about this.\n\nFigure 1: Please, consider clarifying the number of excluded reports per each specified criterion regarding the 473 excluded reports; no clarification (in numbers) has been made for them as for the 86-excluded full-text reports.\n\nFigure 1: Please, give the readers more information about the nine additional sources. Which was your strategy, how did you found them? Did all arrived from the other articles´ reference lists?\nResults:\nAre MIDAS (presented in Table 1), and ARCOS-IV and LAS-1 (presented in Table 3) examples of studies not yet published?\n\n(In Table 1, the fourth column, there is space available (two rows are already used) for you to write “Number of participants” or “No.” instead of using the symbol “#”.)\n\nTable 2: I like the use of different colours, and red and green are instantaneous to understand. However, in color blindness, the most common difficulty is to distinguish between red and green. Perhaps you can choose another colour combination. In the online version of Robot Reviewer report, I think that the table Risk of bias has a more easy to read layout than Table 2 in the main manuscript.\n\n(Table 3: Consider using “Number” or ”Number of” or “No.” or “No. of” instead of “#” in the third and the fifth column?)\n\nTable 3, sixth column, “Stroke type”: Please, review the use of “/”. Should you use “and”, “,” or delete the “/” somewhere? Regarding Kuppuswamy and NotFAST: “First”, but which stroke type?\n\n(Table 4, fourth column: I suggest you to use “Post-stroke fatigue finding” instead of the use of the abbreviation “PSF finding”. Avoiding unnecessary abbreviations makes reading easier.)\n\n(In Table 5, consider if you should specify “Finding” to “Post-stroke fatigue finding” in the light blue heading (in line with the selected sub-heading in Table 4).)\n\nIn the data commentary related to Table 6, you state that the Canadian guideline was the only one with comprehensive recommendations on post-stroke fatigue. In Table 6, under the heading “Recommendation” the reader is referred to page 15-16 of the guideline. I suggest that you present vital parts of the content in Table 6, in addition to this reference.\n\nI lack information on age and gender of the patients in the different studies from which the review is based. Is it possible to add this information?\n\nIs it possible to give the reader even more specific information on which quantitative measures the post-stroke fatigue findings are based? (Although the level of significance is arbitrary set and statistically significance results does not need to be clinically meaningful for the patients.)\nDiscussion:\nI suggest a sentence about time-dependent concerns regarding the construct (post-stroke fatigue) in the research studies conducted years after stroke and its potential significance for validity.\n\nInternational recommendations regarding outcome measures related to, and time points for measuring, post-stroke fatigue in research studies on recovery after stroke would probably reduce the heterogeneity of studies and facilitate further summarises and updates. You could possibly include that post-stroke fatigue might be an issue for future Stroke Recovery and Rehabilitation Roundtable (SRRR) work.\n\nYou have a separate paragraph regarding limitations. Have you thought about including a sentence related to strengths of your study, in a paragraph in close proximity to “Limitations”?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5650", "date": "25 Aug 2020", "name": "Ghazaleh Aali", "role": "Author Response", "response": "We are grateful to the expert reviewer for their interest in the topic and for spending time in reviewing this manuscript. We value their positive and thorough comments and have revised the manuscript accordingly. Method Comment: As written, I am uncertain about limitations to language. According to the manuscript, there were no limitations to language. However, in the extended data, English language seems to be a limitation in the search strategies. Please, clarify so there is no doubt about this. Reply: Thank you for this comment. We initially intended to have no language restrictions but, as we followed the methods from an existing scoping review, we needed to apply a language limitation. This has now been corrected to: \"We ran a search to include studies in English language only…\". Comment: Figure 1: Please, consider clarifying the number of excluded reports per each specified criterion regarding the 473 excluded reports; no clarification (in numbers) has been made for them as for the 86-excluded full-text reports. Reply: We followed the PRISMA reported guideline and flow diagram which does not specify recording or reporting detailed reasons for exclusion at the Title/Abstract Screening step (Liberati et al. 2009; DOI 10.1136/bmj.b2700); however, as it is mandatory to report the reasons for exclusion in the Full Text Screening step, we have done this. Comment: Figure 1: Please, give the readers more information about the nine additional sources. Which was your strategy, how did you found them? Did all arrive from the other articles' reference lists? Reply: We have now included this statement under the Selection of studies section: \"we also investigated the reference lists of the relevant studies to identify additional relevant studies \". Results Comment: Are MIDAS (presented in Table 1), and ARCOS-IV and LAS-1 (presented in Table 3) examples of studies not yet published? Reply: No. These studies were completed and have reported their results, so they met our eligibility criteria. If a study was 'ongoing' at the time of our review, we did not report them in the table because \"Ongoing studies or protocols with no results\"was one of our exclusion criteria. If a study had no specific name, we used the last name of the first author and the year of publication as the study name in the tables. Comment: In Table 1, the fourth column, there is space available (two rows are already used) for you to write \"Number of participants\" or \"No.\" instead of using the symbol \"#\". Reply:We have corrected this now to \"No. of\". Comment: Table 2: I like the use of different colours, and red and green are instantaneous to understand. However, in colour blindness, the most common difficulty is to distinguish between red and green. Perhaps you can choose another colour combination. In the online version of Robot Reviewer report, I think that the table Risk of bias has a more easy to read layout than Table 2 in the main manuscript. Reply: We followed Cochrane's Risk of Bias tool and therefore also followed their reporting method. However we recognise that some of our readers might be colour-blind and so we are using plus signs and question marks in addition to the colour coding. We have also now changed the colours from red/green to grey/white. Comment: Table 3: Consider using \"Number\" or \"Number of\" or \"No.\" or \"No. of\" instead of \"#\" in the third and the fifth column? Reply:We have corrected these to \"No. of\". Comment: Table 3, sixth column, \"Stroke type\": Please, review the use of \"/\". Should you use \"and\", \",\" or delete the \"/\" somewhere? Regarding Kuppuswamy and NotFAST: \"First\", but which stroke type? Reply: We replaced \"/\" with\" ,\". We used type of stroke as reported in the studies. Thus, although 'First' does not refer to a specific stroke type, this was what was reported and therefore what we presented under 'type'. Since this is a scoping review, we did not contact specific researchers to clarify these details. Comment: Table 4, fourth column: I suggest you to use \"Post-stroke fatigue finding\" instead of the use of the abbreviation \"PSF finding\". Avoiding unnecessary abbreviations makes reading easier. Reply: We have now corrected this to 'post-stroke fatigue'. Comment: In Table 5, consider if you should specify \"Finding\" to \"Post-stroke fatigue finding\" in the light blue heading (in line with the selected sub-heading in Table 4). Reply: We have now added 'post-stroke fatigue'. Comment: In the data commentary related to Table 6, you state that the Canadian guideline was the only one with comprehensive recommendations on post-stroke fatigue. In Table 6, under the heading \"Recommendation\" the reader is referred to page 15-16 of the guideline. I suggest that you present vital parts of the content in Table 6, in addition to this reference. Reply: We did consider this approach initially. However, for the following reasons we decided not to report the text for this guideline: 1. there are two pages of content that could be paraphrased and summarized into the table but because of the volume of the content (even after summarizing and paraphrasing) it would require official copyright permission from the publisher of the guideline. Aside from the process of obtaining such permission, this will require payment to the publisher which is not included within our grant. 2. We found the content of these two pages relevant, well-written, and important, and we have consequently intentionally referred the reader to this source, rather than paraphrasing and losing important detail. Comment: I lack information on age and gender of the patients in the different studies from which the review is based. Is it possible to add this information? Reply: If it is not reported in our review, it means that this information was missing from the primary study. Since this is a scoping review, we did not consider contacting each researcher separately. However, we have highlighted the fact than even very basic but important demographic information has not been reported by researchers of the primary studies. Not including key demographic information is an important issue to highlight, because it needs to be addressed in future studies Comment: Is it possible to give the reader even more specific information on which quantitative measures the post-stroke fatigue findings are based? (Although the level of significance is arbitrary set and statistically significance results does not need to be clinically meaningful for the patients. Reply: Reporting/calculating effect sizes is not routine practice or part of reporting guidelines for conducting scoping reviews. However, we thought this revision could add to the value of the interventional studies so we reported these statistics in Table 4. Since this is not a systematic review, we did not consider contacting the researchers for complete information and we did not calculate or combine the effect sizes. Instead we reported the statistics as they were reported in the original studies. Discussion Comment: I suggest a sentence about time-dependent concerns regarding the construct (post-stroke fatigue) in the research studies conducted years after stroke and its potential significance for validity. Reply: Thank you. We have added the following: \"One issue worth considering is whether the construct of PSF holds for fatigue experienced in research participants recruited years after their stroke, and whether this fatigue is a function of other issues. Future systematic reviews could address this issue by conducting sensitivity analyses comparing studies that include participants many years after their stroke with those including participants immediately after their stroke.\" Comment: International recommendations regarding outcome measures related to, and time points for measuring, post-stroke fatigue in research studies on recovery after stroke would probably reduce the heterogeneity of studies and facilitate further summaries and updates. You could possibly include that post-stroke fatigue might be an issue for future Stroke Recovery and Rehabilitation Roundtable (SRRR) work. Reply: Thank you for this suggestion. We have made a comment about this being an important area for future research but do not want to identify an individual group or organization to take this forward. We added: \"In addition, harmonisaton of studies requires standard international guidelines regarding outcome measurements and time points for measuring PSF in a standard way to create homogenous and collective body of evidence.\". Comment: You have a separate paragraph regarding limitations. Have you thought about including a sentence related to strengths of your study, in a paragraph in close proximity to \"Limitations\"? Reply:Thank you for this comment. We have changed the heading to 'Limitations and Strengths' and added three strengths of the review in this section: A. Public and open sharing of our methods so that anyone can update our review, B. Utilizing automation tools such as Rayyan and RobotReviewer to save time and resources and C. The fact that this was A multi-disciplinary review team." } ] }, { "id": "62109", "date": "04 May 2020", "name": "Nicola Hancock", "expertise": [ "Reviewer Expertise Stroke rehabilitation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the invitation to review this interesting scoping review, focussing on an important area of current research highly relevant to multiple aspects of life after stroke- that of Post-Stroke Fatigue, PSF.\nThe current review updates the work of Hinkle et al. 2016, and the rationale for doing so is clearly stated by the authorship team. That the review here generated a further 24 studies (and six sets of guidelines including PSF) since 2016 further demonstrates the growing impetus of work in this area. The objective stated is rather non-specific, but this is acceptable in such a scoping review that serves as a summary of recent evidence. It is unsurprising that this review concludes that further research in this area is required, and the authors make relevant suggestions as to the characteristics of future research that are clearly based on the findings of this review.\n\nThe review is clearly written, and the methods used and ensuing findings have been reported with transparency and considerable attention to detail. Search strategies are available via an embedded link. This paper makes a very useful contribution and provides a foundation for further work.\n\nThe following minor comments and suggestions may be of use to the authors:\nThe use of the word ‘following’ in the opening two section headings of the methods might be worth reconsidering, readability of this phrase in a title is challenging (page 3).\n\nIn Figure 1, suggest clarifying ’34 not new/covered’- does this mean 34 records removed as covered in the previous review? (page 4).\n\nSection on data extracted from clinical practice guidelines, please clarify ‘study name and year’- does this refer to the name and year of the guidelines or the included studies from which the guidelines was written? (page 5).\n\nWas any specific tool used to assess risk of bias in the non-randomised studies? If so, this should be stated in the methods text. If not, some justification would be helpful.\n\nIn table 3, the final column is not entirely clear- is this mean time after stroke onset for included participants? Simple clarification in the column heading or legend would address this (page 7/8).\n\nSuggest rephrasing ‘probably the most effective interventions’ to deliver a clearer message here (page 9).\n\nIn the discussion, the section on online platforms is somewhat unexpected- possible delivery via online mechanisms does not seem to have arisen prior to this point, though I apologise if I have missed this. Perhaps a line to place this paragraph in context might help the interpretation here? (page 10).\n\nAs the authors focus on possible reasons for the use of the Fatigue Severity Score (FSS) in one section of the discussion, it might be helpful to include a line about the validity/reliability of this measure at this point.\n\nThere are a few very minor typographical and grammatical errors.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "5651", "date": "25 Aug 2020", "name": "Ghazaleh Aali", "role": "Author Response", "response": "We thank the expert reviewer for spending time on our review and for providing helpful comments. We have made amendments to the manuscript based on these comments. Comment: The use of the word 'following' in the opening two section headings of the methods might be worth reconsidering, readability of this phrase in a title is challenging (page 3). Reply:Thank you. We have now changed these to 'Methods from an existing review' and 'Scoping review methods'. Comment: In Figure 1, suggest clarifying '34 not new/covered'- does these mean 34 records removed as covered in the previous review (page 4)? Reply:We have now clarified this in the text.We excluded studies that had been covered in the previous review. We also excluded the new studies that were not covered in the previous review if they repeated the findings reported in the previous reviews. We included new studies that were repeating findings from the previous review if they had a larger sample size or a new factor or intervention so were adding to the existing knowledge. This is compatible with the 'scoping' element of this review. Comment: Section on data extracted from clinical practice guidelines, please clarify 'study name and year'- does this refer to the name and year of the guidelines or the included studies from which the guidelines was written (page 5). Reply: The 'Citation'column refers to the reference publication and publication date of the guideline. Comment: Was any specific tool used to assess risk of bias in the non-randomised studies? If so, this should be stated in the methods text. If not, some justification would be helpful. Reply:Assessing risk of bias is not usually a feature of scoping reviews. However, because of availability of the automation tool (i.e. RobotReviewer) for RCTs and to provide additional training to two PhD students who were involved in this review, we added risk of bias assessment only for the RCTs. We did not assess non-RCTs for risk of bias because our resources were limited. We have now addressed this in the text: \"Because of the ‘scoping’ nature of this review and lack of time and resources, we did not assess the risk of bias for non-RCTs.\" Comment: In Table 3, the final column is not entirely clear- is this mean time after stroke onset for included participants? Simple clarification in the column heading or legend would address this (page 7/8). Reply:It is both. Because of the lack of standards in reporting these data, some studies reported time period after stroke and some reported only mean and standard deviation.We have now detailed this under the table. Comment: Suggest rephrasing 'probably the most effective interventions' to deliver a clearer message here (page 9). Reply: We have changed 'the most effective' to 'the future'. Comments: In the discussion, the section on online platforms is somewhat unexpected- possible delivery via online mechanisms does not seem to have arisen prior to this point, though I apologise if I have missed this. Perhaps a line to place this paragraph in context might help the interpretation here (page 10)? Reply:Thank you. In the second paragraph of the Results we note that the delivery was mostly face-to-face and individually rather than online. Comment: As the authors focus on possible reasons for the use of the Fatigue Severity Score (FSS) in one section of the discussion, it might be helpful to include a line about the validity/reliability of this measure at this point. Reply:We have added a sentence about validity/reliability: \"Bearing in mind that both these PSF measurement scales are valid and reliable…\". We therefore tried to raise the other 'possible' reasons for the frequent use of this specific scale in this field. Comment: There are a few very minor typographical and grammatical errors. Reply: Thank you. We have now addressed these and apologise for overlooking these." } ] } ]	1	https://f1000research.com/articles/9-242
https://f1000research.com/articles/9-1038/v1	25 Aug 20	{ "type": "Research Article", "title": "The factor structure of the Center for Epidemiological Study - Depression Scale in people with multiple sclerosis", "authors": [ "Ian I. Kneebone", "Chris Fife-Schaw", "Lawrence T. Lam", "Roshan das Nair", "Chris Fife-Schaw", "Lawrence T. Lam", "Roshan das Nair" ], "abstract": "Background: Depression is common in multiple sclerosis (MS); however, its assessment is complicated by biological processes. In this context it is important to consider the performance of depression screening measures including that their factor structure is consistent with expectation. This study sought to identify the factor structure of the Center for Epidemiological Study - Depression Scale (CES-D) in people with MS (PwMS). Methods: Participants (N = 493) were those who had consented to take part in a large three-phase longitudinal study of depression in PwMS. CES-D questionnaires completed at phase 1 of the study were utilised. An error in the questionnaire meant it was most appropriate to consider data for 19 of the 20 CES-D questionnaire items. The data was split into two samples by a random selection process to create an exploratory, model development sample and a validation sample. The first sample was subject to confirmatory factor analysis. Following examination of model fit and specification errors, the original model was modified. The revised model was tested in the confirmation sample to assess reproducibility. Results: The analysis results supported the original four factor solution for the CES-D, that is: Depressed Affect, Positive Affect, Somatic Complaints/Activity Inhibition, and Interpersonal Difficulties. Conclusions: The CES-D appears to have a coherent structure with which to examine depression in PwMS.", "keywords": [ "Multiple Sclerosis", "Depression", "Center for Epidemiological Study - Depression Scale", "Factor Analysis" ], "content": "Introduction\n\nMultiple sclerosis (MS), a disease of the central nervous system, involves a variety of debilitating physical, sensory, cognitive and emotional symptoms. People with MS (PwMS) are typically diagnosed aged 20–40 years and often face psychosocial consequences including disruptions to life goals, education/employment, income, relationships, leisure activities, and daily living activities1. Indeed, MS is considered the leading cause of disability in young adults in the developed world2. Furthermore, the chronic and unpredictable course of MS and side effects of MS-related medications have profound social and psychological consequences3.\n\nDepression is common in PwMS with a point prevalence rate of up to 40% and up to 50% experiencing it at some time post diagnosis4,5. Depression is also associated with higher suicidal ideation and attempts in pwMS6 and often disrupts relationships and reduces compliance with MS disease-modifying treatments7. The negative sequelae associated with depressive symptoms in MS also include decreased perceived cognitive function8, increased fatigue9, and sleep difficulties10.\n\nDepressive symptoms in MS may not only be caused by the psychosocial adjustment to the illness and its affects but in relate to biological aspects of the disease11. Organic changes, including in, neuroendocrine function, inflammatory process and brain associated brain damage likely to play a role12–14.\n\nDepression is assessed by clinical interviews but is more often assessed (especially in research contexts) through self-report questionnaires. There is a significant overlap between the somatic symptoms common to depression and MS symptoms, principally fatigue, poor sleep and concentration. This overlap has led to concerns over the accuracy of assessment of depression in PwMS15. Furthermore, some self-report measures of depression include questions about health and work difficulties, which are also impacted by MS-related physical disability16. As such, levels of depression in MS may be over- or underestimated, particularly when using self-report measures.\n\nDespite the Center for Epidemiological Studies - Depression Scale (CES-D)17 originally being developed for use with the general community18 it has become to widely used in clinical research and practice settings, including with PwMS19. The original CES-D is a 20-item self-reported scale that has been shown to measure depressive symptoms across four domains: Depressed Affect, Positive Affect, Somatic Complaints/Activity Inhibition, and Interpersonal Difficulties18. The four original latent factors have been replicated in numerous populations20. However, a number of other studies have yielded different CES-D factor structures. For instance a three-factor solution was evident in Chinese adolescents21 and as many as five factors has been found in a random sample of adults in the USA22. Some studies have also shown the presence of variation in the items comprising each factor23,24 and the magnitude of item loadings has varied across clinical groups25. Variations such as these may affect the sensitivity and specificity of this instrument in detecting depressive symptoms in different populations and question the test’s validity. With respect to PwMS, Amtmann et al.26 confirmed acceptable inter-item reliability and convergent/discriminant validity of the CES-D in a sample of 455 patients. However, in this study, confirmatory factor analysis was only used to consider the presence of a single factor, depression, i.e., unidimensionality. One study considered the multi-dimensional factor structure of the French version of the CES-D in people with MS, confirming the initially identified four factor structure27.\n\nConsistent with concerns that different populations can produce different factor analytic structures for measures28,29 and this pertains to validity30, the current study aimed to assess whether the four-factor model of depression is supported in PwMS in an English language version of the CES-D.\n\n\nMethods\n\nThe research was approved by the University of Surrey Advisory Committee on Ethics [ACE/99/30/Psych]. Participants were those who had provided written, informed consent to take part in a large three-phase longitudinal study of depression in PwMS commencing in 199931 Participants were required to have a diagnosis of MS and be 18 years or older. Only participants under the age of 65 years were included in the current study. Participants were a convenience sample who self-referred to the study following the publication of an article in an MS magazine available to people in the United Kingdom. Data, including the CES-D, were collected yearly by postal survey using a prepaid system, on three occasions. The CES-D data collected at phase 1 are reported here. No power analysis to determine sample size was calculated a priori.\n\nThe CES-D17 requests the self-reporting of depressive symptoms experienced over the previous week. The 20 items are rated on a ‘0’ to ‘3’ scale, with a higher rating indicating greater symptom frequency. Scores can range from 0 to 60. The CES-D is considered to be relatively unaffected by somatic variables32 and has been used in studies considering depression in PwMS19.\n\nData analysis was performed using SPSS v25. The data set was split into two by a random selection process to create an exploratory model development sample and the validation sample. In the exploratory stage, a model based on Radloff’s17 original specification of the CES-D was tested using confirmatory factor analysis (CFA). Model fit and the degree of specification errors were examined and modifications of the original model made as indicated and re-tested in the exploratory sample. This specification search involved examination of modification indices, identification of non-significant paths and conceptual acceptability. In the second stage, the revised model was tested in the confirmation sample to assess reproducibility. Models were estimated using maximum likelihood estimation and adequacy of fit was assessed using criteria proposed by Hu and Bentler33 for CFA models. These criteria of goodness of fit are a non-normed fit index/Tucker Lewis index (NNFI/TLI) >0.95, comparative fit index (CFI) >0.95, root mean square error of approximation (RMSEA) <0.06 and standardized root mean square residual (SRMR) <0.08. For completeness we also report the traditional chi-square fit index and the reduced chi-squared statistics (χ2/df), which should ideally be less than 234. To determine internal reliability of each factor Cronbach’s alpha coefficients were calculated. Due to a typographical error in one of the items (item 10: ‘I felt fearful’ was printed as ‘I felt tearful’) in the questionnaire useda, which was only observed after data collection; this item had to be removed, and the analyses are reported on the 19 items. The exploratory and confirmatory factor analyses were performed again on the full 20 items.\n\n\nResults\n\nA total of 493 participants were recruited (n = 399 women). The age range was 22 years to 65 years (mean, X = 45.8 years, SD = 9.25). MS diagnoses sub-types were as follows: 45% had ‘relapsing remitting type’, 20% had ‘secondary progressive type’, 10% has ‘primary progressive type’, and 19% ‘did not know’ their MS sub-type, with 5% ‘missing data’. Over half (62.9%) of participants scored >16 on the CES-D, the cut off indicating ‘significant depressive symptoms’ (X = 22.1, SD = 12.57, Range 0–59).\n\nSince there was only one sample for factor analyses, both exploratory and confirmatory, the original data set consisting of 493 patients was split into two using SPSS’s random selection process. Only cases with complete data on all 20 CES-D items (n = 472) were retained and as a result, a model specification sample with 235 participants was generated and the remaining 237 were used as the validation sample.\n\nFollowing Radloff’s17 exploratory factor analyses we assessed the fit of our data to her original model with the omission of item 10. Table 1 gives the fit indices for this initial model (Model 1). These indices suggest that the model, while not unreasonable in terms of the absolute size of the indices, is mis-specified. A specification search suggested that the two Somatic Symptoms items ‘I felt everything that I did was an effort (CESD7)’ and ‘I could not get going (CESD20)’ shared variance not entirely captured by the Somatic Symptoms factor (MI = 10.21) so a second model with this term added was assessed. Fit indices for this model (Model 2) are presented in Table 1. On Hu and Bentler’s33 four criteria this model fits acceptably and the χ2/df criterion is also satisfactory. No further modifications were made as these either made trivial improvements in fit or did not make sense theoretically.\n\nThe re-specified model was then assessed in the confirmation sample (Model 3) and as seen in Figure 1, the fit indices suggest a satisfactory fit of this modified four-factor model. Indeed, the fit indices are slightly better in the confirmation sample than the exploratory one. The modification made in the re-specification process did not imply factorially complex items nor that the basic four-factor CES-D model was substantively incorrect, so we estimated the standard Cronbach’s Alpha reliability coefficients for the implied CES-D subscales in the confirmation sample. These were 0.81 for Positive Affect, 0.87 for Depressed Affect, 0.73 for Somatic Symptoms and 0.79 for Interpersonal Problems.\n\nFigures are standardised maximum likelihood estimates.\n\nFor the 20-item scale, following Radloff’s17 exploratory factor analyses we assessed the fit of our data to her original model. Table 2 gives the fit indices for this initial model (Model 1). These indices suggest that the model, while not unreasonable in terms of the absolute size of the indices, is mis-specified. A specification search suggested a substantial correlation between the error terms associated with the depressed affect items ‘I felt tearful (CESD10)’ and ‘I had crying spells (CESD17)’ needed to be modelled (MI = 51.92) so a second model with this term added was assessed. This correlated error term reflects the incorrect wording of Item 10 we identified in the Johnston et al.35 version of the CES-D we used. Fit indices for this model (Model 2) are presented in Table 2. On three of Hu and Bentler’s33 four criteria this model fits acceptable in the χ2/df is also satisfactory. The addition of this path in the model suggests that these two items share variance (r = 0.48) in this PwMS sample that is not captured solely by Depressed Affect. Further inspection of model mis-fit suggested that the two Somatic Symptoms items ‘I felt everything that I did was an effort (CESD7)’ and ‘I could not get going (CESD20)’ also shared variance not entirely captured by the Somatic Symptoms factor (MI = 11.08). Indices for this model (Model 3) are presented in Table 2 and Figure 2 and suggest a good fit against the criteria. No further modifications were made as these either made trivial improvements in fit or did not make sense theoretically.\n\nFigures are standardised maximum likelihood estimates.\n\nThe re-specified model was then assessed in the confirmation sample (Model 4) and the fit indices suggest a satisfactory fit of this modified 4-factor model. Indeed, the fit indices are slightly better in the confirmation sample than the exploratory one. The modification made in the re-specification process did not imply factorially complex items nor that the basic 4-factor CES-D model was substantively incorrect, so we estimated the standard Cronbach’s Alpha reliability coefficients for the implied CES-D subscales in the confirmation sample. These were 0.81 for Positive Affect, 0.87 for Depressed Affect, 0.73 for Somatic Symptoms and 0.79 for Interpersonal Problems.\n\n\nDiscussion\n\nThe four-factor structure of the CES-D for the 19-item scale was supported by the factor analyses reported here. On the basis of these results, the English version of the CES-D has factorial validity in PwMS. Despite the potential contribution of neuropathology to symptoms, it does appear to have a coherent structure with which to examine depression in PwMS. Consistent with Shafer20, it supports the potential for considering the four factors in research such as that which might consider whether these factors vary as part of the natural course of co-existent depression or indeed that considers whether treatments might differentially affect the factors.\n\nThe main limitation of the current study is the typographical error we found in the questionnaire. We did not initially spot the typographical error (item 10 reads “I feel tearful” when it should read “I feel fearful”), and only noticed it after we conducted our initial analyses. It should be noted the compendium of instruments from which the CES-D was obtained is commonly available: it is held in many university and clinical departments. On this account we have contacted the First author of the compendium so notifications might ensue.\n\nInterestingly, after a brief search for studies that give details of the CES-D items used we note that other studies36–39 have also included this (or a similar) version of the questionnaire, meaning their results will need to be re-evaluated. In fact, one Rasch analysis study using the CES-D in a rheumatoid arthritis sample38 found differential item functioning on this item (which the authors present as “I felt tearful”) for age and gender. The authors do suggest that this finding be replicated before a decision is made to remove this (and one other) item from the scale.\n\nWe could not complete a full review of the literature to determine how many other studies have used a version of the CES-D that includes the typographical error, and the impact this has on the findings from these studies. One of the problems is that not all authors report where they obtained the scale from, instead only citing the original reference of the scale. We were, however, able to spot the typo by looking for the word ‘tearful’ when item 10 is mentioned in the paper. On account of the error in the questionnaire, our modelling requires replication with all the full version of the CES-D. Nonetheless, despite this our result at this point are sufficient for us to be confident of how this this instrument would likely perform, that is, consistent with the four-factor solution.\n\n\nData availability\n\nConsent was not obtained from participants for the sharing of their data, meaning that access to the data is restricted. Those wishing to access the data can apply for access. The data custodian is Prof Chris Fife-Schaw, University of Surrey (c.fife-schaw@surrey.ac.uk). Access will be provided to researchers at accredited institutions.", "appendix": "Footnotes\n\naWe used the photocopy-permitted master copy of the scale from the Measures in Health Psychology: A User's Portfolio (Johnston, Wright, & Weinman, 1995). An erratum for this measure has now been issued.\n\n\nAcknowledgements\n\nWe wish to express our thanks to the MS Society of Great Britain and Northern Ireland for their assistance in obtaining participants for this study. Dr Helen Wain and Dr Sue Guerrier supported general administration and data analysis. Dr Emma Dunmore supported design of the longitudinal study project.\n\n\nReferences\n\nMullins LL, Cote MP, Fuemmeler BF, et al.: Illness intrusiveness, uncertainty, and distress in individuals with multiple sclerosis. Rehabilitation Psychology. 2001; 46(2): 139. Publisher Full Text\n\nBrowne P, Chandraratna D, Angood C, et al.: Atlas of multiple sclerosis 2013: a growing global problem with widespread inequity. Neurology. 2014; 83(11): 1022–1024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson HR, Nair RD: The psychological impact of the unpredictability of multiple sclerosis: a qualitative literature meta-synthesis. British Journal of Neuroscience Nursing. 2013; 9(4): 172–178. Publisher Full Text\n\nSiegert R, Abernethy D: Depression in multiple sclerosis: a review. J Neurol Neurosurg Psychiatry. 2005; 76(4): 469–475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkokou M, Soubasi E, Gourzis P: Depression in multiple sclerosis: a review of assessment and treatment approaches in adult and pediatric populations. ISRN Neurol. 2012; 2012: 427102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrenner P, Burkill S, Jokinen J, et al.: Multiple sclerosis and risk of attempted and completed suicide–a cohort study. Eur J Neurol. 2016; 23(8): 1329–1336. PubMed Abstract \| Publisher Full Text\n\nFeinstein A: Multiple sclerosis and depression. Mult Scler. 2011; 17(11): 1276–1281. PubMed Abstract \| Publisher Full Text\n\nMessinis L, Kosmidis MH, Lyros E, Papathanasopoulos P: Assessment and rehabilitation of cognitive impairment in multiple sclerosis. Int Rev Psychiatry. 2010; 22(1): 22–34. PubMed Abstract \| Publisher Full Text\n\nvan Kessel K, Moss-Morris R, Willoughby E, et al.: A randomized controlled trial of cognitive behavior therapy for multiple sclerosis fatigue. Psychosom Med. 2008; 70(2): 205–213. PubMed Abstract \| Publisher Full Text\n\nLunde HMB, Aae TF, Indrevåg W, et al.: Poor sleep in patients with multiple sclerosis. PLoS One. 2012; 7(11): e49996. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer A, Heesen C, Gold SM: Biological outcome measurements for behavioral interventions in multiple sclerosis. Ther Adv Neurol Disord. 2011; 4(4): 217–229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGold SM, Krüger S, Ziegler KJ, et al.: Endocrine and immune substrates of depressive symptoms and fatigue in multiple sclerosis patients with comorbid major depression. J Neurol Neurosurg Psychiatry. 2011; 82(7): 814–818. PubMed Abstract \| Publisher Full Text\n\nRocca MA., Parisi L, Pagani E, et al.: Regional but not global brain damage contributes to fatigue in multiple sclerosis. Radiology. 2014; 273(2): 511–520. PubMed Abstract \| Publisher Full Text\n\nZorzon M, Zivadinov R, Nasuelli D, et al.: Depressive symptoms and MRI changes in multiple sclerosis. Eur J Neurol. 2002; 9(5): 491–496. PubMed Abstract \| Publisher Full Text\n\nHind D, O’Cathain A, Cooper, CL, et al.: The acceptability of computerised cognitive behavioural therapy for the treatment of depression in people with chronic physical disease: a qualitative study of people with multiple sclerosis. Psychol Health. 2010; 25(6): 699–712. PubMed Abstract \| Publisher Full Text\n\nMohr DC, Goodkin DE, Likosky W, et al.: Identification of Beck Depression Inventory items related to multiple sclerosis. J Behav Med. 1997; 20(4): 407–414. PubMed Abstract \| Publisher Full Text\n\nRadloff LS: The CES-D scale: A self-report depression scale for research in the general population. Appl Psychol Meas. 1997; 1(3): 385–401. Publisher Full Text\n\nCole JC, Rabin AS, Smith, TL, et al.: Development and validation of a Rasch-derived CES-D short form. Psychol Assess. 2004; 16(4): 360–72. PubMed Abstract \| Publisher Full Text\n\nPandya R, Metz L, Patten, SB: Predictive value of the CES-D in detecting depression among candidates for disease-modifying multiple sclerosis treatment. Psychosomatics. 2005; 46(2): 131–134. PubMed Abstract \| Publisher Full Text\n\nShafer AB: Meta-analysis of the factor structures of four depression questionnaires: Beck, CES-D, Hamilton, and Zung. J Clin Psychol. 2006; 62(1): 123–146. PubMed Abstract \| Publisher Full Text\n\nWang M, Armour C, Wu Y, et al.: Factor structure of the CES-D and measurement invariance across gender in mainland Chinese adolescents. J Clin Psychol. 2013; 69(9): 966–979. PubMed Abstract \| Publisher Full Text\n\nThorson JA, Powell FC: The CES-D: Four or five factors? Bull Psychon Soc. 1993; 31: 577–578. Publisher Full Text\n\nAssari S, Moazen-Zadeh E: Confirmatory Factor analysis of the 12-item center for epidemiologic studies Depression scale among Blacks and Whites. Front Psychiatry. 2016; 7: 178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi H, Fogg L, Lee, EE, et al.: Evaluating differential item functioning of the CES-D scale according to caregiver status and cultural context in Korean women. J Am Psychiatr Nurses Assoc. 2009; 15(4): 240–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams, CD, Taylor TR, Makambi K, et al.: CES-D four-factor structure is confirmed, but not invariant, in a large cohort of African American women. Psychiatry Res. 2007; 150(2): 173–180. PubMed Abstract \| Publisher Full Text\n\nAmtmann D, Kim, J, Chung, H, et al.: Comparing CESD-10, PHQ-9, and PROMIS depression instruments in individuals with multiple sclerosis. Rehabil Psychol. 2014; 59(2): 220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerdier-Taillefer MH, Gourlet V, Fuhrer R, et al.: Psychometric properties of the Center for Epidemiologic Studies-Depression scale in multiple sclerosis. Neuroepidemiology. 2001; 20(4): 262–267. PubMed Abstract \| Publisher Full Text\n\nComrey AL: Common methodological problems in factor analytic studies. J Consult Clin Psychol. 1978; 46(4): 648–59. Publisher Full Text\n\nSimón A: Effects of selective sampling on a factor analysis. The Journal of General Psychology. 1979; 101(2): 259–264. Publisher Full Text\n\nMokkink LB, Terwee CB, Patrick DL, et al.: The COSMIN checklist manual. 2012. Accessed November 2019. Reference Source\n\nKneebone II, Guerrier S, Dunmore E, et al.: A longitudinal examination of the hopelessness theory of depression in people who have Multiple Sclerosis. Behav Neurol. 2015; 2015: 190405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDevins GM, Orme CM, Costello CG, et al.: Measuring depressive symptoms in illness populations: Psychometric properties of the Center for Epidemiologic Studies Depression (CES-D) scale. Psychology and Health. 1988; 2(2): 139–156. Publisher Full Text\n\nHu LT, Bentler PM: Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct Equ Modeling Multidiscip J. 1999; 6(1): 1–55.Publisher Full Text\n\nHayduk LA: Structural equation modeling with LISREL: Essentials and advances. The John Hopkins University Press: Baltimore, MY. 1987. Reference Source\n\nJohnston M, Wright S, Weinman J: Measures in health psychology: a user's portfolio. NFER - Nelson. 1995. Reference Source\n\nSchroevers MJ, Sanderman R, Van Sonderen E, et al.: The evaluation of the Center for Epidemiologic Studies Depression (CES-D) scale: depressed and positive affect in cancer patients and healthy reference subjects. Qual Liife Res. 2000; 9(9): 1015–1029. PubMed Abstract \| Publisher Full Text\n\nTakeuchi H, Hiroe T, Kanai T, et al.: Childhood parental separation experiences and depressive symptomatology in acute major depression. Psychiatry Clin Neurosci. 2003; 57(2): 215–219. PubMed Abstract \| Publisher Full Text\n\nCovic T, Pallant JF, Conaghan PG, et al.: A longitudinal evaluation of the Center for Epidemiologic Studies-Depression scale (CES-D) in a rheumatoid arthritis population using Rasch analysis. Health Qual Life Outcomes. 2007; 5: 41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCovic T, Pallant JF, Tennant A, et al.: Variability in depression prevalence in early rheumatoid arthritis: a comparison of the CES-D and HAD-D Scales. BMC Musculoskelet Disord. 2009; 10: 18. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71819", "date": "07 Oct 2020", "name": "Daphne Kaklamanou", "expertise": [ "Reviewer Expertise I have done work looking at the reliability and validity of depression inventories for PwMS and looked into user experience of depression inventories in PwMS." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 article that is looking at the factor structure of the CES-D scale for PwMS. The study has a really good sample size to complete the analysis and draw conclusions from.\nOverall the introduction seems to cover most of the literature in the area, although the systematic review (Hind et al., 20161) on the reliability and validity of depression inventories for PwMS is missing, so the authors might want to check that they have not missed anything. Also it would be good to explain why the authors chose the CES-D scale rather than other depression inventories.\nFor the Method section, there seems to be some detail, but more detail of the CES-D scale factor structure and the items would be good and would help understand the discussion in terms of the factors. In terms of the participants, it is clearly stated that this is a study that has taken place over a long period of time but providing a bit more detail (1) on the exact time/year; (2) over how long did they collect the data that are analysed in the study; (3) were there any other measurements collected e.g. other depression measures or the Gold standard the interview. At some in the method section the authors state that \"the CES-D data collected at phase 1 are reported here\", I presume they mean the current study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "76797", "date": "01 Feb 2021", "name": "Bilge Piri Cinar", "expertise": [ "Reviewer Expertise I am an expert in the field of MS. In all my studies", "I definitely consider the presence of depression in patients. In our MS clinic", "we routinely use different depression scales in practical life." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis depression scale is not used very often in MS. For this reason, it is recommended that the reason for choosing this scale should be briefly explained in the introduction part of the comparison studies with other scales.\n\nWhen the models in the article are examined in the source where values such as SRMR and RMSEA are referred, the models seem sufficient. The general outline of the study is well-designed and the statistical approach has its strengths in general.\n\nIt is recommended to indicate whether the sample size has been calculated or not.\n\nAccording to a general rule, it is stated that the number of items recommended for the study group to use the factor analysis technique or the sample size five times the number of observed variables (Child, 20061). According to Kline (19942), although it is recommended to keep the item (variable) ratio 10: 1 for the sample size, it is stated that this ratio can be reduced, but should be at least 2: 1. It is recommended to give information about the sample size in the method section.\n\nThe suitability of the dataset for factor analysis is recommended to be detailed in the statistics section (normal distribution ...).\n\nIt would be appropriate to write explanations and abbreviations under the tables and figures.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1038
https://f1000research.com/articles/9-353/v1	12 May 20	{ "type": "Brief Report", "title": "Apolipoprotein E expression pattern in human induced pluripotent stem cells during in vitro neural induction", "authors": [ "Hyunah Lee", "Paulina Nowosiad", "Lucia M. Dutan Polit", "Jack Price", "Deepak P. Srivastava", "Sandrine Thuret", "Hyunah Lee", "Paulina Nowosiad", "Lucia M. Dutan Polit", "Jack Price", "Deepak P. Srivastava" ], "abstract": "Apolipoprotein E (APOE) is a multifunctional protein that plays significant roles in important cellular mechanisms in peripheral tissues and is as well expressed in the central nervous system, notably by adult neural stem cells (NSCs) in the hippocampus. Evidence from animal studies suggest that APOE is critical for adult NSC maintenance. However, whether APOE has the potential to play a similar role in human NSCs has not been directly investigated. To address this question, we conducted a focused study characterising APOE gene and protein expression in an in vitro model of neural differentiation utilising human induced pluripotent stem cells. We found that APOE gene expression was dramatically decreased as the cells became more differentiated, indicating that APOE expression levels reflect the degree of cellular differentiation during neural induction. Furthermore, qualitative analysis results of immunocytochemistry showed that intracellular localisation of APOE protein becomes more pronounced as neural differentiation progresses. Taken together, our findings suggest a potential role for APOE in human NSC maintenance and justify further investigations being carried out to understand whether changes in APOE levels can directly impact the neurogenic capacity of human stem cells.", "keywords": [ "Induced pluripotent stem cells", "Neural stem cells", "Directed differentiation", "Apolipoprotein E" ], "content": "Abbreviations\n\nAD (Alzheimer’s disease); APOE (Apolipoprotein E); iPSCs (induced pluripotent stem cells); NSCs (neural stem cells)\n\n\nIntroduction\n\nApolipoprotein E (APOE) is a pleiotropic protein that plays an important role in lipid metabolism (Mahley & Rall, 2000) and is highly expressed in the brain (Elshourbagy et al., 1985). Although the primary function brain APOE is lipid transport, its expression is also found in other cell types outside the context of lipid metabolism (Liao et al., 2017). For example, a recent single-cell RNA sequencing study on human post-mortem Alzheimer’s disease (AD) brains showed that activated microglia (relevant to the disease state) express high levels of APOE unlike naïve microglia (relevant to healthy/homeostatic state) in the prefrontal cortex, indicating that APOE expression is associated with immune function (Mathys et al., 2019). Furthermore, neuronal APOE can also be expressed at high levels under stress conditions such as brain injury although APOE expression is normally low in healthy neurons (Mahley & Huang, 2012; Xu et al., 2006). Interestingly, APOE is highly expressed in nestin/glial fibrillary acidic protein (GFAP) double-positive neural stem cells (NSCs) in the adult hippocampus of mice, and one of the phenotypes characterised in APOE-null mice is the premature depletion of NSC pool in the hippocampus, suggesting that NSC maintenance requires APOE expression (Yang et al., 2011).\n\nAlthough the existing literature suggest that APOE plays an important role in stem cell maintenance, one should note that the majority of these findings were generated from rodent models. Since NSCs obtained from different species have been shown to behave in fundamentally different ways (Mertens et al., 2013; Otani et al., 2016; Ray & Gage, 2006), characterisation of APOE expression in ‘human’ NSCs should be done prior to investigating its exact function. However, such evidence has not been reported to this date. To reduce this knowledge gap, we conducted a short study examining the expression pattern of APOE gene and protein in human induced pluripotent stem cells (iPSCs) undergoing neural induction in vitro. We found that gene expression is the highest in cells at the earliest stage of neural induction, whereas protein expression becomes more localised intracellularly, indicating that APOE expression pattern changes according to the differentiation state of cells.\n\n\nMethods\n\nA list of materials used in this study is presented in Table 1.\n\nCTR_M3_36S human induced pluripotent stem cell (iPSC) line was reprogrammed from keratinocytes obtained from a neurotypical male. Keratinocytes were reprogrammed by introducing a set of Sendai virus encoding human OCT4, SOX2, KLF4, and C-MYC (Yamanaka factors) using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher) according to the manufacturer’s instructions. The virus was a gift from Dr. Mahito Nakanishi (AIST, Japan).\n\nCells were regularly tested for mycoplasma and certified mycoplasma-free. iPSCs were maintained in Essential 8™ medium (Thermo Fisher) without antibiotics at 37°C, 5% CO2, 5% O2 in 6-well NUNC™ plates (Thermo Fisher) coated with Geltrex™ (Thermo Fisher). Passaging of iPSCs lines were done with Versene (EDTA) solution (Lonza) according to the manufacturer's instructions. Passaging ratio for iPSC maintenance was kept between 1:6 and 1:18.\n\niPSC colonies approaching 80% confluence were passaged at 3:2 ratio on 6-well NUNC™ plates coated with Geltrex™ on D-2/-1 and maintained at 37°C, 5% CO2, 5% O2 for 24–48 hrs until they approached 100% confluence. Directed differentiation began on D0 by changing Essential 8™ medium to neural induction medium and incubating the cells at 37°C, 5% CO2, 20% O2. Neural induction lasted for 7 days. To prepare neural induction medium, N2:B27 was first prepared by mixing the N2 medium (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) (Sigma Aldrich) supplemented with 1X GlutaMAX™ (Thermo Fisher) and 1X N-2 supplement (Thermo Fisher)) and the B27 medium (Neurobasal® medium (Thermo Fisher) supplemented with 1X GlutaMAX™ and 1X B-27 supplement (Thermo Fisher) or 1X B-27 without vitamin A supplement (Thermo Fisher)) at 1:1 ratio. The following small molecule inhibitors were added to N2:B27 to make the neural induction medium: 100 nM LDN193189 (Sigma Aldrich) and 10 µM SB431542 (Sigma Aldrich) for dual SMAD inhibition (DSi); 100 nM LDN193189, 10 µM SB431542, and 2 µM XAV939 (Sigma Aldrich) for dual SMAD inhibition plus Wnt/β-catenin inhibition (DS-Wi); and 100 nM LDN193189, 10 µM SB431542, 2 µM XAV939, and 1 µM Cyclopamine (LC Laboratories) for dual SMAD inhibition plus Wnt/β-catenin plus sonic hedgehog inhibition (DS-WHi). Neural induction medium was used from D0 to D7, and N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire directed differentiation period.\n\nNeural passaging 1, 2, and 3 were performed with Accutase (Thermo Fisher) on D7, D12, and D15/16, respectively. Briefly, cells were washed with room temperature HBSS and treated with Accutase at 37°C, 5% CO2, 5% O2 for 3–4 minutes. Cold Accutase was used for neural passagings 1 and 2, and room temperature Accutase was used for neural passaging 3. Cells in Accutase were then collected with a P1000 pipette. Extra care was taken during neural passagings 1 and 2 where P1000 pipetting was done no more than 5 times when cells in Accutase were collected. Collected cells were then mixed with room temperature DMEM/F12 (twice the volume of Accutase used) so that Accutase could be deactivated, and centrifugation was performed twice to wash off the Accutase from cells. Centrifugation was done at 900 revolutions per minute (RPM) for 2 min during neural passaging 1 and 2, and at 1250 RPM for 2 min during neural passaging 3. After centrifugation, cells were plated on new 6-well NUNC™ plates coated with Geltrex™. Passaging ratios were 1:1 for neural passaging 1 and 2, and 2:3 for neural passaging 3. To ensure cell survival 10 µM Y-27632 (Sigma Aldrich), a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, was mixed with the plating medium at each neural passaging and then removed after 24 hrs.\n\nGenomic DNA was extracted from iPSCs using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions. The APOE locus containing the rs429358 and rs7412 SNPs was amplified with Taq DNA Polymerase (QIAGEN) according to the manufacturer's instructions. Briefly, the reaction mix containing 1X PCR Buffer, 1X Q-Solution, 10 mM dNTP mix (0.2 mM final concentration), primers (forward and reverse each at 0.4 µM final concentration), 1.25 units Taq polymerase, and 1 µg of genomic DNA was incubated at 95°C for 4 mins to activate the Taq polymerase. Then, 35 cycles of ‘denaturation at 94°C for 30 secs, annealing at 68°C for 30 secs, elongation at 72°C for 1 min’ was performed. Then, final extension was done at 72°C for 10 mins. This polymerase chain reaction (PCR) was done with S1000 Thermal Cycler (Bio-Rad). The primers used for APOE genotyping (Table 2) were previously designed by Henderson and colleagues (Henderson et al., 2002), and they are able to generate PCR products that can be visualised easily by gel electrophoresis after HhaI enzyme (Thermo Fisher) digestion. Amplified PCR products were digested with 1 unit of HhaI digestion enzyme and gel electrophoresis was performed using a 3% agarose gel containing 0.5 µg/mL ethidium bromide. Raw dataset is available as an underlying data via Figshare (doi: 10.6084/m9.figshare.12199721.v1).\n\nTotal RNA was extracted from D7, D12, D15/16, and D18/19 cells that were not used for neural passaging with TRIzol® reagent (Thermo Fisher) according to manufacturer’s instructions and eluted in 25-30 µL of diethyl pyrocarbonate (DEPC)-treated water. Reverse transcription of total RNA into complementary DNA was performed using SuperScript® III First-Strand Synthesis System (Thermo Fisher) according to the manufacturer's instructions. Briefly, the random hexamers were annealed to total RNA at 25°C for 10 mins, then the synthesis was performed at 50°C for 50 mins, and then the reaction was terminated at 85°C for 5 mins. The final product was diluted to 5 ng/µL of total RNA converted to cDNA using DEPC-treated water. S1000 Thermal Cycler (Bio-Rad) was used for reverse transcription.\n\nFor gene expression analysis, real-time quantitative polymerase chain reaction (qPCR) was performed using the HOT FIREPol® EvaGreen® qPCR Mix (Solis Biodyne) according to the manufacturer’s instructions. Briefly, the reaction mix containing the HOT FIREPol® EvaGreen® qPCR Mix, primers (forward and reverse each at 0.2 µM final concentration), and cDNA was incubated at 95°C for 15 mins to activate the HOT FIREPol® DNA polymerase, then 45 cycles of ‘denaturation at 95°C for 30 secs, annealing at 60°C for 30 secs, elongation at 72°C for 30 secs’ was performed. Melting curve analysis was done on each gene based on the melting profile generated every 1°C increment between 60°C and 95°C. MJ Research PTC-200 Thermal Cycler (Bio-Rad) was used for qPCR. The sequence of primers are presented in Table 2. CT values of APOE were normalised to that of GAPDH, and relative expression of APOE across samples were quantified using the 2-ΔΔCt method where D7 sample was used as a reference for each differentiation lineage. Raw dataset is available as an underlying data via Figshare (doi: 10.6084/m9.figshare.12136944).\n\nCells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™ X-100 in 1X Tris-buffered saline (TBS) for 15–30 minutes, and then blocked with 5% normal donkey serum in TBS for 30 minutes. Primary antibodies were incubated at 4°C overnight followed by 3 washings with TBS. Secondary antibodies conjugated with fluorescent dyes were incubated at room temperature for 1 hours followed by 2 washings with TBS. Nuclei were stained with 5 µg/mL Hoechst® 33342 solution (Thermo Fisher) for 30 seconds immediately prior to imaging, and cells were washed with TBS 2 times after nuclear staining. All primary antibodies were diluted in 5% normal donkey serum in TBS, secondary antibodies in 1% normal donkey serum in TBS, and Hoechst® 33342 solution in TBS. Imaging was done with IX 70 inverted epifluorescence microscope (Olympus) connected to AxioVision imaging software (version 4.4). Scale bars were inserted on the images using ImageJ software (version 1.49c). Raw dataset is available as an underlying data via Figshare (doi: 10.6084/m9.figshare.12199745.v1).\n\nGraphPad Prism (version 8.4.2.679) was used for statistical analysis. The statistical significance of the mean differences between groups were analysed by one-way analysis of variance (ANOVA) followed by Bonferroni correction for multiple testing. The mean, standard error of measurement (SEM), and number of biological replicates are reported. P-value < 0.05 was considered significant to reject the null hypothesis that the differences observed between groups is due to random variation.\n\n\nResults\n\nTo characterise the expression of APOE in human stem cells undergoing neural induction, an iPSC line derived from a neurotypical male with APOE3 homozygous genotype (CTR_M3_36S cell line) (Figure 1; (Lee, 2020b)) (Deans et al., 2017) were differentiated into neural lineages. Genotyping of CTR_M3_36S was performed using the method developed by Henderson and colleagues (Henderson et al., 2002), and CTR_M3_36S was confirmed to be homozygous for APOE3 by comparing the data with that of an APOE3 homozygous cell line (CTR_M1_04) that reported by Henderson and colleagues (Henderson et al., 2002, Figure 1). Neural induction into dorsal forebrain progenitors was performed using modified dual SMAD inhibition protocols (Cocks et al., 2014; Deans et al., 2017; Kathuria et al., 2018; Yu et al., 2014), where combinations of small molecule inhibitors were used to inhibit bone morphogenetic protein (BMP), transforming growth factor (TGF)-β, Wnt/β-catenin, and sonic hedgehog signalling pathways from D0 to D7 of neural induction (Figure 2A).\n\nCTR_M3_36S human iPSC line derived from a neurotypical male is homozygous for APOE3 (denoted as M3 in this figure). CTR_M1_04 human iPSC line that was known to be homozygous for APOE3 was used as control (denoted as M1 in this figure). HhaI-digested PCR amplicons were run on a 3% agarose gel, and the band loci were compared with the data previously reported by Henderson and colleagues who developed this genotyping method (Henderson et al., 2002). The band loci for both CTR_M3_36S and CTR_M1_04 lines match with the homozygous APOE3 data reported by Henderson and colleagues (see Figure 1 of Henderson et al., 2002).\n\nA) Schematic diagram of directed differentiation. CTR_M3_36S iPSCs were maintained in stem cell maintenance medium after replating (D-2/-1). On D0 neural induction began by changing the stem cell maintenance medium to neural induction medium. N2:B27 was used from D8 onwards. Medium was changed every 24 hrs throughout the entire differentiation period. Neural passaging 1, 2, and 3 were carried out on D7, D12, and D15/16, respectively. Total RNA extraction was made on cells that were not used for neural passaging on D7, D12, D15/16, and D18/19. Neural induction medium composition for each differentiation lineage and N2:B27 medium composition are also shown. B) APOE gene expression is reduced along neural induction regardless of lineage. Real-time qPCR was performed on CTR_M3_36S iPSCs undergoing directed differentiation at D7, D12, D15/16, and D18/19. APOE expression was normalised to that of GAPDH. D7 samples were used as reference samples for each lineage. One-way ANOVA with Bonferroni correction. n = 3. Mean (bars) with S.E.M. (error bars) shown. **** ANOVA p-value < 0.0001. ns: non-significant after Bonferroni correction. DSi: dual SMAD inhibition. DS-Wi: DSi plus Wnt/β-catenin inhibition. DS-WHi: DS-Wi plus sonic hedgehog inhibition. C) APOE protein is more localised intracellularly in differentiated cells. Representative images of cells at D12 and D18/19 expressing SOX2 (NSC marker) and TBR2 (NPC marker), respectively. Insets show images of SOX2/TBR2 in green and APOE in red. Scale bars indicate 50 µm unless stated otherwise.\n\nGene expression analysis revealed that APOE expression was the highest at D7, and the drastic down-regulation of APOE from D7 > to D18/19 was observed regardless of the combination of small molecule inhibitors used from D0 to D7 (p < 0.0001) (Figure 2B, underlying data (Lee, 2020a)). Immunocytochemistry showed that D12 and D18/19 cells expressed SRY-box transcription factor 2 (SOX2), a NSC marker, and T-Box Brain Protein 2 (TBR2), a neural progenitor cell (NPC) marker, respectively, for all combinations of small molecule inhibitors used from D0 to D7. Qualitative analysis of immunocytochemistry results revealed that ApoE became more localised to the intracellular region at D18/19 compared to D12 (Figure 2C; (Lee, 2020c)).\n\n\nDiscussion\n\nUnlike the existing animal models of APOE deficiency and humanised APOE expression where genetic modifications were introduced globally (whole body) rather than specifically to NSCs, the in vitro model used in this study allowed us to examine APOE expression pattern exclusively in stem cells that were pushed towards the neural lineage. Our findings demonstrate that in cells at the earliest stage of neurodevelopment, 1) human APOE gene expression is high, and 2) APOE protein is not clearly localised at the intracellular region. Various combinations of small molecule inhibitors did not alter these patterns of expression.\n\nAlthough further investigations will be needed to understand the exact role of APOE in neurodevelopment, the existing literature seems to suggest that APOE can be both downstream and upstream of stem cell maintenance. For example, several chromatin precipitation studies have shown that POU class 5 homeobox 1 (POU5F1), SOX2, Kruppel like factor 4 (KLF4), MYC proto-oncogene (MYC) and Nanog Homeobox (NANOG) all bind to the promoter region of APOE, suggesting that APOE expression could be directly regulated by such stem cell maintenance factors (ENCODE Project Consortium, 2004; ENCODE Project Consortium, 2011; Kim et al., 2008; Lachmann et al., 2010; Liu et al., 2008; Marson et al., 2008). However, other evidence suggests that APOE itself could be a direct regulator of cell fate determination. Meyer and colleagues (Meyer et al., 2019) showed that changing the APOE genotype from ε4 (AD risk factor) to ε3 (neutral genotype) in human NPCs can suppress premature neuronal differentiation and maturation via increasing the transcription repressor activity of RE1 silencing transcription factor (REST). Interestingly, APOE mRNA levels were lower in ε4 NPCs compared to ε3 NPCs, suggesting higher APOE gene expression is indeed likely to be associated with the undifferentiated state of NPCs. As a follow-up to our findings and the existing literature, we propose that further investigations should be carried out to elucidate the role of APOE in stem cell maintenance. For example, one could examine whether prolonged expression and/or overexpression of APOE gene in human NPCs can suppress further differentiation in these cells.\n\nIn this study, qualitative analysis was performed on APOE immunocytochemistry results. As the cells became more differentiated from NSCs to NPCs, APOE localisation pattern became more clearly intracellular. To validate this observation, co-localisation analysis with cytoskeletal proteins (such as Tubulin beta-3 chain and Microtubule-associated protein 2) or with plasma membrane proteins (such as N-Cadherin) will be needed in future investigations. Furthermore, APOE has been shown to exist in both secreted and intracellular forms in the existing literature (Huang & Mahley, 2014). Therefore, it will also be important to examine which form of APOE is produced at each differentiation stage. It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells. Interestingly, Gan and colleagues previously reported that APOE is indeed secreted by NSCs as well as NPCs, and secreted APOE was found to play a vital role in regulating NSC survival and neurosphere formation (Gan et al., 2011). Further investigations on changes of secreted and intracellular protein levels throughout neural differentiation will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state. This will in turn provide more definitive clues to whether APOE plays a stage-dependent role in neurodevelopment.\n\nIn conclusion, we report that human APOE gene expression levels are highly correlated with the undifferentiated state of cells during directed differentiation in vitro, and ApoE protein is more clearly localised in the intracellular region as the cells become more differentiated. Combining our observations and previous evidence reported in the literature, we speculate that APOE has an important role in stem cell maintenance and propose that further investigations should be carried out to investigate the exact underlying mechanisms such as 1) whether APOE is an upstream or downstream factor of stem cell maintenance, and 2) whether APOE4 genotype and APOE loss-of-function would produce similar phenotypes.\n\n\nData availability\n\nFigshare: raw data for qPCR. https://doi.org/10.6084/m9.figshare.12136944.v1 (Lee, 2020a)\n\nThis project contains the following underlying data:\n\n- Lee et al. raw data for qPCR.csv (C(t) values, efficiency of amplification, and values calculated for normalised gene expression analysis for APOE.)\n\nFigshare: raw data for Genotyping. https://doi.org/10.6084/m9.figshare.12199721.v1 (Lee, 2020b)\n\nThis project contains the following underlying data:\n\n- Lee et al. raw data for Genotyping.TIF (Gel image used in Figure 1. Genomic DNA from human iPSCs amplified for the APOE locus, then digested with HhaI enzyme. Run on 3% agarose gel.)\n\nFigshare: raw data for D12 and D18/19 immunocytochemistry. https://doi.org/10.6084/m9.figshare.12199745.v1 (Lee, 2020c)\n\nThis project contains the following underlying data:\n\n- 01 Lee et al. raw data for DS D12 Hoechst.tif\n\n- 02 Lee et al. raw data for DS D12 SOX2.tif\n\n- 03 Lee et al. raw data for DS D12 APOE.tif\n\n- 04 Lee et al. raw data for DS D18-19 Hoechst.tif\n\n- 05 Lee et al. raw data for DS D18-19 TBR2.tif\n\n- 06 Lee et al. raw data for DS D18-19 APOE.tif\n\n- 07 Lee et al. raw data for DS+Wi D12 Hoechst.tif\n\n- 08 Lee et al. raw data for DS+Wi D12 SOX2.tif\n\n- 09 Lee et al. raw data for DS+Wi D12 APOE.tif\n\n- 10 Lee et al. raw data for DS+Wi D18-19 Hoechst.tif\n\n- 11 Lee et al. raw data for DS+Wi D18-19 TBR2.tif\n\n- 12 Lee et al. raw data for DS+Wi D18-19 APOE.tif\n\n- 13 Lee et al. raw data for DS+WHi D12 Hoechst.tif\n\n- 14 Lee et al. raw data for DS+WHi D12 SOX2.tif\n\n- 15 Lee et al. raw data for DS+WHi D12 APOE.tif\n\n- 16 Lee et al. raw data for DS+WHi D18-19 Hoechst.tif\n\n- 17 Lee et al. raw data for DS+WHi D18-19 TBR2.tif\n\n- 18 Lee et al. raw data for DS+WHi D18-19 APOE.tif\n\n(Images were taken with IX70 inverted epifluorescence microscope (Olympus) connected to AxioVision imaging software 4.4. ImageJ 1.49c was used to generate TIFF files.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank Dr Graham Cocks for the insightful discussion regarding the project. We also thank Rupert Faraway, Matthew J. Reid, and James Williams, past members of the iPSC technicians’ team of Jack Price group, for reprogramming, performing quality-control, and providing guidance for maintaining the CTR_M3_36S iPSC line used in this study. We also thank Dr. Mahito Nakanishi (AIST, Japan) for providing the Yamanaka factors Sendai virus that was used for reprogramming.\n\n\nReferences\n\nCocks G, Curran S, Gami P, et al.: The utility of patient specific induced pluripotent stem cells for the modelling of Autistic Spectrum Disorders. Psychopharmacology (Berl). 2014; 231(6): 1079–1088. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeans PJM, Raval P, Sellers KJ, et al.: Psychosis Risk Candidate ZNF804A Localizes to Synapses and Regulates Neurite Formation and Dendritic Spine Structure. Biol Psychiatry. 2017; 82(1): 49–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElshourbagy NA, Liao WS, Mahley RW, et al.: Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets. Proc Natl Acad Sci U S A. 1985; 82(1): 203–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nENCODE Project Consortium: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 2011; 9(4): e1001046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nENCODE Project Consortium: The ENCODE (ENCyclopedia of DNA elements) project. Science. 2004; 306(5696): 636–640. PubMed Abstract \| Publisher Full Text\n\nGan HT, Tham M, Hariharan S, et al.: Identification of ApoE as an autocrine/paracrine factor that stimulates neural stem cell survival via MAPK/ERK signaling pathway. J Neurochem. 2011; 117(3): 565–578. PubMed Abstract \| Publisher Full Text\n\nHenderson JN, Crook R, Crook J, et al.: Apolipoprotein E4 and tau allele frequencies among Choctaw Indians. Neurosci Lett. 2002; 324(1): 77–79. PubMed Abstract \| Publisher Full Text\n\nHuang Y, Mahley RW: Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol Dis. 2014; 72 PtA: 3–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKathuria A, Nowosiad P, Jagasia R, et al.: Stem cell-derived neurons from autistic individuals with SHANK3 mutation show morphogenetic abnormalities during early development. Mol Psychiatry. 2018; 23(3): 735–746. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim J, Chu J, Shen X, et al.: An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 2008; 132(6): 1049–1061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLachmann A, Xu H, Krishnan J, et al.: ChEA: transcription factor regulation inferred from integrating genome-wide ChIP-X experiments. Bioinformatics. 2010; 26(19): 2438–2444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee H: raw data for qPCR. figshare. Dataset. 2020a. http://www.doi.org/10.6084/m9.figshare.12136944.v1\n\nLee H: raw data for Genotyping. figshare. Dataset. 2020b. http://www.doi.org/10.6084/m9.figshare.12199721.v1\n\nLee H: raw data for D12 and D18/19 immunocytochemistry. figshare. Dataset. 2020c. http://www.doi.org/10.6084/m9.figshare.12199745.v1\n\nLiao F, Yoon H, Kim J: Apolipoprotein E metabolism and functions in brain and its role in Alzheimer’s disease. Curr Opin Lipidol. 2017; 28(1): 60–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu X, Huang J, Chen T, et al.: Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Cell Res. 2008; 18(12): 1177–1189. PubMed Abstract \| Publisher Full Text\n\nMahley RW, Huang Y: Apolipoprotein e sets the stage: response to injury triggers neuropathology. Neuron. 2012; 76(5): 871–885. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahley RW, Rall SC: Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet. 2000; 1: 507–537. PubMed Abstract \| Publisher Full Text\n\nMarson A, Levine SS, Cole MF: Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 2008; 134(3): 521–533. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathys H, Davila-Velderrain J, Peng Z, et al.: Single-cell transcriptomic analysis of Alzheimer’s disease. Nature. 2019; 570(7761): 332–337. PubMed Abstract \| Publisher Full Text\n\nMertens J, Stüber K, Wunderlich P, et al.: APP processing in human pluripotent stem cell-derived neurons is resistant to NSAID-based γ-secretase modulation. Stem Cell Reports. 2013; 1(6): 491–498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer K, Feldman HM, Lu T, et al.: REST and Neural Gene Network Dysregulation in iPSC Models of Alzheimer’s Disease. Cell Rep. 2019; 26(5): 1112–1127.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOtani T, Marchetto MC, Gage FH, et al.: 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size. Cell Stem Cell. 2016; 18(4): 467–480. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRay J, Gage FH: Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells. Mol Cell Neurosci. 2006; 31(3): 560–573. PubMed Abstract \| Publisher Full Text\n\nXu Q, Bernardo A, Walker D, et al.: Profile and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus. J Neurosci. 2006; 26(19): 4985–4994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang CP, Gilley JA, Zhang G, et al.: ApoE is required for maintenance of the dentate gyrus neural progenitor pool. Development. 2011; 138(20): 4351–4362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu DX, Di Giorgio FP, Yao J, et al.: Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Reports. 2014; 2(3): 295–310. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63366", "date": "08 Jun 2020", "name": "Robert J. Williams", "expertise": [ "Reviewer Expertise Molecular and Cellular Neuroscience" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nIt has previously been shown that APOE regulates neural stem cell (NSC) maintenance in rodent models but until now this has not been demonstrated or validated in human cells. The authors address this important question through use of human induced pluripotent stem cells (APOE3 homozygous) and monitor APOE status throughout neural differentiation. APOE gene expression was notably much lower following induction of differentiation and coincident with this was an apparent change in the cellular distribution of APOE with the authors describing a much more pronounced intracellular localisation of APOE.\n\nMajor comments: This is a very focused piece of work and although the findings are quite preliminary they are very interesting and worth reporting particularly because of the human cell context. A key question going forward will be whether the APOE4 genotype influences NSC maintenance or differentiation as this could have clear implications for neurogenesis in Alzheimer’s Disease. The gene expression data presented here is clear and the changes in APOE are quite dramatic but the immunostaining is rather descriptive and is not quite as convincing.\nSpecific comments:\nThe resolution of the images in the composite cell panels in Fig 2 is not great and it is difficult to clearly see or judge the intracellular expression described. Can these be improved at all?\n\nFor the gene expression changes, data is provided for D7, D12 and D18, yet for the cell staining only D12 and D18 are shown. Is D7 cell data not available to present here?\n\nThe most obvious change at the protein level is the apparent increase in cellular APOE staining between D12 and D18 (certainly for DSi and DS-Wi) and as Hoe33342 has been performed I would have thought some quantitative data could be provided here which would help support the conclusions made.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5837", "date": "24 Aug 2020", "name": "Sandrine Thuret", "role": "Author Response", "response": "Comment 1. The resolution of the images in the composite cell panels in Fig 2 is not great and it is difficult to clearly see or judge the intracellular expression described. Can these be improved at all?The authors would like to thank the reviewer for this comment. We agree that the resolution of the images in Figure 2 is not as high as the original data when the manuscript is viewed/downloaded online. In the updated manuscript, these images are shown in a separate figure (Figure 3). The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D18/19 cells. In addition, as indicated in the ‘Data availability’ section of the submitted manuscript, the original data with higher resolutions for all images can be found on Figshare, a public repository for scientific data, which can be accessed via the following DOI: https://doi.org/10.6084/m9.figshare.12199745.v1. We are confident that the patterns of APOE immunocytochemistry (ICC) reported in the manuscript can be confirmed with greater detail from the raw data available on Figshare. Comment 2. For the gene expression changes, data is provided for D7, D12 and D18, yet for the cell staining only D12 and D18 are shown. Is D7 cell data not available to present here?The authors would like to thank the reviewer for mentioning this important aspect of the ICC experiment reported in our manuscript. While the authors confirm that the ICC experiments were conducted for APOE on D7 cells, the data were not included in the manuscript due to the following reasons. According to the differentiation protocol, the cells were maintained at high density approaching near 100% confluence from D0 to D7. We observed that this inadvertently diminishes the quality of immunocytochemistry images for D7 cells, since clear boundaries of nuclei could not be easily identified with epifluorescence microscopy and further complicated the downstream quantification process. The possibility of dissociating D7 cells and plating them on to a different surface for better image quality and quantification was considered briefly. However, such additional handling was not done to the cells so that any potential source of artefacts that could mask the true state of D7 cells can be ruled out in our experiments. While the use of epifluorescence microscopy in our study can be seen as a clear limitation, APOE immunostaining patterns of D7 cells was not qualitatively different from that of D12 cells in our observations. Further investigations using three-dimensional imaging techniques such as confocal microscopy will enable better imaging and quantification of densely packed cells on D7. The ‘Discussion’ section in the updated manuscript now includes a new paragraph regarding this aspect.“One limitation of this study is that the time-dependent changes of differentiation markers such as SOX2 and TBR2 were not examined alongside APOE. It is worth noting, however, that TBR2 was shown to be capable of suppressing SOX2 expression during differentiation of NSCs to NPCs ( Hodge et al., 2012). Given this information, it is unlikely that TBR2-positive cells observed in this study at D18/19 will simultaneously express high levels of SOX2. However, time-dependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells. Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study. High-resolution microscopy techniques (such as confocal microscopy) would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction (D0-D7). Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.” Comment 3. The most obvious change at the protein level is the apparent increase in cellular APOE staining between D12 and D18 (certainly for DSi and DS-Wi) and as Hoe33342 has been performed I would have thought some quantitative data could be provided here which would help support the conclusions made.The authors would like to thank the reviewer for the comment on the quantification of ICC images. We now include a quantification of the images in the updated manuscript. The percentage of SOX2+ cells and TBR2+ cells at D12 and D18/19, as well as the percentage of SOX2+ and TBR2+ cells that showed immunostaining patterns for intracellular APOE were quantified using CellProfiler v3.1.9. The quantified data presented as bar graphs can be found in Figure 3; the analysis procedure can be found in the ‘Methods (Immunocytochemistry)’ section; and the analysis pipeline as well as the raw data for quantification can be found on Figshare which can be accessed by the following DOI: https://doi.org/10.6084/m9.figshare.12781064.v1. The results indicate a higher percentage of intracellular APOE/TBR2+ cells at D18/19 compared to intracellular APOE/SOX2+ cells at D12, supporting our conclusions made in the manuscript prior to the inclusion of these quantification data." } ] }, { "id": "65480", "date": "06 Jul 2020", "name": "Noelia Urbán", "expertise": [ "Reviewer Expertise Adult neurogenesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nAPOE is highly expressed by neural stem cells (NSCs) in the hippocampus of adult mice, particularly in the quiescent state, and has been associated with stem cell maintenance. However, no data is available on the expression pattern of APOE in human neural stem cells. The authors monitor APOE expression by QPCR and immunocytochemistry during the neuronal differentiation of iPSCs using three different types of neural induction media. They report a dramatic reduction in APOE mRNA levels during differentiation, as well as a change in the cellular distribution of APOE protein.\nComments:\nAlthough preliminary, the changes in APOE expression are an interesting and important observation. However, while the QPCR data is convincing and very robust, the immunocytochemistry studies should be further analysed/improved in order to draw any strong conclusions. The images presented are not of very good quality, and if judging by them, APOE expression rather seems to increase globally during differentiation, with few cells expressing high levels at D12 and most cells expressing moderate levels at D18/19. My main specific recommendations are:\nIf possible, provide higher magnification/higher quality images of APOE stainings, including also the other time points during differentiation. Day 7 would be particularly important to include, since it displays the highest levels of expression by QPCR.\n\nAPOE stainings should be quantified to support the conclusions that its expression decreases during differentiation and there is a change in the protein localization. If this is not possible, the conclusions should be toned down and further experiments suggested in the discussion (for example, protein quantification by WB and cellular fractionation and quantification of protein in the media to assess intracellular protein localization and secretion, respectively).\n\nIn the discussion, it should be noted that iPSC-derived NSCs might not fully resemble adult NSCs. A brief discussion of what is known about the expression of APOE in NSCs during development would be very useful.\n\nOther comments/suggestions:\nSox2 (neural stem and progenitor marker) and Tbr2 (neuronal progenitor marker) stainings are presented only at one stage each. It would be very informative to see if/how these two markers change over the course of the differentiation protocol. If not possible by immuno, a QPCR for these genes would also be enough to show the trends of expression during differentiation.\n\nPlease specify in the methods how long the cells are fixed in PFA.\n\nEither in the introduction or the discussion, it could be noted that astrocytes express very high levels of APOE in the brain. This is important because it links APOE expression to the astroglial nature of adult NSCs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5838", "date": "24 Aug 2020", "name": "Sandrine Thuret", "role": "Author Response", "response": "<>Comment 1. If possible, provide higher magnification/higher quality images of APOE stainings, including also the other time points during differentiation. Day 7 would be particularly important to include, since it displays the highest levels of expression by QPCR.The authors would like to thank the reviewer for this comment. We agree that the resolution of the images in Figure 2 is not as high as the original data when the manuscript is viewed/downloaded online. In the updated manuscript, these images are shown in a separate figure (Figure 3). The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D18/19 cells. In addition, as indicated in the ‘Data availability’ section of the submitted manuscript, the original data with higher resolutions for all images can be found on Figshare, a public repository for scientific data, which can be accessed via the following DOI: https://doi.org/10.6084/m9.figshare.12199745.v1. We are confident that the patterns of APOE immunocytochemistry (ICC) reported in the manuscript can be confirmed with greater detail from the raw data available on Figshare.While the authors confirm that the ICC experiments were conducted for APOE on D7 cells, the data were not included in the manuscript due to the following reasons. According to the differentiation protocol, the cells were maintained at high density approaching near 100% confluence from D0 to D7. We observed that this inadvertently diminishes the quality of immunocytochemistry images for D7 cells, since clear boundaries of nuclei could not be easily identified with epifluorescence microscopy and further complicated the downstream quantification process. The possibility of dissociating D7 cells and plating them on to a different surface for better image quality and quantification was considered briefly. However, such additional handling was not done to the cells so that any potential source of artefacts that could mask the true state of D7 cells can be ruled out in our experiments. While the use of epifluorescence microscopy in our study can be seen as a clear limitation, APOE immunostaining patterns of D7 cells was not qualitatively different from that of D12 cells in our observations. Further investigations using three-dimensional imaging techniques such as confocal microscopy will enable better imaging and quantification of densely packed cells on D7. The ‘Discussion’ section in the updated manuscript now includes a new paragraph regarding this aspect.“One limitation of this study is that the time-dependent changes of differentiation markers such as SOX2 and TBR2 were not examined alongside APOE. It is worth noting, however, that TBR2 was shown to be capable of suppressing SOX2 expression during differentiation of NSCs to NPCs ( Hodge et al., 2012). Given this information, it is unlikely that TBR2-positive cells observed in this study at D18/19 will simultaneously express high levels of SOX2. However, time-dependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells. Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study. High-resolution microscopy techniques (such as confocal microscopy) would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction (D0-D7). Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.”We do not have the data for other time points during differentiation yet, and we agree with the reviewer that it would be highly informative to examine more time points. It would be particularly interesting to examine the time-course of APOE expression changes from the stem cell stage to the mature neuronal/glial stage. We hope that the data reported in our manuscript can serve as a foundation to such experiments to be conducted in the future. Comment 2. APOE stainings should be quantified to support the conclusions that its expression decreases during differentiation and there is a change in the protein localization. If this is not possible, the conclusions should be toned down and further experiments suggested in the discussion (for example, protein quantification by WB and cellular fractionation and quantification of protein in the media to assess intracellular protein localization and secretion, respectively).The authors would like to thank the reviewer for the comment on the quantification of ICC images. We now include a quantification of the images in the updated manuscript. The percentage of SOX2+ cells and TBR2+ cells at D12 and D18/19, as well as the percentage of SOX2+ and TBR2+ cells that showed immunostaining patterns for intracellular APOE were quantified using CellProfiler v3.1.9. The quantified data presented as bar graphs can be found in Figure 3; the analysis procedure can be found in the ‘Methods (Immunocytochemistry)’ section; and the analysis pipeline as well as the raw data for quantification can be found on Figshare which can be accessed by the following DOI: https://doi.org/10.6084/m9.figshare.12781064.v1. The results indicate a higher percentage of intracellular APOE/TBR2+ cells at D18/19 compared to intracellular APOE/SOX2+ cells at D12, supporting our conclusions made in the manuscript prior to the inclusion of these quantification data.To incorporate the reviewer’s suggestions, we have clearly stated in the ‘Discussion’ section of our updated manuscript that further investigations using other techniques should be done to quantify the levels of APOE protein. The following changes have been made in the ‘Discussion’.“In this study, qualitative analysis was performed on APOE immunocytochemistry results. As the cells became more differentiated from NSCs to NPCs, APOE localisation pattern became more intracellular. To validate this observation, however, additional experiments with a more direct quantitative approach should be conducted. For example, APOE protein levels in various subcellular compartments could be measured and compared by performing Western Blot. Since APOE has been shown to exist in both secreted and intracellular forms ( Huang & Mahley, 2014), it will be interesting to see which form of APOE is produced at each differentiation stage. It is possible that more APOE is secreted in undifferentiated cells compared to differentiated cells, which may not be fully captured using immunocytochemistry techniques performed on fixed cells. Interestingly, Gan and colleagues previously reported that APOE is indeed secreted by NSCs as well as NPCs, and secreted APOE was found to play a vital role in regulating NSC survival and neurosphere formation ( Gan et al., 2011). Therefore, further investigations on secreted and intracellular APOE using quantitative approaches will be able to clarify whether cells indeed produce different forms and levels of APOE depending on its differentiation state. This will in turn provide more definitive clues to whether APOE plays a stage-dependent role in neurodevelopment.”Furthermore, the conclusions have been amended as follows to reflect the changes in the ‘Discussion’.“In conclusion, we report that human APOE gene expression levels are highly correlated with the undifferentiated state of cells during directed differentiation in vitro, and ApoE protein is localised more in the intracellular region in cells at later stages of differentiation. Combining our observations and previous evidence reported in the literature, we speculate that APOE has an important role in stem cell maintenance and propose that further investigations should be carried out to validate our findings including methods that were not employed in this study. Moreover, it would be interesting to examine the exact underlying mechanisms such as 1) whether APOE is an upstream or downstream factor of stem cell maintenance, and 2) whether APOE4 genotype and APOE loss-of-function would produce similar phenotypes.” Comment 3. In the discussion, it should be noted that iPSC-derived NSCs might not fully resemble adult NSCs. A brief discussion of what is known about the expression of APOE in NSCs during development would be very useful.The authors would like to thank the reviewer for this comment on highlighting the differences between iPSC-derived NSCs and adult NSCs. To incorporate this aspect into our updated manuscript, we have now added a new paragraph in the ‘Discussion’ section as follows.“Since NSCs derived from iPSCs in vitro may not fully resemble the developmental and postnatal NSCs found in vivo, APOE expression should be further investigated in animal models of brain development as well. The most direct evidence of in vivo APOE expression in NSCs to this date comes from a study by Yang and colleagues, where Nestin-positive NSCs in the mouse developing dentate gyrus was isolated using fluorescence-activated cell sorting, and APOE expression was examined from as early as postnatal day 7 (P7) ( Yang et al., 2011). NSCs at P7 had low expression of APOE which increased with the age of mice, and the deletion of APOE had detrimental effects on the maintenance of stem cells in the dentate gyrus. Although these findings clearly demonstrate the importance of APOE in brain development, the study had limitations in that prenatal NSCs were not examined, and functional studies of APOE were based on global rather than conditional knockouts. Furthermore, Yang and colleagues’ data cannot be directly compared with our dataset due to species difference and the lack of detailed characterisation of NSCs in this study. To address this knowledge gap, more data from both in vitro and in vivo samples derived from various species should be generated and compared against each other. We hope that our focused study has laid a strong foundation to such collaborative investigations that may be conducted in the future.” <>Comment 1. Sox2 (neural stem and progenitor marker) and Tbr2 (neuronal progenitor marker) stainings are presented only at one stage each. It would be very informative to see if/how these two markers change over the course of the differentiation protocol. If not possible by immuno, a QPCR for these genes would also be enough to show the trends of expression during differentiation.The authors would like to thank the reviewer for pointing out the expression changes of SOX2 and TBR2. While we have not examined the time-dependent changes of these markers in this study, the authors can confirm that SOX2 and TBR2 expression was consistently observed at D12 and D18/19 by other experienced members of the lab using the differentiation protocols reported in this study. These data (although not shown in this study) were generated by qPCR, ICC, and microarray experiments that collectively show the expression of SOX2 and TBR2 similar to the ICC data reported in our manuscript. While we are confident with the SOX2 and TBR2 expression pattern in our study, we agree with the reviewer that APOE expression should be examined alongside the differentiation markers. To incorporate this into our updated manuscript, we have now included a new paragraph in the ‘Discussion’ section as follows to suggest that further investigations should be conducted to address this aspect.“One limitation of this study is that the time-dependent changes of differentiation markers such as SOX2 and TBR2 were not examined alongside APOE. It is worth noting, however, that TBR2 was shown to be capable of suppressing SOX2 expression during differentiation of NSCs to NPCs ( Hodge et al., 2012). Given this information, it is unlikely that TBR2-positive cells observed in this study at D18/19 will simultaneously express high levels of SOX2. However, time-dependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells. Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study. High-resolution microscopy techniques (such as confocal microscopy) would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction (D0-D7). Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.” Comment 2. Please specify in the methods how long the cells are fixed in PFA.The authors can confirm that the cells were fixed for 10 mins at room temperature with 4% PFA, and this has been now clearly stated in the ‘Methods’ section. We thank the reviewer for pointing this out.“Cells were fixed with 4% paraformaldehyde for 10 mins at room temperature, permeabilized with 0.1% Triton™ X-100 in 1X Tris-buffered saline (TBS) for 15–30 minutes, and then blocked with 5% normal donkey serum in TBS for 30 minutes. Primary antibodies were incubated at 4°C overnight followed by 3 washings with TBS. Secondary antibodies conjugated with fluorescent dyes were incubated at room temperature for 1 hours followed by 2 washings with TBS. Nuclei were stained with 5 µg/mL Hoechst® 33342 solution (Thermo Fisher) for 30 seconds immediately prior to imaging, and cells were washed with TBS 2 times after nuclear staining. All primary antibodies were diluted in 5% normal donkey serum in TBS, secondary antibodies in 1% normal donkey serum in TBS, and Hoechst® 33342 solution in TBS. Imaging was done with IX 70 inverted epifluorescence microscope (Olympus) connected to AxioVision imaging software (version 4.4). Scale bars were inserted on the images using ImageJ software (version 1.49c). CellProfiler (version 3.1.9) was used to quantify the percentage of cells immunopositive for SOX2, TBR2, and APOE at the intracellular regions. Raw dataset for the images is available as an underlying data via Figshare (doi: 10.6084/m9.figshare.12199745.v1). Raw dataset for the quantification is available as an underlying data via Figshare (doi: 10.6084/m9.figshare.12781604.v1).” Comment 3. Either in the introduction or the discussion, it could be noted that astrocytes express very high levels of APOE in the brain. This is important because it links APOE expression to the astroglial nature of adult NSCs.The authors would like to thank the reviewer’s comment on the existing evidence of APOE expression in astrocytes. To incorporate this aspect into our updated manuscript, the ‘Introduction’ section has been amended as follows.“Apolipoprotein E (APOE) is a pleiotropic protein that plays an important role in lipid metabolism ( Mahley & Rall, 2000) and is highly expressed in the brain mainly by glial cells ( Elshourbagy et al., 1985, Boyles et al., 1985). Although the primary function of APOE is lipid transport, its expression is also found in other cell types outside the context of lipid metabolism in the brain ( Liao et al., 2017). For example, a recent single-cell RNA sequencing study on human post-mortem Alzheimer’s disease (AD) brains showed that activated microglia (relevant to the disease state) express high levels of APOE unlike naïve microglia (relevant to healthy/homeostatic state) in the prefrontal cortex, indicating that APOE expression is associated with immune function ( Mathys et al., 2019). Furthermore, neuronal APOE can also be expressed at high levels under stress conditions such as brain injury although APOE expression is normally low in healthy neurons ( Mahley & Huang, 2012; Xu et al., 2006). Interestingly, APOE is highly expressed in nestin/glial fibrillary acidic protein (GFAP) double-positive neural stem cells (NSCs) in the adult hippocampus of mice, and one of the phenotypes characterised in APOE-null mice is the premature depletion of NSC pool in the hippocampus, suggesting that NSC maintenance requires APOE expression ( Yang et al., 2011).”" } ] }, { "id": "65479", "date": "10 Jul 2020", "name": "Jade Marsh", "expertise": [ "Reviewer Expertise Molecular and cellular neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nPrevious studies in mice have shown that APOE is highly expressed by neural stem cells (NSCs) and that it has an isoform-dependent regulatory role in their maintenance. Whether this function extends to human NSCs is not known. Here, the authors begin to address this knowledge gap by characterising the expression of APOE in APOE3/APOE3 human induced pluripotent stem cell (iPSC)-derived NSCs undergoing differentiation. Through qPCR and immunocytochemistry (ICC) techniques, the authors show a reduction in APOE gene expression during the differentiation of NSCs to NPCs and observe a change in the cellular distribution of APOE.\n\nGeneral comments:\nThis short study is concise and provides preliminary but important data to contribute to our understanding of the role of APOE in NSCs of human origin. Although the qPCR data is robust and clearly demonstrates a significant decrease in APOE gene expression as NSCs differentiate, the qualitative assessment of the ICC data is not as convincing.\n\nSpecific comments:\nFig 2 C: APOE appears to be more widely expressed at D18/19 for all three NSC lineages (DSi, DS-Wi and DS-WHi). Quantification of the signal intensities of APOE and the differentiation markers should be carried out to validate the conclusion that APOE expression decreases as NSCs differentiate.\n\nFig 2 C: It may be more helpful and striking to compare D18/19 images with D7 images instead of D12 since APOE mRNA levels are highest at this time point. Notably, D7 is also used as the baseline for the qPCR data.\n\nThe authors describe an increase in intracellular localisation of APOE following NSC differentiation – providing higher magnification images may reveal changes in APOE distribution more clearly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5839", "date": "24 Aug 2020", "name": "Sandrine Thuret", "role": "Author Response", "response": "Comment 1. Fig 2 C: APOE appears to be more widely expressed at D18/19 for all three NSC lineages (DSi, DS-Wi and DS-WHi). Quantification of the signal intensities of APOE and the differentiation markers should be carried out to validate the conclusion that APOE expression decreases as NSCs differentiate.The authors would like to thank the reviewer for the comment on the quantification of ICC images. We now include a quantification of the images in the updated manuscript. The percentage of SOX2+ cells and TBR2+ cells at D12 and D18/19, as well as the percentage of SOX2+ and TBR2+ cells that showed immunostaining patterns for intracellular APOE were quantified using CellProfiler v3.1.9. The quantified data presented as bar graphs can be found in Figure 3; the analysis procedure can be found in the ‘Methods (Immunocytochemistry)’ section; and the analysis pipeline as well as the raw data for quantification can be found on Figshare which can be accessed by the following DOI: https://doi.org/10.6084/m9.figshare.12781064.v1. The results indicate a higher percentage of intracellular APOE/TBR2+ cells at D18/19 compared to intracellular APOE/SOX2+ cells at D12, supporting our conclusions made in the manuscript prior to the inclusion of these quantification data. Comment 2. Fig 2 C: It may be more helpful and striking to compare D18/19 images with D7 images instead of D12 since APOE mRNA levels are highest at this time point. Notably, D7 is also used as the baseline for the qPCR data.The authors would like to thank the reviewer for mentioning this important aspect of the ICC experiment reported in our manuscript. While the authors confirm that the ICC experiments were conducted for APOE on D7 cells, the data were not included in the manuscript due to the following reasons. According to the differentiation protocol, the cells were maintained at high density approaching near 100% confluence from D0 to D7. We observed that this inadvertently diminishes the quality of immunocytochemistry images for D7 cells, since clear boundaries of nuclei could not be easily identified with epifluorescence microscopy and further complicated the downstream quantification process. The possibility of dissociating D7 cells and plating them on to a different surface for better image quality and quantification was considered briefly. However, such additional handling was not done to the cells so that any potential source of artefacts that could mask the true state of D7 cells can be ruled out in our experiments. While the use of epifluorescence microscopy in our study can be seen as a clear limitation, APOE immunostaining patterns of D7 cells was not qualitatively different from that of D12 cells in our observations. Further investigations using three-dimensional imaging techniques such as confocal microscopy will enable better imaging and quantification of densely packed cells on D7. The ‘Discussion’ section in the updated manuscript now includes a new paragraph regarding this aspect.“One limitation of this study is that the time-dependent changes of differentiation markers such as SOX2 and TBR2 were not examined alongside APOE. It is worth noting, however, that TBR2 was shown to be capable of suppressing SOX2 expression during differentiation of NSCs to NPCs ( Hodge et al., 2012). Given this information, it is unlikely that TBR2-positive cells observed in this study at D18/19 will simultaneously express high levels of SOX2. However, time-dependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells. Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study. High-resolution microscopy techniques (such as confocal microscopy) would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction (D0-D7). Further investigations with improved imaging capacity will therefore allow us to characterise APOE during the earlier stages of neural induction and hint at potential mechanisms underlying its role in neurodevelopment.”Comment 3. The authors describe an increase in intracellular localisation of APOE following NSC differentiation – providing higher magnification images may reveal changes in APOE distribution more clearly.The authors would like to thank the reviewer for this comment. We agree that the resolution of the images in Figure 2 is not as high as the original data when the manuscript is viewed/downloaded online. In the updated manuscript, these images are shown in a separate figure (Figure 3). The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D18/19 cells. In addition, as indicated in the ‘Data availability’ section of the submitted manuscript, the original data with higher resolutions for all images can be found on Figshare, a public repository for scientific data, which can be accessed via the following DOI: https://doi.org/10.6084/m9.figshare.12199745.v1. We are confident that the patterns of APOE immunocytochemistry (ICC) reported in the manuscript can be confirmed with greater detail from the raw data available on Figshare." } ] } ]	1	https://f1000research.com/articles/9-353
https://f1000research.com/articles/9-491/v1	02 Jun 20	{ "type": "Case Report", "title": "Case Report: Use of hydroxychloroquine and N-acetylcysteine for treatment of a COVID-19 positive patient", "authors": [ "Carlos Puyo", "Danielle Kreig", "Venugopal Saddi", "Essam Ansari", "Oliver Prince", "Danielle Kreig", "Venugopal Saddi", "Essam Ansari", "Oliver Prince" ], "abstract": "There is worldwide concern for lack of specific therapy against the novel Betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This case report presents the results of a pharmacological intervention aimed at modulating the inflammatory effects of coronavirus disease 2019 (COVID-19), in an effort to avoid the use of mechanical ventilation. A COVID-19 positive patient was admitted with multisystem organ dysfunction, including acute respiratory insufficiency, and was treated with a combination of low oral doses of hydroxychloroquine and intravenous N-acetylcysteine (NAC). The combination therapy resulted in noticeable clinical improvement and a quantifiable decrease of several of the inflammatory markers measured, in particular ferritin levels, C-reactive protein (CRP) and lactic acid. He also developed pulmonary embolism (PE) and deep vein thrombosis (DVT), both known side effects of COVID-19 infection. Following thrombolysis and heparinization his clinical evolution continued a positive trend until discharge.\nThe therapeutic approach utilized in this case suggests that early intervention not only decrease acute organ dysfunction but also may decrease the need for mechanical ventilation in COVID-19 positive patients.", "keywords": [ "COVID-19", "Hydroxychloroquine", "N-Acetylcysteine" ], "content": "Background\n\nThe outbreak of pneumonia in Wuhan, China has been correlated with a novel coronavirus, COVID-19, isolated in January 20201. Human to human transmission has reached pandemic levels with cases infecting millions of individuals resulting in significant morbidity and mortality. Although multiple therapies have been proposed against the COVID-19 virus, no clear consensus exists on the best approach for treatment2,3.\n\nThe human body requires an efficient innate immune system in the airway mucosa to respond to viral or bacterial antigens and preserve tissue homeostasis. COVID-19 enters the human airway in a process reminiscent of other viruses4,5. The virus invades healthy cells, replicates, and leads to cellular necrosis6. Neutrophils are essential for a proper innate response to antigens derived from cellular necrosis7. We previously demonstrated that restoring the capacity of the innate immune system by modulating neutrophil activity with hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) was sufficient to ameliorate local tissue effects of cellular necrosis and inflammation7 HCQ is a well-known therapy for certain inflammatory autoimmune diseases such as rheumatoid arthritis and lupus erythematosus and has significant impact on Toll-like receptor 9 (TLR-9) activity8. NAC has been used as an antioxidant, as a modulator of inflammatory responses due to its actions on NF-κβ9, as a mucolytic agent, and for the treatment of acetaminophen-induced liver failure10. In a patient with COVID-19 infection, we used oral low-dose HCQ in combination with intravenous NAC in an effort to modulate the inflammatory response secondary to COVID-19.\n\n\nCase report\n\nWe describe a 54-year-old Caucasian male patient, with past medical history significant for hypertension, hyperlipidemia, and obesity, who tested positive for COVID-19 by reverse-transcriptase-polymerase-chain-reaction (RT-PCR) 11 days prior to his admission on mid April, 2020 (Table 1) at Holy Family Hospital in Methuen, Massachusetts. Upon presentation, he was admitted to the Intensive Care Unit with shortness of breath, body aches, fever, diaphoresis, tachypnea, low oxygen saturation of 92% requiring oxygen supplementation via non-rebreather mask, elevated lactic acid of 7.6 (0.5–2.2 mmol/L), and hyperglycemia with blood glucose of 402 (<100 mg/dL fasting). His vital signs included initial blood pressure of 92/62 mmHg (72 MAP), respiratory rate of 48 bpm, heart rate 120-130 bpm, and temperature of 97°F.\n\nInitial laboratory work-up showed elevations of inflammatory markers common to patients diagnosed with COVID-197–9 (Table 2); particularly lymphocytes 900 (850-3900 cells per mm3), high-sensitivity C-reactive protein 149.2 (1.0–3.0mg/L), D-dimer 16.47 (<0.5 μg/ml FEU), lactate dehydrogenase 1579 (102–266 U/L), and serum ferritin 23713 (30–400 ng/L). A noticeable decrease in lactic acid 7.6 to 2.4 (0.5–2.2 mmol/L) was observed in the first 24 hours of treatment. He also showed signs of multi-system end-organ damage, as evidenced by elevations in alanine aminotransferase 1017 (14–63 U/L), aspartate amino transferase 852 (15–41 U/L), and serum creatinine 1.4 (0.6–1.4 mg/dL). At presentation, lung auscultation was remarkable for scattered rales, chest radiograph was unremarkable, and abdominal exam was normal.\n\n* Day 0 denotes assessments on the morning prior to N-acetylcysteine administration\n\n† Not determined\n\nDespite escalating oxygen requirements, intubation was delayed as the patient was assessed to be stable. The patient was prescribed HCQ 400 mg, given as a single oral dose, and NAC intravenously at 75 mg/kg over 4 hours, then 35 mg/kg over 16 hours, followed by 17 mg/kg over 24 hours on Day 2. Prophylactic anticoagulation was started with subcutaneous heparin. An additional 200 mg dose of HCQ was given on Day 2. No cardiac arrhythmia was noticed with either dose of HCQ, with the highest measured corrected QT interval documented at 0.49 (0.36–0.44 seconds).\n\nThis patient initially experienced progressive clinical improvement; however, bilateral pulmonary embolism (PE) and right lower extremity popliteal deep venous thrombosis were diagnosed in the setting of persistently elevated D-dimer. A heparin infusion was started, and PE embolization was complicated by severe hypoxemia requiring mechanical ventilation. After three days of mechanical ventilation and catheter-directed thrombolysis, he was successfully extubated and transferred to a general medicine floor on Day 7. The patient was discharged home on Day 12 with stable vital signs, normalizing laboratory values, and on therapeutic anticoagulation with rivaroxaban. COVID-19 RT-PCR prior to discharge was negative.\n\n\nDiscussion\n\nCOVID-19 infection is characterized by multisystem organ involvement as illustrated in the present case yet no universally accepted standard therapy is available. It is theorized that COVID-19 causes the human immune system to overcompensate in response to infection and inflict collateral damage on itself, as evidenced by the abnormalities in inflammatory markers11–13 COVID-19 also appears to increase the risk of thrombotic events14,15. Previous work has shown that HCQ and NAC can modulate the innate immune system7,8, as well as reduce hypercoagulability and inhibit thrombosis16,17. We recognize that HCQ has been associated with a higher risk of cardiac abnormalities and fatal heart rhythms18; however, our low-dose strategy allowed us to take advantage of its potential benefits and long half-life. NAC has been shown to be safe at doses up to 980 mg/kg over 48 hours when used for acetaminophen overdose10. Because of this, we theorized that the administration of HCQ and NAC would be well-tolerated and have favorable effects on patient outcomes.\n\nAfter the patient above received HCQ/NAC, clinical improvement was observed, and laboratory reports followed a similar pattern. Interestingly, use of low-dose HCQ in combination with intravenous NAC appeared to positively influence this patient’s clinical course. HCQ, with its lysosomal activity and impact on TLR-9, and NAC, with its anti-inflammatory activity via NF-κβ modulation, antioxidant activity, and glutathione replenishment, may provide a therapeutic combination capable of enhancing the activity of the innate immune system to combat viral invasion.\n\n\nConclusions\n\nEarly therapeutic intervention with modulators of the innate immune system such as HCQ (low doses) and NAC, appear to mitigate the effects of multisystem organ dysfunction observed in COVID-19 positive patients. Avoidance of mechanical ventilation may represent a secondary benefit of this therapy. A randomized clinical trial is warranted to further evaluate the benefits of HCQ/NAC combination for COVID-19 treatment.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanders JM, Monogue ML, Jodlowski TZ, et al.: Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review. JAMA. 2020; 323(18): 1824–1836. PubMed Abstract \| Publisher Full Text\n\nBhimraj A, Morgan RL, Shumaker AH, et al.: Infectious Diseases Society of America Guidelines on the Treatment and Management of Patients with COVID-19. Clin Infect Dis. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen FS: How Viruses Invade Cells. Biophys J. 2016; 110(5): 1028–1032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobert J: Mason Pathogenesis of COVID-19 from a cell biology perspective. Eur Respir J. 2020; 55(4): 2000607. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarga Z, Flammer AJ, Steiger P, et al.: Endothelial cell infection and endotheliitis in COVID-19. Lancet. 2020; 395(0234): 1417–1418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuyo CA, Earhart A, Staten N, et al.: Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF-κB activity. J Leukoc Biol. 2019; 105(3): 577–587. PubMed Abstract \| Publisher Full Text\n\nEwald SE, Lee BL, Lau L, et al.: The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature. 2008; 456(7222): 658–662. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpapen H, Zhang H, Demanet C, et al.: Does N-acetyl-L-cysteine influence cytokine response during early human septic shock?. Chest. 1998; 113(6): 1616–1624. PubMed Abstract \| Publisher Full Text\n\nHeard K, Rumack BH, Green JL: A single-arm clinical trial of a 48-hour intravenous N-acetylcysteine protocol for treatment of acetaminophen poisoning. Clin Toxicol (Phila). 2014; 52(5): 512–8. PubMed Abstract \| Publisher Full Text\n\nZhou F, Yu T, Ronghui D, et al.: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet. 2020; 395(10229): 1054–1062. PubMed Abstract \| Publisher Full Text\n\nHenderson LA, Schulert GS, Caricchio R, et al.: On the alert for cytokine storm: Immunopathology in COVID-19. Arthritis Rheumatol. 2020. PubMed Abstract \| Publisher Full Text\n\nVelavan TP, Meyer CG: Mild versus severe COVID-19: Laboratory markers. Int J Infect Dis. 2020; 95: 304–307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBikdeli B, Madhavan MV, Jimenez D, et al.: COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-up. J Am Coll Cardiol. 2020; S0735-1097(20)35008-7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlok FA, Kruip MJHA, van der Meer NJM, et al.: Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb Res. 2020; S0049-3848(20)30120-1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoone BA, Murthy P, Miller-Ocuin J, et al.: Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. BMC Cancer. 2018; 18(1): 678. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCraver BM, Ramanathan G, Hoang S, et al.: N-acetylcysteine inhibits thrombosis in a murine model of myeloproliferative neoplasm. Blood Adv. 2020; 4(2): 312–321. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorba MGS, Val FFA, Sampaio VS, et al.: Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial. JAMA Netw Open. 2020; 3(4): e208857. PubMed Abstract \| Publisher Full Text" }	[ { "id": "65157", "date": "09 Jul 2020", "name": "Bin Cao", "expertise": [ "Reviewer Expertise Pulmonary and critical care medicine", "Infectious diseases" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPuyo et al. presented the clinical course of a severe COVID-19 patient treated by hydroxychloroquine, N-acetylcysteine and supportive care. The insufficient details and case report nature of the study hamper a reliable causal inference for the efficacy and safety of hydroxychloroquine and N-acetylcysteine. Given the information provided in the manuscript, the influence of other factors (natural disease progression, effects of other treatments, etc.) cannot be excluded. Although we are in urgent need of treatment for COVID-19 in such a pandemic, inappropriate interpretation of the result may be misleading and cause unnecessary resources wasting at this time point. It should be very cautious to try to associate the recovery of the patient with these two drugs.\nSpecific comments are listed below:\n\n1. According to the background and discussion part, the authors’ inference for the potential efficacy of the two drugs are mainly established on evidence from pre-clinical experiments. However, clinical evidences from COVID-19 or other respiratory infectious diseases should also be mentioned and discussed in the manuscript.\nFor hydroxychloroquine, the results of several studies have been released. The RECOVERY trial was reported to find no clinical benefit from use of hydroxychloroquine in hospitalized COVID-19 patients (28-day mortality, length of hospitalization, etc.)1. A published RCT in BMJ from Tang et al found that the use of hydroxychloroquine did not increase the proportion of virus negative conversion2. Also, the interim analysis of WHO solidarity trial showed little or no reduction in the mortality of hospitalized COVID-19 patients in the hydroxychloroquine arm and WHO thus discontinued the hydroxychloroquine arm for hospitalized patients3. Although the dosage of hydroxychloroquine varies in different trials and might be higher than the dosage used in this case report (400mg/600mg per day), it is hard to attribute the efficacy to hydroxychloroquine given the accumulating clinical evidences. The authors should provide clinical evidences in the manuscript in discussion to defend their hypothesis.\n\nFor N-acetylcysteine, the authors also mainly provide evidence from pre-clinical experiments. If there is no direct evidence from COVID-19, the authors should carefully review the literature and present possible indirect clinical evidence from severe pneumonia or viral pneumonia (if there is any).\n\n2. Details of the case: The authors only described in detail about hydroxychloroquine and N-acetylcysteine and details of other treatments were not provided. The authors should provide the details of all the important treatments to give readers a whole picture of the patient’s disease course. For example:\nCOVID-19 patients are often presented with normal or decreased white blood cell (WBC) counts and lymphocyte counts4,5. This patient had elevated WBC count, elevated neutrophil count and normal lymphocyte count (according to the provided normal range). Though the procalcitonin was lower than 0.5 ng/mL, the possibility of co-bacterial infection could not be excluded. The authors should provide results of lower respiratory tract etiological testing (sputum bacteriology, etc.) and blood culture. Besides, whether the patient received antibiotics and (if so), the details of antibiotics should also be described.\n\nAccording to the clinical information provided in the manuscript, the patient’s qSOFA score at admission was 2 (Blood Pressure 92/62mmHg; Respiratory Rate 48 times per mintue). The patients showed signs of sepsis or even septic shock. However, the provided information was not enough, and I am wondering whether the diagnosis of sepsis was made across the disease course and whether guideline-based treatment was given. More details should be described. Besides, if sepsis-related treatment is given, the role of hydroxychloroquine and N-acetylcysteine should be further questioned.\n\n“Prophylactic anticoagulation was started with subcutaneous heparin”. The dosage of heparin should be specified.\n\n3. Please distinguish between the disease name “COVID-19” and the virus name “SARS-CoV-2”, and use the right word in the manuscript. For example, “COVID-19 positive patient” should be revised to “COVID-19 patient”; “COVID-19 enters the human airway in a process reminiscent of other viruses” should be revised to “SARS-CoV-2 enters…”; “COVID-19 infection” should be revised to “SARS-CoV-2 infection”; “COVID-19 RT-PCR” should be revised to “SARS-CoV-2 RT-PCR”.\n\nThe objective of the case report is to present hypothesis for the potential efficacy of hydroxychloroquine and N-acetylcysteine. However, given the rationale mentioned above, at this stage, the information provided in the manuscript did not support such hypothesis.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5833", "date": "24 Aug 2020", "name": "Carlos Puyo", "role": "Author Response", "response": "August 18, 2020 Drs. Bin Cao, M.D., and Vui Heng Chong, M.D. Reviewers F1000Research Dear Drs, Cao and Chong: We are very grateful for your insightful comments on our Case Report “Use of hydroxychloroquine and N-Acetylcysteine for treatment of a COVID-19 patient”. We have given careful consideration to the points raised by you during the review process. The case report has been adjusted to reflect our comments as indicated in the manuscript using a tracking system. We look forward to your response and hope to complete the publication of our Case Report in F1000Research. Sincerely, Carlos A. Puyo, M.D., FCCP Specific comments are listed below: 1. According to the background and discussion part, the authors’ inference for the potential efficacy of the two drugs are mainly established on evidence from pre-clinical experiments. However, clinical evidences from COVID-19 or other respiratory infectious diseases should also be mentioned and discussed in the manuscript. For hydroxychloroquine, the results of several studies have been released. The RECOVERY trial was reported to find no clinical benefit from use of hydroxychloroquine in hospitalized COVID-19 patients (28-day mortality, length of hospitalization, etc.)1. A published RCT in BMJ from Tang et al found that the use of hydroxychloroquine did not increase the proportion of virus negative conversion2. Also, the interim analysis of WHO solidarity trial showed little or no reduction in the mortality of hospitalized COVID-19 patients in the hydroxychloroquine arm and WHO thus discontinued the hydroxychloroquine arm for hospitalized patients3. Although the dosage of hydroxychloroquine varies in different trials and might be higher than the dosage used in this case report (400mg/600mg per day), it is hard to attribute the efficacy to hydroxychloroquine given the accumulating clinical evidences. The authors should provide clinical evidences in the manuscript in discussion to defend their hypothesis. Response to reviewers: The release of several studies promoting or discrediting the use of chloroquine or hydroxychloroquine for the treatment of COVID-19 has created confusion in the medical community. Here, the reviewer duly noted studies showing lack of or questionable therapeutic benefit1,2, however, there is no mention of studies that have shown benefit3,4,5,6,7. The RCT by Tang, et al, contains several shortcomings among them the use of an open label instead of a double blind approach, potentially introducing bias by the researchers. Similarly, the underpowered sample size and enrollment of patients in more advance stages of the disease likely influence negatively their assessment of the true potential value of HCQ in the disease progression. In the RECOVERY trial mentioned by the reviewer, the doses of HCQ 2400 mg in 24 hours followed by 1600 mg daily for at least 9 days, are indeed higher than for malaria treatment, thus exposing patients to unnecessary risks. It is unclear what scientific bases were utilized to determine the exceedingly high doses of HCQ administered. The Indian Council of Medical Research alerted the WHO about the excessive doses of HCQ (4 times higher) used in the RECOVERY trial, thus, possibly introducing bias in the comparative analysis. The RECOVERY trial also has some interesting findings regarding their unexplained very high mortality data in the control group (23.6%), compared to the 12.7% and 13.5% reported by others in treating high acuity hospitalized patients4,8. Interpretation of HCQ studies is difficult, in part due to the variety of end points utilized by the researchers. For instance some studies used mortality or pneumonia1, while others used negative viral conversion2 as the end points of the studies, thus making a comparative analysis on the effects of HCQ a complex one. In some cases reputable journals (Lancet and others) had to RETRACT anti-HCQ studies9-10 for a variety of reasons, further confounding the data analysishttps://c19study.com/. Discussions regarding the clinical use of HCQ11 need to account for the intracellular activity of this compound to avoid toxicity and to attain therapeutic benefit. Infections due to SARS-CoV-2 alter cellular homeostasis leading to cytotoxicity and cell death. HCQ may impact viral activity at multiple levels by blocking viral entry into the cells, interfering with endosomal maturation, and lysosomal transport5. Furthermore, HCQ may also serve as an immune-modulator through its action on cytokine activity12. The human oral absorption of HCQ/CQ reach significant concentrations in various tissues including the lungs, and has been reported to be 200-700 times higher than plasma levels13. HCQ given at a dose of 6-6.5mg/kg per day would generate serum levels of 1.4-1.5 mM in humans14, sufficient to inhibit SARS-CoV-2 infection3,5. Also the prolong half-life of HCQ (1440 hours) and the distribution on intercellular compartments (1300 hours)15, needs to be accounted as a factor in prescribing the treatment. Ultimately, the therapeutic goal in our treatments is to contain the viral invasion and prevent amplification of the infectious and inflammatory damage. Generation of reactive oxygen species (ROS) capable of inducing cellular apoptosis, necrosis and tissue destruction has been documented in viral infections16, and SARS-CoV-2 is not an exception. We have shown in an in vitro sterile injury model of cellular necrosis (neutrophils) that HCQ was capable of modulating ROS, and Toll-like receptor 9 (TLR9) activity mediated by the released of Damage Associated Molecular Patterns (DAMPS) in particular mitochondrial DNA (mtDNA)17. Other studies have shown that cellular injury mediated by viral invasion may release other DAMPS such as High Mobility Group B1 (HMGB1) during cellular damage18. Activity of HCQ on other inflammatory pathways such as cyclic-GMP-AMP (cGAMP) synthase (cGAS), or by interfering with interferon activity for example, needs to be analyzed as factors involved in generation of acute or chronic symptoms induced by viral infections19. We are in urgent need to use HCQ at effective low doses as indicated by various basic science research groups thus preventing the occurrence of clinical toxicity observed worldwide. There is no sufficient evidence for or against completely eliminating HCQ as a therapeutic intervention during SARS-CoV-2. HCQ in combination with NAC needs to be considered a possible option to treat viral diseases. Studies that have used HCQ need to be analyzed for their potential benefit in post-viral chronic disease. No clinical benefit from use of hydroxychloroquine in hospitalised patients with COVID-19. University of Oxford. 2020. Reference Source Tang W, Cao Z, Han M, Wang Z, et al.: Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ. 2020. Publisher Full Text Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0 Arshad S, Kilgore P, Chaudhry ZS, et al. Treatment with Hydroxychloroquine, Azithromycin, and Combination in Patients Hospitalized with COVID-19 [published online ahead of print, 2020 Jul 2]. Int J Infect Dis. 2020;97:396-403. doi:10.1016/j.ijid.2020.06.099 Wang M., Cao R., Zhang L. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30(3):269–271. Gao J., Tian Z., Yang X. Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends. 2020;14(1):72–73. Chen Z, Hu J, Zhang Z, et al. Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial. medRxiv 2020. doi: 10.1101/2020.03.22.20040758. Rosenberg ES, Dufort EM, Udo T, et al. Association of Treatment With Hydroxychloroquine or Azithromycin With In-Hospital Mortality in Patients With COVID-19 in New York State. JAMA. 2020;323(24):2493–2502. doi:10.1001/jama.2020.8630 Mehra MR, Desai SS, Ruschitzka F, Patel AN RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. Lancet. 2020; (published online May 22.) https://doi.org/10.1016/S0140-6736(20)31180-6 Funck-Brentano C, Salem J-E. RETRACTED: Chloroquine or hydroxychloroquine for COVID-19: why might they be hazardous?.Lancet. 2020; (published online May 22.)https://doi.org/10.1016/S0140-6736(20)31174-0 Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0 van den Borne BE, Dijkmans BA, de Rooij HH, le Cessie S, Verweij CL. Chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin 6, and interferon-gamma production by peripheral blood mononuclear cells. J Rheumatol. 1997;24(1):55-60. POPERT AJ. CHLOROQUINE: A REVIEW Rheumatology, Volume 15, Issue 3, August 1976, Pages 235–238, https://doi.org/10.1093/rheumatology/15.3.235 Laaksonen AL, Koskiahde V, Juva K. Dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. A clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine. Scand J Rheumatol. 1974;3(2):103-108. doi:10.3109/03009747409115809 Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020;16(3):155-166. doi:10.1038/s41584-020-0372-x Tan YJ, Lim SG, Hong W. Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses. Cell Microbiol. 2007;9(11):2552-2561. doi:10.1111/j.1462-5822.2007.01034.x Puyo CA, Earhart A, Staten N, et al. Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF-κB activity. J Leukoc Biol. 2019;105(3):577-587. doi:10.1002/JLB.5A0718-254RR Moisy D, Avilov SV, Jacob Y, et al. HMGB1 protein binds to influenza virus nucleoprotein and promotes viral replication. J Virol. 2012;86(17):9122-9133. doi:10.1128/JVI.00789-12 Shu C, Li X, Li P. The mechanism of double-stranded DNA sensing through the cGAS-STING pathway. Cytokine Growth Factor Rev. 2014;25(6):641-648. doi:10.1016/j.cytogfr.2014.06.006 For N-acetylcysteine, the authors also mainly provide evidence from pre-clinical experiments. If there is no direct evidence from COVID-19, the authors should carefully review the literature and present possible indirect clinical evidence from severe pneumonia or viral pneumonia (if there is any). Response to reviewers: N-Acetylcysteine (NAC) is an antioxidant, precursor of reduced glutathione, and modulator of transcription nuclear factor-κβ (NF-κβ) activity with a potential therapeutic profile for use in prevention or as adjuvant treatment for SARS-CoV-2. Viral infections induce cytotoxicity, cell death, overproduction of ROS and cytokines, thus promoting cellular and organ damage. NAC interferes with viral replication of human coronaviruses HCoV-229E1, influenza H5N12, human immunodeficiency virus (HIV)3, and respiratory syncytial virus (RSV)4, among others. Recently, it was shown that NAC inhibited SARS-CoV-2 replication by binding to Cys-145, which is an active site for the main protease (Mpro) involved in viral activity5,6. Several in vitro studies have shown other potential mechanisms of NAC anti-viral activity that include: NF-κβ modulation (cytokine modulation)7, interference with angiotensin II type 1 receptors8, direct inhibition of furin (protease involved in the viral fusion process) 9, and interaction with disulfite bonds10. Pneumonia is a severe manifestation of SARS-CoV-2, SARS-CoV-1, and Middle East Respiratory Syndrome (MERS-CoV), with all sharing similar CT-scan findings of bilateral patchy infiltrates or ground glass opacities. NAC may modulate the effects of viral pneumonia induced by the cytotoxic effects of viral proliferation, cellular apoptosis, necrosis and excessive production of ROS that promote release of proinflammatory cytokines7,10. Other proposed mechanisms of action related to NAC include preservation of T cell activity and inhibition of the NOD-LRR- and pyrin domain-containing protein 3 (NLRP3) inflammatory pathway in monocytes and macrophages during viral respiratory infections11,12. Promising results have been reported with the use of NAC for influenza, ARDS and VAP as measured by length of hospital stay and disease severity13,14. The therapeutic potential for NAC during viral infections and in particular in SARS-CoV-2 is supported by in vitro and indirectly by clinical studies (coronavirus, influenza). We are proposing the use of NAC during viral infections as adjuvant therapy with broad impact on viral activity, with low toxicity and cost. Certainly, further studies are necessary to document the cellular activity as well as the clinical benefits that NAC may provide. The combination of low dose HCQ (total dose 600mg) and short-term NAC administered IV had a positive impact on the treatment of this patient and merits evaluation in large scale randomized clinical studies. The administration of both compounds orally during preintubation periods may attenuate viral symptomatology and possibly decrease the need for intubation. Poppe M, Wittig S, Jurida L, et al. The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. Plos Pathogens. 2017; doi.org/10.1371/journal.ppat.1006286 Geiler J, Michaelis M, Naczk P, et al. N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus. Biochemical Pharmacology. 2010;79:413–420 Ho W, Douglas SD. Glutathione and N-Acetylcysteine suppression of human immunodeficiency virus replication in human Monocyte/Macrophages in vitro. AIDS Research and Human Retroviruses. 1992;1249-1253. Mata M, Sarrion I, Armengot M, et al. Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-Acetylcysteine. PLOS One. 2012;7(10) e48037 Zhang L, Lin D, Sun X, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020;368, 409–412 Guthappa R. Molecular Docking Studies of N-Acetyl Cysteine, Zinc Acetyl Cysteine and Niclosamide on SARS Cov 2 Protease and Its Comparison with Hydroxychloroquine. Chemarxiv. 2020;doi.org/10.26434/chemrxiv.12161493.v1 Paranjpe A, Cacalano NA, Hume WR, Jewett A. N-acetyl cysteine mediates protection from 2-hydroxyethyl methacrylate induced apoptosis via nuclear factor kappa B-dependent and independent pathways: potential involvement of JNK. Toxicol Sci. 2009;108(2):356-366. doi:10.1093/toxsci/kfp010 Ullian ME, Gelasco AK, Fitzgibbon WR, Beck CN, Morinelli TA. N-acetylcysteine decreases angiotensin II receptor binding in vascular smooth muscle cells. J. Am. Soc. Nephrol. 16(8), 2346–2353 (2005) Jorge-Aarón RM, Rosa-Ester MP. N-acetylcysteine as a potential treatment for novel coronavirus disease 2019 [published online ahead of print, 2020 Jul 14]. Future Microbiol. 2020;10.2217/fmb-2020-0074. doi:10.2217/fmb-2020-0074 Schoeman D, Fielding BC. Coronavirus envelope protein: current knowledge. Virol. J. 16(1), 1–22 (2019). Poe FL, Corn J. N-Acetylcysteine: A potential therapeutic agent for SARS-CoV-2 [published online ahead of print, 2020 May 30]. Med Hypotheses. 2020;143:109862. doi:10.1016/j.mehy.2020.109862 Rangel-Méndez JA, Moo-Puc RE. N-acetylcysteine as a potential treatment for novel coronavirus disease 2019 Published Online:14 Jul 2020https://doi.org/10.2217/fmb-2020-0074 De Flora S, Grassi C, Carati L. Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-tern N-acetylcysteine treatment. Eur Respir J 1997; 10: 1535-1541. DOI: 10.1183/09031936.97.10071535 Sharafkhah M, Abdolrazaghnejad A, Zarinfar N, et al. Safety and efficacy of N-acetylcysteine for prophylaxis of ventilator-associated pneumonia: a randomized, double blind, placebo-controlled clinical trial. Mojtaba Med Gas Res. 2018;8(1):19-23 2. Response to reviewers: The clinical manifestations of viral or bacterial infections as initially observed in this patient with a qSOFA score of 2, multiorgan dysfunction and systemic inflammatory response syndrome (SIRS) as indicated by various inflammatory markers, are consistent with high risk of death. Fluctuation of neutrophils and lymphocyte counts during bacterial or viral infections are used in general to support empiric antibiotic therapy. However, in the case presented and due to the absence of identifiable bacteria (see case report addendum) and with a positive SARS-CoV-2 RT-PCR a decision to treat the viral infection was made. In this case initial neutrophilia and borderline lymphopenia would suggest a bacterial process, however low PCT indicates otherwise, thus antibiotic therapy was not warranted. Inflammatory markers as seen in the current case (CRP, Ferritin), highlights the acuity of the process, and the decreased observed over a few days correlated with the clinical improvement of this patient. CRP in particular is an acute phase reactant, indicative of inflammation, produced in the liver and may support the diagnosis of sepsis if other indicators of infection are also present. In this particular case there was a disconnect between elevated CRP and low PCT, raising the possibility of an inflammatory process rather than an infectious condition. Secondarily, the initial cultures reported negative findings, observation supporting a non-infectious inflammatory process. Although co-infections may occur during viral infections, the possibility of a therapeutic benefit of the 3 doses of antibiotics administered empirically (Azithromycin 500 mg IV 1 dose, Vancomycin 2750 mg IV divided doses), is difficult to assertain since cultures were reported negative. The only positive culture revealed was Serratia Marcescens 6 days after ICU admission and was treated with levofloxacin. Several markers of inflammation and the clinical improvement observed was dramatic after the introduction of HCQ/NAC in the treatment of this patient. Aside from the presence of deep venous thrombosis and pulmonary embolism every other inflammatory parameter measured had a positive progression. There is no clear distintion between viral or bacterial respiratory infections based only on clinical manifestations such as fever, cough, shortness of breath, and fatigue, therefore the symptomatology exhibited by this patient was considered SARS-CoV-2 infection, in the absence of positive bacterial cultures. We remain confident that the use of HCQ/NAC altered positively the clinical evolution of this patient and would suggest that this therapy merits further evaluation in randomized trials, with focus on early treatment of the SARS-CoV-2 infection." } ] }, { "id": "67021", "date": "30 Jul 2020", "name": "Vui Heng Chong", "expertise": [ "Reviewer Expertise Internal medicines with interest in gastroenterology", "hepatology and infectious diseases" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a case of COVID-19 with respiratory compromised that was tipped over by pulmonary embolism from lower limb venous thrombus. The patient also had hepatic compromised in this case probably hemodynamic related given the admission parameters with hypotension and metabolic acidosis. Hepatitis secondary to COVID-19 is a possibility. The focus of the authors in this case report is that initiation of HCQ and NAC, the authors theorized had contributed to the recovery of the patient.\nThere is a lack of information; not certain if there were additional therapies provided? Antimicrobials (for sepsis given the high CRP), lopinavir/ritonavir, azithromycin, and inotropes. Based on the information provided, the patient also had septic shock and co-infection must be considered.\nThere is strong evidence and is now widely accepted that HCQ does not work in COVID-19. NAC use in COVID-19 is novel and there are several publications that hypothesis its role. NAC replenishes glutathione stores and helps mitigate the oxidative injuries during inflammatory process not just for COVID-19 but also other diseases.\nThere is nothing concrete in the information presented that supports the authors' claim as the recovery may well have been related to the hemodynamic supportive provided.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5834", "date": "24 Aug 2020", "name": "Carlos Puyo", "role": "Author Response", "response": "August 18, 2020 Drs. Bin Cao, M.D., and Vui Heng Chong, M.D. Reviewers F1000Research Dear Drs, Cao and Chong: We are very grateful for your insightful comments on our Case Report “Use of hydroxychloroquine and N-Acetylcysteine for treatment of a COVID-19 patient”. We have given careful consideration to the points raised by you during the review process. The case report has been adjusted to reflect our comments as indicated in the manuscript using a tracking system. We look forward to your response and hope to complete the publication of our Case Report in F1000Research. Sincerely, Carlos A. Puyo, M.D., FCCP Specific comments are listed below: 1. According to the background and discussion part, the authors’ inference for the potential efficacy of the two drugs are mainly established on evidence from pre-clinical experiments. However, clinical evidences from COVID-19 or other respiratory infectious diseases should also be mentioned and discussed in the manuscript. For hydroxychloroquine, the results of several studies have been released. The RECOVERY trial was reported to find no clinical benefit from use of hydroxychloroquine in hospitalized COVID-19 patients (28-day mortality, length of hospitalization, etc.)1. A published RCT in BMJ from Tang et al found that the use of hydroxychloroquine did not increase the proportion of virus negative conversion2. Also, the interim analysis of WHO solidarity trial showed little or no reduction in the mortality of hospitalized COVID-19 patients in the hydroxychloroquine arm and WHO thus discontinued the hydroxychloroquine arm for hospitalized patients3. Although the dosage of hydroxychloroquine varies in different trials and might be higher than the dosage used in this case report (400mg/600mg per day), it is hard to attribute the efficacy to hydroxychloroquine given the accumulating clinical evidences. The authors should provide clinical evidences in the manuscript in discussion to defend their hypothesis. Response to reviewers: The release of several studies promoting or discrediting the use of chloroquine or hydroxychloroquine for the treatment of COVID-19 has created confusion in the medical community. Here, the reviewer duly noted studies showing lack of or questionable therapeutic benefit1,2, however, there is no mention of studies that have shown benefit3,4,5,6,7. The RCT by Tang, et al, contains several shortcomings among them the use of an open label instead of a double blind approach, potentially introducing bias by the researchers. Similarly, the underpowered sample size and enrollment of patients in more advance stages of the disease likely influence negatively their assessment of the true potential value of HCQ in the disease progression. In the RECOVERY trial mentioned by the reviewer, the doses of HCQ 2400 mg in 24 hours followed by 1600 mg daily for at least 9 days, are indeed higher than for malaria treatment, thus exposing patients to unnecessary risks. It is unclear what scientific bases were utilized to determine the exceedingly high doses of HCQ administered. The Indian Council of Medical Research alerted the WHO about the excessive doses of HCQ (4 times higher) used in the RECOVERY trial, thus, possibly introducing bias in the comparative analysis. The RECOVERY trial also has some interesting findings regarding their unexplained very high mortality data in the control group (23.6%), compared to the 12.7% and 13.5% reported by others in treating high acuity hospitalized patients4,8. Interpretation of HCQ studies is difficult, in part due to the variety of end points utilized by the researchers. For instance some studies used mortality or pneumonia1, while others used negative viral conversion2 as the end points of the studies, thus making a comparative analysis on the effects of HCQ a complex one. In some cases reputable journals (Lancet and others) had to RETRACT anti-HCQ studies9-10 for a variety of reasons, further confounding the data analysishttps://c19study.com/. Discussions regarding the clinical use of HCQ11 need to account for the intracellular activity of this compound to avoid toxicity and to attain therapeutic benefit. Infections due to SARS-CoV-2 alter cellular homeostasis leading to cytotoxicity and cell death. HCQ may impact viral activity at multiple levels by blocking viral entry into the cells, interfering with endosomal maturation, and lysosomal transport5. Furthermore, HCQ may also serve as an immune-modulator through its action on cytokine activity12. The human oral absorption of HCQ/CQ reach significant concentrations in various tissues including the lungs, and has been reported to be 200-700 times higher than plasma levels13. HCQ given at a dose of 6-6.5mg/kg per day would generate serum levels of 1.4-1.5 mM in humans14, sufficient to inhibit SARS-CoV-2 infection3,5. Also the prolong half-life of HCQ (1440 hours) and the distribution on intercellular compartments (1300 hours)15, needs to be accounted as a factor in prescribing the treatment. Ultimately, the therapeutic goal in our treatments is to contain the viral invasion and prevent amplification of the infectious and inflammatory damage. Generation of reactive oxygen species (ROS) capable of inducing cellular apoptosis, necrosis and tissue destruction has been documented in viral infections16, and SARS-CoV-2 is not an exception. We have shown in an in vitro sterile injury model of cellular necrosis (neutrophils) that HCQ was capable of modulating ROS, and Toll-like receptor 9 (TLR9) activity mediated by the released of Damage Associated Molecular Patterns (DAMPS) in particular mitochondrial DNA (mtDNA)17. Other studies have shown that cellular injury mediated by viral invasion may release other DAMPS such as High Mobility Group B1 (HMGB1) during cellular damage18. Activity of HCQ on other inflammatory pathways such as cyclic-GMP-AMP (cGAMP) synthase (cGAS), or by interfering with interferon activity for example, needs to be analyzed as factors involved in generation of acute or chronic symptoms induced by viral infections19. We are in urgent need to use HCQ at effective low doses as indicated by various basic science research groups thus preventing the occurrence of clinical toxicity observed worldwide. There is no sufficient evidence for or against completely eliminating HCQ as a therapeutic intervention during SARS-CoV-2. HCQ in combination with NAC needs to be considered a possible option to treat viral diseases. Studies that have used HCQ need to be analyzed for their potential benefit in post-viral chronic disease. No clinical benefit from use of hydroxychloroquine in hospitalised patients with COVID-19. University of Oxford. 2020. Reference Source Tang W, Cao Z, Han M, Wang Z, et al.: Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ. 2020. Publisher Full Text Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0 Arshad S, Kilgore P, Chaudhry ZS, et al. Treatment with Hydroxychloroquine, Azithromycin, and Combination in Patients Hospitalized with COVID-19 [published online ahead of print, 2020 Jul 2]. Int J Infect Dis. 2020;97:396-403. doi:10.1016/j.ijid.2020.06.099 Wang M., Cao R., Zhang L. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30(3):269–271. Gao J., Tian Z., Yang X. Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends. 2020;14(1):72–73. Chen Z, Hu J, Zhang Z, et al. Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial. medRxiv 2020. doi: 10.1101/2020.03.22.20040758. Rosenberg ES, Dufort EM, Udo T, et al. Association of Treatment With Hydroxychloroquine or Azithromycin With In-Hospital Mortality in Patients With COVID-19 in New York State. JAMA. 2020;323(24):2493–2502. doi:10.1001/jama.2020.8630 Mehra MR, Desai SS, Ruschitzka F, Patel AN RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. Lancet. 2020; (published online May 22.) https://doi.org/10.1016/S0140-6736(20)31180-6 Funck-Brentano C, Salem J-E. RETRACTED: Chloroquine or hydroxychloroquine for COVID-19: why might they be hazardous?.Lancet. 2020; (published online May 22.)https://doi.org/10.1016/S0140-6736(20)31174-0 Liu, J., Cao, R., Xu, M. et al. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6, 16 (2020). https://doi.org/10.1038/s41421-020-0156-0 van den Borne BE, Dijkmans BA, de Rooij HH, le Cessie S, Verweij CL. Chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin 6, and interferon-gamma production by peripheral blood mononuclear cells. J Rheumatol. 1997;24(1):55-60. POPERT AJ. CHLOROQUINE: A REVIEW Rheumatology, Volume 15, Issue 3, August 1976, Pages 235–238, https://doi.org/10.1093/rheumatology/15.3.235 Laaksonen AL, Koskiahde V, Juva K. Dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus. A clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine. Scand J Rheumatol. 1974;3(2):103-108. doi:10.3109/03009747409115809 Schrezenmeier E, Dörner T. Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology. Nat Rev Rheumatol. 2020;16(3):155-166. doi:10.1038/s41584-020-0372-x Tan YJ, Lim SG, Hong W. Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses. Cell Microbiol. 2007;9(11):2552-2561. doi:10.1111/j.1462-5822.2007.01034.x Puyo CA, Earhart A, Staten N, et al. Endotracheal intubation results in acute tracheal damage induced by mtDNA/TLR9/NF-κB activity. J Leukoc Biol. 2019;105(3):577-587. doi:10.1002/JLB.5A0718-254RR Moisy D, Avilov SV, Jacob Y, et al. HMGB1 protein binds to influenza virus nucleoprotein and promotes viral replication. J Virol. 2012;86(17):9122-9133. doi:10.1128/JVI.00789-12 Shu C, Li X, Li P. The mechanism of double-stranded DNA sensing through the cGAS-STING pathway. Cytokine Growth Factor Rev. 2014;25(6):641-648. doi:10.1016/j.cytogfr.2014.06.006 For N-acetylcysteine, the authors also mainly provide evidence from pre-clinical experiments. If there is no direct evidence from COVID-19, the authors should carefully review the literature and present possible indirect clinical evidence from severe pneumonia or viral pneumonia (if there is any). Response to reviewers: N-Acetylcysteine (NAC) is an antioxidant, precursor of reduced glutathione, and modulator of transcription nuclear factor-κβ (NF-κβ) activity with a potential therapeutic profile for use in prevention or as adjuvant treatment for SARS-CoV-2. Viral infections induce cytotoxicity, cell death, overproduction of ROS and cytokines, thus promoting cellular and organ damage. NAC interferes with viral replication of human coronaviruses HCoV-229E1, influenza H5N12, human immunodeficiency virus (HIV)3, and respiratory syncytial virus (RSV)4, among others. Recently, it was shown that NAC inhibited SARS-CoV-2 replication by binding to Cys-145, which is an active site for the main protease (Mpro) involved in viral activity5,6. Several in vitro studies have shown other potential mechanisms of NAC anti-viral activity that include: NF-κβ modulation (cytokine modulation)7, interference with angiotensin II type 1 receptors8, direct inhibition of furin (protease involved in the viral fusion process) 9, and interaction with disulfite bonds10. Pneumonia is a severe manifestation of SARS-CoV-2, SARS-CoV-1, and Middle East Respiratory Syndrome (MERS-CoV), with all sharing similar CT-scan findings of bilateral patchy infiltrates or ground glass opacities. NAC may modulate the effects of viral pneumonia induced by the cytotoxic effects of viral proliferation, cellular apoptosis, necrosis and excessive production of ROS that promote release of proinflammatory cytokines7,10. Other proposed mechanisms of action related to NAC include preservation of T cell activity and inhibition of the NOD-LRR- and pyrin domain-containing protein 3 (NLRP3) inflammatory pathway in monocytes and macrophages during viral respiratory infections11,12. Promising results have been reported with the use of NAC for influenza, ARDS and VAP as measured by length of hospital stay and disease severity13,14. The therapeutic potential for NAC during viral infections and in particular in SARS-CoV-2 is supported by in vitro and indirectly by clinical studies (coronavirus, influenza). We are proposing the use of NAC during viral infections as adjuvant therapy with broad impact on viral activity, with low toxicity and cost. Certainly, further studies are necessary to document the cellular activity as well as the clinical benefits that NAC may provide. The combination of low dose HCQ (total dose 600mg) and short-term NAC administered IV had a positive impact on the treatment of this patient and merits evaluation in large scale randomized clinical studies. The administration of both compounds orally during preintubation periods may attenuate viral symptomatology and possibly decrease the need for intubation. Poppe M, Wittig S, Jurida L, et al. The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. Plos Pathogens. 2017; doi.org/10.1371/journal.ppat.1006286 Geiler J, Michaelis M, Naczk P, et al. N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus. Biochemical Pharmacology. 2010;79:413–420 Ho W, Douglas SD. Glutathione and N-Acetylcysteine suppression of human immunodeficiency virus replication in human Monocyte/Macrophages in vitro. AIDS Research and Human Retroviruses. 1992;1249-1253. Mata M, Sarrion I, Armengot M, et al. Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-Acetylcysteine. PLOS One. 2012;7(10) e48037 Zhang L, Lin D, Sun X, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020;368, 409–412 Guthappa R. Molecular Docking Studies of N-Acetyl Cysteine, Zinc Acetyl Cysteine and Niclosamide on SARS Cov 2 Protease and Its Comparison with Hydroxychloroquine. Chemarxiv. 2020;doi.org/10.26434/chemrxiv.12161493.v1 Paranjpe A, Cacalano NA, Hume WR, Jewett A. N-acetyl cysteine mediates protection from 2-hydroxyethyl methacrylate induced apoptosis via nuclear factor kappa B-dependent and independent pathways: potential involvement of JNK. Toxicol Sci. 2009;108(2):356-366. doi:10.1093/toxsci/kfp010 Ullian ME, Gelasco AK, Fitzgibbon WR, Beck CN, Morinelli TA. N-acetylcysteine decreases angiotensin II receptor binding in vascular smooth muscle cells. J. Am. Soc. Nephrol. 16(8), 2346–2353 (2005) Jorge-Aarón RM, Rosa-Ester MP. N-acetylcysteine as a potential treatment for novel coronavirus disease 2019 [published online ahead of print, 2020 Jul 14]. Future Microbiol. 2020;10.2217/fmb-2020-0074. doi:10.2217/fmb-2020-0074 Schoeman D, Fielding BC. Coronavirus envelope protein: current knowledge. Virol. J. 16(1), 1–22 (2019). Poe FL, Corn J. N-Acetylcysteine: A potential therapeutic agent for SARS-CoV-2 [published online ahead of print, 2020 May 30]. Med Hypotheses. 2020;143:109862. doi:10.1016/j.mehy.2020.109862 Rangel-Méndez JA, Moo-Puc RE. N-acetylcysteine as a potential treatment for novel coronavirus disease 2019 Published Online:14 Jul 2020https://doi.org/10.2217/fmb-2020-0074 De Flora S, Grassi C, Carati L. Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-tern N-acetylcysteine treatment. Eur Respir J 1997; 10: 1535-1541. DOI: 10.1183/09031936.97.10071535 Sharafkhah M, Abdolrazaghnejad A, Zarinfar N, et al. Safety and efficacy of N-acetylcysteine for prophylaxis of ventilator-associated pneumonia: a randomized, double blind, placebo-controlled clinical trial. Mojtaba Med Gas Res. 2018;8(1):19-23 2. Response to reviewers: The clinical manifestations of viral or bacterial infections as initially observed in this patient with a qSOFA score of 2, multiorgan dysfunction and systemic inflammatory response syndrome (SIRS) as indicated by various inflammatory markers, are consistent with high risk of death. Fluctuation of neutrophils and lymphocyte counts during bacterial or viral infections are used in general to support empiric antibiotic therapy. However, in the case presented and due to the absence of identifiable bacteria (see case report addendum) and with a positive SARS-CoV-2 RT-PCR a decision to treat the viral infection was made. In this case initial neutrophilia and borderline lymphopenia would suggest a bacterial process, however low PCT indicates otherwise, thus antibiotic therapy was not warranted. Inflammatory markers as seen in the current case (CRP, Ferritin), highlights the acuity of the process, and the decreased observed over a few days correlated with the clinical improvement of this patient. CRP in particular is an acute phase reactant, indicative of inflammation, produced in the liver and may support the diagnosis of sepsis if other indicators of infection are also present. In this particular case there was a disconnect between elevated CRP and low PCT, raising the possibility of an inflammatory process rather than an infectious condition. Secondarily, the initial cultures reported negative findings, observation supporting a non-infectious inflammatory process. Although co-infections may occur during viral infections, the possibility of a therapeutic benefit of the 3 doses of antibiotics administered empirically (Azithromycin 500 mg IV 1 dose, Vancomycin 2750 mg IV divided doses), is difficult to assertain since cultures were reported negative. The only positive culture revealed was Serratia Marcescens 6 days after ICU admission and was treated with levofloxacin. Several markers of inflammation and the clinical improvement observed was dramatic after the introduction of HCQ/NAC in the treatment of this patient. Aside from the presence of deep venous thrombosis and pulmonary embolism every other inflammatory parameter measured had a positive progression. There is no clear distintion between viral or bacterial respiratory infections based only on clinical manifestations such as fever, cough, shortness of breath, and fatigue, therefore the symptomatology exhibited by this patient was considered SARS-CoV-2 infection, in the absence of positive bacterial cultures. We remain confident that the use of HCQ/NAC altered positively the clinical evolution of this patient and would suggest that this therapy merits further evaluation in randomized trials, with focus on early treatment of the SARS-CoV-2 infection." } ] } ]	1	https://f1000research.com/articles/9-491
https://f1000research.com/articles/9-1034/v1	24 Aug 20	{ "type": "Study Protocol", "title": "Effect of riboflavin supplementation on blood pressure and possible effect modification by the MTHFR C677T polymorphism: a randomised trial in rural Gambia", "authors": [ "Modou Jobe", "Mary Ward", "Bakary Sonko", "Abdul Khalie Muhammad", "Ebrima Danso", "Helene McNulty", "Andrew M Prentice", "Mary Ward", "Bakary Sonko", "Abdul Khalie Muhammad", "Ebrima Danso", "Helene McNulty" ], "abstract": "Introduction: Emerging evidence links a functional polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene (rs1801133) with hypertension in adults. This variant reduces the affinity of MTHFR for its cofactor flavin-adenine dinucleotide (FAD) which is derived from riboflavin. Previous work has demonstrated a blood pressure (BP)-lowering effect of riboflavin in Irish adults with the MTHFR 677TT variant. We hypothesize that the almost-universal severe riboflavin deficiency seen in rural Gambia mimics the BP phenotypic effect of the TT variant and exacerbate the effect of the CT variant. We will test this in a randomised, placebo-controlled trial, whether intervention with riboflavin can decrease BP in adults in rural Gambia. Methods: This is a phase 2 recall-by-genotype randomised single-blind placebo-controlled riboflavin supplementation trial. We will use the Keneba biobank to recruit approximately 102 individuals aged between 18-70, previously genotyped for the MTHFR C677T polymorphism and identified as carrying the T allele; these individuals will be age- and sex-matched to a similar number of homozygotes for the C allele. The participants will be randomised to a 16-week supplementation trial of 5 mg/day riboflavin or placebo, supplied every 14 days. The primary outcome, BP, will be measured at baseline and at weeks 8 and 16. Blood samples, collected at baseline and week 16, will be analysed for riboflavin, homocysteine, red cell folate, cobalamin (vitamin B12) and pyridoxine (vitamin B6). Discussion: The study will evaluate the role of riboflavin supplementation in BP control within a population with high levels of riboflavin deficiency and will test a possible gene-nutrient interaction with the MTHFR C677T polymorphism. If improvements in BP are observed in this study, and proven in subsequent large-scale interventions, riboflavin could offer a cost-effective, safe and accessible option for the prevention and control of hypertension in this population. Trial registration: ClinicalTrials.gov Identifier NCT03151096. Registered on 12 May 2017.", "keywords": [ "hypertension", "blood pressure", "MTHFR", "riboflavin", "clinical trial" ], "content": "Abbreviations\n\nAE: adverse events; BMI: body mass index; BP: blood pressure; CRF: case report forms; CTSO: clinical trial support office; DAG: data access group; DM: data manager; EGRAC: erythrocyte gluthathione reductase activation coefficient; US FNB: United States Food and Nutrition Board; FAD: flavin-adenine dinucleotide; GCP: Good Clinical Practice; HIGH: Hepcidin and Iron in Global Health; MRCG at LSHTM: Medical Research Council Unit The Gambia at London School of Hygiene and Tropical Medicine; MTHF: methylene tetrahydrofolate; MTHFR: methylene tetrahydrofolate reductase; QC: quality check; PI: principal investigator; REDCap: Research Electronic Data Capture; RNI: Recommended Nutrient Intake; SAE: severe adverse events; SEM: structural equation modelling; SSA: sub-Saharan Africa.\n\n\nIntroduction\n\nHypertension is a global public health problem affecting 1 billion people worldwide and up to 46% of adults in sub-Saharan Africa (SSA)1. Despite a generally very low body mass index (BMI) in the rural Kiang West district of The Gambia, the prevalence of high blood pressure (BP) (>140/90) has recently been estimated as 18.3% in adults over 18 years2. Hypertension arises from a combination of multiple lifestyle and genetic factors (many of which probably remain unknown). Its treatment requires an individualized combination of drug therapy and lifestyle changes3,4. The treatment of hypertension is generally lifelong and exerts a heavy economic burden on individuals and their families as well as on national economies. The identification of safe cost-effective treatment interventions without adverse side effects would be hugely advantageous, particularly in low-income settings with high prevalence of hypertension such as SSA.\n\nEmerging evidence from clinical and genome-wide association studies (GWAS) links a functional polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene (rs1801133), encoding the folate-metabolising enzyme MTHFR, with blood pressure (BP) and hypertension in adults5,6. This enzyme catalyses the conversion of 5,10-methylenetetrahydrofolate (MTHF) to 5-MTHF. Variation at rs1801133 is relatively common and has 3 genotypes; homozygous “normal” wild-type CC, heterozygous CT and homozygous “variant” TT genotypes. Of these genotypes, the homozygous variant TT is strongly associated with a higher BP. The precise mechanism by which MTHFR C677T is associated with BP remains unclear.\n\nVitamin B2 (riboflavin) is converted to flavin-adenine dinucleotide (FAD) which acts as the essential co-factor for the MTHFR enzyme. Molecular studies demonstrate that the decreased activity of the variant enzyme is caused by reduced affinity for the redox cofactor FAD7; hence its original name as the ‘heat-labile’ variant. Work at Ulster University has demonstrated, in three separate randomised controlled trials to date, that BP is highly responsive to intervention with riboflavin and that this response is dependent on MTHFR genotype8–10. In all of these trials, riboflavin supplementation resulted in significant reductions in both systolic and diastolic BP in adults with the homozygous variant TT genotype, while at baseline (pre-intervention) we observed intermediate BP values in those with the heterozygous CT genotype compared to adults with CC (lower) or TT (higher) genotypes.\n\nIn the Keneba biobank11, a total of 2114 individuals between the ages of 18 and 70 were genotyped for rs1801133 of whom 1951 (92.3%) and 163 (7.7%) had the CC and CT genotypes respectively, while none had the TT genotype. We hypothesize that the almost universal severe riboflavin deficiency seen in most Kiang West residents of The Gambia (assessed historically12) and more recently13 will a) mimic the effect of the TT variant and b) exacerbate the effect of the CT genotype based upon the following argument. In vitro studies show that the TT variant is only 30% saturated with FAD compared to CC and the CT is 84% saturated14. Riboflavin deficiency is a major problem in Kiang West district with typical values for erythrocyte gluthathione reductase activation coefficient (EGRAC; gold-standard riboflavin biomarker) of >2.0 (compared with indicative values of <1.2, 1.2-1.4 and >1.4, defining normal, suboptimal and deficient status, respectively), indicating that EGR is only 50% saturated by FAD. By analogy, we expect these levels of severe deficiency to lower levels of MTHFR activity by ~50% in CC the genotype and a further 16% in CT variants. This is the basis for the anticipated efficacy of riboflavin supplementation.\n\nThe adult recommended nutrient intake (RNI) for riboflavin is 1.3 mg/day for men and 1.1 mg/day for women15. There is no known toxicity (due to a renal threshold whereby unused riboflavin is excreted rapidly in the urine) and the US FNB has noted that there have been no known adverse effects, even at intakes of 400 mg/day for at least 3 months16. We propose to use just 5 mg/day to replenish body stores and attempt to saturate the CT genotype enzyme as well as aligning the dose with previous trials.\n\nThe possibility that riboflavin deficiency represents a new, easily-correctible causal factor in hypertension in SSA would require further large-scale interventions if this study yields encouraging results.\n\n\nMethods/study design\n\nThe primary objective is to compare the effect of 16-weeks of riboflavin supplementation (5 mg/day) versus placebo on systolic and diastolic BP.\n\nThe secondary objectives are:\n\na) to test for possible effect modification in CC vs CT genotypes for the MTHFR C677T polymorphism on BP response to intervention\n\nb) to describe the cross-sectional associations at baseline between BP (continuous variable and proportion with hypertension, >140/90 mmHg) and riboflavin status (assessed by the EGRAC functional biomarker test) and interaction with MTHFR genotype\n\nThe primary outcome is change in BP at 8 and 16 weeks compared to baseline in response to riboflavin supplementation versus placebo. We will also determine the response of the riboflavin biomarker to intervention, determined (before and after riboflavin supplementation) using the EGRAC method, considered to be the gold standard method to assess biomarker status of riboflavin.\n\nThis is a phase 2 recall-by-genotype, randomised single-blind placebo-controlled micronutrient supplementation trial. We will invite all individuals between the ages of 18 and 70 in the Keneba biobank with the CT genotype and a similar number of age- and sex-matched CC homozygotes to participate in the study (Figure 1).\n\nThe study protocol was reviewed by the Scientific Coordinating Committee of the MRCG at LSHTM and has received ethics approval from the Joint Gambia Government/MRC Ethics Committee (SCC1518). Approval was also obtained from the National Medicine Regulatory Authority of The Gambia.\n\nParticipants must meet all of the inclusion criteria and none of the exclusion criteria to be eligible to participate in the trial.\n\nInclusion criteria\n\nAged between 18–70 years\n\nHas provided appropriate ethical consent for involvement in studies relating to genetics.\n\nHas available genotype data in the Keneba biobank needed for the current study\n\nAvailable for the duration of the intervention period\n\nExclusion criteria\n\nTaking vitamin B/multivitamin supplements\n\nOngoing pregnancy as confirmed by participant\n\nHistory of digestive, hepatic, renal, hematological disorders or dementia\n\nEpilepsy or taking anti-epileptic medications\n\nGlucose-6-phosphate dehydrogenase (G6PD) deficiency\n\nSensitization of community elders, youth and women groups and respective village residents within the Kiang West district was conducted by the study team prior to recruitment of study participants. During community sensitization, community elders of respective villages were asked to identify literate people to act as impartial witnesses during the consenting process of illiterate potential participants.\n\nIndividual consent for the study will sought by trained field assistants after explaining the project in full detail, covering all aspects of the study as laid out in the approved study information sheet. Literate participants will be given a copy of the information sheet while illiterate participants will have the full information sheet read to them in the language they understand in the presence of an impartial witness. The fieldworkers answer any questions that arise, and participants are also be given the possibility to obtain further clarifications and explanations by speaking to one of the study investigators if they wish. Participation is entirely voluntary and individuals who are not able to give consent are not enrolled. If participants agree to participate, written informed consent is then obtained. Assessment of informed consent understanding is performed using a specially designed form of 10 questions derived from the participant information sheet (available as Extended data17).\n\nThe Keneba biobank is used to identify potential participants. After the exclusion criteria are applied and non-consenters are taken into account, we aim to enrol at least 102 participants (see sample size calculations below) with the CT genotype and a similar number of age and sex-matched participants with the CC genotype. A randomisation code, written in Stata, would select each of the participants with the CT genotype and match them to participants with the CC genotype as randomly and equally as possible. This will ensure that all participants with the CT genotype would be age and sex-matched to several possible individuals with the CC genotype. Since each consented CT genotype participant has several possible matches with the CC genotype, consent would be sought until it is obtained from any of the possible matches. Thus if the first participant approached with the CC genotype consents to participate, all other possible matched participants would not be sought after for their consent.\n\nFollowing consent, the participants’ Biobank IDs would be linked to their Study IDs and allocated to receive either riboflavin or placebo by block randomization (of varying block sizes). To ensure balance between the two genotypes, we shall randomly allocate 50% of those with the CT genotype to receive the riboflavin supplement. A programme will perform the randomisation in a 1:1 ratio and produce randomization envelopes to be used. This randomisation is such that for each CT genotype participant that receives the riboflavin supplement the matched participant with the CC genotype is automatically allocated the placebo and vice versa. The randomisation codes and database are accessible only to an independent statistician and data manager.\n\nField nurses and field assistants measuring BP and dispensing investigational products to participants, and laboratory staff will be blinded to the genotype of the study participants and to the identity of the treatment arm to which a participant is assigned from the time of randomization to the time of unblinding. Details of matching will also not be revealed to them. The placebos are designed to be indistinguishable from those containing active drugs. The products will be pre-packed with the participant’s name and study ID on a weekly basis by an unblinded study nurse and clinical trial assistant following a study specific protocol and handed over to field assistants who will distribute them to study participants. Double-blinding is not possible because subjects randomized to the riboflavin will have yellow urine, a harmless outcome that will be explained to them at the start of the study and told not to discuss this with any member of the study and other MRCG at LSHTM staff.\n\nIn this trial, we will ensure that sealed envelopes containing the individual treatment allocation are kept at the Medical Research Council Unit The Gambia at London School of Hygiene and Tropical Medicine (MRCG at LSHTM) field station in Keneba in case of the need for emergency unblinding. The code would be broken in cases of severe adverse events for which the product needs to be known for individual participants.\n\nThe blinding code will be kept by the independent statistician and will only be broken after the database lock.\n\nAt the initial visit, socio-economic data and medical history will be obtained by a field staff using a questionnaire. Anthropometric measurements (height, weight, waist and hip circumference, skin-fold thickness and body composition by bioelectrical impedance analysis) will be taken. Thereafter, BP will be measured with an automated Omron 705IT device (Omron, Kyoto, Japan) in triplicate, each in a seated position and 5 minutes apart, using a standard protocol by the same investigator who will be blinded to genotype and treatment group. The mean of the second and third measurements will be used for analysis. A 10ml blood sample will be collected for laboratory assessment of vitamin B status. During the intervention phase of the trial, we will supplement participants with 5mg/day of riboflavin or a matching placebo for 16 weeks. The pills will be supplied every 10 days with instructions to return any unused pills. Another BP measurement following similar protocol as before will be taken at 8 weeks as well as at 16 weeks post- intervention. Finally, a 10ml blood sample will be collected 16 weeks post-intervention. Blood samples will be analysed for riboflavin (by EGRAC), homocysteine, red cell folate, cobalamin and pyridoxine. Participants will be followed-up after the intervention period for a further 3 weeks to monitor AEs. Details of the study schedule are summarised in Table 1.\n\nThe investigational products to be used in this study are manufactured by Advanced Molecular Research, Calgary, Canada (https://aor.ca/) following Good Manufacturing Processes and quality control procedures. The products are in the form of capsules, are physically indistinguishable from each other and having the following excipients: microcrystalline cellulose, hypromellose (capsule shell) and chlorophyll (color of the capsule shell) with the supplements (as opposed to placebo) containing 5mg of riboflavin. 10 green capsules are packed in a clear plastic blister and stored as per manufacturer instructions at the MRCG at LSHTM Field Station in Keneba. All used blisters and unused pills will be recovered for accountability purposes as required by local regulations.\n\nThe trial will be monitored by personnel of MRCG at LSHTM Clinical Trial Support Office as per the sponsor’s regulations. An initiation visit between the monitors, Principal Investigator (PI) and study team was conducted to approve the start of the study. The purpose of this visit is to review ethical approval, the trial master file and required documents (logs, study specific procedures, data collection tools), investigational products, and ensure that study specific training and appropriate facilities are available. An interim visit will be conducted 1 month after the start of the study and then after 6 months. These visits will include the following: review of current status and recruitment rate, verify that the study is conducted in accordance with approved protocols and regulatory requirements and perform source data verification. A close out visit will be conducted at the end of the study to ensure completeness of all essential documents and archiving arrangements, and appropriate distribution of all remaining study products and supplies.\n\nThe trial will provide 5-mg riboflavin pills or a matching placebo. Riboflavin is safe and because it is water soluble, any excess will be excreted via the urine. No adverse events were observed in studies by our collaborators8–10 and the US FNB has noted that there have been no known adverse effects of consuming 400 mg/day (80 fold higher than our proposed dose) for at least 3 months16.\n\nThe only potential adverse risk identified in this study relates to blood collection, i.e. pain or discomfort, bruising and infection. The total amount of blood to be collected is 20 ml (10 ml at baseline and at the end of the study) which does not pose any health risk. Our study nurses are sufficiently trained and standard protocols will be used to avoid infection.\n\nParticipants who will receive the riboflavin supplements will have their riboflavin stores improved and possibly replenished. Other participants who were randomized to receive placebo will be offered riboflavin supplementation at the end of the study.\n\nFormat and scale of data: The database software to be used in this trial is REDCap. Trial staff trained on GCP and electronic case report forms (eCRF) will be authorized to collect data and other trial specific documentation. Training on REDCap was conducted and they will continue to be updated through in-house training. A web and mobile version of the application installed on mobile tablet devices, smart phones, laptops and PCs will be used at each stage of data collection.\n\nThe trial data manager and database developer in consultation designed the eCRFs with input from the PI/sub-investigator and research clinician according to MRCG at LSHTM’s CRF Development standard operating procedure. The eCRFs have built-in quality check (QC) process which aims to minimize the rate of errors/missing fields and enhance data quality. Other quality control measures will include running queries by the DM that will flag discrepancies in the form of missing values, records and outliers. The database developer developed all data entry screens and all reporting functionalities. The final data format can be downloaded to Excel, PDF, and common statistical packages (SPSS, SAS, Stata, R).\n\nThe eCRFs will include question fields which are either single or multiple checked, drop-down single selection answers, binary (yes/no) fields, numerical fields and free text. REDCap branching tools will be utilised to guide data collection making sure data is collected on the required activity at each visit. For instance, sample collection and processing data should be allowed to be collected only at visits 00 and 11. Study time points after recruitment and randomisation are categorically numbered 0–14 (Table 1). Call lists will be generated to help organize study visits. Strict validation and verification rules will be adhered to when completing the eCRFs. All data collected on mobile tablets collected offline will be synced directly on MRCG at LSHTM dedicated servers accessible only by users within a given Data Access Group (DAG) managed by the software. User access groups for the trial will be granted to trial staff based on individual responsibilities and privileges.\n\nSystem validation: All validation, range logical checks will be applied as required to maintain data integrity. Demographic and clinical data will be imported into the study database but all personally identifiable fields will be marked in REDCap to prevent them from being exported as part of datasets. Data export options from the clinical database include CDISC ODM XML, a vendor neutral, platform independent format for interchange and archive of data collected in clinical trials. This should ensure sharing and long-term validity of data.\n\nAll data collection instruments will be continuously reviewed at the various stages of development to ensure that data collected meet the required standards in line with study analysis plan. Data entry screens are designed with rigid validation and logical checks, skip patterns and validations to accurately guide data collection. Study specific procedures (SSPs) and the protocol will guide data collection at the field and in the lab.\n\nInstructions will be embedded on the eCRFs. As part of data supervision, status of the record’s data form completeness, denoted with a colour; red incomplete; yellow unverified; green complete will allow data to be firstly submitted in complete status. The supervisor ensures these records are verified and record status changed to verified in a timely manner.\n\nData validation: Validation checks will be enforced real-time at each stage of data collection which will be programmatically built into the database application to ensure accuracy, consistency and completeness. Primary data will be generated from the field and lab which shall be verified by the study PI or delegate and supervisors.\n\nOther QC checks will be routinely conducted to ensure data collected is of high quality. Participants and visits will be programmed using REDCap calendar to ensure that they are seen on scheduled time points.\n\nMandatory fields must be completed or flagged for data entry to proceed. Data fields are mainly univariate, allowing only one acceptable value or option. Multivariate checks will be implemented where required. Date fields will be formatted as dd-mm-yyyy and pre-determined ranges applied. Every field will be formatted with the right input masks (e.g. visit number can be masked as regexp: /\\d {2}/, to allow two-digits like 01).\n\nData coding: An up-to-date data dictionary will document the study metadata. The data dictionary specifies the database structure normalised to an appropriate level. It specifies database tables, variable names, description types an all properties including all data coding. Categorical and float data will be coded using standard conventions (e.g. 1 = Yes; 0 = No) and range checks on numerical data will ensure plausible data is collected in line with the analysis plan. XML features in REDCap keeps a downloadable up-to-date study metadata.\n\nData capture: For audit purposes, all new and modified records will be logged with the username of the person collecting the data. Data will be captured at each stage using REDCap mobile application installed on tablets that allow data to be collected offline and synced later through a Wi-Fi or mobile data connections. Appropriate rights are allocated and managed by the DM to users. The ability to view any data will be limited and permissions granted by the data manager through DAGs.\n\nQuery resolution: Due to the real time nature of data collection, queries/errors are expected to be easily detected and resolved immediately. It will be mandatory on all study staff to raise and attend to queries in a timely fashion. In addition, the monitors may raise queries with the team using the same process. A log of all queries handled by the data manager or delegate at any point can show how many queries are raised, resolved, or pending.\n\nData transfer and consolidation: Extracted data requested by the study team will be in the form of CDISC ODM XML or Excel. This will be made available to the recipient through the appropriate medium or as requested by PI. It will be carried out as and when required. Data to be transferred to study PIs would be zipped and password-protected. All data transfers will conform to MRCG at LSHTM data transfer procedures.\n\nSafety data reconciliation: Serious adverse events (SAEs) are least expected in this trial but they will be assessed by research clinician and immediately logged and reported to the Clinical Trials Support Office (CTSO) by email alerts. Standard questionnaires will used to report and follow-up SAEs.\n\nProtocol deviations will be reported and kept in the Trial Master File.\n\nDatabase security: All persons dealing with personal data are responsible for ensuring the security and safety of these data. Strict access controls will apply both to the servers and the database. Access to the database is controlled by the Data Manager and any requests for access would be documented.\n\nStudy closure: Lost to follow-ups would be assessed and after the final trial monitoring visit is completed, the following targets are expected to be met:\n\nThat all data is entered into the study REDCap database and all records marked as complete\n\nAll AEs reported and SAEs followed\n\nAll queries are resolved and data cleaning completed\n\nSource document verified\n\nAccess rights revoked\n\nThe database would be locked in due course and all accesses denied.\n\nSample size rationale:This pilot trial is constrained by the number of available eligible and consenting adults carrying the MTHFR 677 CT variant. We estimate this will be 102 to be matched to an equal number of CC controls. Using standard deviation estimates from our own and published literature (14 mm for systolic BP and 10 mm for diastolic BP) we will have 95% confidence (at p<0.05) of detecting differences of 3.9 mmHg for systolic BP and 2.8 mmHg for diastolic BP.\n\nStatistical analysis plan:A preliminary evaluation of data will be conducted to identify issues that will affect our approach to the primary analysis. This will specifically include the following:\n\nsummarize the amount of missing data for key variables,\n\nsummarize time between baseline and follow-up key measurements\n\nexamine the distributional characteristics of key variables in order to detect potential data entry/reduction errors\n\nWe will also carry out tabulation of descriptive statistics for key variables (including missing observations) and histograms, quantile plots of baseline levels and follow-up measures and changes in those measures will be assessed.\n\nWe will also describe the study process to assess whether there is a difference between the trial results compared to the pre-trial plans. “Table 1” describing the baseline characteristics of the riboflavin and placebo arm of the trial as well as a CONSORT diagram will also be constructed.\n\nThe statistical approached to be used to evaluate the primary and secondary study end points are discussed below:\n\n\nPrimary analysis\n\nWe shall use regression to compare differences in BP at week 16 between the two arms of the trial after adjusting for the BP at week 0. The models are summarized below\n\nWe would perform diagnostics to investigate normality and heretoskedasticity so as to perform a transformation as necessary.\n\nThe analyses on BP would be for systolic and diastolic separately. We shall use structural equation modelling (SEM) to predict a latent variable that could reduce the dimension from two to one variable that could reasonably summarize overall change in BP. This would be done by predicting a latent variable for both systolic and diastolic BP at week 0 and week 16 before comparing the two arms using regression as summarized in the model below.\n\nAssessing the relationship between the systolic and diastolic BP with the overall BP would be assessed to see if the relationship is clinically plausible. If it is, then it can be used to test whether, holistically, BP at week 16 is different between the riboflavin arm and the placebo arm, adjusting for overall BP at week 0.\n\nMissing data: Missing data less than 5% (below the 102 participants required for adequate power in the sample size calculation) would be considered acceptable to do a complete case analysis. In the event that the missing data is or above 5% we shall use Multiple Imputation to minimize bias.\n\nInterpretation: We shall reject the null hypothesis that there is no difference between the riboflavin arm and the placebo arm at a 5% significance level. A statistically significant result between the arms where the mean BP (separated or overall) for participants that received riboflavin is lower than the mean of the participants that received placebo would mean that riboflavin successfully lowers BP 16 weeks after administration.\n\n\nSecondary analyses\n\nWe shall use regression to ascertain whether there is effect modification of the intervention due to the genotype. The models are summarized below\n\nSimilarly diagnostics would be performed to assess none of the assumptions of regression were violated and ensure that proper methods would be used to address any violations.\n\nInterpretation: We shall reject the null hypothesis that there is no effect modification at a 5% significance level.\n\nWe shall use regression to ascertain whether there is an association between EGRAC based riboflavin status and systolic and diastolic BP at both week 0 and week 16. This is when we are considering the BPs as continuous. The models are summarized below.\n\nThe models that use a BP cut-off of 140 mmHg for systolic and 90 mmHg for diastolic are similar to the ones above.\n\nDiagnostics would be performed to assess and correct violation of any regression assumptions as appropriate.\n\nWe shall also assess the effect modification by the MTHFR genotype using both continuous and dichotomized BP measurements. These models would also be diagnosed and violations addressed accordingly.\n\nInterpretation: We shall reject the null hypothesis that there is no effect modification at a 5% significance level.\n\n\nDiscussion\n\nHigh BP is a public health problem, especially in SSA. The causes are not fully understood in the vast majority of cases and commonly result from a combination of lifestyle and genetic factors. The treatment of high BP is often very expensive and sometimes not affordable especially in poor communities. The development of low-cost, effective treatments would therefore be hugely beneficial.\n\nIt is possible that the severe riboflavin deficiency observed in the Kiang West district and possibly in other parts of SSA may be contributing to the rising prevalence of high BP, especially among those with a genetic predisposition for developing hypertension owing to MTHFR 677CT genotype. The study will represent the first step in helping us to understand if riboflavin supplementation helps to control blood pressure in this population group. If improvements in BP were achieved in this study and proven in large-scale intervention, it would offer a novel means of controlling hypertension that would be safer, more accessible and more cost-effective than current treatment options.\n\n\nTrial status\n\nA total of 122 participants have been randomised into the study at the time of submission.\n\n\nDeclarations\n\n\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: RiboBP_SPIRIT Checklist.docx. https://doi.org/10.6084/m9.figshare.12627902.v117.\n\nFile ‘Prentice_SCC 1518_ICD_version4.0.docx’ contains the participant information sheet used in this study.\n\nFigshare: SPIRIT checklist for ‘Effect of riboflavin supplementation on blood pressure and possible effect modification by the MTHFR C677T polymorphism: a randomised trial in rural Gambia. https://doi.org/10.6084/m9.figshare.12627902.v117.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to express gratitude to Traj Nibber of Advanced Orthomolecular Research, Calgary, Canada for providing the investigational products to be used in this study. We would like to thank the entire study team: Kabiru Ceesay, Ousubie Jawla, Seedy Singhateh, Lamin Ceesay, Buba Ceesay, Siaka Gaye, Omar Darboe, Omar Jallow, Wandifa Cham, Ansumana Kombo, Alhassan Colley, Sainey Beyai and Buba Sonko and to the entire MRCG at LSHTM staff at Keneba for their hard work and dedication. We are also grateful to the Clinical Trial Support Office at MRCG at LSHTM for monitoring this trial and for their helpful suggestions. Special thanks go to all participants as well as impartial witnesses in making this trial a success.\n\n\nReferences\n\nWorld Health Organization: A Global Brief on Hypertension. 2013; Geneva: World Health Organization. Reference Source\n\nJobe M, Agbla SC, Prentice AM, et al.: High blood pressure and associated risk factors as indicator of preclinical hypertension in rural West Africa: A focus on children and adolescents in The Gambia. Medicine (Baltimore). 2017; 96(13): e6170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMancia G, Fagard R, Narkiewicz K, et al.: 2013 ESH/ESC guidelines for the management of arterial hypertension: The Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). Eur Heart J. 2013; 34(28): 2159–219. PubMed Abstract \| Publisher Full Text\n\nWhelton PK, Carey RM, Aronow WS, et al.: 2017 Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults. A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017. Reference Source\n\nYang B, Fan S, Zhi X, et al.: Associations of MTHFR gene polymorphisms with hypertension and hypertension in pregnancy: A meta-analysis from 114 studies with 15411 cases and 21970 controls. PLoS One. 2014; 9(2): e87497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcNulty H, Strain JJ, Hughes CF, et al.: Riboflavin, MTHFR genotype and blood pressure: A personalized approach to prevention and treatment of hypertension. Mol Aspects Med. 2017; 53: 2–9. PubMed Abstract \| Publisher Full Text\n\nYamada K, Chen Z, Rozen R, et al.: Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci U S A. 2001; 98(26): 14853–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorigan G, McNulty H, Ward M, et al.: Riboflavin lowers blood pressure in cardiovascular disease patients homozygous for the 677C-->T polymorphism in MTHFR. J Hypertens. 2010; 28(3): 478–86. PubMed Abstract \| Publisher Full Text\n\nWilson CP, McNulty H, Ward M, et al.: Blood pressure in treated hypertensive individuals with the MTHFR 677TT genotype is responsive to intervention with riboflavin: Findings of a targeted randomized trial. Hypertension. 2013; 61(6): 1302–8. PubMed Abstract \| Publisher Full Text\n\nWilson CP, Ward M, McNulty H, et al.: Riboflavin offers a targeted strategy for managing hypertension in patients with the MTHFR 677TT genotype: A 4-y follow-up. Am J Clin Nutr. 2012; 95(3): 766–72. PubMed Abstract \| Publisher Full Text\n\nHennig BJ, Unger SA, Dondeh BL, et al.: Cohort profile: The Kiang West Longitudinal Population Study (KWLPS)-a platform for integrated research and health care provision in rural Gambia. Int J Epidemiol. 2017; 46(2): e13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBates CJ, Powers HJ: Studies on micronutrient intakes and requirements in The Gambia. J Hum Nutr Diet. 1989; 2(2): 117–24. Publisher Full Text\n\nDominguez-Salas P, Moore SE, Cole D, et al.: DNA methylation potential: Dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women. Am J Clin Nutr. 2013; 97(6): 1217–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrosst P, Blom HJ, Milos R, et al.: A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995; 10(1): 111–3. PubMed Abstract \| Publisher Full Text\n\nVitamin and mineral requirements in human nutrition: report of a joint FAO/WHO expert consultation, Bangkok, Thailand, 21–30 September 1998. Reference Source\n\nInstitute of Medicine. Food and Nutrition Board: Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press; 1998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJobe M, Ward M, Sonko B, et al.: RiboBP_SPIRIT Checklist and informed consent document.docx [Internet] figshare. 2020; [cited 2020Jul11]. http://www.doi.org/10.6084/m9.figshare.12627902" }	[ { "id": "74570", "date": "20 Nov 2020", "name": "Daniel J Hoffman", "expertise": [ "Reviewer Expertise Energy metabolism", "body composition", "growth", "international nutrition." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rationale for this study is very clear; hypertension is highly prevalent in low-income countries and effective methods to prevent this disease are warranted. The authors propose to test the impact of riboflavin supplementation on blood pressure by studying adults who may be at increased risk for hypertension due to a common polymorphisms in a gene linked to hypertension.\nThe authors have provided a highly detailed explanation of their rationale with sufficient supporting literature. The protocol is very thorough and demonstrates the expertise of the investigators. There is no doubt that the study will generate sufficient data to test the proposed hypotheses. A review of the statistical analyses shows a complete understanding of appropriate tests to be conducted and the models presented are clear and reasonable. While there was some discussion about the sample size constraints due to the prevalence of the polymorphism studied, the authors are cognizant of this challenge and have incorporated it into their design and analyses.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "141606", "date": "01 Jul 2022", "name": "Alexandre S. Silva", "expertise": [ "Reviewer Expertise Genetics", "nutrition", "exercise and hypertension" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a research project registered in ClinicalTrials.gov Identifier NCT03151096, and which the authors chose to publish in F1000Research. The proposal has a plausible justification in terms of clinical grounds and epidemiological relevance. I verify some methodological issues that were not clear, so I present them below as constructive criticism accompanied by suggestions:\nIn the primary and secondary objectives, it is not clear whether the study will be carried out with hypertensive patients.\n\nGiven that the prevalence of the CT genotype is very low (7.7%), I think the authors need to present a sample calculation considering this population. For this, I suggest calculating the effect size of a previous study with the same supplement in a hypertensive population. This information is presented in the item “Statistical considerations”, however I think the information is incomplete. If effect data already exist, both the size of this effect and the estimated sample size can be predicted to give a greater sense of feasibility for the research project.\n\nRegarding feasibility, I ask the following question: are the 102 subjects in each CC and CT group all hypertensive? After disregarding those who did not accept to participate in the study or who did not meet the inclusion criteria, will the sample size be sufficient?\n\nThe absence of expected prevalence precisely for the TT genotype (variant) needs a brief discussion to justify carrying out the study in the chosen population.\n\nAll CT will be invited. Considering a much larger population size of CC, how will the 102 guests be chosen (randomized) in this most prevalent group? Does not pairing the participants of the CC group according to the characteristics of the CT group violate the principle of probability of participation in the study for the entire CC population?\n\nI think that all the criteria presented as exclusion are, in fact, inclusion criteria (no taking vitamin B, for example). If people taking vitamin B supplements don't enter the study, how could they be excluded? As I understand it, the exclusion criteria are those that determine that a participant needs to leave the study because something with the potential to change the results of the study has happened. For example, not complying with the supplementation/placebo protocol, women becoming pregnant during the intervention period, having important dietary changes during the intervention period, etc. Clearly defining the exclusion criteria is important to demonstrate the integrity and transparency of the study.\n\nI suggest clarifying whether the participants may (or may not) be users of antihypertensive drugs before the start of the intervention. I also suggest that both nutritional behavior and medication administration be monitored throughout the intervention period and let this be an exclusion criterion.\n\nWill the plan for statistical analysis of secondary variables (riboflavin, homocysteine, red cell folate, cobalamin, and pyridoxine) be the same as for blood pressure analysis? I suggest stating this in the text.\n\nI think the discussion does not contribute much to the understanding of the project. The text of this section is very short and the information is already presented in the introduction.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-1034
https://f1000research.com/articles/9-679/v1	06 Jul 20	{ "type": "Research Article", "title": "Genomic evidence of multiple SARS-CoV-2 introductions into Morocco", "authors": [ "Bouchra Chaouni", "Imane Allali", "Sofia Sehli", "Wajih Rhalem", "Abdellah Idrissi Azami", "Nihal Habib", "Salsabil Hamdi", "Lahcen Wakrim", "Abderrahmane Bakkali", "Mustapha Mahmoud", "Laila Bouguenouch", "Najib Al Idrissi", "Youssef Bakri", "Saaïd Amzazi", "Said Barrijal", "Hassan Ghazal", "Bouchra Chaouni", "Imane Allali", "Sofia Sehli", "Wajih Rhalem", "Abdellah Idrissi Azami", "Nihal Habib", "Salsabil Hamdi", "Lahcen Wakrim", "Abderrahmane Bakkali", "Mustapha Mahmoud", "Laila Bouguenouch", "Najib Al Idrissi", "Youssef Bakri", "Saaïd Amzazi", "Said Barrijal" ], "abstract": "Background: The recent emergence of a novel coronavirus (SARS-CoV-2) has caused serious public health concerns due to its rapid dissemination worldwide. A total of 8,931 positive cases had been reported in Morocco by the 16th of June 2020. Methods: To better understand the SARS-CoV-2 pandemic in this North African country, we analyzed the complete genome sequences of the virus related to Morocco by constructing a phylogenetic tree and creating a variant network using the available Moroccan and other sequences in dedicated databases. Results: Phylogenetic and variant network analyses of SARS-CoV-2 strains from early patients with COVID-19 in Morocco showed multiple spatiotemporal introductions from Italy (ten), France (seven), Spain (one) and Portugal (one). This is consistent with the assumption that the early infections in Morocco were imported, mainly from Europe. The 17 virus strains form two independent phylogenetic clusters and provide evidence for early community-based transmission following the initial introductions of the virus. We then catalogued 13 novel mutations in the SARS-CoV-2 isolates from Moroccan patients. Interestingly, the recurrent missense variant A>G at position 23,403 in the spike gene, known to be associated with virus severity, has been identified in all Moroccan isolates. Conclusions: These primary findings testify of the importance of the genomic surveillance strategies as a means of understanding the virus spread dynamics, counteracting the pandemic outbreak, and drawing useful lessons for dealing with any future emerging infectious pathogens.", "keywords": [ "Coronavirus", "SARS-CoV-2", "Covid-19", "Morocco", "Molecular Epidemiology", "Genomics Surveillance", "Phylogenetics", "Variant Network Analysis" ], "content": "Introduction\n\nFollowing its onset in December 2019, several cases of a new respiratory illness were reported in the city of Wuhan, Hubei province, China1. The disease, named coronavirus disease 2019 (COVID-19), was later on confirmed as caused by a novel coronavirus that was subsequently called the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)2. On the 12th of March 2020, the ongoing SARS-CoV-2 outbreak was declared as a pandemic by the World Health Organization (WHO)3. As of June 16th 2020, there have been 8,228,025 confirmed cases with 444,442 deaths around the world (John Hopkins Center (JHC))4. Currently, Morocco has reported 8931 confirmed cases and 212 deaths associated with COVID-19 (JHC update of June 16th 2020). To gain further understanding of the molecular epidemiology of the outbreak in Morocco, we conducted a phylogenetic and a Variant Network Analyses of the full-genome sequences of 21 SARS-CoV-2 strains, 19 were isolated from Covid-19 patients in Morocco, one sequence was isolated from a patient in Melilla and another one was isolated from a Moroccan patient in Cadiz, Andalusia.\n\n\nMethods\n\nThe Morocco related-sequences used in this study have been deposited in the GISAID database by the Laboratory of the Royal Gendarmerie of Morocco (LRAM) (one sequence), the Pasteur Institute of Morocco (IPM) (17 sequences), the Anoual Laboratory in Casablanca (one sequence), and the SeqCovid Spanish Project (two sequences). The sequences were collected from the GISAID database and analyzed in comparison with 500 selected genomes, that were collected between the 15th of February 2020 (2 weeks before the first detected case in Morocco) and the 30th of March 2020 (2 weeks after the international borders of Morocco were closed; lockdown). Only high-quality sequences have been included in this study. All sequences and metadata are provided as Extended data5 and can be found at the GitHub repository https://github.com/covidmor/covid19_morocco.\n\nThe Moroccan sequences were mapped to the Wuhan reference genome (NC_045512) using Bowtie 2.3.5.16. Variants were called using mpileup 1.7 and bcftools 1.97. The variants were annotated using the China National Center for Bioinformation Annotator. Variant network analysis was performed using Gephi 0.9.2.\n\nA total of 500 sequences from different countries were retrieved from the GISAID database (www.gisaid.org) and aligned using Muscle 3.8.1551 multiple sequence alignment8; see Extended data, metadata5 for details on each. A maximum likelihood model was created using RaxML 8.2.129 with a bootstrap of 100 using the Wuhan reference genome (NC_045512) as outgroup. The phylogenetic tree was generated using Figtree 1.4.410.\n\n\nResults and discussion\n\nTo put the complete genomes of SARS-CoV-2 that were isolated from 19 Moroccan patients, a patient from Melilla and a Moroccan patient from Cadiz (Andalusia, Spain), into the context of the global pandemic, they were aligned together with the dataset of 500 SARS-CoV-2 complete genomes from different countries. Extended data, Figure 15 shows the estimated maximum likelihood phylogeny. To have a good presentation of the phylogeny, we created a circular presentation subset of 120 sequences from our dataset (collected from 18th of February 2020 (15 days before the first Moroccan case) to 15th of March 2020 (Date of closing international borders in Morocco)) (Figure 1b).\n\n(a) Maximum likelihood phylogeny, constructed using complete SARS-CoV-2 genomes from the GISAID database. The blue sequences represent the Moroccan isolates (19), a strain isolated from Melilla and a strain isolated from a Moroccan patient in Cadiz, Andalusia. The phylogenetic tree is divided into four clades: the red part for the GR strains, the green one for the GH strains, the yellow one for the G strains, and the pink one for the S strain. This Figure represents selected sub-tree extracts from the extended phylogenetic tree in Extended data, Figure 15. A1–3: represent clusters of Moroccan strains originated from Italy and belonging to the GR clade; B: represents a cluster of Moroccan strains from a French origin and belonging to the GH clade; C: represents the Moroccan strain isolated from Ouarzazate, it originated from Portugal and belongs to the G clade; D: represents a cluster of Moroccan strains originated from France and belonging to the G clade; E: represents a Moroccan strain that originated from Italy and it belongs to the G clade; F: represents a Moroccan strain from a Spanish origin and belonging to the G clade, G: represents the sequence isolated from Melilla belonging to the G clade with a Spanish origin, H: represents the sequence isolated from Cadiz; it belongs to the S clade and it’s close to strains from Spain. (b) Circular representation of the phylogenetic tree of a subset of 120 sequences collected before closing Moroccan international borders, constructed using complete SARS-CoV-2 genomes from the GISAID database. The blue sequences represent the Moroccan isolates (19). The phylogenetic tree is divided into three clades: the red part for the GR strains, the green one for the GH strains, and the yellow one for the G strains. The clade S containing the Cadiz sequence is absent as we removed this sequence from this phylogenetic analysis.\n\nThe extended phylogenetic tree is subdivided into seven clades that correspond to the main seven SARS-CoV-2 strain types GR (red), GH (green), G (yellow), S (pink), V (magenta), L (light gray) and O (others; dark gray). The four strain types S, G, GR, and GH are scattered all over the world (Extended data, Figure 2)5. According to GISAID (update of the 12th of June 2020), they made their first appearance in February 2020, being G the strain type that gave origin to the GH and GR ones, while the S strain was mainly found in Spain11.\n\nEvery dot represents a sequence, and each edge on the network connects, using a shared variant, a sequence to one or more other sequences. The red circle group Moroccan sequences originated from Italy, the green one group sequences originated from France, the purple one assemble sequences originated from Spain, and the blue one represents the sequence originated from Portugal; we used different color gradients to separate sequences that belong to different clades in the same cluster. A: Strain isolated from Cadiz, it belongs to the S clade and it shares no variants with Moroccan strains, including the Spanish ones. M: represents the strain isolated from Melilla, it belongs to the G clade in a cluster of Spanish sequences. IPM3: is a Moroccan strain isolated by Pasteur Institut of Morocco (IPM), it belongs to the G clade and it’s coming from Spain. O: represents the strain isolated by LRAM from Ouarzazate, it's the only sequence with a Portuguese origin and it belongs to the G clade. IPM9: represents a sequence collected by IPM, it originated from Italy and belongs to G clade. C; IPM1; IPM2; IPM7; IPM10; IPM13; IPM15-17: represent a strain isolated by Anoual laboratory in Casablanca (C) and sequences collected by IPM, they belong to the GR clade and they all are from Italian origin. IPM4; IPM11-12: represent strains isolated by IPM, they originated from France and they belong to the GH clade. IPM5-6; IPM8; IPM14: represent strains isolated by IPM they are from a French origin and they belong to the G clade.\n\nZooming into the trees (Extended data, Figure 1)5, Figure 1a shows how the Moroccan isolates group into four independent clusters. In the GR clade, the sequences GISAID IDs: EPI_ISL_459973, EPI_ISL_459966 and EPI_ISL_459965 (the first infected patient in Morocco) cluster together with sequences from Italy (GISAID ID: EPI_ISL_417922), Thailand (GISAID ID: EPI_ISL_430837), Mexico (GISAID ID: EPI_ISL_452139), and Iceland (GISAID ID: EPI_ISL_417829) (Figure 1a: A1–3). Interestingly, the hosts of these four sequences were all travelling in Italy during the same period (Extended data, Metadata)5. This suggests that the first Moroccan patient (GISAID: EPI_ISP_459965), that returned to Morocco from recent travel to Bergamo, Lombardy (the most affected region in Italy), and the three other individuals from Mexico, Iceland and Thailand (GISAID: EPI_ISL_452139, EPI_ISL_417829 and EPI_ISL_430837, respectively) were probably infected by the same person in Italy (GISAID: EPI_ISL_417922). From this cluster, we infer, based on the sample collection dates (Extended data, Metadata)5, that the first Moroccan patient (GISAID: EPI_ISP_459965) probably infected the two other Moroccan patients whose infecting virus sequences are found in the same cluster (GISAID: EPI_ISL_459973 and EPI_ISL_459966). The Moroccan sequences (GISAID IDs: EPI_ISL_458150, EPI_ISL_459980 and EPI_ISL_459984) that cluster together in the GR clade, are close to sequences from Iceland (GISAID IDs: EPI_ISL_417861, EPI_ISL_417863 and EPI_ISL_417852) whose carriers have travel history to Italy. Furthermore, all these three Icelandic sequences are related to one Italian sequence (GISAID ID: EPI_ISL_460081). For their part, the Moroccan sequences EPI_ISL_459983, EPI_ISL_459977 and EPI_ISL_459978 cluster with another sequence from Italy (GISAID ID: EPI_ISL_452186). Surprisingly, many sequences from the GR clade were isolated from patients with known travel history to Italy (Figure 1a: A1–3). Therefore, we can conclude that all the viral sequences in this clade, where 9 Moroccan viral sequences clustered, were of Italian origin.\n\nThe four Moroccan sequences that cluster together within the G type strain (GISAID IDs: EPI_ISL_459968, EPI_ISL_459974, EPI_ISL_459972 and EPI_ISL_459981) were close to French sequences (GISAID ID: EPI_ISL_417333, EPI_ISL_443270 and EPI_ISL_416752). These four Moroccan sequences were collected the 17th of March, the 20th of March, the 20th of March and the 19th of April 2020, respectively. Interestingly, the third Covid-19 case in Morocco was a French male tourist, in his 30s that arrived in Morocco on the 7th of March 2020 as reported by the Moroccan Health authorities. He, therefore, was most likely the source of the Moroccan viral strains that belong to the G type. The case reported on the 19th of April 2020 (sequence with the GISAID ID: EPI_ISL_416752) seems to be the result of a local community transmission.\n\nThe Moroccan strain isolated from Ouarzazate (South of Morocco) (GISAID ID: EPI_ISL_451400) is close to the Portuguese sequences (GISAID ID: EPI_ISL_413648 and EPI_ISL_453947) in the G type suggesting a third introduction path to Morocco from Portugal. For their part, the Moroccan sequence (GISAID ID: EPI_ISL_459967), that also belongs to the G type, is close to the Spanish sequences (GISAID IDs: EPI_ISL_455349 and EPI_ISL_455332) making Spain another point of introduction to Morocco. Furthermore, the Moroccan sequence (GISAID ID: EPI_ISL_459975) is closely related to the Italian G strain (GISAID: EPI_ISL_417921), which represents another Italian introduction to Morocco. It’s also similar to a sequence from Iceland (GISAID ID: EPI_ISL_417730), where the host patient has travel history to Italy (Figure 1a: E) and could have been either infected in Italy or by another asymptomatic carrier coming from there.\n\nThree other viral sequences isolated from Moroccan patients (GISAID IDs: EPI_ISL_459976, EPI_ISL_459982 and EPI_ISL_459979) appear in the clade of the GH strain types where they cluster with French sequences (GISAID IDs: EPI_ISL_416748 and EPI_ISL_416501) (Figure 1a: B) suggesting a further introduction to Morocco, once more from France.\n\nThe viral sequence isolated from a patient in Melilla (GISAID ID: EPI_ISL_455344) is clustered within the G type, with Spanish sequences (GISAID IDs: EPI_ISL_419235, EPI_ISL_450337 and EPI_ISL_419236). While the sequence isolated from a Moroccan patient in Cadiz, Andalusia (GISAID ID: EPI_ISL_452463) (Figure 1a: G) that belongs to the S type, also clusters with Spanish sequences; however, this sequence is quite far from the other Spanish related sequences. Thus, supporting the apparent neutrality of the host’s genetics for the transmission and potential selection on the type of strain.\n\nFinally, similarity analysis of the Moroccan sequence (GISAID ID: EPI_ISL_467299) collected on May 21st, 2020 and sequenced by the LRAM Laboratory, is close to the first identified French cluster, suggesting that the virus strain circulating in Morocco did not experience any major mutations and, once again, confirms the local community-based transmission of the virus. This sequence was not included in the overall analysis, as it was deposited when the writing of this work was finalizing.\n\nThus, the phylogenetic analysis shows that the virus outbreak in Morocco was likely the result of multiple introductions. We can highlight four independent introductions to Morocco from Italy, France, Spain, and Portugal. This finding has obvious implications for the epidemiological tracing of the pathogenic agent’s initial introduction that caused the current outbreak in Morocco. Generally, the viral sequences isolated from Moroccan patients showed close relationships primarily to European strains. The geographic nearness and tourist and migratory connections, therefore, play a key role in the spread of the virus. We caution that further analyses are needed to evaluate the statistical robustness of the interference suggested herein.\n\nTo further understand the evolution of the SARS-CoV-2 virus within the Moroccan population and trace the infection pathways, we performed a Variant Network Analysis using Gephi. We used the complete sequences of the 21 SARS-CoV-2 genomes, 19 from Morocco, and two sequences from Melilla and Andalucia (Figure 2). The variant network in Figure 2 shows three main clusters, one isolated sequence from Portugal and another one from Cadiz sharing no variants with the other sequences. The first cluster comprising the following viral strains, all closely related to each other and diagnosed in Casablanca (west Morocco) (GISAID ID: EPI_ISL_458150), IPM_1 (GISAID ID: EPI_ISL_459965), IPM_2 (GISAID ID: EPI_ISL_459966), IPM_13 (GISAID ID: EPI_ISL_459980), IPM_15 (GISAID ID: EPI_ISL_459978), IPM_7 (GISAID ID: EPI_ISL_459973), IPM_16 (GISAID ID: EPI_ISL_459977), IPM_10 (GISAID ID: EPI_ISL_459984) and IPM_17 (GISAID ID: EPI_ISL_459983) were closely related to each other. This is in agreement with the phylogenetic tree where all these sequences belong to the GR clade and appear to have been originated from Italy (Figure 1 and Extended data, Figure 1)5. The second cluster (Blue) made out of the sequences labeled IPM_5 (GISAID ID: EPI_ISL_459974), IPM_6 (GISAID ID: EPI_ISL_459968), IPM_8 (GISAID ID: EPI_ISL_459972) and IPM_14 (GISAID ID: EPI_ISL_459981) belong to the clade G and has a French sequence (GISAID ID: EPI_ISL_418222) as a root in the phylogenetic tree. The sequences IPM_4 (GISAID ID: EPI_ISL_459976), IPM_11 (GISAID ID: EPI_ISL_459982) and IPM_12 (GISAID ID: EPI_ISL_459979) belong to the GH clade and seem to have originated from another French sequence (GISAID: EPS_ISL_418219). Interestingly, this sequence belongs to the G clade and is close to a strain that was isolated from an Icelandic patient that was most probably infected during his stay in Italy (GISAID ID: EPI_ISL_417730). Another separated Moroccan sequence, labelled IPM_3 (GISAID ID: EPI_ISL_459967), is closer to Spanish sequences (GISAID ID: EPI_ISL_455349, EPI_ISL_455332). The sequence of the viral strain from Ouarzazate (GISAID ID: EPI_ISL_451400) is closely related to a Portuguese sequence (GISAID ID: EPI_ISL_413648), just as shown in the phylogenetic tree. Yet, all these sequences belong to the G clade, which has an Italian sequence as a root. Hence, the Variant Network Analysis is in accordance with the phylogenetic tree and further supports our deduction of multiple SARS-CoV-2 introductions to Morocco from at least four European countries. Thus, while geography marks the variation that the virus undergoes in a way that we can identify geographically separated strains, population interconnection, via travel and migratory flows, seems more important than the geographical nearness for the worldwide spread of the virus.\n\nTo further test the multiple introductions to Morocco from the aforementioned four European countries, we performed a Variant Network analysis using all the Moroccan sequences as one group and compared them with randomly selected sequences from different countries (Italy, France, Spain and Portugal, in addition to Austria, Australia, and Brazil). These results show that the sequences detected during the early stages of the epidemic in Moroccan share variants with Italian (seven variants), French (four), Portuguese (three), and Spanish (three) strains. They also share one variant with strains from Austria, one with Brazil and another one with Australia (Figure 3). Interestingly, there was one shared variant between all countries located in the Spike gene (23403A>G) which induces the amino acid change D614G; it began spreading in Europe in early February and, when introduced to new regions, it rapidly became the dominant form. This mutation is suspected to increase the transmissibility of the virus12.\n\nEach dot represents a group of 19 sequences randomly selected from each country. The Moroccan node represents all 19 sequences isolated in Morocco. Morocco shares more variants with Italy (seven), France (four), Portugal (three) and Spain (three) than with Brazil (one), Austria (one) and Australia (one).\n\nThe genome-wide SNPs and the corresponding amino-acid positions and variations of the virus proteins are described in Table 1. Our results showed that all 20 sequences including the sequence coming from the Melilla patient, shared four mutations in common and 13 novel mutations exclusively present in the 19 sequences isolated in Morocco (Table 2). The four common mutations happened to be the same recurrent mutations described in several reports13. The mutation in the leader sequence (241C>T) is one of the most common mutations in the SARS-CoV-2 genome; it affects an important genomic site for discontinuous sub-genomic replication. The mutation in the 5’UTR genomic region co-evolved with two other mutations, 14408C>T and 23403A>G. They both affect critical RNA replication proteins (241C>T, 14408C>T) and the ACE2 receptor binding protein S (23403A>G). We noticed that these four mutations are prevalent in the virus isolates from Europe, where the infections seem to be more severe. In fact, Yin et al.14 suggested that these mutations could increase the transmissibility of the virus. The sharing of these mutations between European and Moroccan isolates further indicates that strains circulating in Morocco came mainly from Europe. Moreover, at least one variant of the Moroccan sequences of SARS-CoV-2 is shared with one of the other countries (Table 3). The mutations found in the sequences identified in Morocco, and the identification of amino acid change, should be further investigated in order to understand whether they affect the transmission and clinical characteristics of the SARS-CoV-2 virus circulating in Morocco.\n\n\nConclusions\n\nPhylogenetic and variant network analyses of SARS-CoV-2 sequences from early patients with COVID-19 in Morocco showed multiple spatiotemporal introductions, primarily from Italy (ten), France (seven), Spain (one) and Portugal (one). The results also provide evidence for early community-based transmission. A variant calling analysis allowed us to catalogue new mutations in SARS-CoV-2 isolates from Morocco. Interestingly, the recurrent missense variant A>G at position 23,403 in the spike gene known to be associated with virus severity has been identified in all Moroccan isolates. However, the lack of demographic and clinical information on most of the sequences of the Moroccan isolates deposited in the database prevented us from inferring the potential link between the mutations and clinical effects of the strains. Also, the number of Moroccan genomes available at the time of this analysis hampers robust statistical inferences. Still, the results of the present work offer interesting insights into how the virus got into Morocco and primary lessons as to understanding the dynamics of the initial introductions and local transmission of SARS-CoV-2 in Morocco. This being a global pandemic, the conclusions of this study, taken together with those of recent similar studies such as those done in Egypt15, South Africa16 and Brazil11, might be applicable to other developing countries. The results of the methodology of genomic surveillance that we developed, and the methodology itself would be helpful in the case of resurgence or the emergence of any new pandemic-causing pathogen. Integrative analysis of SARS-CoV-2 should promote our understanding of the virus dynamics and interactions with hosts and environments. The method can even be further extended to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens17.\n\n\nData availability\n\nSequences analyzed in this study were downloaded from the GISAID database. The identity of each sequence is given in the Extended data, Metadata file5.\n\nZenodo: Genomic evidence of multiple SARS-CoV-2 introductions into Morocco. http://doi.org/10.5281/zenodo.39094635.\n\nThis project contains the following extended data:\n\nExtended data Figure 1 (PDF). (Full size image of the phylogenetic tree seen in Figure 1.)\n\nExtended data Figure 2 (PNG). (Full genome tree derived from all outbreak sequences.)\n\nExtended data Labels (PDF). (Labels of the Moroccan sequences)\n\nExtended data Metadata (PDF). (Metadata for the sequences assessed in this study.)\n\nAll supplementary material and figures can be found at GitHub: https://github.com/covidmor/covid19_morocco.\n\nExtended data are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWu P, Hao X, Lau EHY, et al.: Real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in Wuhan, China, as at 22 January 2020. Euro Surveill. 2020; 25(3): 2000044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUddin M, Mustafa F, Rizvi TA, et al.: SARS-CoV-2/COVID-19: Viral Genomics, Epidemiology, Vaccines, and Therapeutic Interventions. Viruses. 2020; 12(5): 526. [cited 2020 Jun 15]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO announces COVID-19 outbreak a pandemic. 2020. [cited 2020 Jun 15]. Reference Source\n\nJohns Hopkins Center: Coronavirus COVID-19 (2019-nCoV). 2020. [cited 2020 Jun 16]. Reference Source\n\ncovidmor: covidmor/covid19_morocco: Genomic evidence of multiple SARS-CoV-2 introductions into Morocco (Version 1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3909463\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4): 357–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5): 1792–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006; 22(21): 2688–90. PubMed Abstract \| Publisher Full Text\n\nRambaut A: FigTree Version 1.4.4. 2020. [cited 2020 Jun 15]. Reference Source\n\nde Jesus JG, Sacchi C, Claro I, et al.: First cases of coronavirus disease (COVID-19) in Brazil, South America. 2020. Reference Source\n\nKorber B, Fischer WM, Gnanakaran S, et al.: Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2. bioRxiv. 2020; 2020.04.29.069054. Publisher Full Text\n\nPachetti M, Marini B, Benedetti F, et al.: Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med. 2020; 18(1): 179. [cited 2020 Jun 4]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYin C: Genotyping coronavirus SARS-CoV-2: methods and implications. Genomics. 2020. [cited 2020 Jun 15]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKandeil A, Mostafa A, El-Shesheny R, et al.: Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Egypt. Microbiol Resour Announc. 2020; 9(22): e00489–20. [cited 2020 Jun 15]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMavian C, Pond SK, Marini S, et al.: Sampling bias and incorrect rooting make phylogenetic network tracing of SARS-COV-2 infections unreliable. Proc Natl Acad Sci U S A. 2020; 117(23): 12522–12523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGussow AB, Auslander N, Faure G, et al.: Genomic determinants of pathogenicity in SARS-CoV-2 and other human coronaviruses. bioRxiv. 2020; 2020.04.05.026450. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "66513", "date": "13 Jul 2020", "name": "Keith Crandall", "expertise": [ "Reviewer Expertise Phylogenetics", "population genetics", "molecular evolution", "bioinformatics", "infectious disease" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: This paper reports on new sequence data on the SARS-CoV-2 outbreak in Morocco. Through phylogenetic analyses they identify 4 distinct introductions to Morocco from distinct European origins followed by local transmissions. The analysis are OK, but need some additional work (see below). Specifically, there are no bootstrap analyses discussed to provide confidence in the resulting phylogenetic relationships (the main result of the paper). The authors use an excellent sampling strategy, focusing on Moroccan sequences and then those available from two weeks before the first known case in Morocco and two weeks after the closing of the boarders. Their data are deposited with GISAID and thereby publicly available (with some effort and approval), but they also provide their entire working set on GitHub. Excellent! This is the way to do open science which is so critical for the COVID studies. However, the nomenclatural approach is confusing and the phylogenetic presentation could use some help. There are a lot of different things going on with this analysis (lots of different metadata to focus on) and the authors could do a better job of making things more clear in their presentation. Below are some ideas to help with both the analyses and interpretation.\n\nAbstract/Results spike gene variant associated ‘with virus severity’ – this doesn’t make sense to me. What does it mean to have a severe virus? Do you mean severity in clinical outcomes?\n\nMethods/Data Set – you say 500 sequences were selected from GISAID both before and after the initial case in Morocco. I cannot, however, get a feel for the relative split or the relative timeline. It would be really helpful to have a graph that had time along the x-axis (perhaps by week or something) and then different colors for the pre-GISAID, post-GISAID, and Moroccan sequences.\n\nMethods/Phylogenetic analysis – the authors state the ‘model was created using RaxML’. First, RAxML doesn’t create models. It estimates phylogenies. Second, it needs a model to do this. What model was used in this analysis? There is an updated version of ModelTest, ModelTest-NG https://academic.oup.com/mbe/article/37/1/291/5552155 that helps with optimizing a model of evolution for a data set to then feed into RAxML. I recommend the authors use this tool to justify a model choice, report the model, and then use it for the ML estimate. Then ‘FigTree’ doesn’t ‘generate’ a tree, it is a tool for phylogeny visualization. I would recommend iToL as an alternative. iTOL also allows for more legible mapping of metadata on the phylogeny: https://itol.embl.de/ Given that the phylogeny seems to be the main result, the authors should conduct a bootstrap analysis with at least 1000 replicates and show bootstrap support values on the phylogeny as an assessment of confidence.\n\nIn general, no details of the analyses are provided and it is therefore impossible to replicate the study. The authors simply list software. They need to be more explicit about how they actually ran their analyses.\n\nSome pretty specific information is provided on patients, e.g., ‘French male tourist’ with a date of the case report. I’m wondering if these individuals are consented to participate in the research and be effectively identified. This seems pretty specific. There is no information about consent.\n\nResults/Phylogenetic analyses – the phylogenetic results are difficult to read. The image is low resolution, so if I zoom in on the pdf, it just gets blurry. There are a different nomenclatures being thrown about (A1, A1, GR, GH, etc.) and it is hard to find these groups on the tree and or their significance. There are no bootstrap values, so there is no confidence in the presented relationships. Again, if the authors us iTOL, they can make a more effective presentation with better labels on the tree. I don’t see the pink clade on the main tree at all.\n\nIn the text, everything is presented in terms of GISAID accessions, which is nice for track but not for presentation. It would be better if the authors relabeled things in the supplementary table with more accessible labels like Moroc1, Franc1, Spain1, etc., and then not list every isolate in a clade in the text. It would be even more informative if you included isolation date in the name, e.g., Moroc1_3_21. This would allow readers to immediately interpret the phylogeny. As it is, it is uninformative with respect to isolation date and difficult to identify isolates from different countries.\n\nThen in the sequence variant network analysis, the nomenclature seems changed again. Now there are things called ‘IPMx’. I don’t understand why this network analysis is not done in the context of the other non-Moroccan sequences. Given the phylogenetic result that the Moroccan sequences don’t form a cluster by themselves, but rather cluster with other sequences with distinct geographic origins, it makes no sense to analyze them by themselves here. You are just picking up variants that differentiate the geographic clades and assigning them (incorrectly) to the Moroccan sequences. It would be much more powerful to compare the Moroccan sequences to the others WITHIN EACH CLADE as this allows for effectively independent replicates of viral adaptation to Moroccan hosts. Then you can look for convergently evolved changes associated with the Moroccan isolates.\n\nTables 1, 2, & 3 should identify the nonsynonymous changes (e.g., show the amino acid replacement in the same way you show the nucleotide substitution).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-679
https://f1000research.com/articles/9-1028/v1	24 Aug 20	{ "type": "Software Tool Article", "title": "CRI iAtlas: an interactive portal for immuno-oncology research", "authors": [ "James A. Eddy", "Vésteinn Thorsson", "Andrew E. Lamb", "David L. Gibbs", "Carolina Heimann", "Jia Xin Yu", "Verena Chung", "Yooree Chae", "Kristen Dang", "Benjamin G. Vincent", "Ilya Shmulevich", "Justin Guinney", "Vésteinn Thorsson", "Andrew E. Lamb", "David L. Gibbs", "Carolina Heimann", "Jia Xin Yu", "Verena Chung", "Yooree Chae", "Kristen Dang", "Benjamin G. Vincent" ], "abstract": "The Cancer Research Institute (CRI) iAtlas is an interactive web platform for data exploration and discovery in the context of tumors and their interactions with the immune microenvironment. iAtlas allows researchers to study immune response characterizations and patterns for individual tumor types, tumor subtypes, and immune subtypes. iAtlas supports computation and visualization of correlations and statistics among features related to the tumor microenvironment, cell composition, immune expression signatures, tumor mutation burden, cancer driver mutations, adaptive cell clonality, patient survival, expression of key immunomodulators, and tumor infiltrating lymphocyte (TIL) spatial maps. iAtlas was launched to accompany the release of the TCGA PanCancer Atlas and has since been expanded to include new capabilities such as (1) user-defined loading of sample cohorts, (2) a tool for classifying expression data into immune subtypes, and (3) integration of TIL mapping from digital pathology images. We expect that the CRI iAtlas will accelerate discovery and improve patient outcomes by providing researchers access to standardized immunogenomics data to better understand the tumor immune microenvironment and its impact on patient responses to immunotherapy.", "keywords": [ "genomics", "cancer", "immunology", "systems biology", "R", "Shiny" ], "content": "Introduction\n\nImmuno-oncology (IO) is one of the most promising areas of cancer research, with IO-based treatments demonstrating high efficacy within certain cancer types and subsets of patients1–4. To broaden the utility of these therapies to more patients, fundamental research is required to improve our understanding of tumor-immune interactions—allowing the next-generation of therapeutics and treatment strategies to emerge4. Advances in the IO field are impeded by the inaccessibility of IO study data and results and lack of data standardization, limiting the ability to easily compare results across studies. This has led to the underutilization of existing data, unnecessary study duplication, and failure to achieve rapid consensus in the field5. With the vast increase in the number and scope of IO projects expected in the coming years combined with widespread adoption of genomics and other high dimensional technologies, these problems will be compounded going forward.\n\nWe developed the Cancer Research Institute (CRI) iAtlas portal (https://www.cri-iatlas.org) to integrate IO research data, with the goal of providing an interactive, exploratory hub for the IO research community. In doing so, we hope to improve the accessibility and utility of critical resources generated from IO studies. iAtlas is a set of analytic modules—hosted on the web—for studying interactions between tumors and the immune microenvironment. These modules allow researchers to explore associations among a variety of immune characterizations as well as with genomic and clinical phenotypes.\n\nThe initial release of iAtlas (April 5, 2018) provided a rich resource to complement analysis results from The Cancer Genome Atlas (TCGA) Research Network on the TCGA data set comprising over 10,000 tumor samples and 33 tumor types6 (“The Immune Landscape of Cancer”; here referred to as “Immune Landscape”). This study identified six immune subtypes that span cancer tissue types and molecular subtypes, and found that these subtypes differ by somatic aberrations, microenvironment, and survival. Per-sample characterizations included total lymphocytic infiltrate (from DNA methylation as well as H&E imaging data), estimated cell type fractions, immune gene signature expression, MHC/HLA type and expression, antigen presentation machinery, T cell and B cell receptor repertoire inference, viral/microbial characterization, associations with pathway disruption and activity, and other analysis results. The Immune Landscape6 manuscript reported on the most novel and potentially therapeutically salient statistical associations between these immune subtypes and the results of the immune characterization. We have continued to develop and evolve the CRI iAtlas application; here, we report the technical design and implementation of iAtlas up to and including the recently released version 1.27. This version includes new features requested by users including: (1) user-defined loading of sample cohorts, (2) a tool for classifying expression data into immune subtypes, and (3) integration of TIL mapping from digital pathology images.\n\n\nMethods\n\niAtlas is a web-based application to enable data exploration for clinicians, biologists, and informaticists. The inputs and architecture of the application are described below.\n\nThe iAtlas app uses structured data and outputs from the Immune Landscape6 study and the TCGA PanCancer Atlas initiative8, which harmonized TCGA data, ensuring uniform quality control and sample inclusion, batch effect detection, normalization across platforms, combination mutation calling from multiple centers, and robustly compiled clinical and outcome data. A key source of iAtlas data is the table summarizing tumor-sample and immune characterizations for 11,080 TCGA patient participants of the TCGA, Table S1 of the Immune Landscape6 manuscript, here termed the “PanImmune Feature Matrix”. Auxiliary data were sourced from files available on this manuscript’s data page at the NCI Genomic Data Commons, from the TCGA PanCancer Atlas Data Mirror, and from the TCGA PanCancer Atlas working space in Synapse (see Data availability). iAtlas data were formatted as data frames (tables) and stored as “feather” files (https://github.com/wesm/feather) on the application server for fast loading (Table 1).\n\nAnnotation and browsing of the PanImmune Feature Matrix: iAtlas includes a Data Description page with details on all variables presented in individual modules, with the ability for users to “drill down” on related groups of variables to understand how values were derived. Variables are listed in a text-searchable table containing the name of the variable, the ‘Variable Class’, the unit (if applicable), and whether the variable is numeric or categorical. A ‘Variable Class’ is the name of a group of variables that are of similar type and are often the result of one particular analysis. Clicking on a row exposes a list of all variables in the ‘Variable Class’ and provides links to text descriptions of the analysis methods used to generate the variables.\n\niAtlas is powered by Shiny9 and makes extensive use of Shiny Modules10 to organize code into composable units (Figure 1). Each iAtlas Analysis module is designed as a Shiny module, allowing simple integration of new analytical functionality. iAtlas uses the tidyverse11 family of R packages (e.g., dplyr12, tidyr13, purrr14, stringr15, tibble16) as well as the wrapr17 package to assist with tidy evaluation. These functions power the data transformations of internal tabular data that are then used to create the interactive plots (i.e., with the plotly18 graphing library) and data tables (via the DT19 wrapper to the DataTables library) seen through the iAtlas modules. We also make heavy use of the crosstalk20 package to enable event-driven updates to the application state. The core iAtlas application is hosted on https://shinyapps.io.\n\n(A) Structured data from immunogenomic analyses, including the Immune Landscape6 study and expanding over time, are organized and stored as flat (i.e., feather) files within Synapse and made available alongside the application code in GitHub. (B) Tabular data from feather files are read from disk into memory to drive all operations related to sample groupings, sample-level immune characterizations (readouts), and more granular—and high dimensional—assay measurements. (C) The core application code is built as a catalog of Shiny Modules, each of which encapsulates logic for data transformation and visualization related to a scientific theme or assay type. (D) Analysis modules, tools, and data description views are hosted in a unified application on shinyapps.io; the layout and connectivity between modules in the iAtlas Explorer space are managed by the shinydashboard21 library.\n\nThe main feature of the iAtlas interface is the iAtlas Explorer (Figure 2, found under the EXPLORE tab), which provides several Analysis modules to explore and visualize results. Each module supports a type of exploration, with interactive views and controls to enhance and extend the results and analytics as initially described in the Immune Landscape6 study. The layout of pages and sections within the iAtlas Explorer is driven by the shinydashboard21 package.\n\nA range of Analysis modules (blue boxes above) are available that span from clinical to molecular and imaging data types. Within each module, interactive controls allow researchers to expand views, exposing underlying data and results. Settings are available (green box above) to select the sample groupings (TCGA Study, Disease Subtype, or Immune Subtype) which then propagate through modules.\n\nWithin each module in iAtlas, results are displayed relative to Sample Groups, corresponding to defined study cohorts. Several Sample Groups options are pre-loaded in the tool: first, TCGA tumor type (TCGA Study), which are the standard TCGA tumor types collected and designated by the TCGA. Second, TCGA tumor subtypes (TCGA Subtype), a compendium of further subdivision of TCGA studies into molecular subtypes according to publications by the TCGA Research Network22. Finally, a division of tumor samples into distinct patterns of immune response in cancer (Immune Subtypes) is provided6. The choice of Sample Groups is global across all modules but can be updated at any time via the Select Sample Groups element in the side menu. We also allow users to upload custom-grouped samples and analyze those with iAtlas modules. The selection of a sample group defines the samples utilized in all analysis modules. For convenience, group annotations can be displayed in visualizations within each module.\n\nSample Group Overview: View summary information for user-selected sample cohort groups. There are currently three sections: Custom Groups, Group Key, and Group Overlap. Respectively, these sections permit loading of user-defined sample groups, review of detailed annotations of sample groups in a table, and display of overlap between different types of groupings in a mosaic plot.\n\nTumor Microenvironment: Explore immune cell proportions in sample groups with two sets of faceted bar charts, one for overall cellular proportions (i.e., leukocyte, stromal, and tumor fraction) and one for computed immune cell proportions (e.g., monocytes, CD8+ T cells, naive B cells).\n\nImmune Feature Trends: Visualize how immune readouts vary across sample groups. Violin or box plots show the distribution of individual values across samples in each group, while heatmaps and scatter plots can be used to explore the correlation between any pair of variables within each group.\n\nClinical Outcomes: Quantify the relationship between immune response and disease outcome, in terms of either overall survival (OS) or progression free interval (PFI)23. Results are displayed as Kaplan Meier plots as well as heat maps showing the concordance index between variables and survival.\n\nImmunomodulators: Explore the expression of genes coding for immunomodulating proteins6, which include therapeutically important immune checkpoint proteins. Immunomodulators are organized by grouping into three categories: Gene Family (such as “TNF”, “MHC Class II”, “Immunoglobulin”, or “CXC chemokine”), Super Category (such as “Ligand”, “Receptor”, or “Antigen presentation”), and Immune Checkpoint (classified as “Inhibitory” or “Stimulatory”). Violin and box plots are again used to present distributions, and a table provides additional metadata about immunomodulator genes.\n\nDriver Associations: Test and visualize associations between mutations and IO-related response variables. In the Immune Landscape6 study, we reported somatic driver alterations that are correlated with increases or decreases in overall immune cell content, or with the fraction of individual immune cell types. These and other variables can be selected to calculate the significance of relationships in each sample group and view results in a volcano plot.\n\nTIL Maps: We used the results of a recently reported method to assess which spatial regions of hematoxylin and eosin (H&E) whole slide images show evidence of tumor-infiltrating lymphocytes (TILs)24. The method, which uses deep learning, was applied to thousands of H&E slides of the TCGA, allowing slides to be characterized in terms of TIL density and patterns.\n\nIntegration with Landscape of IO Drug Target Development: CRI has compiled and published comprehensive overviews describing ongoing immunotherapy drug trials, including targets, agents, and tumor sites and has made summaries available in an online resource, the Immune-Oncology Landscape (IO Landscape ) (www.cancerresearch.org/IO-landscape)25–28. The iAtlas and the IO Landscape resource have been interlinked, enabling researchers to more readily understand the relationship between targeted proteins in IO therapy and the behavior of those targets in tumor tissue.\n\nIn IO Target Gene Expression Distributions, the distribution of gene expression values for the selected IO target, by sample group, is displayed in violin plots. Clicking on the expression distribution (violin plot) of a particular sample group, a histogram of the values is displayed.\n\nThe IO Target Annotations section provides a searchable table with IO targets and associated annotations. In the rightmost column, a link is provided to a view of the IO Landscape page, the selected target is highlighted in summary barcharts showing the number of agents and cancer types being studied for that target.\n\nIn the opposite direction, clicking on targets in the barcharts in the IO Landscape on CRI web pages brings up the target gene expression in iAtlas.\n\niAtlas Tools are accessible via the TOOLS tab on the iAtlas Portal. Modules in this space of the portal enable users to “bring their own data” for processing through immunogenomic algorithms that drive some of the results presented in the Analysis modules described above.\n\nImmune Subtype Prediction: This tool performs classification of RNA-seq data into one of six immune subtypes as described in the Immune Landscape6 study. Using a new ensemble model based on XGBoost29, researchers can upload their own data for classification30. Each member of the ensemble was trained on a random subset of previously reported immune subtypes6 and features (described below) based on gene expression data from the TCGA PanCancer Atlas Initiative8. All code and methods have been confirmed as reproducible. An R package is available on GitHub (https://github.com/CRI-iAtlas/ImmuneSubtypeClassifier)30.\n\nThe submitted expression data—subsetted to the 485 genes that comprised the 5 signatures that produced the immune subtypes—are used to generate robust features of three types: quartiles, binary gene-pairs, and signature-pairs. For example, given a single sample, genes are binned into quartiles and given a bin label (quartile features). Then, similar to the “Top Scoring Pairs” classifier31, genes are paired, and given binary values depending on whether (gi > gj) for two gene expression values, gi and gj. Lastly, signature-pair features are calculated using the five immune subtype signatures, where smn = ∑ij(gim > gjn)/k, where gim is gene i from signature m, gjn is gene j from signature n, and k is the number of gene pairs considered resulting in a value between 0 and 1. The features are computed independently for each sample, and do not require normalization across samples. These features are given to a trained XGBoost classifier which returns a probability of being in any of the six subtypes. Lastly, a “best call” is made with a final trained XGBoost classifier using the six probabilities as input. To validate the robustness of the classifier, TCGA data were processed using four different software pipelines and normalization, showing that classification performance was independent of the gene expression quantification method30. Along with a downloadable table of results, visualizations are also provided. This tool is a convenient way for researchers to apply the methods of the Immune Landscape6 study to their own data without difficult statistical coding.\n\n\nOperation\n\nTo use iAtlas, access the web app via https://www.cri-iatlas.org. The software can also be run locally on all platforms (Windows, Mac, Linux). To run the Shiny app locally, a working R installation with necessary libraries is required and an installation of RStudio is recommended.\n\nTo install and run the app locally:\n\n1. Clone shiny-iatlas GitHub repository\n\n\n\n2. Open shiny-iatlas.Rproj in RStudio\n\n3. Install packages. In the RStudio console, run:\n\n\n\n4. Start the app by running:\n\n\n\n\nUse cases\n\nOne of the initial motivations behind iAtlas was to provide an interactive platform that is able to reproduce figures published in the Immune Landscape6 manuscript but expands that with the ability to generate variations of those figures, for other choices of tumor samples and immune readouts of interest. As an example, in order to reproduce Figure 4A from the Immune Landscape6 publication, which shows the correlation of DNA damage measures with the fraction of leukocytes in the tumor, we began by selecting the EXPLORE tab. We then opened the Immune Feature Trends module and selected the “Immune Subtype” option under Select Sample Groups in the Explorer Settings panel in the left menu. In the ensuing module page, at the Correlations section (Figure 3), we selected the “DNA Alterations” under Select or Search for Variable Class, “Leukocyte Fraction” under Select or Search for Response Variable, and the “Spearman” method under Select or Search for Correlation Method (each a separate dropdown menu). This produced a heatmap identical in content to Figure 4A in the Immune Landscape6 publication. However, the heatmap provides additional information on underlying data via interactivity: by clicking on a heatmap-cell, the underlying data is displayed in a scatterplot. Hovering a cursor over a point in the scatter plot reveals sample-level information.\n\nTop right: Original manuscript figure panel from the Immune Landscape6study. Bottom left: Equivalent figure generated in iAtlas. By selecting a specific heatmap cell (highlighted), the underlying data is displayed (Bottom right), using the selections shown. Individual points can be selected to get sample IDs and additional information (blue box).\n\nSelection for PD-L1 expression distributions displayed as violin plots within molecular subtypes of breast cancer, according to “PAM-50” classification. Elevated expression is seen in the HER2 subtype.\n\nTable 2 lists the particular manuscript figures (from the Immune Landscape6 publication) that can be reproduced or adapted to specific research questions.\n\niAtlas Analysis module (Column 1) and examples of figure panels (Column 2) in the Immune Landscape6 manuscript that can be generated using the module. Researchers can use this as a starting point for tailoring figures to their own interests.\n\nWith the iAtlas portal, scientists can explore and answer new questions based on specific research interests. For example, we asked: “What is the expression level of PD-L1, a therapeutically important protein, in subtypes of breast cancer?” To answer this question, from the landing page, we first selected the “TCGA Subtype” sample group, followed by the “Breast Invasive Carcinoma (BRCA)” study subset. Next, we selected the Immunomodulators module (Figure 4). Based on a very quick scan of the drop down, we didn’t see any names that matched our gene of interest, so we scrolled further down on the page to view the table of ‘Immunomodulator Annotations’. By typing in the first few letters of a gene name (e.g., “PD...”) into the ‘Search’ field, the table was filtered to a set of matching genes, and we could see that “PD-L1” is the Friendly Name for the gene “CD274” (the approved gene symbol on genenames.org). After returning to the Select or Search for Variable drop down menu above and selecting “CD274 (PD-L1)”, we were able to see a display of violin plots showing the distributions of gene expression across BRCA molecular subtypes. We could then visually compare distributions between subtypes, noticing for example the elevated expression level in the Her2 subtype compared to Basal breast cancer. These comparisons can guide further characterization not only of how gene expression can differ between TCGA subtypes of breast cancer, but also how these subtype-specific differences might correlate with clinical outcomes, as investigated in other studies32–34. Using this module and others, the researcher has the ability to answer new questions which could lead to developments in oncology research.\n\nIn order to classify any tumor-derived gene expression samples into immune subtypes6,30, users can select the TOOLS tab (top right), which leads to an interface containing notes, several links and the controls. In order to classify new data, we submitted data as a text file, in this case tab separated, with the first column containing gene IDs and later columns containing samples. A provided example file can be found in the description text. The first row of the data was a header containing sample IDs. Gene IDs can be either HGNC gene symbols (preferred), Entrez ID, or Ensembl identifiers. The locally available data was selected using the Browse button, and the file delimiter was selected, along with gene ID type, using drop down menus. Hitting the GO button produced classifications, signature scores, and cluster probabilities, which were reported in a table that was downloaded as a csv, xlsx, or pdf file. In addition, a barplot with the frequency of predicted subtypes for the submitted data was displayed.\n\nAll data required to run the application and describe the Use Cases are available in GitHub and archived with Zenodo7.\n\n\nConclusions\n\nCRI iAtlas is a platform that facilitates analysis and exploration of the tumor immune microenvironment by making IO-related data and tools accessible to the research community. iAtlas builds upon the comprehensive TCGA analysis of tumor-immune interactions on 10,000 tumors and illustrates how commonalities and differences of the immune response across 33 tumor types can provide clues for advancing therapeutics. iAtlas provides researchers with the tools to dive deeper into immunogenomic and clinical data and to develop and refine hypotheses regarding tumor-immune interactions that will empower researchers to gain insight and design the next generation of immuno-oncology treatment strategies.\n\n\nData availability\n\nOriginal data files from the TCGA PanCancer Atlas publication can be found in the NCI Genomic Data Commons (https://gdc.cancer.gov/about-data/publications/panimmune) or the TCGA PanCancer Atlas Data Mirror (https://isb-cancer-genomics-cloud.readthedocs.io/en/latest/sections/PanCancer-Atlas-Mirror.html.\n\nZenodo: CRI iAtlas (Version 1.2.0). https://doi.org/10.5281/zenodo.39267577.\n\nFolder ‘Data’ contains all data required to run the application and describe Use Cases. This is also available on GitHub.\n\nLicense: Apache License 2.0.\n\n\nSoftware availability\n\nSource code is available from GitHub: https://github.com/CRI-iAtlas/shiny-iatlas.\n\nSource code for the specific version described at the time of publication: https://github.com/CRI-iAtlas/shiny-iatlas/releases/tag/v1.2.0.\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.39267577.\n\nHosted iAtlas application on shinyapps.io: https://isb-cgc.shinyapps.io/shiny-iatlas.\n\nPinned version of the hosted iAtlas app described at the time of publication: https://isb-cgc.shinyapps.io/iatlas_v1-2.\n\nLicense: Apache License 2.0.", "appendix": "Acknowledgements\n\nWe are grateful to the Cancer Research Institute for supporting this work. We thank all collaborators in the TCGA PanCancer Atlas Immune Response Working Group, whose careful and thorough work generated the immune readouts displayed in iAtlas, and thank the NCI TCGA Program Office, Research Network, and PanCancer Atlas initiative for laying the foundation to this work. We thank Tai-Hsien Ou Yang, Eduard Porta-Pardo, Jun Tang, Vanessa Lucey, and Jill O’Donnell-Tormey, and the iAtlas user community for helpful suggestions and discussion on features and modules included in iAtlas. We also thank the TCGA participants who contributed samples used in this work.\n\n\nReferences\n\nMellman I, Coukos G, Dranoff G: Cancer immunotherapy comes of age. Nature. 2011; 480(7378): 480–489. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFridman WH, Pagès F, Sautès-Fridman C, et al.: The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012; 12(4): 298–306. PubMed Abstract \| Publisher Full Text\n\nLiu XS, Mardis ER: Applications of Immunogenomics to Cancer. Cell. 2017; 168(4): 600–612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinnewies M, Roberts EW, Kersten K, et al.: Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018; 24(5): 541–550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaruch EN, Berg AL, Besser MJ, et al.: Adoptive T cell therapy: An overview of obstacles and opportunities. Cancer. 2017; 123(S11): 2154–2162. PubMed Abstract \| Publisher Full Text\n\nThorsson V, Gibbs DL, Brown SD, et al.: The Immune Landscape of Cancer. Immunity. 2018; 48(4): 812–830.e14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEddy J, Gibbs D, Lamb A, et al.: CRI iAtlas (Version 1.2.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3926758\n\nHutter C, Zenklusen JC: The Cancer Genome Atlas: Creating Lasting Value beyond Its Data. Cell. 2018; 173(2): 283–285. PubMed Abstract \| Publisher Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. R package version 140. 2019. Reference Source\n\nCheng W: Modularizing Shiny app code. 2020. Reference Source\n\nWickham H, Averick M, Bryan J, et al.: Welcome to the Tidyverse. JOSS. 2019; 4(43): 1686. Publisher Full Text\n\nWickham H, François R, Henry L, et al.: dplyr: A Grammar of Data Manipulation. R package version 083. 2019. Reference Source\n\nWickham H, Henry L: tidyr: Tidy Messy Data. R package version 100. 2019. Reference Source\n\nHenry L, Wickham H: purrr: Functional Programming Tools. R package version 033. 2019. Reference Source\n\nWickham H: stringr: Simple, Consistent Wrappers for Common String Operations. R package version 140. 2019. Reference Source\n\nMüller K, Wickham H: tibble: Simple Data Frames. R package version 213. 2019. Reference Source\n\nMount J, Zumel N: wrapr: Wrap R Tools for Debugging and Parametric Programming. R package version 192. 2019. Reference Source\n\nSievert C, Parmer C, Hocking T, et al.: plotly: Create Interactive Web Graphics via “plotly.js”. R package version 490. 2019. Reference Source\n\nXie Y, Cheng J, Tan X: X: DT: A Wrapper of the JavaScript Library “DataTables”. R package version 09. 2019. Reference Source\n\nCheng J: crosstalk: Inter-Widget Interactivity for HTML Widgets. R package version 100. 2016. Reference Source\n\nChang W, Ribeiro BB: shinydashboard: Create Dashboards with “Shiny”. R package version 071. 2018. Reference Source\n\nColaprico A, Silva TC, Olsen C, et al.: TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data. Nucleic Acids Res. 2016; 44(8): e71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu J, Lichtenberg T, Hoadley KA, et al.: An Integrated TCGA Pan-Cancer Clinical Data Resource to Drive High-Quality Survival Outcome Analytics. Cell. 2018; 173(2): 400–416.e11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaltz J, Gupta R, Hou L, et al.: Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images. Cell Rep. 2018; 23(1): 181–193.e7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTang J, Shalabi A, Hubbard-Lucey VM: Comprehensive analysis of the clinical immuno-oncology landscape. Ann Oncol. 2018; 29(1): 84–91. PubMed Abstract \| Publisher Full Text\n\nTang J, Hubbard-Lucey VM, Pearce L, et al.: The global landscape of cancer cell therapy. Nat Rev Drug Discov. 2018; 17(7): 465–466. PubMed Abstract \| Publisher Full Text\n\nTang J, Pearce L, O’Donnell-Tormey J, et al.: Trends in the global immuno-oncology landscape. Nat Rev Drug Discov. 2018; 17(11): 783–784. PubMed Abstract \| Publisher Full Text\n\nYu JX, Hubbard-Lucey VM, Tang J: Immuno-oncology drug development goes global. Nat Rev Drug Discov. 2019; 18(12): 899–900. PubMed Abstract \| Publisher Full Text\n\nChen T, Guestrin C: XGBoost: A Scalable Tree Boosting System. In: Proceedings of the 22Nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining. KDD’16. New York, NY, USA: ACM. 2016; 785–794. Publisher Full Text\n\nGibbs DL: Robust classification of Immune Subtypes in Cancer. bioRxiv. 2020. Publisher Full Text\n\nGeman D, d’Avignon C, Naiman DQ, et al.: Classifying gene expression profiles from pairwise mRNA comparisons. Stat Appl Genet Mol Biol. 2004; 3: Article19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang C, Cao S, Li N, et al.: PD-1 and PD-L1 correlated gene expression profiles and their association with clinical outcomes of breast cancer. Cancer Cell Int. 2019; 19: 233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPadmanabhan R, Kheraldine HS, Meskin N, et al.: Crosstalk between HER2 and PD-1/PD-L1 in Breast Cancer: From Clinical Applications to Mathematical Models. Cancers (Basel). 2020; 12(3): 636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurozumi S, Inoue K, Matsumoto H, et al.: Clinicopathological values of PD-L1 expression in HER2-positive breast cancer. Sci Rep. 2019; 9(1): 16662. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70105", "date": "05 Oct 2020", "name": "Vincent Rouilly", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors introduce iAtlas, a web-based application that allows to browse a rich diversity of immune profiles, generated from the public TCGA dataset, and published in the landmark Immune Landscape study. Further than simply giving the possibility to replicate the published figures, the application provides a great flexibility to explore the entire PanImmune feature matrix through sophisticated and interactive tools.\n\nThe modular architecture and the features of the software are clearly explained. Detailed instructions are provided. And, all the necessary information is given to run the application. Its source code repository on github is well structured. The documentation is very comprehensive, as it gives ample information on the underlying methods, as well as how to extend the software application.\nIt is a very valuable resource for the Immuno-oncology community.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "73636", "date": "11 Nov 2020", "name": "David L. Goode", "expertise": [ "Reviewer Expertise Cancer Genomics", "Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents CRI iAtlas, a website with a suite of interactive tools for exploring and visualizing tumor immune microenvironment data collected by The Cancer Genome Atlas project.\n\nIt is based on robust, well-established R packages that are widely used for filtering, analysing and displaying large, multi-faceted data sets of the type used here.\nThe design and implementation of the website are well described and the underlying code and data provided in readily accessible public repositories.\nCRI iAtlas will provide a valuable resource for the cancer immunology field. The tools it provides will enable a range of users to quickly and easily access and visualize complex genomics and immunology data sets. The interface is user friendly and well documented, particularly for those without bioinformatics training.\n\nThe detailed instructions certainly made it easier to understand what each tool did the first time I tried to use them. However, they also kind of clutter the interface. It would be nice to be able minimize those panels without minimizing the entire module.\nIt would also be helpful to search the Driver Associations data for interactions involving certain genes of interest instead of having to scan through the volcano plots point by point.\nBut those are minor quibbles. Overall this is a slick and powerful online tool that will be welcomed by the field.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "73635", "date": "04 Dec 2020", "name": "Nathan E. Reticker-Flynn", "expertise": [ "Reviewer Expertise Tumor immunology", "systems biology", "immunotherapy", "models of cancer", "metastasis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript, Eddy et al. describe the interactive data portal iAtlas that accompanies the authors’ 2018 publication in Immunity, “The Immune Landscape of Cancer”. In that seminal paper, the authors present a compendium of analyses they performed to interrogate the diversity of immune states across human cancers using publicly available data from The Cancer Genome Atlas (TCGA). While the Immunity manuscript described the identification and characterization of six distinct “Immune Subtypes” and highlighted examples of key distinguishing features of the data sets, the accompanying iAtlas tool enables users to explore these rich analyses on their own, using parameters of their choosing. Comprising over 11,000 patients across 33 cancer types, these data represent an exceptionally comprehensive patient cohort from which a wide range of immunological insights can be gleaned.\niAtlas, which is available as both a web-based Shiny app and for offline implementation in R from GitHub, is a remarkably powerful and user-friendly portal through which researchers can easily interrogate the TCGA datasets for all of the immune analyses presented in their manuscript. The platform allows users to group their analyses by examining differences across TCGA cancer types, tumor subtypes (e.g. Luminal A vs. Her2 breast cancers), or their six defined Immune Subtypes. Furthermore, they have included the ability for users to define their own subsets and perform all of the included analyses across these subsets. The analyses within iAtlas include cellular readouts (e.g. degree of immune infiltration, fractions of particular leukocyte populations), molecular readouts (e.g. proliferation rates, gene expression, SNVs, CNAs, etc.), lymphocyte receptor (TCR/BCR) repertoires, network analyses, and tissue-level analyses (e.g. architectural characteristics). These variables can be investigated in the context of a wide range of parameters (e.g. clinical outcomes, correlations to driver gene mutations, etc.), designated as distinct modules, rendering the tool incredibly powerful with a near limitless number of potential questions to investigate in the field of tumor immunology. iAtlas is a remarkable tool with an extremely rich associated dataset whose elegant and straight-forward implementation render it accessible to researchers of all computational abilities.\nI have no major concerns with this manuscript. What follows are a list of comments, minor concerns, and suggestions that should not preclude acceptance of the manuscript but rather serve as potential items to include in future releases of iAtlas:\nIn the Cell Type Fractions of the Tumor Microenvironment module, it would be helpful to include an option for the user to select which CIBERSORT immune types to include as a custom “Cell Fraction Type”. For example, a user could select only memory resting CD4 T cells, memory activated CD4 T cells, naïve CD4 T cells, Tregs, and Tfh if they wanted to easily visualize the relative fractions of particular Th subsets within the CD4 compartment across groups. In some instances, it may be more biologically meaningful to compare these shifts within broad subsets without having to account for changes in unrelated populations (e.g. M2 macrophages).\n\nIn the Tumor Microenvironment module, when comparing leukocyte fraction to the total stromal fraction, there are typically a small number of samples where the leukocyte fraction exceeds the total stromal fraction, denoted as “estimation artifacts”. What is the source of these artifacts? It may help to include a description of this in the legend.\n\nAs a general comment, in a variety of instances it might be informative if there would be a way to include statistics between groups, though I recognize that doing so could be computationally intensive and invalid in many instances. Nonetheless, given the large sample sizes I am often left wondering if modest differences between groups are statistically meaningful and where inferences can be drawn (e.g. an increase in Treg fraction from 0.024 in the “Inflammatory” subtype to 0.029 in the “TGF-b Dominant” subtype).\n\nThe Data Description tab might be aided by including a sentence or phrase within the table stating what each variable is without requiring following all of the methods links. For example, I am assuming the BCR/TCR Shannon incorporates both the richness and evenness to depict the diversity of the repertoire, but it might be helpful to state the meaning of the terms in the table.\n\nFor the Clinical Outcomes, it might be useful to include “Immune Subtypes” as a variable when analyzing TCGA Study so that users can evaluate the survival probabilities depending on immune subtype for a given cancer.\n\nThere appears to be a minor issue wherein no results can be displayed for the Concordance Index of the Clinical Outcomes analyses for most, if not all, variables when using TCGA Study for the Sample Groups.\n\nThe TIL Maps module is quite impressive and the ability to examine the histology from each of the patients along with the calculated metrics is an outstanding feature. Nonetheless, when one clicks one of the violin plots, the annotation lists seems only to display an individual patient rather than the entire list of patients for that group.\n\nThe Immune association with driver gene analysis is a powerful tool whose insights have far-reaching implications and represent an active area of research in tumor immunology. While the interactive volcano plot is highly informative and useful for identifying genes of interest, it would also be useful to be able to run the analysis in the opposite direction. If possible, it would be informative to be able to select a driver gene and see where it lies in the various volcano plots and which of the variables are correlated with the mutation.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1028
https://f1000research.com/articles/9-1016/v1	21 Aug 20	{ "type": "Research Article", "title": "Parental risk perceptions of child exposure to thirdhand smoke and related factors", "authors": [ "Nirun Intarut" ], "abstract": "Background: Thirdhand smoke (THS) exposure is linked to lung cancer, asthma, and chronic diseases, especially in children. The parental risk perception of THS exposure in children has rarely been reported. The objective of this study was to test the association between sociodemographic factors and parental risk perceptions of child exposure to thirdhand smoke (PRPCETS) in residential homes with a child or children aged from one to five years old. Methods: This study used secondary data from the Smoke Free Home intervention trial. 336 participants were included and analyzed. PRPCETS was assessed by self-administered questionnaire. Multiple logistic regression was used to test the factors related to parental risk perception to the harm of thirdhand smoke. Results: The overall prevalence of disagreement that THS might be harmful to children was 22.02% (95% CI: 17.59%, 26.45%). Factors associated with PRPCETS were being over 50-years-old (OR: 2.15; 95%CI: 1.05, 4.41); attending school for more than six years (OR: 2.08; 95%CI: 1.07, 4.08); being unemployed (OR: 6.98; 95% CI, 1.41, 34.71); and the number of smokers in the home ≥2 persons (OR: 2.48; 95%CI: 1.41, 4.36). Conclusions: Our findings show the factors related to PRPCETS as follows; aged over 50, duration of school attendance less than six years, no job status, and having ≥2 smokers in the home. Further studies should investigate parental knowledge of and attitude towards thirdhand smoke exposure.", "keywords": [ "thirdhand smoke exposure", "parental risk perception", "risk factors", "child health" ], "content": "Introduction\n\nThird hand smoke (THS) means residual smoke emitted from tobacco products that is inhaled by a third party1. Smoke from tobacco products, secondhand smoke, will become embedded on the surface of walls, carpets, furniture, clothing, flooring, vehicles or toys2. Exposure to THS induces health problems such as lung cancer development3,4 and asthma5. Exposure to THS happens in a place that a smoker has previously smoked, such as in houses or cars. A study reported that the home is one of places that THS can occur if there is smoking in the home. Evidence shows that some homes remain contaminated with THS for six months6. The results show that infants or children stay, play and climb in the home, and they have a chance to be exposed to THS.\n\nReviews show few studies have been conducted to test factors related to parental risk perception concerning the harm to the child of THS exposure. Therefore, this study tested factors related to parental risk perceptions of child exposure to thirdhand smoke (PRPCETS).\n\n\nMethods\n\nThe Mahasarakham University Institutional Review Board (IRB) approved the study. The study identification number is 113/2561. Written consent forms were distributed and provided to all participants, and they signed these consent forms.\n\nThis study used secondary data from the Smoke Free Home study project. This project aimed to give smoke-free home information to parents of children aged 1–5 years that reside in the home. A cluster randomized controlled trial was carried out from February 2019 to October 2019 in Roi-Et province, Thailand, The trial was registered at the Thai Clinical Trials Registry (TCTR) with the code TCTR20190213001. Briefly, four primary health care facilities were randomly recruited. Of those, all 47 villages within each of the four health care facilities were selected to be part of the study setting. The targeted settings were screened using the Health Data Center (HDC), the health information database that collects data from the community and hospitals in Thailand. In the HDC, we recruited families according to the following criteria; families with a child aged 1–5 years, where the parent does not smoke, with a smoker in their home. The baseline characteristics and smoking behavior was collected by trained research teams using self-administered questionnaire at participant’s home. The trained research teams interviewed a parent of children or a child about their smoke-free home status, exposure to secondhand smoke, THS risk perception, and smoking behavior in the home. If the eligibility criteria (healthy child aged 1–5 years, the parent is a non-smoker, and a smoker smoked in their home) were met, they were invited to the intervention study. In total, we screened 336 households. Of those, 305 were included in the intervention study. In this cross-sectional study, we use the screening data of 336 participants to explore the parental risk perceptions of child exposure to thirdhand smoke.\n\nThe outcome, PRPCETS, was assessed by asking the parent of children the question “Are you aware that breathing in a room that has had someone smoking in it previously, can affect the health of babies and children?” The response to the question was ranked from 1 to 4 (1: absolutely disagree, 2: disagree, 3: agree, 4: absolutely agree). We divided the answers into ‘Agree’ (which included; 3: agree and 4: absolutely agree) and ‘Disagree’ (which included 1: absolutely disagree, 2: disagree).\n\nWe also collected demographic data as follows; age in years, gender, duration of school attendance in years, occupation, marital status, income (Thai Baht, THB/month), number of smokers in the home, number of children in the home (under five years), and co-use of smoking and drinking alcohol. The age of the children’s parents was categorized as 18–40 years, 40–50 years, and >50 years. Duration of school attendance was categorized as 0–6 years, and >6 years. Occupation was categorized as agricultural, merchant, government officer, and no job. Marital status was categorized as married and divorced/other. Income (THB) was assessed and categorized as <10000THB/month, and ≥10000THB/month. Data on the number of smokers in the home was also collected and categorized as one person and ≥2 persons. Number of children in the home aged one to five years was categorized as one person and ≥2 persons. In addition, co-use of drinking alcohol in the home was assessed by a sequence of two questions; “In the past 1 month, has there been alcohol drinking in the home?” Reponses were no or yes. If they answered yes, they were asked “During alcohol drinking, was there smoking?”. The answers were ‘yes, every time’, ‘yes, sometimes’, and ‘no, not at all’. We categorized this variable as alcohol drinking but no smoking (when they answered yes to alcohol drinking but reported no smoking at all), co-use (alcohol drinking and smoking), and no (no alcohol drinking).\n\nAll factors were presented as frequencies and percentages. For univariate analysis, we tested the association between factors and PRPCETS using the chi-square statistic. We also used multiple logistics regression in the multivariate analysis, and the final model was adjusted for candidate confounder factors. All statistics were performed in R version 3.6.17 and epiDisplay version 3.5.0.1 package8.\n\n\nResults\n\nThe overall prevalence of ‘Disagree’ answers to the PRPCETS question was 22.02% (95% CI: 17.59%, 26.45%). Of 336 participants included and analyzed in this study, 60.4% were aged 18–40 years, 92.3% were female parents, 77.1% worked in agriculture, 79.2% were married, and 67.3% had one person smoking at home9. Regarding the number of children in the home, 82.7% had ≥2 children, and 53.6% reported that tobacco was smoked during alcohol drinking in the home. We compared the distribution of the prevalence of PRPCETS for each factor. There was a statistically significant difference between factors and PRPCETS as follows; age (p value: 0.012), the number of years of school attendance (p value: 0.009), occupation (p value: 0.002), and the number of smokers in the home (p value: <0.001). Table 1 shows the results of the univariable analysis and prevalence distribution among factors.\n\nIn Table 2, results of the multiple logistic regression analysis is shown after adjustment for potential confounders. The group aged over 50 years had an increased risk of disagreement with the PRPCETS question (OR: 2.15; 95%CI: 1.05,4.41). Parents who had attended a school for more than six years had an increased risk of disagreeing with the PRPCETS question (OR: 2.08; 95%CI: 1.07, 4.08). Being unemployed (OR: 6.98; 95% CI, 1.41,34.71) was positively associated with disagreement with the PRPCETS question. In addition, having ≥2 smokers in the home (OR: 2.48; 95%CI: 1.41, 4.36) was positively associated with disagreement with the PRPCETS question.\n\n\nDiscussion\n\nOur results show the association between age, the number of years school attendance, occupation status, the number of smokers in the home and PRPCETS. However, statistical significance was not observed for gender, marital status, income per month, the number of children in home, and concurrent smoking and drinking in home.\n\nPRPCETS was correlated with the age of parents. Our results show that older parents tended to disagree with the statement that THS might be harmful to children. This effect might be because older parents have received little information on the harms of THS exposure. In addition, the major tobacco control strategies included little information on THS exposure in the promotion of tobacco control10.\n\nSeveral studies show that high education level might protect against exposure to second-hand smoking11–13, but there is little evidence to support this. Our results reveal that a high level of education protected against disagreement with the PRPCETS question. Parents who had the opportunity to study at a high level at school who were exposed to tobacco control campaigns at school or on the internet, radio, or television14–16 might have increased knowledge of or more positive attitudes towards the risk of THS exposure.\n\nOccupation status is one of socioeconomic factors that is associated with tobacco control research17. Our analysis showed that parents who have no job tended to disagree with the statement concerning the harms of THS exposure to their children. The effect described, usually, the parents who stayed at home to look after their child or children and had no job.\n\nIn households with smokers, our results show that households with more than one smoker were at greater risk of parents who disagreed with the PRPCETS question. Home is a one place that THS occurs because people have previously smoked inside18–20. Our hypothesis is that in households that have many smokers and have children, there is a chance that a smoker smoked in their home or close to home such as by a wall or door. When this event has occurred frequently, parents might think that THS is not an important matter when they cannot smell tobacco smoke.\n\nOur study has several limitations. Our analysis used the data from a smokefree home intervention project that was carried out in the northeast of Thailand. Therefore, the prevalence observed might not represent parents with a child or children aged under five years in the rest of the country. In addition, our questionnaire did not assess parent’s knowledge of and attitude towards the harms of THS exposure. This measurement might impact the risk perception of harms of THS exposure. The strength of the study is that this might be the first study in Thailand to investigate the factors related to parental risk perception concerning the harms of child THS exposure.\n\n\nConclusion\n\nResults show the factors related to parental risk perception of child harm from THS exposure as follows; aged more than 50 years, duration of school attendance less than six years, having no job, and having many smokers in the home (≥2 persons). Further studies should investigate parents’ knowledge of and attitude towards THS exposure.\n\n\nData availability\n\nHarvard Dataverse: thirdhand smoke parental risk perception: https://doi.org/10.7910/DVN/N188CL9\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe acknowledge the health volunteer workers and staff of the Faculty of Medicine, Mahasarakham University, in collecting data. In addition, we also thank Dr. Adrian Roderick Plant for assistance with manuscript presentation.\n\n\nReferences\n\nMatt GE, Quintana PJE, Destaillats H, et al.: Thirdhand Tobacco Smoke: Emerging Evidence and Arguments for a Multidisciplinary Research Agenda. Environ Health Perspect. 2011; 119(9): 1218–1226. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJacob P 3rd, Benowitz NL, Destaillats H, et al.: Thirdhand Smoke: New Evidence, Challenges, and Future Directions. Chem Res Toxicol. 2017; 30(1): 270–294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHang B, Mao JH, Snijders AM: Genetic Susceptibility to Thirdhand-Smoke-Induced Lung Cancer Development. Nicotine Tob Res. 2019; 21(9): 1294–1296. PubMed Abstract \| Publisher Full Text\n\nThomas JL, Guo H, Carmella, SG, et al.: Metabolites of a tobacco-specific lung carcinogen in children exposed to secondhand or thirdhand tobacco smoke in their homes. Cancer Epidemiol Biomarkers Prev. 2011; 20(6): 1213–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong JH, Li Z, Shi J, et al.: Passive Smoking Exposure from Partners as a Risk Factor for ER+/PR+ Double Positive Breast Cancer in Never-Smoking Chinese Urban Women: A Hospital-Based Matched Case Control Study. PLoS One. 2014; 9(5): e97498. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatt GE, Quintana PJE, Zakarian, JM, et al.: When smokers quit: exposure to nicotine and carcinogens persists from thirdhand smoke pollution. Tob Control. 2017; 26(5): 548–556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna, Austria. 2019. Reference Source\n\nChongsuvivatwong V: epiDisplay: Epidemiological Data Display Package. 2018. Reference Source\n\nIntarut N: thirdhand smoke parental risk perception. Harvard Dataverse, V1, UNF:6:B27xA3sw2lWsCH4tDNy+bw== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/N188CL\n\nChung-Hall J, Craig L, Gravely S, et al.: Impact of the WHO FCTC over the first decade: a global evidence review prepared for the Impact Assessment Expert Group. Tob Control. 2019; 28(Suppl 2): s119–s128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim KH, Teh CH, Mohamed MHN, et al.: Exposure to tobacco secondhand smoke and its associated factors among non-smoking adults in smoking-restricted and non-restricted areas: findings from a nationwide study in Malaysia. BMJ Open. 2018; 8(1): e017203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTripathy JP: Secondhand smoke exposure at home and public places among smokers and non-smokers in India: findings from the Global Adult Tobacco Survey 2016-17. Environ Sci Pollut Res Int. 2020; 27(6): 6033–6041. PubMed Abstract \| Publisher Full Text\n\nPetersen AB, Thompson LM, Dadi GB, et al.: Factors associated with secondhand tobacco smoke in the home: an exploratory cross-sectional study among women in Aleta Wondo, Ethiopia. BMC Public Health. 2016; 16(1); 910. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim JK, Glantz SA: The rise and fall of tobacco control media campaigns,1967-2006. Am J Public Health. 2007; 97(8): 1383–1396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuipers MAG, Beard E, West R, et al.: Associations between tobacco control mass media campaign expenditure and smoking prevalence and quitting in England: a time series analysis. Tob Control. 2018; 27(4): 455–462. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSangthong R, Wichaidit W, Ketchoo C: Current situation and future challenges of tobacco control policy in Thailand. Tob Control. 2012; 21(1): 49–54. PubMed Abstract \| Publisher Full Text\n\nBang KM, Kim JH: Prevalence of cigarette smoking by occupation and industry in the United States. Am J Ind Med. 2001; 40(3): 233–239. PubMed Abstract \| Publisher Full Text\n\nHennessy M, Bleakley A, Mallya G, et al.: The effect of household smoking bans on household smoking. Am J Public Health. 2014; 104(4): 721–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStafylis C, Rachiotis G, Katsioulis, A, et al.: Prevalence and determinants of smoking and secondhand smoke exposure in a rural population of central Greece: a cross-sectional study. Rural Remote Health. 2018; 18(2): 4218. PubMed Abstract \| Publisher Full Text\n\nPhetphum C, Noosorn N: Prevalence of secondhand smoke exposure at home and associated factors among middle school students in Northern Thailand. Tob Induc Dis. 2020; 18: 11. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70190", "date": "01 Sep 2020", "name": "Supot Kamsa-ard", "expertise": [ "Reviewer Expertise I am a instructor in university", "which included more than 10 years experiences in public health research and cancer epidemiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEntitled: Parental risk perceptions of child exposure to thirdhand smoke and related factors\n\nReviewer comments: Minor comments:\n\nMeasurement section Paragraph 2: Please give more information for a variable as Duration of school attendance. What does it mean?\n\nResult section Paragraph 2: There is number of decimals, 1 or 2. It should be consistency in the hole full text.\n\nTable 1\nIs it importance for report the p-value for comparison between disagree to harms THS exposure and agree to harms THS exposure? Because of the table 1 for baseline characteristics. Do not need for hypothesis testing.\n\nFor continuous variable such as Age, Income should report mean (standard deviation), and median (minimal and maximal) as well.\n\nHeading title, please insert n (%) in each column.\n\nTable 2 Statistics methods, it should describe model fitting strategies such what type of method for fitting model, for example forward or backward method. In additional, the table for demonstrate on Crude analysis still for important that we explore for multiple analysis. Why not report the crude analysis table?\n\nConclusion section Please concise conclusion, it is not repeat the result. For instance, which is factor that the most important for PRPCETS.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "159878", "date": "03 Feb 2023", "name": "Varduhi Hayrumyan", "expertise": [ "Reviewer Expertise We are public health researchers. One of our main areas of research is Tobacco Control." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments Introduction\nThe introduction should include information on the topic of research in the study setting (Thailand): smoking as a public health issue, second-hand smoke exposure in general and particularly in private settings such as homes and cars.\n\nThirdhand smoke (THS) exposure should be described as an issue currently under the focus of international public health research. What is known about perceptions, attitudes, and knowledge of THS? What about any studies on this conducted among parents of young children?\n\nDescribe the factors associated with the PRPCETS discussed in the literature globally and their importance and implications. It is mentioned that “Reviews show few studies have been conducted to test factors related to parental risk perception concerning the harm to the child of THS exposure.” What do those few studies conclude and how this study will feel the gap in the literature?\n\nThe author included a variable regarding alcohol consumption. How is alcohol consumption connected with the research questions? A justification should be provided for including this in the analysis.\n\nThe rationale of the study should be strengthened to demonstrate its importance for both the international public health community and Thailand.\nMethods\nIt is mentioned that the study used secondary data from a project. However, in the “Ethical approval and informed consent” paragraph it is mentioned that “Written consent forms were distributed and provided to all participants, and they signed these consent forms.” In case of studies using secondary data, consent requirements are exempt as the studies usually pose no risk to study participants. It is possible the author described the ethical considerations of the parent study (trial) instead of describing the current study.\n\nIs the parent study published or described elsewhere? If yes, it would be helpful to include an appropriate citation.\n\nIt is written, “In the HDC, we recruited families according to the following criteria; families with a child aged 1–5 years, where the parent does not smoke, with a smoker in their home.“ The author should clarify whether both parents were non-smokers or only one parent and if the “smoker in the home” is another family member living in the home or others (e.g. guests). How “a smoker smoked in their home” eligibility criteria was defined? How it was decided whether the mother or the father should participate in the interview?\n\nIt is not clear why families with smoker parents were excluded from the study. Justification is needed.\n\nThe categorization of educational status (0-6 years and >6 years) in the measurements section is not clear to someone outside of the local context.\n\nHow multiple logistic regression models were constructed? What strategies were used?\nResults\nConsistency while reporting decimal points should be maintained throughout the paper. A suggestion would be to keep one decimal point in the text.\n\nThe confidence intervals of the outcome variable are presented with percentages (“95% CI: 17.59%, 26.45%”) others without.\n\nOverwhelming majority of interviewed parents were females (92.3%). This is a limitation of the study given that male parents’ perceptions could be different. Also needs to be discussed in the discussion section.\n\nTable 1 needs to be checked for typos.\n\nThe potential confounders mentioned in the following sentence could be specified: “In Table 2, results of the multiple logistic regression analysis is shown after adjustment for potential confounders.”\n\nTable 2 does not include p values. Should be added.\n\nRegression analysis could be better interpreted.\n\nThis sentence in the Results section “Parents who had attended a school for more than six years had an increased risk of disagreeing with the PRPCETS question (OR: 2.08; 95%CI: 1.07, 4.08).” contradicts the table. Maybe you meant “Parents who had attended a school less than six years” …\nDiscussion\nThe explanation of why older parents tended to disagree with the PRPCETS question is poor. Needs more discussion.\n\nThe explanation of occupation status as a significant factor associated with PRPCETS is not clear.\n\nSmoker in the home factor is not well explained as well. The discussion section overall needs revisions for language clarity.\n\nAny evidence/discussion on why other hypothesized factors such as gender, marital status, income per month, the number of children in the home, and concurrent smoking and drinking in the home were not revealed as factors in this study? Is there any literature on these things?\n\nNo implication of the study findings was made. What is the value of the current study? What gaps in the literature if fills? What are the conclusions and lessons learned?\n\nThe study identified groups that are risky in terms of disagreeing that THS is dangerous. What could be done with this information? This should be discussed.\n\nThe Conclusion section needs to be rewritten. It is just briefly repeating the study findings now.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1016

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.