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https://f1000research.com/articles/12-1224/v1	27 Sep 23	{ "type": "Method Article", "title": "Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model", "authors": [ "Max J Cumberland", "Jonas Euchner", "Amar J Azad", "Nguyen T N Vo", "Paulus Kirchhof", "Andrew P Holmes", "Chris Denning", "Katja Gehmlich", "Jonas Euchner", "Amar J Azad", "Nguyen T N Vo", "Paulus Kirchhof", "Andrew P Holmes", "Chris Denning" ], "abstract": "Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.", "keywords": [ "Engineered Heart Tissue (EHT)", "3D cardiac model", "induced pluripotent stem cell derived cardiomyocytes (iPSC-CM)", "induced pluripotent stem cell derived cardiac fibroblasts (iPSC-CF)", "cardiac co-culture", "cardiac fibrosis" ], "content": "\n\n\n\nScientific benefits\n\n\n\n• Generation of a more physiologically relevant human cardiac model\n\n• Utility as a genetically homogenous system for disease modelling of cardiac arrhythmia, heart disease and cardiomyopathy\n\n• Capacity in exploring cellular dynamics integral to cardiac fibrosis\n\n3Rs benefits\n\n\n\n• Engineered heart tissue models can replace the use of small animal models of cardiac arrhythmia, cardiomyopathy and heart failure\n\n• Acquiring data from the small animal models used in cardiovascular research often requires the animal to undergo multiple moderate severity procedures which require repeated anaesthesia, such as electrocardiograms and mini pump/telemeter implantation\n\n• As mouse physiology is vastly different to human hearts, insights from mouse models cannot always be extrapolated to human hearts. In contrast, engineered heart tissue models use human cells with human physiology\n\n• Many of the pathogenic variants modelled in mouse lines cause chronic and severe illness\n\nPractical benefits\n\n\n\n• The relatively low cost in comparison to mouse models\n\n• Induced pluripotent stem cells are relatively easier to genetically manipulate than in vivo models\n\n• Ability to generate healthy human quiescent cardiac fibroblasts\n\n• Reproducible methods for the derivation of cardiomyocytes and cardiac fibroblasts from hiPSCs\n\n• Accessibility of cardiac cells with a consistent genome\n\nCurrent applications\n\n\n\n• Disease modelling of cardiac arrhythmia, cardiomyopathy and heart failure\n\n• Exploring physiological interplay between cardiac fibroblasts and cardiomyocytes\n\nPotential applications\n\n\n\n• Low throughput drug screening platform for cardiotoxicity\n\n• Use as a human model to investigate the reversibility of cardiac fibrosis\n\n\nIntroduction\n\nLimited availability of healthy human heart tissue combined with its inherent nature as a non-proliferative tissue type has meant that heart disease researchers have historically been reliant on in vivo models. Large animal models used in heart disease research, for example dogs, pigs, and sheep, are broadly analogous to humans in cardiac physiology and anatomy but are heavily restricted by cost, throughput, and the sentiment of the general public (Camacho et al., 2016). Small animal models commonly used in cardiovascular research, such as the mouse and zebrafish, offer a cheaper but drastically less physiological and anatomical alternative. Zebrafish offer distinct advantages including optical transparency and rapid development but are limited in their capacity as a cardiac model by their two-chambered heart and temperature-dependent action potential (Vornanen and Hassinen, 2016). Significant differences in the intracellular electrophysiology of mouse cardiomyocytes, caused by contrasting expression and activation of the delayed rectifier and transient outward K+ currents as well as the voltage-gated sodium and calcium channels, are broadly illustrated in their comparatively rapid heart rate of between 500-700 bpm (Xu et al., 1999; Niwa and Nerbonne, 2010; Blechschmidt et al., 2008). Consequently, the modelling of cardiac diseases such as arrhythmia, cardiomyopathy and heart failure, which often manifest through concomitant electrical and structural remodelling, is often hampered in mice due to distinct species-dependent differences.\n\nThe co-development of human-induced pluripotent stem cell (hiPSC) technology and CRISPR-Cas 9 genome editing provides a new type of genetically modified disease model based on human cells (Ma et al., 2018; Mosqueira et al., 2018). The utility of induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has caused a shift in the approach many cardiovascular researchers take to cardiac disease modelling, with many groups opting to replace or supplement the use of small animal models with comparatively cheaper stem cell-based models (Cumberland et al., 2022). Consequently, hiPSC-CM have been used to successfully model pathogenic variants that predispose individuals to cardiac diseases, including arrhythmia and cardiomyopathy (Lin et al., 2015; Goktas Sahoglu et al., 2022; Jung et al., 2022; Reyat et al., 2020; Schulz et al., 2023).\n\nThe incorporation of hiPSC-CM into cardiovascular disease modelling has however not come without difficulty. Early attempts at differentiating cardiomyocytes produced variable and electrophysiologically immature cells with depolarized diastolic membrane potentials, slow action potential upstroke velocities and large pacemaker currents (Knollmann, 2013). Optimisation and development of differentiation protocols enabled the derivation of chamber-specific cardiomyocytes to explore diverse disease aetiologies but offered marginal advancements in variability and maturity (Jeziorowska et al., 2017; Devalla et al., 2015). Accordingly, innovative maturation strategies were developed to increase the maturity of the cells produced. Current methods include: micropatterning, metabolic maturation, electrical stimulation, soft-substrate culture, co-culture, and engineered 3D models (Jimenez-Vazquez et al., 2022; Feyen et al., 2020; Yoshida et al., 2018; Tzatzalos et al., 2016; Machiraju and Greenway, 2019).\n\nWith greater electrophysiological, metabolic, structural, and functional maturity, 3D cardiac models offer greater potential in understanding the nuances of complex cardiac diseases such as heart failure and cardiomyopathy (Correia et al., 2018; Shadrin et al., 2017; Ulmer et al., 2018; Vučković et al., 2022). Furthermore, the concurrent ease in which contractile force and transmembrane potential can be monitored in 3D cardiac models presents great potential for their future incorporation into in vitro cardiotoxicity studies (Gintant et al., 2019). A number of 3D cardiac models have been generated to examine cardiac physiology in disease including, but not limited to: two-post engineered heart tissues (EHTs), ring-shaped engineered heart muscle (EHM), cardiac patches, cardiac micro-, and biowires, and Novoheart (Tiburcy et al., 2017; Ronaldson-Bouchard et al., 2018; Zhang et al., 2013; Thavandiran et al., 2013; Zhao et al., 2019). Although differing substantially in size and shape, all the 3D cardiac models listed above attempt to better recapitulate the cell-to-cell and cell-to-matrix interactions present in the myocardium and encourage the longitudinal alignment of cardiomyocytes upon a scaffold (Smith et al., 2017).\n\nDespite occupying the majority of the volume of the mammalian heart, cardiac myocytes are estimated to account for less than 50 % of the total cell number (Zhou and Pu, 2016). Major non-myocyte cell types in the heart include cardiac fibroblasts and endothelial cells as well as ancillary populations of immune cells and autonomic neurones. Therefore, the incorporation of non-myocyte cell populations into cardiac modelling systems should facilitate the structural, metabolic, and electrophysiological maturity of hiPSC-CM and aid in generating a more physiologically relevant microenvironment (Yoshida et al., 2018; Kim et al., 2010). Up to now, cell types commonly incorporated into 3D cardiac models include endothelial cells, mesenchymal stem cells, and cardiac fibroblasts (Tadano et al., 2021; Tulloch et al., 2011; Zhang et al., 2017).\n\nCardiac fibroblasts are a common cell type in human ventricles (circa 20 % by number, [Pinto et al., 2016]) and are integral to the architecture, alignment, and electromechanical properties of the myocardium in health and disease. Quiescent or inactive induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CF) can be derived from hiPSCs and used effectively as a stand-alone model of cardiac fibrosis (Zhang et al., 2019). The trans-differentiation of the quiescent cardiac fibroblast to myofibroblast occurs prior to and during the development of cardiac fibrosis and is often difficult to prevent in vitro as the cells cultured on plastic dishes are subject to a Young’s modulus up to a million times stiffer than the native myocardium (Landry et al., 2019; Acevedo-Acevedo and Crone, 2015). This pathological transition is substantially stimulated by the profibrotic signalling cytokine TGF-β1. Suppression of TGF-β1 can be achieved in vitro using small molecule inhibitors such as SB 431542 and can be used on in vitro models to control the activation status of quiescent cardiac fibroblasts (Law and Carver, 2013).\n\nAn amalgamated 3D cardiac model consisting of hiPSC-CF and hiPSC-CM presents the opportunity for the generation of genetically homogenous (isogenic) systems capable of investigating the pathophysiological interplay present in cardiac diseases such as cardiomyopathy and arrhythmia. Here we describe a modified protocol for the generation and incorporation of quiescent hiPSC-CF into an engineered heart tissue model.\n\nIn this study we describe efficient protocols for the differentiation of hiPSC-CM and quiescent cardiac fibroblasts. Methods are outlined detailing the incorporation of these cells into EHTs with improved contractile function and tissue compaction and potential use exploring the pathophysiological interplay between hiPSC-CM and hiPSC-CF in cardiac fibrosis. In the UK, 51,427 procedures were carried out on mice in 2021 for research on the cardiovascular, blood and lymphatic system (Home Office Report on Annual Statistics of Scientific Procedures on Living Animals Great Britain 2021). Assuming that 5 % of these mice were used to study cardiac arrhythmia, heart failure or cardiomyopathy, use of the model proposed could lead to the direct replacement of 2500 mice per year in the UK alone.\n\n\nMethods\n\nInduced pluripotent stem cells Kolfc2 (WTSIi018-B) were maintained on six-well plates coated with Geltrex (1:100; for details of reagents and suppliers see Table 1) according to the manufacturer’s protocol. Cells were cultured in mTeSR Plus media at 37 °C and 5 % CO2 and passaged at 70-80 % confluency. Cells were passaged by removing the media in the well, washing once with PBS and adding TrypLE Express Enzyme (1X), phenol red for 3 minutes at 37°C. The TrypLE Express was removed and the cells were gently washed off the surface of the well with 1 mL of warm mTeSR Plus medium containing 10 μM Rock Inhibitor (Y-27632). Cells were passaged according to a seeding density of 20,000 cells per cm2 and cultured in 10 μM Rock Inhibitor (Y-27632) mTeSR Plus media for the first 24 hours before switching to mTeSR Plus. The Rho-kinase inhibitor (Rock Inhibitor), Y-27632 was added to prevent dissociation-induced apoptosis. It should be removed 24 hours after the passaging of the cells to maintain iPSC pluripotency.\n\nThe method for differentiation of hiPSC-CM was broadly adapted from the protocol outlined in Smith et al. (2018) (Figure 1). hiPSCs were seeded onto six-well plates coated with Geltrex and cultured in 2 mL per well of mTeSR Plus. The medium was changed on the cells every 48 hours until the cells reached 60 % confluency. The medium was then changed for 24 hours with StemPro-34 SFM (1X) supplemented with 2 mM L-Glutamine, 1 ng/mL Recombinant Human BMP-4 Protein and 1:100 Geltrex. The cells were subsequently changed with StemPro medium supplemented with 10 ng/mL BMP-4, 8 ng/mL Activin A and 2 mM L-Glutamine and incubated for 48 hours (day 0). The medium was changed with RPMI 1640 Medium with B-27 Supplement, minus insulin, 10 μM XAV 939 and 10 μM KY 02111 for 48 hours (day 2). The cells were subsequently changed with RPMI 1640 Medium with B-27 Supplement (50X), 10 μM XAV 939 and 10 μM KY02111 for 48 hours (day 4). The medium on the cells was changed with RPMI 1640 Medium with B-27 Supplement (50X) every other day. Atrial differentiation was achieved through the addition of 1 μM retinoic acid to the media on days 4 and 6. At 12 days after the initiation of differentiation, glucose starvation was performed to purify the population of cells for cardiomyocytes. This was achieved by changing the medium of the cells with RPMI 1640 Medium supplemented 1:50 with no glucose with B-27 Supplement (50X) for 48 hours. Cells were dissociated at day 15 for incorporation into EHT models. This media was removed from the well(s), the well was washed once with 1 mL of PBS and the cells were incubated in 2 mL of TrypLE Select 10× for 30 minutes at 37 °C. The cells were then washed from the surface of the plate with pre-warmed DMEM-F12.\n\nSchematic representation of the hiPSC-CM differentiation protocol. Mesodermal specification is achieved through the activation of the Wnt signalling pathway using the recombinant proteins: Bone morphogenic protein- 4 (BMP-4) and Activin A. Subsequent inactivation of the Wnt signalling pathway by small molecules XAV 939 and KY02111 enables the differentiation of cardiac progenitor cells into induced pluripotent stem cell derived cardiomyocytes. Differentiation can be driven towards atrial cardiomyocyte specification through the addition of retinoic acid on days 4 and 6 only. The medium on the cells was changed every 48 hours after day 6 with RPMI 1640 with B27 + Insulin. Selection is achieved from days 12-14 by culturing the cells in RPMI 1640 No Glucose with B27 + insulin for 48 hours. Created with Biorender.com.\n\nThe method for the differentiation of quiescent induced pluripotent stem cell-derived cardiac fibroblasts was broadly adapted from the protocol outlined in Zhang et al. (2019) (Figure 2). Induced pluripotent stem cells (Kolfc2/WTSIi018-B) were seeded at 30,000 cells/cm2 in a T25 flask pre-coated with 1:100 Geltrex for 2 hours at 37 °C, in 10 μM Rock Inhibitor (Y-27632) mTeSR Plus medium. The cells were cultured for 48 hours prior to the medium being changed with RPMI 1640 supplemented with B27 minus insulin and 6 μM CHIR-99021 for 48 hours. The medium was then changed with RPMI 1640 supplemented with B27 minus insulin for 24 hours. The cells were subsequently treated with a RPMI 1640 with B27 minus insulin and 5 μM IWR1 for 48 hours. The cells were dissociated by washing with 3 mL PBS and then treating with 5 mL accutase enzyme for 3 minutes at 37 °C. The cells were removed from the flask, diluted with 5 mL pre-warmed RPMI 1640 and spun down at 200 g for 3 minutes. The cells were resuspended in Advanced DMEM/F-12 supplemented with 5 μM CHIR-99021 and 2 μM Retinoic Acid and seeded at 20,000 cells/cm2 in a Geltrex coated T25 flask. The cells were incubated in this medium for 3 days and then subsequently changed to Advanced DMEM/F-12 for a further 4 days. The cells were passaged and plated into a Geltrex coated T25 flask at a density of 20,000 cells/cm2 in Fibroblast Growth Medium 3 supplemented with 10 ng/mL FGF2 and 10 μM of the TGF-β1 inhibitor SB431542. The cells were cultured in this media for 6 days, with media changes taking place every 48 hours. Following the 6 days, the cells can be assessed for purity using immunocytochemistry. It is recommended that the cells are expanded, passaged at 70-80 % confluency for 2 passages and cryopreserved for use in future experiments in serum free freezing medium. Cells were passaged by washing once with PBS, and then incubating in TrypLE™ Express for 3 minutes at 37 °C. Cells can be dislodged from the surface of the flask by gently tapping the sides of the container. Following the detachment of the cells, a 2 × volume of DMEM/F12 was added to dilute the TrypLE. The TrypLE/cell mix was centrifuged at 300 g for 3 minutes. The cells were resuspended in Fibroblast Growth Medium 3 supplemented with 10 ng/mL FGF2 and 10 μM SB431542 and seeded at a density of 10,000 cells/cm2.\n\nSchematic representation of the hiPSC-CF differentiation protocol. Mesodermal specification is achieved through the activation of the Wnt signalling pathway using CHIR-99021. Subsequent inactivation of the Wnt signalling pathway by the small molecule IWR1 allows the generation of cardiac progenitor cells. Culture in CHIR-99021 and retinoic acid drives the cells towards the derivation of cardiac fibroblasts. Cardiac fibroblasts are maintained in medium supplemented with FGF2 and the TGF-β1 inhibitor SB 431542 to prevent activation into myofibroblasts. Created with Biorender.com.\n\nProtocol for EHT construction\n\nHere we describe a step-by-step protocol for the construction of four EHTs (one strip) containing iPSC-CF. The reagents required are listed in Table 2.\n\n\n\n• 1 mL of 10x DMEM\n\n• 1 mL Gibco Horse Serum, heat inactivated, New Zealand origin (ThermoFisher Scientific, 11540636)\n\n• 100 μL Penicillin-Streptomycin (ThermoFisher Scientific, 11548876)\n\n• 2.9 mL sterile Tissue-Culture H2O\n\n\n\n• Reconstitute in PBS to 10 mM, divide into 50 μL aliquots and store at -20 °C\n\n\n\n• 100 mL DMEM, high glucose (ThermoFisher Scientific, 11965084)\n\n• 10 mL FBS (Sigma-Aldrich, F9665-50ML)\n\n• 1 mL Penicillin-Streptomycin (ThermoFisher Scientific, 11548876)\n\n• 1 mL L-Glutamine (ThermoFisher Scientific, 25030081)\n\n\n\n• 45 mL DMEM, high glucose (ThermoFisher Scientific, 11965084)\n\n• 500 μL Penicillin-Streptomycin (ThermoFisher Scientific, 11548876\n\n• 50 μL Insulin (10 mg/mL)\n\n• (Merck Millipore, 91077C-250MG)\n\n• 50 μL Aprotinin (33 mg/mL)\n\n• (Sigma-Aldrich, A1153-100MG)\n\nFilter sterilise and store at 4°C\n\n\n\n• Other brands of 24 well plate have differing dimensions and will reduce success rate of EHT formation\n\n\n\n• Single cell dissociation is required for the generation of a homogenous EHT\n\n\n\n• Autoclave sterilised and pre-chilled.\n\n\n\n1. Ensure there is premade sterile 2 % agarose in the fridge\n\n2. Prepare and filter sterilise 2X DMEM mix, and leave on ice\n\n3. Ensure there is premade NKM medium and chill on ice\n\n4. Ensure there is premade EHT medium\n\n5. Prewarm 40 mL of sterile DMEM-F12 in the water bath\n\n\n\n1. Remove media and wash 1-2 confluent wells of a six-well plate containing iPSC-CM with room temperature PBS once\n\nNOTE: 1 × confluent well of hiPSC-CM in a 6 well plate should yield approximately 4 × 106 cells\n\n2. Add 2 mL of TrypLE Select Enzyme (10X) per well of a 6 well plate and incubate for 30 minutes at 37 °C\n\n3. Transfer the detached cells to a 50 mL centrifuge tube and wash off any attached cells with 1 mL per well of pre-warmed DMEM-F12\n\n4. Add DMEM-F12 to the cell mix to give a total volume of 10 mL\n\nNOTE: Centrifugation of iPSC-CM takes place later, when both cell types have been combined\n\n5. Perform a cell count\n\n\n\n1. Remove the media and wash a T25 flask (80 % confluency) containing iPSC-CF with PBS\n\nNOTE: A T25 flask at 80 % confluency should yield approximately 600,000 iPSC-CF\n\n2. Add 3 mL TrypLE Express and incubate for 3 minutes at 37 °C\n\n3. Gently tap the flask on all sides to dissociate cells from the surface of the flask\n\n4. Transfer the 3 mL of TrypLE/cell mix to a 50 mL centrifuge tube\n\n5. Rinse the flask with 7 mL of pre-warmed DMEM-F12 before combining it with the TrypLE containing cells\n\n6. Perform a cell count\n\n\n\n1. Combine required volumes of hiPSC-CM (4 × 106 cells) and hiPSC-CF (6 × 105 cells) cell mixes in a centrifuge tube and make up to 30 mL with pre-warmed DMEM-F12\n\n2. Spin down the tube containing both iPSC-CM and iPSC-CF at 100 g for 10 minutes\n\n3. Place thrombin, Fib-Ap, Rock Inhibitor and Geltrex on ice\n\n4. Following centrifugation, gently place the cells on ice\n\nNOTE: Be careful not to disturb the pellet\n\n\n\n1. Microwave 2 % agarose until completely molten\n\n2. Pipette 1.6 mL of molten agarose per well in a column of a Nunc 24 well plate\n\n3. Insert a Teflon spacer into a column of the Nunc 24 well plate containing molten agarose to create agarose moulds for a silicon rack of four EHTs\n\nNOTE: Wells of a 24 well plate can differ in dimensions. The spacers and silicon racks are optimised for use in Nunc Cell-Culture Treated 24 well plates\n\n4. Remove the Teflon spacers from the now solidified agarose moulds after 15 minutes\n\nNOTE: Agarose moulds can deteriorate and crack over time. Cast EHTs in the agarose moulds 10 minutes after removal of the Teflon spacers\n\n5. Place the silicon racks into the moulds, ensuring central alignment\n\n6. NOTE: Misaligned silicon racks increase the risk of snagging during removal from the moulds\n\n\n\n1. Aliquot 3 μL of the thrombin into 4 × 0.5 mL microcentrifuge tubes and chill on ice (4 tubes for 4 EHTs)\n\nNOTE: Briefly spin down the tubes containing the thrombin\n\n2. Add 368.55 μL of NKM medium to a prechilled 1.5 mL microcentrifuge tube\n\n3. Prewarm the Fib-Ap mix in your hands (to enable accurate pipetting) and then add 11.25 μL to the NKM medium\n\nNOTE: Complete dissolution of the Fib-Ap mix is paramount to successful generation of EHTs.\n\n4. Add 24.75 μL of 2X DMEM\n\n5. Add 0.45 μL of Rock Inhibitor\n\n6. Add 45 μL of geltrex\n\n7. Resuspend the cell pellet containing both hiPSC-CM and hiPSC-CF in the EHT mix\n\nNOTE: Everything should be kept on ice at this stage.\n\n\n\n1. Add 100 μL of the EHT cell mix to a 3 μL aliquot of thrombin and then quickly pipette the mix in between the two silicon posts resting inside the agarose mould\n\nNOTE: Minimise risk of air bubble formation by only pipetting down to the first stop of the pipette\n\n2. Repeat step 1 for each EHT\n\nNOTE: Be careful not to knock or disturb the 24 well plate holding the solidifying EHTs\n\n3. Gently triturate the EHT cell mix intermittently (every 4 EHTs) to ensure homogeneity\n\n4. Gently place the lid on the 24 well plate and place in a 37 °C incubator for 2 hours\n\n\n\n1. Warm NKM medium in a 37 °C water bath\n\n2. Add 1.5 mL of EHT medium to each well of a column of wells in a Nunc 24 well plate and place in the 37 °C incubator\n\n3. After 2 hours, gently pipette 350 μL of prewarmed NKM medium on top of the agarose mould in each well\n\nNOTE: Pipetting on top of the agarose mould prevents disruption to the EHT\n\n4. Place in the incubator for a further 10 minutes\n\n5. Move the plate from side to side to encourage dislodging of the EHTs\n\n6. Carefully but firmly remove the EHT strips from the agarose moulds and place into the wells containing the prewarmed EHT medium\n\nNOTE: Removal of the strips should be performed in a single action as prolonged and gentle removal of the EHTs from the agarose moulds can increase the risk of snagging and malformation\n\n8. Maintain the EHTs by transferring the silicon racks into wells containing fresh EHT medium every 48 hours.\n\nhiPSC-CF were cultured in T25 flasks for 72 hours in Fibroblast Growth Media 3 supplemented with 10 ng/mL FGF2 + 10 μM SB 431542 or 10 ng/mL FGF2 + 10 ng/mL TGF-β1 (R&D Systems, 240-B-002/CF). RNA extraction was performed using the Direct-zol RNA Miniprep Kit (Zymo Research, R2050) according to the manufacturer’s protocol. cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, 4368814) according to the manufacturer’s protocol. RT-qPCR was performed using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific, 4444557) following the manufacturer’s protocol and on a QuantStudio 5 Real-Time PCR System, 384-well (ThermoFisher Scientific, A28140). RT-qPCR was performed using TaqMan Gene Expression Assay (FAM) (ThermoFisher Scientific, 4331182) for ACTA2 (Hs00426835_g1) and COL1A1 (Hs00164004_m1). All reactions were multiplexed and normalised to the housekeeping gene GAPDH using Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe, primer limited) (ThermoFisher Scientific, 4326317E). 10 ng of cDNA were used per reaction. Transcript levels were measured from three samples of iPSC-CF, with each sample being obtained from cells at different passages. Cycle threshold values were obtained from three technical replicates of each sample. Relative gene expression was calculated using the comparative cycle threshold method (Schmittgen and Livak, 2008).\n\nInduced pluripotent stem cell derived cardiac fibroblasts\n\nhiPSC-CF were split into wells of a Geltrex-coated 24 well plate (10,000/well) and cultured for 72 hours in Fibroblast Growth Media 3 supplemented with 10 ng/mL FGF2 + 10 μM SB 431542 or 10 ng/mL FGF2 + 10 ng/mL TGF-β1. Cells were fixed for 14 minutes at room temperature using 4 % paraformaldehyde Solution (ThermoFisher Scientific, J19943.K2). Fixed cells were blocked for 1 hour in blocking buffer consisting of PBS, Fetal bovine serum (5 %), Bovine Serum Albumin (1 %) (Merck Millipore, A9418-10G) and Triton X-100 (0.5 %) (Merck Millipore, X100-100ML). Primary antibodies were added at dilutions listed in Table 4 in blocking buffer for 2 hours at room temperature. This was followed by 3 washes in blocking buffer and the addition of secondary antibodies (Table 4) for 1 hour in the dark at room temperature. DAPI (ThermoFisher Scientific, D1306) was added with the secondaries at 0.1 μg/mL to visualise nuclei. Samples were washed 3 times in blocking buffer and then once with PBS. Samples were imaged using a EVOS M5000 Imaging System (ThermoFisher Scientific, AMF5000).\n\nEngineered heart tissues\n\nEHTs constructed with and without the addition of hiPSC-CF were removed from posts after 15 days in culture (30 days post initiation of differentiation), washed three times in PBS and fixed overnight at 4 °C in 4 % paraformaldehyde solution. A scalpel was used to longitudinally section EHTs. Sectioned pieces of EHTs were blocked overnight in blocking buffer. Primary antibodies were added in blocking buffer, at the dilutions listed in Table 4, overnight at 4°C. Samples were washed three times in blocking buffer before the addition of secondary antibodies for 4 hours in the dark at room temperature. DAPI was added with the secondaries at 0.1 μg/mL to visualise nuclei. Tissue sections were placed onto coverslips (VWR, 631-0170) and mounted onto cover slides using Hydromount Histology Mounting Media (Scientific Laboratory Supplies, NAT1324). Images were acquired using a Zeiss LSM780 Confocal laser scanning microscope equipped with a C-Apochromat 63x/1.20 W objective.\n\nEHTs constructed with and without hiPSC-CF were cultured for 15 days in EHT media (30 days after initiation of differentiation) prior to recording and analysis. 10 second videos of each EHT was recorded at 1080p/30 fps using an inverted camera with a macroscopic lens (3 EHTs with iPSC-CF, 3 EHTs without iPSC-CF). Videos were converted into tif files and analysed using the ImageJ Plugin TrackMate for displacement over time. All EHTs with contractile function were included in the analysis. Contractile function was defined as at least two macroscopically visible contractions within 15 seconds. All EHTs constructed with iPSC-CF demonstrated contractile function. Only three of the nine EHTs constructed (one of three batches, three EHTs per batch) without the addition of iPSC-CF showed contractile function. The analysis of contractile function was performed blinded to the presence or absence of hiPSC-CF. Outputs were semi-automated and included contraction duration, time to peak, relaxation time, total contraction amplitude and beat rate. For converting displacement into force, elastic bending of the pillars was assumed with a measured Young’s modulus of 3.0 MPa, yielding a conversion factor of 0.26 mN/mm.\n\n\nResults\n\nInduced pluripotent stem cells were differentiated into cardiac fibroblasts using the protocol outlined in materials and methods. Following differentiation, the activation status and plasticity of the cells was assessed using immunofluorescence. The cells were cultured in the presence and absence of 10 ng/mL TGF-β1 for 72 hours. The cells were then fixed and stained for vimentin, αSMA and collagen 1 with the results shown in Figure 3A. Vimentin is a cardiac fibroblast marker irrespective of activation status whilst αSMA and collagen are predominantly expressed in activated or myofibroblasts. The hiPSC-CF cultured in the absence of TGF-β1 were positive for vimentin but negative for αSMA, indicating a quiescent cardiac fibroblast phenotype. The cells cultured without the TGF-β1 inhibitor, SB 431542, and in the presence of 10 ng/mL TGF-β1 were positive for vimentin and αSMA and showed greater expression of collagen. The gene expression of ACTA2 (the transcript which encodes αSMA) and COL1A1 (a transcript which encodes collagen) was assessed using RT-qPCR as markers for cardiac fibroblast activation. Expression analysis was performed on three biological samples, with three technical repeats performed for each sample. No data were excluded from the analysis. The results are shown in Figure 3B. The hiPSC-CF showed a three-fold increase in the mean expression of COL1A1 following TGF-β1 treatment (-TGF-β1: M = 1, SD = 0.21, +TGF-β1 M = 2.9, SD = 0.47). The mean expression of ACTA2 in the hiPSC-CF increased approximately five-fold following TGF-β1 treatment (TGF-β1: M = 1, SD = 0.13, +TGF-β1 M = 4.9, SD = 1.59).\n\nhiPSC-CF were cultured in the standard media (FGM3 + 10 ng/ml FGF2 + 10 μM SB 431542, ‘-TGF-β1’) and with 10 ng/mL TGF-β1 instead of the SB 431542 (‘+TGF-β1’) for 72 hours to assess activation status. A: The cells were fixed and stained for vimentin (top red), Collagen (bottom red) αSMA (green) and DAPI (blue, indicating nuclei). Scale bar = 150 microns. B: RNA was extracted, and RT-qPCR was performed on the cDNA for ACTA2 and COL1A1.\n\nN = 3 (three biological samples with 3 technical replicates per sample are plotted).\n\nA workflow delineating the generation of EHTs from hiPSCs is displayed in Figure 4. EHTs containing hiPSC-CM and hiPSC-CF started twitching approximately 48 hours after construction, with maximal beating capacity being reached after approximately 14 days in culture. Those generated in the absence of hiPSC-CF started twitching approximately 96 hours after construction and showed diminished contractile function throughout the duration of culture. EHTs constructed with and without hiPSC-CF demonstrated marked differences in shape and structure after 72 hours in culture as shown in Figure 5. EHTs constructed with hiPSC-CF showed tightening around the silicon posts whilst hiPSC-CM-only EHTs remained more block-like throughout time in culture.\n\nAn outline of the workflow to generate of EHTs with and without the addition of hiPSC-CF. The EHT shown on the top right was generated without the addition of hiPSC-CF. The EHT shown on the bottom right was constructed with the addition of hiPSC-CF. Created with Biorender.com.\n\nEHTs were constructed with and without the addition of hiPSC-CF according to the protocol outlined above. A and B show side by side comparisons. C - Close-up of an EHT with hiPSC-CF and hiPSC-CM demonstrated compaction after 72 hours, which was not observed in EHTs generated from hiPSC-CMs alone (D). Scale bar = 4 mm.\n\nAt day 30 after the initiation of differentiation (15 days in EHT form), 1080p/30 fps videos of EHTs with and without hiPSC-CF were analysed using the Open-source ImageJ plugin TrackMate (Figure 6). All EHTs which demonstrated contractility were included in the analysis (Section 3.8). EHTs with and without hiPSC-CF demonstrated similar contraction durations and relaxation times of approximately 400 and 240 ms respectively. The mean time to peak time of the EHTs containing hiPSC-CF was similar to that of their hiPSC-CM-only counterparts at approximately 170 ms. The average contraction amplitude of the EHTs containing hiPSC-CF was approximately 30 % greater than that of the EHTs consisting of hiPSC-CM only. EHTs constructed with hiPSC-CF demonstrated a marked decrease in spontaneous beat rate.\n\nEHTs constructed with and without the addition of hiPSC-CF were analysed to assess contractile activity. Frames of the videos of the EHTs were analysed using the ImageJ Plugin TrackMate for displacement over time, the box represents the analysis window where movement of the pillar was tracked (A). Contractile profiles were generated (a representative profile of an EHT with and without iPSC-CF is shown) (B) and used to ascertain Contractile Duration, Relaxation Time, Time to Peak, Contraction Amplitude and Spontaneous Beat Rate (C). N = 3 EHTs. Scale bar = 4 mm.\n\nEHTs consisting of hiPSC-CM and hiPSC-CM/hiPSC-CF were cultured for 15 days after construction (30 days after initiation of hiPSC-CM differentiation). The tissue was fixed and sectioned using a scalpel and then stained for DAPI, titin and vimentin to assess composition and alignment (Figure 7). Vimentin is a key marker of cardiac fibroblasts whilst titin is an integral component of the sarcomeres of cardiomyocytes. The hiPSC-CM demonstrated longitudinal alignment in EHTs constructed with and without hiPSC-CF (Figure 7A). Cardiac fibroblasts were present in EHTs constructed with hiPSC-CF and were absent in the hiPSC-CM only tissues.\n\nEHTs were constructed with and without iPSC-CFs. Tissues were fixed, sectioned and stained for Titin and DAPI (A) and titin, vimentin and DAPI (B). Scale bar = 20 microns.\n\n\nDiscussion\n\nIn this study, we described efficient and reproducible methods for the generation of hiPSC-CM and hiPSC-CF. Furthermore, we described the subsequent integration of the cells into co-culture EHTs with improved contractile function. This approach has potential to explore the complex interplay between cardiomyocyte and cardiac fibroblast signalling involved in pathophysiology. The hiPSC-CF generated were quiescent under the culture conditions outlined in the protocol, with plasticity demonstrated through the addition of the pro-fibrotic cytokine TGF-β1. EHTs constructed with the addition of hiPSC-CF exhibited anisotropic alignment, decreased beat rate, increased tissue compaction and enhanced contractile force.\n\nThe importance of non-myocyte cell types in 3D cardiac constructs is well established and has been widely discussed in previous publications (Kofron and Mende, 2017). EHTs consisting of both hiPSC-CM and hiPSC-CF demonstrated an increase in tissue compaction following just 72 hours in culture and a decrease in beat rate after two weeks in culture. Tissue compaction plays an important role in determining contractile capability and likely occurred in the EHTs constructed with hiPSC-CF due to the regulatory function that cardiac fibroblasts have on extracellular matrix (Rivera-Arbeláez et al., 2022; Thavandiran et al., 2013). A decrease in beat rate was observed from EHTs that were constructed with hiPSC-CF. This is consistent with previous studies that have demonstrated lower beating rate frequencies in hiPSC-CM/fibroblast co-culture models and is likely due to increased CM maturity and decreased funny current expression (Ronaldson-Bouchard et al., 2018).\n\nEHTs constructed in this study from metabolically purified populations of hiPSC-CM, without the addition of hiPSC-CF, demonstrated diminished or no contractile function. Functional and contractile EHTs consisting solely of hiPSC-CM were generated during the course of this study but with a significantly reduced rate of success. The presence of non-myocyte cell types in the tissue population has previously shown to be beneficial to the successful generation and improved contractility of 3D cardiac models (Naito et al., 2006; Ravenscroft et al., 2016). Some studies have robustly demonstrated the successful generation of EHTs using CM populations with a purity of 92-100 % (Mannhardt et al., 2016, 2020). Differences in stem cell line, hiPSC-CM differentiation, handling, and maturation may help explain contrasting success rates of hiPSC-CM only EHT construction. The further development of 3D cardiac models consisting of defined populations of cardiac myocytes and stromal cells, e.g., fibroblasts, will help to reduce batch-to-batch variability and contribute to the development of more reliable in vitro modelling systems.\n\nThe relative ease by which hiPSCs can be genetically engineered has allowed researchers to efficiently interrogate the effects pathogenic variants have in the cardiac myocyte. The development of isogenic EHTs consisting of hiPSC-derived cardiomyocytes and cardiac fibroblasts provides a more physiological model in which mutations can be explored and presents increased opportunities for exploring variants with pathological effect in both cell types (Zou et al., 2022). Furthermore, such models represent an exciting prospect for the future incorporation into pre-clinical drug screening and may eventually help relieve the current reliance on in vivo models.\n\nDifferences in the intrinsic electrophysiological properties of mouse cardiac myocytes have hampered efforts to model complex cardiovascular diseases. Furthering our understanding of currently enigmatic aspects of human cardiac disease, such as the dynamic interplay between cardiac fibrosis and electrical remodelling, likely requires physiological and ultimately human models, such as the EHTs applied in this study. Improvements to human in vitro modelling systems are required to understand the complexities of cardiac pathology and will likely aid in the reduction and/or replacement of animal models in cardiac research.", "appendix": "Data availability\n\nFigshare: qPCR Data of iPSC-CF Activation (ACTA2, COL1A1).xlsx, https://doi.org/10.6084/m9.figshare.23639205.v1 (Cumberland et al., 2023a).\n\nFigshare: Raw Images of EHTs constructed with and without iPSC-CF, https://doi.org/10.6084/m9.figshare.23978340.v1 (Cumberland et al., 2023b).\n\nThis project contains the following underlying data:\n\n‐ EHT -iPSC-CM TTN.tif\n\n‐ EHT +iPSC-CM TTN.tif\n\n‐ EHT Vim:TTN -iPSC-CF.tif\n\n‐ EHT Vim:TTN +iPSC-CM.tif\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAcevedo-Acevedo S, Crone WC: Substrate stiffness effect and chromosome missegregation in hIPS cells. J. Negat. Results Biomed. 2015; 14: 1–7. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "210539", "date": "13 Oct 2023", "name": "Claudia Crocini", "expertise": [ "Reviewer Expertise cardiac biology", "imaging", "optogenetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Cumberland et al., describes a protocol to generate the EHT using cardiomyocytes and cardiac fibroblasts derived from the same hiPSCs.\nThe work is not particularly new but is well-described.\nMy comments:\nI would like to see the force expressed as mN/mm2 measuring the diameter of EHTs. I know that the EHT community doesn't do it but the physiology community does not agree. The raw values could be showed in a table or in the supplement.\n\nFigure 7: It would be better to see the EHTs with the vimentin and TTN signals splitted (instead of the supplement). Also a maximum intensity projection of a z-stack could help to avoid seeing blue nuclei with very faint green/red signal.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "11023", "date": "13 Apr 2024", "name": "Max Cumberland", "role": "Author Response", "response": "We thank the reviewer for their comments. I would like to see the force expressed as mN/mm2 measuring the diameter of EHTs. I know that the EHT community doesn't do it but the physiology community does not agree. The raw values could be showed in a table or in the supplement. We have converted force into mN/mm2 and added the graph to Figure 6. This exacerbates the differences in contractile force produced between EHTs with and without the addition of iPSC-CFs. We have also attached the raw values in a spreadsheet in the supplement. This includes pillar displacement, diameter, and cross-sectional area. Figure 7: It would be better to see the EHTs with the vimentin and TTN signals splitted (instead of the supplement). Also a maximum intensity projection of a z-stack could help to avoid seeing blue nuclei with very faint green/red signal. We have split the channels showing the vimentin, titin and nuclei staining. We now show them as a separate panel in Figure 8. Unfortunately, a z-stack was not captured so we are unable to perform a maximum intensity projection but welcome the comment for future use." } ] }, { "id": "221605", "date": "22 Nov 2023", "name": "Emma Louise Robinson", "expertise": [ "Reviewer Expertise heart failure", "epigenetics", "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\nCongratulations to the authors on a thorough and well-described methods paper on how to generate an engineered heart tissue comprised of iPSC-derived CMs and CFs. The text and figures are clear and the article flows very well.\nOne suggestion is to highlight, in a line or two, the novelty of this EHT system over other similar EHT methods published.\nAnother is to comment on the omission of demonstrating responsiveness of the iPSC-CMs to pathological stimuli, such as isoproterenol or endothelin-1 increasing ANF and BNP production, for example. If this EHT system is to be used to model pathological remodeling and heart failure, this would have been nice. I am unsure how much time and effort it would take to add this data or whether the authors have some relevant data already along these lines. There is a nice demonstration of the activation of iPSC-CFs to TGF-Beta.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "11024", "date": "13 Apr 2024", "name": "Max Cumberland", "role": "Author Response", "response": "We thank the reviewer for their comments. 1. One suggestion is to highlight, in a line or two, the novelty of this EHT system over other similar EHT methods published. We have added a short description regarding the novelty of the EHT system to the discussion. 2. Another is to comment on the omission of demonstrating responsiveness of the iPSC-CMs to pathological stimuli, such as isoproterenol or endothelin-1 increasing ANF and BNP production, for example. If this EHT system is to be used to model pathological remodeling and heart failure, this would have been nice. I am unsure how much time and effort it would take to add this data or whether the authors have some relevant data already along these lines. There is a nice demonstration of the activation of iPSC-CFs to TGF-Beta. The model was designed for future use assessing pathological remodelling in heart disease. Unfortunately, at this time, we are unable to show data demonstrating the responsiveness of the model to pathological stimuli but hope that the model can be used in the future to this effect." } ] }, { "id": "221607", "date": "30 Jan 2024", "name": "Mike Dodd", "expertise": [ "Reviewer Expertise cardiac metabolism", "epigenetics", "diabetes and hypoxia." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCumberland and colleagues have produced a really good protocol on the generation of more mature EHT using a combination of iPSC- cardiomyocytes and fibroblasts. It flows really well and I find it easy to follow, supported by good figures and details of reagents.\nOne of the areas where iPSC-CM models still need to improve is in the metabolic maturity to better replicate the adult heart. With the co-culturing of cardiac fibroblast with cardiomyocytes in this EHT system it would be useful to know whether there is an improvement in metabolic markers. Whilst something like Clarke electrodes for measuring metabolic rate would be excellent, I would like to see some qPCR data on metabolic genes. Some of the key genes in the transition between fetal (immature) and adult cardiac models would be increase GLUT4, PPARA and fatty acid oxidation gene expression and possibly decrease in pyruvate kinase 2 and hexokinase 1. I would be interested to see the differences in these profiles between the two EHT models, to see if the CF are increasing metabolic maturity. Otherwise this is an excellent piece of work.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "11025", "date": "13 Apr 2024", "name": "Max Cumberland", "role": "Author Response", "response": "We thank the reviewer for their comments and agree that better metabolic maturity of iPSC-CM models is still required to help recapitulate the human heart in health and disease. Unfortunately, at this time we are unable to provide expression data on the metabolic maturity of the model containing iPSC-CFs. We hope that future studies employing such a model will investigate markers of metabolic maturity." } ] } ]	1	https://f1000research.com/articles/12-1224
https://f1000research.com/articles/12-861/v1	20 Jul 23	{ "type": "Research Article", "title": "Levels of polycyclic aromatic hydrocarbon in the soil around typical automobile repair workshops in Nigeria", "authors": [ "Olusola Adedayo Adesina", "Oluwatomi Atinuke Fakayode", "Mayowa Adeoye Lala", "Abiodun John Adewale", "Jacob Ademola Sonibare", "Oluwatomi Atinuke Fakayode", "Mayowa Adeoye Lala", "Abiodun John Adewale", "Jacob Ademola Sonibare" ], "abstract": "Background: This study determined the levels of polycyclic aromatic hydrocarbons (PAHs) in the soil around typical automobile repair workshops in Nigeria. Risk assessment associated with human contact with the soil was carried out using hazard quotient (HQ) and incremental life cancer risk (ILCR) from human unconscious ingestion and dermal contact with the soil. Methods: Soil samples were obtained at different automobile workshops in Ado-Ekiti, Western Nigeria. The PAHS in the samples were extracted using dichloromethane and extracts were cleaned up silica gel open column chromatography. Analysis of PAHs in the soil was done using a gas chromatograph coupled to a mass selective detector operated on electron ionization mode. Results: The results showed the mean PAHs concentration at the sampling locations ranged from 5.58 – 6.4 μg/g\n\nand the mean ∑ carcinogenic PAHs was 58.4 μg/g , equivalent to 59.39 % of total PAHs observed. The mean Toxicity equivalence (TEQs) ranged from 0.02 - 6.680 μg/g . Benzo (a) pyrene and dibenzo(a,h)anthracene have the highest toxicity equivalent. The total ILCRs from accidental ingestion and dermal contact in adults were 1 ×10^(-3) and 9.8 ×10^(-5) , for adults and children respectively; both are higher than the permissible limit stipulated by the World Health Organization. The HQs obtained are several folds higher than 1. This implies high carcinogenic and non-carcinogenic risks for children and adults. Conclusions: The study revealed the levels of PAHs and risks associated with human contact with the soil around automobile repair workshop.", "keywords": [ "PAHs", "ambient air", "automobile workshop", "Nigeria" ], "content": "Introduction\n\nIndiscriminate discharging of used engine oil into the surroundings, especially around automobile workshops, remains a major problem in most parts of Nigeria and this constantly affects the ecosystem (Odjegba and Sadiq, 2002; Emoyan et al., 2020). The soil around automobile workshops is usually contaminated with spent engine oil which contains heavy metals and polycyclic aromatic compounds (PAHs) and other pollutants. PAHs constitute two or more fused aromatic rings solely made from carbon and hydrogen (Arey and Atkinson, 2003). PAHs are emitted from the exhaust of automobiles as well as by-products from petroleum ranging from lubricating oil, gasoline, diesel, and several others put to constant use in automobile workshops, and these have high chances of spilling into and degrading the soil (Kidman and Boehlecke, 2011; Muze et al., 2020).\n\nPAHs, which are the products of incomplete combustion, are transported into the crankcase and concentrated in lubricating oil in the engine of the car (Pruell and Quinn, 1988; Akintunde et al., 2015). Additionally, emissions from the exhausts of the cars being repaired at these workshops can be washed down through rain to the soil around the workshop. Attention has been drawn to the health risks associated with human contact with soil contaminated with PAHs (Wcisło, 1998; Adeniyi and Afolabi, 2002; Dong and Lee, 2009; Kwon and Choi, 2014). PAHs are found to have a negative effect on human health as they can lead to cancer, as well as having a mutagenic and teratogenic effect (Boström et al., 2002).\n\nThe presence of PAHs in the spent engine oils disposed in the environment during maintenance poses a serious threat to the environment (Ololade, 2014). The lack of required knowledge on proper handling of these hydrocarbon products during repairs and vehicle servicing in automobile workshops is a major problem, especially in Nigeria (Sharifi et al., 2007). Akintunde et al. (2015) studied the reproductive effects of used engine oil on male rats, and their results showed used engine oil has the potential to hamper male rats' germ cell development and also affect other testicular activities in producing viable spermatozoa. Their study also concluded that soil contaminated with spent engine oil poses a great reproductive risk to humans in areas where there is high exposure. The Agency of Toxic Substances and Diseases Registry rates PAHs as ninth on the list of compounds dangerous to human health. Organisms are also affected by the toxic effect of PAHs as they affect the functioning process of cellular membranes (Abdel-Shafy and Mansour, 2016). Clients and workers at automobile workshops are exposed to PAHs through contact with the soil and ambient around the workshop.\n\nFew or no studies are available on the level of PAHs around automobile repair workshops, especially in Sub-Saharan Africa countries such as Nigeria. Hence this study focuses on determining the levels of PAHs around automobile repair workshops, this is with a view to determining the risk associated with human contact with these pollutants.\n\n\nMethods\n\nThe study area locations are shown in Table 1. In order to assess the implication of automobile repair workshops’ activities on the soil levels of PAHs, soil samples were taken from five different local automobile repair workshops (locations A-E) in Ado-Ekiti, Southwestern Nigeria. Ado-Ekiti is the capital of Ekiti state in Southwestern Nigeria and is a city on 7° 37′ 15.9996″ N and 5° 13′ 17.0004″ E.\n\nThe top layer of soil samples (0–15 cm) was collected around the automobile workshops. The samples were wrapped in foil papers after collection and transported to the laboratory for analysis. At the laboratory, the impurities were then removed by hand picking, and then sieved through a 2 mm sieve shaker. The samples were then oven dried using a universal drier at 80 oC to constant mass, and they were then kept in aluminum foil bags and stored at -20 °C in the freezer.\n\nThe soil samples were initially spiked with recovery standards of PAHs to monitor the integrity of the treatment. The sample was then extracted with dichloromethane (DCM) using a Soxhlet extractor at 30°C for 8 hrs to enable PAHs trapped on the PUF disk and soil to dissolve in DCM. A clean-up procedure was done to remove unwanted compounds in the matrix by using silica-alumina column chromatography. The column was prepared by adding about 10 to 15 mm plug of glass wool to a chromatograph column and stuffing it down using a glass rod, then alumina and silica were added (1:2). The column packing was partially deactivated using a methanol-DCM solution (1:3) for better recovery. The sample extract was decanted into the column and eluted with 40 mL 1:1, DCM: hexane (Adesina et al., 2018). The extracted samples were then concentrated using a rotary evaporator (Büchi®), to gently remove the solvent from the extract in order to bring down the volume of extract to 5ml. The resultant extracts were later analyzed for PAHs.\n\nQuantification of PAHs present in the sample was carried out using gas chromatography (GC) (Agilent 7890) with a mass detector (Agilent 5975) operated in a selected ion monitoring mode and using electron impact ionization (EI). The chromatographic column dimensions are 30 m × 0.25 mm, and the internal diameter of 0.25 μm film thickness. The temperature program for the analysis was set as 90°C (1.0 min), 30°C/min, 250°C, 4°C/min, and 330°C (5 min). Determination of the concentration of PAHs was done using the internal standard method. The internal standards are naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12, used to quantify the amount of PAHs in the extract.\n\nApart from normal samples, field and laboratory blanks were also taken and treated the same way as the samples to ensure high integrity of the data. Soil blank samples were taken at locations far from the workshops, that have not been contaminated by automobile repair activities. Determination of instrument detection limits (IDLs) and method detection limits (MDLs) followed Norlock et al. (2011) procedure. Standard solutions were prepared for PAHs and six surrogates which were analyzed four times in the SIM mode using GC-MS. The PAH concentrations in each calibration solution were recalculated from the regression equation obtained using all six calibration standards. The average of the four replicates with the lowest detectable concentration was taken as the IDL. MDLs were calculated by multiplying the standard deviation of the replicates by the one-side t statistic at the 99%. Before the extraction, 20 ng of phenanthrene d10 recovery standard (RS) was used to spike the sample and the recovery range of the PAH was between 80% and 90%. Concentrations of PAHs in the field blanks were below the detection limit for all targeted compounds and no blank correction was carried out.\n\nToxic equivalent\n\nThe potential toxicity of the PAHs is calculated by multiplying the individual concentration, C, with the toxicity equivalence factor (TEF) (eq. i) (Van den Berg et al., 2006).\n\nILCR from ingestion, inhalation, and dermal contact with PAHs contaminated soil are calculated using eq. ii, iii, iv, respectively. Also, the non-carcinogenic associated risk is assessed using the hazard quotient index (HQ) which is the ratio of the estimated to the reference dose using eq. v (USEPA, 1991, 2011).\n\nC is the PAH concentration in the soil solid residue (mg kg−1). IRing is the soil ingestion rate (100 mg d−1 for adults and 200mg d −1for children). IRinh is the inhalation rate (20 m3/day was assumed for adults while 9.6 m3/day was assumed for children). Table 2 shows the exposure parameters and factors used for the study.\n\n\nResults and discussion\n\nIn this study, 16 different USEPA priority PAHs were analyzed in soil samples: naphthalene (Naph), acenaphthylene (Acy), acenaphthene (Ace), fluorene (Fln), phenanthrene (Phe), pyrene (Pyr), fluoranthene (Flt), anthracene (Ant), benzo [e] pyrene (BeP), benzo [a] pyrene (BaP), benzo [b] fluoranthene (BbF), benzo [a] anthracene (BaA), chrysene (CHR), benzo [k] fluoranthene (BkF), indeno[1,2,3-cd] pyrene (InP), and dibenzo [a,h] anthracene (DAh). Table 3 shows the concentrations of PAHs at different automobile workshops. The results showed BbF and Bkf have the highest concentrations. The concentration of BbF ranged from 4.0 – 31.03 μg/g with a mean concentration of 17.30 μg/g. BkF’s concentration ranged from 4.12-27.92 μg/g with a mean concentration of 17.53. These two compounds contribute 36 % of the total PAHs found in the soil. A high concentration of this compound implies that the source of the PAHs contamination of the soil is largely from petroleum products such as spent engine oil. Another compound with a high concentration is Fln, with a range of 3.96 – 18.13 μg/g and a mean concentration of 10.81 μg/g, which is 11 % of the total PAHs. Fln is one of the Middle Molecular Weight PAHs formed by the combustion of petroleum products such as emission from the exhaust of the vehicle. This could be the reason for the high concentration of this compound around the soil analyzed. BaP concentration is usually used as an indicator of PAH carcinogenic activity due to its stability. The result obtained showed a BaP mean concentration of 5.39 μg/g with a range of 0.26-11.5 μg/g, which shows carcinogenic activities. The mean concentration of other carcinogenic PAHs observed in the soil is benzo [a] anthracene (2.45 μg/g), chrysene (3.24 μg/g), benzo [b] fluoranthene (17.30 μg/g) benzo [k] fluoranthene (17.53μg/g) and indeno (1, 2, 3-cd) pyrene (5.83 μg/g). Generally, the result showed the mean ∑ carcinogenic PAHs observed is 58.4 μg/g which is equivalent to 59.39% of total PAHs.\n\nPAHs are also classified based on the number of aromatic rings in each compound. The following ring compounds were present in this analysis: 2-ring PAHs (naphthalene), 3-ring PAHs (acenaphthylene, acenaphthene, fluorene, anthracene, and phenanthrene), 4-ring PAHs (pyrene, fluoranthene, chrysene, and benzo [a]anthracene), 5-rings PAHs (benzo [b] fluoranthene, benzo [a] pyrene, benzo [e] pyrenes, dibenzo [a,h] anthracene, and benzo [k]fluoranthene) and 6-rings PAHs (indeno [1,2,3-cd]pyrene). Figure 1 shows the distribution of PAHs based on the number of rings. 5-ring PAHs have the highest percentage at 39.37%, followed by 4-ring PAHs at 23.77%. 3-ring PAHs have a percentage contribution of 23.23%, 6-ring PAHs have a percentage contribution of 11.67%, and 2-ring PAHs have the lowest contribution at 1.97%. PAHs classification can also be by molecular weight: low molecular weight (LMW), consisting of 2- and 3-ring PAHs, middle molecular weight (MMW) consisting of 4-ring PAHs, and high molecular weight (HMW), consisting of 5- and 6-ring PAHs. Based on the results in Figure 2, LMW accounts for 25.2% of the total PAHs, MMW accounts for 23.77% and HMW accounts for the highest percentage of 51.03%.\n\nThe TEQ approach is used to determine the toxic potency of complex mixtures, the ILCR is used to determine the probability of developing cancer as the result of exposure to a specific carcinogen, and the hazard quotient is the ratio of the potential exposure to a substance and the level at which no adverse effects are expected. Table 3 shows the TEQ of the PAHs in all the soil samples analyzed. DhA has the highest toxicity with a mean value of 6.68 μg/g. BaP, which is usually used as an indicator of PAHs contamination, has a toxicity of 5.4 μg/g. Other compounds with high TEQ are BbF and BkF with mean values of 1.73 and 1.75 μg/g, respectively.\n\nThe ILCR is used to assess the probability of developing cancer by having contact with soil from the automobile workshop. Table 4 shows the ILCR and HQ values obtained from the study. The ILCR values from accidental ingestion of soil from these workshops are 3.9×10−4 and 4.4×10−4 for adults and children respectively, while ILCR values for dermal contact with the soil are 6.9×10−4 and 5.4×10−4. The value is higher than the permissible limit of 10−6 stipulated by the World Health Organization. This implies that, with exposure to this soil for a particular period, there is a probability of developing cancer. However, the ILCR values for accidental inhalation of the soil are 3.2×10−8 and 8×10−9, for adults and children respectively. This indicates the chances of developing cancer from inhalation of this soil are slim. The combination of ILCR from accidental ingestion, inhalation and dermal contact gives 1×10−3 and 9.8×10−5, for adults and children respectively. These values are higher than the permissible limit.\n\nTable 4 also shows the hazard quotient, which is several folds higher than 1, the permissible limit, which implies that there is great non-carcinogenic risk associated with exposure to this soil.\n\n\nConclusion\n\nThis work studied the levels of polycyclic aromatic hydrocarbons in the soil around typical automobile repair workshops in Nigeria. The study also assessed the risks associated with human contact with the soil. The results showed the mean PAHs concentration at the sampling locations ranged from 5.58 – 6.4 μg/g. The total toxicity equivalents at various locations range from 8.57 to 16.6 μg/g. The value of summation of ILCR and HQs from ingestion, inhalation, and dermal contact with PAHs contaminated soil is higher than the permissible limit stipulated by the World Health Organization. This study revealed the risk associated with human contact with soil around automobile workshops in Nigeria.", "appendix": "Data availability\n\nZenodo: Levels of polycyclic aromatic hydrocarbon in the soil around typical automobile repair workshops in Nigeria. https://doi.org/10.5281/zenodo.7939076 (Adesina et al., 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAbdel-Shafy HI, Mansour MS: A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egypt. J. Pet. 2016; 25(1): 107–123. Publisher Full Text\n\nAdeniyi AA, Afolabi JA: Determination of total petroleum hydrocarbons and heavy metals in soils within the vicinity of facilities handling refined petroleum products in Lagos Metropolis. Environ. Int. 2002; 28(1-2): 79–82. PubMed Abstract \| Publisher Full Text\n\nAdesina OA, Sonibare JA, Diagboya PN, et al.: Spatiotemporal distributions of polycyclic aromatic hydrocarbons close to a typical medical waste incinerator. Environ. Sci. Pollut. Res. 2018; 25(1): 274–282. PubMed Abstract \| Publisher Full Text\n\nAdesina OA, Fakayode OA, Lala MA, et al.: LEVELS OF POLYCYCLIC AROMATIC HYDROCARBON IN THE SOIL AROUND TYPICAL AUTOMOBILE REPAIR WORKSHOPS IN NIGERIA. [Dataset]. Zenodo. 2023. Publisher Full Text\n\nAkintunde WO, Olugbenga OA, Olufemi OO: Some Adverse Effects of Used Engine Oil (Common Waste Pollutant) On Reproduction of Male Sprague Dawley Rats. Med. Sci. 2015; 3(1): 46–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArey J, Atkinson R: Photochemical reactions of PAH in the atmosphere.Douben PET, editor. PAHs: An ecotoxicological perspective. 2003; 4(47 – 63). Publisher Full Text\n\nBoström CE, Gerde P, Hanberg A, et al.: Cancer risk assessment, indicators and guidelines for polycyclic aromatic hydrocarbons in ambient air. Environ. Health Perspect. 2002; 110(3): 451–489. Publisher Full Text Reference Source\n\nDong TT, Lee BK: Characteristics, toxicity, and source apportionment of polycyclic aromatic hydrocarbons (PAHs) in road dust of Ulsan, Korea. Chemosphere. 2009; 74: 1245–1253. PubMed Abstract \| Publisher Full Text\n\nEmoyan OO, Onocha EO, Tesi GO: Concentration assessment and source evaluation of 16 priority polycyclic aromatic hydrocarbons in soils from selected vehicle-parks in southern Nigeria. Scientific Africa. 2020; 7(2): e00296–e00213. Publisher Full Text\n\nFerreira-Baptista L, De Miguel E: Geochemistry and Risk Assessment of Street Dust in Luanda, Angola: A Tropical Urban Environment. Atmos. Environ. 2005; 39(25): 4501–4512. Publisher Full Text\n\nKidman RL, Boehlecke R: Evaluating petroleum hydrocarbon-contaminated soil. WM 20 conference. Phoenix; 2011.\n\nKumar B, Verma VK, Kumar S, et al.: Probabilistic Health Risk Assessment of PolycyclicAromatic Hydrocarbons and Polychlorinated Biphenyls in Urban Soils from a Tropical City of India. J. Environ. Sci. Health A Tox Hazard Subst. Environ. Eng. 2013; 48(10): 1253–1263. PubMed Abstract \| Publisher Full Text\n\nKwon HO, Choi SD: Polycyclic aromatic hydrocarbons (PAHs) in soils from a multi-industrial city, South Korea. Sci. Total Environ. 2014; 470-471: 1494–1501. Publisher Full Text\n\nMuze NE, Opara AI, Ibe FC, et al.: Assessment of the geo-environmental effects of activities of auto-mechanic workshops at Alaoji Aba and Elekahia Port Harcourt, Niger Delta, Nigeria. Environ. Anal. Health Toxicol. 2020; 35(2): 1–12. Publisher Full Text\n\nNorlock FM, Jang J, Zou Q, et al.: Large-Volume Injection PTV-GC-MS Analysis of Polycyclic Aromatic Hydrocarbons in Air and Se diment Samples. J. Air Waste Manage. Assoc. 2011; 52(1): 19–26. Publisher Full Text\n\nOdjegba VJ, Sadiq AO: Effects of spent engine oil on the growth parameters, chlorophyll and protein levels of Amaranthus hybridus L. Environmentalist. 2002; 22(1): 23–28. Publisher Full Text\n\nOlolade IA: An assessment of heavy metal contamination in soils within auto-mechanic workshops using enrichment and contamination factors with geo – accumulation Indexes. J. Environ. Prot. 2014; 05: 970–982. Publisher Full Text\n\nPeng, Chen W, Liao X, et al.: Polycyclic Aromatic Hydrocarbons in Urban Soils of Beijing: status, Sources, Distribution and Potential Risk. Environ. Pollut. (Barking, Essex: 1987). 2011; 159(3): 802–808. Publisher Full Text\n\nPruell RJ, Quinn JQ: Accumulation of polycyclic aromatic hydrocarbons in crankcase oil. Environ. Pollut. 1988; 49:(2): 89–97. Publisher Full Text\n\nSharifi M, Sadeghi Y, Akbarpour M: Germination and growth of six plant species on contaminated soil with spent oil. Int. J. Environ. Sci. Technol. 2007; 4(4): 463–470. Publisher Full Text\n\nSoltani N, Keshavarzi B, Moore F, et al.: Ecological and Human Health Hazards of Heavy Metals and Polycyclic Aromatic Hydrocarbons (PAHs) inRoad Dust of Isfahan Metropolis, Iran. Sci. Total Environ. 2015; 505: 712–723. PubMed Abstract \| Publisher Full Text\n\nUnited States Environmental Protection Agency, USEPA: Exposure factors handbook (Final). Washington: 2011.\n\nUSEPA: Guidelines for developmental toxicity risk assessment.1991. Reference Source\n\nWcisło E: Soil contamination with polycyclic aromatic hydrocarbons (PAHs) in Poland – a review. Pol. J. Environ. Stud. 1998; 7(5): 267–272.\n\nVan den Berg M, Linda SB, Michael D: The 2005 World Health Organization re-evaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 2006; 93: 223–241. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "191176", "date": "08 Aug 2023", "name": "Daniel O. Omokpariola", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors evaluated the health risk assessment of PAHs in automobile workshops in Ado Ekiti with a mandate to assess the influence in the location, as I have some assertions which are not correct or may need to be worked on:\n1. The last sentence of your introduction \"Few or no studies are available... in sub-Saharan African countries\" is not correct because a clear check on google scholar states that over 100 papers have been published and data are available in Nigeria and sub-Saharan countries. Remodifying that statement is advised.\n2. The materials and methods are okay within the scope of the work.\n3. The results and discussion does not provide a relationship and impact of these PAHs components to cause health based impact. More discussion is needed in relation to previous works available.\n4. The conclusion is not adequate, as there are no recommendation or summary of the study in tandem to adults to children influences and how these automobile workshops can work effectively and dispose waste oils.\nThanks.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "214475", "date": "27 Oct 2023", "name": "Akanimo N. Ekanem", "expertise": [ "Reviewer Expertise Environmental toxicologist. I mostly monitor organic pollutants in the environment and their health implications." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe statement “Few or no studies are available on the level of PAHs around automobile repair workshops, especially in Sub-Saharan Africa countries such as Nigeria” is not actually true because there are a lot of recent data on the subject matter such as Ekanem et al. (2019), Abugu et al. (2023), Daniel et al. (2023) etc.\n\nThe methodology is not explicit. In the soil sampling section, the apparatus used, sampling method and time of sampling is not mentioned. In the sample pretreatment section, quantity of solvent used for extraction is not mentioned. The references in the methods section are old. The author should review literature for the latest references.\n\nIn the discussion section, the authors could have presented a comprehensive table showing the concentrations of PAHs in all the samples analysed before presenting the statistical table. This would have enabled the reader to have comprehensive information about the study area.\n\nThe authors have not been able to drive home their point so as to draw conclusions on the health implication of PAHs. After comparison with the WHO standards, the authors would have made a conclusion whether the soil has low, moderate or high contamination and make recommendations.\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? Partly", "responses": [ { "c_id": "10512", "date": "10 Nov 2023", "name": "Olusola Adesina", "role": "Author Response", "response": "The authors evaluated the health risk assessment of PAHs in automobile workshops in Ado Ekiti with a mandate to assess the influence in the location, as I have some assertions which are not correct or may need to be worked on: 1. The last sentence of your introduction \"Few or no studies are available... in sub-Saharan African countries\" is not correct because a clear check on google scholar states that over 100 papers have been published and data are available in Nigeria and sub-Saharan countries. Remodifying that statement is advised. Response: This has been remodified. 2. The materials and methods are okay within the scope of the work. 3. The results and discussion does not provide a relationship and impact of these PAHs components to cause health based impact. More discussion is needed in relation to previous works available. Response: ILCR and HQ are the indexes used to show the contamination of PAHs 4. The conclusion is not adequate, as there are no recommendation or summary of the study in tandem to adults to children influences and how these automobile workshops can work effectively and dispose waste oils. Response: This has been modified in the revised manuscript. Thanks." }, { "c_id": "10513", "date": "10 Nov 2023", "name": "Olusola Adesina", "role": "Author Response", "response": "The statement “Few or no studies are available on the level of PAHs around automobile repair workshops, especially in Sub-Saharan Africa countries such as Nigeria” is not actually true because there are a lot of recent data on the subject matter such as Ekanem et al. (2019), Abugu et al. (2023), Daniel et al. (2023) etc. Response: This part has been deleted in the revised manuscript. The methodology is not explicit. In the soil sampling section, the apparatus used, sampling method and time of sampling is not mentioned. In the sample pretreatment section, quantity of solvent used for extraction is not mentioned. The references in the methods section are old. The author should review literature for the latest references. Response: Authors appreciate this comment apparatus used for sampling has now been included, quantity of solvent used has now been included in the revised manuscript. Authors want to state that standard method for extraction and analysis of PAHs were duly followed The authors have not been able to drive home their point so as to draw conclusions on the health implication of PAHs. After comparison with the WHO standards, the authors would have made a conclusion whether the soil has low, moderate or high contamination and make recommendations. Response: Authors have taken care of this in the revised manuscript." } ] } ]	1	https://f1000research.com/articles/12-861
https://f1000research.com/articles/12-300/v1	20 Mar 23	{ "type": "Research Article", "title": "Moringa oleifera fruit extract as a potential antioxidant against liver injury by 2-Nitropropane induction in obese male mice model: pre-clinical study", "authors": [ "Maria Selvester Thadeus", "Tiwuk Susantiningsih", "Hikmah Muktamiroh", "Cut Fauziah", "Mila Citrawati", "Agneta Irmarahayu", "Sri Wahyuningsih", "Yanti Harjono Hadiwiardjo", "Hany Yusmaini", "Meiskha Bahar", "Fajriati Zulfa", "Diana Agustini", "Aulia Chairani", "Tiwuk Susantiningsih", "Hikmah Muktamiroh", "Cut Fauziah", "Mila Citrawati", "Agneta Irmarahayu", "Sri Wahyuningsih", "Yanti Harjono Hadiwiardjo", "Hany Yusmaini", "Meiskha Bahar", "Fajriati Zulfa", "Diana Agustini", "Aulia Chairani" ], "abstract": "Background: Moringa oleifera fruit extract contains beneficial chemical compounds. This study was conducted to observed the power of antioxidant against liver injury by 2-Nitropropane induction in an obese male mice model. Methods: This research was in vivo laboratory experimental study with a post-test control design group only. The population was obese male mice models, Swiss strain, aged 6–8 weeks, weighing between 60–80 gr. The research sample was determined by Federer's formula for a complete randomized design experimental test, group N (control), O1 (induced by 2-Nitropropane intraperitoneal (i.p) once), O2 (induced by 2-Nitropropane i.p twice), P1 (induced by 2-Nitropropane i.p. once and gavage with M. oleifera fruit extract 500mg/kg bodyweight (BW) once a day), P2 (induced by 2-Nitropropane i.p. twice and gavage of M. oleifera fruit extract 500mg/kg BW once a day), and P3 (induced by 2-Nitropropane i.p. twice and gavage of vitamin C 500mg/kg BW once a day). Antioxidant potential parameters were measured by levels of malondialdehide (MDA), glutation (GSH), 8-hydroxy-2'-deoxyguanosine (8-OHdG), catalase activity, manganese superoxide dismutase (MnSOD), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT). This research was held at the Biochemistry laboratory of Medicine Faculty, UPN Veteran Jakarta in May–September 2020. Analysis was carried out using SPSS version 20.0. The parameters were tested using ANOVA. Results: MDA levels decreased, GSH increased, 8-OHdG decreased, catalase activity increased, MnSOD activity increased and SGOT, SGPT levels decreased. M. oleifera fruit extract was statistically proven to be a candidate for potential antioxidant against liver injury of 2-Nitropropane induction in obese male mice model. Conclusions: M. oleifera fruit extract was statistically evident as an antioxidant substance that reduces oxidative stress in acute liver injury caused by 2-Nitropropane induction.", "keywords": [ "2-Nitropropane induction", "liver injury", "Moringa oleifera fruit extract", "obese male mice model", "potential antioxidant" ], "content": "Introduction\n\nIndonesia has abundant natural wealth, spread from the west to the east.1 One of the natural resources is plants as a source of traditional medicine. Moringa oleifera fruit is the Moringa plant's fruit, and contains beneficial chemical compounds.2 The vitamin content in M. oleifera includes vitamin C, vitamin A, vitamin B1 and B2. The antioxidant content includes flavonoids, quercetin, isothiocyanates and glucosinolates.3,4 In addition, Moringa fruit also has anti-sensitivity, anti-inflammatory, and anti-rheumatic, anti-hypertensive, anti-diabetic, anti-cancer properties.3–5\n\nM. oleifera has antimicrobial, antibacterial, antifungal and anthelmintic properties.5–9\n\nAntioxidants are chemical ingredients that can donate one or more electrons to free radicals so that free radicals can be mutated.7,8 There are two antioxidants: natural antioxidants and synthetic antioxidants. Synthetic antioxidants are industrial products.10–12\n\nNatural antioxidants can protect the body against damage caused by reactive oxygen species that can inhibit the occurrence of degenerative diseases and can inhibit lipid peroxidase in the body.13 Vitamin C as an antioxidant exogen can be used to reduce oxidative stress. It can be an effective antioxidant in inhibiting free radicals with a suitable dose and its chemically capable of reacting with most radicals free and oxidants in the body.14,15 In 2016, obesity was estimated to exceed 250 million people worldwide.16 Obesity was the third leading cause of cancer after tobacco and alcohol.15 Experts estimate that obesity causes about 20% of all cancer cases.14,15,17\n\nEpidemiological studies show that obesity was associated with an increased risk of cancer.16,18 One of the cancers thought to occur due to obesity was hepatocellular carcinoma.13,17,19 Obesity causes dysregulation of growth signals such as increased insulin and leptin.20–22 Obesity causes angiogenesis, tumorigenesis and chronic inflammation.15,23 Experts suspect obesity is also associated with the incidence of hepatocellular cancer.15,24,25 The chemical 2-Nitropropane can induce hepatocellular.26 2-Nitropropane (2NP) can trigger the mechanism of hepatocarcinogenesis.17,24,27\n\nResearch by Metwally et al. (2017) in obese female rats given M. oleifera at a dose of 600 mg/kgBW orally for 12 weeks is a good candidate for therapy for symptoms of metabolic syndrome.8\n\nThere have been many studies reporting on M. oleifera fruit. However, only a few studies have reported on the potential of M. oleifera fruit extract as a potent antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in obese male mice models. Can M. oleifera fruit extract treat liver injury by 2-Nitropropane induction in an obese male mice model? This research observed the compound antioxidant of M. oleifera fruits extract against liver injury by 2-Nitropropane induction in an obese male mice model. M oleifera fruits extract can treat the liver injury by 2-Nitropropane induction in an obese male mice model.3,5\n\n\nMethods\n\nThis study protocol was reviewed and approved by The Health Research Ethics Committee (HREC) Pembangunan Nasional Veteran University Jakarta, approval number B/1822/4/2019/KEPK on April 23th 2019. This study was held May–September 2020, and all authors provide a statement indicating that all efforts were made to ameliorate harm to animals. Researchers agreed to use the minimum number of experimental animals using the Ferderer formula. The experimental animals were treated according to the 5 F principle. The experimental animals were terminated with ketamine xylazine anesthesia, then buried.\n\nThe research design was an experimental laboratory study with a post-test control group only design. The indicators seen on antioxidant potential were by seeing malondialdehyde (MDA), glutathione (GSH), 8-Hydroxy-2′-deoxyguanosine (8-OhdG), catalase activity, manganese superoxide dismutase (MnSOD), serum glutamic pyruvic transaminase (SGPT), and serum glutamic oxaloacetic transaminase (SGOT). The research location was in the Biochemistry Laboratory Faculty of Medicine Universitas Pembangunan Nasional Veteran Jakarta. The population was male obese mice models Swiss strain aged 6–8 weeks weighing between 60–80gr obtained from the Animal Laboratory of University Institut Pertanian Bogor, Bogor West Java. Determination of the research sample using the Federer formula for a completely randomized design experimental test.\n\nInclusion criteria: mice in healthy condition (active and no anatomical abnormalities), weight 60–80 gr, age 6–8 weeks, line: Swiss, gender: male. Exclusion criteria: sick and die during the process of adaptation and treatment.\n\nMaterials: Cages, food and drink container, scales, analytical scales, logbook and stationery, S-30 spectrophotometer ThermoFisher®, homogenizer Tokyo Rikaikai®, micropipette, minor surgical instruments, one cc syringe, three cc syringe, vacutainer, micro tip, microtubes vortex, microtube rack, and cuvettes. Materials: high fat high protein feed, 2-Nitropropane (2NP) liquid From Aldrich®, MDA Assay kit Elabscience®, GSH Assay kit Elabscience®, 8-OHdG Assay kit Elabscience®, Catalase activity Assay kit Elabscience®, MnSOD kit RanSOD®, SGPT Assay kit Elabscience®, SGOT Assay kit Elabscience®, ice, Ketamine-xylazine liquid AgrovetMarket®.\n\nMice were obtained from the Animal Laboratory of University IPB Bogor, the health of the mice was monitored every day, and their body weight was weighed before conducting the study. After going through a laboratory adaptation process for seven days, obesity was induced in mice aged 4–5 weeks by feeding them high-fat diets for 4 weeks until the average body weight of obese mice was 60–80g. Then the mice were placed in a plastic cage with a lid made of ram wire and covered with husk, fed with high-fat feed pellets and drinking water was given ad libitum. The cage environment was made so that it was not humid, with adequate ventilation and adequate lighting where the light time is 14 hours and the dark was 10 hours.\n\nThe sample consisted of 36 Swiss obese male mice models divided into six groups. Each group consisted of six mice: group N (obese mice control), O1 (obese mice induced of 2-Nitropropane 20 mg/kgBW intraperitoneal (i.p.) once), O2 (obese mice induced of 2-Nitropropane 20mg/kgBW i.p. twice), P1 (obese mice induced of 2-Nitropropane 20 mg/kgBW i.p. once and gavage with Moringa oleifera fruit extract 500 mg/kgBW once a day), P2 (obese mice induced of 2-Nitropropane 20 mg/kgBW i.p. twice and gavage of Moringa oleifera fruit extract 500 mg/kgBW once a day), and P3 (obese mice induced of 2-Nitropropane 20 mg/kgBW i.p. twice and gavage of vitamin C 500 mg/kgBW once a day). After induction, the mice were treated with betadine.\n\nThis study using obesity models of mice; a possible thing that can happen is the sudden death of mice which causes the need to use new mice so the number of mice increases. Each mouse that has undergone surgery and liver removal will be buried. The mice were well cared for, and fed according to the standard. After completing the research, the rats were necropsied and buried. During the study there were no sick mice or mice that had adverse effects, so no mice were excluded.\n\nAfter 10 weeks, the mice were fasted, then necroption under anesthesia: xylazine 0.1 mg/kgBW + ketamine 0.03 mg/kgBW by i.p., then euthanized. Prior to necropsy, the rats were anesthetized using ketamine xylazine until completely asleep and then dissected. The blood samples were taken from the heart’s left ventricle with a 3cc syringe by cardiac puncture. The blood was placed in a vacutainer, and the serum was stored at -80°C. The liver was taken and weighed then examined for biochemical markers. The dissected mice will be put into a special animal bag, then buried properly. The liver tissue will be measured for MDA, GSH, 8-OHdG, Catalase activity, MnSOD, SGPT, and SGOT levels.\n\nThe dose of 2-Nitropropane (130263-5ml Aldrich®) of 0.02 mg/kgBW was given intraperitoneally to induce oxidative stress and liver injury in male obese mice models.27\n\nThe research used M. oleifera fruit extract from Lampung Province was a concentrated preparation obtained by removing the active ingredients of M. oleifera fruits. A total of 500 g of dried simplicia M. oleifera fruits with 70% ethanol solvent maceration got 55.2 g of M. oleifera fruits to extract.\n\nA homogenate was made by putting 100 mg of rat liver tissue into a 1.5 ml Eppendorf tube, then adding 1 ml of phosphate buffered saline (PBS) (0.01 M pH 7.4). This was crushed using a homogenizer for 90 seconds (maximum time) at a speed of 3500 rpm (the crushed samples were placed in the ice floes until they are all crushed) using Tokyo Rikaikai homogenizer. Next, the Eppendorf tube was inserted and centrifuged for 10 minutes at 3500 rpm. To obtain the supernatant from liver tissue, the supernatant was taken and transferred to a new Eppendorf tube. After that, the Eppendorf tube was put in the refrigerator with a minimum temperature of -20°C overnight.\n\nExamination of liver MDA levels using the Will method28 from Assay kit Elabscience® at the Biochemistry Laboratory, Faculty of Medicine, University of Indonesia. A total 10 μL MDA Color Reagent solution was added into each well of MDA Standard, Blank Control, and Test Samples. The reaction mixture was incubated at room temperature for 10–30 minutes and 40 μL of Reaction Solution was added to make the total assay volume to 100 μL/well. The final reaction mixture was incubated at room temperature for 30–60 minutes. The end-point absorbance was measured with an absorbance plate reader with path-check correction at optical density (OD) 695–700 nm. Absorption readings were taken using a spectrophotometer (Thermo scientific® ab233471 Lipid Peroxidation (MDA) Assay Kit (Colorimetric)). Furthermore, to get the concentration results is to plot the absorbance data of the sample into a standard curve.23\n\nHomogenate supernatant as much as 40 μL entered a 1.5 ml tube, then 210 μL of 5% trichloroacetic acid was added and mixed using a 3–5 second vortex. Then the solution was centrifuged at 5000 rpm for 10 minutes, then take the supernatant and transferred to a 2 ml Eppendorf tube. Then 800 μL of PBS pH 8.0 was added and mixed by vortex until homogenous. After that, 25 μL 5,5′-Dithiobis 2-nitrobenzoic acid (DTNB) reagent was added so that the yellow-colored compound benzoate would be formed due to the binding of the hydryl sulfur (SH) group by DTNB. This was then incubated at room temperature for 1 hour in a dark room, then the absorbance was read using a spectrophotometer (Thermo scientific®) at the wavelength (λ) = 412 nm. All samples were measured by using the Duplo principle (measurement twice); to avoid errors in calculations.23,28\n\nWe took 10 mg of homogenate dissolved in 0.1M phosphate buffer pH 7.5 was homogenized and then centrifuged at 1000 × g for 10 minutes. The supernatant was purified using a Deoxyribonucleic acid (DNA) (Quick-DNATM MiniPrep Plus) extraction kit. The DNA was digested using nuclease P1 following the manufacturer's instructions. The pH was adjusted to 7.5–8.5 using 1 M Tris buffer. One unit per 100 g of Alkaline phosphatase DNA was added and incubated at 37°C for 30 minutes. This was heated up for 10 minutes and placed on ice until used.29–31\n\nMnSOD activity was determined biochemically using the RanSOD® kit. It determined the total SOD activity utilizing the degree of inhibition of this formazan color as measured by a 505 nm spectrophotometer A. The sample was diluted 20 times using PBS 0.01 M pH 7. 25 μL of standard/diluted sample was put into the cuvette, then 20 μL of mixed substrate mixture was added, mixed well, and the xanthine oxidase enzyme was added. The researcher read the light absorption with a spectrophotometer at a wavelength of 505 nm in the first 30 seconds after the enzyme was added (A1) and 3 minutes later (A2).32,33\n\nThe liver of obese mice were weighed with an analytical balance of 100 mg and then inserted into a tube. Then PBS was added to as much as 1 mL/100 mg of the liver.32,34\n\nThe liver and PBS tube were crushed using a homogenizer for 90 seconds. After thrashing, the tube was put into a centrifuge with 3500 rotations per minute for 10 minutes. The supernatant was then taken and moved it into a different tube. The tube was stored for one night at a temperature of -20oC. Catalase enzyme activity was expressed in U/mL. One unit means the amount of enzyme that catalyzes the reaction of 1 μM substrate per minute. Sample absorption value with formula:\n\nThe Eppendorf tube was taken from the refrigerator and 0.1 ml of the supernatant + 1 ml of the SGOT/SGPT reagent Tris was inserted into the cuvette and placed in the spectrophotometer (λ = 340 nm, temperature = 37°C) then mixed using a micropipette (the timer runs when the SGOT/SGPT reagent enters the first time). The start button on the spectrophotometer was clicked and the absorbance was read after 1 minute and 3 minutes.2\n\nAnalysis was carried out using SPSS version 20. The tests were started with tests of normality and homogeneity. Parametric tests followed data that were normally distributed and homogeneous. Parametric test with one-way analysis of variance (ANOVA) and if different significantly, was followed by Bonferroni's post hoc test for multiple comparisons.\n\n\nResults\n\nThe results of the phytochemical from Balai Penelitian Tanaman Rempah dan Obat (BALITRO) Bogor showed the content of extract substances are alkaloids, tannins, flavonoids, and glycosides. The extracts have quercetin content, saponin and triterpenoid substances. The statistical analysis was conducted using SPSS version 20. Parametric test with ANOVA and Bonferoni’s post hoc test for multiple comparation.\n\nFigure 1 shows that the M. oleifera fruits extract in groups P1 and P2 had an average MDA level of 1.729 nmol/mL and 1.559 nmol/mL, which was lower than group O1 and O2; the P3 group given vitamin C has an average MDA level of 1.766 nmol/mL, which was increased compared to the control group N and group O1, with an average MDA level of 1.541 nmol/mL and 1.825 nmol/mL. The GSH level of group O1 was decreased to 0.654 nmol/mL compared to the N group, O2 group. The group that was given M. oleifera fruits extract had an average GSH level of 0.765 nmol/mL, compared to group P1 and the group P2 of 0.865 nmol/mL. The GSH level of the P1 and P2 groups decreased compared to the O2 group. These data on the MDA level have been analyzed further using Post Hoc Test.\n\nM. oleifera fruit extract as a candidate for potential antioxidant against for liver injury induced 2-Nitropropane in male obese mice models. The orange bars show MDA levels, the yellow bars show GSH levels.\n\nThe average liver 8-OHdG levels in groups O1 and O2 was increased (12.763 ng/mL and 15.249 ng/mL) compared to group N 7.832 ng/mL (Figure 2). In group P1, P2 and P3 the average liver 8-OHdG was decreasing to 10.233 ng/mL, 8.012 ng/mL and 8.534 ng/mL. The catalase enzyme activity in the male obese mice model in group N shows 1.98 u/mL higher than all O1, O2, P1 P2 and P3 groups. In the O1 and O2 groups, there was a decrease in catalase activity of 1.782 u/mL and 1.436 u/mL compared to the other groups. The group given M. oleifera fruits extract had an average catalase activity enzyme level of 1.762 u/mL and 1.824 u/mL. The MnSOD activity level in group O1 was decreased compared to group N, and then more decreased in group O2, as seen on Figure 2, than other groups. The MnSOD activity enzyme level increased in the P1 (0.876 u/mL) and P2 (0.882 u/mL) groups which were given M. Oleifera fruits extract. The MnSOD activity enzyme level also increased in the P3 group (0.681 u/mL) which was given Vitamin C.\n\nSGOT and SGPT levels in the liver of each group showed a decrease in SGOT and SGPT levels in P1, P2 and P3 compared to O1 and O2 (Figure 3). This figure showed a significant decrease in SGOT and SGPT levels in groups that were given M. oleifera fruits extract than groups N.\n\nM. oleifera fruit extract as a candidate for potential antioxidant against for liver injury induced 2-Nitropropane in male obese mice models.\n\n\nDiscussion\n\nMoringa fruit obtained from the Moringa plant is a fruit with many benefits because it contains beneficial chemical compounds. The nutritional content in M. oleifera fruit includes vitamin C, vitamin A, vitamin B1 and vitamin B2, including flavonoids, quercetin, isothiocyanates and glucosinolates.6–8 M. oleifera fruits have potential as antioxidants and prevent the spread of cancer cells, and can reduce cholesterol levels, carbohydrates, calcium, phosphorus, magnesium and potassium.1,9–11 M. oleifera fruit extracts are effective as an antimicrobial, anti-sensitivity, anti-inflammatory, antirheumatic, anthelmintic, anti-cancer, antihypertensive, antibacterial, antifungal, antidiabetic, and antioxidant.2–4\n\nM. oleifera fruits extract contains flavonoids as antioxidants.2,35 The antioxidant activity of flavonoids comes from their ability to donate hydrogen atoms and the ability to chelate metal.18 M. oleifera fruits extract was also anti-obesity because it can down-regulate leptin and resistin mRNA expression while at the same time up-regulating the expression of adiponectin mRNA.8 Vitamin C as an antioxidant agent given as a positive control in group P3 can be compared with M. oleifera fruits extract. The results of the research show that M. oleifera fruits extract have a significant difference as a candidate for antioxidant properties for decreasing oxidative level of induction of 2-Nitropropane in male obese mice models.\n\nThe results of this study showed that there was a decrease in MDA levels in P1 and P2 groups that were given M. oleifera fruits extract. The level of GSH increased in the groups O1 and O2. So, it can be concluded that M. oleifera fruits extract can inhibit oxidative stress due to the induction of 2-Nitropropane. As a positive control in group P3, vitamin C also does the same as an antioxidant exogen. So, this study proved that M. oleifera fruits extract has the ability as an exogenous antioxidant.\n\nThe level of 8-OHdG also decreased when groups P1 and P2 were given M. oleifera fruits extract compared to groups O1 and O2. So, we can see that the administration of M. oleifera fruits extract can repair damaged cells caused by 2-Nitropropane induction. Likewise, MnSOD and catalase activity increased when groups P1, P2 and P3 were given M. oleifera fruits extract. These data indicate an increase in the activity of MnSOD36 and catalase enzymes assisted by the presence of antioxidant candidates and expenses from the M. oleifera fruit extract.6,32,33\n\nInduction of 2-Nitropropane can cause liver cell damage and acute liver injury, which was characterized by increased levels of SGOT and SGPT.18,27 When given M. oleifera fruits extract, there was a decrease in liver SGOT and SGPT levels in groups P1 and P2 compared to groups O1 and O2. These findings indicate that M. oleifera extract can be a potential antioxidant candidate in liver damage due to 2-Nitropropane induction.18\n\nThis study was in line with Nafi,37 Bilqis,19 Anis,18 Fiqih,21 Rahmi38 and Susantiningsih's38 research that M. oleifera fruit extract has high antioxidant levels and deserves to be called a potential antioxidant candidate in 2-Nitropropane-induced obese male mice through the parameters. The findings of increased levels of MDA, SGOT and SGPT in liver tissue indicate that 2-Nitropropane induction causes acute liver injury. This research answers the objectives, to observe the contains of antioxidants M. oleifera fruits extract against liver injury by 2-Nitropropane induction in an obese male mice model.\n\n\nConclusion\n\nM. oleifera fruit extract was statistically proven to be used as a candidate for potential antioxidant against liver injury of induction 2-Nitropropane in male obese mice model by looking at the levels of MDA, GSH, etc. 8-OHdG, Catalase activity, MnSOD activity and SGOT and SGPT levels. Antioxidant substances in M. oleifera fruit extract can significantly reduce oxidative stress in acute liver injury caused by 2-Nitropropane induction. The limitation of this research is the induction of 2-Nitropropane which requires special skills. Further clinical research on healthy human subjects is needed.", "appendix": "Data availability\n\nFigshare: Moringa oleifera Fruit Extract As a Potential Antioxidant Against Liver Injury By 2-Nitropropane Induction In Obese Male Mice Model: Pre-Clinical Study. https://doi.org/10.6084/m9.figshare.19655757.v2. 39\n\nThis project contains the following underlying data:\n\n• Data Moringa oleifera fruit.xlsx. (Moringa oleifera fruit as a candidate for potential antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in male obese mice models.)\n\n• Raws Data DokMar.xlsx (The data of MDA and GSH levels, 8-OHdG, MnSOD levels, SGPT and SGOT level).\n\n• Raw Data yang diminta Reviewer oleh MST TS.xlsx (Raw data for Moringa oleifera fruit as a candidate for potential antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in male obese mice models.)\n\nFigshare: ARRIVE checklist for ‘Moringa oleifera fruit extract as a potential antioxidant against liver injury by 2-Nitropropane induction in obese male mice model: pre-clinical study’ https://doi.org/10.6084/m9.figshare.19655757.v2. 39\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nThe authors would like to express their gratitude to LPPM Universitas Pembangunan Nasional “Veteran” Jakarta for supporting the research.\n\n\nReferences\n\nRiastiwi I, Damayanto IPGP, Ridwan R, et al.: Moringa oleifera distribution in java and lesser Sunda islands which are attributed with annual rainfall. 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PubMed Abstract \| Publisher Full Text\n\nIida T, Ito Y, Kanazashi M, et al.: Effects of psychological and physical stress on oxidative stress, serotonin, and fatigue in young females induced by objective structured clinical examination: Pilot Study of u-8-OHdG, u-5HT, and s-HHV-6. Int. J. Tryptophan Res. 2021; 14: 117864692110484. Publisher Full Text\n\nKataoka T, Shuto H, Naoe S, et al.: Radon inhalation decreases DNA damage induced by oxidative stress in mouse organs via the activation of antioxidative functions. J. Radiat. Res. 2021; 62(5): 861–867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIghodaro OM, Akinloye OA: First line defense antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defense grid. Alexandria J. Med. 2019; 54 (4): 287–293.\n\nHardiany NS, Sadikin M, Siregar N, et al.: The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells. Med. J. Indones. 2017; 26(1): 19–25. Publisher Full Text\n\nGlorieux C, Calderon PB: Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach. Biol. Chem. 2017; 398(10): 1095–1108. PubMed Abstract \| Publisher Full Text\n\nYati SJS, Candra IN: Potensi aktivitas antioksidan metabolit sekunder dari bakteri endofit pada daun Moringa oleifera L. ALOTROP, Jurnal Pendidikan dan Ilmu Kimia. 2018; 2: 82–87.\n\nMiar A, Hevia D, Munoz-Cimadevilla H, et al.: Manganese superoxide dismutase (SOD2/MnSOD)/catalase and SOD2/GPx1 ratios as biomarkers for tumor progression and metastasis in prostate, colon, and lung cancer. Free Radic. Biol. Med. 2015; 85: 45–55. PubMed Abstract \| Publisher Full Text\n\nNafi MST: Moringa oleifera Leaf Extract and Infusion Effect on GSH Levels in Male Mice (Mus musculus) Liver With Obesity Induced by 2-Nitropropane. Proceeding International ICHD.2019.\n\nRahmi MAST: Chemopreventif Effect of Extract and Infusion of Moringa oleifera Leaves on Manganese Superoxide Dismutase (MnSOD) Liver Level Induced by 2-Nitropropane in Mice (Mus musculus) with Obesity Proceeding International ICHD.2019.\n\nThadeus MS, Susantiningsih T, Hadiwiardjo YH: Moringa oleifera Fruit Extract As a Potential Antioxidant Against Liver Injury By 2-Nitropropane Induction In Obese Male Mice Model: Pre-Clinical Study. Dataset. figshare. 2022. Publisher Full Text" }	[ { "id": "204632", "date": "26 Sep 2023", "name": "Terisha Ghazi", "expertise": [ "Reviewer Expertise Biochemistry and molecular biology", "including oxidative stress", "DNA damage", "and epigenetics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Thadeus et al. investigated the antioxidant potential of Moringa oleifera fruit extract against 2-nitropropane-induced liver injury in obese male mice. The findings are interesting; however, there are some flaws that should be addressed:\nIn the methods section, the value of p that was considered statistically significant needs to be mentioned.\n\nThe authors mention throughout the manuscript that their findings were statistically significant but, in the results, there is no indication of the level of significance. The p values need to be included for each result obtained and the level of significance needs to be denoted by the use of \", , or **\" on the graphs.\n\nOnce the appropriate statistical significance is shown, the correct conclusions can be drawn. Statistical significance is required to verify the current conclusions made by the authors.\n\nThere are also many spelling and grammatical errors. The authors should have the manuscript proofread by an English language expert.\n\nMice and rats are not the same. Yet, it is used interchangeably in the manuscript. This needs to be corrected.\n\nIn the discussion, the authors merely repeat their results. The results need to be discussed in the context of other previously published literature. Are the findings in this study similar or different from other published 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? 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? Partly", "responses": [ { "c_id": "10305", "date": "06 Oct 2023", "name": "Maria Selvester Thadeus", "role": "Author Response", "response": "We greatly appreciate your constructive criticism.We hope that every bit of your suggestions will contribute to helping us better our future articles. Many thanks so much." }, { "c_id": "10352", "date": "06 Oct 2023", "name": "Maria Selvester Thadeus", "role": "Author Response", "response": "Dear Reviewer We want to take this opportunity to let you know our sincere appreciation for your feedback. Suggestions and criticism are very constructive for our upcoming research and articles. Thank you very much. Best Regards Maria Selvester Thadeus" } ] }, { "id": "204636", "date": "02 Oct 2023", "name": "Wahyunia Likhayati Septiana", "expertise": [ "Reviewer Expertise Stem cells", "Liver organoid", "scaffold." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe researchers mentioned using obese mice with a body weight of 40-60 grams. What data indicates that these mice were obese? Certain indicators are frequently used to assess the level of obesity and the impact of anti-obesity drugs. These variables include blood pressure, body mass index, insulin tolerance test, total body fat, adiposity index, Lee index, fat pad mass, and magnetic resonance imaging and dual-energy X-ray absorptiometry.\n\nThis study employed 2-Nitropropane to induce oxidative stress and liver injury in male obese mouse models. The study involved two groups, O1 and O2, which were given 2-Nitropropane once and twice. It is advisable to include the dosing schedule because it is not clear how much time passed between the two doses and how long after 2-Nitropropane and M. oleifera extract were administered.\n\n2-Nitropropane can induce liver injury. Is there data indicating that this induction was successful? Can the authors provide a histopathological description of the liver?\n\nThe results of this study, both from the graphs and the result descriptions, do not show which data is significantly different between groups. No p-values are mentioned. How can the authors claim that there are significant results?\n\nWhat is the basis for administering a dose of 500mg/kgBW? Have there been any previous studies on this? The method section does not cite any previous research.\n\nThe GSH level decreased more in group P2 compared to O2. However, in group P1 with O1, this decrease was not as pronounced. Why did this happen? The same pattern also occurred in the levels of MDA, SGOT, SGPT, and 8oHDG. Please explain this in the discussion. This raises the question of whether M. oleifera might be more effective when given in cases of more severe liver injury.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "209938", "date": "18 Oct 2023", "name": "Taghred Saber", "expertise": [ "Reviewer Expertise Neurotoxicity", "genotoxicity", "Hepatotoxicity", "Nephrotoxicity", "Cardiotoxicity", "Testicular toxicity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 that this manuscript is totally relevant for the field and brings interesting results that deserve consideration. However, certain points need to be revised before the manuscript could be considered for indexing.\nThe laboratory conditions of the animal house should be provided.\n\nThe solvent of moringa oleifera extract should be provided.\n\nThe level of statistical significance (P value) should be mentioned under the title of Statistical Analysis.\n\nStatistical comparison should be provided in the results and foot note of the figures.\n\nThe authors should mention the result values as mean ± standard error.\n\nFoot note of all figures should include the value of P.\n\nFigure legends are short and incomprehensible. It is necessary to expand and rewritten the legend for these 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-300
https://f1000research.com/articles/12-1519/v1	28 Nov 23	{ "type": "Systematic Review", "title": "Research on emotional intelligence among Indian teachers: A Systematic Review and meta-analysis of its correlation with health parameters and impact of gender", "authors": [ "Mamta Pandey", "Deepti Sharma", "Deepti Sharma" ], "abstract": "Background Emotional intelligence is the self-perception related to identification and regulation of emotions. Several studies have been done among Indian teachers evaluating emotional intelligence in relation to demographic, professional and various psychological parameters, but the variety of scales, teacher types, and conflicting results makes it difficult to draw any meaningful conclusions from this heterogeneous data. The present work aims to synthesize the available data by both qualitative and quantitative analysis and is the first such attempt to include only Indian studies in this field. The main objectives were to determine the correlation of emotional intelligence with teachers’ health parameters and to study the gender difference in emotional intelligence.\n\nMethods After a thorough literature search in Google, Google scholar, Scopus, Web of science and Pubmed, fifty-five Indian studies were selected which empirically examined teachers’ emotional intelligence, either alone or in association with another parameter evaluating teachers’ psychological health and performance. After qualitative assessment of major findings, quantitative analysis was performed. Three separate meta-analysis were carried out. The first one with fifteen effect sizes among 3291 participants evaluated correlation with personal health parameters. The second with nineteen effect sizes in 4165 participants evaluated correlation with professional health parameters. The third with twenty-six studies involving 6005 participants assessed effect of gender.\n\nResults The results show that almost all studies have used a trait measure, teachers’ emotional intelligence is positively correlated with both personal and professional health parameters and gender has no effect on emotional intelligence.\n\nConclusion Major limitations are a very high degree of heterogeneity of the data, incomplete description of the scales, inadequate randomization and small sample sizes in many studies. The results indicate the importance of emotional intelligence in both personal and professional life of teachers and no effect of gender preparing a solid base for future research.", "keywords": [ "Emotional intelligence", "teachers", "trait EI", "education system", "personal health", "professional health", "gender effect on EI" ], "content": "Introduction\n\nEmotional intelligence (EI) has gained wide popularity since its inception around three decades back, both among popular media and research community. The emotional aspect of human intelligence is such an attractive concept that practicing psychologists, policymakers, motivational speakers, or business managers, all are drawn towards it and have carried forward the concept to a wider audience and general population. While the actual term is relatively recent, most authorities agree that the concept is very old. If Goleman traces its early origins in the writings of Aristotle (Goleman, 1995), then Bar-On gives credit to Darwinian theory of evolution for inspiring his research (Bar-On, 2006). In modern times, Thorndike’s ‘social intelligence’ can be taken as the first scientific enquiry seriously looking into the realm of non-intellective factors (Thorndike, 1920), which were further elaborated by many later researchers. The landmark studies of Mayer and Salovey in 1990s (Salovey and Mayer, 1990; Mayer, Salovey and Caruso, 2004), the spectacular success of Goleman’s subsequent book and theoretical research of Bar-On established the solid scientific framework for this construct. The three basic models proposed by these three groups of scholars, along with definitions and measuring tools based on them have been extensively discussed in literature previously. The most important development since the early elaboration of three fundamental models is the distinction between ability EI and trait EI. This distinction is now standard in international scientific literature but less well recognized in Indian studies and needs emphasis. According to Petrides and Furnham (2000a), who highlighted this at the turn of the century, Ability EI tests measure constructs related to an individual’s theoretical understanding of emotions and emotional functioning, whereas Trait EI questionnaires measure typical behaviors in emotion-relevant situations and self-rated perception. The main differentiating feature of this classification is that EI type is best defined by method of measurement: Measures that are based on self-report items are termed “trait EI” whereas measures that are based on maximum performance items are termed “ability EI”. Later research has shown that trait EI is better correlated with psychological, health, organizational or behavioral parameters than ability EI (Petrides et al., 2016). The term “mixed” is now frequently used in the literature to refer to EI tools that measure a combination of traits, social skills and competencies and overlaps with other personality measures (O’Connor et al., 2019).\n\nA simple literature search of EI studies in India reveals that there is a plethora of tests developed by Indian scholars. This is both a boon and a bane for EI research in India. While it shows the huge interest shown by Indian scholars for this rapidly developing field as well as the depth of research aptitude and methodology, it also creates a situation where most of these tests are used by very few studies and thus lack the widespread validity and reproducibility required of a rigorous scientific tool. It is hardly surprising that none of these tools have gained wide recognition outside India, as it’s simply not possible to project any one of them as a standard Indian scale. A detailed analysis of these scales, including their strengths and weaknesses and uses in Indian studies is beyond the scope of this review, but a brief introduction of three of the commonly used tests is presented here. The first one is Emotional Competence Scale (ECS) developed by Sharma and Bhardwaj in 1993 with publication of its psychometric properties in 1994 (Bharadwaj and Sharma, 1994). It seems to be the first such scale in India but now not available online and its conceptual framework seems somewhat different from the later models of emotional intelligence. The two most common Indian scales are Emotional intelligence Scale (EIS) developed by Hyde, Pethe and Dhar (Hyde and Pethe, 2001) and Emotional Quotient test (EQ test) by Singh and Chaddha (Singh, 2006). Both were first published around the same time in 2001.\n\nThis is a self-rated test comprising 34 questions. In the manual describing the scale, the authors note that they had come across only two international scales, one by Cooper and Sawaf (1997) and another by Goleman (1995). A lot of questions in this scale pertain to the behaviour or perception of an individual working within an organization and the related issues of motivation, encouragement and decision making etc. Being a self-report measure, it would be classified as measure of trait EI, although like Schutte Self-report Emotional intelligence test or SSEIT (Schutte et al., 1998), it is primarily based on a definition of EI which is more in sync with the ability model. Being available in both English and Hindi language, it is not surprising that it received widespread acceptance and is one of the most commonly used tests by Indian scholars, both in education and other sectors as well. A brief comparison with the almost similar SSEIT shows that the later test is more generalized in its approach and suitable for different populations while the former is a suitable test mainly for working executives. While SSEIT is more focused on how one ‘feels’, EIS seems more focussed on how one ‘acts’ under certain emotional situations.\n\nSingh and Chadha developed two different tests which were published in the book by D Singh in 2001, named Emotional Intelligence at Work: A Professional Guide. As the name of the book itself suggests, these tests are also more suitable for working professionals, although the authors have tested the validity of the scales in different sample populations. The first scale, called Emotional Quotient test, which is also the more popular and used scale is based on maximum performance with right and wrong answers (ability measure) comprising 22 questions based on emotional situations. The second one, called Emotional Intelligence Scale, comprises 60 questions and is a self-report measure with responses in Likert type scale ranging from strong disagreement to strong agreement (trait measure). Being a lengthy and less rigorously tested measure, it is very rarely used by other scholars. The only relevant reference in the book is of Goleman’s book, and the writing style and practical examples give a clue to the obvious influence of that book, but there is no mention of any other international tests in the book.\n\nIt appears that both these groups had not come across the three other important and later on more widely used tests developed around the same time, the EQ-i by Bar-On in 1997, the SSEIT by Nicola Schutte and others in 1998 and the MEIS by Mayer, Caruso and Salovey in 1999, which was the precursor of MSCEIT (Mayer Salovey Caruso Emotional Intelligence Emotional Intelligence test) (Ackley, 2016). In this respect, the two Indian scales assume more importance as it was a big scholarly feat.\n\nStarting with the landmark meta-analysis by Van Rooy and Viswesvaran (2004), which not only examined the psychological bearing of EI and similarity and variance with cognitive intelligence and personality but also assessed its predictive role with various life outcomes, there have been several attempts by various scholars to present the ever-increasing data in a systematic way. Schutte et al. (2007) established that EI had a positive correlation with mental, psychosomatic and physical health and trait measures had better correlation than ability measures. Martins et al. (2010) carried forward this research and showed that EI is better correlated with mental and psychosomatic health than physical health and TEIQue showed strongest association among all trait measures. Joseph and Newman (2010) proposed a cascading model of EI and tried to integrate ability and mixed models. They also looked at gender difference in EI scores, which has been a subject of many studies with contrasting results (Van Rooy et al., 2006), and showed that while women fared better than men in ability measures, for self-report measures, there was no gender difference. In recent years, many meta-analyses have been published linking EI with students’ academic performance (Maccann et al., 2019; Sánchez-Álvarez et al., 2020) life satisfaction (Hartanto and Helmi, 2021), and adolescents’ subjective well- being (Llamas-Díaz et al., 2022). To the best of our knowledge, only two meta-analyses have been conducted by Indian researchers so far in EI domain. Shukla and Srivastava (2016) examined the association of EI with demographic and occupational parameters like job stress and satisfaction, while Manimozhi and Srinivasan (2018) assessed the correlation of EI with students’ academic achievement. Both of them included some Indian studies. There has been a lot of research among Indian teachers with respect to their EI and its relation with demographic, personal and professional attributes but no meta-analysis has been attempted to synthesize their results and present their findings in a coherent manner. This systematic review and meta-analysis is the first attempt in this direction with inclusion of only Indian studies in this field, and aims to fill an important research gap regarding the impact of teachers’ EI on their health and performance.\n\nA lot of studies and subsequent reviews and meta-analyses have examined the effect of EI on students’ personal and professional metrics, but no such work has been done regarding Indian teachers. Individual studies have reported conflicting results regarding the association of EI with factors like teacher effectiveness, job satisfaction or mental health and the role of age, professional qualification or gender. The inceasing body of research in this field now needs a systematic collation and synthesis of available data to gain insightful knowledge from this seemingly conflicting literature. The role of gender on EI is one such issue which needs to be explored in both large scale studies as well as systematic meta-analysis to reconcile the conceptual framework with solid evidence. Studies in other sectors, both in India and abroad have also reported similarly conflicting results regarding gender (Bar-On et al., 2000; Petrides and Furnham, 2000b; Extremera et al., 2006; Van Rooy et al., 2006; Brackett et al., 2006; Malouff and Bhullar, 2009; Fernández-Berrocal et al., 2012; Pooja and Kumar, 2016; Meshkat and Nejati, 2017). The impat of EI on teachers’ performance and health also needs emphasizing so that policy makers and school administrators become aware regarding the needs and interventions in this regard.\n\nStudy objectives\n\nThis review paper was written primarily with two objectives. First, to collect and collate all available Indian studies on emotional intelligence done among teachers and to analyze the data qualitatively and present a systematic synthesis of major findings. Second, where available and feasible, quantitative meta-analysis would be attempted to gain more insight into the research done in this field. PRISMA guidelines were followed for screening and inclusion of studies for systematic review as well as for presenting the major findings of meta-analysis. The PRISMA checklist for abstract and review (2020) for the present work is available as underlying data (Pandey, 2023), with details provided at the end of this paper. Similarly, SAGER guidelines were followed for reporting and analyzing all relevant data regarding gender. The main focus for quantitative analysis was on the impact of demographic and professional attributes on EI and teachers’ personal and professional parameters associated with EI. The main objectives are listed as follows:\n\n1. To collect, tabulate and analyze all available studies of EI done among Indian teachers in the last decade.\n\n2. To study the relationship of demographic and professional factors like age, gender, educational qualification and level of teaching on EI and perform meta-analysis of available studies.\n\n3. To study the relationship of EI with other parameters concerned with personal and professional health of teachers.\n\nHypotheses\n\nBased on the literature review and objectives of the present study, the following hypotheses were formed.\n\nH01. Emotional intelligence of teachers has no relation with personal and professional health parameters.\n\nH1 (alternative). Emotional intelligence of teachers is positively correlated with personal and professional health outcomes.\n\nIt is well established that EI is positively correlated with various professional outcomes like students’ academic performance and mental and psychological health. Organizational parameters like job satisfaction, performance, occupational stress, have also been associated with EI. Teacher’s EI can affect both their personal and professional life and can have far reaching consequences on the life of students as well. We propose that EI of teachers will have a significant correlation with parameters associated with both personal and professional attributes like teaching effectiveness and attitude, job satisfaction, stress, burnout, and mental health etc.\n\nH02. There is no effect of age, gender and educational qualification on teachers’ emotional intelligence.\n\nH2 (alternative). There is an effect of age, gender and educational qualification in teachers’ emotional intelligence.\n\nThe conceptual definition of trait EI places it near personality and quite distinct from cognitive intelligence. As such, it should not change with age, experience or educational qualification. Regarding gender, although women are thought to be better at handling and expressing emotions, the studies with different EI measures have reported contrasting results. As this is the first meta-analysis of Indian studies among teachers with different scales, we make the null hypothesis that gender should not affect EI.\n\n\nMethods\n\nThe following eligibility criteria and search strategies were used for screening and inclusion of studies.\n\nAll studies available in English language on Google, Google Scholar, PubMed, Scopus and WOS databases, done among Indian teachers published between 2009 and 2022 were screened for the review. The keywords used were “India, Indian, emotional intelligence, trait EI, ability EI, teachers, educators, primary/secondary/college teachers, professional institutes, education system and educational institutes”. Both the authors independently searched and screened available papers and met at regular intervals to exclude duplicate papers.\n\n\n\n(1) Original, empirical studies which measured EI of teachers, either alone or in association with other parameters.\n\n(2) The year 2009 was taken as a cut-off period to make data more relevant and current as initial screening revealed first studies from that year.\n\n\n\n(1) Studies which reported the following parameters and conducted relevant statistical analysis: age, gender, educational qualification.\n\n(2) Studies which conducted a correlation analysis between EI and a second parameter associated with psychological heath or occupation.\n\nAfter initial screening of papers, subsequent papers were searched in the references of those papers. All efforts were taken to collect as many studies as possible. Both the authors screened all abstracts and for those found suitable, full papers were retrieved. Duplicate papers were carefully excluded. All relevant papers were included for qualitative analysis and out of those, after further screening, only certain papers with relevant statistical analysis were included for quantitative meta-analysis. To reduce the chances of missing studies due to publication bias, all the references in a given paper were screened for any relevant studies and carefully assessed. Despite that, some of the studies might have been missed, and studies in print only journals with no full text available online were not included.\n\nStudies were coded for type of teachers used as sample population (trainee teachers, school teachers, and college teachers), type of EI scale used (ability, trait, mixed), demographics (age and gender), educational qualification, and teachers’ personal or professional attributes (second parameter in relation to EI). All such parameters were classified as personal or professional attributes depending upon whether they were primarily concerned with teachers as individuals (e.g. personality, stress, life satisfaction, spiritual intelligence) or professionals (e.g. occupational stress, job satisfaction, teacher efficiency and attitude etc.). This was done both for presenting the seemingly unrelated datasets from different studies in a coherent and relatable way and for the purpose of meta-analysis. Like previous work in this field, categorization of different parameters into broad categories helps in building proper evidence and can be used by future researchers to plan their studies in a more systematic way. Both the authors independently coded the studies and any difference was resolved after discussion.\n\nMeta-essentials, a set of free workbooks available online with Excel sheets was used for all statistical analysis (Suurmond et al., 2017). There are standard formats for different types of analysis in this software with readymade excel sheets which require only the correct input at various places and changes in settings as per the requirement of the meta-analysis. For correlation coefficients, it uses Fisher’s r to z transformation for calculation of combined effect size. This avoids the skewness of correlation coefficient and it can be assumed to be normally distributed so that tests of significance can be applied in a meaningful way. Data are presented as summary statistics, meta-analysis with combined effect size calculation, heterogeneity data, Forest plot for graphical representation of effect sizes, subgroup analysis where applicable and publication bias in the form of funnel plot and Egger’s regression. For subgroup analysis, due to the small number of studies, the ‘tau pooled over subgroup’ setting was used. This is done to avoid calculating an imprecise tau square within subgroups as is likely the case when the number of studies is small. For calculation of effect size in meta-analysis of gender effect on EI, the software automatically calculates Hedges’ g for effect size calculation which accounts for the small sample bias which is seen with Cohen’s d. A random effect model was used for calculation of effect size. Although all the studies have been done in India and all use teachers as subjects, there are still differences in the level of educational institutes (from primary level to college/university/professional level), type of institute (government versus private sector), demographics, educational qualifications, sample size and method of sampling (randomized or convenient). Moreover, the various tests used to measure EI and other parameters add to the heterogeneity of the data. Therefore, fixed effect model was not used.\n\nPublication bias analysis\n\nBias due to non-publication of studies with either negative or insignificant results makes the conclusion of any systematic review and meta-analysis less robust. Several methods to study publication bias are available which constitute an essential part of all systematic reviews. We have evaluated this bias with the help of two most used tools for this purpose. The first one is a funnel plot analysis of all studies used for meta-analysis, which simply plots effect size against either sample size or standard error. The presumption being that if bias is present, the plot is skewed towards right and shows a linear, rather than a funnel shape. The second tool is Egger regression analysis with calculation of regression intercept (zero in case of no bias) with p values.\n\nSensitivity analysis\n\nSensitivity analysis was intended for two possible scenarios to look for the robustness of the results. The first one with removal of studies with very large effect sizes as these may skew the results and are often a common cause of selection bias. The second sensitivity analysis would be carried out with removal of studies with smaller sample sizes. The purpose of carrying out sensitivity analysis is to see whether altering the methodology or study characteristics changes the overall result of the meta-analysis.\n\nAlthough no formal tool would be used for critical appraisal or of risk of bias assessment and certainty of evidence as all the studies intended for review are supposed to be observational in design with no intervention, a subjective assessment of methodology would be done to evaluate whether the studies used randomization and how.\n\n\nResults\n\nA total of seventy-four studies were identified through the initial literature search. For four studies, only abstracts were available, two were review articles and six papers were conceptual. Two papers had emotional intelligence in the title but it was not the exact parameter studied empirically through a specific tool. Five studies were excluded due to either a very small sample size, or incomplete details about methodology, measuring scales, statistical analysis or final results. The remaining fifty-five studies were included for qualitative analysis. For the purpose of meta-analysis, studies were further grouped on the basis of adequate data regarding impact of age, gender and educational qualification on EI and correlation with another parameter. Ten studies had adequate data for studying correlation of EI with personal health parameters, which were included in first meta-analysis. Eighteen studies had adequate data for studying correlation of EI with professional health parameters, which were included for the second meta-analysis. Thirty-three studies evaluated gender difference in EI, out of which seven did not have an adequate data set. The remaining twenty-six studies were included in the third meta-analysis. Only six studies examined the relationship of EI with age with only four having adequate data. Similarly, there were only seven studies which evaluated the impact of educational qualification on EI and out of them, only five studies had adequate data for analysis. Therefore, no meta-analysis was conducted for age and educational qualification. Figure 1 depicts the process of inclusion and Table 1 presents the summary characteristics of all the studies.\n\nStudies with results in italics could not be included in meta-analysis due to inadequate data.\n\nAbbreviations: ATW – attitude towards work, B – burnout, Edu – educational qualification, EE – employee engagement, F – female, FIW – family interference with work, GI – general intelligence, JS – job satisfaction, LS – life satisfaction, (M)A – maladjustment, MF – meaningfulness, M – male, MH – mental health, NS – non-significant, OS – occupational stress, P – personality, Per – Perseverance, Pers – personal, Prof – professional, PD – professional development, PW – Psychological well-being, S – significant, SA – self-actualization, SE – self-efficacy, SoI – social intelligence, SpI – spiritual intelligence, SR – self-reliance, SSEIT – Schutte Self-report Emotional Intelligence Test, Te – teaching efficiency, TE – teacher effectiveness, WIF – work interference with family, WS – workplace spirituality.\n\nFrom primary level to professional institutes, different studies have used educators from different level of teaching. Maximum studies (12) have been done on secondary school teachers. Although B.Ed. students are technically not teachers, a lot of studies (10) have been done among them considering them as trainee teachers. Notably, only 5 studies have taken mixed participants from two or more type of institutes. Among government and private sector, 17 studies have taken participants from both sectors while 6 studies each took participants from only one sector. The remaining 30 have not specified this.\n\nTests of emotional intelligence (Can change to same font as prev heading)\n\nThere is a lot of heterogeneity in the measurement tool used to assess emotional intelligence. The most commonly used scale is the indigenous scale developed by Hyde, Pethe and Dhar (14 studies with actual mean scores reported in 11) followed by the Teacher’s emotional intelligence inventory developed by Mangal and Mangal (7 studies). Only 12 studies have used international scales with 3 using Wong and Law scale (WLEIS, Law, Wong and Song, 2004)), 3 using SSEIT, while 1 each using Trait EI questionnaire-Short form (TEIQue-SF), Bar-On’s Emotional quotient test, Boyatzis and Goleman’s modified EI test, Clyde Winter EI test, Weisinger Emotional Intelligence Inventory and Leadership toolkit EI scale, although the exact version of Bar-On and Goleman scales have not been specified and some of the papers have not listed the exact reference for these tools.\n\nAmong the studies utilizing the scale by Hyde et al., the majority of the participants had a moderate to high EI score with a range of 113.53-150.39. Compared to the mean scores given by the developers of the scale (68, Hyde and Pethe, 2001), the scores in these studies are much higher, almost double in all studies. This can be interpreted in two ways. First, the EI of teachers, as measured by this scale, is very high as compared to general population. Or, given the much larger total sample (the developers used 200 subjects), the mean EI scores found in these studies should be considered “normal” and may be set as new reference point, as the developers have also suggested. That the teachers, as a profession, possess high EI has been seen in other studies, but more population-based studies using the same scale in different professionals can truly prove this point convincingly.\n\nOne of the biggest limitations in the majority of the studies is a lack of description of sample collection. Only a handful of studies have used randomized sampling and the rest have either used a purposive convenient sampling or not described the sampling method. Those which have used randomized sampling have not described in detail how the randomization was done.\n\nThirty-six studies evaluated another parameter along with EI (in some cases more than one parameter), either in relation to EI or independently. Twelve were categorized as concerning personal health, twenty-four were categorized as concerning professional health and four studies had both. The most common parameter studied is teacher effectiveness (8 studies), closely followed by job satisfaction (7). The other common parameters studied in relation to EI are occupational stress (4) personality (3), self-actualization (2), burnout (2), mental health (2), and general intelligence (2), etc. Most of these parameters had a significant correlation with EI, but not uniformly. Among the eight studies on teacher effectiveness, only 4 had a clear significant, positive correlation with EI, 2 had no correlation, and in three, results were not clear. Among the seven studies on job satisfaction, 4 had a significant positive correlation with EI, one had correlation with some aspects only, one had no correlation and in one study the results were not clearly described. All three studies on self-actualization showed a positive correlation with EI and all 3 studies on occupational stress reported a negative correlation with EI.\n\nTwenty-eight studies assessed the correlation of EI with another parameter with Pearson correlation coefficient, sometimes with more than one parameter or subgroup analysis resulting in multiple r values from a single study. In studies which calculated correlation of EI with multiple parameters, all effect sizes were included. For those studies which did the correlation with either subgroups (gender, type of teachers), or components of EI, or used two different scales for the same parameter, all effect sizes were averaged to avoid giving undue weightage to one sample and study. Some studies assessed another parameter with EI but did not conduct the correlation analysis or used another statistical analysis than Pearson r and therefore were not included in meta-analysis. Some of the studies could not be included either due to different statistical tools used or using component measures which could not be averaged due to opposing nature of parameter studied or multiple subcomponents.\n\nSubgroup analysis was intended for type of teachers (trainee, school or college) and type of EI scale (ability, trait, mixed) but as almost all studies used trait measure for EI, subgroup analysis was carried out for teacher type only.\n\nThree different meta-analysis were carried out. The initial two for evaluating correlation of EI with personal and professional health parameters and the third for effect of gender on EI.\n\nFrom ten studies examining the correlation of EI with parameters associated with personal health, fifteen effect sizes in 3291 participants were deduced and used for meta-analysis. Table 2 lists all the correlations with confidence intervals and relative weights. Table 3 shows the combined effect size calculated from random effect model and also gives heterogeneity data.\n\nFigure 2 depicts the Forest plot for the same analysis with individual studies shown in blue circles with their calculated confidence interval and the bottom line showing combined effect size in green with confidence interval in black line and prediction interval in green colour.\n\nIt is evident from this meta-analysis that there is a large and significant correlation of EI with personal health of teachers with a combined effect size of 0.45 (95% CI, 0.12 to 0.69). The confidence interval, z value and p values (<0.05) all suggest that the correlation is significant rejecting the null hypothesis (H01) and confirming the alternative hypothesis (H1). As per Cohen (Carson, 2012), effect size of 0.2 is small, 0.5 is medium and > 0.8 is large. The heterogeneity analysis shows a Q statistic of 1097.47 and a p value of 0.000 denoting significant heterogeneity, which is also evident from the different effect sizes as shown in Forest plot. While a few studies have reported a negative correlation, the majority of studies have reported a positive correlation with one study reporting a correlation coefficient of 0.98 (blue circle on right with no black line). The high value of I2 (98.7%) again shows the very high level of heterogeneity. The large prediction interval (-0.70 to 0.95) suggests that the studies included for meta-analysis are very different from each other and it is difficult to draw any straightforward conclusions regarding significant effect sizes. The main reason for this is the limited number of studies and small sample sizes. Other reasons of heterogeneity could be socio-cultural factors, regional differences (for example North and South India), educational and financial backgrounds etc. One parameter, which was available for all studies is the type of teachers included as sample population. This was subjected to a subgroup analysis to see whether it is a significant factor, or if there is a similarity among studies with similar teacher types.\n\nThe results of subgroup analysis, as shown in Table 4, reveal two things. First, despite these subgroups being made on the presumption that there may be differences in teachers according to their level of teaching, there will be some similarity within groups. But the high degree of heterogeneity as expressed by I2 (98%, 99%, 95% for groups college, school and trainee respectively) suggests that even within these subgroups, there is a lot of differences between study samples. Second, the differences between subgroups are not significant (p value of 0.823). Both of these conclusions indicate that level of teaching is not a significant factor affecting the correlation of EI with teachers’ personal health and there are other factors contributing to heterogeneity of studies.\n\nFrom eighteen studies examining the relationship of EI with teachers’ professional health parameters, nineteen effect sizes among 4165 participants were deduced and included in meta-analysis. Table 5 shows the correlation coefficients of EI with parameters associated with professional health along with their confidence intervals and relative weights. Table 6 shows the combined effect size with confidence and prediction interval and heterogeneity data. The study by Anjum and Swathi (2017) reported a correlation of 1, which was changed to 0.99 for proper calculation as some deductions are not possible with this coefficient level. Figure 3 shows the Forest plot for this meta-analysis.\n\nThe above analysis shows a significant correlation of EI with professional health of teachers with a combined effect size of 0.42 (95% CI, 0.14-0.64). Similar to previous results, there is a lot of heterogeneity among studies, as suggested by an I2 value of 97.5%. The Z value of 3.03 and low p values (<0.05) suggest that the combined effect size is significant rejecting the null hypothesis (H01) and confirming alternative hypothesis (H1). The forest plot also depicts the significant combined effect size in the bottom line with green circle and confidence interval in black. As can be seen from this plot, the difference among the results of studies is very marked, such that while some studies show a negative correlation, others have reported very high positive correlation with one study reporting it as 0.99 (blue circle on extreme right without a black line). This again makes the prediction interval very large (-0.47 – 0.89), making the statistical significance of combined effect size less strong.\n\nFor subgroup analysis, only two groups were defined (school and college) as there was only one study with trainee teachers.\n\nAs shown in Table 7, there is still a very high degree of heterogeneity within these subgroups (I2 of 97% and 98% in college and school subgroup) and there is no significant difference in pooled effect sizes between groups (p value of 0.169). One interesting aspect of these results is that while for personal health parameters, the effect size is more for college teachers as compared to school teachers, the reverse is true for professional health parameters. The higher Q (1.89 versus 0.39) with lower p value (0.169 versus 0.823), although not statistically significant, may suggest a trend which can be explored in future studies with larger sample size.\n\nThe general inference which can be drawn from both these meta-analyses of available research is that although teachers’ EI is significantly correlated with both personal and professional health parameters, the high level of heterogeneity resulting in a large prediction interval suggests that larger studies with bigger sample size are still needed so that the strength of association and significance of results can have a more meaningful interpretation.\n\nStudies evaluating the effect of gender on EI have reported conflicting results, with five studies reporting higher EI in female teachers as compared to males, five studies reporting higher EI in males as compared to females and sixteen studies reporting no effect of gender on EI, while twenty-nine studies did not specifically look into this relationship. Six studies evaluated the effect of gender on EI, but had inadequate data. Finally, twenty-six studies with appropriate datasets were included in meta-analysis involving 6005 participants. Table 8 presents the summary statistics and calculation of effect size in the form of Hedges’ g with confidence interval and relative weights. Only means, standard deviations and sample size were used for calculation of Hedges’ g, t values are extracted from studies but not used for this calculation. Table 9 shows the result of meta-analysis with combined effect size with confidence intervals, along with heterogeneity data. Figure 4 shows the Forest plot for the meta-analysis. For all parameters, first column (1) denotes values for males and second column (2) denotes values for females.\n\nThe above meta-analysis clearly shows that the combined effect size, or the difference between male and female teachers’ EI scores is negligible (0.04) which is too small an effect to be either statistically significant (p value more than 0.05) or have any practical significance. The Forest plot, as depicted in Figure 4 shows that with the exception of a few studies showing a strong effect on either direction, the majority of studies have reported a very small difference among genders. This result confirms our second null hypothesis (H02) and rejects alternative hypothesis (H2) and concludes that there is no significant difference between the emotional intelligence of male and female teachers. The heterogeneity data shows an I2 value of 71%, which, although high, is much lower than the previous two meta-analyses.\n\nPublication bias analysis for all three meta-analyses are summarized together with a depiction of funnel plots in Figures 5, 6 and 7 with Egger regression analysis in Tables 10, 11 and 12.\n\nCareful analysis of these funnel plots suggests that, although there was no statistically significant bias in all three meta-analysis, as there is no linear relationship between the x and y axes and effect sizes are almost equally distributed on both sides of the combined effect size in a near funnel shape, there is still some bias present in all three. This is obviously more apparent in the first two plots with presence of a single study with very strong positive correlation, which somewhat skews the shape of the funnel. The same conclusion can be drawn from analysis of Egger’s intercept which is highest in the second meta-analysis and least (near zero) in the third meta-analysis. Although the p-value is insignificant in all three, the near perfect funnel shape and lowest value of Egger’s intercept make the third meta-analysis least affected by publication bias. The obvious reasons are larger number of studies in this analysis. Therefore, the general inference which can be drawn from study of publication bias is that the third meta-analysis, studying the effect of gender presents the most reliable data in terms of publication bias.\n\nFor all three meta-analyses, two alterations were planned namely in sample size and effect size. In the first analysis, studies with a sample size of 100 or less and then less than 200 were removed. For effect size alteration, in the first two meta-analysis, studies with effect size greater than 0.9 and then greater than 0.75 were removed. In the third meta-analysis, this was reduced to effect size greater than 0.5 after an evaluation of the results. The purpose of these alterations is to see how sensitive the final results are to these changes and what is the robustness of the results. As can be seen from Table 13, there is little or no difference in heterogeneity and p values with these changes. In the third meta-analysis, the change in effect size is also minimal. However, in the first two meta-analysis, the effect size drops to some extent with exclusion of studies with a sample size less than 200 and by exclusion of studies with greater effect sizes. As the sample size is inversely related to standard error (which can be a surrogate for the accuracy of results), the results of sensitivity analysis point to the fact that although the relationship between studied parameters is positive and significant, the pooled effect size of more accurate studies is somewhat smaller. This, coupled with a high degree of heterogeneity is further argument for designing future studies with strong methodologies. The sensitivity analysis for the third meta-analysis clearly suggests the robustness of the results, similar to the conclusion of publication bias analysis.\n\nTo see whether the heterogeneity is only because of inclusion of studies with negative effects, a tentative sensitivity analysis was done excluding such studies (not shown in the table), but did not affect the heterogeneity (93% in both), which implies that even among the studies with positive effects, there is marked heterogeneity.\n\nGiven the combination of low sample size in many studies resulting in low precision, high risk of bias due to absence or inadequate randomization, marked heterogeneity and high level of publication bias, the overall certainty of results can be labelled as low.\n\nTable 14 summarizes the results of all three meta-analyses. Because of least amount of heterogeneity and publication bias and previously discussed sensitivity analysis, the third meta-analysis examining the effect of gender on EI has the most reliable and robust conclusion.\n\n\nDiscussion\n\nThe aim of writing this review was to present a qualitative synthesis of the available literature on EI among Indian teachers. We have tried to include as many studies as possible, but despite that, it may be possible that some studies have been missed, especially if they are not easily accessible online. The review shows clearly that a robust data exists regarding EI in the education sector with studies done among different types of teachers with a reasonable number of participants in some of the studies. The salient features are as follows.\n\nMaximum studies have been conducted on secondary school teachers, closely followed by trainee teachers. However, very few studies have taken participants from more than one type or level of educational institutes. Some of the studies had adequate sample size but not all studies detailed the sampling methods, demographic details, educational and other professional background and studied the relationship of these factors with EI. The most commonly utilized EI scale was the indigenous tool developed by Hyde et al., followed by teacher EI inventory by Mangal. Although the majority of scholars and tool developers have not specifically mentioned this, the majority of tools are trait measures. The ease of administration and short time duration, especially in the digital age, along with better correlation with adaptive and other psychological outcomes are the main reasons for this trend. All studies using the Hyde scale have reported moderate to high level of EI scores of teachers, which needs comparative analysis with other professions with the same tool. A positive correlation with teacher effectiveness, job satisfaction, attitude, self-actualization, life satisfaction, physical and mental health, and negative correlation with occupational stress and burnout has been reported which emphasizes the role of EI in both personal and professional life of teachers. There was inadequate explanation for some of the associations like general intelligence, professional qualifications or level of teaching reported in some studies which did not discuss the findings in detail, especially given the evolving conceptual framework of trait EI which places it as a separate entity than cognitive intelligence bearing no relation with educational qualification or conventional forms of intelligence. Lack of detailed references especially regarding the measurement tool used, inadequate description of EI tool and inadequate discussion of results are some of the major limitations detected in some of the studies.\n\nBeing the first such attempt regarding Indian studies on EI, all efforts were put to include maximum number of studies to make the data as representative as possible. The exclusion was done only either in case of too small a sample size or marked deficiency in methodology or statistical analysis. Fifty-five studies were available for the qualitative analysis and three broad conclusions can be drawn from them. First, trait measures have been used by almost all researchers but the individual studies have marked heterogeneity in the scale used with inadequate information about some of the scales being used. Second, role of demographic factors (age) and professional (educational) qualification has been rarely studied and data on only the effect of gender on EI is available but with conflicting results. And finally, a lot of parameters concerning personal and professional health of teachers have been studied in relation to EI with some common features, but lacking a clear understanding and unifying inference.\n\nMeta-analytical methods were employed subsequently to synthesize the major findings of the included studies. After evaluation of all the available data independently by both the authors, it was decided that three meta-analyses should be carried out. To address the issue of the second parameter studied in relation to EI and to cumulatively calculate combined effect size of the relationship between EI and the different parameters studied, all such parameters were grouped into two broad categories of personal and professional health parameters and then two different meta-analyses were carried out to test the hypothesis of whether EI is significantly associated with personal and professional health of teachers. Fifteen effect sizes were deduced from ten papers evaluating correlation of EI with personal health parameters and included in first meta-analysis. Nineteen effect sizes were deduced from eighteen papers evaluating correlation of EI with professional health parameters. Random effect modelling with Fisher’s r to z transformation was used to calculate combined effect size. The results of these meta-analyses show that although a significant relationship exists between EI and parameters concerned with both personal and professional health, irrespective of the level of teaching, the results are to be interpreted with the understanding that a high degree of heterogeneity exists between the studies which makes the level of significance less robust. The reasons for heterogeneity could be small sample size in many studies, inherent differences in demographic and professional parameters of sample population, different methodologies and statistical tools used and marked variations in correlation coefficients reported. One reason for apparent heterogeneity could be incorrect grouping of these parameters into personal or professional health parameters, but the more likely reason is an inherent difference in results, best exemplified by studies evaluating correlation of EI with Teacher Effectiveness. Among the seven effect sizes deduced from five papers, three were statistically insignificant while one showed a strong positive correlation. This suggests that larger studies with better methodologies are still needed to reduce such heterogeneity.\n\nThe third meta-analysis was carried out to study the effect of gender on teachers’ EI. Twenty-six papers had adequate data regarding sample size of both genders, mean EI scores and standard deviations. Hedges’ g was used as effect size. The results showed that there is no significant difference in EI between male and female teachers. The combined effect size of 0.04, coupled with a lesser degree of heterogeneity and the least amount of publication bias among the three meta-analyses implies the relatively more robust conclusion regarding lack of gender effect on EI.\n\nPublication bias analysis was carried out by Funnel plot and Egger’s intercept regression.\n\nThe low publication bias can be attributed to two factors. First, the less stringent inclusion criteria with exclusion of limited number of studies may have resulted in data with some compromise in quality but broader inclusion results in less selection bias. As this is the first such exclusive meta-analysis of Indian studies, more emphasis was placed on inclusion of maximum number of studies. Future researchers may attempt to carry out such analysis with more stringent inclusion criteria with more strict emphasis on methodology and reporting of results. The second reason for less bias is related to a policy decision. Early attempts in the field of meta-analysis included unpublished dissertations to reduce bias. In India, the University Grants Commission (UGC) has made it mandatory to publish papers related to one’s research work in journals as part of thesis submission before awarding doctorate degrees. Thus, looking for unpublished dissertations in thesis repositories is mostly not productive as all the theses will have an accompanying paper which must have been published in a journal.\n\nThe results of sensitivity analysis mirror the findings of publication bias analysis. Even by excluding studies with lesser participants or higher effect size, the results of the third meta-analysis remain unchanged, while there was a small change detected in first two meta-analyses.\n\nLack of detailed description on sampling method used, low level of randomization and lack of description of randomization methods are some of the biggest limitations in these studies which result in high risk of bias, imprecision and low certainty of evidence derived from the review.\n\nOne of the obvious areas of future research is designing studies with larger sample size and clearly defined tools and methodologies. The relationship of age, educational level and other professional qualification with EI needs further research. An attempt can be made to do a comparative study of an Indian scale with some internationally recognized EI measure which can help to give it an international recognition and acceptance. Pre-defined parameters for teachers’ health and performance and their correlation with EI in larger and possibly longitudinal studies will further consolidate the EI research domain.\n\nHigh level of heterogeneity reduces the strength of conclusions from any meta-analysis. More elaborate delineations of various demographic and professional parameters are needed in future studies to analyse different subgroups to reduce this. Lesser stringent criteria for inclusion in review may have resulted in some compromise with quality of data, but care was taken to include only those studies in meta-analysis which reported all relevant metrics. Elaborate discussion regarding tools and results in each study would have improved the qualitative results of the review but every attempt was made to synthesize as much information as possible.\n\n\nConclusion\n\nIntelligent use of emotions is always desirable, whether in a student, teacher, corporate executive or home-maker. The usefulness of determining the role of demographic, academic and psycho-social factors in determining emotional intelligence and its impact on the health and performance of individuals goes beyond any one sector, and can help administrators and policy makers in improving the work environment, productivity and performance by adequate training programs and by specific target interventions for those who require special attention. The present work summarizes the major findings from studies done among Indian teachers and concludes that emotional intelligence positively impacts the personal and professional life of teachers. There is no difference between genders regarding trait EI measures. Longitudinal studies examining the impact of any intervention to improve EI are needed to support the argument that such interventions must be made part of routine teacher training programs which are already in place. This review can also serve as a useful baseline data for future researchers in EI domain, both in India and abroad.", "appendix": "Data availability\n\nFigshare: Data for systematic review and meta-analysis. https://doi.org/10.6084/m9.figshare.24212460.v2 (Pandey, 2023).\n\nThis project contains the following underlying data:\n\n- Data-total.xlsx (List of all studies with relevant details for statistical analysis)\n\nThis project contains the following extended data:\n\n- PRISMA checklist for abstract and review (2020).docx\n\n- Fig -1 - prisma flowchart for study inclusion.docx\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\nReferences\n\nAckley D: Emotional intelligence: A practical review of models, measures, and applications. Consulting Psychology Journal. 2016; 68(4): 269–286. 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Publisher Full Text\n\nPaul ASH, Thavaraj HS: IJEMR – April 2015 - Vol 5 Issue 4 - Online - ISSN 2249 – 2585 Print - ISSN 2249-8672 Emotional Competency Emotional Maturity Emotional Sensitivity Emotional Intelligence. 5(4).2015.\n\nPetrides KV, Furnham A: On the dimensional structure of emotional intelligence. Personality and Individual Differences. 2000a; 29: 313–320. Publisher Full Text\n\nPetrides KV, Furnham A: Gender Differences in Measured and Self-Estimated Trait Emotional Intelligence. Sex Roles: A Journal of Research. 2000b; 42(5): 449–461. Publisher Full Text\n\nPetrides KV, Mikolajczak M, Mavroveli S, et al.: Developments in Trait Emotional Intelligence Research. Emotion Review. 2016; 8(4): 335–341. Publisher Full Text\n\nPonmozhi DD, Ezhilbharathy T: Emotional Intelligence of School Teachers. IOSR Journal of Research and Method in Education (IOSRJRME). 2017; 07(03): 37–42. Publisher Full Text\n\nPooja P, Kumar P: Demographic Variables and Its Effect on Emotional Intelligence: A Study on Indian Service Sector Employees. Annals of Neurosciences. 2016; 23(1): 18–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradhan RK, Jandu K, Samal J, et al.: Does practicing spirituality at workplace make teachers more engaged? Examining the role of emotional intelligence. International Journal of Ethics and Systems. 2022; 39(ahead-of-print): 859–874. Publisher Full Text\n\nPugazhenthi P, Srinivasan P: Impact of Teaching Tfficiency through Emotional Intelligence on the performance of B. Ed teacher trainees. Global Journal for Research Analysis. 2018; 7(2): 396–397.\n\nRajam Jeya Devi R, Kumar S: Relationship between emotional intelligence and job satisfaction of primary school teachers. Paripex - Indian Journal of Research. 2018; 7(8): 62–63.\n\nRani MS, Prasad UD: Emotional Intelligence and Attitude towards Work of Teachers: A Performance Improvement Model. Pacific Business Review International. 2016; 9(5): 1–11.\n\nRawat A: A Study of the Relationship between the Emotional Intelligence and Self -actualization of. B.Ed. Teacher trainees. Paripex - Indian Journal of Research. 2017; 6(2): 210–212.\n\nReddy MR: Emotional Intelligence of Teachers working in B. Ed. Colleges of Rayalaseema Region of Andhra Pradesh State. International Journal of Creative Research Thoughts. 2017; 5(4): 1743–1754.\n\nSalovey P, Mayer JD: Emotional intelligence. Imagination, Cognition and Personality 1990; 9: 185–211. Publisher Full Text\n\nSánchez-Álvarez N, Berrios Martos MP, Extremera N: A Meta-Analysis of the Relationship Between Emotional Intelligence and Academic Performance in Secondary Education: A Multi-Stream Comparison. Frontiers in Psychology. 2020; 11: 1517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSandhu D, Agrawal M: A study of component gender in teacher effectiveness, personality and emotional intelligence amongst high school educators. International Journal on Emerging Technologies. 2020; 11(2): 973–977.\n\nSchutte NS, Malouff JM, Hall LE, et al.: Development and validation of a measure of emotional intelligence. Personality and Individual Differences. 1998; 25(2): 167–177. Publisher Full Text\n\nSchutte NS, Malouff JM, Thorsteinsson EB, et al.: A meta-analytic investigation of the relationship between emotional intelligence and health. Personality and Individual Differences. 2007; 42(6): 921–933. Publisher Full Text\n\nShanthini, Kumar V, Nawaz N: Assessment of emotional intelligence among the primary schools teachers: A comparative study. International Journal of Scientific and Technology Research. 2020; 9(3): 3834–3837.\n\nSharma S: Adjustment Among Working Women in Relation To Emotional Intelligence, Spiritual Intellligence and Life Satisfaction. International Journal of Educational. 2017; 8(63022): 1–11.\n\nSharma V, Singh SP, Kumari H: Demographic Predictors of Emotional Intelligence and Job Satisfaction among Teaching Professionals in Indian Higher Education Sector. Globus- An International Journal of Management and IT. 2016; 8(1): 1–5.\n\nShukla A, Srivastava R: Meta Analysis of the Relationship Between Emotional Intelligence and Different Behavioral Intentions. Research Journal of Business Management. 2016; 10: 58–73. Publisher Full Text\n\nSingh B, Kumar A: Effect of Emotional Intelligence and Gender on Job Satisfaction of Primary School Teachers. Eurasian Journal of Educational Research. 2016; 5(1): 1–9. Publisher Full Text\n\nSingh D: Emotional intelligence at work – A professional guide. NewDelhi: Response Books; 2006.\n\nSingh N, Singh S: Emotional intelligence of pupil teachers in relation to their gender and locality. International Journal of Research in Social Sciences. 2018; l8(6): 830–840.\n\nSoanes DG, Sungoh SM: Influence of Emotional Intelligence on Teacher Effectiveness of Science Teachers. Psychology. 2019; 10(13): 1819–1831. Publisher Full Text\n\nSripal P, Paramasivam T: Emotional Intelligence College among Teachers in Coimbatore. Shanlax International Journal of Management. 2016; 4(1): 1–5.\n\nSurana A, Rawat A: A Study Of The Emotional Intelligence of B. Ed. Teacher Trainees. Journal of Teacher Education and Research. 2014; 9(1): 88–97.\n\nSuresh K, Srinivasan P: A Study of Teacher’s Intelligence and Emotional Intelligence on Students’ Mental Health among Higher Secondary School of Thanjavur District. American Journal of Educational Research. 2018; 6(6): 869–876. 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. Research Synthesis Methods. 2017; 8(4): 537–553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorndike EL: Intelligence and its uses. Harper’s Magazine. 1920; 140: 227–235.\n\nToor A, Kang T: Relationship of Emotional Intelligence with Occupational Stress Across Gender and Designation. Indian Journal of Health And Wellbeing. 2018; 9(3): 335–341. Reference Source\n\nToor K: A study of social and emotional intelligence of secondary school teachers. International Journal of Behavioral, Social and Movement Sciences. 2013; 2(4): 22–34.\n\nUniyal DD, Rawat S: Exploring Emotional Intelligence in School and College Teachers. International Journal of Scientific and Research Publications (IJSRP). 2020; 10(06): 510–520. Publisher Full Text\n\nVan Rooy DL, Dilchert S, Viswesvaran C, et al.: Multiplying intelligences: Are general, emotional, and practical intelligences equal?Murphy KR, editor. A critique of emotional intelligence. Mahwah, NJ: Erlbaum; 2006; pp. 235–262.\n\nVan Rooy D, Viswesvaran C(V): Emotional intelligence: A meta-analytic investigation of predictive validity and nomological net. Journal of Vocational Behavior. 2004; 65: 71–95. Publisher Full Text" }	[ { "id": "228959", "date": "24 Jan 2024", "name": "Monu Bhargava", "expertise": [ "Reviewer Expertise Financial Literacy", "research methodology", "HR", "Banking" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 interesting. The research paper is well-written and structured appropriately. Objectives and discussion are matched. Research gap is mentioned clearly. However, in my opinion, the research paper should Although the use of PRISMA technique has been mentioned in the objective part, it has not been appropriately projected in the paper. therefore, PRISMA flowchart can be included to explain the methodology and to reduce textual information. Can add a test to measure \"the degree of inconsistency. Hypothesis testing results should be clearly explained in a separate section: hypothesis testing results. Grammatical and spelling mistakes must be taken care of.\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": "11002", "date": "13 Apr 2024", "name": "Mamta Pandey", "role": "Author Response", "response": "1. PRISMA guidelines have been diligently adhered to and both the abstract and complete manuscript were drafted as per the PRISMA checklist for systematic review and meta-analysis 2020. The journal itself demands strict adherence to these guidelines and the checklist has been provided at the the end as part of extended data. The flowchart showing the inclusion of studies has also been made as per the guidelines, Because of the already lengthy manuscript, other diagrams and flowchart showing all methodological points like heterogeneity analysis, publication bias analysis, subgroup analysis and sensitivity analysis were not presented separately. 2. Tests of inconsistency are inherent part of any meta-analysis and we have presented them with both tabular and textual form as heterogeneity analysis with special mention of I2 as the main parameter used to represent inconsistency or heterogeneity. The high level of heterogeneity in initial two meta-analysis and its implication has also been discussed. 3. Thanks for pointing out this lapse. We have now added a separate subheading titled ' summary of results of hypothesis testing' which briefly summarizes the results regarding acceptance or rejection of hypothesis. 4. Spelling mistakes and grammatical errors have been checked again." } ] }, { "id": "228963", "date": "31 Jan 2024", "name": "Ravi Kant", "expertise": [ "Reviewer Expertise ICT in Education", "Educational Psychology", "Eductional Technology", "Internet 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\nThis paper is a good attempt to explore the status of research on emotional intelligence in India. The following are some points suggested to the author-\n1. The paper is too long in comparison to the normal length of the research paper. 2. The rationale behind choosing the Indian research only, needs a more logical background. 3. Methodology is sufficient and clear for a layman. 4. I am unable to understand why researchers used two different methods in a single paper i.e. meta-analysis and survey. 5. References are relevant to the present study.\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": "11008", "date": "13 Apr 2024", "name": "Mamta Pandey", "role": "Author Response", "response": "1. The criticism about paper being too long is well taken. The intension to put together a comprehensive database of all EI research among Indian teachers and collating and synthesizing that data resulted in a lengthy manuscript. Given the requirements for systematic reviews and meta-analysis as per international guidelines, various analytical methods, though presented briefly have still made the paper lengthier. 2. We deliberately focused our literature survey to Indian studies as the main purpose behind this research was to study the status of Indian research in EI domain and compare it to international studies as well as to present the available data to an international research community so that scholarly work in this filed, which seldom gets recognition in isolation, receives the due recognition. The fact that several meta-analysis by foreign researchers have not included Indian studies was another reason to present an exclusive Indian data in this field. 3. The reason for doing multiple analysis was twofold. First , as part of the accepted protocol, before conducting any quantitative analysis, a qualitative assessment and synthesis of data is required to see whether enough datasets are available for further analysis. So , the initial literature search, done as per the PRISMA guidelines was very broad, and as previously mentioned, done with the objective of including all relevant studies in this field. Second, for the purpose of meta-analysis, specific objectives and hypothesis were framed and only those studies were included which had all relevant statistical data. These data were presented in three different meta-analysis. This process, although cumbersome and making the paper very lengthy, resulted in a comprehensive presentation of all Indian data." } ] } ]	1	https://f1000research.com/articles/12-1519
https://f1000research.com/articles/12-1400/v1	24 Oct 23	{ "type": "Review", "title": "Models for the retention of duplicate genes and their biological underpinnings", "authors": [ "Raquel Assis", "Gavin Conant", "Barbara Holland", "David A. Liberles", "Malgorzata M. O'Reilly", "Amanda E. Wilson", "Raquel Assis", "Gavin Conant", "Barbara Holland", "Malgorzata M. O'Reilly", "Amanda E. Wilson" ], "abstract": "Gene content in genomes changes through several different processes, with gene duplication being an important contributor to such changes. Gene duplication occurs over a range of scales from individual genes to whole genomes, and the dynamics of this process can be context dependent. Still, there are rules by which genes are retained or lost from genomes after duplication, and probabilistic modeling has enabled characterization of these rules, including their context-dependence. Here, we describe the biology and corresponding mathematical models that are used to understand duplicate gene retention and its contribution to the set of biochemical functions encoded in a genome.", "keywords": [ "gene duplication", "probabilistic modeling", "theoretical biology", "Markov model", "synteny", "phylogenetic analysis" ], "content": "Introduction\n\nRichard Feynman left the quotation, “What I cannot create, I do not understand” on his blackboard at the time of his death. Creation in mathematical modeling is writing down a model that describes a process. The retention of duplicate genes over long evolutionary periods involves mechanisms and processes in population genetics, evolution, molecular biology, ecology and biochemistry. Here we describe the current state of modeling in the field of duplicate gene retention.\n\nThe genome can experience duplications of its content across a range of sizes, from incomplete duplications of single genes to small-scale events (single gene duplicates through tandem duplication or retrotransposition events) to larger scale events that involve multiple genes or even whole genomes. These events are broadly classified as whole genome duplications (WGD) or small scale duplications (SSD): the two types have several key differences. WGD duplicates are duplicated together with their interacting partners, and the population (and cellular) genetic model for the initial fixation of WGD duplicates is less straightforward than for SSDs. WGD events can be either allopolyploidies or autopolyploidies (see below). SSD duplicates typically do not see their interaction partners duplicated and initially have a frequency of 1/2N in a diploid population. SSD duplicates are typically assumed to begin as identical copies, but this isn’t always the case.1–6\n\nOnce duplicated, the accumulation of nonfunctionalizing mutations leading to the eventual loss of one copy through neutral processes can be naturally modeled with an exponential decay distribution.7–10 Several factors can lead to duplicate pairs losing their redundancy and falling under the action of natural selection; when this change occurs that pair will deviate from the neutral expectation of exponential loss.10–12 In general, it has been suggested by Wagner (2005)13 that the expression cost of a duplicated gene leads to a fitness cost to its possessor, and a similar argument could be made for a replication cost, especially for organisms that have limitations on genome size. Such a limitation would occur when selection acts to minimize replication time in log phase growth.\n\nMore generally, duplicates can be retained as a mechanism to gain extra expression.14 An example of this in humans and other mammals appears to be the convergent duplication and retention of copies of the amylase gene.15 Trypanosomes seem to regulate gene expression more generally through gene duplication, with very few transcription factors in their genomes.16,17 There is also a selective pressure to retain duplicates that physically interact in stoichiometric balance to prevent misassembly of imbalanced heteromultimers or pathological interactions among the exposed hydrophobic surfaces of those multimers.18–22 Mechanisms of duplicate preservation that involve changes of function through mutation accumulation include subfunctionalization,23,24 the partitioning of functions among copies from the pre-duplication ancestral state, and neofunctionalization,14 the acquisition of a new beneficial function. Multiple models for all of these processes exist and are described below together with their biological assumptions.\n\n\nGene Duplicability\n\nThese different mechanisms of retention are differently applicable to different genes. This has given rise to the notion of gene duplicability, that some genes are inherently more duplicable than other genes.8,25,26 For a gene to be retained after duplication, it ultimately needs to be either subfunctionalizable or neofunctionalizable.10 To be subfunctionalizable, a gene must have at least two modular functions (biochemical activities, including binding, or modular expression domains) such that there are mutations that can disable one subfunction without damaging others. The probability of eventual subfunctionalization for two identical duplicate copies scales as 1-(0.5)^(f-1), where f is the number of functions. The probability of neofunctionalization is harder to quantify, but either new selectable biochemical functions or expression domains must be evolvable. At the coding sequence function level, this is influenced by the fold, the number of binding partners, including those that are obligate heteromultimerization partners,20,21 and the type of function encoded.27–33 The network position and expression level also influence gene duplicability.26,31,34–38 Empirically, there is a class of “duplication-resistant” genes where natural selection apparently acts against the maintenance of both copies.33 Different genomes might have different proportions of genes that are duplicable as well as that are subject to dosage constraints.39\n\nIn analyzing the retention of genes following two rounds of whole genome duplication, the Atlantic salmon genome paper40 presented a conditional probability analysis suggesting that the gene duplicability hypothesis predicts that genes retained after one round of genome duplication might be more likely to be retained after the second round. However, prior analyses from plants tended to suggest that the factors that favor the retention of duplicates after a first polyploidy tend to be attenuated in subsequent polyploidies.41 Support for the hypothesis from the test in Atlantic salmon was also lacking, but there is more complexity to the process, including changing gene duplicability, the time-dependence of the retention process, and other factors42,43 and probabilistic models that can be used as an expectation for different hypotheses are described below.39\n\n\nThe Biological Considerations as Building Blocks for Models\n\nTo model duplicate gene retention, one must describe what is mutable and selectable. Protein encoding genes must function as proteins after transcription and translation. They are expressed at a given concentration in specific places and at specific times. They then carry out various functions: binding, catalysis, or transport in interaction with other molecules in the cell. This is what we mean by function. The expression domains for a protein are a quantitative description of where and when expression occurs. There are cases where duplication is used as a mechanism of amplifying expression level, although this seems to be a temporary situation in most organisms, with trypanosomes being a possible exception.\n\n\nModeling Expression Evolution of Duplicate Genes as a Stand-Alone Process\n\nThough classical models for the retention of duplicate genes often consider their levels of sequence divergence, gene expression data provide a promising source of underutilized information. In particular, gene expression data are now widely available for many species and often consist of measurements across multiple conditions, which can include tissues, sexes, and developmental stages. These measurements are an attribute of function, as knowledge of where and when a gene is expressed provides insight into its biological roles. Indeed, Ohno proposed that the first step of functional divergence between duplicate genes is their expression divergence.14 Thus, gene expression is a trait that can be exploited to understand gene function and, in the case of duplicate genes, the divergence between their functions.\n\nMany early studies compared expression levels between duplicate genes, finding that divergence between copies is often widespread, rapid, and asymmetric.11,44–46 Yet expression divergence between duplicate genes does not provide information about the exact mechanisms of their retention. For instance, both neofunctionalization and subfunctionalization result in functional divergence between gene copies. Thus, it is important to compare the expression profiles of both copies to that of the ancestral single-copy gene, as this can elucidate how each copy has changed since duplication. Such an approach was developed about a decade ago47 and later implemented as the software CDROM.48 Applications of this approach have uncovered widespread neofunctionalization in Drosophila,47 mammals,49 honeybees,50 and grasses.51\n\nHowever, a key shortcoming of the approach of Assis and Bachtrog (2013)47 is that it does not account for stochastic changes in gene expression arising from phenotypic drift.52 This obstacle can be overcome by modeling gene expression evolution on the phylogenetic tree relating a pair of duplicates and their single-copy ancestor. There is a natural hierarchy of models for describing how gene expression evolves along the branches of a phylogenetic tree. The simplest is Brownian motion (BM), which models phenotypic drift without making any assumption of selection for a particular expression level.53 The next level of the hierarchy is an Ornstein-Uhlenbeck (OU) process with stabilizing selection for a particular expression level.54–58 Finally, one can utilize an OU process with a shift reflecting positive selection for an optimal expression level.59–63 Another conceivable approach is to model the genotype driving expression evolution, but this requires an understanding of the combinatorial role of promoters and enhancers regulating expression.64,65 which is currently beyond our modeling capabilities.66\n\nWith this in mind, researchers have recently begun to employ OU models for studying the expression evolution of duplicate genes.22,67 Additionally, DeGiorgio and Assis (2021)67 developed CLOUD, which also predicts retention mechanisms of duplicate genes by overlaying their OU model with a neural network.67 Though likelihood ratio tests (LRTs) have classically been used for similar tasks with single-copy genes,22,56–59,68 machine learning approaches present several advantages, such as the optimization of model fit to training data, direct evaluation of performance on independent test data, and ability to make predictions from data with correlated or conflicting signals.69 Further, such methods make predictions solely from data,69 which can be advantageous when the underlying evolutionary model is unknown. Indeed, CLOUD demonstrates excellent predictive performance, outshining CDROM in classifying retention mechanisms while also being able to predict parameters corresponding to expression optima and strengths of selection and drift.67\n\nStill, much remains to be done in this area. For one, many advanced machine learning algorithms have yet to be explored in this context. Moreover, though expression data can provide a lot of useful information about a gene, this does not necessarily mean that we should neglect other complementary sources, such as its sequence or protein structure. An additional advantage of machine learning is that it is not weighed down by additional information, as correlated or conflicting signals can be reduced or even removed through regularization. However, the problem lies in extending the underlying OU model to accommodate diverse pieces of information. Last, one can argue that the most important extension of such work is to accommodate more species and gene copies. Currently, most researchers do not have access to expression data for multiple of the same conditions in many species, but this is soon to change as the cost of sequencing continues to decrease. And of course, it is critical to assay the fates of gene families with more than two members, as many such families are prominent across study systems and may be key to understanding adaptation.\n\n\nModeling the Evolutionary Cost of Gene Duplication\n\nAnalyses of duplicated genes often start from the premise that gene duplications are selectively neutral, creating redundant copies that can potentially degrade through degenerative mutations.7,70 The patterns of which genes do and do not tend to survive in duplicate and the dosage-balance hypothesis (see above) already suggest that gene duplications are not all necessarily selectively neutral at birth. As mentioned above, Wagner has extended this argument by showing that, at least in microbial organisms, the gene expression costs associated with an extra gene copy are rarely if ever expected to be selectively neutral.13,71 His model considers the per-time unit cost, in terms of high energy phosphate bonds, of expressing a duplicated gene for different ranges of mRNA and protein levels, finding that, for reasonably large values of the effective population size of microbial species, those energy costs are large enough to discount the hypothesis that the duplication’s fate is primarily driven by neutral evolution.72 The principle that excess gene expression has measurable negative fitness effects due to the costs of transcription and translation has been elegantly experimentally explored in E. coli.73\n\n\nDosage Imbalance Cost\n\nIt is well established that maintaining stoichiometric balance with interacting partners is an important driving force to preserve duplicate genes in genomes while waiting for other preservation mechanisms to act. The mechanistic driving force behind this is thought to be the prevention of the accumulation of exposed hydrophobic residues that populate binding interfaces and can lead to misinteractions that might be deleterious to cells. An explicit model that relates fitness to the expected concentration of surface hydrophobic residues has been generated and used to explore how this model enables the transition to subfunctionalized states, with opposite trends observed after whole genome duplication and smaller scale duplication.74 This model is a mechanistic update over a previously described hazard function model that didn’t model this underlying biochemistry.75\n\n\nPopulation Genetic Considerations\n\nIn Eukaryotic organisms, the baseline state for most chromosomes is diploidy. Either across the whole genome or for individual loci, this diploid state is disrupted after gene duplication. Functional tetraploidy has meiotic implications that are not present for smaller scale duplication events (see40 for a discussion in Atlantic salmon). Over time, whole genome duplication events return to a state of functional diploidy and may start that way for alloduplication events with the chromosome sets already diverged, as may have been the case for Xenopus76 and Brassica species.77,78\n\nHowever, so far, this divergence characterization has been viewed without the underlying population-level dynamics. While more complex for whole genome duplication events, smaller scale duplicates in diploid organisms begin with a frequency of 1/2N and must fix before they are lost if they are going to be retained. The neutral expectation for eventual fixation for such duplicates is that they will fix with a probability equal to their frequency. Classical population genetics then gives a time-dependent expectation for the frequency based upon the age of the duplicate. Stark et al. (2021)79 have presented a population genetic model to evaluate if the age-dependent frequency is unexpected for a duplicate evolving neutrally. The power of the approach was evaluated using a Moran Model, with two selective parameters, one for selection on the duplicate itself, which can be positive when total gene dosage amplification is beneficial or negative due to factors like expression and replication cost, and the other for selection on changed function (for example neofunctionalization).\n\nIn using this model with actual segregating duplicates, the frequency of a duplicate in a population can be measured through population genomic sequencing. However, the age of the duplicate is the next question. For SNPs, population geneticists examine the length of tracts of identity by descent to estimate the age of an allele (e.g. duplicate locus) (see80), but a much simpler approach based upon pairwise pS values between copies may be possible. Application of these approaches to real data hasn’t been performed yet to evaluate their performance.\n\nThose considerations are used to evaluate selection on the copy itself. Selection on the sequence of the duplicate might be measured by a more rich data source and parametrization or in a simpler manner by examining ratios like pN/pS or in using tests like the McDonald-Kreitman Test applied to duplicates.81\n\n\nInterspecific and Phylogenetic Models\n\nMoving from intraspecific to interspecific analysis of gene duplicates, including models that run on a single genome, early work from Lynch and Conery (2000)7 and from Lynch, Force, and coworkers8,23,82 has been pioneering. The first model presented by Lynch and Conery (2000)7 modeled duplicate gene retention expectations with a simple exponential distribution. This assumes that no matter how long a gene has been in a genome, the instantaneous probability of loss is constant, which is not consistent with retention mechanisms, but is useful as a non-functionalization null model. Konrad et al. (2011)10 and Teufel et al. (2016)75 described sets of hazard functions that did not have the property of time-independent hazard functions and Zhao et al. (2015)83 presented an age-dependent birth-death process inspired by this framework. Yohe et al. (2015)84 presented a theoretical gene tree-species tree reconciliation framework using the Konrad et al. (2011)10 model, but this was never implemented as software. Arvestad et al. (2009)9 presented a formal probabilistic gene tree-species tree reconciliation framework using the exponential distribution model and generated software for this. Others have created similar software packages.85,86 Additional innovations to this framework have included the treatment of synteny,87 and species level processes.88\n\nContemporaneously with the Lynch and Conery (2000)7 modeling, Force et al. (1999)23 presented a more mechanistic framework for subfunctionalization and neofunctionalization as processes. This was formalized as a Markov Model by Stark et al. (2017)89 and expanded upon by Wilson and Liberles (2023)74 to enable consideration of dosage balance. Multi-scale Markov models reflect a further step in this trajectory.\n\n\nMulti-Scale Markov Models\n\nA wide range of multi-scale Markov models for the evolution of gene families have been studied in the literature. Models with states that record very detailed information about biology are suitable for simulation-based analysis. However, such models may not be useful for theoretical analysis, due to the size of their state space. On the other hand, models with simplified state space are useful for in-depth theoretical and numerical analysis which often leads to novel biological insights. Both types of models provide powerful tools for the analysis, and the choice of one over another may depend on the type of biological questions one might want to answer.\n\nAs an example, Stark (2017)90 suggested a simulation model for the evolution of the family of genes, in which detailed information is recorded within a binary-matrix of 0’s and 1’s such that each row corresponds to a gene and each column corresponds to its function. Later, Diao et al. (2022)91 applied the binary-matrix model of Stark (2017)90 in their simulation-based analysis which led to some interesting biological insights. Their results suggested that when the rate of gene duplication dominates the rate of gene loss, then the distribution of tree shapes is close to following the uniform ranked tree shape (URT) distribution (i.e. the distribution for a constant birth-death process). However, when the opposite is true then gene trees are more balanced than the URT distribution.\n\nNext, Stark et al. (2017)89 constructed a model with a simplified state space, for the theoretical analysis of the evolution of gene duplicates, see Figure 1. The manageable size of the state space allowed them to derive analytical expressions for the rates of subfunctionalization and pseudogenization. This led to the result, earlier predicted by classical models, that models with subfunctionalization provided a better fit to the age distribution of duplicate genes than models with a constant rate. Further, Diao et al. (2020)92 developed a more advanced model for the theoretical analysis of the evolution of a family of gene duplicates, based on the application of a level-dependent Quasi-Birth-and-Death process (QBD). The state (n,m,k) of their QBD model consists of the variables n and m representing the number of genes and the number of the redundant genes respectively, and the variable k which, in a simplified manner, records the remaining information about the family. The authors took advantage of both types of models. They used the simulation-based binary-matrix model of Stark (2017)90 to obtain detailed outputs and then fitted the parameters of their QBD to data obtained from these outputs. Next, they derived biological insights by computing metrics based on the expressions from the theory of QBDs, such as the stationary distribution of observing various states within the model and the distribution of the time it takes for the family to lose a gene. Soewongsono et al. (2023)93 then applied this QBD model to a more general problem of reconciliation, in which the task is to find a mapping of a gene tree into a species tree, to maximize the likelihood. The authors provided an algorithm to compute the likelihood of the reconciliation, given the available incomplete data.\n\nIn the above example of transitions with z=4, the regions hit by null mutations are in red, and the regions protected by selective pressure are in yellow. This figure is adapted from Stark et al. (2017),89 which was published under an open access license.\n\n\nModeling Asymmetric Divergence and Gene Conversion in Duplicated Genes\n\nAnother angle to evaluate duplicate gene retention mechanisms involves an examination of evolutionary symmetry. In fact asymmetric divergence between pairs is relatively common.94 This result is particularly striking when one considers that many tests have low power to detect asymmetry.95 The test that was employed uses an outgroup gene from a relative lacking the duplication to polarize the differences between the two paralogs, forming a triplet tree. Maximum likelihood was then used to compare a symmetric model, where dS (K), dN (Ka), or dN/dS (also Ka/Ks or omega) was constrained to be equal for both paralogs to an alternative model where the divergence statistic is allowed to differ between paralogs.94 It is tempting to attribute to neofunctionalization cases where one paralog has accelerated evolution relative to the other, but in fact many different modes of evolution can induce asymmetry.96 In fact, asymmetry in divergence is arguably more interesting for the light it sheds on other questions regarding duplicate evolution, such as the differences seen between duplicated genes created by different mechanisms3 or as a means for detecting recent gene conversion. We have used the same triplet-based approach to show that duplicated ribosomal proteins created by the ancient polyploidy in bakers’ yeast have experienced very recent gene conversion events, such that ancient polyploidy-produced paralogs in one species are more similar to each other than those copies are to their orthologs in a closely related species, despite the fact that the divergence time between the ortholog pairs is probably ten-fold less than the time since the paralogs were formed.97\n\nGene conversion refers to several mutational mechanisms that can allow part of the sequence of one member of a gene family to overwrite the corresponding region in another paralog, effectively erasing some of the divergence between those two paralogs.98 Because such events violate the assumption of independent evolution between paralogs, they are difficult to treat with standard models. Ji et al. (2016),99 have described a codon model of evolution that jointly considers the paired codons from two paralogous genes, incorporating a parameter t modeling the frequency with which conversion events alter the paralogs’ sequences. This model confirms the surprisingly high rate of gene conversion among the yeast ribosomal proteins, which had previously and incorrectly been taken to represent the more general rate of gene conversion among yeast paralogs.100\n\n\nWhole Genome Duplication: Duplicate Losses, Modeling and Synteny\n\nWhile approaches such as standard time-independent birth and death models can be applied to duplicate genes produced by whole-genome duplications (WGD), or polyploidies, there are complexities and opportunities introduced by WGD events that benefit from models that are specific to them. Polyploidy refers to a variety of events that result in eukaryotic cells with more than two copies of the genome.101 Polyploid lineages are formed relatively often but most quickly go extinct.102 However, great trunks of the eukaryotic tree of life descend from surviving ancient polyploidy events, including all vertebrates and flowering plants, as well as lineages of yeasts, ciliates and other plants.103\n\nPolyploid individuals can form through the merger of genomes from the same species, known as autopolyploidy, or of distinct species, referred to as allopolyploidy.104 The relative frequency of formation of these two types of polyploids many be approximately equal,105 but because allopolyploidy confers the potential benefits of both polyploidy and hybridization, there is reason to suspect that most surviving ancient polyploidy events were allopolyploidies.105–107\n\nThe phrase whole-genome duplication is potentially slightly misleading because it suggests that all genes in the genome are duplicated. Initially they are. However, for any reasonably old polyploidy event, many or even most of the duplicated genes will have been lost.107,108 Probably most of these losses occur through the fixation of loss-of-function mutations in one copy by genetic drift, a process common to duplicates of all types.7 As described, selection from various sources can also play a role.\n\n\nAnother Class of Models for Evolution After Polyploidization Events\n\nThe question of demonstrating that a particular genome has an ancient polyploidy in its history is a complex one109 and somewhat distinct from our concerns here. However, one obvious consequence of a polyploidy is the production of a group of duplicated genes that were all formed “at the same moment.” In principle, a neutral measure of paralog divergence, such as the number of synonymous substitutions per synonymous site (Ks) should be able to detect a polyploidy through the excess of duplicates with similar KS values.7,45 While the actual practice of detecting polyploidy events in this way is one that requires care,110 it has been an extremely illuminating approach. For instance, in a pioneering study, Maere et al. (2005),111 were able to fit a mixture of age models to the Arabidopsis thaliana genome and detect three different ancient polyploidies in its history. They further showed convergent retention of genes of similar function in duplicate after these different events.111 To do so, they modeled three distinct processes: 1) a basal continuous rate of single gene duplication, 2) a set of between one and three ancient polyploidy events and 3) continuous losses of duplicates created by 1 and 2. They evolved these three processes in simulated discrete Ks time intervals and fit the simulations to the observed set of Ks values from duplicated genes found in the A. thaliana genome.\n\nMaere et al.’s (2005)111 approach is elegant but challenging to implement: other analyses of a similar form have instead fit mixtures of models to the observed duplicate divergences, combining a basal steady-state duplicate birth-death model with one or more discrete events duplicating the entire genome (with the potential for the immediate removal of some of these duplicates).110,112 Such approaches allow for testing hypotheses regarding the number of polyploidy events in the lineage of a genome, but the results require some caution in their interpretation due to the relatively modest information provided by Ks values.110\n\n\nPolyploidy and Gene Synteny\n\nOur discussion so far has considered signals such as gene tree topologies and divergence measures which are applicable to all types of duplicated genes. However, for the specific case of a polyploidy, another type of highly informative data are present: the gene order patterns among the duplicated and non-duplicated genes. These patterns are commonly referred to as gene synteny. They were critical in identifying the first ancient polyploidy found in a eukaryotic genome113 and have been used in many subsequent analyses of polyploid genomes.114–116 Figure 2 illustrates the principle that a genome duplication, in contrast to single-gene duplications, produces duplicated genes that preserve the gene order present in the unduplicated ancestor. Indeed, these patterns can identify ancient polyploidies even in the limiting case where all of the duplicated genes were subsequently lost, provided an outgroup genome lacking the polyploidy is available and the degree of chromosomal rearrangement is not too large. Hence, synteny is often considered the best evidence of the presence of an ancient polyploidy, even if formal tests using it are hard to develop.110,117\n\nShown in the center in gray are the ten genes as they are found in the genome of the spotted gar (L. oculatus), which lacks the TGD. The lines joining pairs of genes indicate that these genes are neighbors in the genome (i.e., they are in synteny). After the TGD, some duplicates survive in all (pink) or some (tan) genomes, while others have been returned to single copy, either from the subgenome with more surviving genes (blue) or than with fewer (green). Numbers at the top of each column/pillar are the orthology confidence estimates from POInT. Genes are shown with their Ensemble identifiers124 for reference. This figure is an original figure produced by the authors for this review article.\n\nOne example of the power of combining syntenic information with models of duplicate gene gain and loss can be seen with POInT (the Polyploid Orthology Inference Tool)118,119 Assuming that the duplicate products of a WGD are known through syntenic information (as in Figure 2), one can use the generic discrete character evolutionary model of Paul Lewis (2001)120 to model the preservation or loss of duplicate copies in different genomes that share this WGD (Figure 3A). Briefly, the loss model presumes all loci (or pillars) start in a duplicated state D and then can undergo fates such as loss (resulting in states S1 or S2) or becoming fixed (Df). Such a model can be applied to the duplicate presence and absence data for a group of genomes sharing the polyploidy. However, the difficulty arises that the orthology assignments between those genomes is unknown. POInT hence computes the likelihood of the observed gene presence/absence data at each pillar for all possible orthology relationship under a duplicate loss model. It then uses a hidden Markov model to condition that set of likelihoods at the current pillar i on those from pillars 0..i-1- using a transition matrix Q.121 The elements of Q are determined by whether or not synteny is preserved between i-1 and i in each genome.107\n\nA) Following Lewis (2001),120 a discrete state model M allows an ancestral position to be duplicated (D), single copy (S1 or S2) or a fixed duplicate (Df). Transitions between these states occur at rates proportional to model parameters 𝛼,ɛ, and γ. Losses occur along an assumed phylogenetic tree t with branch lengths l1..lt. The extant genomes are phased into a series of homologous columns or pillars: each genome may have one or two homologs present at a pillar (a state for complete homolog absence will be added to future versions of POInT). Different parental subgenomes within an extant genome can be distinguished (orange verses tan) but subgenome identities between the genomes are unknown. B) For N = 2 polyploid genomes, there are 2N possible orthology relations. At each pillar i, we can compute the likelihood of the observed gene presence and absence data for a given orthology pattern XX using the model M and the tree t: Lixx\|M,t. C) Using the synteny relationships, the values Li00\|M,t .. Li11\|M,t can be conditioned on Li-100\|M,t .. Li-111\|M,t with a transition probability matrix Θ. The elements of Θ depend on Θi,g, where i is the pillar number and g is the genome. If synteny in maintained between pillars i and i+1 for genome g, Θi,g= ΘM, a global constant estimated by maximum likelihood (0≤ΘM≤1). Otherwise Θi,g=0.5, meaning the orthology pattern at i is independent of that at i-1. This equation can be applied recursively to compute the likelihood of the entire dataset with standard hidden Markov model approaches121: the ⨀ operator represents an element-wise vector product. The tree branch lengths and model parameters are estimated from the data by maximum likelihood using standard numerical techniques.125 This figure is an original figure produced by the authors for this review article.\n\nOnce such a framework is in place, standard likelihood ratio tests122 can be used to test hypotheses about the evolution of polyploid genomes such as what fraction of the duplicates appear to have become fixed118 or whether one of the two parental genomes from an allopolyploid is favored when duplicates are lost.119 This second pattern, termed biased fractionation, is likely indicative of an allopolyploidy106 and raises questions as to whether the subgenomes of allopolyploid hybrids are functionally compatible.77\n\n\nConcluding Thoughts\n\nA number of models have been generated that describe different levels of duplicate gene retention with different levels of mechanistic detail and as standalone models for individual problems, or as models that are integrated with other problems. Two key elements of duplicate gene retention are coding sequence function modeled using summary statistics (like dN/dS) or with Markov models describing increasing layers of complexity and expression evolution. These can also include models for syntenic position. While some of the Markov models attempt to integrate the two layers of evolution reflecting coding sequence function and expression in a sophisticated manner, other approaches either use a simpler unifying factor, like [P] in a biophysical model or treat them independently or without differential specification. Work in these directions is making substantial progress in capturing biological realism. Modeling of duplicate gene retention can converge with the broader modeling frameworks being developed for the genotype-phenotype map. While much of this modeling is in the realm of additive statistical association, the field of computational systems biology includes modeling frameworks that add another layer to the genotype-phenotype map that haven’t been touched much at the boundaries of the duplicate gene retention modeling field. Mechanistic models for gene expression evolution will also be fruitful in this field. There is a lot of room to keep expanding these modeling frameworks as genomic and other omic data accumulates for species and underlying populations and as biological domain-specific modeling improves that can improve mechanistic duplicate gene retention models.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nKatju V, Lynch M: The structure and early evolution of recently arisen gene duplicates in the Caenorhabditis elegans genome. Genetics. 2003 Dec; 165(4): 1793–1803. 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New York: Cambridge University Press; 1992." }	[ { "id": "218598", "date": "22 Nov 2023", "name": "Nadia El-Mabrouk", "expertise": [ "Reviewer Expertise Computational biology", "phylogeny", "gene/species tree reconciliation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting review on the dynamic of gene retention and loss, depending on gene structure, gene function, the mechanism creating the duplicated copies, and other biological constraints. It presents various mathematical models characterizing such gene duplicates retention/loss dynamics in a context-dependent way. In particular, the difference between gene duplicates evolution depends on whether gene copies result from small scale duplication or whole genome duplication (WGD). The last part of the paper also explains the probabilistic and algorithmic ways of predicting WGD events.\nMy main criticism is that the review intermingles a variety of biological concepts and mathematical models, with no clear subdivision. The introduction should clearly explain the objective of the review and how it is organized. The content should be subdivided into coherent subsections. I would suggest adding a table with all the various constraints for gene duplicability and retention listed in the different sections, summarizing which of them are considered by which model/paper.\nIn addition, it would be interesting to discuss gene retention as a function not only of small-scale duplication versus whole-genome duplication, but also as a function of the type of small-scale events: duplication of a single gene versus duplication of larger segments, tandem duplication versus retrotransposition.\nFew other comments:\nMathematical formulae should be better written. This may be a problem linked to F1000Research's writing constraints? Writing “1-(0.5)^(f-1)” or event 1/2N is unclear. Is it 1/(2N) or (1/2)N? Although the target audience should be familiar with most of the biological and mathematical terms used, some are less obvious and should be explained, such as “heteromultimerization partners”. On page 7, correct the sentence: “The relative frequency of formation of these two types of polyploids many be approximately equal”. I guess “many” should be replaced by “may”. On the same page, 6th line before end: “As described, selection from various sources can also play a role.” As described where? First part of Figure 2: I guess the segments, for each genome, above and below that for L. oculatus correspond to the two paralogous syntenies? That should be specified. Caption of Figure 3: “Transitions between these states occur at rates proportional to model parameters ?, ɛ, and γ”. “?” should be corrected.\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": "10983", "date": "22 Mar 2024", "name": "David Liberles", "role": "Author Response", "response": "Response to Professor El-Mabrouk, Assis et al. This is an interesting review on the dynamic of gene retention and loss, depending on gene structure, gene function, the mechanism creating the duplicated copies, and other biological constraints. It presents various mathematical models characterizing such gene duplicates retention/loss dynamics in a context-dependent way. In particular, the difference between gene duplicates evolution depends on whether gene copies result from small scale duplication or whole genome duplication (WGD). The last part of the paper also explains the probabilistic and algorithmic ways of predicting WGD events. >We thank Dr. El-Mabrouk for these comments and review. My main criticism is that the review intermingles a variety of biological concepts and mathematical models, with no clear subdivision. The introduction should clearly explain the objective of the review and how it is organized. The content should be subdivided into coherent subsections. I would suggest adding a table with all the various constraints for gene duplicability and retention listed in the different sections, summarizing which of them are considered by which model/paper. >We have now added a paragraph to the introduction on the organization of the work. We have also added the suggested table. In addition, it would be interesting to discuss gene retention as a function not only of small-scale duplication versus whole-genome duplication, but also as a function of the type of small-scale events: duplication of a single gene versus duplication of larger segments, tandem duplication versus retrotransposition. >A discussion along these lines has been added. Few other comments: Mathematical formulae should be better written. This may be a problem linked to F1000Research's writing constraints? Writing “1-(0.5)^(f-1)” or event 1/2N is unclear. Is it 1/(2N) or (1/2)N? >This and other formula have now been written more formally. Although the target audience should be familiar with most of the biological and mathematical terms used, some are less obvious and should be explained, such as “heteromultimerization partners”. >An explanation of this term has been added. On page 7, correct the sentence: “The relative frequency of formation of these two types of polyploids many be approximately equal”. I guess “many” should be replaced by “may”. >This has now been corrected. On the same page, 6th line before end: “As described, selection from various sources can also play a role.” As described where? >This sentence has now been clarified. First part of Figure 2: I guess the segments, for each genome, above and below that for L. oculatus correspond to the two paralogous syntenies? That should be specified. >This has now been specified in the figure legend. Caption of Figure 3: “Transitions between these states occur at rates proportional to model parameters ?, ɛ, and γ”. “?” should be corrected. >This has now been corrected." } ] }, { "id": "228194", "date": "02 Jan 2024", "name": "Arturo Becerra", "expertise": [ "Reviewer Expertise Early evolution of 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\nThe authors have provided a succinct yet comprehensive overview of the primary models governing the retention of duplicate genes—a critical biological process. The manuscript is well-crafted and explores a subject of significant originality, mainly as there are few or no existing text reviews of this nature. However, I have a minor concern and a suggestion:\nThe text appears densely packed given the format constraints, resulting in brief coverage of specific models, such as the Dosage Imbalance Cost. While I understand the limitations imposed by the format and the challenge of expanding on each model, I propose including a table or images that summarize the characteristics of these models, both mathematically and biologically. This addition would greatly aid in providing readers with a more precise and concise understanding of the models.\nMinor point. Page 7, third paragraph, last line: \"including all vertebrates and flowering plants, as well as lineages of yeasts, ciliates and other plants.\"\nChange by: ... including all vertebrates, flowering plants, lineages of yeasts, and ciliates.\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": "10984", "date": "22 Mar 2024", "name": "David Liberles", "role": "Author Response", "response": "Response to Professor Becerra The authors have provided a succinct yet comprehensive overview of the primary models governing the retention of duplicate genes—a critical biological process. The manuscript is well-crafted and explores a subject of significant originality, mainly as there are few or no existing text reviews of this nature. >We thank the reviewer for this comment. However, I have a minor concern and a suggestion: The text appears densely packed given the format constraints, resulting in brief coverage of specific models, such as the Dosage Imbalance Cost. While I understand the limitations imposed by the format and the challenge of expanding on each model, I propose including a table or images that summarize the characteristics of these models, both mathematically and biologically. This addition would greatly aid in providing readers with a more precise and concise understanding of the models. >The other reviewer also made a suggestion for such a table, and one has now been added. Minor point. Page 7, third paragraph, last line: \"including all vertebrates and flowering plants, as well as lineages of yeasts, ciliates and other plants.\" Change by: ... including all vertebrates, flowering plants, lineages of yeasts, and ciliates. >A change along these lines has been made." } ] } ]	1	https://f1000research.com/articles/12-1400
https://f1000research.com/articles/11-894/v1	04 Aug 22	{ "type": "Research Article", "title": "Factors related to university teaching that influence academic success of international medical students in China", "authors": [ "Qinxu Jiang", "Hugo Horta", "Mantak Yuen", "Qinxu Jiang", "Mantak Yuen" ], "abstract": "Background: Academic success of international medical students enrolled in Chinese universities is of great significance, because it directly influences their performance in the license exam and in obtaining a job. Insufficient research has been conducted on academics’ awareness of factors related to teaching that affect their students’ academic success.\n\nMethods: Semi-structured interviews were conducted with academics (N=36) from November 2020 to January 2021 at two medical universities in China. Each interview, lasting between 30 to 70-min, was audiotaped, transcribed verbatim and analyzed using thematic analysis.\n\nResults: The important teaching factors that academics perceived to influence the success of students are: (i) style of pedagogy, (ii) addressing students’ language difficulties and differences, (iii) teaching resources management, (iv) attributes of the academics, (v) supervision and guidance by the academics, (vi) rapport between the academic and student, (vii) linking teaching content to license exams, (viii) classroom discipline management, and (ix) assessment style.\nConclusions: University faculties and departments that are involved in teaching international medical students need to ensure that academic staff are provided with ongoing professional development and resources to enhance teaching quality. The nine areas identified above should provide priority topics for such staff training.", "keywords": [ "academic success", "international medical students", "China", "medical education", "university teaching" ], "content": "Introduction\n\nOver the past two decades, the internationalization of China’s universities (Dang et al., 2021; Jin & Horta, 2018) has seen medicine emerge as the most popular program after Chinese language for international students to pursue in Chinese universities (Zhou, 2016). The Bachelor of Medicine and Bachelor of Medicine (MBBS) program taught in English medium is supported and supervised by China’s Ministry of Education (MOE) as well as being designed and implemented by universities (Huang, 2014). An example of this is the release in 2020 of the document Quality Control Standards of Basic Medical Education (in English Medium) for International Students in China. This is from the China Education Association for International Exchange and the International Medical Education Committee, entrusted by the International Department of the MOE. The objectives of this standards document were to further standardize MBBS education across Chinese universities, effectively improve training quality and promoting a sustainable development of MBBS education.\n\n45 universities are authorized by the MOE to recruit international students for medical education (Xie, 2021). The World Health Organization (WHO) recognizes all these universities in the Directory of World Medical Schools. It means international graduates holding a Chinese MBBS degree are eligible to attend license exams in their home country. Examples include the Medical Council of India Screening Test, or the United States Medical Licensing Examination (USMLE), or the UK Professional and Linguistic Assessment Board (PLAB). This is perceived by students as one of the great advantages of studying MBBS in China—in addition to other perceived benefits that include less demanding admission requirements, affordable cost, and a safe, stable society (Zhou, 2016).\n\nTypically, the MBBS program takes six years to complete, with five years devoted to courses in natural science, Chinese language, basic science medicine, and clinic subjects, and one year of internship in Chinese hospitals or in students’ home country.\n\nMost of the MBBS students originate from low-income countries from Asia and Africa, where the demand for health professionals is high (Liu et al., 2017). The majority of the students choose to return to their home country for career development after completing the MBBS program—except for some students who continue further studies (Huang, 2019; Fan et al., 2020). Jiang and Sun (2007) found that 87.4% of MBBS students planned to engage in medicine-related careers after graduation to make a significant addition to the health workforce in their home country, or they would migrate to other high-income countries (Li & Sun, 2019; Han & Guo, 2009). The quality of MBBS education in China will likely influence their future license exam performance and the employment position they obtain. The reputation gained by these courses is the key to sustainable development of medical education for international students (Zhou, 2016).\n\nMedical license exams are fundamental to evaluate the learning of medical schools and their graduates (Han & Guo, 2009). To some extent, the later success rate of MBBS graduates reflects whether China’s MBBS education meets international standards (Mao et al., 2012). In this respect, the results to date have been sub-standard when compared to those in several countries. For example, China has the most Indian students compared to other countries, who are required to pass the Foreign Medical Graduate Examination (FMGE) to practice medicine in India (Muthyanolla, 2022). From 2015 to 2020, the total number of students sitting the FMGE from China was 43,632, but the average passing rate was only 12.51% (Muthyanolla, 2022). Similarly, the passing rate of Nepalese students from China in Nepal Medical Council (NMC) Examination from 2016-2018 was only 34.3%, behind that of Nepalese graduates from Nepal (80.3%), Philippines (74.8%), Bangladesh (60.9%), Pakistan (58.4%), India (49.2%), Russia (44.6%), and Ukraine (40.9%) (Aryal, 2018). These results indicate that the majority of China-educated MBBS graduates do not have the expected level of professional knowledge and skills.\n\nRecent studies, while acknowledging the accomplishments of medical education for international students in China, have pointed out there are still several problems to be addressed. These problems are mainly identified as: lack of a unified training program for international medical students (Liu et al., 2021b), language barriers between lecturers and students (Ding, 2016; Huang, 2019), ineffective teaching pedagogy (Yang et al., 2019a), insufficient well-qualified and experienced academics, and poor teaching resources (Ding, 2016; Huang, 2019; Wang et al., 2018), absence of robust quality assurance system (Liang & Liu, 2019), classroom discipline issues (Zeng & Sheng, 2019), and poor student quality (Ma et al., 2018; Huang, 2019). It should be noted that many of these studies were based on the authors’ own working experience and subjective reflection, so lacked empirical evidence. A few Chinese studies adopted a more objective quantitative method by surveying international students’ opinions, but the views of academic staff were rarely solicited. The study reported here was designed to address this important issue by interviewing academics.\n\nTo the best of our knowledge, this is the first study in China delving into the beliefs of academic staff concerning pedagogical factors that influence success of international medical students. The findings may contribute to a better understanding of challenges faced by international medical students in China, and offer a direction for improving the quality of teaching in these universities. This is important for ensuring that graduates have the necessary expertise—and is very important for the reputation of the universities.\n\n\nMethods\n\nThis study was approved by Ethics Committee of Xuzhou Medical University (XZMU20200028) and Institutional Review Board of Nanjing Medical University (NJMUIRB774) in [September] 2020. All participants gave written and oral informed consent to participate in the study.\n\nThe study was performed in two medical universities (A and B) located in Jiangsu Province, eastern China. Both universities had over 15 years’ experience in teaching international MBBS students, so it was reasonable to regard them as relevant sites from which to gather perspectives of academics engaged in teaching. A similar MBBS curriculum was adopted at these two universities, in line with the requirement by the MOE. MBBS students and Chinese students took separate courses and did not mix in the classroom. The courses for international students were taught principally through the English medium. The majority of their international students were from developing countries in Asia and Africa, and some students were far from proficient in English.\n\nPurposive sampling was adopted in this study. Criteria for selecting interviewees were: academic titles, years of teaching, gender, and course taught. The aim was to obtain a heterogeneous sample from the medical teaching body at the two universities. Academics were approached individually by phone call and WeChat invitation with the help of two “gatekeepers”. The target sample size before recruitment was around 30, but had to be increased until data saturation was reached (Morse, 1991). A total of 40 academics were approached, but 2 of them declined the interview invitation and another 2 initially agreed but later withdrew by not responding to the researcher’s text messages.\n\nIn total, 36 academics (17 males, 19 females) were recruited, including 2 teaching Chinese, 3 teaching nature science, 11 teaching clinical subjects, and 20 basic medical science. The participants had between 2 to 13+ years of experience teaching international students; and all were responsible for teaching the same subject to both Chinese and international students.\n\nSemi-structured interviews were conducted with each participant from November 2021 to January 2022. Only the first author and participants were present for each individual interview. Academics from University A participated in face-to-to-face interviews in their office or a small meeting room, where it was perceived to be comfortable and quiet, while online interviews were conducted with academics from University B (due to the coronavirus pandemic). Both face-to-face and online interviews were audio-recorded. Field notes were taken during the interviews. The interview questions, which were pilot-tested, addressed participants’ views on factors affecting student success, and their recommendations for improving teaching quality at classroom level and organizational level.\n\nBefore the interview, academics were debriefed concerning the purpose and significance of the study, and the likely length of the interview. Interviews were conducted in a quiet and comfortable environment. In conformity with ethical requirements, all interviewees were assured of anonymity and agreed to audio recording and dissemination of findings. Each interview, conducted in Chinese, lasted for around 30 to 70 minutes.\n\nA thematic approach was used to analyze data (Braun & Clarke, 2006), with recorded audios later transcribed verbatim in Chinese. The Chinese transcripts were coded manually by the primary researcher, who immersed herself in the data and noted down her initial ideas on emergent themes. Initial codes were generated based on identified key words and phrases. Next, the codes were summarized into potential themes for each participant. The primary researcher translated the codes and themes into English. Then the researcher team discussed, adjusted and reached a consensus on final relevant categories. Trustworthiness was guaranteed by member checking, peer auditing and self-reflection. During the whole research process, the primary researcher kept a self-reflective journal to minimize the effect of her own background and work experience on the interpretation of the data.\n\n\nResults\n\nAn overwhelming majority (86%) of academics perceived their teaching methods and practices to be a major influential factor on international students’ learning. They perceived a mismatch between their teaching approach and the learning characteristics of students. Most academics still relied on traditional teacher-centered lecturing, and tended to read verbatim from slides without properly explaining further or adding practical examples that could enhance the students’ comprehension of the subject. Some academics had noticed that lectures as a learning approach may not be suitable to international students’ learning needs. One academic teaching clinical medicine commented that in a lecture on clinical manifestations and diagnosis of diseases, the students were more concerned about the treatment and recovery from diseases. The academic suggested:\n\nStudents enjoyed learning about clinical treatments because they may think that it would be useful for their clinic work in the future. However, treatment is only briefly introduced in this class.\n\n(Participant 25)\n\nUnlike Chinese students, international students asked more questions and would interrupt the class to express opinions. This made the academics think more about how to communicate with them, and how to improve teaching quality. The majority of academics felt that much of the current teaching was spoon-feeding information and lacking interaction. Lecturers were failing to grasp students’ attention and hold their interest in class, which often affected their exam performance. Interviewees felt that teaching innovations such as case-based learning (CBL) and problem-based learning (PBL) should be used to teach international students, because this type of teaching mode was more heuristic, which would encourage students to learn more actively. They suggested CBL and PBL would increase the involvement of international students in learning activities. They could also make more frequent use of individual and group presentations and discussions, encourage asking questions, guiding students to think, and trying flipped class teaching (setting preparation assignments on a lecture topic before presenting the lecture). However, academics also felt that implementing these changes would be a challenge for them, because it would place more demands on their language ability, subject knowledge, ability to listen and respond to students, and control the class. It was pleasing to note that one of the few academics who already involved international students in interactive class learning activities reported positive changes in performance of these students:\n\nInternational students are not interested in the traditional lecturing we use to teach Chinese students. Very few students listen carefully in class. I usually ask international students to summarize the content of the previous lesson in front of the whole class. They would become very active to participate and prepare well. I think international students like to show themselves in public. If I take advantage of this characteristic of theirs, I can stimulate their passion for learning.\n\n(Participant 31)\n\nCompared to Chinese students, international students were relatively weaker in basic knowledge and learning. Academics acknowledged the diverse academic abilities of international students and their different culture backgrounds and agreed that teaching approaches should be adjusted to these student characteristics. They also believed that it would be important for them to give culture-relevant examples in class that students were familiar with, make timely summaries of key knowledge points, give practice assignments after class, cover exam points, and provide timely feedback on assignments. An academic teaching radiology gave the example below:\n\nFor international students, you have to start from the basics. For example, in my [radiology] class, there is a CT value, high or low. High is bright, low is dark. You have to explain this foundation setting first; otherwise, they would become confused. Doing this sort of thing could help them, but would require academics to take more time to prepare for class.\n\n(Participant 19)\n\nInterviewees reported that real clinical cases needed to be integrated into basic medicine lectures, to help international students understand them better. They added that international students learn better with case examples. Academics without deep clinical experience but who are teaching biochemistry, microbiology, immunology, and parasitology found this to be a challenge. They suggested it was necessary for them to provide more than theory and facts by including relevant clinical knowledge to facilitate the learning of their students. An academic teaching biochemistry, with a background in biology, stated in this regard:\n\nStudents have asked me some clinical questions, which shows that they have this demand. This demand actually reminds me that I should include clinical knowledge in my teaching. This could make my class more attractive and make them realize that my course is very helpful to their clinical study in the future and can help solve clinical problems. In addition, it can make students realize the importance of my course, at least for now, and improve their learning enthusiasm. I’ve already started learning relevant clinical knowledge.\n\n(Participant 6)\n\nAcademics also stated that their lecture slides probably need to be more interesting to attract students’ attention. The academics stated that there may be a need to use different teaching aids beyond writing on blackboard—such as videos, audios, and animations to make the classes more dynamic and understandable for students. An academic teaching pharmacology mentioned how he utilized online technology to make the learning in his class more effective.\n\nI like to go to YouTube to find some videos about the mechanism of drugs to show in the class. Maybe my explanations are not very good, but the videos explain very well, which can help deepen students’ understanding. Moreover, I feel that playing videos when students are sleepy can increase their attention.\n\n(Participant 22)\n\nAfter teaching pedagogy, the language barrier was identified as a significant challenge in improving the learning of international students. In discussion, 81% of the interviewees indicated that language differences and difficulties are negatively influencing teaching and learning quality. They also offered potential solutions. These Chinese academics spoke English as a foreign language, while international students spoke English as their first or second language (albeit far from perfectly). This is not a harsh criticism of either group, but merely highlights the actual situation. Mutual communication was constrained because the academics were not necessarily fluent in English, and the students were not necessarily fluent either. Academics found it difficult to understand students’ English accents and expressions, which may not be standard American or British English, particularly with students from India, Nepal, and Pakistan.\n\nAcademics revealed that while they could generally command medical English for in their own field after years of teaching, they still struggled with daily-life English and cross-disciplinary English. Limited English vocabulary outside their field affected their teaching, and was one of the main reasons why they often resorted to reading verbatim from slides rather than explaining concepts in their own words, and why they avoided engaging interactively with students. Consequently, students become bored during class, and sometimes were annoyed by poor communication. An academic teaching surgery compared teaching Chinese and international students concerning language in theory lectures.\n\nIf it is a Chinese class, the academic has many methods to teach. He has eloquence and rich experiences; he can solicit and quote from others when giving lectures. However, in case of teaching foreign students, due to the language barrier, academics cannot express themselves 100% smoothly. They can only communicate with students in the simplest way, so they cannot always respond appropriately to students. If they cannot attract students, students will lack interest in the class. Naturally, they do not devote themselves to learning.\n\n(Participant 35)\n\nAcademics teaching clinical medicine who were also responsible for clerkship and internship at hospitals, indicated that it was hard to translate some Chinese words concerning a patient’s symptoms to English for the benefit of the students. Sometimes the translation made by an academic would not exactly match the symptoms. This situation was further exacerbated when international students could not comprehend explanations delivered in daily-life Chinese. Therefore, the effectiveness of clerkship and internship learning was adversely affected because of language barriers, as this academic teaching internal medicine explains:\n\nTake the symptoms of angina pectoris for example. When I talk about angina pectoris to Chinese interns, I will use Xuzhou dialect, which is very easy to understand. However, it is difficult for me to convey the clinical symptoms reflected by most patients to foreign students through a more appropriate word. Because the pain reflected by the patient is not the oppressive feeling mentioned in the textbook. We cannot simply transfer the knowledge from books to students, but we have to combine it with our clinical practice. However, sometimes, it is very difficult to translate properly.\n\n(Participant 25)\n\nOver half of the interviewees (58%) stated that issues with teaching resources also affected teaching and learning. Four sub-issues emerged as part of this theme.\n\n3.1 Demand of textbook and teaching materials\n\nTwelve academics complained that they lacked easy access to necessary textbooks, English question banks, and other relevant teaching materials such as reference books and videos. Universities did not mandate or provide any particular textbook for academics to use, and academics had to search for textbooks, or create their own materials by drawing on different English and Chinese sources. When the textbook of the academic was different from students’ own books, they could have difficulty understanding lectures. Sometimes, students were not assigned any textbook or relevant learning materials. Moreover, academics responsible for different modules or units in a course would often use different textbooks, which led to inconsistencies. Academics felt it is necessary for experts to develop good textbooks to help students understand and review lecture knowledge; and they suggested universities should purchase specialized English books, medical videos, and question banks for use by staff and students. One academic teaching gynecology commented on her difficulty with textbooks:\n\nOur Clinical College once gave us some textbooks, but I think the versions of those books are relatively old. I have not seen updated books later. In those books, old methods of screening gynecological cervicitis, which were used one or two decades ago, were even introduced.\n\n(Participant 27)\n\n3.2 Need for collaborative lesson planning\n\nSeven academics felt that it was important to engage in collective lesson planning for intellectual communication. This is lacking at present, but may be an important procedure for a teaching team to share experiences and discuss which knowledge points are the most important for students to master. In these planning sessions, application of flipped class teaching could be debated, and how to properly create synergies among different topics taught by different academics. Moreover, with collective lesson planning, new academics could be given a demonstration on how to teach international students by more experienced academics. Academics inexperienced in teaching international students expressed their needs to receive teaching tips on pedagogies, and have feedback from more experienced lecturers. However, in most departments, this collective lesson planning and guidance was lacking, particularly clinical medicine subjects because the teachers were busy with their own clinical and research work:\n\nTheoretically, there should be collective lesson planning, but it cannot be done in practice … Teachers have their own understandings of knowledge and lecturing methods, and collective lesson preparation has little impact on them … Basically, teaching has been weakened and become a mere formality. There is no teaching demonstration for new teachers any more. The supervision mechanism has gradually disappeared. Now the overall orientation no longer takes teaching as the main evaluation index, but mainly takes SCI publications.\n\n(Participant 24)\n\n3.3 Weak teaching supervision system\n\nSeven academics reported that universities lacked a sound supervision system for teaching quality, so academics might well conclude that it makes no difference if one teaches well or not. Without a system that has rewards for good performance and penalties for poor performance, academics would have no incentive to teach well. Strengthening supervision of teaching by including regular observation by experts and spot checking of lecture quality was something that some academics desired.\n\nThe School of International Education needs to establish a system or assessment standard, and implement rewards and punishments in each teaching and research department. If there is no difference between good and bad teaching, teachers will not be motivated.\n\n(Participant 33)\n\n3.4 Inadequate clinical teaching resources\n\nFive senior clinical academics mentioned there was a shortage of well-qualified persons to teach theory, clerkship and mentoring internships in hospitals. In some departments, due to high turnover of staff, there was lack of a stable teaching team responsible for international students. Recently hired academics and new doctors who had minimal or no prior teaching experience were asked to teach these students. Their teaching would be limited to reading their notes or slides aloud, without explanation or discussion. Academics with teaching experience hoped universities and hospitals would attach more importance to consistency in teaching international students, and to introduce incentives to encourage young, qualified doctors to participate in teaching. There was a perceived need to train new academics for future teaching work:\n\nDuring the clerkship, we had two teachers to teach 25 international students. The venue space and teachers were not enough … Internship was more like clerkship learning just for a visit in the case of foreign interns, because some teachers could not speak English and they were very busy. Foreign interns could not operate due to doctor-patient relationship concern. They could not receive much guidance … You know, this is a problem.\n\n(Participant 24)\n\nThree academics also mentioned that their course syllabus was obsolete. A senior academic who teaches pediatrics said it was imperative to discuss with international students to know which diseases they needed to learn about for work in their own country. The course syllabus could then be updated accordingly. This would make it easier for students to relate to the content being taught in class. In this regard, academics suggested their departments should evaluate their existing syllabus, and improve it where necessary by making it more relevant.\n\nApproximately 50% of interviewees disclosed that the attributes of academics, such as their dedication, passion for teaching, attitude, sense of responsibility, and subject knowledge played a pivotal role in ensuring teaching quality that helps students learn. The academics interviewed commented that much more time and effort is needed to prepare lessons for international students because of the modifications required. Some perceived the workload of preparing a lesson in English was three to ten times greater than preparing a lesson for Chinese students. For instance, they would have to write a lesson script beforehand to read in aloud in class, because it was too challenging to improvise their English during the session. This type of language barrier would not occur in the Chinese class.\n\nThe interviewees admitted that many academics were not motivated to engage in teaching international students because universities prioritized research over teaching. There were no incentives for academics to devote time and effort to areas that would not advance their careers. Therefore, they felt more inspired to do research and were much less driven to teach international students, even though they were qualified. Moreover, some academics might become apathetic about teaching, which would affect the quality of their contribution. A typical observation was:\n\nAll materials for lesson preparation must be in English. It is already very difficult to prepare lessons in Chinese. I have to prepare lessons in English and practice my manuscript … In this current university environment, which generally attaches importance to scientific research rather than teaching, everyone is willing to focus on their own research rather than pay attention to teaching. Research awards are more tempting… When it comes to professional rank evaluation, papers are more useful than a teaching competition award.\n\n(Participant 3)\n\nDespite the common perception of heavy workload, challenges, and dissatisfaction with a lack of incentives, some academics maintained a personal commitment to their teaching out of a sense of responsibility and self-improvement.\n\nTo maintain the enthusiasm of academics in teaching, it is not about monetary stimulation, but a sense of value and honor. Academics are not unsentimental people … Many academics are dissatisfied, but they continue to teach even if they are not satisfied. This is dedication, awareness and sentiment … I don’t want to talk about interests, but the school should give me some other aspects that emotionally make me feel honored to teach international students.\n\n(Participant 4)\n\nWhen asked about what incentives universities could provide to motivate academics, the interviewees claimed that instead of placing MBBS education in a peripheral position, university management should attach more importance to MBBS education and to the academics who provide it. They expressed strong need for continuous professional learning to improve pedagogies, and assistance with oral English through formal or informal learning opportunities at home or abroad. Several academics claimed that a university should count their teaching performance as part of any evaluation for promotion, rather than solely basing it on number of publications. This would ensure that academics would become more motivated and enthusiastic to teach. A few academics also desired funding opportunities to implement teaching innovations in the MBBS program, but it was not possible. Two academics hoped the university could count their class hours as double or three times more than Chinese class hours, because they spent much more time on lesson preparation and had a need of class hours for academic promotion.\n\nWhen asked what academics could do to help students improve their academic performance, nearly 50% of the interviewees indicated that there was a need for them monitor their students’ progress more closely, and to push underperforming students harder to be responsible for their study. Students could be lazy and lack self-discipline. Academics felt that if they could devote more time into supervising low-achieving students and talking with them after class to encourage them, the students may feel they are being cared about and be more motivated to study. An academic teaching cell biology highlighted the importance of encouraging and caring about students:\n\nStudents are still children. If academics communicate with them more, they will feel pressure and study under pressure … In the first year of my teaching, I dedicated all my energy to push them. I tried my best to memorize all the students' names and faces. They felt my care. Several students still have been in contact with me since graduation … I was busy with my own life and family, but if I had spent more time at that time, I could have saved a few more students from failing.\n\n(Participant 21)\n\nMoreover, six academics felt it was not only important to impart knowledge and skills to students, but also moral and ethical education, values and responsibility in class. They stated that it was critical to educate students to cherish learning opportunities in China, correct their learning attitude, and be responsible for medicine. By doing so, students’ intrinsic motivation could be stimulated with a sense of respect and responsibility, as stated by an academic teaching anatomy:\n\nIn anatomy class, the specimens are donated by others. No matter the country in the world, the donors are the worthiest of respect. As a medical student, you should respect them and patients, because they, ordinary people, have given us the opportunity to learn and explore medicine. You should treat patients well in the future. Why did they donate their bodies? Because they hoped you could become a good doctor.\n\n(Participant 28)\n\nApproximately 31% of the interviewees mentioned that getting to know about students and their cultures and establishing good teacher-student rapport would be conducive to better communication and teaching. In particular, communication between academics and students could help build emotional connections and understanding in class. For example, without knowledge of students’ lives and cultures, any academic may accidentally say culturally insensitive statements that can cause discomfort. An academic teaching orthopedics with more than ten years’ teaching experience with international students shared how he successfully developed a good relationship with students by incorporating students’ cultures into class.\n\nOnce, as soon as I finished watching the Indian film Dangal, I quoted 10 classic sentences into my slides to motivate my students in class. They became very interested when they saw these quotes. “Wow, this is our Indian movie.” Sometimes, I talk about my feelings about Nepalese festivals in class. They feel excited and think the teacher acknowledges their culture… If students like you, they will learn your course well.\n\n(Participant 18)\n\nAn academic teaching anesthesiology mentioned that academics teaching clinical subjects generally knew less about their students, and thus they failed to establish good rapport. This made their lectures seem rigid and lacking in attention to the students as individuals. The academics teaching clinical medicine suggested that universities should arrange activities to increase communication between staff and students. This is particularly important for younger academics.\n\nOver a quarter (28%) of the interviewees pointed out that one of the primary reasons why many international students lack interest in learning is probably because the current course content and resources do not correspond closely enough to the demands made in the license exams. Academics usually teach the knowledge that they think is important, and this may not precisely match a curriculum based on the license exam. What is required is a blending of both academics’ specialist knowledge and a prescribed curriculum. This issue was critical, because students would be more motivated to learn things if they perceived them to be useful to them in passing examinations, and in their working life. However, some academics argued that it would be difficult to address license exam needs of students from diverse countries, where exam content may vary. In this situation, it seemed essential to develop a more contextualized curriculum, as explained by an academic teaching anatomy:\n\nOne of the key points of our teaching is to help students pass license examination. If a student does not pass the exam, he/she will not even qualify for matching hospitals. This is true of both Chinese students and foreign students. Teaching and license examination cannot be separated. If we want to help students pass the license exam, teaching activities should play a guiding role … The passing rate of license exam is the most important measure standard of a medical university. Practice is the sole criterion for testing truth.\n\n(Participant 2)\n\nChinese academics felt frustrated and perplexed by international students’ certain behaviors in class. International students were often late for class, left class in the middle for no reasons, listened to music, used smartphones, or would chat with others and made noise. These behaviors negatively influenced the mood and positivity of academics towards international students, as by comparison such discipline problems would rarely occur in Chinese students’ class. Seven academics (19%) stated that it might be students’ own cultural habits, but they should follow Chinese rules. These academics dedicated certain time to disciplining students in class to make students more serious about study. However, more academics emphasized that it was the student administrators’ responsibility to strengthen student discipline management. One academic teaching hygiene explained how she managed class discipline to push students learn.\n\nIn sharp contrast to Chinese students, foreign students pay too little attention to class discipline… For example, for morning class at 8 a.m., some students cannot come until 9 a.m. I wondered why. Later, I went to Pakistan for a visit. When I went to a university, I understood that this was their habit. However, in China, if you want to obtain a Chinese degree, you have to follow Chinese rules … I count the check-in and lateness into class participation score, and I will call the roll five minutes in advance. Sometimes I close the back door in class for fear that they might sneak out. Sometimes I put roll call on the second quarter of class to prevent someone from sneaking out after signing in … It is like fighting a battle of wits and courage with them.\n\n(Participant 30)\n\nSix academics (17%) stated that the current assessment system was problematic, mentioning the exam evaluation system and the final elimination system.\n\n9.1 Exam evaluation system\n\nThere is no formative assessment but only a summative assessment based on final exam performance. Academics suggested that the university add formative assessments such as a mid-term exam or in-class quizzes, because it could help academics to follow students’ progress and difficulties, and push students to study harder, and decrease the final exam pressure on students.\n\nOne academic teaching pathophysiology mentioned that the evaluation of international students’ academic performance should take into account of their characteristics rather strictly follow the same evaluation system of Chinese students. This academic felt that international students were more interested in practical learning in the laboratory and generally outperformed Chinese students, so the university could consider increasing the weight of practical score into their final course score.\n\nAcademics also mentioned they wanted to change the assessment methods, but in most cases, it was beyond their control unless the changes were approved at the organizational level after going through complicated application procedures. One academic teaching parasitology exemplified how their department improved their assessment method based on students’ feedback.\n\nStudents came to complain that the exam was unfair because some students did not attend class, just memorized key points the week before the exam and got higher marks. After receiving their feedback, we improved. We took into their formative performance as a part, and the final exam accounted for only 50%. Answering questions and signing in to class are all part of the formative performance. I think we should add formative assessment and tell students to listen to the whole class. Our purpose is not to let them fail in the exam, but to guide them to learn something. Students understand this. Finally, almost all students passed my course.\n\n(Participant 29)\n\n9.2 Final elimination system\n\nSeven academics pointed out university assessment and evaluation systems were less strict with international students. It was perceived as a loose entry and exit to the course, which would have no pressure on students to study. These academics suggested the university should implement a final elimination system to delay graduation or fail those who are disqualified for graduation and a MBBS degree.\n\nThe university must strictly enforce the examination system and cannot deliberately make exceptions in international students’ favor. Schools should dare to weed out unqualified students, so that they will be afraid. Once they are afraid, most of them will study hard.\n\n(Participant 10)\n\n\nDiscussion\n\nFrom the standpoint of academics, this study revealed critical factors that are worthy of attention with respect to academic success of international students in China. Nine key themes emerged covering challenges that include teaching pedagogy, English language, teaching resources management, teacher attributes, teacher supervision and guidance, teacher-student rapport, detachment of teaching content from license exams, class discipline management, and assessment styles.\n\nIt is evident that teaching methods and language barrier were the most frequently reported factors that impacted Chinese academics’ teaching effectiveness and international medical students’ academic success. This finding is in keeping with that reported in the systematic review by Dang et al., (2021), in that these are the most common challenges facing Chinese academics in English-medium-instruction (EMI) programs. The finding of teaching method as the most dominant factor perceived relevant to student academic success, is aligned with the findings of Helm and Guarda (2015). They reported that in an Italian university, most lecturers in programs where English is the medium instruction needed to improve their teaching competences. The literature also indicates that the pedagogical abilities of academics seem to be greatly influenced by language barriers (Ding, 2016; Huang, 2019; Zhan, 2017). Yang et al. (2019b) found EMI teachers in a Chinese university, more than non-EMI teachers, became more didactic and less interactive in class, which then hindered teaching and learning. Despite this, improved English skill does not necessarily equate to improved pedagogical changes (Dafouz, 2018; Dang et al., 2021; Helm & Guarda, 2015). The finding of this study suggests that while it is important for the university to address the oral English abilities of academics in both English related to a specific discipline and English in daily life, it is more imperative to focus on improving the pedagogy of these academics as key to academic success of international medical students. Universities should offer ongoing professional learning in teaching approaches such as PBL and CBL for pre-service and in-service academics, taking full consideration of the needs, ability and interests of international medical students (Liu et al., 2021b; Saleh et al., 2012; Yang et al., 2019a). Meanwhile, universities should strongly encourage and support teaching innovations with sufficient funding.\n\nThis study uncovers that limited access to teaching resources largely inhibits teaching and learning. The lack of uniform English textbooks and relevant teaching materials for international students has also been identified in many universities in China (Huang; 2019; Jiang et al., 2014; Yang et al., 2019a; Zeng & Sheng, 2019), and in other developing countries, such as in a Ukrainian university where EMI programs were taught to domestic students (Goodman, 2014). It is acknowledged that textbooks are still the main carrier of subject content, therefore, the selection and use of textbooks will directly affect the teaching and students’ knowledge attainment (Li & Cui, 2010; Li et al. 2013). Compared with the rich curriculum integrated teaching resources in developed countries in Europe and the United States, China still lacks a set of English medical teaching materials with complete independent intellectual property rights and in line with the characteristics of its medical education in terms of pertinence and adaptability (Wang et al., 2018). The dire shortage of English teaching resources has limited the sustainable development of medical education for international students in China (Wang et al., 2016). Although some Chinese universities have started to write teaching textbooks on their own, and have made progress, there are also some problems. For example, the English level of Chinese teachers is uneven, and the writing style cannot be unified (Wang et al., 2018).\n\nIn alignment with existing literature (Ding, 2016; Huang 2019; Yang et al., 2019a; Zeng & Sheng, 2019), the findings of this study show that lack of collaborative lesson planning for MBBS classes, shortage of clinical teaching resources, and weakened supervision mechanisms are prevailing in Chinese universities. With regard to the issue of perceived obsolete syllabus, Zhou (2016) shows that Tianjin Medical University has added relevant teaching content according to changes of the disease spectrum in the country of origin. For instance, contents such as Schistosoma aegypti and Schistosoma mansoni which are prevalent in Africa were added to the course of Human Parasites. The case of Tianjin Medical University implies that organizational support plays a critical role in guaranteeing the quality of MBBS education.\n\nBlömeke et al. (2016) found the quality of academics in terms of their experience, education background, motivations, professional knowledge and self-efficacy to predict student achievement. Similarly, the finding of this study suggests that the attributes of academics such as passion for teaching, professional responsibility, dedication, attitude, and content knowledge are perceived to have an effect on teaching quality. As indicated by the interviewees, academics are often demotivated to teach international students because of a lack of emotional, material, and career incentives. Lacking incentives seems a prevalent phenomenon, as reported by other universities in different provinces in China—to name a few, Liaoning Medical College (Ding, 2016), Three Gorges University (Zeng & Sheng, 2019), and Zhengzhou University (Zhang, 2016). This problem was also found in a study in Sweden (Stenfors-Hayes, 2010). This suggests that university policy makers need to be attentive to the opinions and needs of academics if they aim to formulate an appropriate reward system that can promote teaching quality for student success.\n\nThe finding concerning supervision and guidance for students and its effect on student success is new to the literature. Chinese academics subconsciously assumed a parental role to discipline international students, which implies their commitment to education. Moreover, moral and ethical education for international students during and outside lectures has been recognized in Chinese studies (Liu et al., 2021a; Wang & Huang, 2019). This may be more relevant to a Chinese context because of the influence of Confucian ideology in Chinese education that academics should not only impart knowledge and skills but also bear an obligation to cultivate the mind (Ye, 2001).\n\nSimilar to the findings of previous studies in China (Yang & Lei, 2021; Zhao, 2021) and Iraq (Saleh et al., 2012), teacher-student rapport is an overlooked dimension in MBBS teaching. Lack of good teacher-student rapport, due to differences in language, cultural background, and religious beliefs, could make the dynamics within a classroom dull and rigid (Yang & Lei, 2021). Building a good relationship between academics and their students could help foster a classroom environment that is favorable to learning. This requires academics to learn more about the life and learning characteristics of international students, and to communicate with them more after class to establish a harmonious and supportive teacher-student relationship.\n\nThe issue of teaching course content that is detached from license exam requirements has been recognized in some Chinese studies (Han & Guo, 2009; Huang, 2019). The universities with more capabilities have taken action to integrate license exam content in their teaching. For example, the MBBS curriculum at Zhejiang University is internationalized by reference to UK medical education standards and clinical executive standards in Tomorrow’s Doctors (Fan et al., 2020), United States Medical Licensing Examination, and Professional and Linguistic Assessment Board. Li and Cui (2010) from Dalian Medical University suggest introducing license exam tutoring by inviting foreign academics to teach Chinese academics by studying exam questions and content; but this was seen as separate from regular teaching. This difference between the two cases implies that university staff should certainly prepare students for license exams, but also shape the curriculum to include topics in curricula tested in license exams (e.g. FMGE).\n\nClassroom disciplinary issues with international students are common and can negatively influence the lecturing style of academics. This was also identified in other studies (Jin et al., 2009; Zeng & Sheng, 2019). Some academics take disciplinary action with problem students while others ignore the behavior but feel uncomfortable with the lack of control. In a survey of learning experience in a medical university in Southwest China, half the MBBS students felt academics were not strict enough with class discipline (Yang & Lei, 2021), probably because of language barriers. Yet, the finding of this study suggests that academics should try to ensure good disciplinary management for the sake of well-behaved students. Action needs to be taken with misbehaving students to focus their attention on attendance and listening during lectures.\n\nThe finding on the effect of assessment style on international medical students’ academic achievement has scarcely been discussed in the literature. In one study conducted in Ningxia Medical University, Zhang et al. (2020) focus on how the implementation of formative assessment in their MBBS microbiology course helped improve class attendance and learning attitude, develop creativity and self-directed learning habits, increase teacher-student communication, and make evaluation fairer and more objective. This case shows the advantages of revising assessment styles. Notably, Zhao et al. (2020) claim that the current assessment of students in medical colleges and universities in China is generally based on summative assessment (at the end of the course), and that there is a lack of understanding of and attention to formative (ongoing) assessment and evaluation during the course. Summative assessment is seen by the interviewed academics as a relatively single and weak assessment method that could reduce students’ enthusiasm for learning. Assessment only at the end of a course makes it difficult to monitor progress in students’ growth in professional knowledge (Zhan, 2017). Therefore, it is important for universities to transform assessment styles appropriately to help international medical students learn.\n\nIn congruence with other studies (Wang, 2013; Zhang & Hu, 2019), it was noted that there was often a tendency in Chinese universities to graduate international students by lowering exam difficulty or pass mark, or modifying the students’ marks to help them graduate. These behaviors not only severely undermine student quality but also affect the international reputation and status of Chinese higher education. There needs to be a system that deliberately holds back any student who is not mastering the knowledge and skills taught in the MBBS program.\n\n\nConclusion and limitations\n\nThe findings of this study show that teaching-related factors play a critical role in influencing international medical students’ academic success. The problems identified here, when associated with other literature, suggest that the same problems are prevalent across other Chinese universities that host international medical students. It seems that although China MOE attaches great importance to development of MBBS education quality, there are still many issues in implementing its policies at the university level. These may be related to the importance and support each university attaches to the MBBS program, as well its own capabilities to teach the program effectively. The findings highlight that university policy-makers should devote more attention to the quality and development of MBBS education. Academics are the implementers of internationalization of education and they are the developers of educational resources (Ding, 2016). Universities have the responsibility to support and meet the needs of academics in terms of professional learning and in provision of incentives, as well as being attentive to their recommendations for how to improve teaching quality. Efforts at individual level, department level, and university level should be jointly made to ensure high teaching quality, which is the key factor to promote student academic success.\n\nThis study was conducted at two sites, but caution with generalization of the findings is needed. They may be relevant to other Chinese universities offering courses to international students, but not to other settings. In future studies, this research will be extended to investigate the perceptions of academics on the salient student-related factors that may affect their academic success.\n\n\nData availability\n\nThe datasets generated and/or analyzed during the current study are not publicly available due to privacy agreements between the researchers and the interviewees. Making the interviews available – considering the relatively low number of international students at both Medical Universities – would likely compromise the anonymity of the respondents, thus breaking the ethical consent agreement that was established. Requests for data can be considered and if considered reasonable (e.g. having a professor position at a university) are available from the corresponding author Hugo Horta (horta@hku.hk) or Qinxu Jiang (e-mail: jiangviolet86@hotmail.com). English language partial transcripts will be provided.\n\nfigshare: Interview protocol.docx. https://doi.org/10.6084/m9.figshare.20290566.v1 (Jiang, 2022).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAryal BP: Docs trained in China, Russia, Philippines fail licensing exam more often. My Republica;2018, September 7.Reference Source\n\nBlömeke S, Olsen RV, Suhl U: Relation of student achievement to the quality of their teachers and instructional quality. IEA Research for Education. Springer International Publishing;2016; (pp. 21–50).\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual. Res. Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nDacre JE, Fox RA: How should we be teaching our undergraduates? Ann. Rheum. Dis. 2000; 59(9): 662–667. 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(Chinese)." }	[ { "id": "193769", "date": "04 Sep 2023", "name": "Muhammad Azeem Ashraf", "expertise": [ "Reviewer Expertise 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 article is focused on an important topic, and limited data is available on this topic, especially in English. The paper is written well and applies appropriate methods to collect and analyze the data. Authors may consider a few suggestions to improve the article.\n\n1. Introduction provides good information about the context, quality, structure, and previous literature on medical education in China. The third paragraph is better merged with the other paragraph, as a one sentence paragraph looks strange. It would add more value to the section if some background information were provided on medical teachers training in other countries. The recent two decades have seen a massive flow of Chinese medical professionals to go abroad for higher studies. And these medical professionals with foreign qualifications take a central position in international medical education.\n\n2. Methods: It is written well and provides sufficient information on methods applied in the research. A table on participants' background information is necessary. It will help readers get the participants' essential information while reading their reflections in the results section. Did any participants have foreign qualifications or working or learning experience in a foreign country?\n\n3. It would be interesting if the comparison is provided between teachers who have had foreign learning/working experience and teachers who have never been to foreign learning/working. Did teachers reflect on the challenges of diverse classrooms (with students from different backgrounds/nationalities/religions) and students' English proficiency?\n\n4. Practical and theoretical implications are better reported in the article, as the study is important to policy-making institutions to improve the teaching and learning of medical education in universities.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10995", "date": "22 Mar 2024", "name": "Hugo Horta", "role": "Author Response", "response": "Responses to the comments from F1000 paper: Reviewer #1 Comment: 1. Introduction provides good information about the context, quality, structure, and previous literature on medical education in China. The third paragraph is better merged with the other paragraph, as a one sentence paragraph looks strange. It would add more value to the section if some background information were provided on medical teachers training in other countries. The recent two decades have seen a massive flow of Chinese medical professionals to go abroad for higher studies. And these medical professionals with foreign qualifications take a central position in international medical education. Response: We are extremely thankful for this comment. The location of the third paragraph was misplaced by mistake. Now it has been placed where it should be and the text flows much better. Many thanks. We are highly appreciative of this insightful comment regarding the training of medical teachers. Recent and relevant literature on this topic has been added in a new paragraph in the Introduction to address this issue. Comment: 2. Methods: It is written well and provides sufficient information on methods applied in the research. A table on participants' background information is necessary. It will help readers get the participants' essential information while reading their reflections in the results section. Did any participants have foreign qualifications or working or learning experience in a foreign country? Response: Thanks for this good suggestion. We have added a table with the participants’ demographic information. As the reviewer can observe from the table, most of the participants had education, working or training experience in a foreign country, predominantly in the USA. Comment: 3. It would be interesting if the comparison is provided between teachers who have had foreign learning/working experience and teachers who have never been to foreign learning/working. Did teachers reflect on the challenges of diverse classrooms (with students from different backgrounds/nationalities/religions) and students' English proficiency? Response: This is a very good suggestion. We carefully reviewed our data again and found out that there were no major differences between teachers who had overseas learning or working experience and those who had not on the challenges they faced. The teachers in our study seemed to face overall very similar challenges and barriers in terms of diverse classrooms and international students’ English proficiency. Comment: 4. Practical and theoretical implications are better reported in the article, as the study is important to policy-making institutions to improve the teaching and learning of medical education in universities. Response: Thank you very much." } ] }, { "id": "193776", "date": "04 Sep 2023", "name": "Myla Arcinas", "expertise": [ "Reviewer Expertise e-learning", "social research methods", "impact studies", "population", "health and migration studies", "youth and women studies", "organizational development and human resource management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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's objectives are clearly outlined and underscore the importance of investigating the academic success of international medical students within Chinese universities. The direct influence of this success on licensing exams and future job prospects adds solid motivation for the research. Moreover, the study aims to address a noted research gap regarding the awareness of teaching-related factors that impact students' academic achievements, which is a relevant and worthwhile endeavor.\nThe chosen methodology, involving semi-structured interviews with 36 academics from two medical universities in China, is suitable for capturing the nuances of the research topic. The timeframe of November 2020 to January 2021 allows for comprehensive data collection over a reasonable period. The interviews' audio recording, verbatim transcription, and subsequent thematic analysis highlight the rigor in data handling and analysis, ensuring transparency and accuracy in the findings.\nThe thematic analysis results provide valuable insights into teaching factors perceived by academics as influential for student success. The identified themes demonstrate a comprehensive exploration of the issue, encompassing elements such as teaching style, language support, resource management, academic attributes, mentoring, and assessment practices. This breadth enhances the credibility of the study's findings and adds depth to its implications. However, it is essential to note that the themes generated can still be improved and clustered into three to create depth and quality in the discussion section.\nTeaching Methodology/Pedagogy and Content Alignment\n\nEncompassing challenges in teaching pedagogy\n\nAlignment of teaching content with license exams\nThis cluster focuses on the interrelation between teaching methodology and content alignment with licensure exams. Both elements are integral to effective instruction and student preparation. Teaching pedagogy influences content, and aligning content with licensure exams ensures students are adequately prepared for assessments. The combined cluster highlights the synergy between pedagogical strategies and content relevance.\nEducator Qualities/Attributes and Guidance\n\nAddressing teacher attributes\n\nSupervision and guidance by teachers\n\nEstablishment of teacher-student rapport\nThis cluster centers around the educators' role in student development. Teacher attributes influence their ability to provide effective guidance, mentorship, and establish rapport with students. By grouping these themes, the study delves into how educators' personal qualities impact their capacity to provide meaningful Supervision, guidance and build strong relationships with students.\nResource Management and Learning Environment\n\nManagement of teaching resources\n\nClassroom discipline management\n\nAssessment methodologies\nThis cluster revolves around the practical aspects of creating a conducive learning environment. Management of teaching resources, classroom discipline, and assessment methodologies collectively influence the overall learning experience. Efficient resource management contributes to effective instruction, while classroom discipline and assessment methods shape the learning atmosphere. Grouping these themes underlines the importance of maintaining an organized, supportive learning environment. Given the current themes presentation, the conclusions drawn from the findings are pragmatic and actionable, emphasizing the need for continuous professional development and resource provision for academic staff. The recommendation for staff training aligns well with the identified teaching factors and offers practical guidance for institutions seeking to enhance teaching quality. The clarity of these conclusions contributes to the study's relevance and potential impact on teaching practices.\nWhile the study's strengths are evident, a few aspects could be considered for improvement. A broader geographical scope or the inclusion of more diverse medical universities might have added insights into potential variations. Additionally, exploring potential research limitations or biases, such as the interviewees' backgrounds or the context of the two chosen universities, would have enhanced the study's transparency and thoroughness.\nIn summary, the study's objectives are well-established and significant within the academic context. The methodology is appropriate for the research aim, and the results' thematic analysis provides a comprehensive understanding of teaching factors affecting student success. The study's conclusions offer practical recommendations for improving teaching quality. While the study's strengths are evident, considering a broader scope and addressing potential limitations would further enhance its credibility and impact. It is important to note that clustering of the themes generated could definitely improve the discussion section and the overall quality of the paper.\nAdditional Points for Improvement of the Quality of the Paper:\nIn the Abstract, Methods section, it is crucial to explicitly mention the use of purposive sampling and state specific criteria employed in selecting informants.\n\nIn the paper, additionally, within the methods section, clarify the criteria used to identify the two medical universities covered in the study.\n\nWhen describing the study's sources of data, avoid using \"participants\" unless it pertains to scenarios wherein focused group discussions or participant observation was used as method of data collection. In employing semi-structured interviews for data collection, the appropriate terminology is \"informant.\"\n\nPlease exercise caution in providing qualitative interpretation derived from descriptive statistics presented in the paper. For instance: :\nInstead of \"An overwhelming majority (86%),\" employs the term \"most\" as it correctly corresponds to the range of 75% to 90% in relation to descriptive statistics.\n\nFor the statement \"Approximately 50% of interviewees,\" provide the actual frequency count and rephrase it to \"Nearly half (mention the frequency count).\"\n\nSimilarly, for expressions like \"nearly 50% of the interviewees,\" provide the precise frequency count, for instance, \"Nearly half or majority (mention the frequency).\"\n\nWhen discussing \"Approximately 31% of the interviewees,\" explicitly mention the factual frequency count.\n\nWhen commencing a sentence with a numerical value, spell out the number. For instance, instead of writing \"45 universities are authorized by the MOE…,\" use \"Forty-five universities are authorized by the MOE…\"\n\nThe overall quality of the paper can be further improved through thorough proofreading and editing. For example, consider:\n\"Nine key themes emerged covering challenges: teaching pedagogy, English language, teaching resources management, teacher attributes, teacher supervision and guidance, teacher-student rapport, detachment of teaching content from license exams, class discipline management, and assessment styles.\"\nIt can be written this way -\nNine prominent themes surfaced, encompassing challenges spanning teaching pedagogy, English language proficiency, management of teaching resources, teacher attributes, Supervision and guidance by teachers, establishment of teacher-student rapport, alignment of teaching content with license exams, classroom discipline management and assessment methodologies.\n\nTwo themes can be combined into one theme - Theme: teacher attributes\nTheme: Supervision and guidance by teachers\nCombined Theme: Teacher attributes and Supervision, or Teacher attributes and Guidance\nCombining these two themes into a single category of \"Teacher Attributes and Supervision/Guidance\" is justified by the intrinsic relationship between these aspects within the context of education. Teacher attributes inherently influence the quality of Supervision and guidance they provide students. Teachers' personal qualities, experience, and expertise significantly impact their effectiveness in guiding and mentoring students. By grouping these themes, the study can more comprehensively explore how specific teacher attributes contribute to the efficacy of their Supervision and guidance, leading to a deeper understanding of educators' role in student success.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10994", "date": "22 Mar 2024", "name": "Hugo Horta", "role": "Author Response", "response": "Responses to the comments from F1000 paper: Reviewer #2 Comment: 1.The study's objectives are clearly outlined and underscore the importance of investigating the academic success of international medical students within Chinese universities. The direct influence of this success on licensing exams and future job prospects adds solid motivation for the research. Moreover, the study aims to address a noted research gap regarding the awareness of teaching-related factors that impact students' academic achievements, which is a relevant and worthwhile endeavor. Response: Thank you very much for your encouraging acknowledgement of the significance of our study. We are delighted that the reviewer feels that our work provides a valuable contribution to the advancement of knowledge. Comment: 2. The chosen methodology, involving semi-structured interviews with 36 academics from two medical universities in China, is suitable for capturing the nuances of the research topic. The timeframe of November 2020 to January 2021 allows for comprehensive data collection over a reasonable period. The interviews' audio recording, verbatim transcription, and subsequent thematic analysis highlight the rigor in data handling and analysis, ensuring transparency and accuracy in the findings. Response: Many thanks for this positive assessment of our work and our methodological choices, as well as the collection and analysis of the data. Comment: 3.The thematic analysis results provide valuable insights into teaching factors perceived by academics as influential for student success. The identified themes demonstrate a comprehensive exploration of the issue, encompassing elements such as teaching style, language support, resource management, academic attributes, mentoring, and assessment practices. This breadth enhances the credibility of the study's findings and adds depth to its implications. However, it is essential to note that the themes generated can still be improved and clustered into three to create depth and quality in the discussion section. Response: We are extremely thankful again, this time for the positive remarks concerning the analysis. We have tried to re-write the analysis thematically into three themes as the reviewer suggested but ended up reverting some of the changes and decided to have four themes, which are independent even if somewhat overlapping with one another. This – in our opinion – makes the text to flow better and for the readers to have a clearer understanding of the different facets of the analysis. In this regard, we have structured better the sub-themes of the themes presented, many thanks! Comment: 4. Teaching Methodology/Pedagogy and Content Alignment Encompassing challenges in teaching pedagogy Alignment of teaching content with license exams This cluster focuses on the interrelation between teaching methodology and content alignment with licensure exams. Both elements are integral to effective instruction and student preparation. Teaching pedagogy influences content, and aligning content with licensure exams ensures students are adequately prepared for assessments. The combined cluster highlights the synergy between pedagogical strategies and content relevance. 5. Educator Qualities/Attributes and Guidance Addressing teacher attributes Supervision and guidance by teachers Establishment of teacher-student rapport This cluster centers around the educators' role in student development. Teacher attributes influence their ability to provide effective guidance, mentorship, and establish rapport with students. By grouping these themes, the study delves into how educators' personal qualities impact their capacity to provide meaningful Supervision, guidance and build strong relationships with students. 6. Resource Management and Learning Environment Management of teaching resources Classroom discipline management Assessment methodologies This cluster revolves around the practical aspects of creating a conducive learning environment. Management of teaching resources, classroom discipline, and assessment methodologies collectively influence the overall learning experience. Efficient resource management contributes to effective instruction, while classroom discipline and assessment methods shape the learning atmosphere. Grouping these themes underlines the importance of maintaining an organized, supportive learning environment. Given the current themes presentation, the conclusions drawn from the findings are pragmatic and actionable, emphasizing the need for continuous professional development and resource provision for academic staff. The recommendation for staff training aligns well with the identified teaching factors and offers practical guidance for institutions seeking to enhance teaching quality. The clarity of these conclusions contributes to the study's relevance and potential impact on teaching practices. Response: Thank you very much for the reviewer’s positive view on the relevance and content of themes 4, 5 and 6. Comment: 7. While the study's strengths are evident, a few aspects could be considered for improvement. A broader geographical scope or the inclusion of more diverse medical universities might have added insights into potential variations. Additionally, exploring potential research limitations or biases, such as the interviewees' backgrounds or the context of the two chosen universities, would have enhanced the study's transparency and thoroughness. Response: We agree that the inclusion of more diverse medical universities such as from different provinces in China would have enhanced the rigor in data and added insights into potential variations. This is one of the limitations of our study that we addressed in the paper. Practically it was a bit difficult to involve more universities especially from other provinces, but we aim to try to make this happen in our future studies. According to the comment of the other reviewer, we provided a table with the informants’ demographic information and their backgrounds. Meanwhile, potential research limitations are now addressed in the Conclusion section. Comment: 8. In summary, the study's objectives are well-established and significant within the academic context. The methodology is appropriate for the research aim, and the results' thematic analysis provides a comprehensive understanding of teaching factors affecting student success. The study's conclusions offer practical recommendations for improving teaching quality. While the study's strengths are evident, considering a broader scope and addressing potential limitations would further enhance its credibility and impact. It is important to note that clustering of the themes generated could definitely improve the discussion section and the overall quality of the paper. Response: There are several limitations of the study. This study was conducted at two sites, but caution with generalization of the findings is needed. They may be relevant to other Chinese universities offering courses to international students, but not to other settings. Both research sites were located within one single province, although representative of the general MBBS education in other universities in the same province, a broader geographical scope or the inclusion of more diverse medical universities from other regions or provinces might have added insights into potential variations. Additionally, as the data was sourced from subjective opinions of the academics, potential biases may exist. We have regrouped the themes according to your insightful suggestions in the discussion of the findings. Comment: Additional Points for Improvement of the Quality of the Paper: 1. In the Abstract, Methods section, it is crucial to explicitly mention the use of purposive sampling and state specific criteria employed in selecting informants. Response: Thank you for this helpful comment. We have now added the use of purposive sampling and specific criteria employed in selecting our informants in the Abstract. Comment: Additional Points for Improvement of the Quality of the Paper: 2. In the paper, additionally, within the methods section, clarify the criteria used to identify the two medical universities covered in the study. Response: Many thanks. The criteria used to identify the two medical universities have been clarified in the Methods section. Comment: Additional Points for Improvement of the Quality of the Paper: 3. When describing the study's sources of data, avoid using \"participants\" unless it pertains to scenarios wherein focused group discussions or participant observation was used as method of data collection. In employing semi-structured interviews for data collection, the appropriate terminology is \"informant.\" Response: Thank you for this important point. All the “participants” have been replaced with the terminology of “informants” now in the paper. Comment: Additional Points for Improvement of the Quality of the Paper: 4. Please exercise caution in providing qualitative interpretation derived from descriptive statistics presented in the paper. For instance: Instead of \"An overwhelming majority (86%),\" employs the term \"most\" as it correctly corresponds to the range of 75% to 90% in relation to descriptive statistics. For the statement \"Approximately 50% of interviewees,\" provide the actual frequency count and rephrase it to \"Nearly half (mention the frequency count).\" Similarly, for expressions like \"nearly 50% of the interviewees,\" provide the precise frequency count, for instance, \"Nearly half or majority (mention the frequency).\" When discussing \"Approximately 31% of the interviewees,\" explicitly mention the factual frequency count. Response: We appreciate this comment and decided to adopt it in the paper. Changes have been made in relevant places. Comment: Additional Points for Improvement of the Quality of the Paper: 5. When commencing a sentence with a numerical value, spell out the number. For instance, instead of writing \"45 universities are authorized by the MOE…,\" use \"Forty-five universities are authorized by the MOE…\" Response: Corrected. Thank you. Comment: Additional Points for Improvement of the Quality of the Paper: 6. The overall quality of the paper can be further improved through thorough proofreading and editing. For example, consider: \"Nine key themes emerged covering challenges: teaching pedagogy, English language, teaching resources management, teacher attributes, teacher supervision and guidance, teacher-student rapport, detachment of teaching content from license exams, class discipline management, and assessment styles.\" It can be written this way - Nine prominent themes surfaced, encompassing challenges spanning teaching pedagogy, English language proficiency, management of teaching resources, teacher attributes, Supervision and guidance by teachers, establishment of teacher-student rapport, alignment of teaching content with license exams, classroom discipline management and assessment methodologies. Response: Thank you very much for this point and for your good example of rewriting this paragraph. We agree that some proofreading and editing can improve the quality of the paper. Therefore, we have sent the paper for thorough proofreading and editing with an online academic proofreading service. Comment: Additional Points for Improvement of the Quality of the Paper: 7. Two themes can be combined into one theme - Theme: teacher attributes Theme: Supervision and guidance by teachers Combined Theme: Teacher attributes and Supervision, or Teacher attributes and Guidance - Combining these two themes into a single category of \"Teacher Attributes and Supervision/Guidance\" is justified by the intrinsic relationship between these aspects within the context of education. Teacher attributes inherently influence the quality of Supervision and guidance they provide students. Teachers' personal qualities, experience, and expertise significantly impact their effectiveness in guiding and mentoring students. By grouping these themes, the study can more comprehensively explore how specific teacher attributes contribute to the efficacy of their Supervision and guidance, leading to a deeper understanding of educators' role in student success Response: There is now a teacher attributes and guidance theme, which includes the sub-themes “addressing teaching attributes”, “supervision and guidance by teachers”, and “establishment of teacher-student rapport” . Many thanks for the fantastic suggestion." } ] } ]	1	https://f1000research.com/articles/11-894
https://f1000research.com/articles/11-1441/v1	06 Dec 22	{ "type": "Case Study", "title": "Analysis of Albania's diplomatic relations with the Soviet Union during the years 1948-1959", "authors": [ "Manjola Xhaferri", "mirela tase" ], "abstract": "Background: This paper aims to shed light on a very intense period of relations between Albania and the Soviet Union (USSR), focusing on the period from 1948 with the intensification of Albania's relations with the USSR until the termination of diplomatic relations in 1959. The main objective of this paper is to know the truth of the relations of the USSR and its behavior with Albania. Methods: The paper focuses on analyzing this period as a case study, selecting three levels (context-actors-outcomes). Evaluation of the period (1948-1959) is done through the identification of key indicators and the implications of the relationship in domestic and international contexts. The sources used include documents, interviews, films, newspapers, and books. We requested permission to use the Archival resources of the Albanian Central State Archives (AQSH), where they made some accessible non-state secret resources workable only within the archive available to us. Results: The facts show that the relations between Albania and the USSR benefitted Albania more. particularly with a lot of economic support from the USSR, which helped Albania emerge from crisis and build the so-called ‘socialist state’. Through these relations and contributions, Albania took its place in the international arena. We find that this may be because of the geographical position of Albania, as the construction of the Pashaliman naval base and the bay of Vlora indicates. Conclusions: This study tries to demystify this relationship that has had a lot of ambiguity cast over it. Documents of the AQSH do not provide an unbiased overview, as they were compiled with an ideologically charged spirit, making it difficult for researchers to reach conclusions without ideological input from both sides. We hope that we will have the opportunity to access restricted documents to provide a clearer picture of the relations between Albania and the USSR.", "keywords": [ "Relations", "diplomacy", "Soviet Union", "Analysis", "Albania" ], "content": "Introduction\n\nThis paper corresponds to one of the most important periods of history regarding the national and international policy of the Albanian state, especially the isolated political and state cooperation of Albania with the Soviet Union. Albanian-Soviet relations were very important, as they determined not only the orientation of Albanian domestic policy but also the foreign behavior that Albania had to maintain in the international arena, especially in relations with Western allies and neighbors.\n\nThis paper refers specifically to the period from 1948, when the Albanian-Soviet relations took their official form and capacity, until 1959 when these relations were broken. Two figures in the Soviet Union and Albania are key during this period. Enver Hoxha (October 16, 1908–April 11, 1985) was an Albanian communist politician who led the Albanian state from 1944 to 1985. Enver Hoxha was the chairman of the Democratic Front of Albania as commander-in-chief of the Albanian Armed Forces from 1944 to 1985. He led as Prime Minister of Albania during the years 1944–1954. At different times served as Minister of Foreign Affairs and Defense. He became a member of the Communist Party of Albania after its formation in 1941, and in 1943 he was elected its first secretary. During his period time, lieth the iron defense of Stalinism ideology, the elimination of any kind of external or internal opposition, through methods that included the death penalty; the transformation of the country towards an industrial and self-sufficient economy, state atheism; the creation of secret police for the political suppression of the population called the State Security and, in recent years, the international isolation into which Albania plunged. Also prominent was the cult of the individual with him at the center. After his death, his cult was honored by building statues in almost every city of Albania, headed by the one in the center of Tirana, as well as the construction of a museum dedicated to him, in the Albanian capital. He is also known as the organizer of the victory over German fascism. He carried out forced industrialization of the country and forced collectivization. In the late 1920s–30s, he destroyed and supposed his potential enemies, which became the initiator of mass terror. To better explain the reasons and causes of the establishment of these relations, many internal and external political situations have been touched upon, which go beyond the defined time limits of our study. The main purpose of this paper is to analyze the conditions in which these relations are established and their unusual, bilateral cooperation in the political, economic, international, and military fields. What are very important to include are the moments of establishing the Albanian-Soviet relations, their development, and completion in the most peaceful way possible. The paper aims to approach this relationship from a new perspective of study, to understand its true nature. What we will try to clarify in this paper are some research questions that orient us towards clarification by understanding the truth of the facts. specifically, we will examine:\n\n• How much space was devoted to Albania in the international communist movement?\n\n• How important was Albania to the Soviet Union?\n\nThere was not only the economic growth in the years 1950–1960 to consider, but what was an unusual phenomenon for the poor Albanian people, coming from a backward economic tradition, was the social development of the Albanian society, where we will focus on distinct areas: housing, employment, health, the fight against illiteracy, etc. What we noticed in this study is that education, as well as culture, increased the trust in the communist government in Albania, which was part of the perfect implementation of the Soviet model, not only politically but also socially. The whole ‘Sovietization’ of the Albanian state and society was used by the Union of Soviet Socialist Republics (USSR), as with all countries of the former Eastern bloc, to realize its hegemony in the international arena. In conclusion, I would summarize with the expression from “Stalin was the political muse for Enver Hoxha, this worship that he had continued until the end of his life and refused to be de-Stalinized. This turned out to be fatal in relations with the Soviet Union and all other countries and ended in the greatest isolation of Albania until the fall of the regime in 1990”.1\n\n\nResearch questions and hypotheses of the paper\n\nAlbania’s relationship with the Soviet Union has been very complex. Analyzing documents that belong to the period but also evidence of the time creates a series of research questions to verify whether Albania’s relationship with this superpower has been as truthfully presented to the public so far, or if things are not as they seem. The research questions to address this are:\n\na. Has Albania been completely dependent on the Soviet Union?\n\nb. Was Albania really in the spotlight of the Soviet Union as the idea was created in Albania over the years?\n\nc. Was it possible for Enver Hoxha to behave differently with the Soviet Union and what has been Albania’s strong point in relations with the Soviet Union?\n\nd. What was hidden in the telegrams, radiograms, and secret letters exchanged between the ambassadors, the officials of the delegations, and even those of the great leaders of both countries, and what was the language used by them? These sources were accessed only by the Central State Archive source of the Foreign Ministry. Some of the items were restricted as they were considered state secrets, but we hope that after 50 years after Hoxha’s passing away, these archives will open and give us access to use them in the future. It will be a challenge for us to read more secret documents required to continue this study in the future.\n\ne. Did Khrushchev have a plan to attack Albania and could he do so, taking into account the country’s geographical position?\n\nThese and other questions will be answered within this paper. The aim of the paper is to prove whether the relationship with this superpower has been as it has been presented to us so far. Albania was an important factor in relations with the Soviet Union or the things not as they seemed.\n\nIn all question that we are raising our focus is to analyze a) the impact of these relations, b) the individual cults of leadership in both Albania and the Soviet Union, c) the ideological divergences and impact in bilateral relations, and d) the economic dependence on relations between Albania and the Soviet Union.\n\nThis study offers three hypotheses that address the key problem. The main problem of the hypotheses is the clarification in this bilateral relationship, which has had a lot of light and shade obscuring it. This paper emphasized giving an unbiased overview of the situation, taking into account the ideological load of the documentary sources of that period. We tried, through hypotheses, to provide an assessment and undoubtedly a clearer insight into the relations with the Soviet Union. According to our judgment, Albania was an unimportant country for the Soviet Union, from the way, Enver Hoxha benefited from this relation to building the socialist state and giving it a name in the international arena, not only for himself as the Albania head, but also Albania as a whole. In each section, this study tries to show the veracity of these hypotheses.\n\nThe first and main hypothesis of the paper is: The Soviet Union did not have the geopolitical or economic interest in Albania, as it was thought at the time. The rapprochement of the Soviet Union with Albania came mainly due to the breakdown of relations between the Soviet Union and Yugoslavia, and Albania with the latter. Without international economic and political support, Albania found in the Soviet Union the economic and political support it needed to fill the vacuum created by the break from Yugoslavia.\n\nThe second hypothesis of the paper is that Albania’s relations with the Soviet Union never emerged from the tutelage of dependence and preferences. Although within the same ideological context, Albania’s relations with the Soviet Union reflected the relations of the powers, and for this reason, they were always dominated by pressure; blackmail on the one hand, and humility and obedience on the other.\n\nThe third hypothesis is about the Yugoslavs, as Albania has never been a priority for the Soviet Union. In any case, it would have to be dealt with depending on the ups and downs of Soviet-Yugoslav relations. This did not change in both Stalin and Khrushchev’s time. So, Albania was not measured by the strength, position, and close connections that existed between Yugoslavia and the Soviet Union, although, in some cases, Enver Hoxha claimed that the connection between his country and the Soviet Union was strong and inseparable. This claim was mainly made during the break-up of the Yugoslavia-Soviet Union relations. But this showed the opposite, it was just a facade, as Hoxha exploited this fact for his political interests, and even the fanaticism he expressed towards Stalinism was an excuse for the fear that he had for the longevity of his power and of being replaced by anyone including another Albanian leader who might come to power.\n\n\nMethods\n\nThe research model on which the paper is based is that of the case study, focusing on the context of international relations. This paper follows a combined research strategy as outline below:\n\n\n\n‐ Evaluation of the period (1948–1959) through the identification of key indicators, their values, and impact on the analysis of the case study.\n\n‐ Evaluation of the implications of the relationship in the domestic and international context through the analysis of the behavior of the actors, their discourse, and strategic documents.\n\nThis model is mainly used to answer the research questions posed in the previous section. The basis of the research work was the research and study of documents and testimonies of the protagonists as local and foreign literature about the problems related to the Eastern bloc in the years 1948–1959 (Some protagonists of the drives have died and, some are in the form of memories that we have quoted in our work). In terms of literature, world-renowned works examined which have described and analyzed the model of the Stalinist dictatorship in Albania and the behavior of the dictator Enver Hoxha in the international arena. These are works translated by the Albanian National Library and the Central State Archives. Also, a special place involves the documents of the central committee of the Albanian Labor Party, as well as the works of Enver Hoxha,2–5 which include valuable documents. As we have mentioned in the methods session, for the realization of this work, in addition to publications by different authors and periodicals from the period of communism, we also used archival sources, which are not available to the public in digital form. The only way to use it is to study on the premises of the central archive of the state. The sources used in the archives belong to the 1953–1959 years. We have integrated all the sources used in the paper, mainly in the History of Albania-Soviet Union Relations section. To use the same sources from other researchers below, we are giving a way to identify them.\n\nThe first archival source of the Central State Archive is the manuscript of the Ministry of Foreign Affairs entitled: “History of Relations between Albania and the Soviet Union,” file number I-8, entitled New Albania, number pages 48, the year 1953. This archival document contains several pages devoted to the beginnings of Diplomatic Relations between Albania and the Soviet Union and the continuation until their final breakdown. These documents list the ‘good things’ that the Soviet Union has done for Albania. “The leaders of the Soviet people, Lenin and Stalin, have repeatedly exposed the aggressive policy of the imperialist powers for the fragmentation of the world on the one hand and Albania on the other.\n\nThe result of this source is that the Soviet Union has supported Albania in several political and financial moments. Great political support was when the leader of the Bolshevik Revolution, Lenin, was on Albania’s side, opposing the bargains of the great powers at the Conference of Ambassadors in London in 1913. Another support of the Soviet Union, vis-à-vis Albania, was the financial one during the time of Stalin or later to Nikita Khrushchev. So Albania, even though it was not the main focus of the policies of the Soviet Union, was constantly helped financially by the three leaders of the Soviet Union, Lenin, Stalin, and Khrushchev. They used Albania as a strategic place for their interests in the Balkans\n\nThe second archival source of the Central State Archives is the manuscript of the Ministry of Foreign Affairs entitled: “History of Relations between Albania and the Soviet Union,” with file number I-19, entitled XX Congress of the Communist Party of Soviet Union, number of pages 118, the year 1956. This archival document contains the proceedings of the 20th Congress of the Communist Party in BS. For the first time, Khruschev openly criticizes Stalin’s cult of the individual.\n\nThe result of this source is 20th congress bells rang in the ears of the Albanian leader as an alarm. The influence of the 20th Congress of the Communist Party of the Soviet Union was huge. We cannot say what Enver Hoxha’s reaction was to Khrushchev’s statements, but what he did publicly was that first, he did not hesitate to come out against Stalin’s cult of the individual.\n\nThe third archival source of the Central State Archives is a manuscript of the Ministry of Foreign Affairs entitled: “History of Relations between Albania and the Soviet Union,” with file number I-6 entitled “Letter sent by Enver Hoxha to the Central Committee of the Communist Party of the Soviet Union and specifically to Nikita Khrushchev on April 14, 1958”, page number 3, the year 1958. Enver Hoxha wrote enthusiastically about the decision taken by the Central Committee of the Communist Party of the Soviet Union regarding the non-sending of the Soviet delegation to the Seventh Congress of the League of Communists of Yugoslavia.\n\nThe result of this source is that relations with the Soviet Union after Stalin’s death could never function independently of Yugoslavia’s relations with the Soviet Union.\n\nThe fourth archival source of the Central State Archives was a manuscript of the Ministry of Foreign Affairs entitled: “History of Relations between Albania and the Soviet Union,” with file number I-92, entitled “Nikita Khrushchev’s visit in May 1959”, page number 85, 1959 year.\n\nDuring his visit to Albania, Nikita Khrushchev gave a speech at a large rally with the workers of the “Stalin” textile factory, which was a gift from the Soviet government to Albania.\n\nThe result of this source is that Khrushchev’s visit was not as successful as the Communist Party leader Enver Hoxha defined it. Khrushchev addressed the government with critical notes regarding the development of the country, where he asked for an account of how the financing made by the Soviet Union invested, where Albania should be a country many times more developed than with all these natural resources that God had given them.\n\nThe source for the documents used in this study are available in the Archive of the Ministry of Foreign Affairs (https://www.arkiva.gov.al). Access to the documents is only available as hard copies. All sources in hard copy are referenced or cited in the manuscript.\n\nMost of the resources are provided by the State Archives. All sources used in the central archive of the state are referred to in the manuscript. In order to use any source in the archives, authorization is required through a request for the topic of the resources that you need to use. After the approval of the authorization, the archive staff will make available the resources that are available for use. The search for sources is in cooperation with the archive staff, who addressed us to use the files related to the documents about the diplomatic relations between Albania and the Soviet Union. In the review procedure for previously used sources, the researcher, through a request, must provide arguments for previously used documents and what conclusion will be drawn from the review.\n\nThe collection of data and the realization of the study includes a two-year period 2018–2020. All the resources are provided to us in the Albanian language, where without a doubt the resources in foreign languages have been translated by the translation department within the State Archives.\n\nThe criteria for the documents to be included in the work is that the line of relations must be maintained without accusations and allusions, taking into account the form of the regime at that time, the way the communist system built diplomatic relations, without nationalistic spirit, or unilateral political influences, thus preserve the professional ethics of the researcher (historian).\n\nComparative research consists of ascertaining the similarities and differences between different phenomena, in this case, to better understand and interpret the behaviors of the states (Albania and the Soviet Union) and their certain policies, in this case regarding the relations between the two countries. Comparisons can be made between different fields, factors, and actors, or even between different countries or organizations. In terms of time, they can be at different times, diachronically or synchronously, at the same time. Comparative studies can be intra-state, inter-regional, or global. The empirical approach occupies a special place in the methodology of scientific research in political sciences. In this case, she tries to interpret the reality of international politics throughout the years under study as objectively as possible through observations, experiences, instruments, and certain scientific methods.\n\nThe case analysis of the Yugoslav influence in relations with the Soviet Union will continue to remain a case study in international relations. Concerning the Yugoslavs, Albania has never been a priority for the Soviet Union. In any case, it would have to be treated depending on the ups and downs of Soviet Union-Yugoslavia relations. This did not change both under Stalin and Khrushchev. Disputes within the communist bloc, such as those between Yugoslavia and the Soviet Union, or Albania and Yugoslavia and the Soviet Union, showed that there could be divergence even when countries follow the same strategy and ideology. The reason for these divergences in these cases has been sovereignty as an important issue of national character. Regarding Albania, the facts show that the national interest fueled Enver Hoxha’s ego and struggle for power. He used it for his own sake, to maintain his jurisdiction.\n\nThe methodological limitations of the paper relate to the difficulty of balancing the documentation available to us due to the different perspectives between the parties, and the documentation of Albanian-Russian relations. Russia continues to be quite conservative in publishing documentation for this period. The Albanian materials that we have at our disposal and belong to this period (1948–1959) have strong ideological and propaganda loads. Finding materials and resources has not been difficult because academic publications on this topic are numerous and with abundant material. I would emphasize mainly the sources in the Central State Archive, where there are thousands of manuscripts, such as personal letters or diplomatic agreements between the two countries. The voluminous number of domestic documentation, the data available, and the difficulties in selecting the most appropriate statements or documents are other limitations. It is also worth noting the limited number of analyses by third parties, due to the small weight of Albania-Soviet Union relations, international relations, etc. To minimize the consequences that come from such limitations, the paper focuses on using analysis in the context of this period as a case study, selecting three levels (context-actors-outcomes) to help place the paper in an internal coherence. The actors are the Soviet Union-Albania-Yugoslavia. Also focuses on a defined number of indicators of the implications of bilateral relations such as stakeholder lectures and key strategic documents. The primary documents used in the central archive of the state have been provided by the institution. For the sources used in these archives, authorization requires a request for the topic of the resources. The search for sources was in cooperation with the archive staff, who addressed us to use the files related to the documents about the diplomatic relations between Albania and the Soviet Union. It is worth noting the time limit for this purpose (1948–1959). As we emphasized above, a special place had occupied by the documents of the central committee of the Work Party of Albania, as well as the works of Enver Hoxha, which include valuable documents. Although with a high ideological charge, these sources have given this paper a picture of how diplomatic relations were built between the countries of the former communist bloc. More details are included in the methodology section of the abstract.\n\n\nOverview of the history of Albania-Soviet Union relations\n\nThe relations of the Albanian people with the peoples of Eastern Europe begin with the Russo-Turkish war of 1768–1774, a war which started as a result of the encouragement from France to the Turks.6 According to a document of the Central Archive of the Albanian state, it turns out that in the fight the Turkish sailors fought many Albanians, who came from Albanian lands which at that time were under the occupation of the Ottoman Empire, referring to a document of 1953.6 These Albanian fighters were decorated with medals of bravery for the contribution given by the Russian state.6 According to the documents discovered in the archives of the Albanian state, it turns out that the Albanian volunteers took part in many battles alongside the Russians against the Turks. Enver Hoxha, in the early documents of the communist era, finds evidence in these times for the molding of Russian-Albanian friendship.6\n\nAccording to these documents, during the Ottoman occupation many Albanians, finding it impossible to live in subjugation, emigrated. Some settled in Russia. Several others settled in several villages in the Zaporizhzhia region of Ukraine.7 According to many documents in the Russian archives referred to by Russian scholars who have dealt with this topic, it turns out that these Albanian fighters were from the south of Albania, mainly from the province of Himara.8 In one of his works, Shaqir Vukaj,9 the current Albanian ambassador to Moscow but also a historian, writes about the Moscow myth. According to him, although the Albanian-Russian relations have been little studied, it seems that in the –fifteenth to eighteenth centuries the myth of Moscow was raised in Albania, primarily as a savior of Christians who suffered under the Ottoman yoke. Some Albanian bishops went to Moscow not only to ask for Russia’s interests in the Balkans are early, but it was Peter the Great who, with his call in 1711, made Russia directly involved in Balkan developments.8\n\nLater Lenin in his 1915 work entitled ‘Socialism and War’,7 opposing the argument that war was not made to plunder but to defend the homeland, wrote among other things: “Only hypocrites hide this. England plundered the German colonies and Turkey, Russia, Galicia and Turkey, France is looking for Alsace Lorraine and even the left bank of the Rhine, with Italy a treaty has been concluded on the division of the spoils (of Albania and Asia Minor)”.7\n\nIn archival documents belonging to the years of dictatorship, all this is interpreted as early support of Russia to Albania. These documents, mainly from the 1950s, list the ‘benefits’ that the Soviet Union has done for Albania; “The leaders of the Soviet people and all mankind, Lenin, and Stalin have constantly unmasked the aggressive policy of the imperialist powers to divide”.7\n\nEnver Hoxha dedicated the liberation of Albania primarily to the war of the peoples of the Soviet Union, the Soviet army, and the Bolshevik party. At least this is what the leader of the Albanian communists himself says in one of the documents of the central archive of the Albanian state.7\n\nHowever, as enough has been written about this topic, even according to the scholar William Griffith, Hoxha was placed at the head of the Albanian communists, thanks to the Yugoslav contribution on November 8, 1941.10 With the help of the British and Yugoslavs, the Albanian communists managed to finally a long and successful partisan war.11 Even though Enver Hoxha in his writings tried to make the friendship with the Soviets clear as early as possible, the facts show that Albania until 1948 remained a Yugoslav satellite more than a Soviet one. As a country, Albania did not attend Comin-tern meetings and was represented by Yugoslavia instead. However, like all communists who came to power with their forces, the Albanian communist leadership pursued the extreme left ‘Stalinist’ policy both inside and outside the country. But the Yugoslavs were allowed to negotiate with the Albanians, perhaps more than Moscow wanted. We recall here the creation of a large federal state which would include Albania, which was opposed by Stalin.1 From the first days after the liberation, Albania’s foreign policy oriented created diplomatic relations with the countries that aligned themselves in favor of the anti-fascist war against the Nazi-fascist bloc. The country with its first diplomatic relations was the Soviet Union, from which Albania did not break away until the death of Stalin. The economic period of domination of Albania by the Soviet Union began in September 1948, when the first economic agreement was signed by them. It was finalized during Enver Hoxha’s visit to Moscow in 1949. After the signing of this agreement, other Soviet Union (USSR) satellites also started to support Albania.10 By 1949, 38 percent of the Albanian state’s internal revenue came from loans as aid grants from the Soviet Union blocked several countries.10 Meanwhile, an intensive program, especially in industry,10 began under Soviet Union leadership. During 1951–1955 years, periodic industrial production increased by three times.10 But in 1953s, Stalin’s death and Khrushchev’s approach to Belgrade in 1955 threatened Enver Hoxha with de-Stalinization and the emergence of Yugoslavia. It was led by a reduction of aid to Albania in the 1953–1955 years, both in machinery and other investments.10\n\n\nAlbania’s relations with the Soviet Union after Stalin’s death\n\nThe period of political and economic domination of Albania by the Soviet Union began in September 1948, when the first economic agreement was signed. It was finalized during Enver Hoxha’s visit to Moscow in 1949. In 1953, the death of Stalin and the coming to power of Khrushchev, as well as his rapprochement with Belgrade in 1955, threatened the policy of Enver Hoxha’s relations with the Soviet Union. Enver Hoxha’s relationship with Stalin, although limited to a few meetings, seemed to be loyal, at least on the part of the Albanian leader. Enver Hoxha was a Stalinist follower and remained so until Stalin’s death. On March 5, 1953, Stalin died. His statue in the center of Skanderbeg Square, in the Albanian capital of Tirana, was turned into a place of pilgrimage. In his book ‘The Bend and Fall of Albanian Tyranny’, Spartak Ngjela describes the atmosphere after Stalin’s death as dramatic. As a witness, he says that for three consecutive days, the leadership block, the whole big house of the central committee which seemed shocked by the news of Stalin’s death.12 The public lined up on that day in front of the statue to already honor the memory of Stalin so soon after his death. But life would go on through with the heavy weight of this great loss to world communism. Although with the anxiety about the ideological and political line that the Soviet Union would follow in the future, Enver Hoxha would continue to praise this great country and call it a source of inspiration for further achievements.13\n\n\nDisputes between Albania and the Soviet Union 1955–1957\n\nThe first disputes between the Soviet Union began after the death of Stalin and the coming to power of Nikita Khurshov. This is because the new leader of the Soviet Union criticized the cult of Stalin and openly expressed the reform of the Soviet Union. From the documents already open in the Russian archives, it appears that the content of Nikita Khrushchev’s famous speech had already been decided at this meeting of the central committee.13 The 20th Congress of the Communist Party of the Soviet Union convened on February 14, 1956 and lasted until the 25th of that month.13 The most important event of this congress was the de-Stalinization of the Soviet Union, which the first secretary of the Soviet Communist Party, Nikita Khrushchev, announced in a long speech in which he analyzed his entire political career as Stalin’s leader.13 The speech was held in an audience of people chosen and approved by Khrushchev himself. For the first time, Stalin was officially criticized for the cult of the individual and blamed for its consequences in the society of the Soviet Union. Khrushchev would begin his speech with the words: “Dear comrades, in the report of the party’s central committee for the twentieth congress and a considerable number of speeches delivered by delegates even during previous plenary sessions, much was said about the cult of the individual and its harmful consequences”.14\n\nKhrushchev further tried to explain that this report was not intended to judge and evaluate Stalin’s life and activity; he stressed that everyone worldwide recognizes Stalin’s role in implementing the socialist revolution, in the civil war, and in his efforts to build socialism in the Soviet Union. According to Khrushchev, the central committee after Stalin’s death analyzed what had happened to him.15 This was the first speech of its kind in terms of the content and feedback it brought. Never before had a senior Soviet leader spoken out against Stalin and his deeds. According to Khrushchev, Stalin greatly popularized himself as a great strategist, instilling in the minds of the people in all possible ways the version that all the victories won by the Soviet people in the great patriotic war was the result of Stalin’s manliness, bravery, genius, and no one else.16\n\nEnver Hoxha did not hesitate to give his opinion on this issue. The official organ of the Albanian Labor Party, The people’s voice on April 14, 1956, was opened with the main article entitled ‘Marxism – Leninism’ which13 teaches us that the people are the creator of history. This article was signed by Enver Hoxha. In it, he expressed: “Stalin made mistakes that cost the Soviet people and the cause of socialism. The Communist Party of the Soviet Union rightly condemned the cult of the individual created for J.V. Stalin in the last years of his life activity and who did so much damage to the Soviet Union”.13 It must be said that J.V. Stalin, at a time after the Communist Party of Soviet Union and the Soviet people achieved great victories that led to the triumph of socialism, began to put himself above the party and the people, to leave the masses and so mistakes were made that cost the Soviet people a lot. and the question of socialism.13\n\nEnver Hoxha did not hesitate to criticize Khrushchev, his idol, as harshly. Stalin was Hoxha’s idol until his death, but for political reasons and personal interests, after the death of Stalin, Enver Hoxha temporarily criticized the figure of Stalin, in order to maintain relations with the Soviet Union, but this did not last long and Albania broke relations with the Union. The Soviet Union oriented its foreign policy toward China. In the pages of The people’s voice, Enver Hoxha writes further:\n\nOn the pages of the “Voice of the People” newspaper, Enver Hoxha writes further: “In the condemnation that the 20th Congress made of the cult of the individual, in the open and courageous criticism that it made of its damages, the Communist Party of the Soviet Union and all communist and labor parties of all countries conclude practical importance.”13 Although Enver Hoxha tried to follow Khrushchev’s path, again the first sparks of distrust and paranoia brought about by the restoration of a friendship between the Soviet Union and Yugoslavia had begun to emerge. The effects of the 20th congress of the BS KP were strongly felt in the Tirana conference of the Albania Social Party (ASP) Voices against the cult of the individual increased and Enver Hoxha’s power was more endangered than ever. The effects of the 20th congress of the Communist Party of the Soviet Union was strongly felt at the Tirana conference on the Socialist Party of Albania. Voices against the cult of the individual increased and Enver Hoxha’s power was more endangered than ever. Hoxha interrupted his vacation in Vlora and was at the Tirana conference, where the rebellion was huge, but he managed to reduce the discontent and reverse the situation. The next step was to arrest the parties. Just as they were leaving the conference hall, not a few, but 44 communists were arrested and sentenced. Researcher Ana Lalaj writes that Enver Hoxha himself referred to the Tirana conference, describing it as one of the most difficult moments of the Socialist Party of Albania.17\n\n\nNikita Khurshov’s visit to Albania and the improvement of relations between the two parties during the years 1958–1959\n\nRelations with the Soviet Union after Stalin’s death could never function independently of Yugoslavia’s relations with the Soviet Union. In a letter to the Central Committee of the Communist Party of the Soviet Union and specifically to Nikita Khrushchev on April 14, 1958, Enver Hoxha wrote enthusiastically about the decision taken by the Central Committee of the Communist Party of the Soviet Union not to send the Soviet delegation to the congress of the seventh of the League of Communists of Yugoslavia16; “The Central Committee of the Albanian Labor Party is fully in solidarity with your decision on this issue. And he fully agrees with your Marxist-Leninist assessment of the draft program of the League of Communists of Yugoslavia”.16 It was a direct quote since Enver Hoxha saw the relationship of the Soviet Union with Yugoslavia as a danger for the fading of bilateral cooperation between Albania and the Soviet Union. Enver Hoxha says:’’ Yugoslavia was an obstacle to realizing the projects and returning Albania as a strategic country in the Balkan Region. Soviet delegation not participating in the seventh congress of the association of communists of Yugoslavia enthused Enver Hoxha, and he expressed this in the letter he sent to Nikita Khrushchev on April 14, 1958. It raised Hoxha’s hope for further relations, between the two countries Albania-Soviet Union.16\n\nThe culmination of Albania’s relations with the Soviet Union, with too many analysts or witnesses of the time, was Nikita Khrushchev’s two-week visit to Albania. This visit lasted from May 25 to June 4 and was the first visit by such a senior Soviet leader. She seemed enthusiastic but also defiant.18 The Prime Minister and Chairman of the Communist Party of the Soviet Union arrived in Tirana in May 1959 with a large contingent of Soviet officials and was enthusiastically received by the Albanians, from high officials to the common people. The Soviet delegation during its stay in Albania made a trip throughout the country, visiting a range of industrial enterprises, agricultural cooperatives, state-owned agricultural enterprises, and research and teaching institutions.19 In total, Khrushchev visited almost all the cities of Albania as, in addition to Tirana, he went to Shkodra, Durres, Korca, Vlora, etc. Khrushchev’s visit to Albania marked a historical moment since the first time a senior leader of the Soviet Union had visited a small country like Albania. This achievement undoubtedly paid tribute to the party and its leader Enver Hoxha, who proudly expressed the success of Khrushchev’s visit to Albania. In his book, Spartak Ngjela shows the moment like this: “I went out to see what to do with the school because all the schools in Tirana scattered along the entire road which started from Zogu I Zi and reached the Brigade Palace, where was Khrushchev’s two-week residence. It was frenzied enthusiasm everywhere. All the people were shouting The Soviet Union as well as Enver - Hurshov names”.12\n\nDuring his visit to Albania, Nikita Khrushchev delivered a speech at a large rally with workers at the Stalin textile factory, which was a gift from the Soviet government to Albania. The history of the construction of this great work called Kombinat begins in the second five-year period of the Albanian communist government. Kombinat was built in 1948 and undertaken by the Soviet government. As for Khrushchev’s speech at the ‘Stalin’ combine, we will extract a short fragment where he openly threatens the Western countries by stating a thing. “You will build a missile base in Albania if one builds in Italy and Greece from the United States of America.20 He said that Albania was uniquely suited to build a missile base The factory was Stalin’s gift to the Albanian people, so it got its name “Stalin” textile factory. The textile factory “Stalin“ was inaugurated by Enver Hoxha on November 8, 1951. It employed about 4,600 employees and each of the specialists and staff managers was trained in the Soviet Union. Radio Moscow reported that the car of the Soviet leader on the way to the combine was occasionally stopped by workers who wanted to hug the leader, the representative of a friendly country, and shake hands with him.20\n\nDespite this, Khrushchev’s visit to Albania in 1959 certainly did not go well. Hurshov’s visit to Albania, despite the enthusiasm, brought with it a series of criticisms directed at the Albanian leader as a note of protest for the transparency of the investment of the donations that the Soviet Union had awarded to Albania. During his visit to Albania, Hurshov noticed a lot of poverty in the country, saying: “God has given this country all the opportunities and natural beauty to develop, you [referring to the Albanian citizens] should know the value”. Historians of the time also distinguish those disputes that existed despite statements that seemed to be on the same wavelength.21,22 Only a year later at the Moscow meeting in November 1960, Khrushchev was greatly surprised by the confrontation with angry Albanians. So, in Albania’s relations with the Soviet Union, the show had to be distinguished from the essence. In the central archive of the Albanian state, we found a series of top-secret (archive documents) about the time when Nikita Khrushchev’s remarks are listed during his visit to Albania. For instance, on May 25,1959, Khrushchev said that in connection with the construction of the small metallurgical plant, one should first calculate how much a ton of metal would cost. Either for the construction of the new textile factory or the increase of the capacity of the ‘Stalin’ factory, the question must be seen whether other countries can accept take the Albanian cotton that was poor quality and give in exchange good quality cotton.16 What Khrushchev constantly advised was to ensure the math was done well, an area in which Enver Hoxha’s power seemed to falter.\n\n“Economy and agriculture are going well and people are working. The country can become very rich and live without support. Albania has good development prospects because it has mines, good land, and climate, and sea. We should not start from the fact that they grow here all cultures to meet all the needs of the country but to cultivate those cultures for which there is more benefit”.16\n\nDuring his visit to Albania, Khrushchev was fascinated by the bay of Vlora. Enver Hoxha remembers this moment like this: “….What a miracle is here. The ideal base could be built for our submarines. To excavate and throw these antiquities into the sea [they were talking about the archeological objects of Butrint], let’s drill this mountain and get to the other side. We will have the most ideal and safest base in the Mediterranean. From this place, we can paralyze and attack everything. The Pashaliman base was a pipe dream for the Soviets. More precisely, the dream of all Russian tsars was coming true. Their fleet would finally emerge in the Mediterranean. It would realize through Albania….”.23\n\n\nResults\n\nThe findings in this paper are intended to be realized by analyzing data and realization of the study includes a two-year period 2018–2020. All the resources are provided to use in the Albanian language, where without a doubt the resources in foreign languages have been translated by the translation department within the State Archives. The number of documents are over 200 pages from the archives and over 10 books reference by Albanian and foreign authors.\n\nIn our research, we have used the state archive related to the relations between Albania and the Soviet Union. Enver Hoxha’s writings and some foreign or Albanian authors who were present at this time, giving their data and impressions, also helped our work. Based on this literature, we tried to bring an analysis of these relationships. Albania’s relations with the Soviet Union are still mostly undiscovered as long as many documents in the Russian archives are not available for study. However, like any study or work carried out in different periods, this one cannot set the last stone in terms of these relationships. Maybe it could be an attempt to demystify or an attempt to clarify something in this relationship that has had a lot of light and shadow. The documents of the Central State Archive of Albania do not give an impartial overview of the situation as they have been compiled with a load of ideological spirit and therefore do not present an accurate picture of the situation on both sites. Publications about Albanian-Soviet relations have been numerous in Albania but not in Russia. There are very few studies on this part of the history of the Soviet Union. The Russians have written a lot about relations with many countries of the world (not only with superpowers) but there is a marked lack of clarification of relations with our country.\n\nThe facts show that economic relations were very valuable. The value is explained by the fact that the economic aid that the Soviet Union gave to Albania led to the construction not only of infrastructure but also enterprises such as the ‘Stalin’ textile factory and many others like these, which led to the employment of thousands of workers and the increase to some extent in the standard of living Albania also took a place in the international arena thanks to the contribution of the Soviet Union. There is no doubt that without their support, Albania was a bankrupt country just as we emerged from friendship with Yugoslavia. Why was all this done? We hypothesize that this was because of the geographical position of Albania. The construction of the Pashaliman naval base and the fascination of the Soviet leadership with the Gulf of Vlora was an indication of this.23 However, the Soviets fled this country without much fuss, even though over the years it was proclaimed in Albania that we were quite coveted by the Soviets because of their geostrategic goals. Pashaliman’s base builds on an agreement and a highly secret document between the government of the People’s Republic of Albania and that of the Union of Soviet Socialist Republics. The document found in the archive of the Ministry of Foreign Affairs has the following content:\n\n“…The Government of the People’s Republic of Albania and the government of the Union of Soviet Socialist Republics, to strengthen the defense of the People’s Republic of Albania, felt the need to conclude this agreement between them and purpose was appointed their plenipotentiaries: the chief of to the Albanian General Staff Major Arif Hasko and the Chief of the General Staff of the Soviet Naval Military Fleet Admiral Fokin V.A…”.3 The agreement consisted of seven articles.\n\nAs for the de-Stalinization and liberalization of the country after the 20th Congress of the Communist Party of the USSR, which was dictated to Hoxha by the Soviet leader, history has shown that Hoxha maintained personal power at all costs. Tito’s Yugoslavia and the opinion that existed in the latter about Hoxha openly threatened Enver Hoxha and his power. The reasons for the split are manifold. On the one hand, in Yugoslavia, the restoration of Soviet-Yugoslav relations played a very important role in the development of relations between Albania and the Soviet Union. However, we cannot say with conviction that Albania and the Soviet Union would have maintained their friendship if these relations had not been restored. Or if Albania would not have lined up in favor of China. But we have also seen this lineup in the light of the return of Soviet relations with Yugoslavia, because exactly when it felt threatened by this return, Albania turned its eyes away from China and vice versa, China found that there could be little support in Eastern Europe.\n\nConsidering that Enver Hoxha wanted to install the Stalinist regime in the country and then keep it alive as long as possible to reach the desired conclusions, the world’s criticism of Stalinism must be carefully considered. Enver Hoxha’s desired conclusions were for Albania to maintain relations with the Soviet Union as long as possible, for many reasons; economic, political, social, cultural, etc. At the same time, Enver Hoxha wanted to eliminate any relationship between Yugoslavia and the Soviet Union, as he saw it as a danger for Albania. So, the restoration of Yugoslavia-Soviet Union relations would mean the elimination of Albania in the diplomatic policy of the Eastern Bloc. Enver Hoxha claimed to be the only allied country of the Soviet Union in the Balkan region. Through theory and secondary documents, we manage to make a non-superficial study, but to unite the pieces of a puzzle, we must continue combining and clarifying different episodes with a common denominator.\n\nDisputes within the communist bloc, such as those between Yugoslavia and the Soviet Union, or Albania and Yugoslavia and the Soviet Union, showed that there could be great divergences even when countries followed the same strategy and ideology. The cause of divergences in some cases has been sovereignty and important issues of national character. As far as Albania is concerned, the facts show that the national interest ostensibly nurtured the ego and the struggle of Enver Hoxha for power. He simply used it face to face to maintain his power.\n\n\nConclusions\n\nAlbania’s relations with the Soviet Union never emerged from the tutelage of dependence and preferences. Although within the same ideological context, Albania’s relations with the Soviet Union reflected the relations of the powers and for this reason, they were always dominated by pressure, blackmail on the one hand, and bowing and persuasion on the other. Regional developments (relations between the USSR and Yugoslavia) harmed Albania’s relations with the Soviet Union. Communist ideology and the lack of democracy in Albania gave free rein to the communist leadership to maintain the monopoly of diplomacy by focusing on caste interests and communist interests.\n\nThe case of the analysis of Yugoslav influence in relations with the Soviet Union will continue to be a case study in international relations. About the Yugoslavs, Albania has never been a priority for the Soviet Union. In any case, it would have to be dealt with depending on the ups and downs of BS-Yugoslavia relations. This did not change in both Stalin and Khrushchev. Albania never managed to play in the field of international relations between the two greats.\n\nIn ideological assessment, as the Soviet Union embarked on the path of de-Stalinization and liberalization of the country after the XX Congress of the Communist Party of the USSR, a path dictated to Hoxha by the Soviet leader, history has shown that Hoxha retained power at all costs personal. The reasons for the split are manifold. On the one hand, Yugoslavia, the restoration of Soviet-Yugoslav relations, played a very important role in Albania’s relations with the Soviet Union. But we cannot say with conviction that Albania and the Soviet Union would have maintained their friendship if these relations had not been restored. Or if Albania would not have sided with China. But even this lineup in this paper I have seen in the light of the restoration of Soviet relations with Yugoslavia. Because exactly when it felt threatened by this return, Albania turned its eyes away from China and vice versa, China found that there could be little support in eastern Europe. Disputes within the communist bloc, such as those between Yugoslavia and the Soviet Union, or Albania and Yugoslavia and the Soviet Union, showed that there could be great divergences even when countries followed the same line and ideology. The cause of divergences in some cases has been sovereignty and important issues of national character.\n\nAs far as Albania is concerned, the facts show that the national interest ostensibly nurtured the motto of International Relations. But further, numerous documents show that this could be the ‘best cover’ to safeguard the interests and personal power of the leadership. The study, regardless of the issues it addresses, does not aim to exhaust the topic of the paper, but to make a contribution to the objective analysis and to have some impact on the continuation and deepening of the treatment of this topic. We live in a different time, but the phenomena are the same, the phenomena and challenges that the country faced in the period 1949–1959 are similar. Albania’s relations with the Soviet Union are still not fully revealed as long as many documents found in Russian archives are not available for study.", "appendix": "Data availability\n\nThe underlying data for this study is under restriction by the Archival resources of the Albanian Central State Archives and the Central Archive of the Ministry of Foreign affairs. To request access to the same data used in this study, researchers should contact the information office within the premises of the Central Archives (dpa@albarchive.gov.al). The employee of the Information Office orients the researcher about the documentation that must be submitted using archival resources in the internal environments of the archives. To access the archive and the use of resources in the interior, you must submit\n\n1. A work certificate from the institution where you work.\n\n2. A copy of the identity card.\n\n3. A request describes the purpose of using the resources in the Archives, the topic, an explanation time frame for the completion of the study, as well as the contact email address or phone number.\n\nAnswers to requests are typically within 10 days from the day of submission of the documentation according to the legal acts of the archives.\n\n\nReferences\n\nThe Central State Archive, sources of the Ministry of Foreign Affairs’, Albania’s Relations with the Soviet Union, D. B/I-8, fl.11.1953.\n\nHoxha E: Vepra 1 (XI.1941 - X.1943). Tirana:8 Nëntori Publishing House;1983.Reference Source\n\nHoxha E: Vepra 19. (VI.1960 - XII.1960). Tirana:8 Nëntori Publishing House;1975.Reference Source\n\nHoxha E: Vepra 45 (XII.1970 - IV.1971). Tirana:8 Nëntori Publishing House;1985.Reference Source\n\nHoxha E: Vepra 41 (V.1969 - IX.1969). Tirana:8 Nëntori Publishing House;1984.Reference Source\n\nThe Central State Archive source of the Ministry of Foreign Affairs, Albania’s relations with the Soviet Union, D. B/I-8.1953; p.12.\n\nThe Central State Archive, source of the Ministry of Foreign Affairs Albania’s relations with the B. S, D. B/I-8. Fl.13.1953.\n\nAcademy of the Albanian People’s Republic of Albania. Tirana:Institute of History: History of Albania;1983.\n\nVukaj S: The Albanian battalion that fought for Russia, Dita Newspaper.2015, June 18.\n\nThe Central State Archive, source of the Ministry of Foreign Affairs, Albania’s relations with the Soviet Union, F. 14, D. 19, fl.1.1956.\n\nThe Central State Archive, source of the Ministry of Foreign Affairs, Albania’s relations with the Soviet Union, F. 14, D. 6, fl.1.1958.\n\nThe Central State Archive source of the Ministry of Foreign Affairs, Albania’s relations with the Soviet Union, D. 92.1959; p. 83.\n\nDyrmishi D: Political struggle in the leadership of APS 1944-196. Tirana:Toena publishing;2011.\n\nBritannica, The Editors of Encyclopaedia: Twentieth Congress of the Communist Party of the Soviet Union. Encyclopedia Britannica.2022, February 7.Reference Source\n\nHoxha E: Titlist, Historical memories and notes. Tirana:November 8th, 1981.\n\nHoxha E: The Khrushchev Sites, “November 8”. Tirana:1980.\n\nGriffith EG: Albania and the Sino - Soviet rift. Cambridge, Massachusetts:The M.I.T. Press;1963.\n\nLala A: The failed Albanian spring 1956, Historical Magazine, no. 4. Tirana: 2007.\n\nNgjela S: The Bend and Fall of Albanian Tyranny. Tirana:UET Press;2011.\n\nDuka V: History of Albania 1912-2000. Tirana:“Kristalina-a” Publishing House;2007.\n\nPearson O: Albania in the twentieth century The centre for albanian studies. Tirane:2006; vol. 3.\n\nFischer G, George R, Graham L, et al.: Science and ideology in soviet society: 1917-1967.2012.\n\nHoxha E: Superpowers, Political Journal 1959-1984. Tirana:Publishing House;1986 November 8." }	[ { "id": "181069", "date": "02 Aug 2023", "name": "Bekim Sejdiu", "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\nGeneral suggestion\nThe topic of the article is interesting and intriguing. The fact that the authors rely on archival documents adds to the value of the analysis.\nHowever, the Article needs profound revisions before publication.\nTwo types of revisions need to be undertaken:\nFirst, major language corrections and revisions need to be done as well as applying proper citation style and technical format.\nSecond, there should be a clear and consistent link between the stated research questions and hypothesis, with the analysis and findings. In the current draft, this is missing.\nSome major comments are as follows:\n\nAbstract:\n\nAbstract needs to be revised, to follow the usual format for Abstract.\n\nIt is preferable to have explanations about the methodology, results, major argument/findings, in the Introduction, rather than in the Abstract.\n\nNot clear is the objective of this paper to highlight the attitude and intentions of the USSR approach towards Albania in this period? If yes, this needs to be stated clearly in the Abstract.\n\nIt would be useful to add a sentence in the Abstract about the major argument of the paper?\nIntroduction\n\nIntroduction has not been written in a proper/standardized form.\n\nToo much factual/descriptive details about Enver Hoxha in the Introduction.\n\nHypothesis\nHypothesis need to be short, concise and in harmony with the explanation of the research questions.\n\nFrom the hypothesis, it transpires that Albania did not have any importance at all for the Soviet Union, but it was only a burden for Moscow. This is not easy to substantiate? Ideology and geography were intertwined, as a Cold War parameter. In this sense, it would be interesting to dig deeper in the interests of the Soviet Union in Albania. Khrushchev’s visit to Albania in 1959, which is elaborated in the paper, is a strong argument in this direction – particular the interpretation of this visit by Washington as an action intended to thwart the consolidation of nuclear superiority of Americans in the Southern Europe\nOverview of the history of Albania-Soviet Union relations\nThere is no consistency in the historical narrative: from the Ottoman-Russia war of the mid-XIX century, the authors jump to Lenin’s political visions and then to Enver Hoxha?\n\nConsistent analysis about relations between Albanian and Soviet Union is missing.\n\nFurthermore, if the issue under discussing is history of “Albania-Soviet U relations,” the focus needs to be in the Albanian state. This means providing at least a short overview of the position of Russia towards the first Albanian state of 1912/13; Ahmet Zogu’s era and then the Communist Albania (the latter is the major focus).\n\nA clear distinction should be made between the heavy element of propaganda in the Albanian communist historiography – which sought to glorify the ostensibly deep historical ties with Russia – with the real historical facts.\nYugoslav factor\nIt is undisputable that Yugoslav communist party (with emissaries such as Serbian communist Dusan Mugosa and Miladin Popovic), played crucial role in creating and leading from behind, the Albanian Communist Party and, from 1045 – 1948, Albania was a satellite of Belgrade.\n\nIt would be interesting to explore briefly, why Enver Hoxha broke with Tito in 1948, and turned robustly towards Moscow (Albanian Communist state was an ally of\n\nBelgrade, even in suppressing anti-Yugoslav uprising of Albanians in Kosovo, in 1945, lead by Shaban Polluzha)?\nResults\nNo consistency between the analysis in the preceding section and the results?\n\nThere needs to be a stronger and more direct link between the research questions; major hypothesis and the analytical part of the paper. This linkage is very weak, not to say non-existing.\n\nIn this vein, the economic and political dependency of Albania from the Soviet Union needs to be clearly and substantially argued.\n\nFurther, how it is argued in this paper that: “The Soviet Union did not have the geopolitical or economic interest in Albania? This is the first hypothesis?\n\nOr, how does this article address the research question: “Was it possible for Enver Hoxha to behave differently with the Soviet Union and what has been Albania’s strong point in relations with the Soviet Union”?\nLanguage and technical problems in the text\nThe draft needs thorough revision, with regards to the language editing, style of citation and technical aspects.\n\nMany sentences are unclear, not properly formulated and confusing.\n\nBeginning of new, important arguments, to be done in a new sentence with one empty space in between.\n\nSpace between new arguments: not consistent.\n\nCitation style and the titles of the sections: to have a standard format.\n\nTitles of the parts do not follow any standard format.\n\nThe part on methodology, comparative research, limitation, would be OK for a book, but not for an academic journal?\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? No", "responses": [ { "c_id": "10035", "date": "07 Sep 2023", "name": "mirela tase", "role": "Author Response", "response": "These are some comments relating to the review`s report as follows: We want to clarify that the length or brevity of an argument does not affect the construction of hypotheses, despite what the reviewer may have suggested. We have developed three hypotheses that fully align with our research questions. Each hypothesis is thoroughly explained, using both research sources and our well-articulated ideas to establish the significance of the relationships between both sides. As evidence, we can provide the following quote: “We tried, through hypotheses, to provide an assessment and undoubtedly a clearer insight into the relations with the Soviet Union. According to our judgment, Albania was an unimportant country for the Soviet Union (due to the way Enver...) Enver Hoxha benefited from this relation (with the goal of building...) the socialist state and giving it a name in the international arena, not only for himself (head of Albania), but also Albania as a whole.” While we appreciate the reviewer's argument about Khrushchev's visit to Albania in 1959, which suggests that Washington saw the visit as a move to prevent American nuclear superiority in Southern Europe, we must clarify that this argument is not directly related to the theme of our paper, or the three hypotheses presented. However, we will consider it a suggestion to explore in a separate article that addresses this issue. In regard to the Overview of the history of Albania-Soviet Union relations, the reviewer suggested including a brief overview of Russia's position towards the first Albanian state in 1912/13. However, this is irrelevant to our paper's primary focus, which is on the period from 1945 onwards. This suggestion is not directly related to the paper's topic. However, we will try to integrate it into another work that focuses on Albania's relations with BS in the beginnings of the Albanian state and in continuity. Regarding the reviewer's suggestions, we would like to clarify that the connection between the research questions, main hypothesis, and analytical part is not weak but very strong and visible throughout the paper. We emphasized multiple times in the work's results and description that the Soviet Union had no direct political or economic interests in Albania. However, they provided financial assistance during both the Stalin and Khrushchev periods, which benefited Albania's construction of a socialist state. Despite the lack of direct interest from the Soviet Union, the paper provides a clear argument for Albania's economic and political dependence on them, which is also the focus of Enver Hoxha's relations with BS. Regarding the answer to the unexplained question, according to the reviewer, \"Was it possible for Enver Hoxha to behave differently with the Soviet Union, and what was Albania's strong point in relations with the Soviet Union,\" this was very clearly addressed in the paper and mainly in the results. This is included in the conclusion as follows. \"In conclusion, I would summarize with the expression, “Stalin was the political muse for Enver Hoxha, this worship that he had continued until the end of his life and refused to be de-Stalinized. This turned out to be fatal in relations with the Soviet Union and all other countries and ended in the greatest isolation of Albania until the fall of the regime in 1990.” Albania's strong point in Albania-BS relations was Hurshov’s several-day visit to Albania, where he ranked our country as one of the most influential countries and Albania's geopolitical position in the Balkans.\" Following the answer \"Enver Hoxha, if he did not prioritize the longevity of his power and his jealousy towards the relations between BS and Yugoslavia, where he saw the latter as a danger to the end of his relations with BS, he would have no only a vital place in these bilateral relations, but it would give a different look to the relations between these two countries. Enver Hoxha's insistence on preserving the Stalinist cult at all costs, and (his rejection of the criticisms made by (Khrushchev) to Stalin clearly show his fear of reform as the most significant risk of leaving power. Albania's strong point in Albania-BS relations was a several-day visit to Albania, where he ranked our country as one of the most influential countries and Albania's geopolitical position in the Balkans. In final publishing, we will edit the Language by the English editor. Best wishes Authors" } ] }, { "id": "230088", "date": "30 Jan 2024", "name": "Ardian Muhaj", "expertise": [ "Reviewer Expertise History of Albania", "History of the Balkans", "Economic History." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluation: The article defines sufficiently its research question or thesis. The authors effectively set the historical and geopolitical context. The article thoroughly covers the topic, including major events, key figures, and political dynamics. The sources cited are relevant and authoritative with a balanced mix of primary and secondary sources.\nThe method used to analyze Albania's diplomatic relations with the Soviet Union are appropriate and well-executed. Also, the authors have clearly explained of how data and historical documents were collected and analyzed.\nThe article analyzes effectively the diplomatic relations, and the arguments are well-supported with evidence. The discussion offers new insights on the subject. Overall, the historical information is presented accurately, and the article demonstrates a good understanding of the geopolitical dynamics of the period. The article contributes new perspectives to the field of diplomatic history or Albanian-Soviet relations.\nSpecific suggestions: Regarding structure and clarity, the article could have been better organized, allowing readers to follow the argument more easily. The language and style also need a thorough improvement. Regarding references and citations, the sources are properly cited, but the explanation of the content and the procedure to obtain the documents in the State Archive go into too much and unnecessary details. Also, the bibliography should be enriched with more literature on the subject.\nConclusion and Overall Evaluation: The topic of the article is interesting, and the thesis statement is clear and sound. I recommend the article for indexing after the mentioned improvements have been made.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-1441
https://f1000research.com/articles/13-95/v1	09 Feb 24	{ "type": "Research Article", "title": "Management of cultural tourism in Ban Toon, Muang District, Phayao Province, Thailand during the COVID-19 pandemic", "authors": [ "Warach Madhyamapurush", "Krantharat Khawatkun", "Phatpitta Sreesoompong", "Nattawut Somyarone", "Wongduan Sakboonruang", "Krantharat Khawatkun", "Phatpitta Sreesoompong", "Nattawut Somyarone", "Wongduan Sakboonruang" ], "abstract": "Background\nThe research aims to study the process of managing cultural tourism in the community and assess the socio-economic impact on the community with the help of eight key contributors to the project who play an essential role in the implementation of cultural tourism management.\n\nMethods We prepared field notes from interviews and observations, and provided details from data collection (in the form of transcripts). Thematic analysis was employed to answer the research questions using a content analysis tool to obtain the community context in cultural tourism and tourism management dimensions. Participatory Action Research (PAR) was employed to meet the needs of stakeholders and test tourism routes through such routes. The tourism route development was evaluated using descriptive statistics.\n\nResults There is one new cultural tourism route and the process of managing cultural tourism developed community-level organizations with spiritual leaders as the main advisors. The result of the net present value (NPV) analysis is 917,149 baht or 27,792$. The return from the implementation of this project (the internal rate of return (IRR)) is 11.32%. In addition, the ratio of the sum of the present value over the life of the project to the sum of the present value of the cost over the life of the project is 2.45 times, giving the project, if continued, an income 2.45 times greater than its expenditure.\n\nConclusions Cultural tourism management in Ban Toon, Muang District, and Phayao Province during the COVID-19 pandemic can be used as a guideline in other communities with similar contexts.", "keywords": [ "Tourism management", "cultural tourism", "COVID-19 pandemic", "Ban Toon", "Muang District", "Phayao Province" ], "content": "Introduction\n\nThe COVID-19 pandemic from 2020–2022 caused many changes, especially regarding travel. The tourism industry was greatly affected, particularly the main tourist activity of traveling and socializing. The local government should assist the business sector in helping communities develop sustainably in an Albanian location where tourism has been affected by COVID-19 (Alshiqi & Sahiti, 2022). A Spanish case study (Rodríguez-Vázquez et al., 2023) demonstrates that cultural tourism is a more potent draw than other types of tourism. To comprehend the potential of cultural tourism to help resolve the issue and its reaction to the needs of new post-pandemic visitor behavior, it may still be essential to pinpoint the critical factors. The business structure of the tourism sector may need to be modified in light of the current circumstances to handle the crisis, protect the company, and protect jobs. This indicates that a thorough plan is needed to resuscitate the tourism sector. Future research should examine the effects of the COVID-19 outbreak on incoming and outward travel, visitor behavior, employment, and the industry’s overall growth rate (Santus Kumar DEB, Shohel Md. NAFI, 2020).\n\nThe Thailand Safety and Health Administration (TSHA) has released measures in collaboration with the Tourism Authority of Thailand, the Ministry of Tourism and Sports, and the Department of Disease Control in the Ministry of Public Health to protect and foster confidence in public health safety. These initiatives increase visitor comfort levels and make tourist destinations safer (Rodríguez-Vázquez et al., 2023). The COVID-19 epidemic affected many of Thailand’s community tourist attractions, even though many were approved to meet specific standards, according to a study (Rodríguez-Vázquez et al., 2023). These closures resulted in a loss of revenue for the neighborhood. As a result, those who supplied tourist-related services either changed the type of work they were doing or moved to work somewhere else (or both), leaving previously dependent regions on tourism with few other options for income (Kofi Poku Quan-Baffour, 2020).\n\nTourism is essential to the development of Thailand, especially its economic and social aspects. In economics, tourism generates income and promotes the distribution of funds based on the number of tourists to local areas. This, in turn, enhances job creation and employment in various occupations, directly and indirectly. Societally, tourism contributes to the development of public utilities and various facilities that help enhance the quality of life throughout the community. Tourism also helps communities and localities effectively revitalize and conserve natural and cultural resources. Furthermore, tourism also brings in experienced tourists, who help improve the quality of life and society.\n\nOne of the regions impacted by the decline in tourism brought on by the COVID-19 outbreak is Phayao Province. The province announced an indefinite suspension of tourism activities, including a ban on travelers entering Phayao Province, as well as other measures to address the crisis that arose in the early stages of the pandemic. At the end of 2021, the government announced relief measures that allowed tourism operations to resume, but only with the SHA+ standard.\n\nBecause of its 2010 designation as a national model village with a self-sufficient economy, the Ban Toon community in Muang District, Phayao Province, has high tourism potential. The western area of the community is a source of agricultural learning, producing organic rice and being one of the suppliers of pickled fish in Phayao Province. This has increased the number of tourists interested in and learning about agricultural methods. In addition, the researchers’ spatial survey results showed that the eastern portion of the province had a natural, historical tourist attraction. The ancient port, with cruise activities to see the Red Lotus Sea, is considered one of the most crucial community tourist attractions in Phayao Province. In the two and a half years from 2017 to 2020, there were approximately 3,500 tourists, consisting of study groups visiting Phayao Lake 100 times per year, with roughly ten people per group, totaling 1,000 people per year who came to see the Red Lotus Sea in Phayao Lake. The estimated annual income was as follows: The income from providing educational visits costs 200 baht per person (6.06$), or 200,000 baht per year (6,061$). General tourist groups had expenses related to accommodation (homestay), food, and souvenirs (organic rice), averaging 500 baht per person (15.15$), totaling 1,000,000 baht per year (30,303$). While additional activities specifically to see the Red Lotus Sea earned an average of 100 baht per person (3$), totaling approximately 1,250,000 baht (37,878.79$). However, a survey of tourism conditions in early 2021 found that tourism, which had previously generated an income of approximately 1,250,000 baht per year (37,878.79$), had decreased to only 100,000 baht (30,303$). The number of tourists who had previously visited Phayao Province was estimated to be 3,500 per year. It was found that this number decreased to a mere 200 people per year during the pandemic. As a result, people in the community ceased to operate tourism activities, thus further reducing the likelihood that tourists would visit Phayao Province and the Red Lotus Sea area.\n\nPhayao Province planned a four-year (2017–2021) “Phayao Wellness” initiative. A part of the government’s mandated COVID-19 relief measures was the SHA+ standard for regulating tourism activities. Thus, the question became, during the pandemic, how would the Ban Toon community in Phayao manage cultural tourism so that people could earn a living? The project “Cultural Tourism Management, Ban Toon, Muang District, Phayao Province, during the COVID-19 Pandemic” set out to answer this question. This study aimed to evaluate and develop the process of cultural and tourism management. It includes creating cultural tourism routes and designing cultural products that would facilitate the cooperation of Ban Toon villagers throughout the sub-district. Additionally, the economic and social impacts of the project were assessed.\n\n\n\n1. To study the context and readiness of the Ban Toon Sub-District community area.\n\n2. To develop cultural tourism routes and cultural tourism management.\n\n3. To assess the economic and social impacts of cultural tourism management.\n\nThe concept of tourism systems can be understood by studying related concepts, such as Dickman’s (1996) and Leiper’s (1990) travel composition concepts on tourism systems. Tourism management must consider the host dimension, whereas the source mainly considers the needs of tourists. Madhyamapurush’s (2019) work on the tourism system studied the entire tourism project across three dimensions: the demand side, the response or supply side, and the support side. Madhyamapurush’s study includes the work of Goeldner and Ritchie (2006), defining the components of tourism, or the “tourism phenomenon,” of tourism and tourism management. This includes Kušen’s (2010) work on the tourism system of tourist attractions. A tourism attraction system developed by studying all five researchers attempted to explain tourism links in a global context. Dickman (1996) and Kušen (2010) mainly focused on how attractions should have essential components. Leiper (1990) and Madhyamapurush (2019) linked a knowledge set of tourist needs to create responses to those needs in tourism activities. In addition, Goeldner and Ritchie (2006) and Madhyamapurush (2019) provided an appropriate administrative process or method for demand, supply, and support. These provide a broader picture of the tourism structure and have not yet been effectively applied to community tourism. The study also found that a private agency primarily provides the response side (supply side).\n\nThe concept of tourism management has been developed from a business system to enable the tourism activities of government agencies, the private sector, and community tourism to be able to conduct activities appropriately, using the original management concept of Fayol (1916): POCCC (Planning, Organizing, Commanding, Coordinating, and Controlling). Moreover, it relies on the concept of co-management, the system tourism (Madhyamapurush, 2018) described for the tourism management model used in Phu Hin Rong Kla National Park, Phitsanulok Province, which proposed the idea of cooperation between the government and the tourism community in the national park to direct the work there.\n\nTourists gain experience in tourism through the service provided by the provider and the experience that comes from learning about, getting in touch with, or participating in activities in tourist areas. The study of tourism experience factors was divided into two parts. The first designers to study experience management utilized the concept of Pine and Gilmore (1998). This consisted of four areas of relationship - entertainment, education, aesthetics, and escape – along with two dimensions of participation: participation and relationship with the environment. These concepts were combined with those of Gao, Scott, & Ding (2010) and Cetin & Bilgihan (2015), which consist of outline, atmosphere, memorable story, interaction, service, culture, heritage, and challenges. For the tourist experience, the psychological concepts of Poulsson and Kale (2004) were used, which consisted of novelty, learning, personal relationships, surprise, and participation. The study of the relationship between these two components makes up for Cutler and Carmichael (2010).\n\nThe economic theory (Growth Model) of Harrod-Domar (Besomi, 2001) (Mahawong, 2001) has developed a theory or model of economic and social impact assessment. Economic growth aims to describe the relationship between the two within a particular level of growth. Both concepts hold that investments are at the heart of economic growth. In addition to increasing revenue, there is also the ability to increase efficiency. The production of the economy and maintaining full employment require economic growth that is high enough to absorb savings, which is desired for income investing at the whole employment level. Harrod-Domar assumes that the model is either an industrial unit of production or that the economy as a whole depends on the amount of capital invested in that economic unit.\n\nSource: Researcher's synthesis.\n\n\nResearch methods\n\nThe Human Ethics Committee of the University of Phayao approved the research protocol (approval number UP-HEC 2.2/078/64); the date of approval was August 22, 2021. Participants were given the purpose of the research and asked to participate in the study; they welcomed it. Written consent was obtained from all participants, and they were informed that their anonymized data would be published. All participants also gave their permission and received information about the article’s methodology.\n\nStudy design\n\nThe research was carried out between August 20th and 21st, 2021. General survey data on tourism resources were collected on September 21st. Interviews with stakeholders were conducted at Ban Toon on October 21st. Training on the tourism route and experience was conducted in Ban Toon on November 21st. The selection of tourist volunteers was completed on December 21st. Testing of tourism routes with tourist volunteers and data collection was conducted on January 20th and 22nd. Data were collected to assess the economic and social impact of stakeholders in the community on March 22nd.\n\nSampling\n\nKey informants included local sages, community leaders, community product operators, attraction supervisors, and community development officers of the Ban Toon Sub-District Administrative Organization. They were selected using purposive sampling to obtain accurate cultural information. The selection of volunteer tourists or simulated tourists included announcements for interested applicants to apply via Facebook. The qualifications of volunteers are that they must not be a person in the study area and must have an understanding of the evaluation.\n\nThe study utilized an advertisement posted on Facebook to attract tourists’ interests and encourage their participation. The advertisement was shared by researchers’ colleagues and other associates, expanding its reach and visibility among potential participants. As tourists came across the advertisement on Facebook, they found it interesting and decided to participate in the study on the management of cultural tourism in Ban Toon. By leveraging the power of social media and utilizing networks within their professional circles, researchers were able to effectively promote their studies and generate interest among potential participants. Many tourists discovered the advertisement and found it intriguing. Consequently, they decided to participate in the study.\n\nThe researchers used Facebook as a platform to make announcements because of its widespread reach and the potential to attract a diverse pool of applicants. By utilizing Facebook, we aimed to engage individuals who were interested in participating as volunteers or simulated tourists in the study. To select participants, the researchers employed a method in which they considered the acceptance requests received through Facebook as the basis for their selection process. Individuals who expressed their interest and were formally requested to participate via Facebook were chosen as study participants. As a result, the study’s participant pool was limited to individuals who had submitted an acceptance request through Facebook. The use of Facebook as the primary channel for recruitment and participant selection plays a significant role in determining the composition of the study sample. It is important to note that using Facebook as a recruitment platform may introduce certain limitations such as potential selection bias and limited representation from individuals who may not have access to or actively use the platform. Researchers have considered these factors when interpreting the findings and generalizing the results of the study.\n\nData collection\n\nQuestionnaire: A questionnaire was designed to gather data on the socio-economic impact of cultural tourism on the community. It was distributed to community members, including tourists, and community leaders (Madhyamapurush, 2023).\n\nInterviews: In-depth interviews were conducted with ten key contributors to the cultural tourism management project, including spiritual leaders, community leaders, and tourism business owners. These interviews provided valuable insights into the process of managing cultural tourism in the community (Madhyamapurush, 2023). The interviews were conducted by the research team, which consisted of several individuals with expertise in cultural tourism and socio-economic impact assessment. The interviews were conducted in person at the Ban Toon district. The interviews were semi-structured and followed the COREQ guidelines to ensure that all necessary information was gathered. The interviews were audio-recorded with the participants’ consent, and detailed field notes were taken during and immediately after each interview.\n\nRoute Testing: Participatory Action Research (PAR) was used to test the tourism routes in the community. This approach allowed stakeholders to be involved in the development and testing of the routes. The tourism route development was evaluated using descriptive statistics.\n\nFor the data collection process, the researchers involved ten key contributors to the project who played an essential role in the implementation of cultural tourism management in Ban Toon, Muang District, Phayao Province, Thailand. Key informants, such as local sages, community leaders, community product operators, attraction supervisors, and community development officers from the Ban Toon Sub-District Administrative Organization, were selected using a purposive sample to gather accurate cultural data. A total of ten people were chosen. Additionally, 30 tourists tested the route, with applications accepted for those traveling in November 2021. The data collection process involved several stages. Firstly, the researchers conducted interviews with the key contributors to gather their perspectives and insights on the process of managing cultural tourism in the community during the pandemic. The interviews were conducted in-person and via video conferencing tools such as Zoom. Secondly, the researchers conducted observations of the community to gain a better understanding of the community’s context in cultural tourism and tourism management dimensions. The observations were conducted on-site and involved the researchers actively participating in the community’s cultural tourism activities. Thirdly, the researchers prepared field notes from the interviews and observations that were conducted. The field notes were used to provide details for the data collection process and served as a reference during the analysis stage. Lastly, the data collection process involved the use of Participatory Action Research (PAR) to meet the needs of stakeholders and test tourism routes through such routes. The tourism route development was evaluated using descriptive statistics. Overall, the data collection process involved a collaborative approach between the researchers and the key contributors to the project. The researchers worked closely with the contributors to ensure that the data collection process was comprehensive and accurate.\n\nThe research utilized Participatory Action Research (PAR) to develop a cultural tourism model that meets the needs of tourists and to test the tourism routes. The tourism route assessment form (Madhyamapurush, 2023) was created. The validity of the questionnaire was examined carefully. Three experts examined each of the questionnaire items. The content validity and linguistic suitability were evaluated using an Index of Item Objective Congruence (IOC). An IOC score of 0.70 indicates the permissible range. The questionnaire was then altered in accordance with the experts’ recommendations, and reliability was evaluated using Cronbach’s alpha coefficient. The result of 0.948 was deemed statistically significant.\n\nThe researcher prepared field notes from interviews and observations and provided details from data collection (in the form of transcripts) (Madhyamapurush, 2023). When volunteers arrived, the researcher explained the tourism process. Villagers conduct tourism along the developed tourism route. Researchers created assessment forms for volunteers. The volunteers in this study conducted the assessment of the participants. Researchers conducted interviews on the use of travel routes.\n\nData analysis\n\nMethods for processing data prior to and during analysis:\n\n- Transcription: The interviews were transcribed verbatim, including all pauses, hesitations, and nonverbal cues.\n\n- Data entry: The transcripts were entered into a computer and stored in a secure database.\n\n- Data management and security: The database was password-protected and only accessible to authorized personnel. The data was backed up regularly to prevent loss or corruption.\n\n- Verification of data integrity: The transcripts were cross-checked for accuracy and completeness by two independent researchers.\n\n- Data coding: The transcripts were coded using a content analysis tool to identify themes and patterns related to the research questions.\n\n- Anonymization/deidentification of excerpts: Any identifying information, such as names or locations, was removed from the transcripts to protect the privacy of the participants.\n\n- The researchers involved in data analysis were trained in qualitative research methods and had experience in analyzing data from similar studies.\n\n- The rationale for using this approach was to allow for a more in-depth understanding of the experiences and perspectives of the participants.\n\nDescriptive statistics were used to analyze the collected data, which was done using SPSS Version 27.0. To address the research questions and gain insight into the cultural tourism and tourism management dimensions within the community, a thematic analysis method was employed through a content analysis tool. Content analysis was used to create cultural tourism routes, allowing tourists to travel independently and engage with the Ban Toon community. The development of these tourism routes was evaluated using descriptive statistics.\n\n\nResults\n\nThe study found that “Ban Toon is a community that has cultural road activities, consisting of routes that concretely reflect the distinctive local cultural identity, such as old houses, a market that still maintains a local way of life, temples, ancient sites, religious places, and various artistic activities, as well as local wisdom” (Rodríguez-Vázquez et al., 2023). In addition, the Ban Toon community has its roots in the Tai Yuan ethnic group, a population group that settled in northern Thailand (Chiang Saen) and migrated to the area of the Ban Toon Sub-District until it became a large community with a strong local culture. Various customs are considered a part of the intangible cultural heritage of the Ban Toon community. These consist of the following:\n\n1. Food culture. Ban Toon is an urban community that continues to cook food using traditional methods and culture. The community has outstanding local dishes, such as northern spicy curry and fish curry.\n\n2. Clothing culture. The general style of dress of Ban Toon village men is three-quarter length pants (troops) made of cotton-dyed blue or black. Shirts are round-necked, short-sleeved cotton with small slits on either side (the Mahom shirt). Women dress in a sarong that almost reaches their ankles. The edge of the sarong has a beautiful pattern. The shirt was round-necked with colorful and beautiful patterns.\n\n3. Housing culture. Buildings in the Ban Toon community maintain traditional Lanna (northern style) architecture. Many new residential houses are still in the original style of ancient houses.\n\n4. Traditional culture. The Ban Toon community continues to maintain various traditions, such as setting up the Thamma Luang and the Mahachat Sermon (Vessantara Jataka), held during the festival of floating lanterns in the Yi Peng month or the 12th lunar month.\n\n5. Language culture. The villagers of Ban Toon still use the Northern Thai dialect (Kham Muang language) and the Lanna script. The ancient scriptures of the Ban Toon Tai Moral Community are preserved and sorted into categories.\n\n6. Occupation culture. Most baboon villagers are engaged in agriculture. Local handicrafts also include wickerwork, products made from water hyacinths, lanterns, and ancient flags.\n\n7. Belief culture. The Ban-Toon community is an ethnic community that has migrated from Chiang Saen. Therefore, various beliefs are based on the Lanna culture, such as ancestral spirit raising and traditions to support the buffalo. In addition, there are also important Lanna rituals, namely, the ceremony to forgive or release misfortune, known as Su Kwan, which uses equipment that the villagers call “Satuang” when performing the ritual.\n\n8. Performing art culture. The Ban Toon community has established a cultural learning center to transfer and train youth in the community to preserve Lanna’s traditional Thai music (Salor Saw Sueng).\n\nThe characteristics of the Ban Toon community reflect the agricultural nature of the area, which is connected to Phayao Lake and the Doi Luang National Park. Before the COVID-19 pandemic, visitors preferred agro-tourism activities in the western sub-districts and organic fields near the Doi Luang National Park entrance.\n\nRoutes for cultural tourism are under development. The research team brought information to the initial meetings between community leaders and local communities. Information regarding the context of the area emphasized community participation in developing cultural tourism routes linking tourist sites. Tourism across the district is now more cohesive than it had been in the past. As a result, the tourism route connects the mountains, rice fields, and Phayao Lake based on the promotion slogan: “See the mountain, Walk through the rice fields, Cruise the river” (Dickman, 1996). The cultural attractions of the Ban Toon community consist of Doi Luang National Park, Doi Nok, Huai Toon Reservoir, Phakretnark Waterfall, Ban Bua Bamboo Tunnel, Phu Klong Vineyard, Ban Toon Tai Temple, Ban Bua Temple, Analayo Temple, the clay pot learning center, So Huay House, the Ancient House, Ban Thung Kiew Ancient Ferry, and the local fishing group. The research team brought 30 tourists to the experimental group to test this route. Tourists gave their opinions, as shown in Tables 1 and 2, Figures 1 and 2.\n\nSource: Developed by a group of researchers together with the community.\n\nTables 1 and 2 demonstrate that this tour route can take either 1–3 hours or six hours, indicating that the route can be adjusted to suit tourists’ travel time. The route is only suitable for a single day of travel. Moreover, the route had easy access to tourist attractions, with road routes and signposts averaging a satisfaction rating of 3.93. Facilities in tourist attractions (restaurants, coffee shops, homestay accommodation if desired, toilets, and cell phone signals) averaged 3.77. In contrast, the value and attractiveness of the tourist attractions (tourist activities, beauty, community) averaged 4.13. The environment of the tourist attractions (no pollution, complete ecosystem) averaged 4.33, and tourist attraction management (clarity of signposting and the starting point of the tourism route) averaged 4.23. The overall average of 4.08 implies that the tourists who tested the route had a high level of satisfaction, leading to the conclusion that the cultural tourism route “See the mountain, Walk through the rice fields, Cruise the river” is popular both with people in the community who developed the route and tourists who have come to experience it.\n\nThrough examination of the study of cultural tourism routes, the researchers found that fortune-telling activity is an essential ritual in the community and is commonly performed to increase merit and release bad luck. Understanding the cultural importance of such a ritual, the research team designed an activity in which participants, together with community leaders and others, assembled a merit-making set (Satuang), a cultural product of the Ban Toon community. This encourages tourists who come to experience the culture of the Ban-Toon community along the specified route. The Boon Teung set (Satuang) is a product created by people in the community to sell to people who live or work abroad and, in the coming years, will not be able to attend the ceremony, yet still wish to make merit and release bad luck.\n\nPreparing the tourism route “See the mountain, Walk through the rice fields, Cruise the river” and activities for the Boon Teung (Satuang) event needed to be developed through a committee of a cultural tourism group in the Ban Toon community. The committee includes a mentor and a spiritual leader and chairs the official committee. It includes eight directors from local government agencies, other spiritual leaders, travel leaders, and community leaders who collaborate with tourism and community leaders through participatory work processes.\n\nAn analysis (Table 3) of the assessment of the economic and social impact of the research found that the project will have a net present value (NPV) income result of 917,149 baht (27,792.39$) in its first five years of operation. The internal rate of return (IRR) from the implementation of this project is 11.32%, which is higher than the bank deposit rate, making the project a viable investment. Moreover, when considering the ratio of the present value of results to the sum of the present value of costs throughout the project’s life, indications are that this project will yield up to 2.45 baht (2.45$) for every 1 baht invested (1$). While the net income will be 2.45 times greater than the expenditure, the estimated income will still be less than that of 2017-2020 due to the overall reduction in tourism since the COVID-19 pandemic.\n\n\nConclusion and discussion\n\nThe research team reached the following conclusions by studying cultural tourism management in Ban Toon, Muang District, Phayao Province, during the COVID-19 pandemic.\n\nThe context and readiness of the Ban Toon area, Muang District, Phayao Province, is a community with cultural road activities that consist of routes that reflect and express distinctive local identities, such as old houses. These markets maintain a local way of life, temples, ancient sites, religious sites, traditional culture, local wisdom, and various artistic activities. Furthermore, there is an aesthetic corresponding to the concept of Izidora Marković’s (2017) research on tourism areas, which found that sustainability in tourism management should consider the area’s management to create attractiveness for tourists, including preserving the area’s sustainability. This is consistent with the research of Keeraphan, Boonkhum, and Sungraksa (2016). The authors demonstrated that many Boon Toon environments and tourism resources were ready to host visitors, promoting education and naming a community leader responsible for tourism.\n\nThe development of the cultural tourism route, “See the mountain, Walk through the rice fields, Cruise the river,” connects natural and cultural attractions, as well as tourist attractions across the entire district, consisting of Doi Luang National Park, Doi Nok, Huai Toon Reservoir, Phakretnark Waterfall, Ban Bua Bamboo Tunnel, Phu Klong Vineyard, Ban Toon Tai Temple, Ban Bua Temple, Analayo Temple, the clay pot learning center, So Huay House, Ancient House, Ban Thung Kiew Ancient Ferry, and a local fishing group. Tourism activities can be scheduled to suit tourists’ preferences, which corresponds to Pine and Gilmore’s (1998) concept of experience design, which states that the four boundaries of the relationship are entertainment, education, aesthetics, and escape, along with the two dimensions of participation, participation, and relationship with the environment. This allows travelers to engage with service providers and have experiences that facilitate hands-on learning while participating in activities in tourist areas.\n\nThe management of cultural tourism has been developed in the Ban Toon community cultural tourism group with the establishment of a formal committee consisting of consultants, a spiritual leader, a chairman, and eight committee members from local government agencies working with tourism and community leaders through a participatory work process. This corresponds with Piyaporn Taweekul’s (2001) research, which proposed that sustainable community tourism management relies on specific main components. It includes community needs, opening up to tourism, and participation in tourism management.\n\nAfter analyzing the net present value (NPV), the economic and social impact assessment found that the project will have a net income of 917,149 baht (27,792.39$) over the next five years. The internal rate of return (IRR) analysis showed a rate of 11.32%, which is higher than the bank deposit rate. This makes the project worthy of investment. Additionally, when considering the ratio of the present value of the results throughout the project’s life to the sum of the present value of its costs, the project continues to earn 2.45 times more than expenses.\n\nCultural tourism management in Ban Toon, Muang District, and Phayao Province during the COVID-19 pandemic can be used as a guideline in other communities with similar contexts. Tourism must have the potential and availability of resources and include the participation of people in the community to achieve a distribution of income while thoroughly developing community areas.", "appendix": "Data availability\n\nFigshare: ‘Management of Cultural Tourism in Ban Toon, Muang District, Phayao Province, Thailand during the COVID-19 pandemic, https://doi.org/10.6084/m9.figshare.22683232.v3 (Madhyamapurush, 2023).\n\nThis project contains the following underlying data:\n\n- Underlying Data - 24th April 2023.xlsx\n\nThis project contains the following extended data:\n\n- Questionnaire\n\n- Assessment form\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nCultural tourism management in Ban Toon, Muang District, Phayao Province, received approval in consulting and conducting tourism activities from Phra Chiraphong Woralapho, Acting Abbot of Toon Tai temple. Moreover, we gratefully appreciate the support from teacher Charan Jaiyuen, Chairman of Ban Toon Sub-District Cultural Council, Mrs. Suchit Muangkham, owner of Ban Ma Kao’s ancient house, Mrs. Somporn Mankong, member of the Kom Tung group, Father Noi Somkiat Chamnanya, village doctor of Ban Toon Sub-District, Mr. Peng Setha, of handicrafts for the elderly, and Mr. Somsak Chamnankling, Chairman of the basketry group and President of the organic rice group. In addition, we received cooperation from the Ban Toon Sub-District Administrative Organization, Muang District, Phayao Province.\n\nAuthors of this study would like to extend their gratitude to QUVAE Research and Publications for support during the submission of this article and deposition of the raw data to the online repository system.\n\n\nReferences\n\nAlshiqi S, Sahiti A: The Impact of COVID–19 on Albanian Tourism. Emerg. Sci. J. 2022; 6: 33–41. Special Issue “COVID-19: Emerging Research”, 2022. 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African Identities. 2020; 21: 134–149. Publisher Full Text\n\nKušen E: A system of tourism attractions. Tour. Rev. 2010; 58(4): 409–424.\n\nLeiper N: Tourist attraction systems. Ann. Tour. Res. 1990; 17(3): 367–384. Publisher Full Text\n\nMadhyamapurush W: Tourism Management in Phu Hin Long Kran Nation Park, Phitsanulok. Parichart Journal, Thaksin University. 2018; 31(3): 131–144. Reference Source\n\nMadhyamapurush W: Structure of Tourism System. J. Thai Hosp. Tour. 2019; 14(1): 94–102. Reference Source\n\nMadhyamapurush W: Management of Cultural Tourism in Ban Toon, Muang District, Phayao Province, Thailand during the COVID-19 pandemic. Dataset. figshare. 2023. Publisher Full Text\n\nMahawong S: The development of economic knowledge. Graduate school: Srinakharinwirot University; 2001.\n\nPine BJ II, Gilmore JH: Welcome to the Experience Economy. Harv. Bus. Rev. 1998; 76: 97–105. PubMed Abstract\n\nPoulsson SHG, Kale SH: The experience economy and commercial experiences. Boston: Harvard Business; 2004.\n\nRodríguez-Vázquez C, Castellanos-García P, Martínez-Fernández VA: Cultural Tourism in a Post-COVID-19 Scenario: The French Way of Saint James in Spain from the Perspective of Promotional Communication. Societies. 2023; 13(1): 16. Publisher Full Text\n\nTaweekul P: People’s Participation in Sustainable Tourism Management: A Case Study of Baan Lai Hin, Lai Hin Sub-District, Koh Kha District, Lampang Province. Graduate School: Chiang Mai University; 2001." }	[ { "id": "250190", "date": "18 Apr 2024", "name": "I Gusti Bagus Rai Utama", "expertise": [ "Reviewer Expertise Economic Tourism", "Agritourism", "Destination Management", "and Senior Tourism" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe background of the research is very clear.\n\nThe research methods used are also appropriate.\n\nThe research results are not according to the research objectives and methods that have been set. In this section, the author should present the Management of cultural tourism in Ban Toon, Muang District, Phayao Province, Thailand during the COVID-19 pandemic. The results of financial analysis are only as an additional or supporting analysis.\n\nIn the Conclusion and Discussion section, you should just concentrate on concluding the Management of cultural tourism in Ban Toon, Muang District, Phayao Province, Thailand during the COVID-19 pandemic. For example: how to plan the tour package, what attractions are there and what is attractive to tourists, who is involved in managing it, how to sell it, where is the marketing segmentation, and what is the control system? (POAC: Planning, Organizing, Actuating, and Controlling). Because this research was conducted during the COVID-19 pandemic, how is the health surveillance adaptation system?\n\nIs the work 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? No", "responses": [] }, { "id": "250188", "date": "24 Apr 2024", "name": "Anila Thomas", "expertise": [ "Reviewer Expertise Community initiatives and Tourism development", "sustainability", "SDGs" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The research intends to analyze the techniques associated with handling cultural tourism in the community and investigate the economic and social effects on the community, with the assistance of eight major project contributors who play critical roles in the implementation of cultural tourist management. Literary materials give a more complete image of the tourist system, but sources should be contemporary studies and research. The tourism experience variables, which were broadly discussed in the study, must be properly articulated with the most recent data sources.\nThis study utilized compiled field notes from interviews and observations, gather data specifications (transcripts), and thematic analysis to acquire the community context in cultural tourism and tourist management components. Participatory Action Research (PAR) was used to fulfil the demands of stakeholders and evaluate tourism routes along these routes. Descriptive statistics were used to examine the development of tourism routes.\nThe Ban Toon community in Muang District, Phayao Province, has a strong tourist potential due to its 2010 recognition as a national model village with a self-sufficient economy. The following resources are available:\nThe western region offers agricultural learning opportunities. The eastern region is a natural and historical tourism destination. The ancient port, with cruise operations, is a key community tourist attraction.\nAt the time of the pandemic, residents in the neighbourhood stopped operating tourism businesses, significantly limiting the chance of visitors visiting Phayao Province and the Red Lotus Sea region.\nThe research proposes a new cultural tourism route, and the manner of administering cultural tourism involves community-level groups led by spiritual leaders. The study's findings indicate that the tourists who attempted the route were highly satisfied, leading to the inference that the cultural tourism route \"See the mountain, walk through the rice fields, cruise the river\" is favoured by both locals who originated the route and tourists who have come to appreciate it.\n\nAnother finding from the study is that incorporating the idea of sustainability in tourism management should take into account the area's management in order to attract visitors while also protecting the area's sustainability. The method to sustainable community tourist management is based on certain components, which include community needs, tourism openness, and tourism management involvement.\n\nCultural tourism management in Ban Toon, Muang District, and Phayao Province during the COVID-19 epidemic can provide as a model for other towns with comparable circumstances\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-95
https://f1000research.com/articles/11-1453/v1	08 Dec 22	{ "type": "Research Article", "title": "Molecular docking, ADMET profiling of gallic acid and its derivatives (N-alkyl gallamide) as an anti-breast cancer agent", "authors": [ "Ade Arsianti", "Norma Nur Azizah", "Linda Erlina" ], "abstract": "Background In 2020, breast cancer has become the most common cancer in the world and in Indonesia. Searching for anticancer drugs using computational methods is considered more effective and selective than other methods. Gallic acid and its derivatives (esters and amides) are compounds that have biological activities such as anticancer effects. The purpose of this study was to analyse the molecular modelling and ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) profile of gallic acid derivative compounds (N-alkyl gallamides) as anticancer agents. Methods Target proteins were selected by analysis of protein-protein and drug-protein interactions. Molecular modelling was done by molecular docking. Predictive analysis of the ADMET profile of gallic acid and its derivatives (N-alkyl gallamide) was conducted using Marvin Sketch, Swissadme, protox II, and pkCSM pharmacokinetics. The selected target proteins were JUN, AKT1, CASP3, and CASP7. Results Compounds N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide were the three best gallic acid derivatives based on molecular modelling analysis of target proteins associated with breast cancer. The ADMET profile of the N-alkyl gallamide compound is predictable and shows a good profile as a candidate for anticancer drugs. Conclusion N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide have potential as anti-breast cancer agents.", "keywords": [ "N-Alkyl gallamide", "molecular docking", "ADMET", "breast cancer" ], "content": "Introduction\n\nCancer is ranked second globally in diseases that could lead to death and caused 10 million deaths in 2020. Generally, the most prevalent types of cancer are breast, colorectal, and lung cancers.1,2 Based on Global Cancer Observatory (GLOBOCAN), cancer cases in Indonesia increased in 2020, reaching 396,914 cases with a total death toll of 234,511. The leading forms of cancer were breast and cervical cancers, with the number of cases being 65,858 (16,6%) and 36,633 (9,2%), respectively.3\n\nOver recent years, research on anticancer therapies has been increasing, using various materials as anticancer drugs, which could be obtained via synthetic methods.4–6 Although there are many potential anticancer drugs that have been synthesized, the medical needs are far from fulfilled. Various factors could affect the insufficiency of these drugs to meet these needs, including selectivity limitations of conventional drugs that could lead to toxicity, as well as the presence of metastasis and multi-drug resistance.7,8 The search for selective and effective anticancer agents can be improved by computational methods. Computational methods have been used to significantly accelerate the drug design process. Protein-protein interactions, drug-protein interactions, and molecular modeling are the easiest methods to find and predict new molecules as potential drugs with shorter time and lower costs than conventional methods.9\n\nComputer-based molecular modelling merges informatics methods, medical sciences, and biophysics. The combination of these fields could predict the efficiency of potential therapeutic molecules that are designed before in vitro and pre-clinical tests.10 Characteristics and profiles of adsorption, distribution, metabolism, excretion, and toxicity (ADMET), become one of the important parameters in the discovery and development of drug candidates to determine the feasibility of drug candidates before being developed in the next stage.10\n\nIn recent decades, gallic acid has become a compound that is in great demand as an anticancer therapeutic agent. Gallic acid (3,4,5-trihydroxybenzoic acid) is widely contained in natural ingredients which has been reported to have antioxidant, antifungal, antiviral, anti-inflammatory, and anticancer activities.11 Gallic acid has also been reported to have biological activity against several cancer cell lines such as leukemia, lung cancer, colorectal cancer, and breast cancer cell lines and does not show biological activity against normal lymphocyte cells.12,13\n\nAccording to Silva et al., (2017), gallic acid and its derivatives (alkyl esters) have pro-apoptotic activity and show as non-genotoxic and non-mutagenic agents to prevent chromosomal damage due to chemical exposure.14 However, because gallic acid is a hydrophilic compound, it is more difficult for it to penetrate the hydrophobic cell membrane. Dorothea et al., (2016) concluded that increasing the potential for cytotoxicity to MCF7 breast cancer cells, lipophilicity must be achieved by modifying the structure of gallic acid by adding a lipophilic group to make it more hydrophobic.15 According to in silico study, gallic acid and its derivatives (alkyl ester) can be BRAF inhibitor of colorectal cancer,16 as well as inhibitor of the anti-apoptotic protein Bcl-xL in breast cancer.17,18 In addition, gallic acid derivative compounds (N-alkyl gallamide) have strong cytotoxic activity on MCF7 breast cancer cells, and colorectal cancer cells, HCT 116.19,20 This study aims to design gallic acid derivative compounds (N-alkyl Gallamide), analyze molecular modeling of target proteins associated with breast cancer, and determine the ADMET profile of these compounds as anti-breast cancer agents.\n\n\nMethods\n\nProtein or gene data that are related to the incidence of breast cancer were downloaded from the KEGG (Kyoto Encyclopedia of Genes and Genome) database with keywords human diseases and breast cancer. In addition, apoptosis-related genes were searched and downloaded. The list of gene names was downloaded by copying all the genes into a Microsoft Excel (RRID:SCR_016137) sheet. The list of gene names is shown in repository data with accession number S-BSST934.21\n\nThe list of genes or proteins that were downloaded were then analyzed for interactions between gallic acid as a drug and a list of genes associated with breast cancer and apoptosis as target proteins. This analysis used a website-based tool called STITCH (http://stitch.embl.de/). The list of genes and gallic acid was uploaded to the STITCH webserver with the search multiple names parameter, and the selected organism as Homo sapiens. The confidence score was also regulated in this analysis. The selected confidence score was the high confidence category (0.700). The interaction results were later downloaded in the TSV format. Visualization of drug-protein interactions was performed using Cytoscape 3.9.1 software (RRID:SCR_003032).\n\nSelected proteins that have interactions with gallic acid were then subjected to functional gene ontology (GO) analysis, with the purpose of being able to describe and limit gene functions that apply to all species. The method can effectively identify biological processes related to biological phenomena and is helpful for obtaining more meaningful gene functional information. GO enrichment analysis was performed with the Enrichr database (https://maayanlab.cloud/Enrichr/). Numerous genes that are related to breast cancer and apoptosis pathways were uploaded to the database.\n\nThe macromolecules or target proteins that were used were the crystal structures of the JUN (PDB ID: 2P33), AKT1 (PDB ID:1H10), CASP3 (PDB ID:3KJF), and CASP7 (PDB ID: 5V6U) proteins which were taken from the PDB data bank (http: //www.pdb.org/). Protein preparation was performed using AutoDock 4.2.6 software (RRID:SCR_012746). The protein was separated from the original ligand, and the water molecule was removed from the protein file, which later was ready to be saved in PBD format. Polar hydrogen atoms were added, nonpolar hydrogen atoms were removed, and a gasteiger charge was added to the protein. Binding pockets were defined by a grid map with docking grid sizes of 40x40x40, 50x50x50, and 60x60x60.\n\nA total of 12 gallic acid compounds and their derivatives (N-alkyl gallamide), which were later referred to as test ligands, were drawn using the Marvin Sketch software from ChemAxon (RRID:SCR_004111).22 Test ligands were prepared using AutoDock 4.2.6 software by converting files in SDF format to PBDQT.\n\nThe test ligand's molecular anchorage was determined using the Lamarckian genetic algorithm23 and AutoDock 4.2.6 software with the default settings. The output data was in DPF format, which was then executed via the command prompt by Autogrid4 and the Autodock4 docking process. The AutoDock 4.2.6 software was used to examine the conformation, bond affinity values, and interactions of the docking outcomes. Using Ligplot+ (RRID: SCR_018249) and LigandScout (RRID:SCR_014889) software, the visualization of protein-ligand interactions was examined.\n\nAnalysis of the ADMET characterization profile resulting from molecular modelling was conducted using several tools. Predictive analysis of drug likeness was determined using the web-based tool SwissAdme (http://www.swissadme.ch/). Predictive analysis of adsorption, distribution, metabolism, and excretion was performed using the pkCSM Pharmacokinetics web-based tool (http://biosig.unimelb.edu.au/pkcsm/). Toxicity prediction analysis was conducted using the web-based tool ProTox-II (RRID:SCR_018506) Prediction (https://tox-new.charite.de/protox_II/). Meanwhile, the value of Log D was predicted using Marvin Sketch software (Log D prediction).\n\n\nResults and discussion\n\nDrug-protein interactions were analyzed with the lead compound, which is gallic acid. Furthermore, analysis of the interaction of gallic acid with protein was done with the STITCH network server with a high confidence level of 0.700. A total of 157 nodes (the number of interacting proteins), 1923 edges (the number of associations formed), and four interactions of gallic acid with proteins were obtained. Table 1 shows the results of the analysis of the interaction of gallic acid with proteins at a high confidence level of 0.700. Visualization of gallic acid interaction with proteins using Cytoscape software with a circular display type.24\n\nGallic acid is the leading compound in this study and was used as a drug compound to be analysed for protein-drug interactions. In addition, many studies related to gallic acid as an anticancer agent have been carried out; therefore, research data on gallic acid are widely available in the database. Due to this, it is presumed that the gallic acid derivatives (N-alkyl gallamides) have at least the same type of interaction as gallic acid. The interaction of gallic acid with breast cancer proteins and apoptosis resulted in four interactions (table 1).24 The interaction of gallic acid with four proteins, namely JUN, CASP3, AKT1, and CASP7 had a high combined score. The combined score for each protein are JUN (0,823), CASP3 (0,745), AKT1 (0,742) and CASP7 (0,700). The combined score was obtained bas on the volume of research data in the database, which includes co-expression, co-occurrence, experiment, and text mining scores. The higher the combined score of the interaction, the higher the confidence level.\n\nJUN (c-JUN) is a transcription factor 1 (AP-1) driving protein that binds and activates transcription on the TRE/AP-1 element. Growth factors such as extra or intracellular signals, changes in onco-proteins, and UV light exposure stimulate c-JUN phosphorylation at serine 63/73 and activate c-JUN-dependent transcription. Therefore, activated c-JUN has the potential to play an essential role in carcinogenesis and cancer development.25\n\nAKT, also known as protein kinase B, is a critical element of the PI3K/AKT signalling pathway.26 In addition, AKT regulates cancer characteristics such as tumor growth, as well as survival and invasion of tumor cells. AKT has three different isoforms, those being AKT1, AKT2, and AKT3. The three isoforms are reported to have specific effects on breast cancer. AKT1 plays a role in early tumors, AKT 2 is responsible for tumor development and metastasis, while AKT3 is associated with negative ER (Estrogen Receptor) status. Moreover, AKT1 increases cell proliferation through cell cycle proteins such as 21, p27, and cyclin D, and plays a role in preventing apoptosis through p53.27 AKT1 was also reported to be involved in regulating breast cancer progression tested in vivo in ErbBB-induced mice. In this study, AKT1-deficient breast epithelial tumor cells (MEC) were reduced in size and proliferative capacity with a reduced abundance of cyclin D1 and p27.28\n\nCASP3 and CASP7 are a group of proteins involved in the process of cell apoptosis. More precisely CASP3 and CASP7 are effector caspases along with CASP6. All caspase families are inactive zymogens (pro-caspases) and their activation requires proteolytic activation during apoptosis. Effector caspases are activated by initiator caspases (CASP2, CASP8, CASP9, and CASP10) through cleavage at internal Asp residues leading to disassembly of large and small subunits, whereas inhibitory caspases are activated by dimerization via signals obtained from death receptors.29 In breast cancer, CASP3 is overexpressed and is significantly associated with poor breast cancer-specific survival and provides additional prognostic value in different phenotypes.30 The expression of CASP7, which is a pro-apoptotic protein sterically inhibited by XIAP protein, causes inhibited apoptosis. Higher levels of CASP7 were found in well-differentiated tumors, including ER+ breast tumors. This is due to the presence of an estrogen receptor element located in the CASP7 promoter area. CASP7 expression was significantly associated with estrogen receptor (ERα) expression status and continued to increase at the breast tumor stage.31\n\nThe KEGG pathway analysis based on the interaction of gallic acid with JUN, AKT1, CASP3, and CASP7 proteins indicated several signalling pathways, including the PI3K-AKT signalling pathway, MAPK signalling pathway, estrogen signalling pathway, receptor-mediated extrinsic pathway, and TNF-signalling pathway. These signalling pathways are related to cell proliferation, the cell cycle, and apoptosis.26\n\nSelected target proteins, JUN, AKT1, CASP3, and CASP7 are predicted to play a role in the breast cancer mechanism pathway as a therapeutic pathway for gallic acid and its derivatives summarized by the KEGG database.26 There are four predicted pathways, namely estrogen signalling pathways, PI3K/AKT signalling pathways, extrinsic receptor mediated pathways, and TNF-α signalling pathways. In the estrogen-mediated pathway, gallic acid and its derivatives are expected to bind to JUN proteins that can inhibit JUN activity in the cell cycle process. Whereas in PI3K/AKT signalling pathways, when AKT has been activated then gallic acid and its derivatives in the cell membrane are expected to inhibit both AKT activity and the activity of a downstream protein, namely mTOR so that the process of cancer cell proliferation can be inhibited. In contrast to JUN and AKT, whose activity is expected to be inhibited by gallic acid and its derivatives, the extrinsic and TNF-α signalling pathways are expected to activate CASP3 and CASP7 to immediately carry out the apoptotic process. According to this description, the relationship between proteins that interact with gallic acid based on protein-drug interaction analysis has a role and function in breast cancer related to the growth, development, and apoptosis of breast cancer cells.\n\nBreast cancer protein-associated pathways and apoptosis were obtained by enrichment analysis of the KEGG pathway using the Enrichr database (https://maayanlab.cloud/Enrichr/). One hundred and sixty-nine KEGG pathways were enriched, including breast cancer pathways and deep cancer pathways. Each enriched p value was calculated and compared with the Fisher method, where a p value<0.01 was significantly enriched. The p values were ordered from smallest to largest. The top 10 ontology gene enrichment results are presented in underlaying data.32\n\nGenes that interact with gallic acid directly or indirectly were analyzed for enrichment of the KEEG pathway in order to explore biological pathways involving related genes. The top ten KEEG pathways were significantly enriched with p values<0.001, the pathway in breast cancer was the main biological pathway enriched in related genes. Other biologic pathways in the 10 enriched pathways are associated with cancer, from colorectal cancer to endometrial cancer. The genes involved in the biological pathway of breast cancer were then analyzed for gene ontology (GO) enrichment. GO is a technique for interpreting high throughput molecular data and generating hypotheses about biological phenomena that underlies experiments or interpreting data (genes) using the ontology classification system of genealogy.33\n\nThe functional enrichment analysis of GO proteins that play a role in breast cancer and the mechanism of apoptosis consists of three aspects: cell composition, biological processes, and molecular functions. The analysis was carried out through the Enrichr database (https://maayanlab.cloud/Enrichr/). A total of 73 enrichment results we retrieved in aspects of cell composition, including the nucleus, intracellular membrane-bounded organelle, cyclin-dependent protein kinase holoenzyme complex, and other cell components. The enrichment results on aspects of biological processes showed that 1086 included negative regulation of the apoptotic process, positive gene expression regulation, fibroblast cell proliferation, and other biological processes. Meanwhile, the aspect of the molecular function illustrated 111 functions involving serine/threonine/tyrosine kinase protein activity, phosphatase binding, kinase binding, and other molecular functions. Each enriched p value was calculated and compared with the Fisher method, where p values<0.01 were significantly enriched. The p values were ordered from smallest to largest.34\n\nThere are three categories in GO, namely biological processes, molecular functions, and cellular components. Biological processes refer to the biological goals to which genes or gene products contribute. The process is achieved by means of one or more ordered sequences of molecular functions. Processes often involve chemical or physical transformations. Molecular function is defined as biochemical activity including binding to specific ligands or structures of gene products. Molecular functions simply describe what is done without specifying where or when it occurs. Meanwhile, the cellular component refers to the place in the cell where the gene product is active.35\n\nIn this study, the ontology gene enrichment of the genes inside the breast cancer pathway was significantly enriched with the biological process category called “negative regulation of the apoptotic process”. Negative regulation of the apoptotic process is defined as a series of gene products involved in the breast cancer pathway that inhibit apoptosis or ‘downregulate’ apoptosis. The pathway involved in this biological process is the Wnt canonical signalling pathway. Canonical Wnt signalling is the pathway that is responsible for β-catenin and T cell factor (TCF) or lymphoid enhancer factor (LEF), which is responsible for the proliferation and metastasis of breast cancer cells36 and maintenance of stemness.37 Wnt signaling in breast cancer is activated by loss of the TP53 gene.38\n\nNext, GO in the category of molecular function that was significantly enriched, specifically the biochemical activity of the enzyme “serine/threonine kinase”, will be discussed. Serine/threonine kinase is a member of the protein kinase superfamily that phosphorylates the amino acids serine or threonine. Through phosphorylation of protein kinases, it chemically transfers phosphate from ATP (Adenosine Triphosphate) or GTP (Guanosine Triphosphate) to targeted amino acids with the release of hydroxyl groups from their protein substrates. The phosphorylation process can induce conformational changes in the substrate protein which can disrupt protein-protein interactions. This conformational change influences its protein activity, cellular localization, or association with other proteins. These protein/enzyme kinases have been shown to regulate important molecular pathways in cellular processes including proliferation, metabolism, migration, survival, and apoptosis.39 Uncontrolled kinase activity due to mutation or loss of inhibitory mediators are commonly found in cancers, including breast cancer.\n\nIn breast cancer, serine/threonine kinase activity, also known as protein kinase B/Akt, interacts with breast tumor kinase (Brk) or protein-tyrosine kinase, which is involved in growth and cell survival and is overexpressed in most breast cancers but not in normal breast epithelial cells.40 Recently, one of the serine/threonine kinases (D) family, namely protein kinase D3 (PRKD3) was reported to promote the proliferation, growth, migration, and invasion of cancer cells in several types of tumors including breast cancer and it is said to be an alternative therapeutic target that is promising for cancer treatment.41\n\nThe next category of GO to be discussed is the significantly enriched cellular component called “nucleus”. As previously explained, cellular components describe the sites in the cell where activities occur whereas gene products are active. In this study, many genes in the breast cancer pathway were involved in the process of cell proliferation and interact with transcription factors, where the site of the process was in the cell nucleus. In addition, the significantly enriched cellular component is “intracellular membrane-enclosed organelles” based on the definition of the ontology gene browser. These organelles are organized structures of distinctive morphology and function, bound by single or multiple lipid bilayer membranes and are found within cells. This includes the nucleus, mitochondria, plastids, vacuoles, and vesicles, but excludes plasma membranes. From this definition, there are several places in the cell that are the site of cell proliferation and apoptosis and support the objectives of this study regarding apoptotic agents.\n\nMolecular docking analysis was used to computationally predict the interaction between proteins and the test compound (ligand). The target protein was selected based on the analysis of drug interactions with proteins with a high confidence value. These proteins include JUN (PDB ID: 2P33), AKT1 (PDB ID:1H10), CASP3 (PDB ID:3KJF), and CASP7 (PDB ID: 5V6U) taken from the PDB data bank (http://www.pdb.org/). The protein structure that was used is a 3D crystal structure downloaded in PDB format.\n\nAutoDock 4.2.6 software was used to prepare each protein. The water molecules were removed from the protein, and hydrogen and charge were added. The original protein and ligand were separated. The prepared proteins and ligands were subsequently stored as PDBQT files. The original protein and ligands were used to validate the grid box, which is a location for the attachment of gallic acid molecules and their derivatives. Validation of grid box dimensions began with 40x40x40, 50x50x50, and 60x60x60, or was adjusted to the original ligand size of each protein. The best validation results are chosen when the RMSD reference value is less than 2Å and the binding free energy (ΔG) is low (table 2).\n\nGallic acid and its derivatives, along with tamoxifen used as a control, were then used as test ligands for molecular modeling these proteins with the best grid box. The results of molecular docking are predictive values of binding free energy, inhibition constants, and bond interactions between test ligands and protein amino acid residues. The results of the molecular docking of 13 test compounds to four proteins are shown in table 3.\n\nNext, the best five ranked compounds were selected based on the results of molecular anchoring with the smallest G and Ki values for each protein. A total of nine gallic acid derivatives were obtained which were candidates for further analysis, namely ADMET analysis. ADMET analysis uses the SwissAdme webserver, pkCSM, Marvin Sketch, and the ProtoxII Prediction webserver. Table 4 shows the predicted results of ADMET analysis of gallic acid and the nine gallic acid derivatives N-alkyl gallamide.\n\nBased on the log P characteristic profile which interprets the solubility of the octanol/water phase and the permeability of gallic acid compounds and their derivatives, it can be classified in the biopharmaceutical classification system (BCS) by following the classification system: class I (high solubility and high permeability), class II (low solubility and high permeability), class III (high solubility and low permeability), and class IV (low solubility and low permeability). Predictions of the biopharmaceutical classification system for gallic acid compounds and their derivatives (N-alkyl gallamides) are summarized in table 5.\n\nIn addition, the relationship between pH and Log D, which shows the distribution coefficient of gallic acid compounds and their derivatives, is determined. The purpose of this predictive analysis is to measure the lipophilicity of ionizable compounds, where the partition is a function of pH. Table 6 shows the predicted relationship between pH values and Log D of gallic acid compounds and their derivatives (N-alkyl gallamides).\n\nThe selection of the five best ranked gallic acid derivative compounds was made based on its binding values of ΔG and Ki (ordered from highest affinity). Ligand and amino acid interactions were visualized using LigPlot+ and LiganScout software. The interactions formed between the ligands and the amino acid residues of each protein are in the form of hydrophobic interactions and hydrogen bond interactions. Each interaction formed on the test ligand was compared with the original ligand. An example of visualization of the interaction of amino acid residues with ligands using LigPlot+ and LigandScout can be seen in underlaying data.42 Hydrophobic interactions are shown in red semicircles, while hydrogen bond interactions are denoted by green dashed lines. The numbers on the hydrogen bond indicates the hydrogen bond distance between the amino acid residue and the ligand in angstrom units (Å).\n\nThe absorption of drug compounds and evaluation of drug likeness properties can be predicted by Lipinski's five of rules. These rules describe the molecular properties which are important for the pharmacokinetics of drug candidates. The Lipinski’s data of the drug candidate provides information about its similarity as a ligand of the drug. A good drug candidate will have Lipinski's rule of five data as follows, it has a molecular weight less than 500 Da, it does not have a hydrogen bond acceptor (HBA) of more than 10, it does not have a hydrogen donor bond (HBD) of more than 5, it has a log P value of no more than 5, and it must have a total polar surface area (TPSA) less than 140.43 A complete prediction of the ADMET characteristic profile is shown in table 4.\n\nADMET is used to describe the absorption, distribution, metabolism, excretion, and toxicity of drugs. In-silico ADMET profiling is a useful tool for predicting the pharmacological and toxicological properties of drug candidates, especially at the pre-clinical stage. To improve the prediction of ADMET, an in-silico model has been applied. The use of these models in particular has contributed to drug optimization and the avoidance of late-stage failures, which is also important because such failures lead to unproductive investments of time and money.\n\nThe absorption is predicted based on the nature of solubility in water (Log P), lipophilicity, and absorption in the human intestinal tract (Gi absorption). Based on table 1, the predicted solubility of Log P for all gallic acid compounds and their derivatives (N-alkyl gallamide) is less than 3. This shows that these compounds have good solubility. Log P also describes the role of compound hydration, the higher the log P value, the worse the hydration. The limit of a good log P value ranges from 2 to 3.44 Predicted absorption in the human intestine of gallic acid compounds and their derivatives is high because they can absorb more than 30% of the compound. Gi absorption is also associated with the permeability of drug compounds in the body.\n\nDistribution was predicted using P-glycoprotein (P-gp) substrate, blood-brain barrier (BBB) permeability, and non-segmentation-related descriptors. The descriptors were predicted using pkCSM-pharmacokinetics. P-gp is an ATP-dependent drug extraction pump and is found in various human tissues. All newly synthesized molecules must be P-gp substrates. According to the predicted results in this study, only gallic acid compounds were not predicted as p-gp substrates. The BBB is a complex structure that separates the central nervous system (CNS) from peripheral tissues. To maintain homeostasis in the CNS, the BBB controls the transfer of matter, nutrients, and cells from the blood to the brain and from the brain to the blood. It also participates in the clearance of cellular metabolites and toxins from the brain to the blood.44 The BBB permeability values of our compounds were estimated to range from −0.0996 to −1.156.\n\nThe drug molecules in the plasma are in balance between being bound to serum proteins and unbound. The proportion of drug molecules in plasma that are not bound to proteins is called the “un-bound fraction”. This profile influences renal glomerular filtration and hepatic metabolism, resulting in values for the volume of distribution, total clearance, and drug efficacy.45 The higher the binding of the drug to proteins in the blood, the less efficient it is to spread across cell membranes. In this study, the predictive value of the unbound fraction was large (from a scale of 0 to 1), which means that the binding of the drug to the protein in the blood is not too strong so that it can spread throughout the cell membrane well.\n\nTo make a candidate drug compound, it is also necessary to analyze the pharmacokinetics of the compound against cytochrome P450 (CYP) protein inhibitors. These proteins are marker proteins to determine the effect of the response of an anticancer drug and are metabolic enzymes in the liver. The human CYP enzymes important in drug metabolism are CYP 1A2, the CYP 2C family, CYP 2D6, and CYP 3A4. Therefore, in this study, we investigated whether gallic acid derivative compounds did not have an inhibitory effect on these enzymes. Based on table 1 only N-octyl gallamide compounds had an inhibitory effect on the CYP2D6 enzyme.\n\nExcretion occurs primarily as a combination of hepatic and renal clearance, is associated with bioavailability, and is important for determining dose levels to achieve steady-state concentrations. The predicted excretion values, using the pkCSM-pharmacokinetic total clearance descriptor, ranged from 0.216 to 0.978 ml/min/kg.\n\nIn addition, the toxicity of the drug compound was also analyzed to determine whether the drug compound is toxic or not, which aims to predict the level of safety for its use. The predicted toxicity profile in this study is AOT (acute of toxicity) which is related to LD50. AOT has several classes, namely class 1 (fatal if swallowed, LD50=5 mg/kg), class II (fatal if swallowed, 5<LD50 50), class III (toxic if swallowed, 50<LD50 300), class IV (harmful if swallowed, 300<LD50 2000), class V (possibly harmful if swallowed, 2000<LD50 5000), and class V1 (non-toxic, LD50>5000).46 Based on this category gallic acid and its derivatives fall into class IV.\n\nIt has been discussed previously that the solubility and permeability profile of gallic acid and its derivatives (N-alkyl gallamide) are important in predicting the pharmacokinetic properties of drug candidates. Based on this, this study tries to classify these profiles into the biopharmaceutical classification system (BCS). Based on this classification, gallic acid and its derivatives are classified in class 1 BCS, because all compounds have high solubility and permeability (table 5).\n\nAnother important profile is the relationship between pH and Log D of gallic acid compounds and their derivatives. Log D is the distribution coefficient described for ionizable compounds because log D is a measure of the pH-dependent difference in solubility of all species in the octanol/water system. The importance of predicting the value of log D is to predict the permeability of drug compounds in vivo. The relationship with pH indicates that changes in the pH environment that may occur in orally administered compounds occur in the gastrointestinal tract.47 This is because there is no constant pH in the body so pH is highly considered when predicting in vivo behavior of drug candidates. The pH range that is the primary focus of attention is pH 7.4, which is the physiological pH of blood serum, while the pH of the stomach is pH 1–3 and the ileum is pH 7–8. In this study, predictions of pH and log D values were made for gallic acid and its derivatives (table 6). Based on the prediction results, both gallic acid compounds and their derivatives have varying Log D values. Drug candidate compounds that have a high Log D value are highly lipophilic (Log D at pH 7.4>3.5) tend to have poor water solubility which can interfere with intestinal absorption. Based on this, the log D value of gallic acid compounds and their derivatives can be concluded to have good distribution coefficients as drug candidates\n\nThe determination of the five best gallic acid derivatives based on ADMET analysis could not be determined because all test parameters were met. The selection of five gallic acid derivatives was determined by ordering the binding energy values of G and Ki as a result of molecular docking analysis (table 3). Hydrogen bonding and hydrophobic bonding interaction between gallic acid derivative and amino acid residues in JUN, AKT1, CASP3, CASP7 proteins is presented in table 7 and visualized in underlaying data bellow. The number of hydrogen and hydrophobic bonds is compared with the original ligand interactions. In JUN proteins, in general, there are two hydrogen bonds in the amino acids Met149 and Asn152, this is different from the N-octyl gallamide compound which only has hydrogen bonds in Met149. However, the hydrophobic interactions of the N-octyl gallamide compound with JUN were more than other compounds. In addition, the JUN showed interaction with essential amino acid residues of gallic acid derivative compounds, namely Met149, Leu148, Lys93, Met146, Leu206, Val196, Ile70, and Val78.\n\nIn the AKT1 protein, the hydrogen bonding interactions on the original ligand were more than in the test compound. There were seven hydrogen bonds in the original ligand and three hydrophobic interactions, while the N-ters-butyl gallamide compound has three hydrogen bonds (Glu17, Arg25, Asn53) and five hydrophobic interactions. While the other four compounds have four hydrogen bonds and hydrophobic bonds, with hydrogen bonds at the same amino acid residues (Glu17, Arg25, Asn53, Arg23). Of all the interacting amino acids, there were only two essential amino acids that interacted with the test compound on the AKT1 protein, namely Lys14 and Ile19.\n\nIn CASP3 protein, the N-ters-butyl gallamide compound had more hydrogen bonds with amino acids than the original ligand and four other compounds. Amino acids that have hydrogen bonding interactions in N-ters-butyl gallamide compounds are Glu248, Asn208, and Arg207. Meanwhile, the essential amino acids that interact with the test compound on CASP3 protein are Trp206, Trp214, Phe250, Phe247, and Phe256. The essential amino acid residue that is hydrogen bonded with the N-isoamyl gallamide compound is Trp214.\n\nIn CASP7 protein, hydrogen bond interactions in the native ligand were more than that of the test compound. But uniquely, hydrogen bonds are formed on the same amino acid residues in all test compounds, namely Thr163, which is one of the essential amino acids. The hydrophobic bond interactions formed were also generally the same for all test compounds. The interacting essential amino acids include Phe219, Lys160, Ile159, Phe221, Thr163, and Met294.\n\nBased on the study of the interaction of the test compounds with each target protein, the types of interactions formed are mostly similar to the original ligands for each protein. It can be stated that the test compound is attached to each protein in the same coordinates as the original ligand. In addition, the test compound for gallic acid and its derivatives is expected to have the same activity as the original ligand on each target protein.\n\nIn-silico, the selection of the best compounds derived from gallic acid as candidates for apoptotic agents involves several stages of selection. First, the designed gallic acid derivatives were subjected to molecular docking analysis with the selection criteria being the order of low bond energy (ΔG). The lowest (most negative) binding energies were ranked and the best 5 compounds were selected from each target protein. Second, the five best gallic acid derivatives based on the results of molecular docking analysis were selected based on the characteristic profile of ADMET, with the selection criteria being compounds that have a good ADMET characteristic profile and are non-toxic. However, in this study, the selection based on the characteristic profile of ADMET could not be determined, because gallic acid derivative compounds have good ADMET profiles as drug candidates.\n\nBased on the results of molecular docking and ADMET profiling, the best of three gallic acid derivatives, namely N-octyl gallamide, N-ters-Butyl gallamide and N-isoamyl gallamide have a potency to be further developed as a promising anti-breast cancer agent.\n\n\nConclusion\n\nThere are several protein-protein interactions involved in the incidence of breast cancer and apoptosis against gallic acid compounds with a high level of confidence, namely JUN, AKT1, CASP3, and CASP7. Gallic acid derivative compounds (N-alkyl gallamide) can be characterized based on ADMET properties, and molecular docking predictions can be determined. The best gallic acid derivative compounds based on this study are N-Octyl gallamide, N-Ters-butyl gallamide, and N-Isoamyl gallamide.", "appendix": "Data availability\n\nFigshare: 2D chemicals structure of gallic acid and it derivatives (N-Alkyl gallamide). https://doi.org/10.6084/m9.figshare.21391545.v1. 22\n\nFigshare: Interaction of gallic acid with protein. https://doi.org/10.6084/m9.figshare.21391902.v1. 24\n\nFigshare: Systematic prediction of four target proteins and gallic acid therapeutic pathways. https://doi.org/10.6084/m9.figshare.21391524.v1. 26\n\nFigshare: Top 10 KEGG pathway enrichment analysis results. https://doi.org/10.6084/m9.figshare.21392082.v1. 32\n\nFigshare: Top 10 ontology gene enrichment analysis results. https://doi.org/10.6084/m9.figshare.21392232.v1. 34\n\nFigshare: Visualization of Ligand Interactions with Protein Amino Acid Residues. https://doi.org/10.6084/m9.figshare.21392520.v1. 42\n\nBiostudies: Raw data gene list from KEGG with accession number S- BSST934 21\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\nAcknowledgements\n\nWe would like to thank Kementrian Riset dan Teknologi Pendidikan Tinggi (KEMENRISTEK-DIKTI) Republic of Indonesia for funding our research through PDUPT (Penelitian Dasar Unggulan Perguruan Tinggi) research grant for fiscal year 2021-2022.\n\n\nReferences\n\nGLOBOCAN: All cancers.2020 [cited 2022 Mar 2]. 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Reference Source" }	[ { "id": "175977", "date": "04 Jul 2023", "name": "Damilohun Samuel Metibemu", "expertise": [ "Reviewer Expertise Bioinformatics", "Cancer Biology", "Medicinal Chemistry", "and phyto-medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 recommended that the authors define the full meaning of PDB, SDF, and PDBQT at the point of first use and for all the abbreviations.\n\nWhat is the full meaning of ADMET? Please provide the meaning at the time of first use\n\nThe authors should explain what type of interactions gallic acid and its derivatives make with the protein. Are they inhibitors or agonists?\n\nIn addition to discussing the relevance of the combined scores on the KEGG pathway, the authors should provide references to other studies related to this study. A description of the KEGG pathway is not sufficient.\n\nThe authors are kindly requested to discuss the implications of this study for the different proteins and genes. What are the implications of this study for oncology?\n\nMolecular docking and ADMET profiling results should be discussed in detail, including the implications of each value for potential anticancer compounds of gallic and its derivatives.\n\nIt is noteworthy that in-silico predictions are often not accurate, especially in clinical trials. In order to confirm the results obtained from in-silico screening, I recommend that the authors conduct some in-vitro admit profiling.\n\nThere is too much vagueness in the conclusion. A conclusion should be drawn based on the results of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "9901", "date": "13 Jul 2023", "name": "Norma Nur Azizah", "role": "Author Response", "response": "Thank you for reviewing our article. I have read all the reviews you have provided. We are trying to improve our article according to the review you provided. Best Regard, Norma Nur Azizah" }, { "c_id": "10005", "date": "23 Aug 2023", "name": "Norma Nur Azizah", "role": "Author Response", "response": "To reviewers: Thank you for reviewing our article, we have tried to improve the article according to the suggestions for improvement that have been submitted. The following is a report on the response from the review team that we can provide, hopefully it will be well received. t is recommended that the authors define the full meaning of PDB, SDF, and PDBQT at the point of first use and for all the abbreviations. Respond: we already define the full meaning of PDB, SDF, PSBQT and all abbreviations in our article. What is the full meaning of ADMET? Please provide the meaning at the time of first use. Respond: We think, we already mention full meaning ADMET in the abstract and introductions. The authors should explain what type of interactions gallic acid and its derivatives make with the protein. Are they inhibitors or agonists? Respond: we already explain the type of interaction gallic acid as agonis (page 7) In addition to discussing the relevance of the combined scores on the KEGG pathway, the authors should provide references to other studies related to this study. A description of the KEGG pathway is not sufficient. Respond: We have added a discussion regarding the combined scores and other references related to the study (page 9) The authors are kindly requested to discuss the implications of this study for the different proteins and genes. What are the implications of this study for oncology? Respond: in the end of discussion, we are adding our opinion about implication of this study for oncology (page 26) Molecular docking and ADMET profiling results should be discussed in detail, including the implications of each value for potential anticancer compounds of gallic and its derivatives. Respond: we think the explanation about ADMET has been fully explained in the article. Thank you. It is noteworthy that in-silico predictions are often not accurate, especially in clinical trials. In order to confirm the results obtained from in-silico screening, I recommend that the authors conduct some in-vitro admit profiling. Respond: While waiting for the review process for this article, we have conducted an in vitro test for this study. in this refinement article we have added relevant in vitro data and discussion. There is too much vagueness in the conclusion. A conclusion should be drawn based on the results of the study. Respond: we have changed the conclusion of this article. Thank you." }, { "c_id": "10138", "date": "01 Sep 2023", "name": "Damilohun Samuel Metibemu", "role": "Reviewer Response", "response": "Think the authors have responded well to my suggestions" } ] } ]	1	https://f1000research.com/articles/11-1453
https://f1000research.com/articles/12-677/v1	15 Jun 23	{ "type": "Study Protocol", "title": "Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol", "authors": [ "Raiff Silva", "Stephano Silva", "Vitor Santos", "Jefferson Fernandes", "Raul Oliveira", "Karyna Ribeiro", "Aline Fernandes", "Tatiana Ribeiro", "Stephano Silva", "Vitor Santos", "Jefferson Fernandes", "Raul Oliveira", "Karyna Ribeiro", "Aline Fernandes", "Tatiana Ribeiro" ], "abstract": "Background: Kinematic gait assessment is essential to the gait rehabilitation program after stroke. Portable devices composed of inertial sensors are an alternative for this evaluation. However, knowledge regarding the psychometric properties of these devices is needed to understand their accuracy, especially in evaluation of individuals with movement disorders (e.g., people post stroke). This systematic review aims to analyze the psychometric properties of portable devices that use inertial sensors to assess kinematic gait parameters in people post stroke. We will also investigate which portable device assesses alterations in lower limb angular movements during gait. Methods: We will search for studies in English without publication date restriction, that evaluated psychometric properties of portable devices that use inertial sensors to assess kinematic gait parameters in people after stroke. Searches will be performed in the following electronic databases: Cochrane Central Registry of Controlled Trials (CENTRAL), Medline/PubMed, EMBASE Ovid, CINAHL EBSCO, PsycINFO Ovid, IEEE Xplore Digital Library (IEEE), and Physiotherapy Evidence Database (PEDro). Gray literature will also be searched, including published and unpublished studies (dissertations and theses). The Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN) risk of bias tool will be used to assess the quality of studies that analyzed reliability and measurement error of devices. Expected results: This will be the first review assessing the risk of bias in studies that analyzed psychometric properties of portable devices that use inertial sensors to assess kinematic gait parameters in people post stroke. Then, we hope to elucidate this topic and help the decision-making of clinicians regarding the feasibility of these devices. Finally, we also hope to provide an overview of the characteristics of portable devices that assessed changes in angular lower limb movements during gait in this population. Registration: The protocol was registered in Open Science Framework on May 11th 2023 (https://doi.org/10.17605/OSF.IO/7M6DA).", "keywords": [ "stroke", "gait", "portable devices", "inertial sensors", "psychometric properties", "reliability", "measurement error" ], "content": "Introduction\n\nThe World Health Organization defines stroke as a focal (sometimes global) neurological impairment of vascular origin and sudden onset that lasts more than 24 hours and may lead to death.1 This condition has a global impact: approximately 12.2 million cases and 6.5 million deaths in 2019.2\n\nPeople post stroke commonly present an altered gait, spasticity,3 and sensory and strength deficits (especially in the paretic lower limb) that are not reversible.4 Also, more than half of people post stroke will live with permanent gait alterations,5 hindering function and social interaction.\n\nThe sequelae of stroke contribute to changes in lower limb angular movements. For example, excessive knee extension and reduced hip flexion and dorsiflexion (i.e., the most common changes in paretic lower limbs) may alter gait dynamics, including in the contralateral limb. In this sense, spatial (step and stride lengths), temporal (duration of double support and paretic balance), and spatiotemporal (speed and cadence) parameters are commonly altered.6,7\n\nIn this context, the quantitative assessment of kinematic gait using accurate systems is essential to the gait rehabilitation program after a stroke. For example, optoelectronic systems (gold standard) allow a three-dimensional gait analysis by capturing light from active (light-emitting diodes, LED) or passive markers (reflect light from infrared sources) using cameras. However, these systems are expensive and require a controlled space, precluding the monitoring of patients in natural environments.\n\nPortable devices are an alternative to optoelectronic systems. They are composed of inertial sensors (accelerometers and gyroscopes) that may be used individually or combined with other sensors (magnetometers) to form inertial measurement units (IMUs). These devices acquire information regarding the movement trajectory of one or several body parts during gait and allow the analysis of intra- and inter-individual variations of kinematic gait parameters.8 They also have several advantages: portability, low weight, no wires, low energy consumption, easy installation and handling, and low manufacturing cost.8 For this reason, sensors are widely used for gait analysis because they provide continuous real-time data for clinicians and patients.9\n\nAccelerometers and gyroscopes allow different types of analyses. The former consists of a sensor that moves relative to a fixed base and operates based on Newton’s laws of motion. Mass acceleration of the sensing element, opposite to the transducer, produces an electrical signal proportional to the acceleration. Since the mass is constant, the signal is measured, and acceleration is obtained. On the other hand, gyroscopes collect data on angular movement using active sensors that generate an inertial force (Coriolis force) during rotation. The force acts on the mass sensor and generates a movement, measured by electrodes, representing the rotation rate.10–12\n\nCurrently, portable devices are used to analyze several kinematic gait parameters, such as speed, cadence, balance, asymmetry, and stance times (single and double).13 However, knowledge regarding the psychometric properties of these devices is needed to understand their accuracy, especially in individuals with movement disorders (e.g., people post stroke).\n\nIn this sense, two studies performed recently – Ferraris et al. (2021) and Cimolin et al. (2022) analyzed the agreement between a gold standard system (instrumented 3D- Gait analysis) and a test system (single RGB-D-camera) in the evaluation of gait parameters of patients with stroke and Parkinson’s disease. For this, the authors used tests such as Spearman correlation, intra-class correlation coefficient (ICC) and Bland–Altman plot analysis.14,15 However, studies along these lines are still scarce.\n\nA systematic review by Peters et al. (2021) analyzed studies performed with wireless devices to assess the gait and mobility of people after stroke and few studies on psychometric properties were found.16 However, terms related to psychometrics were not included in the review search strategy. Furthermore, the review did not assess the psychometric properties of these devices.\n\nThe psychometric properties “reliability” and “measurement” error are defined respectively as “the proportion of the total variance in the measurements which is due to “true” differences between patients” and “the systematic and random error of a patient’s score that is not attributed to true changes in the construct to be measured”.17 Although this taxonomy was initially developed for studies with patient-reported outcome measures (PROMs), it is possible to analyze these properties referring to other types of measurement instruments, such as portable devices. Moreover, it is possible to verify the quality of studies that perform this analysis using the “COSMIN Risk of Bias tool to assess the quality of studies on reliability or measurement error of outcome measurement instruments”.18\n\nTherefore, we believe that a review that investigates the psychometric properties of portable devices based on inertial sensors, especially “reliability” and “measurement error”, may allow a more specific analysis of this technology. Another important aspect concerns the altered angular movements of the lower limbs of people after stroke, since the most recent reviews that included inertial sensors did not assess this result.9,16\n\nThis systematic review protocol aims to analyze the psychometric properties of portable devices that use inertial sensors to assess kinematic gait parameters in people post stroke. We will also investigate which portable device assesses alterations in lower limb angular movements during gait in people post stroke and observe their main characteristics (e.g., number and type of inertial components, sensor positioning, and sampling rate of data collection).\n\n\nMethods\n\nThis systematic review protocol was developed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P).19 Our study question was structured based on the recommendation by Munn et al. (2018) for reviews of psychometric properties,20 which covers the following: 1) construct of interest or name of measurement instruments (kinematic gait parameters); 2) population (people post stroke); 3) type of instrument (portable devices that use inertial sensors); and 4) measurement properties (validity, reliability, responsiveness and measurement error).\n\nThis protocol has been registered in Open Science Framework on May 11th 2023 (Registration DOI: https://doi.org/10.17605/OSF.IO/7M6DA).21\n\nTypes of studies\n\nWe will include studies that assessed psychometric properties (validity, reliability, responsiveness and measurement error) of portable devices that use inertial sensors to assess kinematic gait parameters in people post stroke. Only full-text studies, published or not (e.g., theses and dissertations), will be included.\n\nParticipants\n\nWe will include studies conducted with people post stroke of both sexes at the acute, subacute, or chronic stages and aged > 18 years.\n\nInstrument types\n\nStudies that assessed kinematic gait parameters of people post stroke through portable devices that use inertial sensors.\n\nPrimary outcomes\n\nPsychometric properties (validity, reliability, responsiveness and measurement error) related to outcomes obtained by portable devices will be considered the primary outcome.\n\nSecondary outcomes\n\nAngular changes in the sagittal plane of lower limb joints (hip, knee, and ankle) during gait will be considered secondary outcomes.\n\nWe will search for studies in English without publication date restriction.\n\nData sources\n\nSearches will be performed in the following electronic databases: Cochrane Central Registry of Controlled Trials (CENTRAL), Medline/PubMed, EMBASE Ovid, CINAHL EBSCO, PsycINFO Ovid, IEEE Xplore Digital Library (IEEE), and Physiotherapy Evidence Database (PEDro).\n\nA strategy was developed based on descriptors indexed in the Medical Subject Headings Database (MeSH terms) related to: condition (e.g., ‘stroke’ and ‘cerebrovascular accident’), inertial sensors (e.g., ‘inertial sensors’, ‘inertial measurement unit’, and ‘wearable devices’), and psychometric properties (e.g., ‘psychometric properties’, ‘validity’, and ‘reliability’). The terms regarding condition were combined with those for portable devices and psychometric properties using the boolean operator AND. The initial strategy was developed for the MEDLINE/PubMed database and will be translated and adapted to other databases according to codes and syntaxes. Details of the search strategy are shown in the Extended data.22\n\nSearching other resources\n\nWe will search the reference lists of primary studies included in the review to identify relevant studies. The gray literature will also be searched, including published and unpublished studies (dissertations and theses). Moreover, we will contact authors to obtain information from relevant articles and search for additional information regarding the device on the websites of manufacturers.\n\nStudy selection\n\nTwo independent researchers (SS and KR) will use Rayyan software23 to examine the titles and abstracts of studies and exclude irrelevant articles. Full texts of all potentially eligible articles will be retrieved. The same researchers will read the texts, identify studies, record reasons for excluding ineligible studies, and discuss disagreements. A third researcher (RS) will be consulted in case of disagreements. Duplicates will be removed, and the authors of studies will be contacted if more information is needed. The selection process will be recorded following the PRISMA flowchart.24\n\nTwo researchers will independently extract (SS and KR) the following data regarding methods, participants, and characteristics of inertial sensors using a predefined form (see the Extended data)25:\n\n• Methods: objectives, year of publication, study duration, number of study centers and location, assessment environments, psychometric properties assessed, time between repeated measurements, results, and withdrawals.\n\n• Participants: age, sex, anthropometric data (body weight, height, and body mass index), stroke duration and etiology (acute or hemorrhagic), most affected side (right or left), stroke stage (acute, subacute, or chronic), and use of assistive devices.\n\n• Inertial sensors: technology type, sensor components, reports of discomfort and inadequacy, kinematic parameters, location of sensors on the body, angular data (hip, knee, and ankle joints), cost of equipment, and feasibility of implementation.\n\nThe Consensus-based Standards for the Selection of Health Measurement Instruments (COSMIN) risk of bias tool18 will be used to assess the quality of studies that analyzed reliability and measurement error. The reliability box contains six items referring to study design and three regarding statistical methods. The measurement error box contains six items referring to study design and four regarding statistical methods. The checklists of the COSMIN risk of bias tool18 classify items as very good, adequate, doubtful, inadequate, or not applicable. The final score of each checklist is based on the worst score counts.\n\nTo assess the quality of the evidence we will use the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. According to the COSMIN methodology for systematic reviews of PROMs,26 some factors must be considered for studies of measurement properties: 1) risk of bias, 2) inconsistency, 3) indirectness and 4) imprecision (wide confidence intervals). The tool also has four levels of evidence quality: “high”, “moderate”, “low” and “very low”. In the evaluation, we will start from the highest level of evidence quality (high), which may be downgraded depending on the factors: risk of bias, inconsistency, indirectness and imprecision.\n\nResults will be presented and explained in a narrative synthesis, while tables will summarize the information. The extracted data will be explained using descriptive statistics and presented in graphs.\n\nMeasures of central tendency, such as mean and standard deviation, will describe continuous parametric data (sample size and anthropometric characteristics). For non-parametric data, we will use median and interquartile ranges.\n\nA systematic review will be performed according to this protocol and published in a relevant journal in the health area. We also intend to present the results at conferences and academic events.\n\nCurrently, the review is in the phase of “study selection”.\n\n\nDiscussion\n\nThis will be the first review assessing the risk of bias in studies that analyzed psychometric properties of outcomes obtained by portable devices that use inertial sensors to assess kinematic gait parameters in people post stroke. As the low number of studies that analyzed measurement properties may have neglected this topic, we believe our initiative will foster interest in these issues.\n\nIn addition, the characteristics of portable devices that assessed changes in angular lower limb movements during gait will help provide an overview of how this outcome has been assessed since few studies with portable devices addressed this question.\n\nLast, we believe that recent reviews9,16 involving the assessment of gait aspects of people post stroke using portable devices may restrict the scope of this review and be a potential limitation of the study.\n\n\nConclusion\n\nThis study aims to provide evidence regarding measurement properties of portable devices for gait analysis in people post stroke. We hope to elucidate this topic and help the decision-making of clinicians regarding the feasibility of these devices.\n\n\nAuthors’ contributions\n\nR.S.S., T.S.R., and K.M.O.B.F.R, conceived the idea of the study and contributed to the design of the research. R.S.S. wrote the initial draft. All authors contributed to the writing, editing, and approval of the final protocol.", "appendix": "Data availability\n\nNo underlying data are associated with this article.\n\nFigshare: Search strategy for: ‘Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol’, https://doi.org/10.6084/m9.figshare.22787045. 22\n\nThis project contains the following extended data:\n\n• Search strategy.pdf\n\nFigshare: Data extraction form to: ‘Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol’, https://doi.org/10.6084/m9.figshare.22787456. 25\n\nThis project contains the following extended data:\n\n• Data extraction form.pdf\n\nFigshare: PRISMA-P checklist for ‘Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol’. https://doi.org/10.6084/m9.figshare.22782863. 27\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAho K, Harmsen P, Hatano S, et al.: Cerebrovascular disease in the community: results of a WHO collaborative study. Bull. World Health Organ. 1980; 58(1): 113–130. PubMed Abstract\n\nFeigin VL, Stark BA, Johnson CO, et al.: Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet Neurology. 2021 Oct 1; 20(10): 795–820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsu AL, Tang PF, Jan MH: Test-retest reliability of isokinetic muscle strength of the lower extremities in patients with stroke. Arch. Phys. Med. Rehabil. 2002; 83(8): 1130–1137. PubMed Abstract \| Publisher Full Text\n\nSilva RS, Cerqueira MS, Maciel DG, et al.: Rate of torque development of paretic lower limb is an excellent predictor of walking speed in chronic stroke individuals. Clin. Biomech. 2022 Jan 1; 91: 105527. Publisher Full Text\n\nBalasubramanian CK, Clark DJ, Fox EJ: Walking Adaptability After a Stroke and Its Assessment in Clinical Settings. Stroke research and treatment. 2014; 2014: 1–21. Publisher Full Text\n\nBalaban B, Tok F: Gait disturbances in patients with stroke. Pm&r. 2014 Jul 1; 6(7): 635–642. Publisher Full Text\n\nSheffler LR, Chae J: Hemiparetic gait. Physical Medicine and Rehabilitation Clinics. 2015 Nov 1; 26(4): 611–623. PubMed Abstract \| Publisher Full Text\n\nSprager S, Juric MB: Inertial sensor-based gait recognition: A review. Sensors. 2015 Sep; 15(9): 22089–22127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohan DM, Khandoker AH, Wasti SA, et al.: Assessment Methods of Post-stroke Gait: A Scoping Review of Technology-Driven Approaches to Gait Characterization and Analysis. Front. Neurol. 2021 Jun 8; 12: 650024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRueterbories J, Spaich EG, Larsen B, et al.: Methods for gait event detection and analysis in ambulatory systems. Med. Eng. Phys. 2010 Jul 1; 32(6): 545–552. PubMed Abstract \| Publisher Full Text\n\nTarniŢă D: Wearable sensors used for human gait analysis. Romanian J. Morphol. Embryol. 2016 Jan 1; 57(2): 373–382. PubMed Abstract\n\nMuro-De-La-Herran A, Garcia-Zapirain B, Mendez-Zorrilla A: Gait analysis methods: An overview of wearable and non-wearable systems, highlighting clinical applications. Sensors. 2014 Feb; 14(2): 3362–3394.\n\nAnwary AR, Yu H, Vassallo M: An automatic gait feature extraction method for identifying gait asymmetry using wearable sensors. Sensors. 2018 Feb; 18(2): 676. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerraris C, Cimolin V, Vismara L, et al.: Monitoring of gait parameters in post-stroke individuals: A feasibility study using RGB-D sensors. Sensors. 2021 Sep 4; 21(17): 5945.\n\nCimolin V, Vismara L, Ferraris C, et al.: Computation of gait parameters in post stroke and parkinson’s disease: A comparative study using RGB-D sensors and optoelectronic systems. Sensors. 2022 Jan 21; 22(3): 824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters DM, O’Brien ES, Kamrud KE, et al.: Utilization of wearable technology to assess gait and mobility post-stroke: a systematic review. J. Neuroeng. Rehabil. 2021 Dec; 18(1): 1–8. Publisher Full Text\n\nMokkink LB, Terwee CB, Patrick DL, et al.: The COSMIN study reached international consensus on taxonomy, terminology, and definitions of measurement properties for health-related patient-reported outcomes. J. Clin. Epidemiol. 2010 Jul 1; 63(7): 737–745. PubMed Abstract \| Publisher Full Text\n\nMokkink LB, Boers M, Van Der Vleuten CP, et al.: COSMIN Risk of Bias tool to assess the quality of studies on reliability or measurement error of outcome measurement instruments: a Delphi study. BMC Med. Res. Methodol. 2020 Dec; 20(1): 1–3. 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. 2015 Dec; 4(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunn Z, Stern C, Aromataris E, et al.: What kind of systematic review should I conduct? A proposed typology and guidance for systematic reviewers in the medical and health sciences. BMC Med. Res. Methodol. 2018 Dec; 18(1): 1–9. Publisher Full Text\n\nSilva RS, Silva ST, Santos VG, et al.: Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol. OSF. 2023. osf.io/7m6da.\n\nSilva RS: Search strategy for: ‘Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol’. figshare.2023 [cited 2023 May 13]. Publisher Full Text\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan—a web and mobile app for systematic reviews. Syst. Rev. 2016 Dec; 5(1): 1. Publisher Full Text\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. Int. J. Surg. 2021 Apr 1; 88: 105906. PubMed Abstract \| Publisher Full Text\n\nSilva RS: Data extraction form to: ‘Psychometric properties of portable devices used in kinematic gait assessment after stroke: a systematic review protocol’. figshare.2023 [cited 2023 May 13]. Publisher Full Text\n\nMokkink LB, De Vet HC, Prinsen CA, et al.: COSMIN risk of bias checklist for systematic reviews of patient-reported outcome measures. Qual. Life Res. 2018 May; 27(5): 1171–1179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilva RS: Checklist PRISMA-P. figshare.2023 [cited 2023 May 13]. Publisher Full Text" }	[ { "id": "211235", "date": "23 Oct 2023", "name": "Rachel Reoli", "expertise": [ "Reviewer Expertise neurology", "rehabilitation", "psychometric properties", "outcome measures" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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’ objectives were to develop a protocol for a systematic review of current literature to aid in the understanding of 1) the psychometric properties of portable inertial sensor devices for kinematic gait assessment for individuals post-stroke, and 2) how these devices measure lower limb angular movements. This article is timely and relevant given the increased use of external measurement technology to evaluate gait kinematics versus traditional observational methods. The authors proposed methodology referenced established protocols; however, it needs to be fine-tuned to aid their inclusion criteria. By bolstering the below acknowledged strengths and updating the identified weaknesses, the authors would strengthen the protocol and gather more inclusive articles to include in their review.\n\nAbstract:\n\nStrengths:\n\nThe authors clearly state their objectives and are transparent with their search strategies.\n\nWeakness:\n\nThe authors state that this will be the “first review assessing the risk of bias in studies that analyzed psychometric properties of portable devices that use inertial sensors.” however, a systematic review has been published comparing inertial sensors to traditional analysis, see review by Petraglia et al. (20191). Of note, this review contained studies from 2005-2017, so it is important for the authors to perform this current review to include updated literature. The authors do not use the term “bias” in the objectives. If the authors’ objective is to assess bias, they need to include what type of bias they are evaluating and how they will evaluate it. Otherwise, the statement needs to be reworded to accurately address the objectives. In the “expected results” section, the authors state that they would like to address the “feasibility” and the “characteristics” of these devices. It would be helpful to elaborate what this entails. For feasibility - did the authors mean ease of use? The objectives do not address this aspect. As for characteristics - do the authors mean “psychometric characteristics” vs another definition? Please clarify.\n\nIntroduction: Strengths:\n\nThe authors provide a clear description of the gold standard versus portable device options to assess gait kinematics and identify pros and cons of each.\n\nWeakness:\n\nSecond paragraph: the authors state “people post-stroke commonly present an altered gait, spasticity, …\" - it would be clearer to the reader if the authors described the impairments following stroke and then linked them to how they may create an altered gait. Additionally, a definition of traditional gait (see Ranchos Los Amigos Gait Cycle) versus altered gait would be helpful to the reader before transitioning into paragraph three. In paragraph three, a more inclusive definition of “spatial,” “temporal” and “spatiotemporal” parameters would be beneficial. For example, Hollman et al. (20112) use operative definitions for spatiotemporal components. Stenum et al. (20213) use temporal definitions: step time, stance time, swing time, double support time. Overall, more inclusive operative definitions need to be determined and could be used later as search terms. Paragraph 6, unsure why this level of detail between gyroscopes and accelerometers is needed for this scoping review since both are included in the review. Reviewer’s suggestion is to remove paragraph 6. Paragraph 7, the authors state “...to understand their accuracy...” - this statement needs to be clearer, what is meant by accuracy? This would be an opportune time to discuss the psychometric properties to be included and why they are important (e.g. reliabilities, validities, etc). Paragraph 8 and 9 could be combined with the overall goal of supporting the need for this systematic review. Paragraph 9, move the COSIMIN Risk of Bias Tool to the methodology section. Paragraph 10 - only reliability and measurement error are addressed; however, other psychometric properties should be included and defined here (ex// reliability and validity see Zeng et al. (20224), a lower extremity IMU systematic review in 2022). Paragraph 10, last sentence, is the first time the second aim is addressed. This deserves more of a description for importance. It reads as an afterthought. The objective listed immediately prior to the methods is different from the objectives listed in the abstract. It is recommended that the authors clearly define their objectives and remain consistent throughout the manuscript.\n\nMethods: Strengths:\n\nThe authors are transparent with their protocol template (PRISMA-P). Inclusive of population, not biased for stroke chronicity, age (all adults included) or gender. The authors would benefit from the addition of a race component; will all races be included? Additionally, will all stroke neuroanatomical locations be included, e.g. cerebellar?\nWeakness:\n\nThe study design lists additional measurement properties; however, this list is not inclusive for a review. See above introduction comments. Inclusion criteria would benefit from the inclusion of additional psychometric properties. Likewise, will all kinematic gait parameters, or just several, be included? Will assistive device use be included/excluded? Overall, the inclusion/exclusion criteria should be clearly outlined to ensure reproducibility.\n\nInstrument types - the authors could define their instruments here, e.g. accelerometer and/or gyroscopes.\n\nPrimary outcomes: see comments above regarding more inclusive psychometric properties. Data collection - COSMIN Risk of Bias tool was mentioned in the introduction. Will independent researchers use this tool to include/exclude articles? How will this information be shared? Under data extraction, there are bulleted items of information to be extracted from the manuscripts - how will this information be shared? Table format? Quality assessment - only reliability and measurement error are represented here. How will the authors evaluate the other psychometric properties? The GRADE approach does not state to rate everything as high and then reduce as needed. See GRADE instructions how_to_grade.pdf (cochrane.org) https://cgf.cochrane.org/sites/cgf.cochrane.org/files/public/uploads/uploads/how_to_grade.pdf\n\nDiscussion:\n\nRisk of bias is addressed here; however, the authors’ objectives and methodology did not clearly address risk of bias. See above comments. Last paragraph of discussion - unclear what the intent of this sentence is. How will prior reviews restrict the scope of this review? How will this be a limitation? These reviews will not be included in your systematic review. Please clarify.\n\nAs aforementioned, the authors would benefit from honing-in on the methodology, specifically inclusion/exclusion criteria, to ensure they are obtaining the most relevant articles with their search. I would like to thank the authors for their interest and contribution to compiling information for this field of study.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "10773", "date": "13 Apr 2024", "name": "Raiff Silva", "role": "Author Response", "response": "Dear reviewer, We are grateful for all remarks concerning the manuscript and we have considered all points raised in our response letter. All changes were made to the new version of the uploaded text and were highlighted in yellow color. The review process has contributed significantly to improve the quality of our manuscript. We sincerely appreciate your collaboration. Below we seeked to respond to each point identified (our answers are in \"bold\"). Abstract: Weakness: The authors state that this will be the “first review assessing the risk of bias in studies that analyzed psychometric properties of portable devices that use inertial sensors.” However, a systematic review has been published comparing inertial sensors to traditional analysis, see review by Petraglia et al. (20191). Of note, this review contained studies from 2005-2017, so it is important for the authors to perform this current review to include updated literature. Answer - Dear Reviewer, thank you for your valuable comment. We appreciate your attention to detail. In response to your feedback, we would like to clarify that the review conducted by Petraglia et al. (2019) primarily focused on analyzing the validity as one of the psychometric properties of portable devices. In light of this, we have revised our manuscript to explicitly emphasize that our review will be the first to comprehensively analyze the properties of 'reliability' and 'measurement error,' utilizing the COSMIN tool. Additionally, we have underscored, as per your suggestion, the significance of including the most up-to-date literature. The authors do not use the term “bias” in the objectives. If the authors’ objective is to assess bias, they need to include what type of bias they are evaluating and how they will evaluate it. Otherwise, the statement needs to be reworded to accurately address the objectives. Answer - Dear, we appreciate your comment. Although we are analyzing the “risk of bias”, or “methodological quality”, this analysis will allow us to examine whether the reliability and measurement error of the studies is adequate. Therefore, ultimately, we will analyze directly the reliability and measurement error of the studies. So, we have adjusted the summary and body of the text under the \"objectives\" section to align with this approach. In the “expected results” section, the authors state that they would like to address the “feasibility” and the “characteristics” of these devices. It would be helpful to elaborate what this entails. For feasibility - did the authors mean ease of use? The objectives do not address this aspect. As for characteristics - do the authors mean “psychometric characteristics” vs another definition? Please clarify. Answer - Dear reviewer, thank you for your suggestion for clarifying our text. We acknowledge your point that the term \"feasibility\" is broad. In response, we have opted to replace the sentence \"help the decision-making of clinicians regarding the feasibility\" with \"\"help the decision-making of clinicians regarding the use of these devices.\" Concerning the term \"characteristics,\" our intention is to identify devices that analyze angular parameters of lower limbs during gait post-stroke. To better convey this, we have revised the sentence in the text to provide a clearer representation of our objective. Introduction: Weakness: Second paragraph: the authors state “people post-stroke commonly present an altered gait, spasticity, …\" - it would be clearer to the reader if the authors described the impairments following stroke and then linked them to how they may create an altered gait. Additionally, a definition of traditional gait (see Ranchos Los Amigos Gait Cycle) versus altered gait would be helpful to the reader before transitioning into paragraph three. Answer - Thank you for your insightful comment. We have carefully considered your suggestion to establish a connection between impairments following a stroke and observed gait changes. In response, we have revised the text to incorporate this linkage. Additionally, we have included a definition of gait cycles, followed by an explanation of pathological gait to provide a more comprehensive understanding. In paragraph three, a more inclusive definition of “spatial,” “temporal” and “spatiotemporal” parameters would be beneficial. For example, Hollman et al. (20112) use operative definitions for spatiotemporal components. Stenum et al. (20213) use temporal definitions: step time, stance time, swing time, double support time. Overall, more inclusive operative definitions need to be determined and could be used later as search terms. Answer - Dear, thank you for the suggestion. I believe the way the sentence was written was confusing, giving the impression we restricted all the possible spatio-temporal parameters only to those placed in parentheses. Our intention was to provide examples of commonly altered spatio-temporal parameters in gait post-stroke. In an effort to clarify this point, we have revised the sentence accordingly. Paragraph 6, unsure why this level of detail between gyroscopes and accelerometers is needed for this scoping review since both are included in the review. Reviewer’s suggestion is to remove paragraph 6. Answer - Thank you for your feedback on Paragraph 6. We appreciate and agree with your suggestion. Consequently, we have removed paragraph 6 from the manuscript. Paragraph 7, the authors state “...to understand their accuracy...” - this statement needs to be clearer, what is meant by accuracy? This would be an opportune time to discuss the psychometric properties to be included and why they are important (e.g. reliabilities, validities, etc). Answer - Thank you for your valuable comment. In response, we have replaced the previously mentioned sentence, \"...to understand their accuracy...\" with \"...aiding clinicians in making informed decisions regarding its usage...\". Additionally, we have extensively reworked the entire paragraph, incorporating brief definitions of validity, responsiveness, reliability, and measurement error. Paragraph 8 and 9 could be combined with the overall goal of supporting the need for this systematic review. Answer - Thank you for your suggestion. We appreciate your guidance, and we have reformulated and combined the paragraphs to better align it with the study objectives. Paragraph 9, move the COSIMIN Risk of Bias Tool to the methodology section. Answer - Thank you for your suggestion. We appreciate your input, and we have moved the sentence regarding the COSMIN Risk of Bias Tool to the Methods section. Paragraph 10 - only reliability and measurement error are addressed; however, other psychometric properties should be included and defined here (ex// reliability and validity see Zeng et al. (2024), a lower extremity IMU systematic review in 2022). Answer - Dear reviewer, we really appreciate your comment.Our intention is to investigate specific psychometric properties, including validity, responsiveness, reliability, and measurement error. However, for this review, we deliberately focus on reliability and measurement error for two main reasons: 1) The properties included in our search strategy (validity, responsiveness, reliability and measurement error) are the only ones applicable to results measurement instruments such as portable devices. Other properties are applicated in measurements of health-related patient-reported outcomes (HR-PROs); 2) We have chosen to analyze only reliability and measurement error due to the existence of a dedicated analysis tool, specifically the \"COSMIN Risk of Bias tool to assess the quality of studies on reliability and measurement error of outcome measurement instruments.\" Given this, and considering the reviewer's suggestions, we rewrote the objectives seeking to clarify our intentions: to investigate psychometric properties reported in literature and to analyze the reliability and measurement error; additionally, to verify which portable devices assess changes in lower limb angular movements during gait of people after stroke. Paragraph 10, last sentence, is the first time the second aim is addressed. This deserves more of a description for importance. It reads as an afterthought. Answer - Dear Reviewer, We appreciate your comment, and we agree with your suggestion. Accordingly, we have revised this section of the text to emphasize the significance of angular outcomes The objective listed immediately prior to the methods is different from the objectives listed in the abstract. It is recommended that the authors clearly define their objectives and remain consistent throughout the manuscript. Answer - We appreciate your suggestion and accept your considerations. We have aligned the objectives listed in the abstract with those presented throughout the manuscript. Thank you for your valuable feedback. Methods: Strengths: The authors are transparent with their protocol template (PRISMA-P). Inclusive of population, not biased for stroke chronicity, age (all adults included) or gender. The authors would benefit from the addition of a race component; will all races be included? Additionally, will all stroke neuroanatomical locations be included, e.g. cerebellar? Thank you for your valuable suggestion. We have incorporated the information regarding the inclusion of all races into the text, specifically in the \"methods\" section under the \"participants\" subsection. Additionally, we want to clarify that stroke of any etiology, regardless of its neuroanatomical location, will meet our inclusion criteria. Weakness: The study design lists additional measurement properties; however, this list is not inclusive for a review. See above introduction comments. Answer - Thank you for your suggestion to clarify this aspect. We have indeed addressed this issue in response to the 7th comment (referring to paragraph 10). Inclusion criteria would benefit from the inclusion of additional psychometric properties. Likewise, will all kinematic gait parameters, or just several, be included? Will assistive device use be included/excluded? Overall, the inclusion/exclusion criteria should be clearly outlined to ensure reproducibility. Answer - Thank you for your suggestion. As for adding other psychometric properties, we clarify this issue in the text and in the response referring to paragraph 10 (in response to the 7th comment). Additionally, we accept your suggestion regarding the inclusion and exclusion criteria and have further specified these criteria in our text. Instrument types - the authors could define their instruments here, e.g. accelerometer and/or gyroscopes. Answer - We agree with your comment. As requested, we have delimited the study instruments to \"portable devices that use accelerometers, gyroscopes, or Inertial Measurement Units (IMUs)\" in the corresponding text. Primary outcomes: see comments above regarding more inclusive psychometric properties. Answer - Thank you for highlighting this need. We address this issue in the response referring to paragraph 10 (in response to the 7th comment). Data collection - COSMIN Risk of Bias tool was mentioned in the introduction. Will independent researchers use this tool to include/exclude articles? How will this information be shared? Answer - We appreciate your observation regarding the confusing information. To address this, we have relocated the explanation about the COSMIN Risk of Bias tool to the \"methods\" section. Within this section, we have provided clarification on the tool's usage and specified that it will be applied by two independent researchers. Additionally, in the data synthesis section, we have included an explanation about how information is shared, specifically mentioning the \"Table of quality and results of studies on reliability and measurement error\" using the COSMIN risk of bias tool. We hope these adjustments enhance the clarity of our methodology Under data extraction, there are bulleted items of information to be extracted from the manuscripts - how will this information be shared? Table format? Answer - Thank you for your suggestion. We appreciate your input, and in line with your recommendation, we have rewritten the \"data synthesis\" section to provide further clarification on this issue. Quality assessment - only reliability and measurement error are represented here. How will the authors evaluate the other psychometric properties? Answer - This is also a concern of other parts of text. Therefore, this issue was addressed in the response referring to paragraph 10 (in response to the 7th comment). Thank you for the suggestion. The GRADE approach does not state to rate everything as high and then reduce as needed. See GRADE instructions how_to_grade.pdf (cochrane.org) https://cgf.cochrane.org/sites/cgf.cochrane.org/files/public/uploads/uploads/how_to_grade.pdf Answer - We appreciate your comment and the opportunity to clarify this aspect. To assess the quality of the evidence, we will utilize the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) \"modified approach\", as cited in the COSMIN methodology for systematic reviews of Patient-Reported Outcome Measures (PROMs). We clarify this issue in the aforementioned topic of the text. Discussion: Risk of bias is addressed here; however, the authors’ objectives and methodology did not clearly address risk of bias. See above comments. Answer - Dear, we appreciate your comment and adjust this paragraph of the \"discussion\" session in line with the objectives, which were also adjusted. Last paragraph of discussion - unclear what the intent of this sentence is. How will prior reviews restrict the scope of this review? How will this be a limitation? These reviews will not be included in your systematic review. Please clarify. Answer - We appreciate your observation, and we agree that our sentence was written in a way that may have caused confusion. We want to clarify that, given recent reviews on the topic, we deliberately did not address certain aspects of inertial sensors to avoid redundancy. We have revised the sentence to better convey this idea. Thank you for this suggestion." } ] } ]	1	https://f1000research.com/articles/12-677
https://f1000research.com/articles/11-831/v1	26 Jul 22	{ "type": "Research Article", "title": "The role of angiotensin receptor blocker (losartan) on decreasing fibrotic process of corpora cavernosa in priapism model of wistar rats", "authors": [ "Safendra Siregar", "Rulianov Rulianov", "Rangga Adhazi Ksatriapraja", "Dicky Stefanus", "Rulianov Rulianov", "Rangga Adhazi Ksatriapraja", "Dicky Stefanus" ], "abstract": "Background: Priapism induces regulation of Transforming Growth Factor-β1 (TGF-β1) expression and collagen-type-1 deposition. This will replace the normal corpora cavernosa with fibrotic tissue which eventually resulted in erectile dysfunction. It is also known that the fibrosis process of corpora cavernosa is related to Renin-Angiotensin II System (RAS). Angiotensin II receptor blockers (ARB), especially losartan, inhibit the inflammation process and fibrotic tissue formation. This study evaluated the effect of losartan in reducing fibrosis in priapism by evaluating TGF-β1 and collagen-type-1 in cavernous tissue and determined the effect of losartan in preventing fibrosis in priapism model of Wistar rats assessed by the metavir score. Methods: A total of eighteen male Wistar rats mean were divided into five groups. For the priapism models, we applied negative pressure on the penis to make an artificial erection to mimic the priapism process. The control groups were observed and the treatment groups were orally given losartan 15 mg/kg/day. Corpora cavernosa was harvested for TGF-β1 and collagen-type-1 measurement using an enzyme-linked immunosorbent assay (ELISA). The fibrotic tissue of each rat was then collected and assessed histopathologically with the metavir scoring system. Results: Penile TGF-β1 concentration in the losartan-treated group was not significantly different on day 10 and day 28 of observation (p10=0,30; p28=0,17). Meanwhile, collagen-type-1 concentration was significantly lower compared to control group (p10=0,002; p28=0,01). There was a significant difference in metavir scores in rats that received losartan and those who did not (p<0,05). Conclusion: Losartan could suppress the fibrosis process in the priapism model. It could decrease the collagen type 1 deposition during corpora cavernosa tissue regeneration. Based on the metavir score, the group receiving losartan therapy was better than the control group.", "keywords": [ "Angiotensin II receptor blocker", "Losartan", "Collagen", "Transforming Growth Factor", "Priapism", "collagen type I", "fibrosis", "renin-angiotensin II system" ], "content": "Introduction\n\nPriapism is defined as prolonged erection more than four hours after sexual stimulation with orgasm or without any sexual stimulation.1–4 The prevalence of this urology emergency condition was 5,34 cases per 100.000 males per year.2 Based on its pathophysiology, priapism can be classified into the high and low flow.1,2 Low flow priapism correlated with venous occlusion which increases internal corpus cavernous pressure and prevents arterial blood flow.1,5,6 Intervention more than 48 hours prevent structural recovery which leads to erectile dysfunction.1,3,5\n\nDuring hypoxia condition, in the first 12 hours, histological examination showed interstitial edema in the corporal specimen. This condition is followed by endothelial sinusoid injury, inflammation cell infiltration to basal membrane, and platelet aggregation after 24 hours. In 48 hours, thrombus can be found in sinusoids with smooth muscle necrosis and fibroblast proliferation.3–5 Hypoxia-induced growth factors increase during hypoxia environment such as Transforming Growth Factor 1 (TGF beta 1). Overexpression of TGF beta 1 leads to tissue injury and scar formation.1,3 TGF beta 1 recruits fibroblast to form molecular matrices such as fibronectin and collagen to build extracellular matrix and increase protease inhibitor production which inhibits protease degrades the extracellular matrix.7\n\nA study by Costa et al showed that corporal cavernous tissue in the adult penis with a history of priapism had a decreased smooth muscle component with increased type 1 collagen compared to them without priapism history. Various therapy has been carried out to manage the fibrotic condition in the corpora cavernosa, with the hope of treating erectile dysfunction conditions which include medical and surgical therapy. Phosphodiesterase-5 inhibitor (PDE5-I) and pentoxiphylline were an example of medicine while penile implants are one of the surgical options.4 Unfortunately, both medical and surgery focuses more on restoring erectile function than preventing the formation of fibrotic tissue which causes ED.\n\nThe hypoxic state in the corpora cavernosa can be a factor that initiates fibrosis in the corpora cavernosa via the angiotensin II-Smad pathway which is initiated by activation of the renin-angiotensin II system (RAS). Angiotensin II will bind to the AT1 receptor and cause an increase in TGF-β1 and activate the smad pathway. AngII through the AT1 receptor also increases tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) to inhibit matrix metalloproteinase (MMP) which functions in the degradation (turnover) of ECM proteins in the remodeling process. Changes in the histological picture of the corpora cavernosa in the form of a decrease in the number of smooth muscle cells replaced by collagen caused a decrease in the ratio of smooth muscle and collagen and an increase in fibroblasts which could be assessed by Masson’s Trichrome staining with the metavir scoring system.\n\nCurrently, there are several ways of assessing fibrosis in the corpora cavernosa such as the ratio of smooth muscle to collagen, but none of them have assessed the lumen of the cavernosa which is the most important part of the penile erection process, so the metavir scoring system is considered the best for assessing the purpose of this study.8\n\nAngiotensin II receptor blockers (ARBs) are antihypertensive drugs widely used to inhibit the inflammatory process and the formation of fibrotic tissue. The types of ARBs commonly used are losartan, candesartan, eprosartan mesylate, irbesartan, telmisartan, and valsartan.6,7,9 Several studies have demonstrated the ability of ARBs as tissue antifibrosis by inhibiting angiotensin II receptor type 1, thereby reducing the formation of TGF-β1. This can be seen in the study by Yamamoto et al. regarding the effect of ACE-I and ARB in reducing fibrosis in the heart ventricles that occurs due to hypertension.10 Losartan is one of the first-generation ARB drugs that have good antifibrotic properties. The administration of losartan in various studies has been shown to reduce the incidence of heart, liver, and kidney fibrosis.10–12\n\nThe antifibrotic ability of losartan gives hope to overcome the fibrotic process that occurs in various pathological conditions in the human body, including the penis.\n\nThis study aimed to evaluate the antifibrotic effect of losartan in reducing TGF-β1 and type 1 Collagen in the priapism model and to determine the effect of losartan administration on the process of corpora cavernous fibrosis in the priapism model of Wistar rats assessed by the metavir score.\n\n\nMethods\n\nThis was an experimental study. Sample size calculation was done using Federer formula (T-1) (N-1) ≥ 15. Total of eighteen male Wistar rats with an average weight of 328 grams were divided into five groups which are:\n\n• Group I (without ARB therapy): Priapism model rats, sacrificed on day ten\n\n• Group II (without ARB therapy): Priapism model rats, sacrificed on day 28\n\n• Group III (ARB therapy): Priapism model rats given orally losartan, sacrificed on day ten\n\n• Group IV (ARB therapy): Priapism model rats given orally losartan, sacrificed on day 28\n\n• Group V (healthy control): Untreated mice, sacrificed on day 28\n\nThe inclusion criteria were as follows: male Wistar rat, rat bodyweight 300–350 grams, has no congenital or acquired disease, and is active. Other confounders other than our inclusion criteria were not controlled. For the priapism models, we applied negative pressure on the penis to make an artificial erection and hold the base of the penile shaft using a rubber band to mimic the priapism process. After 12 hours, the rubber band was released. The control groups were observed and the treatment groups were orally given losartan 15 mg/kg body weight per day. Half of the groups were euthanized on day ten and the rest on day 28. Following euthanasia, corpora cavernosa was harvested for TGF-β1 and collagen type 1 measurement using ELISA.\n\nThe procedure for making a low flow type of priapism model was carried out on 18 adult male Wistar rats included in the study. The cages were cleaned daily and the temperature was maintained between 26–29 degrees Celsius. The temperature was measured using a thermometer and monitored every morning. Every morning the rat cage was left exposed to the sun from 8 to 10 am, and the cage was cleaned at the same time. After being given the same treatment and resting for a week, the samples were divided into five groups. The manufacture of the priapism model was carried out in collaboration with experienced and certified laboratory personnel using the method that has been carried out and has been published by Sanli, et al.16\n\nThis method was performed on 16 male Wistar rats group I to IV which were anesthetized intraperitoneally by injection of ketamine 75 mg/kg body weight. Meanwhile, two rats in group V were not given any treatment. Furthermore, we performed negative pressure on the penis by placing the tip of the 50 mL catheter tip around the penis and slowly pulling the syringe until the penis is fully erect. The negative pressure exerted was equal to the pull at 20 ccs. Then ligation at the base of the penis by tying a piece of 16 Fr rubber catheter that had been cut 2 mm long. This was done so that hypoxia occurs in the corpora cavernosa of the rat penis resembling priapism.\n\nThe rubber band was then removed after 12 hours to restore blood flow to the corpora cavernosa penis of the rat. Following the removal of the rubber band, groups III and IV were given ARB losartan 15 mg/kg body weight/day orally with the help of NGT number 6 Fr for 10 days and 28 days. The mice in groups I and III were sacrificed after ten days and group II, IV, and V mice were sacrificed after 28 days. Rats were sacrificed by euthanasia injection of ketamine 90 mg/kg body weight intraperitoneally. Surgical removal of the corpora cavernosa was performed in each group. Rats were then exterminated using an incinerator.\n\nThe levels of TGF-β1 and type 1 collagen in the corpora cavernosa tissue samples were measured using the Enzyme-Linked Immunoassay (ELISA) technique. The corpora cavernosa from the penile tissue was separated from other structures such as the urethra, corpora spongiosa, and tunica albuginea. Type 1 collagen in the tissue would stick to the plate which had been coated with type 1 collagen antibody. Before processing, the tissue was rinsed using Phosphate Buffer Saline (PBS) solution (pH 7.4) to remove excess blood and tissue. The tissue was then smoothed and homogenized in PBS solution (pH 7.4) with a glass homogenizer on ice. The tissue could be thawed at 2-8°C or frozen at -20°C if it was to be stored beforehand. Before the procedure, the sample tissue was allowed to reach room temperature and then centrifuged at 2000–3000 RPM for 20 minutes.\n\nThe corpora cavernosa tissue samples were fixed in 10% formaldehyde solution. The sample is then processed and made into paraffin blocks. Paraffin-embedded tissue was sliced into 5-m sections and placed on an object-glass, followed by deparaffinization and rehydration. Hydrogen peroxide was administered for 10 minutes to eliminate endogenous peroxidase activity. The sample was then rinsed using PBS-Triton X 100 (Tx). Hematoxylin-eosin (HE) staining was performed to assess structural changes in the corpora cavernosa. To determine smooth muscle and collagen, cross-sections were stained with Masson’s trichrome (MT) using the MT staining kit. Areas of smooth muscle (red stain) and collagen (blue-green stain) were evaluated in 200× magnification images of tissue using a microscope. Corpora cavernous fibrosis was assessed after Masson’s trichrome staining with the metavir scoring system.\n\nThe data were processed using SPSS version 20.0 with Windows as the operating system. The normality test of the data was carried out with the Shapiro-Wilk test. Because the number of samples was less than 50. The p-value that was considered significant is 0.05. Parametric statistical test to assess the comparison of type I collagen and TGF-β1 between groups receiving losartan therapy with controls at each observation time using the unpaired T-test if the data was normally distributed or the Mann Whitney test if the data were not normally distributed. Non-parametric statistical tests were used to assess meta var scores using the Kruskal-Wallis statistical test with posthoc analysis using the Mann-Whitney U test. This study has been approved by the Health Research Ethics of Universitas Padjadjaran Bandung with ethical protocol number: 329/UN6.KEP/EC/2021.\n\n\nResults\n\nThis study involved 18 male Wistar rats (Rattus norvegicus) with an average weight of 328.1 grams at the beginning of the study. The samples were divided into five groups, namely four rats in group I (a model of priapism without therapy was sacrificed on day ten), four rats in group II (a model of priapism without therapy which was sacrificed on day 28), four rats in group III (a model of priapism without treatment were sacrificed on day 28 with the ARB losartan therapy sacrificed on day ten), four mice in group IV (a model of priapism with the ARB losartan therapy sacrificed on day 28), and two mice in group V (healthy controls). All lived until the end of the study and none of the mice’s penises was necrotic. Each group was selected randomly and there was no significant difference in body weight in all rats (p-value on day 0 = 0.32; p-value on day 10 = 0.84; p-value on day 20 = 0.82; and p-value of day 20 = 0.82; to 28 = 0.54); p < 0.05).\n\nThe average concentration of TGF-β1 in the control group and the losartan ARB therapy group which were examined using the ELISA method on day 10 and day 28 can be seen in Table 1. The concentration of TGF-1 in the corpora cavernosa rats in the control group and the losartan ARB therapy group appeared to increase compared to the healthy control group on both the 10th and 28th-day observations. In the control group, the TGF-β1 concentration increased on day 28 (214.07 ± 41.85 ng/L) compared to day 10 (190.50 ± 10.72 ng/L). Meanwhile, in the losartan ARB therapy group, TGF-β1 concentrations appeared to decrease on day 28 (172.79 ± 29.77 ng/L) compared to day 10 (178.79 ± 17.88 ng/L).\n\nChanges in the concentration of TGF-β1 in all groups can be seen in Figure 1. From the graph, we can see that the concentration of TGF-β1 in the group modeled for priapism (the control group and the losartan ARB therapy group) was higher than the healthy control group. In the control group, the concentration of TGF-β1 tends to increase, while in the ARB losartan group it tends to decrease. If we compare the groups using the priapism model, it can be seen that the concentration of TGF-β1 in the group receiving the losartan ARB therapy is lower than in the control group.\n\nTable 2 shows the average value of type I collagen concentration in the control group and the losartan ARB therapy group on day ten and day 28. The concentration of type I collagen in the control group and the losartan ARB therapy group appeared higher than the healthy control group on both days of observation. the 10th and 28th days. In the control group, the concentration of type 1 collagen increased on day 28 (7.06 ± 0.44 ng/mL) compared to day 10 (6.81 ± 0.41 ng/mL). Likewise in the losartan ARB therapy group, the concentration of type 1 collagen also appeared to increase on day 28 (5.57 ± 1.18 ng/mL) compared to day 10 (5.46 ± 0.28 ng/mL).\n\nIn Figure 2 we can see changes in the concentration of type 1 collagen in all groups. From the graph, it can be seen that the concentration of type 1 collagen in the group modeled for priapism (control group and the losartan ARB therapy group) was higher than the healthy control group. In the group modeled for priapism, the concentration of type 1 collagen was equally likely to increase, but in the ARB losartan group, it was lower than in the control group.\n\nBefore the comparative analysis, a normality test was performed using the Shapiro-Wilk test and all data were normally distributed except for collagen levels in the losartan ARB therapy group on day ten, which was 0.010 (p < 0.05). However, due to the small number of samples, all of them were considered normally distributed.\n\nAnalytical comparative study between TGF-β1 concentration in both groups based on observation time using unpaired T-Test (p < 0.05). From Table 3, although not statistically significant, the difference in the concentration of TGF-β1 on day 10 in the losartan ARB group (178.9 ng/L) compared to the control group (190.50 ng/L) (p = 0.30, mean difference 11.71 ng/L, 95% Confidence Interval). At the time of observation on day 28, there was a difference in the concentration of type 1 collagen in the losartan ARB group (172.79 ng/L) compared to the control group (214.07 ng/L) (p = 0.17, a mean difference of 39.90 ng/L, 95% Confidence Interval).\n\na Independent T-Test.\n\nStatistical analysis of the comparison of the concentration of type 1 collagen in the two groups based on the time of observation using the unpaired T-test (p < 0.05). From Table 4, there was a statistically significant difference in the concentration of type 1 collagen on day 10 in the losartan ARB group (5.46 ng/mL) compared to the control group (6.81 ng/mL) (p = 0.002, mean difference 1.34 ng/mL, 95% Confidence Interval). At the time of observation on day 28, there was also a statistically significant difference in the concentration of type 1 collagen in the losartan ARB group (5.57 ng/mL) compared to the control group (7.06 ng/mL) (p = 0.01, a mean difference of 1.78 ng/mL, 95% Confidence Interval).\n\na Independent T-Test.\n\n* Statistically significant.\n\nTable 5 shows the median metavir scores in the control and treatment groups, both of which were significantly higher at week two than at week four. There was a difference between the group receiving losartan compared to the control group. Changes in the score can be seen in Figure 3, which shows that the increase in metavir scores in the group receiving losartan therapy is lower than in the group that does not.\n\n* Kruskal-Wallis statistical test.\n\nChanges in metavir scores in the losartan therapy group and the control group are shown in Figure 3. Based on the graph, it can be seen that the metavir scores on day ten and day 28 looked different in the group given losartan and not. On day ten, the losartan group appeared to have lower metavir scores than the non-losartan group and closer to the Metavir score in the healthy group. Figure 4 shows the qualitative difference between the corpora cavernous tissue is given losartan and not in the case of priapism. In the corpora cavernosa treated with Losartan, the metavir score was lower.\n\nBased on Table 6, it appears that there is a significant difference between the losartan therapy group and the control group (p = 0.019) on day ten and day 28. Based on Table 5 and Figure 3, there is a difference in metavir scores in the two groups, but the group receiving losartan therapy did better than the group that did not. On day 28, there was a significant difference in metavir scores in mice that received losartan and those that did not. It can be concluded that the administration of losartan can prevent the process of fibrosis in the corpora cavernosa of rats compared to controls that were assessed histopathologically with metavir scores.\n\n* Post Hoc Mann-Whitney U test.\n\n\nDiscussion\n\nIn low-flow priapism, prolonged hypoxia occurs, causing histopathological changes of endothelial damage, exposure of the basement membrane, platelet aggregation, and changes in smooth muscle cells to fibroblasts. This mechanism caused the process of fibrosis in the corpora cavernosa tissue.13–15 The process of tissue fibrosis can occur due to chronic diseases such as chronic disorders, heart failure, liver cirrhosis. Molecularly, this occurs because tissue hypoxia will increase the expression of angiotensin II which stimulates the AT1 receptor causing the synthesis of TGF-β1. TGF-β1 will stimulate fibroblasts to turn into myofibroblasts to produce type I collagen so that type 1 collagen deposits occur in the extracellular matrix and form fibrotic tissue.1,16–18\n\nThis research was conducted in vivo on male Wistar rats. The priapism penis model is made by applying negative pressure to the penis by placing the tip of a 50 mL catheter tip around the penis and gently pulling the syringe until the penis is fully erect. The penis is then ligated by attaching a 16 Fr rubber catheter to create a priapism-like condition and then removed after 12 hours. According to research by Harn et al, evaluation of ARB administration in liver fibrosis model mice on days seven, 14, and 28 showed that fibrosis improvement occurred at week two (day 14) and week four (day 28).19 In general there are three phases in the occurrence of wound healing, namely: the hemostasis phase which lasts from the first day to the 5th day, the proliferative phase from day five to 12, and the remodeling phase which lasts for months. Fibroblast and collagen recruitment was assessed in this study. Therefore, sampling was carried out on day ten during the proliferative phase and day 28 during the remodeling phase.\n\nIn this study, there was an increase in TGF-β1 and type 1 collagen in the groups modeled for priapism (control group and the losartan ARB therapy group). Then we can also note that the increase in the concentration of TGF-β1 in the group receiving losartan therapy was lower than the control group on both the 10th and 28th-day observations. In addition, the concentration tends to decrease on the 28th day of observation compared to the 10th day.\n\nThis decrease in the concentration of TGF-β1 is in line with research by Garg et al that can be helped by several antifibrotic agents, one of which is losartan. In this study, giving losartan 10 mg/kg/day is believed to be able to modulate the expression of fibrosis genes and effectively inhibit AT1 receptors and prevent the formation of TGF-β1 to prevent type 1 collagen deposition, prevent fibrosis, and enhance the tissue regeneration process.20 Administration of losartan as antifibrotic drugs can be used in most chronic inflammatory diseases, especially in heart and kidney disease.21,22 The use of the ARB losartan to prevent fibrosis in acute conditions such as priapism can be a new finding and an attractive alternative in the management of daily practice.\n\nAlthough the levels of TGF-1 on day ten and day 28 in the group receiving losartan were lower than the control, this was not statistically significant when compared to the control group (p day 10 = 0.3 and p day 28 = 0,17). Many factors may have an effect, one of which is the dose and duration of monitoring. If you look at the tendency for TGF-1 concentrations to continue to decrease on day 28 compared to day ten in the treatment group, the administration of losartan with a longer duration can reduce the level of TGF-β1 to a statistical significance.\n\nThe concentration of type 1 collagen also appeared to decrease with the administration of losartan on both the 10th and 28th days of observation. Then through calculations, it was proven that this was statistically significant between the group receiving losartan therapy when compared to the control group (p day 10 = 0.002 and p 28th day = 0.01). This research is in line with research by Varo et al, that administration of angiotensin inhibitors can inhibit type 1 collagen deposits and restore the process of tissue fibrosis.22\n\nThere was no statistically significant difference in TGF beta levels in this study in line with research conducted by Frimpong et al. In his study, it was found that total circulating levels of TGF beta did not change with ARB administration but type 1 collagen levels decreased significantly. It is interesting to note in this study that the decrease in type 1 collagen levels is believed to occur due to a change in the ratio of active and inactive TGF beta of up to 90% which results in a decrease in type 1 collagen deposition and reduced fibrosis.10\n\nIn addition, as we know the formation of type 1 collagen is a result of balancing various fibrotic and anti-fibrotic pathways. One of the cytokines that play a role in type 1 collagen deposition is the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1). Meanwhile, the cytokine that causes the degradation of collagen is Matrix Metalloproteinase (MMP). MMP consists of collagenase, stromelysin, and gelatinase. TIMP-1 is generated by signals from inflammation, while MMP is produced by tissues constantly. TIMP-1 forms a balance with MMP in regulating type 1 collagen levels in tissues. If TIMP-1 is overexpressed, fibrosis will occur. Angiotensin II has a role in increasing TIMP-1 so that giving Losartan as an angiotensin inhibitor, can reduce TIMP-1 concentrations and restore MMP/TIMP-1 balance and able to reduce type 1 collagen deposition and prevent fibrosis by various pathways.\n\nIn this study, the approach used was the ability of losartan to suppress the action of profibrotic cytokines such as TGF-β1, but its ability to increase antifibrotic cytokines such as MMP which can degrade collagen has not been assessed. In addition, the effective dosage and duration of the use of losartan may need to be studied further to obtain maximum results. This can be a good suggestion for further research to determine the effectiveness of losartan in preventing fibrosis in priapism.\n\nSeveral studies have shown a reduction in hepatic fibrosis due to hepatitis C with losartan therapy.23 In a retrospective human study, ACE-I/ARBs were able to reduce liver fibrosis in patients with chronic liver disease. In patients with hepatitis C virus infection who had undergone liver transplantation, the fibrosis stage and fibrosis progression index on liver biopsy were significantly lower in patients treated with ACE-I/ARB than in those not treated with ACE-I/ARB.\n\nHistopathological examination of the liver is the gold standard for fibrosis. In one meta-analysis, there were two randomized controlled trials (RCTs) evaluating fibrosis scores. The results showed that ARB therapy had a trend of positive effects on liver fibrosis as assessed by the Metavir score.24\n\nARB studies in cardiac fibrosis were performed using high doses of Telmisartan (5 mg/kg/day) and low doses (0.5 mg/kg/day) for five weeks. Myocardial fibrosis was significantly decreased in both groups (high dose and low dose). Perivascular fibrosis is also prevented by ARB. The use of ARBs does not decrease the serum angiotensin II concentration. However, aldosterone concentrations were significantly decreased. This suggests that aldosterone changes may be another influential factor in the prevention of cardiac fibrosis and hypertrophy.25\n\nSeveral experimental studies demonstrated the inhibitory effect of AngII on reducing skeletal muscle fibrosis. Losartan can reduce fibrosis and improve skeletal muscle strength in animal models of congenital muscular dystrophy and Marfan syndrome.\n\nThe role of AngII in fibrosis is seen in the presence of excessive induction of renin and AT. In addition, the synthesis of the extracellular matrix increases and AngII induces an increase in the concentration of collagen and fibronectin. Connective tissue growth factor (CTGF) appears as a profibrotic cytokine. CTGF is a mediator that stimulates TGF-β1 resulting in proliferation and activation of fibroblasts.26,27\n\nThe fibrogenic effect of AngII is related to the activation of TGF-β1 signaling. Over-activation of TGF-β1 causes fibrosis in almost all tissues. TGF-β1 stimulates myofibroblast differentiation and extracellular matrix synthesis. TGF-β1 also acts to maintain extracellular matrix proteins by inhibiting the activity of matrix metalloproteinases (MMPs) and inducing the synthesis of tissue inhibitor metalloproteinases (TIMPs).28\n\nAngII inhibitory mechanism can block TGF-β1 signaling. Losartan antifibrotic effect occurs through the mechanism of suppression of TGF-β1 levels via the AngII type I receptor. Using a dose that had minimal effect on mean arterial blood pressure (MABP), it was shown that losartan reduced collagen I levels.29\n\nAnother study also demonstrated a superior antifibrotic effect of ARBs compared to ACE-I on liver tissue fibrosis.30 Long-term administration of ACE-I increases compensation. The accumulation of bradykinin due to ACE inhibition by ACE-I can lead to the development of fibrosis and activate TGF-β1, as well as synthesize extracellular matrix proteins.31\n\nHowever, findings related to the effect of ACE-I/ARB on fibrosis are still contradictory. Another study on liver fibrosis showed that losartan could increase the stage of liver fibrosis in chronic hepatitis C patients. Long-term use of ACE-I/ARBs for hepatitis C patients has not been shown to inhibit the development of liver fibrosis.24,32\n\nContradictions have also been found in studies of the use of ARBs in interstitial lung disease. A study suggests a possible harmful role of renin-angiotensin inhibition in interstitial lung disease. The use of ARBs was associated with a 1.9-fold increase in the likelihood of having lung interstitial abnormalities at ten-year follow-up. It is suspected that ARB-induced compensation causes an increase in renin levels increasing angiotensin II. Renin itself increases the synthesis of collagen and TGF-β1 via an angiotensin II-independent mechanism. However, studies are still limited.33\n\nThere are some limitations of the research. In this study, there was no comparison between the use of losartan and proven anti-fibrotic such as phosphodiesterase-5 inhibitors and pentoxifylline. It is necessary to compare with other anti-fibrotic agents to measure the effectiveness of losartan as an anti-fibrotic. In addition, further research is needed on the minimum dose required to provide anti-fibrotic effects in humans. The side effects of losartan anti-fibrotic doses in humans also need to be evaluated to prevent toxicity and unwanted side effects.\n\n\nConclusion\n\nOral administration of the ARB losartan can reduce the concentration of TGF-β1 in male Wistar rats model priapism although this is not statistically significant and oral administration of the ARB losartan decreased the concentration of collagen type I statistically in male Wistar rats model priapism. Losartan administration also can prevent corpora cavernosa fibrosis in priapism model Wistar rats assessed by metavir score.\n\n\nData availability\n\nMendeley Data. The Role of Angiotensin Receptor Blocker (Losartan) on Decreasing Fibrotic Process of Corpora Cavernosa in Priapism Model of Wistar Rats. DOI: https://doi.org/10.17632/7dpb82y8j5.134\n\nThis project contains the following underlying data:\n\n- Methods: A total of eighteen male Wistar rats mean were divided into 5 groups. For the priapism models, we applied negative pressure on the penis to make an artificial erection to mimic the priapism process. The control groups were observed and the treatment groups were orally given Losartan 15 mg/kg/day. Corpora cavernosa was harvested for TGF-β1 and collagen-type-1 measurement using an enzyme-linked immunosorbent assay (ELISA). The fibrotic tissue of each rat was then collected and assessed histopathologically with the Metavir scoring system\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReporting guidelines\n\nMendeley data. ARRIVE checklist. DOI: https://doi.org/10.17632/7dpb82y8j5.2\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBurnett AL, Bivalacqua TJ: Priapism: Current Principles and Practice. Urol. Clin. North Am. 2007; 34(4): 631–642. PubMed Abstract \| Publisher Full Text\n\nHudnall M, Reed-Maldonado AB, Lue TF: Advances in the understanding of priapism. Transl. Androl. Urol. 2017; 6(2): 199–206. PubMed Abstract \| Publisher Full Text\n\nBroderick GA, Kadioglu A, Bivalacqua TJ, et al.: Priapism: Pathogenesis, epidemiology, and management. J. Sex. Med. 2010; 7(1): 476–500. Publisher Full Text\n\nPartin AW, Dmochowski RR, Kavoussi LR, et al.: Campbell Walsh Wein Urology. 12th ed.Philadelphia:Elsevier;2020.\n\nCosta WS, Felix B, Cavalcanti AG, et al.: Structural analysis of the corpora cavernosa in patients with ischaemic priapism. BJU Int. 2010; 105(6): 838–841. PubMed Abstract \| Publisher Full Text\n\nMunarriz R, Park K, Huang YH, et al.: Reperfusion of ischemic corporal tissue: Physiologic and biochemical changes in an animal model of ischemic priapism. Urology. 2003; 62(4): 760–764. PubMed Abstract \| Publisher Full Text\n\nLue TF: Expression of TGF-β-1 mRNA and ultrastructural alterations in pharmacologically induced prolonged penile erection in a canine model. J. Urol. 1998; 160(6 I): 2263–2266. Publisher Full Text\n\nDickstein K, Timmermans P, Segal R: Losartan: a selective angiotensin II type 1 (AT1) receptor antagonist for the treatment of heart failure. Expert Opin. Investig. Drugs. 1998; 7(11): 1897–1914. PubMed Abstract \| Publisher Full Text\n\nYamamoto K, Mano T, Yoshida J, et al.: ACE inhibitor and angiotensin II type 1 receptor blocker differently regulate ventricular fibrosis in hypertensive diastolic heart failure. J. Hypertens. 2005; 23(2): 393–400. PubMed Abstract \| Publisher Full Text\n\nDiop-Frimpong B, Chauhan VP, Krane S, et al.: Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors. Proc. Natl. Acad. Sci. U. S. A. 2011; 108(7): 2909–2914. PubMed Abstract \| Publisher Full Text\n\nCroquet V, Moal F, Veal N, et al.: Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension. J. Hepatol. 2002; 37(6): 773–780. Publisher Full Text\n\nMoore KL, Dalley AF, Agur AMR: Clinically oriented anatomy. India:Lippincott Williams & Wilkins;2013.\n\nMcAninch JW, Lue TF: Smith & Tanagho’ s General Urology. 18th ed.McGraw Hill;2013; 1–16.\n\nMuneer A, Ralph D: Guideline of guidelines: priapism. BJU Int. 2017; 119(2): 204–208. PubMed Abstract \| Publisher Full Text\n\nSpycher MA, Hauri D: The ultrastucture of the erectile tissue in priapism. J. Urol. 1986; 135(1): 142–147. PubMed Abstract \| Publisher Full Text\n\nLiu Y: Cellular and molecular mechanisms of renal fibrosis. Nat. Rev. Nephrol. 2011; 7(12): 684–696. PubMed Abstract \| Publisher Full Text\n\nSanli O, Armagan A, Kandivali E, et al.: TGF-β1 neutralizing antibodies decrease the fibrotic effects of ischemic priapism. Int. J. Impot. Res. 2004; 16(6): 492–497. PubMed Abstract \| Publisher Full Text\n\nFrantz C, Stewart KM, Weaver VM: The extracellular matrix at a glance. J. Cell Sci. 2010; 123(24): 4195–4200. PubMed Abstract \| Publisher Full Text\n\nHarn HJ, Lin SZ, Hung SH, et al.: Adipose-derived stem cells can abrogate chemical-induced liver fibrosis and facilitate recovery of liver function. Cell Transplant. 2012; 21(12): 2753–2764. PubMed Abstract \| Publisher Full Text\n\nRüster C, Wolf G: Angiotensin II as a morphogenic cytokine stimulating renal fibrogenesis. J. Am. Soc. Nephrol. 2011; 22(7): 1189–1199. PubMed Abstract \| Publisher Full Text\n\nGarg K, Corona BT, Walters TJ: Losartan administration reduces fibrosis but hinders functional recovery after volumetric muscle loss injury. J. Appl. Physiol. 2014; 117(10): 1120–1131. PubMed Abstract \| Publisher Full Text\n\nVaro N, Iraburu MJ, Varela M, et al.: Chronic AT1 blockade stimulates extracellular collagen type I degradation and reverses myocardial fibrosis spontaneously hypertensive rats. Hypertension. 2000; 35(6): 1197–1202. PubMed Abstract \| Publisher Full Text\n\nColmenero J, Bataller R, Sancho-Bru P, et al.: Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C. American Journal of Physiology-Gastrointestinal and Liver. Physiology. 2009 Oct; 297(4): G726–G734. Publisher Full Text\n\nZhu Q, Li N, Li F, et al.: Therapeutic effect of renin-angiotensin system inhibitors on liver fibrosis. J. Renin-Angiotensin-Aldosterone Syst. 2016 Feb 26; 17(1): 1470320316628717. PubMed Abstract \| Publisher Full Text\n\nKawai M, Morimoto S, Komukai K, et al.: The novel angiotensin receptor blocker, telmisartan prevents myocardial hypertrophy and fibrosis in renovascular hypertensive (Goldblatt) rat. J. Mol. Cell. Cardiol. 2008; 2(44): 448.\n\nMeng X-m, Nikolic-Paterson DJ, Lan HY: TGF-β: the master regulator of fibrosis. Nat. Rev. Nephrol. 2016; 12(6): 325–338. Publisher Full Text\n\nZhang C, Meng X, Zhu Z, et al.: Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biol. Int. 2004; 28(12): 863–873. PubMed Abstract \| Publisher Full Text\n\nMurphy AM, Wong AL, Bezuhly M: Modulation of angiotensin II signaling in the prevention of fibrosis. Fibrogenesis Tissue Repair. 2015 Dec; 8(1): 1–7. Publisher Full Text\n\nDiop-Frimpong B, Chauhan VP, Krane S, et al.: Losartan inhibits collagen I synthesis and improves the distribution and efficacy of nanotherapeutics in tumors. Proc. Natl. Acad. Sci. 2011; 108(7): 2909–2914. PubMed Abstract \| Publisher Full Text\n\nKim MY, Baik SK, Park DH, et al.: Angiotensin receptor blockers are superior to angiotensin-converting enzyme inhibitors in the suppression of hepatic fibrosis in a bile duct-ligated rat model. J. Gastroenterol. 2008; 43(11): 889–896. PubMed Abstract \| Publisher Full Text\n\nSancho-Bru P, Bataller R, Fernandez-Varo G, et al.: Bradykinin attenuates hepatocellular damage and fibrosis in rats with chronic liver injury. Gastroenterology. 2007; 133(6): 2019–2028. PubMed Abstract \| Publisher Full Text\n\nAbu Dayyeh BK, Yang M, Dienstag JL, et al.: The effects of angiotensin blocking agents on the progression of liver fibrosis in the HALT-C trial cohort. Dig. Dis. Sci. 2011; 56: 564–568. PubMed Abstract \| Publisher Full Text\n\nGannon WD, Anderson MR, Podolanczuk AJ, et al.: Angiotensin receptor blockers and subclinical interstitial lung disease: the MESA study. Ann. Am. Thorac. Soc. 2019 Nov; 16(11): 1451–1453. PubMed Abstract \| Publisher Full Text\n\nSiregar S, Stravinsky R, Ksatriapraja RA, et al.: The Role of Angiotensin Receptor Blocker (Losartan) on Decreasing Fibrotic Process of Corpora Cavernosa in Priapism Model of Wistar Rats. Mendeley Data. 2022; V1. Publisher Full Text" }	[ { "id": "150826", "date": "03 Oct 2022", "name": "Widi Atmoko", "expertise": [ "Reviewer Expertise Andrology", "male infertility", "male sexual dysfunction", "priapism", "erectile dysfunction", "ejaculatory and orgasmic problem", "endoruology", "urinary tract stone 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\nCongratulations and well done to all the authors for finishing a well-written manuscript. The authors presented an experimental animal study on the effect of Losartan on TGF-β1 expression and collagen type-1 deposition due to low-flow priapism in rats. The topic chosen by the authors is novel and intriguing. The current approach to low-flow priapism has not considered using ARB to prevent fibrosis. Thus the finding of the study will provide novelty to the body of medical literature.\nThe whole manuscript is clear and concise. The title and the abstract adequately represent the study's concept and finding. The objectives of the study are very clear and the methodology is also sound and reproducible. All the findings were correlated to the previous study. The figures are clear and legible.\nHowever, further research will still be needed to confirm its finding due to the small number of samples and the nature of the animal study. Nevertheless, this research will open a new horizon and possibility to a medical or non-surgical approach for low-flow priapism, at least for adjunctive treatment to prevent fibrosis due to priapism.\n\nIs the work 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": "8859", "date": "05 Oct 2022", "name": "Dicky Stefanus", "role": "Author Response", "response": "Thank you for the review, yes we hope this research will make a pilot study with larger sample to other research in the future, Many thanks" } ] }, { "id": "154482", "date": "21 Nov 2022", "name": "Guiting Lin", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript in title of “The role of angiotensin receptor blocker (losartan) on decreasing fibrotic process of corpora cavernosa in priapism model of wistar rats” aims to explore the effect of losartan in reducing fibrosis in priapism by evaluating TGF-β1 and collagen-type-1 in cavernous tissue and determined the effect of losartan in preventing fibrosis in priapism model of Wistar rats assessed by the metavir score. This is a very interesting study. However, there are several issues need be revised extensively.\nThe sample size is too small, there is only a total 18 animals in 5 groups. Only 2 rats in normal control group. The current research findings are not reliable.\n\nPlease provide detail information about the groups, including the animal number in each group.\n\nPlease provide the full name of the abbreviation the first time it appears in the manuscript.\n\nPlease provide the statistical analysis information of the table, such as P value, N, etc.\n\nPlease provide images in high resolution >300 dpi.\n\nFigure 3 should be re-organized, please provide a professional Figure.\n\nFigure 4 is unqualified. Please compare the same area of penile erectile tissue and use the same magnification. Please add the scale bar. More importantly, please provide images for each group.\nProfessional English editing is recommended.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "9618", "date": "11 May 2023", "name": "Dicky Stefanus", "role": "Author Response", "response": "Greetings, I am very sorry for the late response. This study uses the Federer formula to decide the minimum sample size of each group (4 animals). In the normal control group (sham group) we only used 2 samples because we consider the animal welfare law from the Institutional Animal Care and Use Committee (IACUC) guidance. Group 1: sample 1.1, 1.2, 1.3, 1.4 (a model of priapism without therapy which was sacrificed on day ten)Group 2: sample 2.1, 2.2, 2.3, 2.4 (a model of priapism without treatment which was sacrificed on day 28). Group 3: sample 3.1, 3.2, 3.3, 3.4 (a model of priapism with the ARB losartan therapy sacrificed on day ten). Group 4: sample 4.1, 4.2, 4.3, 4.4 (a model of priapism with the ARB losartan therapy sacrificed on day 28). Group 5: sample 5.1, 5.2 (healthy controls). Full name of the abbreviation in this manuscript has been added when it appears for the first time. Tables 1 and 2 don’t need statistical analysis. while Table 3-6 already have their respective statistical analysis results. Figures have been replaced with better resolutions images. Figure 3 has been revised as per the instructions. Figure 4 has been revised as per the instructions." } ] } ]	1	https://f1000research.com/articles/11-831
https://f1000research.com/articles/12-669/v1	14 Jun 23	{ "type": "Research Article", "title": "Understanding the high electronic quantum similarity of a series of ligands used as inhibitors of the SARS-CoV-2 virus by molecular mechanics and density functional theory approaches", "authors": [ "Alejandro Morales-Bayuelo", "Jesús Sánchez-Márquez", "Jesús Sánchez-Márquez" ], "abstract": "Background: A coronavirus identified in 2019, SARS-CoV-2, has caused a pandemic of respiratory illness, called COVID-19. Most people with COVID-19 experience mild to moderate symptoms and recover without the need for special treatments. The SARS‑CoV‑2 RNA‑dependent RNA polymerase (RdRp) plays a crucial role in the viral life cycle. The active site of the RdRp is a very accessible region, so targeting this region to study the inhibition of viral replication may be an effective therapeutic approach. For this reason, this study has selected and analysed a series of ligands used as SARS-CoV-2 virus inhibitors, namely: Darunavir (Daru), Dexamethasona (Dexame), Dolutegravir (Dolu), Fosamprenavir (Fosam), Ganciclovir (Gan), Insoine (Inso), Lopinavir (Lop), Ritonavir (Rito) and Tipranavir (Tipra). Methods: These ligands were analyzed using molecular docking, molecular quantum similarity using four similarity indices like overlap, Coulomb and their Euclidean distances. On the other hand, these outcomes were supported with chemical reactivity indices defined within a conceptual density functional theory framework. Results: The results show the conformations with the highest root-mean-square deviation (RMSD), have π-π stacking interaction with residue LYS621, ARG555 and ASP623, CYS622, ASP760, among others. In the molecular quantum similarity, the highest indices have been obtained in the electronic similarity in comparison with the structural similarity. Conclusions: These studies allow the identification of the main stabilizing interactions using the crystal structure of SARS‑CoV‑2 RNA‑dependent RNA polymerase. In this order of ideas, this study provides new insights into these ligands that can be used in the design of new COVID-19 treatments. The studies allowed us to find an explanation supported in the Density Functional Theory about the chemical reactivity and the stabilization in the active site of the ligands.", "keywords": [ "RNA dependent RNA polymerase SARS-CoV-2 virus", "COVID-19 treatments", "molecular docking", "molecular quantum similarity", "chemical reactivity descriptors", "density functional theory." ], "content": "Introduction\n\nCoronaviruses are the family of viruses to which SARS-CoV-2 belongs, the virus causing the COVID-19 pandemic. They were discovered in the 1960s, but their origin is still unknown.1 Their different types cause different illnesses, from a cold to severe respiratory illness (a severe form of pneumonia). Most coronaviruses are not dangerous and can be treated effectively. In fact, most people contract a coronavirus at some point in their lives, usually during their childhood.1 Although they are more frequent in autumn or winter, they can be found at any time of the year.1–3\n\nThe coronavirus owes its name to the appearance it presents, since it is very similar to a crown or a halo. It is a type of virus present mainly in animals, but also in humans. In recent years,4 three major epidemic outbreaks caused by new coronaviruses have been described. COVID-19/SARS-CoV-2: at the end of December 2019,4,5 the first cases of a new coronavirus were reported in the city of Wuhan (China). Since then, the increase in new infections with the SARS-CoV-2 virus (initially called 2019nCoV), which causes the disease called COVID-19, has been continuous and its transmission from person to person has accelerated.5 Reported cases already far exceed those of the 2002-2003 SARS epidemic.4,5 SARS’s fatality rate is lower than that of other coronaviruses, but many more deaths are occurring (there are already more than 5 million, because the infected people number in the hundreds of millions worldwide (almost 300 million in early January 2022).4–6\n\nThere is no specific treatment for SARS-CoV-2, but multiple drugs, alone or in combination, are being investigated, as well as the use of plasma from patients who have recovered.6–8 The usefulness of the other drugs, which are being administered to patients in clinical trials or for compassionate use, are being studied. For example, Remdesivir is an antiviral drug that was initially developed for the disease caused by the Ebola virus but has also shown in vitro activity against SARS-CoV-2.9 However, the results of this treatment have not been as satisfactory as expected.9,10 Ritonavir/lopinavir is a combination that is usually used against HIV.10 Lopinavir inhibits some enzymes involved in the virus multiplication cycle, while ritonavir acts as a protector of lopinavir because it degrades very quickly.10 The use of hydroxychloroquine against the new coronavirus has been very controversial. The Spanish Agency for Medicines and Health Products (Aemps) warns that this medicine “has been shown to be effective against SARS-CoV-2 in in vitro studies, but there is still no solid scientific evidence on its efficacy against Covid-19 in humans”.11 Finally, Dexamethasone is a corticosteroid that is emerging as an option for the most serious cases of COVID-19, since it could reduce mortality.12\n\nThese compounds are combined with other anti-inflammatory or virus-inhibiting substances, as well as with antibiotics (to treat or prevent secondary bacterial infections) and cytokine inhibitors.13 As for the treatment for infections caused by cold coronaviruses, cases are usually mild and are overcome by following the same steps as with a common cold,13,14 a new alternative to the treatment for the COVID-19 is needed to improve therapeutic alternatives for this disease that has claimed the lives of multiple people around the world. A series of compounds used, tested, and associated as treatments for SARS-CoV-2 have been selected for this study. These compounds are: Darunavir (Daru), Dexamethasona (Dexame), Dolutegravir (Dolu), Fosamprenavir (Fosam), Ganciclovir (Gan), Insoine (Inso), Lopinavir (Lop), Ritonavir (Rito) and Tipranavir (Tipra). They are related to molecular treatment against SARS-CoV-2 in vitro studies, have been analysed in this study using theoretical techniques such as molecular docking, molecular quantum similarity (MQS) and chemical reactivity descriptors within the Density Functional Theory (DFT).\n\nIn our previous publication,15 ligands Ascorbic acid Vitamin C (Asco), Azithromycin (Azythr), Cholecalciferol Vitamin D (Chole) and Hydroxychoroquine (Hidrox); other less known ones such as Abacavir, Acyclovir (Acyc), Amprenavir (Ampre), Baloxavir (Balox), Boceprevir (Boce), Cidofovir (Cido), Edoxudine (Edox) and Emtricitabine (Emitri) were used considering their association for the treatment of SARS-CoV-2. However, in the present study (the second part of an overarching study), a set of entirely different ligands, taking into account the molecular diversity from structural and electronic point of view, have been used to extrapolate and obtain new insights for the SARS-CoV-2 treatment today. The difference between the previous set of ligands15 and the set reported here, is the molecular quantum similarity. The set studied in this work has greater electronic quantum similarity than those presented in Ref. 15, and thus warrants a separate study to specifically investigate the associated impacts of quantum similarity on the results.\n\n\nMethods\n\nTo conduct the docking analysis, the receptor structures discussed in open access References 9–14 for the docking experiment were extricated utilizing the following protocols through the crystal Structure of SARS-CoV-2 RNA-dependent RNA polymerase, using Protein Data Bank (6M71), see also Underlying data, which was adjusted utilizing the protein preparation wizard module of the openly available input file for Schrödinger suite 2017-1. The system preparation has been implemented followed these steps:\n\ni) A key factor on the docking results is the hydrogen bond. For these reasons the hydrogen bond (H-bond) network was optimized, and the protein structure was refined, at physiological pH. This weight was optimized based on the premise that high-resolution structures accurately reflect hydrogen bonding in proteins.\n\nii) The charge of the ligands on the active site is crucial on the stabilization in the active site. Taking this into account, the protonation states were determined using PropKa utility, part of the Schrödinger suite. This program reaffirms the ionic character of compounds and predicts the pKa values of ionizable groups in proteins and protein-ligand complexes based in the 3D structure.\n\niii) The possible correlation effects in the heavy atoms were corrected using the Impact Refinement (Impref) module to execute a restrained molecular minimization with heavy atoms constrained to a low root-mean-square deviation (RMSD) from the initial coordinates.16–18 It is helpful to arrange the observations in serial order of the independent variable when one of the two variables is clearly identifiable as independent.\n\nIn our research group the calculation (docking results) were carried out through the freely available Schrödinger suite using the Glide,19,20 Glide is the program of the Schrödinger suite and was used to obtain the docking results. This program was used with default parameters (that is, the number of poses written per ligand was set to 10,000, and the scaling factors of the vdW radii and the partial atomic charge cut-off were set to the default values 0.80 and 0.15, respectively) and Standard Precision (SP) model has been used for docking outcomes. One of the most important parameters about the docking analysis is the grid. The grid generation implementation has been benchmarked using the target protein of SARS-CoV-2 RNA-dependent RNA polymerase.\n\nMolecular quantum similarity measure\n\nAn important feature associated with structural and electronic point of view is the Molecular Quantum Similarity Measure (MQSM). The MQSM of the systems A and B, known as ZAB, is obtained using the Density Functions (DFs) using Eq. (1).\n\nStudying the nature of the operator Ω(r1, r2) with electronic densities for A and B,21–26 used in Equation 1, provides the information being compared between the two systems while simultaneously designating our measure of similarity. For instance, if the chosen operator is the Dirac delta function (an efficient approach for functions with high peak values, such as the electronic density), i.e., Ω(r1, r2) = δ(r1 - r2).26–33 Another widely used alternative is the Coulomb operator, i.e., Ω(r1, r2) = \|r‌1 - r2\|‌‌-1, resulting in a Coulombic MQSM.34–36\n\nPreviously, several investigations have shown the relationship between quantum similarity and chemical reactivity descriptors.37–47 In addition, the quantum similarity and DFT uses the density function as an object of study of the similarity indexes; specifically, the Coulomb index can be related to electronic factors associated with chemical reactivity.\n\nUsing the Frontier Molecular Orbitals (FMO) and the energy gap, the global reactivity indices, such as chemical potential (μ),48 hardness (ɳ),49 and electrophilicity (ω),48,49 will be calculated.\n\nThese chemical reactivity indices (Eqs. 2-4) give an idea about the stability of the systems. The chemical potential measures the inclination of electrons to leave the equilibrium system,48 whereas chemical hardness measures the resistance of a chemical species to change its electronic configuration.39\n\nElectrophilicity index (ω) measure the stabilization energy of the system when it is saturated by electrons from the external environment and is mathematically defined as48,49:\n\nIn this work, the local reactivity descriptors are the Fukui functions. Equations (5, 6) represents the response of the chemical potential of a system to changes in the external potential.\n\nWhere (f+(r→)) is for nucleophilic attack and (f−(r→)) for the electrophilic attack.50–54\n\nAll the calculations were carried out using the method B3LYP. B3LYP is one of the most used Density-Functional Theory (DFT) approaches. It is capable of predicting molecular structures and other properties according to the experimental data55 and the basis set 6-311G (d,p)56 which is the result of adding a correction to the 6-311G(d) basis set leading to calculations of electronegativity, hardness, reactivity indices and frontier molecular orbitals with a similar quality to those obtained with much larger basis sets (such as Aug-cc-pVQZ and Aug-cc-pV5Z). This method/basis set has been used in combination with Gaussian 16 package57 and GaussView, Version 6.158 a free, alternative software that carries out a similar function is ORCA 5.0.3.\n\n\nResults and discussion\n\nOne of the objectives of this project involves an analysis about molecular coupling of the compounds on the active site. For this reason, a study about the best conformations on the active site of the selected compounds was carried out. Please note that all files associated with the results are available in Underlying data.59\n\nFigure 1 shows the conformation with the highest RMSD, in this conformation Lopinavir has a π-π stacking interaction with residue LYS621 with a length of 1.56Å. On the other hand, with this same residue they have an H-bond with a length of 1.62Å and 1.72Å, respectively. Additionally, this compound has an H-bond with residues ARG553, ARG555 and ASP623 with lengths of 1.56Å, 1.64Å and 1.42Å, respectively.\n\nFigure 2 shows that the Ganciclovir compound has a H-Bond with the residue ARG553 with a length of 1.58Å and ASP452 with a length of 1.62Å. Also, this compound has two H-bonds with the residue ASP760 with lengths of 1.59Å and 1.63Å, respectively.\n\nFigure 3 shows that the Insoine compound has a H-bond with the residue ASP760 with a length of 1.65Å, with the residue ARG553 have two H-bonds with lengths of 1.58Å and 1.61Å, respectively. Finally, it compounds have a H-bond with a length of 1.48Å.\n\nThe Fosamprenavir compound has two a π–π stacking interactions with the residue ARG624 with a length of 1.62Å, another interaction with the residue ARG553 with a length of 1.82Å (see Figure 4). Also have a H-bond with a residue ARG553 with a length of 1.62Å, another H-bond with ARG624 with a length of 1.57Å and with the residue THR556 with a length of 1.61Å.\n\nRitonavir has a -H bond with the residues LYS298 and LYS621 with a length of 1.26Å and 1.43Å (Figure 5). Also has a H-bond with the residue THR687 with a length 1.13 Å, respectively.\n\nDarunavir compound has a π–π stacking interactions with the residue ARG553 with a length of 1.41Å (as can be seen in Figure 6). Also have a H-bond with the residue CYS622 with a length 1.38Å, with the residue LYS621 with a length of 1.68Å and two H-bonds with lengths of 1.48Å and 1.38Å, respectively.\n\nFigure 7 shows that the Tipranavir compound has a π–π stacking interactions with three H-bonds with the residue CYS622 with a length of 1.66Å, with a residue LYS621 with a length of 1.63Å and a π–π stacking interactions with length of 1.46Å.\n\nGanciclovir compound has two π–π stacking interactions with the residue ARG553 with lengths of 1.52Å and 1.61Å (see Figure 8). On the other hand, it compounds have a H-bond with the residue CYS622 with a length of 1.58Å and two H-bonds with the residue ASP760 with lengths of 1.61Å and 1.71Å, respectively.\n\nThe Molecular docking results for Dolutegravir (see Figure 9) involves a π–π stacking interactions with lengths of 1.64Å and two H-bonds with the residues LYS551 and ARG553 with lengths of 1.57Å and 1.59Å, respectively.\n\nIn the Table 1 we can see the structural similarity results for the molecular reaction set. This analysis was developed with the aim the find the common features along the reaction set. From structural point of view the Darunavir (Daru), Dexamethasona (Dexame), Dolutegravir (Dolu), Fosamprenavir (Fosam), Ganciclovir (Gan), Insoine (Inso), Lopinavir (Lop), Ritonavir (Rito) and Tipranavir (Tipra) were analysed, the highest overlap similarity is between the compounds Fosam and Daru (0.343) with a Euclidean distance of 6.946 (see Table 2); Lop vs Fosam (0.414) with a Euclidean distance of 6.694 and Inso vs Gan (0.510) with a Euclidean distance of 0.510. These low values obtained are related with steric effects between structures. Additionally, they do not have a skeleton in common and substitutes groups have hight differences.\n\nDue to the low indices of structural similarity, the electronic similarity has been calculated (see Table 2). These values highest values for electronic similarity are Fosam vs Dexame (0.884) with a Euclidean distance of 36.295; Rito vs Dexame (0.896) with a Euclidean distance of 43.347 (see Table 4), Lop vs Fosam (0.915) with a Euclidean distance of 33.223; and Rito vs Lop (0.881) with a Euclidean distance of 41.726. Unlike the values of structural similarity, the values of electronic similarity are above of 0.5. These values are supported with the Euclidean distances. We think that the structures, despite being structurally different, are electronically very similar.\n\nSince the electronic similarity indices (see Tables 3 and 4) are higher than the structural ones, this section deepens on the electronic effects associated with the highest values of electronic similarity. In the previous section we have studied which ligands have greater structural and electronic similarity. To continue this analysis, from the point of view of chemical reactivity, several ligand pairs have been selected from those with indices indicating greater electronic and structural similarity. Table 5 shows the global parameters chemical potential, chemical hardness, lobal S softness and global electrophilicity that have been calculated in order to compare the chemical reactivity of these ligands, analysing the values of this table it can be concluded that Insoine, Ganciclovir and Fosamprenavir have a set of parameters that are closer to each other. Since the analysis of the global parameters is limited, we will complete it with the comparison of some local descriptor functions. The electrophile and nucleophile Fukui functions (as a measure of reactivity) are then compared using the Frontier Molecular Orbital (FMO) approach. The electrophilic-nucleophilic character of the following functions also shows those molecular areas that are most likely to form charge-donating interactions (basically by charge delocalisation). These types of interactions are important and are difficult to determine using docking analysis.\n\nFigures 10 and 11 show the Fukui f−(r→) and f+(r→) functions corresponding to the Insoine and Ganciclovir ligands. Figure 10 shows a strong similarity between the two functions, which may indicate that both ligands have a similar nucleophilic behaviour and/or that they have a similar tendency to donate electronic charge. On the other hand, Figure 11 shows significantly different descriptor functions so we would expect different electrophilic behaviour for these ligands and/or a very different tendency in relation to the possible charge-withdrawing interactions. The Fukui Functions maps were obtained using Highest Occupied Molecular Oribital (HOMO) maps and the Lowest Unoccupied Molecular Orbital (LUMO) maps).\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases.\n\nFigures 12 and 13 show the Fukui functions for the ligands Lopinavir and Fosamprenavir. In Figure 12 it can be seen that there is no similarity between the two functions, indicating that the ligands have different nucleophilic behaviour and/or show a different trend in electronic charge donation. On the other hand, Figure 13 shows descriptor functions with a certain resemblance so we would expect comparable electrophilic behaviour between these ligands although the similarity is moderate. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases.\n\nFigures 14 and 15 show the Fukui functions for the ligands Fosamprenavir and Darunavir. Figure 14 shows a strong similarity between the two functions, which may indicate that both ligands have a similar nucleophilic behaviour and/or that they have a similar tendency to donate electronic charge. On the other hand, Figure 15 shows very similar descriptor functions so we would expect comparable electrophilic behaviour between these ligands. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nFigures 16 and 17 show the Fukui functions for the ligands Fosamprenavir and Dexamethasone. In Figure 16 it can be seen that there is no similarity between the two functions, indicating that the ligands have different nucleophilic behaviour and/or show a different trend in electronic charge donation. On the other hand, Figure 17 shows descriptor functions with a certain resemblance so we would expect comparable electrophilic behaviour between these ligands although the similarity is moderate.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nFigures 18 and 19 show the Fukui functions for the ligands Ritonavir and Dexamethasone. In Figure 18 it can be clearly seen that there is no similarity between the two functions, so we think that these ligands have different nucleophilic behaviour. On the other hand, Figure 19 shows descriptor functions with a certain resemblance, so we would expect comparable electrophilic behaviour between these ligands, although the resemblance is very limited.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\nIsovalue: 0.002 in both cases. Figure created using Schrödinger suite 2017-1.\n\n\nConclusions\n\nIn this study, the series of compounds, namely Darunavir (Daru), Dexamethasone (Dexame), Dolutegravir (Dolu), Fosamprenavir (Fosam), Ganciclovir (Gan), Insoine (Inso), Lopinavir (Lop), Ritonavir (Rito) and Tipranavir (Tipra), used against SARS-CoV-2 in vitro studies, have been analysed by molecular docking, molecular quantum similarity and chemical reactivity indices to study their active site stabilisation interactions from a structural and electronic point of view.\n\nFrom the molecular docking results, it was observed that Lopinavir, Ganciclovir, Insoin, Fosamprenavir, Ritonavir, Tipranavir, Ganciclovir and Dolutegravir show good active site stabilisation with at least one -H bond in each conformation. To further investigate the active site stabilisation of each ligand, a density functional theory (DFT) analysis of quantum similarity and reactivity was developed.\n\nFrom the structural point of view, the highest overlap similarity is between the compounds Fosam vs Daru (0.343), Lop vs Fosam (0.414) and Inso vs Gan (0.510). From the electronic point of view, the highest Coulomb similarity is between the compounds Fosam vs Dexame (0.884); Rito vs Dexame (0.896), Lop vs Fosam (0.915) and Rito vs Lop (0.881). Finally, with these comparisons, chemical reactivity analysis was performed from a global and local point of view. These Fukui functions were related to charge-donation interactions.", "appendix": "Data availability\n\nProtein Data Bank: SARS-CoV-2 RNA-dependent RNA polymerase. Accession number 6M71; https://www.rcsb.org/structure/6M71.\n\nHarvard Dataverse: Replication data for Study of a series of ligands used as inhibitors of the SARS-CoV-2 virus. https://doi.org/10.7910/DVN/7KFPUT. 59\n\nThis project contains the following underlying data:\n\n- Optimized structure of Darunavir (Daru).\n\n- Optimized structure of Dexamethasona (Dexame).\n\n- Optimized structure of Dolutegravir (Dolu).\n\n- Optimized structure of Fosamprenavir (Fosam).\n\n- Optimized structure of Ganciclovir (Gan).\n\n- Optimized structure of Insoine (Inso).\n\n- Optimized structure of Lopinavir (Lop).\n\n- Optimized structure of Ritonavir (Rito).\n\n- Optimized structure of Tipranavir (Tipra).\n\nEach structure has the following file extensions:\n\n- .out (output file of Gaussian 09 calculations).\n\n- .chk (output file of the Gaussian 09 calculations used to generate the contour maps).\n\n- .gif (input file of the Gaussian 09 calculations).\n\n- .gif.bak (the Gaussian 09 file used to generate the contour maps, i.e. the Highest Occupied Molecular Oribital [HOMO] maps and the Lowest Unoccupied Molecular Orbital [LUMO] maps).\n\n- .mol2 (input file for Schrödinger used to generate the docking results).\n\nThe Gaussian 09 files can be opened by readers using a non-proprietary software, such as ORCA, mentioned previously in the methods. The input files were obtained using Gaussview 6.1, the visualization program of Gaussian 09.\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\nAcknowledgements\n\nAMB thanks Fundación Universitaria Tecnológico Comfenalco.\n\n\nReferences\n\nBanerjee A, Kulcsar K, Misra V, et al.: Bats and Coronaviruses. Viruses. 2019; 11. pii: E41. Publisher Full Text\n\nYang D, Leibowitz JL: The structure and functions of coronavirus genomic 3′ and 5′ ends. Virus Res. 2015; 206: 120–133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong Z, Xu Y, Bao L, et al.: From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses. 2019; 11. pii: E59. Publisher Full Text\n\nGraham RL, Donaldson EF, Baric RS: A decade after SARS: strategies for controlling emerging coronaviruses. Nat. Rev. Microbiol. 2013; 11: 836–848. 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Publisher Full Text" }	[ { "id": "207285", "date": "22 Nov 2023", "name": "Ahmed Metwaly", "expertise": [ "Reviewer Expertise Drug Discovery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have thoroughly reviewed the manuscript entitled \"Understanding the high electronic quantum similarity of a series of ligands used as inhibitors of the SARS-CoV-2 virus by molecular mechanics and density functional theory approaches\" submitted to F1000Research.\n\nThe authors have undertaken a commendable effort to employ molecular mechanics and density functional theory approaches to analyze the ligands' potential as inhibitors of the SARS-CoV-2 virus.\n\nHowever, my primary concern lies in the absence of experimental validation, particularly in vitro studies, to support the computational findings. Despite the availability of the examined compounds and RdRp binding kits, the manuscript lacks crucial experimental data that would enhance the credibility and transnational impact of the study. Experimental validation is essential to confirm the practical applicability of the proposed ligands as SARS-CoV-2 inhibitors.\nGiven the current limitations, I recommend rejecting the manuscript in its present form. I believe that the inclusion of in vitro experimental data would significantly strengthen the study and its potential impact on drug development for COVID-19. I encourage the authors to consider conducting and incorporating such experiments in the next 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "178627", "date": "12 Dec 2023", "name": "Ramon Carbó-Dorca", "expertise": [ "Reviewer Expertise Essentially: Quantum Similarity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study presents in an original way a well-structured study of inhibitors of the COVID virus. The authors give a balanced point of view of the problem using several techniques, among others, quantum similarity analysis. I have been impressed with the amount of work implemented in the paper. I will suggest the publication without changes, except for an editorial revision of the text, just in case there are some incorrections that this referee has not been able to detect.\n\nIs the work 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": "191736", "date": "13 Dec 2023", "name": "Kiran Bharat Lokhande", "expertise": [ "Reviewer Expertise Expertise in Structural Bioinformatics and Computer-Aided Drug Discovery" ], "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\nComments:\n\nTo the Authors:\nDetailed Methodology: It would greatly enhance the reproducibility of your study if you could provide more detailed information regarding the protein preparation process. Specifically, please include information about the pH levels used in the methodology. PDB ID Citation: I kindly request that you cite the PDB ID used in this study following the guidelines provided by the RCSB. This will ensure proper attribution of the structural data used in your research. Experimental Depth: The study primarily focuses on molecular docking, which has its limitations. Since a similar study with MD simulation studies was published two years ago, it is advisable to incorporate high-end atomistic simulations to support your docking results and provide additional depth to your research. MD Simulations: To strengthen your findings, consider conducting at least 1 microsecond MD simulations in triplicate. This will help validate the outcomes of your molecular docking experiments. Article Suitability: Given the preliminary nature of the study and the existence of a similar study with MD simulations, I recommend an extensive revision of the article to either provide novel insights or enhance the depth of the research before considering it for publication.\nTo the Editor:\nI would like to highlight several concerns regarding the manuscript in question: The methodology lacks sufficient detail, particularly regarding the protein preparation process, including the pH levels used. The PDB ID used in the study needs to be cited following RCSB guidelines to ensure proper attribution. The study primarily relies on molecular docking, which may not provide the necessary depth of analysis, especially considering a similar study with MD simulations published two years ago. To address this limitation, the authors should conduct high-end atomistic simulations, such as 1 microsecond MD simulations in triplicate, to strengthen the validity of their findings. In its current form, the article appears to be premature and would benefit from extensive revisions to enhance its suitability for publication. I recommend considering these points in the review process to help the authors improve the quality and depth of their research.\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?\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? No", "responses": [ { "c_id": "10884", "date": "22 Mar 2024", "name": "Alejandro Morales-Bayuelo", "role": "Author Response", "response": "Detailed Methodology: It would greatly enhance the reproducibility of your study if you could provide more detailed information regarding the protein preparation process. Specifically, please include information about the pH levels used in the methodology. Answer: pH 6.2 to 6.8 PDB ID Citation: I kindly request that you cite the PDB ID used in this study following the guidelines provided by the RCSB. This will ensure proper attribution of the structural data used in your research. Answer: PDB ID: 6M71Download ; MMDB ID: 188116 ; PDB Deposition Date: 2020/3/16. Experimental Depth: The study primarily focuses on molecular docking, which has its limitations. Since a similar study with MD simulation studies was published two years ago, it is advisable to incorporate high-end atomistic simulations to support your docking results and provide additional depth to your research. MD Simulations: To strengthen your findings, consider conducting at least 1 microsecond MD simulations in triplicate. This will help validate the outcomes of your molecular docking experiments. Answer: All the docking results were supported by molecular dynamic of 20 ns. To verify the molecular stabilization of the ligands on the active site. Article Suitability: Given the preliminary nature of the study and the existence of a similar study with MD simulations, I recommend an extensive revision of the article to either provide novel insights or enhance the depth of the research before considering it for publication. Answer: The article was revised." }, { "c_id": "10885", "date": "22 Mar 2024", "name": "Alejandro Morales-Bayuelo", "role": "Author Response", "response": "The influence of pH (acidity or alkalinity) on the SARS-CoV-2 virus, which causes COVID-19, is an interesting topic. However, it's important to note that SARS-CoV-2 primarily infects and spreads among human cells, and the virus itself doesn't exist independently in the environment for long periods. The pH level becomes more relevant when considering how the virus interacts with the human body. The human body maintains various pH levels in different parts, and this plays a role in the virus's ability to infect cells. For example: Respiratory Tract: The virus primarily enters the body through the respiratory tract. The pH levels in the respiratory tract can vary, and some studies suggest that certain viruses, including coronaviruses, might be influenced by these pH levels. However, the exact relationship is complex and may not be the sole determinant of infection. Cellular Environment: Once the virus enters human cells, the pH of the cellular environment becomes crucial. Inside cells, the virus needs to release its genetic material and replicate. The pH within cellular compartments can affect these processes. Immune Response: The body's immune response also involves pH-dependent processes. Immune cells often function optimally within specific pH ranges, and any disruption might influence the ability of the immune system to combat the virus. It's important to emphasize that pH is just one of many factors influencing viral infections, and the interplay of these factors is intricate. Factors like temperature, humidity, and the overall health of an individual also contribute to the susceptibility and severity of viral infections." } ] } ]	1	https://f1000research.com/articles/12-669
https://f1000research.com/articles/11-17/v1	07 Jan 22	{ "type": "Method Article", "title": "Smartic: A smart tool for Big Data analytics and IoT", "authors": [ "Shohel Sayeed", "Abu Fuad Ahmad", "Tan Choo Peng", "Abu Fuad Ahmad", "Tan Choo Peng" ], "abstract": "The Internet of Things (IoT) is leading the physical and digital world of technology to converge. Real-time and massive scale connections produce a large amount of versatile data, where Big Data comes into the picture. Big Data refers to large, diverse sets of information with dimensions that go beyond the capabilities of widely used database management systems, or standard data processing software tools to manage within a given limit. Almost every big dataset is dirty and may contain missing data, mistyping, inaccuracies, and many more issues that impact Big Data analytics performances. One of the biggest challenges in Big Data analytics is to discover and repair dirty data; failure to do this can lead to inaccurate analytics results and unpredictable conclusions. We experimented with different missing value imputation techniques and compared machine learning (ML) model performances with different imputation methods. We propose a hybrid model for missing value imputation combining ML and sample-based statistical techniques. Furthermore, we continued with the best missing value inputted dataset, chosen based on ML model performance for feature engineering and hyperparameter tuning. We used k-means clustering and principal component analysis. Accuracy, the evaluated outcome, improved dramatically and proved that the XGBoost model gives very high accuracy at around 0.125 root mean squared logarithmic error (RMSLE). To overcome overfitting, we used K-fold cross-validation.", "keywords": [ "IoT", "Big Data Analytics", "Data Cleaning", "Data Imputation", "Feature Engineering" ], "content": "Introduction\n\nThe Internet of Things (IoT) is reshaping communication with technologies and is becoming a vital part of the development of a smart environment dedicated to make our lives convenient and comfortable.1 Several IoT application sectors like smart homes, smart cities,2 smart healthcare, assisted driving, smart retail, and consumer goods like wearables and smartphones are already available.3–5 IoT is built with electronics hardware, software, and connectivity, which enables device interaction and transfer of data. The IoT ecosystem generates massive amounts of data. This data could be analyzed to make business decisions,6 predict consumer behavior, or to bring solutions to problems that might exist.7 Big Data offers the solutions to handle various types of data on a large scale.\n\nBig Data extends the possibility to conduct extensive and rich analyses utilizing a vast amount of data.4,8 Standard data processing tools are limited in data management capacity, where Big Data goes beyond the capabilities of traditional database management systems (DBMS).9 Big Data comprises a large volume of information that is complex (structured and unstructured) in nature. Data are often being generated in real-time and can be of uncertain provenance.10 New Big Data technologies are being developed to meet the demands for processing massive amounts of heterogeneous data. Big Data management benefits are significant and sometimes far-reaching, and many companies have started operating with Big Data to translate a large amount of data into valuable insights.11\n\nThe bulky and heterogeneous nature of Big Data requires investigation using Big Data Analytics (BDA). These data will yield meaningful outcomes by using methods of dissection in BDA,9 which help to discover concealed patterns, anonymous relationships, trends of the current market situation, consumer preferences and other aspects of data that can assist institutes and companies to make up to date, faster and better decisions for their business. However, the biggest issue with available datasets is the data quality itself. The data quality issues differ depending on the data source; they could be duplicated records, spelling errors or more complex issues relating to unit misuse. A mixture of clean and dirty records in data can mislead to the well-known Simpsons Paradox12,13 in which a pattern appears in a particular dataset but disappears or reverses when datasets are combined. A mixture of dirty and clean data could poorly fit an ML model; Figure 1 shows the different ML models fitting with different sets of mixed data. This would lead to unreliable analysis results. Hence, data pre-processing is an important factor in the data analysis process.\n\nTo make the data suitable for analysis we must clean it. Identifying dirty records and cleaning data sometimes require manual data inspection, which is time-consuming and can be costly. Pre-processing includes several steps, for example, (1) loading the data from the file, (2) cleaning it to fix inconsistencies or errors, (3) encoding the numeric and categorical data types, and finally, (4) the missing value imputation. Missing values can be handled in different ways. Columns or rows containing missing values can be dropped or a value can be imputed in each cell with a missing or improper value. Sometimes, crowdsourcing is used to correct some types of errors, which costs a significant amount of time and human-level works. Some researchers have used statistical computing such as mean, median, sum, among others, and approximate query processing to pre-process data. Some researchers have used sample-based cleaning techniques which can gradually improve data quality. Machine learning is an expanding research area, and is being used in some cases of data cleaning. We propose a hybrid model of the data pre-processing technique called Smartic, which is the combination of sample-based statistical techniques and ML. While sample-based statistical techniques lead to faster execution, ML models provide great accuracy. Our research contribution on Smartic will mitigate challenges related to dirty data cleaning and imputing missing values with better performance accuracy, within a reasonable time frame.\n\nIn this paper we present a tool for IoT data preparation and BDA with ML. After data pre-processing, we carried out some feature engineering. This consists in checking which features are highly informative and which are less informative, and then considering features for the analytic purpose. Highly informative features will usually have the most benefits during feature development, while uninformative features can lead to overfitting. The main sections of this study are listed below:\n\n• A review of the recent literature\n\n• Presentation of a BDA framework\n\n• Discussion of the data preparation issues and solution\n\n• Presentation of techniques to improve analysis performance\n\n• Comparison of different solutions and discussion of the results\n\n• Conclusions and future research directions\n\n\nRelated work\n\nAhmad et al.14 reviewed the recent literature on IoT and BDA. Massive data production in IoT environments, and the versatile nature of the data, make Big Data a suitable solution for IoT systems. They discussed the opportunities for organizations to get valuable insights about their customers and help predict upcoming trends. BDA and ML15 tools like classification, clustering and predictive modeling, provide data mining solutions that create many more opportunities to expose variability, improve decision-making habits and boost performance.16 Cross-domain data gathered from different IoT appliances can be fed into BDA that can provide efficient solutions for different domains.\n\nTo overcome the challenges of collecting, processing, and examining the massive-scale, real-time data produced by smart homes, Bashir and Gill17 offered an analytical framework composed of IoT, Big Data administration, and data analytics. The purpose of data analytics in their study was to automatically maintain the oxygen level consistency, detect hazardous gases or smoke, and control light conditions or quality. The work scheme was executed in the Cloudera Hadoop distribution platform, where PySpark18 was used for big data analysis. The outcomes revealed that the proposed scheme could be used for smart building management with BDA.\n\nIdrees et al.19 proposed a two-step data cleaning method, using Big Data on a network of IoT wireless sensor devices. They attempted to minimize communication cost, save energy, and expand the lifespan of sensors by cleaning and reducing the redundant data. Their proposed two-level data reduction and cleaning approach in IoT wireless sensor networks includes a sensor level and an aggregator level. The aggregator level merged a near- similar data sets by implementing a divide and conquer technique. The reduced data sets were retransmitted to the sink, then the leader cluster algorithm-based cleaning method was applied to remove redundant data.\n\nSalloum et al.20 proposed a Random Sample Partition (RSP) Explore technique, to explore Big Data iteratively on small computing clusters. Their work included three main tasks: statistical estimation, error detection, and data cleaning. They partitioned the entire data into ready-to-use RSP blocks using an RSP-distributed data model. To get samples of clean data, they used block-level samples to understand the data and detect any potential value errors. Their experimental results showed that a sample RSP block cleaning is enough to get an estimation of the statistical properties of any dataset, and the approximate results from RSP-Explore can rapidly converge toward the true values.\n\nGarcía-Gil et al.21 worked on data pre-processing to transform raw data into high-quality, clean data. The quality of the data used in any knowledge discovery process directly impacts the output. They experimented with classification problems due to the presence of noise affecting data quality, particularly a very disruptive feature of data known as incorrect labelling of training dataset. They proposed two Big Data pre-processing techniques with a special emphasis on their scalability and performance traits. The filters they used to remove noisy data were a homogeneous ensemble and a heterogeneous ensemble filter. The results from their experiments show that anyone can retain a smart dataset efficiently from any Big Data classification problem using these proposed filters.\n\nSnineh et al.22 proposed a solution that can be performed in real time to handle the frequent errors of Big Data flows. They proposed a repository for each given domain in their two-step model to store the metadata, cleaning and correction algorithms, and an error log. An advisor was appointed to supervise the system for the first step. The advisor could estimate the algorithm corresponding to error cleaning for a given context. At the second step, the system became autonomous in the selection algorithm procedure based on its learning module. That capability was obtained by using a strategy pattern-based approach. The pattern allowed the building of a family of algorithms, which are interchangeable and evolve independently of the context of use.\n\nJesmeen et al.23 presented a comparison between currently used algorithms and their proposed tool, Auto-CDD, to handle missing values. The developed system improved overall data processing and guaranteed to overcome processing unwanted outcomes in data analysis. Their intelligent tool used Gini index values of random forest for feature selection. Experimental evaluation results showed that the random forest classifier led to a high accuracy on a diabetes dataset from UCI.24 They also imputed the missing values on a student database and performed logistic regression analysis on students’ performance.\n\nShah et al.25 investigated the research gaps in understanding Big Data characteristics generated by industrial IoT sensors, and studied the challenges to processing data analytics. They studied the characteristics of the Big Data generated from an in-house developed IoT-enabled manufacturing testbed. They explored the role of feature engineering for predicting the key process variables in effective machine learning models. The comparison with different levels or extent of feature engineering in between simple statistical learning approaches and complex deep learning approaches, shows potential for industrial IoT-enabled manufacturing applications.\n\nEl-Hasnony et al.26 presented challenges in building an optimal feature selection model for Big Data applications, due to the complexity and high dimensionality of the data sets. They used particle swarm optimization and grey wolf optimization to build a new binary variant of a wrapper feature selection. The optimal solution was found with the help of the K-nearest neighbour classifier and Euclidean separation matrices. The overfitting issue was checked using K-fold cross-validation, and the performance and the effectiveness of the model were validated by conducting statistical analyses.\n\n\nBig data analytics\n\nBDA follows some steps towards getting meaningful insights. Data analytics start with a non-trivial step of problem definition and evaluation. Research on expected gains and costs for reasonable solutions is needed. Generally, a data analytics framework is defined by five main steps:\n\nData acquisition, the key to the data life cycle, defines the data product profile. At this stage, structured and unstructured data are gathered from different sources and different types of unstructured or dirty data are pre-processed. Short data loading times are crucial for BDA due to its naturally exponential growth rate.\n\nThe most essential stage of processing Big Data is to implement a method to extract the necessary data from the loaded, un-structured Big Data. A data analyst spends the most time on cleaning dirty data. Analysing dirty data could lead to erroneous results. To get high-quality data, faulty records, duplicates, unwanted records, and outliers need to be removed. Typos must be fixed and the data requires structuring. An exploratory analysis could investigate the initial characteristics of data and helps refining the hypothesis.\n\nThe cleaned data obtained needs to be aggregated for processing numerical and categorical types of data, followed by data integration. Different types of data in various shapes and sizes obtained from different sources need to be integrated to prepare for analysis. To unify some data features, we may need to convert between formats. For example, one source collecting ratings on a five-star scale, and another source collecting data as “up” and “down” vote only. The response variable could be,\n\nand\n\nBefore integrating both source data, we need to make an equivalent response representation, possibly by converting the first source to the second representation format, considering three stars and above as the positive ratings and the rest of them as the negative ratings. Properly integrated data becomes less complex, more centralized and more valuable.\n\nFrom the perspective of Big Data, the goal is to produce meaningful insights that will be invaluable for business, through the analysis of data which may fluctuate depending on analytics technique and data types. Reports investigating the data must be constructed to help the business for better and faster decision-making.\n\nData interpretation allows to present data in an understandable format for users, for example, presenting data using analysis and modelling results to make decisions by interpreting the outcomes and extracting knowledge. Data interpretation queries are categorized together and indicate the same table, diagram graph or other data demonstration options.\n\n\nProposed method\n\nWe collected data from the UCI repository24 and from publicly available datasets in the Kaggle database.27 We stored these datasets on Kaggle’s server and worked on these data on the database’s kernels. All collected datasets were in CSV (Comma-separated values) format.\n\nData preprocessing is an important phase of data analysis. Raw data is manipulated to make it understandable. This is carried out in several steps, such as cleaning, encoding, imputing, among others. We handled these steps separately.\n\nFor this step, we tried to fix any errors and remove inconsistency. We fixed typos and different representations for any values in a common representation. We used fuzzy matching or edit distance algorithms to remove inconsistency. Outlier detection and removal help to get better accuracy. Figure 2 shows summary statistics (number summary) to represent data, such as, minimum, maximum, median, quartiles (Q1, Q3). The first quartile (Q1) is the middle value between the smallest value and the median (or the 50th percentile, or Q2) of the dataset. A 25% portion of values in the dataset resides below the first quartile.\n\nIQR, or midspread, or middle 50%, is the statistical dispersion equal to the range from lower quartile (25th percentile) to the upper quartile (75th percentile). The values that do not reside within the range of the minimum and maximum value are defined as outliers (Figure 2).\n\nWe prepared statistical data values in numerical and categorical values. The standard statistical types such as numeric and categorical had similar representations in Pandas29 and Python (version 3.10). To treat each feature correctly, we encoded each column as its respective type of data, which helps to apply transformation consistency in further analytical processes.\n\nWe fixed the missing values in this step. We used 0 as the default value for missing numeric data and ‘None’ as the default value for missing categorical data. We used different techniques to impute missing values and train the machine learning model by feeding these imputed datasets. Based on the model performances, we chose the best imputation technique and used it for further analytical process. The implemented algorithmic steps were as follow:\n\nStep 1: Retrieve sample clean dataset (Datasetclean) from the original dataset, excluding missing/incomplete values as much as possible.\n\nStep 2: Order Features (Featureso) based on feature utility scores or mutual scores.\n\nStep 3: Select top features from Featureso and apply step 5.\n\nStep 4: Select the rest of the features from Featureso and apply step 6.\n\nStep 5: For a given feature Fi : label Fi as the target and the rest of the column in Datasetclean as features, and train the ML model to obtain missing or incomplete values for the original dataset.\n\nStep 6: For a given feature Fi : calculate statistical parameters (mean or median) of the Fi column in Datasetclean, and obtain missing or incomplete values for the original dataset.\n\n\nFeature engineering\n\nWe used mutual information (Figure 3) to determine the importance of a feature. For this step, we created new features as well. We used target encoding for categorical features with higher cardinality. Target encoding involves replacing a categorical feature with the average target value of all data points for that category. We used several other techniques of feature engineering for this purpose.\n\nUsing mutual score is a great way to determine a feature’s potential. Feature utility scores help to determine important features and non-important ones as well. Based on scores, we discarded some features for a performance gain.\n\nLabel encoding can be done to transform categorical features, as we are focusing on the tree-ensemble model; this works for both ordered and unordered data categories. Creating new features can be done in several ways such as, taking the product of two numerical features, the square root of a feature, normalize by applying logarithms, determining the group statistics of a feature, etc.\n\nThe unsupervised algorithm k-means clustering can be used to create features as well. Cluster labels or the distance of each entity to each cluster can be used as features. Sometimes, these help to untangle complicated relationships between features, engineered features or targets.\n\nWe can use another unsupervised principal component analysis (PCA) model for feature creation, which can decompose a variational structure. The PCA algorithm gave us loadings which described each component of a variation, and the components which were the transformed datapoints. The loadings can suggest features to create and the components we can directly use as features. Clustering can be done using one or more components.\n\nIt is an encoding of categorical into numeric values derived from the target. It resembles a supervised feature engineering technique. We used mean and median values for this purpose.\n\nA great way of boosting performance is carrying out hyperparameter tuning. For our ML model XGBoost, we set max_depth to 6, learning_rate to 0.01, n_estimators to 1000.\n\nWe adopted the K-fold cross-validation for performance evaluation. Cross-validation divided the data set into training and a testing data set to train the model and test its performance using two distinct data sets. Training and testing on the same data create overfitting issues. To avoid this, we used K-fold cross-validation with a value of 5 for K (Figure 4). All our experimental results were five-fold cross-validated.\n\nThe XGBoost model performance was evaluated using the root mean squared logarithmic error (RMSLE) metric. The formula for RMSLE is represented as follows:\n\nWhere:\n\nn is the number of observations in the dataset\n\npi is the prediction of target\n\nai is the actual target for i.\n\nlog(x) is the natural logarithm of x (logex).\n\n\nExperiments\n\nDiscarding columns or rows is a technique for handling missing values. Our model performance in RMSLE was 0.14249 after discarding columns with missing values.\n\nWe evaluated our XGBoost model using datasets with imputed missing values using different types of imputation techniques. When we filled non-muerical (NAN, not a number) values with a 0, we obtained an RMSLE score of 0.14351, while when filling missing values with the next valid value on the same column, we obtained a score of 0.14348. If we use the statistical mean of a feature column to impute missing values in that column, we notice a performance increment with an RMSLE score of 0.14157.\n\nAs we carried out feature transformation and target encoding based on feature utility scores, we yielded better performances. The use of K-means clustering and PCA led to a better performance as well. We obtained a value of 0.14044 for the RMSLE score.\n\nHyperparameter tuning gave a performance boost in the final performance evaluation. Figure 5 shows the performance improvements after feature engineering and hyperparameter tuning. After fine-tuning some parameters, we obtained our highest RMSLE score with0.12426.\n\n\nResults and discussion\n\nWe calculated the mean RMSLE value of five trials of train/test splits while varying the training dataset size from 0.1 to 0.9 (10% to 90%). In Figure 6(b), we notice that the ML-based missing value imputation technique outperformed all other traditional imputation methods. Imputing 0 in place of the missing value performed worst in our experiment (see Figure 6a). Replacing missing values of any feature column with the median of that column performed slightly better than imputing the mean of that feature column.\n\nAs the ML-based imputation technique outperformed state-of-the-art baseline methods, we evaluated missing value imputation performance of different ML models, such as LinearRegression, DecisionTreeRegressor, LinearSVR, GaussianNB, BaggingRegressor, KNeighborsRegressor, AdaBoostRegressor, XGBRegressor, among others. Although all ML models delivered higher accuracy with the continuous increment of training dataset size, we saw (Figure 7) more uniform and sheer increasing patterns in XGBRegressor and BaggingRegressor models. It proves that with sufficiently large datasets, the XGBRegressor model can outperform the other ML methods. In addition, we noticed that the XGBRegressor model showed a more stable performance with the varying training data size.\n\n\nConclusion\n\nAlmost every data set available may contain missing values, which are essential to analyze and understand the data. Dealing with these types of dirty data is difficult, and getting a robust analytical ML models is more challenging. We used statistical methods equipped to fix the datasets, and the sample-based approximate query processing was integrated to alleviate errors in analysis and predictions. The data fixed using different imputation techniques were fed into ML analytical models, and accuracy was compared against different data preparation techniques. Smartic’s data value imputation was faster than the ML-based missing value imputation model. The ML model, trained on data cleaned using a sample-based technique, showed a significantly better and more stable performance. In the future, evaluation can be done with data collected directly from IoT environments in real time.\n\n\nData availability\n\n\n\n- Ames housing dataset: house sales data in Ames, Iowa between 2006 and 2010. Compiled by Dean De Cock; used for educational purposes. We used a version of that dataset available at https://www.kaggle.com/c/house-prices-advanced-regression-techniques/data\n\n- Diabetes dataset: The dataset represents clinical care at 130 US hospitals between years 1999-2008. This dataset was prepared to predict whether a patient’s re-admission. Dataset available from UC Irvine Machine Learning repository, https://archive.ics.uci.edu/ml/datasets/Diabetes+130-US+hospitals+for+years+1999-2008#\n\nAnalysis code available from: https://github.com/FuadAhmad/smartic\n\nArchived analysis code at time of publication: https://zenodo.org/badge/latestdoi/420156995\n\nLicense: (must be open access) Apache-2.0 License", "appendix": "References\n\nRisteska Stojkoska BL, Trivodaliev KV: A review of Internet of Things for smart home: Challenges and solutions. J. Clean. Prod. 2017; 140: 1454–1464. Publisher Full Text\n\nZanella A, Bui N, Castellani A, et al.: Internet of things for smart cities. IEEE Internet Things J. 2014; 1: 22–32. Publisher Full Text\n\nAsghari P, Rahmani AM, Javadi HHS: Internet of Things applications: A systematic review. Comput. Netw. 2019; 148: 241–261. Publisher Full Text\n\nHariri RH, Fredericks EM, Bowers KM: Uncertainty in big data analytics: survey, opportunities, and challenges. J. Big Data. 2019; 6. 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Publisher Full Text\n\nGe M, Bangui H, Buhnova B: Big Data for Internet of Things: A Survey. Futur. Gener. Comput. Syst. 2018; 87: 601–614. Publisher Full Text\n\nFabris CC, Freitas AA: Discovering Surprising Patterns by Detecting Occurrences of Simpson’s Paradox. Research and Development in Intelligent Systems XVI. 2000.\n\nScarsini M, Spizzichino F: Simpson-type paradoxes, dependence, and ageing. J. Appl. Probab. 1999; 36: 119–131. Publisher Full Text\n\nAhmad AF, Sayeed MS, Tan CP, et al.: A Review on IoT with Big Data Analytics.2021. Publisher Full Text\n\nAl-Garadi MA, Mohamed A, Al-Ali AK, et al.: A Survey of Machine and Deep Learning Methods for Internet of Things (IoT) Security. IEEE Commun. Surv. Tutorials. 2020; 22: 1646–1685. Publisher Full Text\n\nL’Heureux A, Grolinger K, Elyamany HF, et al.: Machine Learning with Big Data: Challenges and Approaches. IEEE Access. 2017; 5: 7776–7797. Publisher Full Text\n\nBashir MR, Gill AQ: Towards an IoT big data analytics framework: Smart buildings systems.2017. Publisher Full Text\n\nPySpark Documentation. Reference Source\n\nIdrees AK, Jaoude CA, Al-Qurabat AKM: Data reduction and cleaning approach for energy-saving in wireless sensors networks of IoT.2020. Publisher Full Text\n\nSalloum S, Huang JZ, He Y: Exploring and cleaning big data with random sample data blocks. J. Big Data. 2019; 6. Publisher Full Text\n\nGarcía-Gil D, Luengo J, García S, et al.: Enabling Smart Data: Noise filtering in Big Data classification. Inf. Sci. (Ny). 2019; 479: 135–152. Publisher Full Text\n\nSnineh SM, Youssfi M, Bouattane O, Daaif A, et al.: Real-Time management model for frequent Big Data errors: Automatic Clean Repository for Big Data (ACR).2018. Publisher Full Text\n\nJesmeen MZH, et al.: AUTO-CDD: Automatic cleaning dirty data using machine learning techniques. Telkomnika (Telecommunication Comput. Electron. Control). 2019; 17: 2076. Publisher Full Text\n\nStrack B, et al.: Impact of HbA1c measurement on hospital readmission rates: Analysis of 70,000 clinical database patient records. Biomed. Res. Int. 2014; 2014: 1–11. PubMed Abstract \| Publisher Full Text\n\nShah D, Wang J, He QP: Feature engineering in big data analytics for IoT-enabled smart manufacturing – Comparison between deep learning and statistical learning. Comput. Chem. Eng. 2020; 141: 106970. Publisher Full Text\n\nEl-Hasnony IM, Barakat SI, Elhoseny M, et al.: Improved Feature Selection Model for Big Data Analytics. IEEE Access. 2020; 8: 66989–67004. Publisher Full Text\n\nDe Cock D: Ames, Iowa: Alternative to the boston housing data as an end of semester regression project. J. Stat. Educ. 2011; 19. Publisher Full Text\n\nGalarnyk M: Understanding Boxplots. (accessed Oct. 21, 2021). Reference Source\n\npandas: Reference Source" }	[ { "id": "136164", "date": "30 May 2022", "name": "Sujatha Krishnamoorthy", "expertise": [ "Reviewer Expertise IoT", "Machine learning", "Computer vision" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the authors for the work that has been carried out, as we are all dealing with the big data day to day in our life, which is very important. Big Data refers to large, diverse sets of information with dimensions that go beyond the capabilities of widely used database management systems, or standard data processing software tools to manage within a given limit. Almost every big dataset is dirty and may contain missing data, mistyping, inaccuracies, and many more issues that impact Big Data analytics performances. One of the biggest challenges in Big Data analytics is to discover and repair dirty data; failure to do this can lead to inaccurate analytics results and unpredictable conclusions. This experimented with different missing value imputation techniques and compared machine learning (ML) model performances with different imputation methods. The authors have proposed a hybrid model for missing value imputation combining ML and sample-based statistical techniques. The authors have used k-means clustering and principal component analysis. Accuracy, the evaluated outcome, improved dramatically and proved that the Boost model gives very high accuracy at around 0.125 root mean squared logarithmic error (RMSLE). To overcome overfitting, we use K-fold cross-validation.\nAs per the quality of the work, the code was impressive and also the source is attached to paper, which is very useful for the people who would like to take up the research in this field.\nThe only suggestion from my side is the flow of the paper - the authors have mentioned “we “in multiple places. Hence, I suggest them to change to passive, which can make readability of the paper clearer.\nThe methods in the data analytics are explained in the section 3 and also the same is clearly explained in the way that is being implemented in the paper.\nThe font in the imputation steps could be formatted to give a unified look. Add a proper citation to the K-fold cross-validation since the data is directly given from the experiment.\nOverall the paper structure is good enough for indexing.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "10960", "date": "22 Mar 2024", "name": "Md Shohel Sayeed", "role": "Author Response", "response": "The manuscript has been revised based on the reviewer's comments." } ] }, { "id": "136161", "date": "23 Jan 2024", "name": "Muzammil Hussain Mohammed", "expertise": [ "Reviewer Expertise Software Engineering", "Cyber Security", "Database and Networking" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this research paper well explains the content.\n\nThe abstract is well defined.\n\nAll the figures are clear and well understood.\n\nRelated work is needed to add some more points to improve understanding. But overall it is ok.\n\nThe proposed method is well explained with equations.\n\nResults and discussion are also explained good.\n\nOverall this paper is well structured and good research work done by the authors.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-17
https://f1000research.com/articles/11-1189/v1	18 Oct 22	{ "type": "Research Article", "title": "Quality of life of Jordanian menopausal working and retired women and its associated factors: a cross-sectional study", "authors": [ "Enas A. Assaf", "Muntaha K. Gharaibeh", "Sawsan Abuhammad", "Mohannad AbuRuz", "Muntaha K. Gharaibeh", "Sawsan Abuhammad", "Mohannad AbuRuz" ], "abstract": "Objective: Life expectancy of Jordanian women has increased, indicating that the number of women entering menopausal age, during the prime of their working life, will also increase. Therefore, assessments of the quality of life (QoL) of working and retired women and factors associated with overall wellbeing, are essential for the provision of quality services and care. Method: A cross-sectional study was conducted with 200 Jordanian women between the ages of 45 to 60 years old. The Utian QOL tool was used to assess the quality of life among menopausal women. Multiple regressions were used to determine predictors for QoL for the whole sample and for each group of working and retired women. Results: The study shows that the total QoL for women was 77.5 ±14.4, with a significant difference (p=.023) in total QoL and the occupational domain (p=.003) between working and retired women. Employed women with fewer chronic diseases and using frequent preventive measures had a higher QoL compared to others. Conclusion: Working itself might be an important indicator for better a quality of life among menopausal women. Better working conditions and more attention from the health care providers for the menopausal changes and the preventive measures could enhance women’s perceived QoL in addition to increasing their productivity.", "keywords": [ "menopausal women", "QoL", "Jordanian" ], "content": "Introduction\n\nMenopause is a normal transitional period experienced by with more than 1.5 million women each year. It is characterized by cessation of the menstrual period and many other disruptive symptoms among which are joint pain, hot flashes, vaginal dryness, insomnia and general tiredness and fatigue.1,2 Among other important symptoms are mood changes and depression, which is correlated with estrogen level decease during the menopausal stage.3\n\nMenopausal age is associated with multiple morbidities and chronic diseases, particularly after the age of 60, such as hypertension, diabetes mellitus arthritis, heart diseases, breast cancer, depression, osteoporosis, chronic obstructive pulmonary diseases, and stroke.4 Therefore, the menopausal stage represents a very important biological milestone in a women’s lifespan; the transition from reproductive to a non-reproductive phase is marked by cessation of menstrual period5 which may have negative effects on the women’s quality of life (QoL).\n\nThe health related QoL was initially defined by WHO based on the concept of subjective perception, cultural context and value scheme.6 In addition, the objective context is related to specific schemes measured for the studied population.7 Women’s QoL, which could be related to several important factors such as biological as represented by hypoestrogenism, psychosocial, and cultural perspectives and might differ based on their attitudes toward menopausal changes and age which culturally could be either defined as positive or negative stage.8\n\nIn a meta-analysis study of 14 studies among Iranian post-menopausal women, it was found that the mean QoL was higher than moderate, while the lowest QoL was reported for the physical and the sexual domain.9 In one Egyptian study using menopausal specific quality of life instrument (MENQOL), it was found that physical symptoms were perceived as the most affecting to women’s QoL, and had the lowest means score.10 Another study in Iran used WHO Quality of Life-BREF (WHOQOL-BREF) and the Menopause Rating Scale (MRS) among pre-menopausal and post-menopausal women; QoL was found to be negatively correlated with menopausal symptoms at all WHOOL domains (physical health, psychological health and social interactions). Moreover, QoL decreased with increasing the severity of menopausal symptoms.11\n\nPrevious studies showed that working women might have better QoL in the menopausal and postmenopausal stages than those unemployed or housewives, as they might have better access to health care, better economic stability, social interaction and empowerment which all might reduce the physical symptoms of menopause.8,11–17 One study measured Serbian women’s QoL using the Utian menopausal QoL scale, which has one specific domain measuring the occupational QoL and found that that employment and economic status were correlated with the occupational domain in QoL.14\n\nBustami et al.,18 conducted a cross-sectional study to assess factors associated with onset of premature/early menopause among 409 Jordanian women. Findings revealed that the mean age of natural menopause (ANM) is 48.5±5.0, and 2.7% of the women experienced premature menopause (ANM <40) and 7.8% early menopause (ANM 40–44). Smoking was highly associated with early/premature menopause, chronic diseases or combination of diseases were associated with average (45–52 years) or late menopause (>52 years). BMI, arthritis pain, hot flushes and inconsistent urination were significantly higher in the early and regular menopause than in premenopausal and perimenopause women.\n\nJaber et al. (2017) examined patterns and severity of menopausal symptoms among 359 women between the age of 45-65 years. The Arabic version of Menopause Rating Scale (MRS) was used. Results showed that the mean age at menopause was 49.4 years, a total of 105 women had regular menstruation (premenopausal), 49 experienced irregular cycles, and 205 had reached menopause. Women who were still menstruating regularly were found to be 1.5 times more likely to suffer from irritability, while women experiencing irregular cycles but were still menstruating, were found to be 1.63 times more likely to suffer from hot flushes, approximately 2 times more likely to suffer from physical and mental exhaustion and more likely to have sexual problems, vaginal dryness, and joint and muscular discomfort.\n\nSeverity of menopausal symptoms of Jordanian women was examined by Gharaibeh et al., (2010) who recruited 350 women between the ages of 45 to 55 years. Authors used the Greene Climacteric Scale to measure the severity of menopausal symptoms. The scale consists of domains of vasomotor symptoms, somatic symptoms, psychological symptoms and sexuality symptoms. Results showed that the mean age of menopause was 48.7 years. A positive association between severity of menopausal symptoms and both educational level and menopausal status; the mean score for severity of menopausal symptoms in perimenopausal women was higher than that for premenopausal women. The scores for the psychological, somatic, vasomotor and sexuality subscales were significantly higher among the perimenopausal, and postmenopausal groups compared to the premenopausal group. Perimenopausal women’s scores for all clusters were higher compared to those of postmenopausal women except for the vasomotor and its symptoms of sweating at nights, and the sexuality and its symptoms of loss of interest in sex, vaginal dryness and pain during intercourse.\n\nAn earlier study by Al-Qutob (2001) investigated types and magnitude problems associated with menopausal using exploratory study with 317 of Jordanian women between 18 and 49 years old and 185 women older than 50 years. The mean and median age of onset of menopause were 47.5 and 49 years. Chronic diseases were found to be present among menopausal women such as urinary tract infection, hypertension, diabetes, iron deficiency anemia, reproductive track prolapse.\n\nIt is clear from the review of the available studies that there is a gap in studies from Jordan that addressed quality of life of menopausal working women. Therefore, the study comes to fill the gap by assessing the quality of life of Jordanian working and retired women and its associated factors. The importance of the study comes from the fact that the life expectancy at birth of Jordanian women has increased from 61.98 years in 1971 to 76.45 years in 2019,19 indicating that the numbers of women in pre-menopause, transitional phase and postmenopausal ages will increase and we are expected to see more working, active women involved in the society and enjoy a positive QoL. Findings of this study will help women live a productive life, and health care professionals who can provide supportive services that enhance working women’s QoL.\n\n\nMethods\n\nThe study was approved by the Institutional Review Board (IRB) of Applied Science Private University, Jordan. The informed participant consent was obtained along with the data collection with maintaining participant privacy, confidentiality, and anonymity which was built into the design and the process of the instrument. The cover letter included with the questionnaire stated the study purpose and that the completion and submitting of the questionnaire was indication of consenting to the study.\n\nA cross-sectional study was conducted with 200 Jordanian women during the period from 5th January to 5th March 2021. Sample size was determined using power analysis software for the following parameters: alpha 0.05, power of 95%, moderate effect size 0.3. The needed sample size was 196 participants; 200 questionnaires were completed and submitted. The main inclusion criteria were working and retired women between the age of 45 to 60 years old. The women were excluded if they suffered from any mental disorders since these will impact the QoL. The women were recruited from main authors or research assistant who employed from the main authors. These women were recruited based on information provided on leaflets, which were distributed in health centers and hospitals. The leaflets included information on the researchers and how to contact them.\n\nWomen were recruited using social media and emails. This research utilized Google Forms for data collection; a link was sent by the authors to participants via social media, such as Facebook, Instagram. Google forms is an application in the form of a template or worksheet that can be used independently or together for the purpose of obtaining information. Google form is very easy to understand and use and is available in the Arabic language. The form took 15 minutes to fill out.\n\nThe questionnaire consisted of three parts; the demographic part which consisted of seven questions on age in years, employment status (employed, retired), social status, level of education, chronic diseases, menopausal stage (pre, menopausal and post-menopausal), and finally number of children. All variables were categorical except for age and number of children, which were continuous. The second part was the menopausal information and preventive measures, which consisted of the perceived menopausal health related information, (very good, moderate, know some, not good), sources of menopausal information (social media, family/friend, TV, health personnel) and health promotion and disease prevention measures (BSE, monogram, pap smear, routine checks). The third part was the QoL measures, the study used the Utian QoL developed by Utian et al. (2018). The tool consists of 23 items divided into four main domains: occupation domain (7 items), health domain (7 items), emotional domain (6 items) and sexual domain (3 items). The original tool was validated with a diverse sample of peri- and postmenopausal women using the Short Form-3620 (well established inventory of QoL). For the QoL domains, confirmatory factor analyses were conducted with a second sample of 270 women. The original scale is in the English language and showed to be valid and reliable. Cronbach’s alpha for the 23-item scale as a whole was 0.830, occupational domain was 0.83, health domain 0.73, emotional domain 0.64 and for the sexual domain 0.79.20\n\nFor our study, the scale was translated to the Arabic language using the World Health Organization standard (translation and back translation process).21 This method consisted of the following steps: forward translation; expert back-translation; pretesting and cognitive interviewing and producing the final version. A panel of three experts in psychiatry, mental health nursing, nursing education and women’s health from Jordan University of Science and Technology and Applied Science University. These experts were chose based on their experience and were tasked with checking the content validity and the process of translation and back translation. The Arabic version was then pilot tested with 25 menopausal women, which were not included in the sample. These women were recruited by research assistants and have the same inclusion and exclusion criteria of the participants of this study and not were entered into the main data of the project. Results of the pilot testing revealed that it is simple to read, and completion of the tool required approximately 10 minutes. The Cronbach reliability coefficients for the translated version for the total scale was 0.85, it was 0.80 for the occupational domain, 0.69 for the emotional domain, 0.62 for the health domain, and 0.50 for the sexual domain.\n\nData was analyzed using the SPSS software for windows version 25 (IBM Corp, USA, RRID:SCR_016479). Descriptive statistics including means, standard deviation, numbers, and frequencies were used to describe the sample characteristics. Means and standard deviation were used to describe each domain and the total QoL. The t-test was used to show the differences in the four domains for working and retired women. Frequencies and percentage were used to describe the response for each item of QoL. Chi-square test was conducted to show the difference in each item for QoL for working and retired women. Multiple regression was conducted to determine predictors for QoL for the whole sample and for each group of working and retired women.\n\n\nResults\n\nTable 1 presents the demographic characteristics of the sample as the mean age of women was 50.5±4.8 years old. Employed women were 117 (58.5) and retired women were 83 (41.5).\n\nWomen reported acceptable information level regarding the menopausal stages and changes 134 (67%). The majority receive their information from social media, rather than their health care providers, 61 (30%). For preventive measures, self-examination breast checks that were done regularly the previous year, was 63 (31.5) and those who had a Mammogram examination once during the previous year was 40 (30.0). As shown in Table 2.\n\nThe total QoL for women was 77.5±14.4, which is higher than the average total universal quality of life (UQoL).16 There is significant difference (p=.023) in total QoL between working (80.5±12.7) and retired women (73.4±15.7). The highest domain mean score was for the occupational domain 25.9±6.4, followed by the emotional domain 22.3 ±4.8, and health domain 21.5±5.0, while the lowest mean score was among the sexual domain 7.6±2.6. There was a significant difference in occupational health (p=.003) between working (27.2±5.3) and retired women (24.1±7.4), see Table 3.\n\nDescriptive statistics was used to describe the women's response to the QoL tool. The highest response was for “I am currently experiencing physical discomfort or pain during sexual activity with my partner” 141 (71%), “I think my work benefits society” 129 (65%), and “I'm not happy with my appearance” 124 (62%). However, the lowest score response was reported for “I do exercise at least 3 times a week” 15 (8%), “I feel like I'm not satisfied with my sex life” 21 (11%), and “I feel in good shape” 38 (19%).\n\nThere was a significant difference in QoL between working and retired women in seven questions. Items were, “I am able to control the important things in my life” (p=.024), “I think my work benefits society” (p=.007), “I am satisfied with my romantic life” (p=.007), “I get anxious frequently” (p=.003), “I am proud of my professional achievements” (p=.006), “I keep setting personal goals for myself” (p=.006), and “I keep setting professional goals” (p=.001) (Table 4).\n\nMultiple regressions test was conducted to determine the predictors of QoL among menopausal women. The model was significant (F= 3.5, p<. 001). This means that many factors predicted QoL among women; these were employment status/currently employed (B=.175, p=.046, r2=.34), number of chronic diseases (B=-.198, p=.004, r2=-.260), and using preventive measures (B=.177, p=.011, r2=.222). Employed women with fewer chronic diseases and using frequent preventive measures had higher QoL compared to others. The three variables explained 54% in the variance of QoL. See Table 5.\n\na Dependent variable: Total QoL.\n\nMultiple regression tests were conducted to determine the predictors of QoL among working and retired menopausal women. These models were significant for working women (F=4.45, p=.001) and retired women (F=6.52, p<. 001). The only significant predictors for QoL for working women were number of chronic diseases (B=-.206, p=.006, r2=-.267) and using of preventive measures (B=.196, p=.003, r2=.194) and income (B=0.204, p=0.037, r2=0.134). This means working women with less chronic diseases and using preventive measures had higher QoL compared to retired women. The only significant predictors for QoL for retired woman were education (B=.199, p=.04, r2=.231), menopausal status (B=.199, p=.04, r2=.222), and using of preventive measures (B=.217, p=.045, r2=.194). This means retired woman with higher education, who reached postmenopausal, and using preventive measures had higher QoL compared with other women. See Table 6.\n\n\nDiscussion\n\nOur study showed that the total mean score for quality of life of working and retired women was more than the UQoL average (77.5±14.4). The highest score was reported for the occupational domain, followed by the emotional domain, with the lowest being among the sexual domains.\n\nThere were significant differences between working and retired women in each of the occupational domains and the total QoL scores. These differences could be to domain questions that showed a significant difference between the two groups of women related to their work benefits to society (p=.007), being proud of their professional achievements (p=.006), setting personal goals for themselves (p=.006), and in setting professional goals (p=.001). This suggests that working serves as a good source of self-satisfaction, planning future goals, sense of achievements and recognition. This is consistent with that of,21 which assessed the QoL between retired and working people, to find out that working has a positive correlation with both social interactions, family interactions and leisure time. As retired women may lack the sense of achievements and may feel that their professional goals and careers have ended. Furthermore, retired women usually face changes in socioeconomic status and lifestyle. Moreover, they might lose the daily socialization with people, work interactions, and challenges of their work environment, which all could enhance the feelings of well-being. This reflects the findings of other studies, on the effect of retirement on ones’ QoL.22\n\nOur study showed no significant differences between working and retired women in the emotional domain. This might be related to the fact that the average age of the study sample of retired women is 53 years old, by which at this age Jordanian women have many responsibilities towards their family needs, through the role of grandmother as well as caring for their elderly parents and in-laws. These responsibilities might give them a great satisfactory feeling and emotional stability that can minimize the bad feelings of retirement, in addition to filling their time with many other responsibilities than work.23 In addition, women going through menopause are likely preoccupied with the menopausal symptoms such as hot flashes which might be a priority issue.24\n\nThe predictors for total QoL among menopausal women in this study were currently working with few chronic diseases, good income, and using frequent preventive measures; these factors explained better QoL compared to others (p=0.046, 0.004, 0.011 respectively). One study that reviewed the relationship between menopausal age and chronic disease and comorbidity, found a strong negative relationship between QoL and chronic diseases.25 It is expected that chronic diseases in general had a negative effect on Qol looking at the overall complaints, limited physical activity and the overall psychological effect.26 In one early study conducted in Jordan, the majority of menopausal women complained of chronic diseases, while only 29% reported that they are feeling “good”.24 Working women appear to be more satisfied socially and have a more meaningful life, and responsibilities. As well as their time being filled with achieving their career plans and objectives,27 reported that Jordanian working women have better leisure time since they have better income. Preventive measures like self-check breast examination, pap smear tests, mammograms among and regular gynecological checkups could be all widely affected by women’s level of education, income and availability of services, which was found in different cultures and studies.28–31 Working women could have a better opportunity to conduct any of the preventive measures than others; the availability of health insurance, better access to health care services and higher awareness of the importance of conducting preventive measures.32 Menopausal QoL was not directly connected previously with using preventive measures, rather it related to health-related issues, problems and disease prevention and how those could affect the overall health of aging women, which was discussed by Pertynska-Marczewska & Pertyński.33\n\nIncome satisfaction reflects women’s access to health services and follow up on health preventive measures as well as a better sense of high self-esteem and empowerment, which was discussed previously with several previous studies.34–36 Women’s employment would increase the overall family income and empowerment, one study among Jordanian women found that women with higher family income reported less severe menopausal symptoms compared to those with low family income.27\n\nOur study shows that retired woman with higher education, who reached postmenopausal, and used preventive measures had a higher QoL compared to other women. Higher educated women have better opportunities and more understanding of the symptoms of menopausal changes, which makes the adaptation a lot easier and more acceptable.24,37–39 A study conducted in Iran found positive correlations between levels of education and occupational status, with better scores of psychological symptoms, they found that women might have a better understanding of menopausal symptoms and better awareness related to the psychological and physiological changes of the menopause.40 Moreover, women in postmenopausal stage reported less severe symptoms compared with pre and peri-menopausal stage. Studies among Jordanian women found that the severity of the menopausal symptoms is positively associated with the women’s level of education, and the menopausal stage.23,27 On the other hand, Jordanian women have positive attitudes towards menopause in that they would have more time to enjoy their Ramadan fasting without any interruptions and pray regularly. Moreover, Jordanian women appreciate that transitional period since it gives them a status of wise women, which would help them to participate in decision making and empower them.23 A higher level of education positively affects women’s health promotion and health prevention behaviors.29\n\nOur study shows that only 19.5% of women received their information about menopausal changes from health care professionals, even though women in this period require more information about the changes in the physiological and psychological status in order to better cope with them.40\n\n\nConclusion\n\nOverall, working women have a total QoL higher than the average UQoL scores in other studies. However, working menopausal women have a significant better QoL than retired women among both total QoL and among the occupational domains. Major predictors for working women for better QoL were employment, fewer chronic diseases and frequent use of preventive measures. While significant predictors for better QoL among retired women were higher level of education, being in the postmenopausal stage, and using preventive measures.\n\nHealth care providers can pay more attention towards the menopausal changes, chronic disease health promotion and disease prevention for working women, to increase their work productivity and achievements. Policy makers should pay more attention towards improving the working conditions that support the transitional period for menopause, to increase women’s productivity and improve their health status during these important life stages.\n\nIncreasing the Jordanian women life expectancy would suggest adding more working years for women, as retired women seem to perceive a lower QoL than working women, therefore this study’s results might be used as a valid indicator for revisiting the retirement age law.\n\nIt’s recommended for further research on women’s perception of the best retirement age and the influence of their menopausal stage on their work productivity.\n\nThis study has limitations that rise from the convenience nonprobability sample that limited the generalizability of the findings. The fact that the study used Google Form for data collection has eliminated women with poor reading comprehension and those who are not familiar with the use of technology or do not have access to computers or mobiles. Findings are also limited to the use of a translated scale developed for women from western cultures, which may not have captured the cultural aspects of the QoL of Jordanian women.\n\n\nData availability\n\nFigshare: Data Set Menopause Women.xlsx. https://doi.org/10.6084/m9.figshare.20822032.v2.41\n\nThis project contains the following underlying data:\n\n• data set menopause.xlsx\n\n• menapause study questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSantoro N, Epperson CN, Mathews SB: Menopausal Symptoms and Their Management. ECL Endocrinology and Metabolism Clinics of North America. 2015; 44(3): 497–515. English. PubMed Abstract \| Publisher Full Text\n\nSantoro N, Roeca C, Peters BA, et al.: The Menopause Transition: Signs, Symptoms, and Management Options. Journal of Clinical Endocrinology and Metabolism. 2020; 106(1): 1–15.\n\nBroker SPPBS: Study of menopausal symptoms, and perceptions about menopause among women at a rural community in Kerala. Journal of Mid-life Health. 2013; 4(3): 182–7. No Linguistic Content. Publisher Full Text PubMed Abstract \|\n\nXu X, Jones M, Mishra GD: Age at natural menopause and development of chronic conditions and multimorbidity: Results from an Australian prospective cohort. Human Reproduction. 2020; 35(1): 203–211. English. PubMed Abstract \| Publisher Full Text\n\nPeacock K, Ketvertis KM, Doerr C: Menopause (Nursing). StatPearls. Treasure Island (FL):StatPearls Publishing;2021. Copyright © 2021, StatPearls Publishing LLC.\n\nThe WG: The World Health Organization quality of life assessment (WHOQOL): Development and general psychometric properties. Social Science & Medicine. 1998; 46(12): 1569–1585. English. Publisher Full Text\n\nPost MW: Definitions of quality of life: what has happened and how to move on. Topics in Spinal Cord Injury Rehabilitation. 2014 Summer; 20(3): 167–180. Epub 2014/12/09. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarmakar N, Majumdar S, Dasgupta A, et al.: Quality of life among menopausal women: A community-based study in a rural area of West Bengal. Journal of Mid-life Health. 2017 Jan-Mar; 8(1): 21–27. Epub 2017/05/02. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharifi K, Tagharobi Z, Sooki Z: Quality of Life among Iranian Postmenopausal Women: A Systematic Review and Meta-Analysis. Galen Medical Journal. 2020; 9: 1649. Epub 2020/03/04. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim ZM, Ghoneim HM, Madny EH, et al.: The effect of menopausal symptoms on the quality of life among postmenopausal Egyptian women. Climacteric. 2020 Feb; 23(1): 9–16. eng. PubMed Abstract \| Publisher Full Text\n\nNazarpour S, Simbar M, Ramezani Tehrani F, et al.: Factors associated with quality of life of postmenopausal women living in Iran. BMC Womens Health. 2020 2020/05/14; 20(1): 104. PubMed Abstract \| Publisher Full Text\n\nIbrahim ZM, Ghoneim HM, Madny EH, et al.: The effect of menopausal symptoms on the quality of life among postmenopausal Egyptian women. Climacteric Climacteric. 2020; 23(1): 9–16. English. PubMed Abstract \| Publisher Full Text\n\nDotlic J, Pimenta F, Kovacevic N, et al.: Menopausal transition in Southern Europe: comparative study of women in Serbia and Portugal. Menopause. 2017 Nov; 24(11): 1236–1245. Epub 2017/06/14. eng. PubMed Abstract \| Publisher Full Text\n\nDotlic J, Kurtagic I, Nurkovic S, et al.: Factors associated with general and health-related quality of life in menopausal transition among women from Serbia. Women Health. 2018 Mar; 58(3): 278–296. Epub 2017/03/17. eng. PubMed Abstract \| Publisher Full Text\n\nBarati M, Akbari-Heidari H, Samadi-Yaghin E, et al.: The factors associated with the quality of life among postmenopausal women. BMC Womens Health. 2021; 21(1): 208. English. PubMed Abstract \| Publisher Full Text\n\nAdhikari BBR: Quality of life among menopausal women in an urban area of Siliguri, West Bengal, India. International Journal of Community Medicine And Public Health. 2019; 6(11): 4964. No Linguistic Content. Publisher Full Text\n\nSmail LSAJG: Menopause-specific quality of life among emirati women. International Journal of Environmental Research and Public Health. 2020; 17(1). eng. Publisher Full Text\n\nBustami M, Matalka KZ, Elyyan Y, et al.: Age of Natural Menopause Among Jordanian Women and Factors Related to Premature and Early Menopause. Risk Management and Healthcare Policy. 2021; 14: 199–207. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDOS: Jordan in figures 2019, Department of Statistics-Jordan.2019.\n\nUtian WH, Janata JW, Kingsberg SA, et al.: The Utian Quality of Life (UQOL) Scale: development and validation of an instrument to quantify quality of life through and beyond menopause. Menopause. 2018; 25(11): 1224–1231. eng. PubMed Abstract \| Publisher Full Text\n\nMenon B, Cherkil S, Ag U: The process and challenges in the translation of World Health Organization Quality of Life (WHOQOL-BREF) to a regional language; Malayalam. Indian Journal of Psychological Medicine. 2012 Apr; 34(2): 149–152. PubMed Abstract \| Publisher Full Text\n\nVercambre MN, Okereke OI, Kawachi I, et al.: Self-Reported Change in Quality of Life with Retirement and Later Cognitive Decline: Prospective Data from the Nurses' Health Study. Journal of Alzheimer's Disease: JAD. 2016; 52(3): 887–898. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGharaibeh M, Al-Obeisat S, Hattab J: Severity of menopausal symptoms of Jordanian women. Climacteric. 2010 Aug; 13(4): 385–394. Epub 2009/09/05. eng. PubMed Abstract \| Publisher Full Text\n\nAl-Qutob R: Menopause-associated problems: types and magnitude. A study in the Ain Al-Basha area, Jordan. Journal of Advanced Nursing. 2001 Mar; 33(5): 613–620. Epub 2001/04/12. eng. PubMed Abstract \| Publisher Full Text\n\nXu X, Jones M, Mishra GD: Age at natural menopause and development of chronic conditions and multimorbidity: results from an Australian prospective cohort. Hum. Reprod. 2020 Jan 1; 35(1): 203–211. eng. PubMed Abstract \| Publisher Full Text\n\nMegari K: Quality of life in chronic disease patients. Health Psychology Research. 2013; 1(3): 27. No Linguistic Content. Publisher Full Text\n\nJaber RM, Khalifeh SF, Bunni F, et al.: Patterns and severity of menopausal symptoms among Jordanian women. Journal of Women & Aging. 2017; 29(5): 428–436. eng. PubMed Abstract \| Publisher Full Text\n\nNiyonsenga G, Gishoma D, Sego R, et al.: Knowledge, utilization and barriers of cervical cancer screening among women attending selected district hospitals in Kigali - Rwanda. Canadian Oncology Nursing Journal = Revue Canadienne de Nursing Oncologique. 2021 Summer; 31(3): 266–274. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhaher EA: Knowledge, Attitudes and Practices of Women in the Southern Region of Saudi Arabia Regarding Cervical Cancer and the Pap Smear Test. Asian Pacific Journal of Cancer Prevention: APJCP. 2019 Apr 29; 20(4): 1177–1184. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwok C, Tranberg R, Lee FC: Breast cancer knowledge, attitudes and screening behaviors among Indian-Australian women. European Journal of Oncology Nursing: The Official Journal of European Oncology Nursing Society. 2015 Dec; 19(6): 701–706. eng. PubMed Abstract \| Publisher Full Text\n\nKısa S, Zeyneloğlu S, Ozdemir N: Examination of midlife women's attitudes toward menopause in Turkey. Nursing & Health Sciences. 2012 Jun; 14(2): 148–155. eng. PubMed Abstract \| Publisher Full Text\n\nBarati M, Akbari-Heidari H, Samadi-Yaghin E, et al.: The factors associated with the quality of life among postmenopausal women. BMC Womens Health. 2021 May 18; 21(1): 208. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPertyńska-Marczewska MPT: Postmenopausal women in gynecological care. pm Menopausal Review. 2021; 20(2): 88–98. No Linguistic Content. Publisher Full Text PubMed Abstract \|\n\nMir Haghjou SNMMPSC: Quality of life and its determinants in postmenopausal women: a population-based study. PNR Applied Nursing Research. 2016; 30: 252–256. eng. PubMed Abstract \| Publisher Full Text\n\nGhorbani R, Nassaji M, Shahbazi A, et al.: Association between quality of life, menopausal status, and sociodemographic factors among middle-aged women in Iran. Journal of the Egyptian Public Health Association. 2015; 90(4): 166–170. PubMed Abstract \| Publisher Full Text\n\nNamazi M, Sadeghi R, Behboodi MZ: Social Determinants of Health in Menopause: An Integrative Review. International Journal of Women's Health. 2019; 11: 637–647. eng. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang Z, Shi J, Liu W, et al.: The influence of educational level in peri-menopause syndrome and quality of life among Chinese women. Gynecological Endocrinology: The Official Journal of the International Society of Gynecological Endocrinology. 2020 Nov; 36(11): 991–996. eng. PubMed Abstract \| Publisher Full Text\n\nLan Y, Huang Y, Song Y, et al.: Prevalence, severity, and associated factors of menopausal symptoms in middle-aged Chinese women: a community-based cross-sectional study in southeast China. Menopause. 2017 Oct; 24(10): 1200–1207. eng. PubMed Abstract \| Publisher Full Text\n\nBanaczek Z, Saracen A: Life satisfaction and self-esteem among women in the menopausal time. Wiadomości lekarskie (Warsaw, Poland: 1960). 2016; 69(2 Pt 2): 174–179. pol. PubMed Abstract\n\nYoshany NMMSSBNMKHF: Association between Lifestyle and Severity of Menopausal Symptoms in Postmenopausal Women. Electronic Journal of General Medicine. 2020; 17(5): em222. No Linguistic Content. Publisher Full Text\n\nAssaf EA, Gharaibeh M: Data Set Menopause Women. figshare. Dataset.2022. Publisher Full Text" }	[ { "id": "191964", "date": "07 Sep 2023", "name": "Camille Cronin", "expertise": [ "Reviewer Expertise Women's 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\nA cross-sectional study of 200 Jordanian women aged 45-60 years using a QOL tool to assess quality of life in menopausal women.\n\nA range of literature is used and some of which is dated, some may correspond to the tools used but other literature should have been more up to date and more international. Menopause is a global issue.\nThe study design look appropriate but I imagine that with the world count the author has had to make a decision to cut certain sections out and this I feel has affected the presentation of tool, and what stats have been chosen to present. Perhaps the author(s) need to be more specific in what they what tp present with corresponding literature to support.\n\nI am not a statistician and unable to comments on the other points.\n\nI do feel this is a contemporary issue to be reporting and this paper is looking at working women and those moving to retirement. I feel again this is not reflective globally as the age of working women is growing and working past the age of 60. In Jordan this may be different but this needs to be contextualised - what is the average retirement age and so on.\nI think the discussion session is short and does not correspond with the amount tot data - perhaps the author has been limited by the word count. Limitations have been reported which is also good.\nI do feel the paper is limited in the literature which I mentioned above.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10978", "date": "22 Mar 2024", "name": "Enas Assaf", "role": "Author Response", "response": "Comments response A range of literature is used and some of which is dated, some may correspond to the tools used but other literature should have been more up to date and more international. Menopause is a global issue. Thank you for your comments, the following literature is added in the introduction : The introduction section has been revised and updated literature was added as follows: This was added in the introduction “Menopausal age is also associated with many psychological changes such as memory loss, lack of self-confidence and issues with body image” and the reference added Camille Cronin, Catherine Hungerford & Rhonda Lynne Wilson (2021) Using Digital Health Technologies to Manage the Psychosocial Symptoms of Menopause in the Workplace: A Narrative Literature Review, Issues in Mental Health Nursing, 42:6, 541-548, DOI: 10.1080/01612840.2020.1827101 . (ref. no…..) This was added in the introduction “Moreover, women at this stage would be at the age of highly skilled and work role model, despite the difficulties and challenges of the menopausal symptoms” reference: House of Commons Women and Equalities Committee, Menopause and the workplace, First Report of Session 2022–23. Available at chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.elaweb.org.uk/sites/default/files/docs/HC%2091%20-%20Menopause%20and%20the%20workplace%20-%20EMBARGO.pdf. acceded on September 12, 2023. Another recent systemic review study ref was cited as no ( ) related to menopausal working women and QoL. S. Theis, S. J. Baumgartner, H. Janka, A. Kolokythas, C. Skala & P. Stute (2023) Quality of life in menopausal women in the workplace – a systematic review, Climacteric, 26:2, 80-87, DOI: 10.1080/13697137.2022.2158729. The following stidies were added in the introduction numbered as (….. ) Krishnapriya, P. B., et al. \"Assess the quality of life among postmenopausal women.\" International Journal of Advances in Nursing Management (2023). Studies in Sauid Arabia ( ref no) Aljarudi, S. , Al-Jumah, H. and Al-Jumah, A. (2023) Evaluating the around and after Menopausal Symptoms and Personal Perception of Quality of Life among Women in Qatif, Saudi Arabia, Attending the Hospital and PHCs. Open Journal of Obstetrics and Gynecology, 13, 280-290. doi: 10.4236/ojog.2023.132029. We added the following sentence highlighted in red as “On the other hand menopausal symptoms shows to have adverse effect on work as what was found in UK study” line Ref Faubion, S. S., Enders, F., Hedges, M. S., Chaudhry, R., Kling, J. M., Shufelt, C. L., ... & Kapoor, E. (2023, June). Impact of menopause symptoms on women in the workplace. In Mayo Clinic Proceedings (Vol. 98, No. 6, pp. 833-845). Elsevier. The study design look appropriate but I imagine that with the world count the author has had to make a decision to cut certain sections out and this I feel has affected the presentation of tool, and what stats have been chosen to present. Perhaps the author(s) need to be more specific in what they what tp present with corresponding literature to support Really appreciated, the cutting will impact the accuracy of the paper I am not a statistician and unable to comments on the other points. Well respect I do feel this is a contemporary issue to be reporting and this paper is looking at working women and those moving to retirement. I feel again this is not reflective globally as the age of working women is growing and working past the age of 60. In Jordan this may be different but this needs to be contextualised - what is the average retirement age and so on. Thank you for the note, this comment was well considered by adding the following in the introduction under the Jordan section In Jordan life expectancy at birth had increased for females to 78.6 in 2020 insert here(worldometer, 2021). However the retirement age for women in Jordan is 55 for women where women can ask for early retirement at early age between 50 and 52 years old and can still benefit from the governmental pension insert ref here (BAKER, 2018). Ref: to be added in the article BAKER, N. (2018). What are the retirement ages around the world? https://www.sbs.com.au/news/what-are-the-retirement-ages-around-the-world#:~:text=Retirement%20ages%2C%20or%20the%20age%20when%20some%20sort,years%20for%20men%20and%2063.7%20years%20for%20women. worldometer. (2021). world lifeexpectancy at birth https://www.worldometers.info/demographics/life-expectancy/#countries-ranked-by-life-expectancy I think the discussion session is short and does not correspond with the amount tot data - perhaps the author has been limited by the word count. Thank you for the note, we actually considered discussing the main points and results looking to the considered limitation Limitations have been reported which is also good. Thank you I do feel the paper is limited in the literature which I mentioned above. Thank you considered and updated introduction section Are all the source data underlying the results available to ensure full reproducibility? Data sources are available in the reference section highlighted" } ] }, { "id": "216514", "date": "21 Nov 2023", "name": "Harmeet Kaur Kang", "expertise": [ "Reviewer Expertise Women Health", "Cardiovascular disease 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\nGeneral Comments: The topic of the paper is deemed important and relevant, focusing on Menopausal Quality of Life (QOL). However, there are notable concerns regarding grammar, sentence formation, and the overall association of Menopausal QOL with menopausal symptoms. The paper lacks an exploration of this critical relationship, which is a significant gap in the study.\nSpecific Comments: Menopausal QOL and Symptoms: The primary outcome variable, Menopausal Quality of Life, is highlighted, but the authors have not adequately addressed the association between Menopausal QOL and menopausal symptoms. This gap diminishes the overall impact of the study, and it is recommended that the authors explore and discuss this relationship in more detail.\nTools and Content Validity: The initial sections of the tools were reportedly self-prepared by the authors; however, there is a lack of information regarding the content validity of these tools. It is essential to provide evidence of the validity of the tools used in the study to ensure the reliability of the results.\nStatistical Tests: A thorough review by a statistician is recommended, particularly for certain tables that appear to lack coherence. The application of statistical tests should be carefully examined to ensure accuracy and relevance. Any discrepancies or inconsistencies in the tables need to be addressed and corrected.\nStrengthening the Discussion Section: The discussion section could be strengthened by providing a more comprehensive analysis of the global perspective on menopausal quality of life. The authors have mainly focused on the Jordanian perspective, neglecting a broader view that could enhance the generalizability and applicability of the study findings.\nGrammar and Sentence Formation: The manuscript requires careful proofreading for grammar and sentence formation. Ensuring clarity in expression and adherence to grammatical rules will significantly enhance the readability of the paper.\nIn conclusion, addressing these concerns will contribute to the overall improvement of the paper. Emphasizing the association between Menopausal QOL and menopausal symptoms, validating the tools used, reviewing statistical tests, broadening the global perspective, and enhancing the clarity of language will collectively elevate the quality of the research article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10977", "date": "22 Mar 2024", "name": "Enas Assaf", "role": "Author Response", "response": "Comments from the reviewer Response General Comments: The topic of the paper is deemed important and relevant, focusing on Menopausal Quality of Life (QOL). However, there are notable concerns regarding grammar, sentence formation, and the overall association of Menopausal QOL with menopausal symptoms. The paper lacks an exploration of this critical relationship, which is a significant gap in the study Thank you very much for your review and your comments. Regarding the menopausal symptoms and its association, this was part of the study but since the main goal was studying the difference between retired and working menopausal women, symptoms show no significance. Therefore, we did not elaborate on that and did not focus on the symptoms rather than the overall quality domains, which would in itself, reflect the symptoms in each of the quality of life studied domain. English editing was considered and checked. Thank you . Tools and Content Validity: The initial sections of the tools were reportedly self-prepared by the authors; however, there is a lack of information regarding the content validity of these tools. It is essential to provide evidence of the validity of the tools used in the study to ensure the reliability of the results. Thank you , content validity was checked in the tool and highlighted in the paper with yellow Statistical Tests: A thorough review by a statistician is recommended, particularly for certain tables that appear to lack coherence. The application of statistical tests should be carefully examined to ensure accuracy and relevance. Any discrepancies or inconsistencies in the tables need to be addressed and corrected All the statistics were carried out by Expert in statistics Strengthening the Discussion Section: The discussion section could be strengthened by providing a more comprehensive analysis of the global perspective on menopausal quality of life. The authors have mainly focused on the Jordanian perspective, neglecting a broader view that could enhance the generalizability and applicability of the study findings. Thank you for your comments, in the discussion each point were analyzed based on both Jordanian and supported by global perspectives taking several international studies from different culture, among which some were highlighted in the text." } ] }, { "id": "223920", "date": "27 May 2024", "name": "Zalikha Al-Marzouqi", "expertise": [ "Reviewer Expertise Maternal 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\nTitle: I think the title is good and reflecting of the study.\nAbstract: Good, just in 'Method' add Cronbach's alpha that you did for your tool in arabic version. Also in 'Results', authors can add some brief information about the findings related to factors associated with overall wellbeing.\nKeywords: Good.\n\nIntroduction: the authors wrote a clear introduction and tough recent studies in literature review. The authors also identified the knowledge gap and the significance of the study.\n\nMethods: the selected design is matching the aim and objective of the study and it is appropriate. Inclusion and exclusion criteria identified, which is good. The tool used was explained in detail. However, the authors need to slightly revise the language and grammar to make it more readable.\n\nDependent variable, the authors should make sure that they mention dependent variables of this study.\nIndependent variables, the authors should make sure that they mention independent variables of this study.\nTool and its validity: Clear and good.\n\nData analysis: good and clear, just to add 2 sentences about data management / control.\n\nResults: regarding the descriptive statistics, it is good and clear. However, I am not a statistician to understand other type of statistics. Thus I can not provide comments.\nDiscussion: good and clear, it covered different angles of the results.\n\nConclusion: no comments, it supports the results of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/11-1189
https://f1000research.com/articles/12-880/v3	04 Aug 23	{ "type": "Review", "title": "Weed science research achievements on maize in Ethiopia: a review", "authors": [ "Takele Negewo", "Nagassa Dechassa", "Amare Fufa", "Tigist Bidira", "Nagassa Dechassa", "Amare Fufa", "Tigist Bidira" ], "abstract": "Recent research into assessing and combatting maize weeds was reviewed based on scientific and working reports from the year 2011 onwards in Ethiopia. This review paper covered two main topics: assessment of major weed types and their impacts, and research results of weed management in the maize cropping system of the country. More than 80 major weed floras dominated by broad leaved weeds were found infesting maize fields in different localities. By competing, toxifying and/or parasitizing maize plants, they reduce the crop germination, growth and productivity. Several experiments indicated that maize yield loss due to uncontrolled weed growth varied from 35 to 87.5%, indicating that weed management is a critical factor for maize production in the country. Hoeing, hand weeding and pre-emergence herbicides application have been the most frequently used weed control practices to minimize the competition effect of weeds. So far, there has been no effective and sustainable individual weed control method for maize fields. Thus, future research should focus on developing integrated weed management that effectively reduce weed infestation and associated impacts, and improve productivity of the crop considering socio-economic condition of a specific maize producing agro-ecology of the country.", "keywords": [ "Ethiopia", "maize", "management", "research achievements", "weed" ], "content": "Introduction\n\nMaize (Zea mays L.) is an important cereal crop in Ethiopia because of its high productivity per unit area, suitability to major agro-ecologies and compatibility with many cropping systems. It is one of the high priority crops mainly cultivated during the rainy season for food, fodder and industrial raw material in the country. According to the Central Statistical Agency of Ethiopia’s (2019) report, out of the total grain crop area (12.7 million hectares), maize took up 18.60% (2.4 M ha) standing next to tef (Eragrostis tef) with 24.17% (3.1 M ha) of occupation. According to the report, the production of maize contributed 30.08% (950,000 tones) of the grain production with a mean productivity of 4 t·ha-1 in the country, which is by far less than its potential yield.\n\nMajor constraints to maize production in Ethiopia include both abiotic and biotic factors. The abiotic factor includes drought, nutrient deficiency, inadequate crop husbandry such as limited land preparation, planting dates and population densities adjustment, fertility management, and harvesting machinery. While the biotic factor includes low yielding varieties, weeds, diseases and insects. Among the biotic stresses, regular and noxious parasitic weeds (Striga species) are the most important limiting factors for maize production. They restrict the crop’s yield by competing for nutrient, water, light and space, may vastly diminish its yield and cause complete failure of the plant due to parasitism and/or allelopathy. There are also indirect effects of weeds infestation on maize fields such as a reduction in land value, creation of inconvenience to agricultural operations and an increase in production costs. Furthermore, maize weeds act as hosts for plant pathogens and harbor insects; thus, aggravating the crop yield loss.\n\nIt has been observed that if no good control is carried out, weeds may cause a 35–87.5% reduction in maize yield (Getahun et al., 2018; Muluadam and Demisew, 2021). Hence, weeds are critical factor in maize production system of Ethiopia, as a result their management is given the top priority compared to other maize pests. Hoeing, hand weeding and/or pre-emergence herbicides technology have been found the most frequently used weed control practices to minimize the competition effect of weeds in maize fields in the country.\n\nDecade-based reports on the status of weed management research in maize in Ethiopia have been made starting from 1980’s; Rezene et al. (1993), Kassa et al. (2002), and Temesgen et al. (2012). Efforts have been made to identify and characterize major weed species, assess crop losses due to the weeds interference, and identify effective management methods to reduce their impact. The objectives of this paper are to review the last decade (2011-2022) maize weeds research achievements, indicate research gaps and suggest future research directions.\n\n\nMajor weeds and their impacts on maize production system\n\nIn maize cropping systems, previous different farm management practices such as crop rotation, fertilizer application, herbicide usage and ploughing frequency are major factors affecting weed species types, distribution and infestation. On the other hand, weed flora are influenced by the current crop population density, sowing geometry, growth duration, growth rhythm, moisture period and fertility status (Ali et al., 2016; Muluadam and Demisew, 2021). The moist and/or hot climate of the maize-growing belts of Ethiopia encourage rapid and abundant growth of weeds, consequently the crop fields are usually heavily infested with the weed plants. Various weed species considerably impede maize production, but their importance varies across maize producing agro-ecologies in the country.\n\nAround 88 major weed floras under 23 plant families have been reported in maize across various localities (Table 1). Aboveground weed flora of 62 species in Abobo district of Gambella (Dinberu et al., 2011), 21 species in Bako (Megersa and Fufa, 2017), 15 species in Jimma (JARC, 2021) and six dominant weed species in north western Ethiopia (Takele et al., 2022) were recorded. According to the findings, majority of the encountered maize associated weed species were annual (71) as compared to perennial (17). Similarly, broad leaved weeds (66) outnumbered grass (18), sedge (2) and parasitic (2) weeds in the crop fields.\n\nWeeds infestation immensely affects maize production and productivity in Ethiopia. Weeds force farmers to spend their effort (time and energy) on managing them; otherwise, they cause a considerable loss and this sadly is a common feature observed in sorghum and maize growing areas of the country (Muluadam and Demisew, 2021).\n\nPrevious studies showed that weeds reduced maize yields by 35–87.5% depending on the crop cultivar types, density and sowing geometry; weed types, density and duration of competition; moisture period, fertility status, and tillage practice (Ali et al., 2016; Muluadam and Demisew, 2021). Weeds interference on maize production system could be in different forms such as competition, allelopathy and/or parasitism.\n\nCompetition\n\nEthiopia, as one of the tropical arid and semi-arid countries confronts constraints due to a high temperature, erratic rainfall pattern, high rate of evapotranspiration, and fast and vigorous growth of weeds in crops (Lulseged et al., 2015; Muluadam and Demisew, 2021). The rapid and vigorously growing weeds aggressively compete with crop plants for scarce soil resources like moisture and organic matter, and sunlight. In maize, weeds compete for growth factors and the crop yield losses due to competition have been estimated at different localities. Keeping maize weed-free throughout the cropping season is preferred to avoid the possible impacts, but considering the management cost it needs to determine the critical period of weed competition. A study by Muluadam and Demisew (2021) showed that maize and sorghum crops are highly sensitive to weed competition, especially during their early growth stage.\n\nWeed rating inversely impacted yield (b = −0.5; p < 0.001) where fields with the lowest weed rating had the highest yield (4.6 t·ha-1) than those with the highest rating (2.3 t·ha-1) (Lulseged et al., 2015). Accordingly, critical weed-free periods of 25–30 days after sowing (DAS) and optimum weed control periods of 30–50 days after emergence (DAE) have been developed and recommended for major weeds of maize, respectively. The critical timing of parthenium removal to avoid 5% and 10% maize grain yield loss was 8 and 17, and 13 and 23 DAE during year 2012 and 2013, respectively (Safdar et al., 2016). The relatively shorter critical period of parthenium competition in maize crop suggested its high allelopathic effect in addition to its competitive behavior.\n\nAllelopathy\n\nAllelochemicals such as phenolics, alkaloids, fatty acids, indoles, terpens and others liberate from weeds through leaf leachates, decomposition of plant residues, volatilization and root exudates that affect crop productivity (Ali et al., 2016). Many studies have revealed the allelopathic effect of different weed species on maize seed germination, stand establishment, growth, yield and the physiology of the crop plants (Table 2). Allelopathic water extracts of Plantago lanceolata, Anagallis arvensis, Medicago polymorpha, Phragmites australis, Ammi visnaga, Silybum marianum, Malcolmia africana, Emex spinosa, Calendula arvensis, Fumaria indica, Convolvulus arvensis and Rumex crispus showed toxicity against maize, sunflower and wheat germination (Khan et al., 2012). Aqueous extracts of all plant parts (shoot, leaf, inflorescence, root and whole plant part) of Parthenium hysterophorus inhibited the germination and growth of maize, demonstrating the allelopathic potential of the weed plant in the crop field (Anteneh, 2015). Residues of leaves, stems, roots and whole plants of Polygonum hydropiper, Amaranthus spinosus, Cyperus esculentus, Cyperus rotundus and Imperata cylindrical, and their mixtures imposed an inhibitory effect on the emergence of maize seedlings (Ali et al., 2016).\n\nWhen added to soil at 5% and 10% concentrations, the inhibition of Lantana camara, Ageratum conyzoides and Eupatorium adenophorum residues on the emergence and shoot growth of wheat, rice and maize were reported by Ali et al. (2016). Eucalyptus (Eucalyptus camaldulensis) leaf extracts (20–100% of a 50 g/L solution) in laboratory and powders (0.5–3% w/w base) in greenhouse were found to have allelopathic effects on seed germination and seedling growth of the tested poaceous crops including maize (Awadallah and Eman, 2017). Similarly, pigweed (Amaranthus viridis) above-ground parts have allelopathic effects on seed germination and seedling growth of the tested poaceous crops including maize (Awadallah et al., 2017). According to Abiyu and Nagappan (2015); and Wakshum et al. (2018), Lantana camara leaf powder significantly inhibited seed germination, speed of germination, shoot and root length, stem thickness and biomass of wheat and maize. Moreover, Calotropis procera (sodom apple) leaves have allelopathic effects on seed germination and seedling growth of the tested poaceous crops including maize (Awadallah and Salwa, 2019).\n\nAllelopathic potential has been also found in many crops like maize, rape seed, niger seed, sunflower, alfalfa, sorghum and wheat, and extracts of these crops contain allelochemicals that release from dead and decaying parts of the plant body (MuahmmadWaheed et al., 2013). If wisely planned, the application of allelopathy in cropping systems is quite effective in managing agricultural pests and improving the productivity of agricultural systems. The extract of maize root residues has been found effective as bio-herbicide in the control of Bidens pilosa (Modupe and Joshua, 2013). Several research findings have found that maize plant produces a number of secondary metabolites that inhibit weed plant growth at high concentrations. Having an allelopathic effect, the incorporation of E. globulus residues as green manure into soil could be a feasible practice to reduce the reliance on synthetic herbicides in maize-based cropping systems (Carolina et al., 2013).\n\nParasitism\n\nNoxious root parasitic weeds like Striga spp. (witch-weeds) are widely spread in semi-arid regions of Africa including Ethiopia. The Striga hermonthica and S. aspera in northwestern and S. asiatica in the highlands of the eastern and southern parts of the country are threatening maize production. They are resulting in a complete crop loss under the worst conditions. According to Nagassa and Belay (2021), Striga spp. cause growth inhibition and yield losses of 20–100% in maize, sorghum, rice, pearl millet, finger millet and sugar cane. Prodigious seed production, prolonged viability of the seeds in soil and the subterranean nature of the early stages of parasitism in the host crops field make the control of the parasite difficult by conventional methods (Berhane, 2016).\n\nIn general, the occurrence of different weed species types and their infestation level vary from field to field mainly based on past farming history on a given field. The heavy infestation of weeds undoubtedly has considerable impacts on the germination, growth, production and productivity of crop plant. The different forms of weed interference on the crop plant indicate that weed management is a critical factor for maize production. Hoeing, hand weeding and pre-emergence herbicides application are the most frequently used weed control practices to minimize effect of weeds in maize fields of the country. However, so far none of them has been found effective and sustainable in controlling weeds in maize fields.\n\nWeeds are always present in the farming system and often the most significant pest of maize in Ethiopia. The level of weed pressure is important to the profitability of the crop production. The carryover effects of proper management practices might be a means of reducing the early weed load and helping to reduce the crop production costs. Adequate weed management knowledge and technology availability are crucial for proper reduction of the impacts of weeds on the crop production. Maize cropping practices such as rotation, proper land preparation, growing of improved varieties, intercropping and soil fertility management are determinant for the effectiveness of the crop production. According to Musa et al. (2014), the impact of technical inefficiency of maize farmers is as high as 66%. It needs to design research and extension packages based on the model farmers’ best practices.\n\nTesfaye et al. (2014) reported that proper weeds management in maize can increase the crop yield by up to 96%. Timely weeding makes the management effort effective than late weeding. It is labor constraint due to overlapping of activities that prevents timely weeding. When weeding delayed, costs of weeding increase and grain yield decline. Research is currently ongoing in the lowland tropics to determine the effect of reducing weed seed production by late weeding, zero tillage, and off-season management on weed pressure. Trials on the effect of farm practices such as crop rotations, intercropping, growing resistant or tolerant varieties, and maize-weed management are also being conducted (WSRS, 2015).\n\nMaize–haricot bean intercropping suppressed weeds, and was more productive and cost-effective than sole crop production in southern Ethiopia (Workayehu and Wortmann, 2011). Use of conservation agriculture-based maize–legume cropping systems was increased soil organic matter, total nitrogen, available phosphorus, and soil water infiltration rate by 37, 54, 12.3 and 327%, respectively, when compared to conventional tillage with maize mono-cropping over three years (Yayeh et al., 2022). By the conservation agriculture-based maize–haricot bean intercropping systems, weed infestation was reduced over the years and maize grain yield increased by 54% over the conventional tillage. Similarly, intercropping maize with forage legumes such as cowpea, silver-leaf and velvet bean has the potential to restore soil fertility and control weeds without reducing the crop grain yield (Marilyn et al., 2022).\n\nSowing an early emerging, fast-growing and competitive maize variety shall be made while cultivating the crop on weed-infested fields. Crop competition in maize may involve the use of competitive varieties that exhibit weed suppression potential. Weed-competitive varieties have a high leaf area index, and other leaf architecture elements that improve light interception by the crop, increasing the weeds shading capacity (Blessing et al., 2016). Another area that has been largely overlooked is the problem of parasitic weeds. Striga spp. inflict considerable losses on maize production, and no resistance has been found within the maize genome unlike for sorghum. Before releasing maize varieties in striga endemic areas, its genotypes must be screening for resistance and/or tolerance to the weed species.\n\nFarmer practices such as proper ploughing and inter-row cultivation are not given strong attention for weed management. The primary objectives of tillage are to prepare a smooth seedbed, alleviate soil compaction and reduce weeds. The clean seedbed is essential to reduce the weed seed bank in soil and reducing post-planting weed pressure. Lack of oxen energy for tillage, force farmers to practice usually minimum tillage that results in high grass weed infestation, thus allowing low labor for land preparation results with the high weed pressure. According to Bakhtiar et al. (2011), the harvested maize grain yield was considerably higher by conventional tillage (2429 kg·ha-1) than zero-tillage (2271 kg·ha-1). The authors also indicated that wheel-hoe weeder is found more effective than manual and oxen labor particularly on properly tilled light soils that the implement can control 85% of the maize weed species.\n\nUnlike many other pests, weeds can be controlled through hand weeding before they damage the crop if sufficient labor is available. Because of the limited resource base, the subsistence farming community relies on hand weeding for the control of weeds. As a result of the overlap of farm operations during the crop season; however, farmers either leave their farms un-weeded or perform weeding late in the season. According to Bakhtiar et al. (2011), a considerably higher grain yield of 2863 kg·ha-1 was recorded by practicing hand weeding compared to plastic or live plants mulching (2145 kg·ha-1) or weedy check (1422 kg·ha-1). However, besides weed pressure reduction, water conservation by mulching makes maize more resilient to the high rainfall variability occurring in some parts such as the Central Rift Valley of Ethiopia (Sime et al., 2015).\n\nThe planting time, seeding pattern, rate and spacing; and fertilizer application played a significant role on maize yield (Lulseged et al., 2015). Lately planted maize experiences greater weed problems, nitrogen deficiency and water deficits usually result in lower grain yields. Then the maize yield reduces markedly and progressively as the time of planting delayed after the onset of the rain. The early-sown crop plants benefit by receiving full-season rainfall, suffering less weed competition and a high soil nitrate level at the beginning of the rains which subsequently declines due to leaching and a slower rate of humus decomposition. The actual utilization of nitrogen by the crop is highly affected by the weedy conditions of the field under which urea is applied.\n\nCompetition in maize may involve techniques such as reduced row spacing and increased planting density that exhibit weed suppressive potential. The row-sowing of maize saves seeds and fertilizer, makes weeding and other agronomic practices easier, and then provides high crop yields. Moreover, increasing the maize plant population from 30,000 to 60,000 plants per hectare was increased the crop yield from 2055 to 2412 kg·ha-1 (Bakhtiar et al., 2011). The row planting of maize preconditions band-application of fertilizers that favors the crop growth more than inter-row weeds and also enhances productivity.\n\nIn Ethiopia, fertilizer application is a good input in improving the maize crop husbandry and farmers was mainly relied on commercial fertilizers to sustain livelihood security. Application of 150 kg·ha-1 NPS (blended nitrogen, phosphorus and sulfur fertilizers) at sowing and 250 kg·ha-1 urea splinted into two; half at knee height and the remained half before flowering stages are recommended for efficient weed control and maize growth improvement (Takele et al., 2022). Farmyard manure applications at the rate of 4 to 5 t·ha-1 also suggested to favor the crop plants more than weeds. Presently, the unaffordable price of inorganic fertilizers has become a burning issue in the fertilizer-use habituated crop production system of the country, indicating the importance of farmyard manure utilization.\n\nHerbicides are preferred over manual weeding when they alleviate labor tension and/or increase yield by controlling weeds better. The effectiveness of various combinations and rates of post and pre-emergence herbicides were compared for their effect on weeds and productivity of maize in Ethiopia. Weed control in maize with herbicides have been suggested by many researchers and research centers of the country (Firehywot et al., 2013; Tesfaye et al., 2014; Megersa and Fufa, 2017; AmARC, 2020; JARC, 2021). The herbicide evaluation woks were compared and confirmed the reliability and promising potential of some products in effectively controlling weeds and enhancing yield of maize (Table 3).\n\nFirehywot et al. (2013) found that twice hand weeding, atrazine or s-metolachlor application at 2 L·ha-1 resulted in higher weed control efficiency (> 94%) than other treatments in maize fields (Table 4). The highest maize grain yield (4664 kg·ha-1) was obtained with twice hand weeding and hoeing followed by atrazine 2 kg·ha-1 (4534 kg·ha-1) and s-metolachlor 2 kg·ha-1 (4490 kg·ha-1) application which were increased by 56, 55 and 54% over uninterrupted weed growth throughout the crop season. In addition, Megersa and Fufa (2017) reported that application of pre-emergence herbicides (Lunar 537.5 EC at 3 L·ha-1, Venus 500 SE at 6 L·ha-1 and s-metolachlor + atrazine at 3 L·ha-1 with 200 L·ha-1 water solution) considerably reduced weed infestation compared to weedy check plot there by boosted maize grain yield. JARC (2021) reported that pre-emergence herbicide Gesparim combi at a rate of 3.5 kg·ha-1 and Lasso + atrazine at 5 L·ha-1 supplemented with one hand weeding at 50–55 DAE kept the maize field weed-free throughout the season and gave the highest maize grain yield (5,593 kg·ha-1). In addition, the report indicated that s-metolachlor+ atrazine at 4 L·ha-1 and alazine 35/20 SE at 5 L·ha-1 were found effective in managing weeds in maize fields of the area.\n\nIn maize fields, integrated management strategy aimed at combining the economically feasible, environmentally safe and socially acceptable weed control options (Takele et al., 2022). This could be the incorporation of two or more weed control methods such as prevention, cultural, biological and/or chemical control to reduce their interference while maintaining acceptable yields of the crop. If various weed management components are integrated in a systematic manner, significant advances in weed management technology can be achieved.\n\nGebisa and Gressel (2007) reported that a combination that included resistant varieties, fertilizer and tied-ridge gave significantly higher sorghum yield followed by one that combined local varieties with fertilizer and tied-ridge in north Wollo. In semiarid areas of the Central Rift Valley, conservation agriculture by covering soil with crop residues reduced soil water evaporation, erosion as well as degradation; water run-off and weed problems, and then improved soil fertility, water infiltration, soil biological activity, soil water availability and maize-bean productivity (Liben et al., 2017).\n\nSimilarly, glyphosates such as herbazed, round-up 36 Sl and weedknock at 2 L·ha-1 rate mixed in 200 L·ha-1 water on 15–18 days before planting application supplemented by hand weeding on 40 DAS provided efficient weed control and then considerably boosted maize grain yield at Bako (Megersa et al., 2018). Twice-hand weeding and a plant density of 53,333 plants per hectare were found to be suitable practices for attaining optimum grain yield for the hybrid maize BH-546 in Assosa district (Getahun et al., 2018).\n\nFurthermore, application of s-metolachlor + atrazine at 2 L·ha-1 in combination with 65 cm inter-rows spacing (IRS) found as the best option for effective weed management in maize (Megersa et al., 2020). The integration of narrower IRS with hand pooling plus hoeing improved weed control efficiency there by enhanced maize grain yield (Table 5).\n\nThe increasing incidence of striga has been attributed to poor soil fertility, low soil moisture and the intensification of land use through continuous cereal cultivation. Thus, potentially successful approaches developed to control this weed include rotation with trap crops or legumes, applying pre-emergence herbicides, growing resistant or tolerant varieties, sowing clean seeds, intercropping with legumes, soil amendments with fertilizer or manure, water conservation, use of push-pull technology and effective microbes (Fusarium oxysporum and arbuscular mycorrhizal fungi) as biological control agent (Nagassa and Belay, 2021). Hence, to successfully reduce infestation pressure and the impact of the parasitic weed, an integrated management strategy needs to be developed considering the ecological and socio-economic conditions of the major maize growing areas of the country.\n\n\nConclusions\n\nThe potential yield of maize varieties is very interesting in Ethiopia. There is a large gap between the potential and actual yields of the crop. Weeds are one of the major biotic factors contributing to the low maize yield in the country. Effective management is substantive in reducing the broad interference of various weed species as well as their negative impact on the crop productivity. Few individual weed control methods identified as part of a package for the improved varieties. Strengthened research activity is needed to ensure that maize yield losses from stresses are minimized through sustainable and strong weed management technology against maize weeds; regular, parasitic and/or allelopathic weeds. Current weed science research information reviewed in this paper is very rare despite the expansion of maize production areas and associated weed infestation problems. Closing the technology gap requires the diffusion of advanced weed management technology against maize weeds. There is a pressing need particularly by smallholder farmers to adopt integrated weed management strategies at different agro-ecologies of the country.\n\nWeed species distribution and interference is dynamic causing consistent maize yield reduction in Ethiopia. However, there is no strong weed science research activity, and then latest information, knowledge and technology that can assist to adequately manage maize weeds in the country. Parasitic and allelopathic effect of weeds are often overlooked, yet they are likely important as potential stress on maize plant physiological functions and productivity. So far there are few individual weed control methods that ineffectively adopted by maize growers, and there is luck of dependable integrated weed management strategy.\n\nThe dynamic nature of weed species’ demands intensive weed science research endeavor in Ethiopia. In the absence of the latest information, knowledge and technology on appropriate weed management measures, it is hardly possible to maximize the grain yield of maize. It could be good to frame future avenues of research that could help to move studies of competition, parasitism and allelopathy into the brooder ecological context. In general, the weed management research at representative major maize growing locations shall be strengthened within the context of developing an integrated weed management approach that considers the social, economic and agro-ecological conditions of a specific locality. Moreover, it needs to upgrade the knowledge and capacity of smallholder farmers by promoting improved weed management technologies.", "appendix": "Data availability\n\nAll data supporting the results are included in the article and no extra source data are necessary.\n\n\nReferences\n\nAhmed MH, Lemma Z, Endrias G: Technical Efficiency of Maize Producing Farmers in Arsi Negelle, Central Rift Valley of Ethiopia: Stochastic Frontier Approach. Agriculture and Forestry. 2014; 60(1): 157–167.\n\nAli Z, Tasawer A, Tahira T: Weeds Cause Losses in Field Crops through Allelopathy. Notulae Scientia Biologicae. 2016; 8(1): 47–56. Publisher Full Text\n\nAmARC (Ambo Agricultural Research Center): Report on completed herbicides verification tests on maize during 2019 to 2020 at Ambo areas.2020; 5.\n\nAwadallah BD, Amna OA, Muna HY: Allelopathic effects of pigweed (Amaranthus viridis L.) on seed germination and seedling growth of some poaceous crops. 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Ethiopia Journal of Weed Management. 2013; 6: 73–84.\n\nSime G, Aune JB, Mohammed H: Agronomic and economic response of tillage and water conservation management in maize, central rift valley in Ethiopia. Soil and Tillage Research. 2015; 148: 20–30. Publisher Full Text\n\nTesfaye A, Amin M, Mulugeta N: Management of Weeds in Maize (Zea mays L.) through Various Pre and Post Emergency Herbicides. Advances in Crop Science and Technology. 2014; 02: 151. Publisher Full Text\n\nWorkayehu T, Wortmann CS: Maize–Bean Intercrop Weed Suppression and Profitability in Southern Ethiopia.2011. Publisher Full Text\n\nWSRS (Weed Science Research Strategy of Ethiopia): A 15 years (2016-2030) plan document developed on weed science research strategy in Ethiopia. Addis Ababa: Ethiopian Institute of Agricultural Research (EIAR); 2015.\n\nYihun K, Debele T, Abera T, et al.: Review of Weed Research in Maize in Ethiopia.Mandefro N, Tanner D, Twumasi-Afriyie S, editors. Enhancing the Contribution of Maize to Food Security in Ethiopia: Proceedings of the Second National Maize Workshop of Ethiopia, 12-16 November 2001 Addis Ababa, Ethiopia: EARO and CIMMYT; 2002; pp. 106–116." }	[ { "id": "220166", "date": "06 Nov 2023", "name": "Abdellatif Boutagayout", "expertise": [ "Reviewer Expertise Agroecological Weed Management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 entitled \"Weed Science Research Achievements on Maize in Ethiopia: A Review\" authored by Negewo et al., offers a comprehensive assessment of weed management in maize production in Ethiopia. The authors emphasized the significance of maize in Ethiopian agriculture, highlighted the primary weed species affecting maize crops, and explored the consequences of these weed infestations on maize production. Furthermore, they delved into various weed management practices and evaluated their effectiveness in controlling weed proliferation. This study provides informative insights into the critical facets of agriculture in Ethiopia.\nSome general and specific assessments and recommendations for the author are as follows:\nClarity and Structure: This article maintains a generally sound structure with a lucid introduction and well-defined section headings. However, there is room for improvement in terms of clarity and structure. One suggestion is to introduce more concise and structured summaries or introductions at the outset of each section. This will enable readers to swiftly grasp the key points without requiring a full reading of the section.\nAdditional Data and Analysis: This article provides a good introduction to the main weed species and their effects on maize. However, it would be useful to include more data and conduct a more detailed analysis. Adding statistics or case studies to support claims would enhance an article's informative value and credibility.\nAddress Research Gaps and Future Directions: While this article briefly touches upon research gaps and future directions, it is advisable to expound on these aspects. A comprehensive elaboration of the research required to bridge these gaps and its practical implementation would be beneficial. Visual Aids: Enhancing the article with visual aids such as charts or graphs to depict data and trends related to weed infestations and management practices could make the content more engaging and comprehensible.\nLanguage and Editing: The manuscript would benefit from additional proofreading and editing. Addressing minor grammatical issues and refining awkward sentence structures will enhance readability.\nSymbol Definitions: The author should consider including a legend or explanation for the symbols and abbreviations used in the tables, particularly in Tables 2 and 3. This will contribute to the clarity of the data presentation.\nCitations and References: It is essential to ensure that all statements and information presented in an article are substantiated by relevant and recent references. Comprehensive and accurate citations are fundamental for establishing the credibility of information.\nBibliographic Research Methodology: A brief description of the bibliographic research methodology employed, including the keywords used and databases targeted, should be included. This will aid readers in understanding how pertinent literature has been gathered.\nComprehensive Citation of Studies: The author should verify that the article thoroughly cites and references all pertinent studies on weed management in corn crops in Ethiopia to ensure the comprehensiveness and academic rigor of the review.\nInclusion of a Summary Table: To align with the article's title, the author is encouraged to incorporate a summary table that offers an overview of diverse studies conducted in Ethiopia concerning weed management in corn. This table should encompass details, such as research objectives, primary findings, key weed species, crop type, study areas, and proper citations for each study.\nRecommendations for Agriculture: An imperative addition would be a section within the article outlining recommendations for agricultural practices, based on the findings of the review. Such recommendations would underscore the practical relevance of this review and elucidate its potential contribution to the development of agriculture in Ethiopia.\nIncorporating these suggestions will not only enhance the article's quality and completeness but also render it more informative and valuable to its readers.\nWe hope that the authors find this review report to be helpful.\nBest regards, Dr. Abdellatif Boutagayout\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	3	https://f1000research.com/articles/12-880
https://f1000research.com/articles/12-394/v1	13 Apr 23	{ "type": "Research Article", "title": "Impact of vitamin D deficiency in relation to the clinical outcomes of hospitalized COVID-19 patients", "authors": [ "Andhika Rachman", "Rizky Rahmaniyah", "Andi Khomeini", "Anggraini Iriani", "Rizky Rahmaniyah", "Andi Khomeini", "Anggraini Iriani" ], "abstract": "Background: Vitamin D deficiency is an emerging public health problem that affects more than one billion people worldwide. Vitamin D has been shown to be effective in preventing and reducing the severity of viral respiratory diseases, including influenza. However, the role of vitamin D in COVID-19 infection remains controversial. This study aimed to analyze the impact of vitamin D deficiency on the clinical outcome of hospitalized COVID-19 patients. Methods: A prospective cohort study was conducted among hospitalized COVID-19 patients at two COVID-19 referral hospitals in Indonesia from October 2021 until February 2022. Results: The median serum 25(OH)D level in 191 hospitalized COVID-19 patients was 13.6 [IQR=10.98] ng/mL. The serum 25(OH)D levels were significantly lower among COVID-19 patients with vitamin D deficiency who had cardiovascular disease (p-value=0.04), the use of a ventilator (p-value=0.004), more severe COVID-19 cases (p-value=0.047), and mortality (p-value=0.002). Furthermore, serum 25(OH)D levels were significantly different between patients with mild and severe COVID-19 cases (p-value=0.019). Serum 25(OH)D levels in moderate and severe COVID-19 cases were significantly different (p-value=0.031). Lower serum 25(OH)D levels were significantly associated with an increased number of comorbidities (p-value=0.03), the severity of COVID-19 (p-value=0.002), and the use of mechanical ventilation (p-value=0.032). Mortality was found in 7.3% of patients with deficient vitamin D levels. However, patients with either sufficient or insufficient vitamin D levels did not develop mortality. Conclusions: COVID-19 patients with vitamin D deficiency were significantly associated with having cardiovascular disease, mortality, more severe COVID-19 cases, and the used of mechanical ventilation. Lower serum 25(OH)D levels were associated with an increased number of comorbidities, COVID-19 severity, and the use of mechanical-ventilation. Thus, we suggest hospitalized COVID-19 patients to reach a sufficient vitamin D status to improve the clinical outcome of the disease.", "keywords": [ "Vitamin D", "25(OH)D", "clinical outcome", "COVID-19" ], "content": "Introduction\n\nCoronavirus Disease-2019 (COVID-19) is a rapidly spreading pandemic disease caused by Severe Acute Respiratory Syndrome Corona-Virus-2 (SARS-CoV-2), the seventh coronavirus that infect humans. This highly contagious virus spreads through phonation and breathing droplets or through direct contact with an infected person.1–3 The disease can exhibit a wide range of symptoms, from asymptomatic to dramatic, such as hypoxia and multiorgan failure.1–5 There is a lack of evidence-based data about the risk factors for the infection, as well as the most effective treatments. Current hospital-based management is focused on the excessive inflammatory response and respiratory support due to the fact that targeted antiviral therapies have not been widely accessible.6\n\nVitamin D is a versatile steroid hormone that plays multiple roles in the body, including the regulation of bone and calcium metabolism.7,8 It also supports the innate and adaptive immune systems against respiratory viruses.7,9 It controls the innate immune system by stimulating the synthesis of antimicrobial peptides such as IL-37, cathelicidins, and defensivins.1,10,11 Vitamin D also modulates adaptive immunity by regulating the formation of inflammatory T helper type 17 (Th17) cells toward the anti-inflammatory regulatory T cells and altering the primary pro-inflammatory cytokines, such as interferon-γ, TNF-α and IL-6.1,7,10–12 This regulation is considered to be less effective in cases of vitamin D deficiency, although it might be obtained if vitamin D had reached a sufficient level.1\n\nDeficient vitamin D is a global health crisis, affecting over a billion people.7,13–17 Vitamin D deficiency was widespread across Southeast Asian countries, despite extensive exposure to sunlight.18 Based on current evidence, vitamin D helps prevent and mitigate the severity of viral respiratory diseases, such as influenza.4,7,19,20 However, the role of vitamin D in COVID-19 infection remains unclear.4,7\n\nUnderstanding the clinical course of COVID-19 is crucial until a viable vaccination becomes widely accessible, due to the lack of specific therapies and the tremendous health and economic impact of the pandemic.1,21 In this situation, deficient vitamin D is a modifiable risk factor due to its safety and affordability.1,22,23 Therefore, the aim of this prospective cohort study was to assess the impact of vitamin D deficiency on the clinical outcome of hospitalized COVID-19 patients.\n\n\nMethods\n\nThis study was a prospective cohort study conducted at two COVID-19 referral hospitals in Jakarta, Indonesia (National Emergency Hospital Wisma Atlet Kemayoran and Dr. Cipto Mangunkusumo General Hospital), from October 2021 until February 2022. The included subjects were confirmed to have COVID-19 and admitted to the hospital; aged 18 years and older. The exclusion criteria were COVID-19 patients with clinically asymptomatic and severely affected COVID-19 patients who arrived using mechanical ventilation prior to admission. This study specifically involved subjects registered with mild, moderate, or severe disease according to WHO interim guidance at admission. The STROBE guidelines (Strengthening the Reporting of Observational Studies in Epidemiology) were followed for this study.\n\nVitamin D status was evaluated by measuring serum 25(OH) D or 25-hydroxyvitamin D levels. Serum 25(OH) D were measured at the time of admission. The results were gathered using Roche Diagnostics’ Cobas e411, a competitive electrochemiluminescent protein binding assay.\n\nAccording to Endocrine Society Clinical Practice Guideline, a serum 25(OH) D level of less than 20 ng/mL (50 nmol/L) was considered as deficient.24 In this study, we divided serum 25(OH) D level into three categories, subjects with serum 25(OH) D levels ≤ 20 ng/mL (≤50 nmol/L) were considered as deficient, serum 25(OH) D levels 21-29 ng/mL (51-74 nmol/L) were considered as insufficient, and serum 25(OH) D levels ≥ 30 ng/mL (≥75 nmol/L) were considered as sufficient.\n\nStatistical Package for the Social Sciences (SPSS) version 27 for Macintosh was used to analyze the data that was collected. The serum 25(OH) D levels between two subgroups were analyzed with either Mann-Whitney U test for 2 subgroups and Kruskal-Wallis test for more than 2 subgroups.\n\nEthical approval for this study was granted by Ethics Committee of the Faculty of Medicine, Universitas Indonesia (ethical approval number: KET533/UN2.F1/ETIK/PPM.00.01/2021) and by the Ethics Committee of Wisma Atlet Hospital Jakarta (029/KERSDCWA/2021). The Declaration of Helsinki was implemented during this study.\n\n\nResults\n\nThis prospective cohort studies included 191 subjects. Before being enrolled, each participant signed a written consent form. The characteristics of the included subjects can be observed in Table 1. From the 191 subjects, 54.5% were female.\n\nSubjects who had a history of diabetes mellitus, peripheral vascular disease, stroke or transient ischaemic index, cardiovascular disease, chronic obstructive pulmonary disease, chronic liver disease, and chronic kidney disease were considered to have comorbidities according to the Charlson Comorbidity Index (CCI).25\n\nThe Coronavirus Clinical Characterization Consortium Mortality Score (ISARIC-4C Mortality Score) was established by the ISARIC-4C consortium as a predictor score for mortality among hospitalized COVID-19 patients. The determinant factors include sex, age, respiratory rate (RR), peripheral oxygen saturation, Glasgow Coma Scale (GCS), urea serum level, and C-reactive protein (CRP) level. In the ISARIC-4C Score, patients are classified into low-risk (0-3), intermediate-risk (4-8), high-risk (9-14), and very high-risk (15) categories.6,26\n\nTable 2 provided the significance levels of serum 25(OH) D level across all included subgroups using the chi-square (χ2) analysis. Vitamin D deficiency was found in 74.4% of COVID-19 patients, including 65.4% of patients under the age of 60 and 11% of patients over the age of 60. Lower serum 25(OH) D levels were associated with an increased number of comorbidities, the severity of COVID-19, and the use of mechanical ventilation. Among 191 patients, mortality was found in 7.3% of patients with deficient vitamin D levels. However, subjects with either sufficient or insufficient vitamin D levels did not develop mortality.\n\nTable 3 showed that serum 25(OH) D levels were significantly lower among COVID-19 patients with vitamin D deficiency who had cardiovascular disease, the use of a ventilator, more severe COVID-19 cases, and mortality. Mortality was found in 9.59% of COVID-19 patients with vitamin D deficiency.\n\na Analyzed using Mann-Whitney U test.\n\nb Analyzed using ANOVA test.\n\nc Analyzed using Kruskal-Wallis test.\n\nd Analyzed using t-test.\n\nFigure 1 presented the post-hoc tests performed on COVID-19 patients who were vitamin D deficiency. Serum 25(OH) D levels were significantly different between patients with mild and severe COVID-19 cases (p-value 0.019). Serum 25(OH) D levels in moderate and severe COVID-19 cases were also significantly different (p-value 0.031).\n\nThe green double-arrow denotes statistically significant difference.\n\nThe red double-arrow denotes non-statistically significant difference.\n\n\nDiscussion\n\nPrior studies indicates that Indonesia has a high prevalence of deficient vitamin D (60%), despite being located in a tropical zone where sunlight is abundant all year around.27–29 Whereas the skin’s absorption of sunlight is established as the primary source of vitamin D, other variables, including age, comorbidities, and skin pigmentation, may alter the vitamin D level.15,29 Based on the skin’s sensitivity to ultraviolet (UV) light, the majority of Indonesians have either Fitzpatrick skin phototype IV (with medium to dark brown) or phototype V (dark brown). A lower vitamin D is associated with darker skin pigmentation due to the higher melanin present in darker skin.18,29,30 Other factors, including haze, altitude, and air pollution, also alter the ultraviolet B radiation.29,31\n\nThe beneficial effect of vitamin D to reduce the severity of respiratory tract infection remains controversial.4,7 The current study investigated the impact of vitamin D deficiency to the clinical outcome of hospitalized patients at two COVID-19 referral hospitals in Indonesia. We found that compared to insufficient and sufficient, those with deficient vitamin D status had more number of comorbidities (Table 2). In COVID-19 patients with deficient vitamin D were significantly associated with cardiovascular disease (Table 3). Our findings was supported by de la Guía-Galipienso et al., that revealed vitamin D deficiency may play a critical role in the initiation of inflammation, myocardial calcification, and endothelial dysfunction, which are risk factors for cardiovascular disease.24,32 The vitamin D receptor (VDR) and the enzyme 1α-hydroxylase, which are necessary for the formation of vitamin D’s active form, are expressed in cardiomyocytes, vascular endothelial cells, fibroblasts, and smooth muscle cells.33–36 Left ventricular hypertrophy, vascular dysfunction, and arterial stiffness have been associated with vitamin D deficiency. A deficiency of the vitamin D receptor causes an increase in left ventricular mass and elevated levels of atrial natriuretic peptide, as well as cardiac metalloproteases and disturbances in homeostasis. Furthermore, the development of fibrotic extracellular matrix induces left ventricular dilation.24,37,38\n\nVitamin D has been shown to have a number of beneficial effects on the cardiovascular system, including natriuretic peptide secretion, inhibition of the renin-angiotensin-aldosterone system (RAAS), anti-hypertrophic effects, and inhibition of cardiomyocyte proliferation.33,39,40 Calcitriol and its analogues activate VDR, which directly suppresses angiotensin I expression and local angiotensin II synthesis in myocardial, kidney tissue, and renal arteries.33,41 Studies have revealed that vitamin D enhances the anti-hypertensive effects of angiotensin 1–7 by inducing the production of angiotensin-converting enzyme 2 (ACE2).33,42,43 MiR-106b-5p, which acts on juxtaglomerular cells to boost renin synthesis, has been shown to be directly influenced by VDR-deficient immune cells.33,43\n\nMoreover, vitamin D affects the progression of HF through modulating the production of metalloproteinases. Evidence strongly suggests that vitamin D has an anti-inflammatory effect by preventing nuclear factor kappa B (NF-κB) and promoting the production of IL-10, which have a significant role in the progression of CVD.33,44,45 Vitamin D deficiency induces arterial stiffness and endothelial dysfunction in blood vessels, which in turn leads to enhanced inflammation, endothelial cell malfunction, and atherogenesis.24,46\n\nThe severity of COVID-19 were significantly associated with the lower serum 25(OH) D levels (Tables 2, 3 and Figure 1). Vitamin D is an immunomodulatory hormone with antibacterial and anti-inflammatory properties, and it plays a crucial role in the immune system. Vitamin D has been reported to exert its effects against COVID-19 by limiting the viral transmission, diminishing viral replication, and optimizing viral clearance.24,47 Vitamin D boosts the innate immune response and protects against excessive inflammation, by increasing anti-inflammatory IL-10 and decreasing pro-inflammatory cytokines and tumor necrosis factor alpha (TNF-α).26,47–51 According to the research of Daneshkhah et al., a lack of vitamin D raises C-reaction protein (CRP) levels, which in turn elevates the risk of a cytokine storm.24,52 The protective effects of vitamin D on the coagulation pathway led to a reduced risk of acute respiratory distress syndrome and thrombosis.47,53–55 Thus, increasing vitamin D levels to adequate levels may help to prevent COVID-19 infection and complications.24,47,48,53,56\n\nTo the best of our knowledge, this is the first study that analyse the ISARIC-4C score in a group of patients with vitamin D deficiency. We found that serum 25(OH) D levels had no significant association with ISARIC-4C Score (Tables 2, 3). In contrast with study by Wellbelove et al. that concluded the ISARIC-4C mortality score is good predictors for 30-day mortality in COVID-19 (AUROC of 0.74–0.88).57 The ISARIC-4C consortium established the ISARIC-4C Mortality Score to predict the mortality of hospitalized COVID-19 patients. Multicentre cohort study was conducted among 74,944 participants at 260 different hospitals. However, the ISARIC-4C has been internally validated but not externally validated. Hence, further study is warranted to fully understand the potential of ISARIC-4C as a prognostic tool to classify patients into specific management groups.6,26,58\n\nSerum 25(OH) D levels were significantly lower among subjects that used the ventilator (Tables 2, 3). Among all patients, mortality was found in 7.3% of patients with deficient vitamin D levels. However, patients with either sufficient or insufficient vitamin D levels did not develop mortality (Table 2). Serum 25(OH) D levels in vitamin D deficiency subjects were significantly lower in the COVID-19 patients with mortality status (Table 3). Our findings were consistent with the cohort study by Angelidi et al., which discovered that lower 25(OH) D levels were associated with increased mechanical ventilation needs and mortality risk among hospitalized patients.59 Prior studies have revealed that vitamin D deficiency has been correlated to a 58% increased risk of acute respiratory infection, prolonged mechanical ventilation, and a 10-fold increase in mortality risk.59,60\n\nAs a steroid hormone, vitamin D interacts with the vitamin D receptor located in the nucleus of cells to have physiologic effects.17,59 The interaction of 25(OH) D with other steroid hormone receptors may have physiological effects similar to glucocorticoids.59,61 Although the underlying mechanisms of vitamin D’s protection against severe COVID-19 are unknown, it is established that vitamin D reduces the production of proinflammatory cytokines such as Th1, TNF-α, interferon-β, IL-6, and promotes the production of anti-inflammatory responses such as T regulatory cells and Th2.49,59,62–64 There are several explanations for vitamin D’s beneficial effects on critically ill patients. Initially, critically ill patients who are given vitamin D supplements will have their plasma vitamin D concentrations restored. Furthermore, vitamin D regulates the synthesis of immune system effector molecules such as β-defensin and cathelicidin, which are both antimicrobial peptide.49,59,65,66 Cathelicidin enhances the production of anti-inflammatory cytokines while decreasing the synthesis of pro-inflammatory cytokines. As a result, vitamin D deficiency could increase the risk of sepsis and inflammation in severely ill patients by diminishing the immune response and modulatory effects on innate immunity.67–72\n\nThe strength of this study lies in the fact that it is the first study to analyze the ISARIC-4C Score in COVID-19 patients with deficient vitamin D. The majority of this study’s data were collected during the Omicron variation’s development and can be utilized to make comparisons to the Delta variant or any other variants. However, this study has several limitations that should be considered to improve the further research. First, this study did not include a healthy control group as a reference population. Second, after patients were discharged, serum 25(OH) D levels were not measured.\n\nHence, despite these limitations, our prospective cohort study demonstrates that hospitalized COVID-19 patients with vitamin D deficiency had a higher risk of using mechanical ventilation and mortality from respiratory failure and other complications. Additionally, a prior meta-analysis revealed that people with a deficient vitamin D level had an increased risk of SARS-CoV-2 infection and COVID-19-related hospitalization. Our data are consistent with the findings of recent pilot studies and a meta-analysis showing that a sufficient vitamin D status is able to reduce COVID-19 severity, indicating that it may be beneficial in minimizing the clinical and economic burden associated with COVID-19.1,73–75\n\n\nConclusion\n\nWe found that lower serum 25(OH) D levels were associated with an increased number of comorbidities, COVID-19 severity, and the use of mechanical ventilation. COVID-19 patients with vitamin D deficiency status were significantly associated with having cardiovascular disease, mortality, more severe COVID-19 cases, and the used of high-flow nasal canule (HFNC) or ventilator. This prospective cohort study doesn’t diminish the significance of the continuing vaccine effort against the health-economic burden of SARS-CoV-2 infection. As a result, we strongly suggest achieving sufficient vitamin D status, which may serve as an important adjuvant strategy to improve clinical outcomes before vaccines become widely available.", "appendix": "Data availability\n\nFigshare: Impact of Vitamin D Deficiency in Relation to the Clinical Outcomes of Hospitalized COVID-19 Patients, DOI: https://doi.org/10.6084/m9.figshare.22145768.v2. 76\n\nThis project contains the following data:\n\n- The data here is only for research paper validation of corresponding author Andhika Rachman entitled: “Impact of Vitamin D Deficiency in Relation to the Clinical Outcomes of Hospitalized COVID-19 Patients”. The raw data consists of subjects characteristics and the levels of serum 25-hydroxy-vitamin D of hospitalized COVID-19 patients.\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\nAcknowledgements\n\nThe authors would like to thank all the participants for their involvement.\n\n\nReference\n\nChiodini I, Gatti D, Soranna D, et al.: Vitamin D Status and SARS-CoV-2 Infection and COVID-19 Clinical Outcomes. Front. Public Health. 2021 Dec 22; 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou M, Zhang X, Qu J: Coronavirus disease 2019 (COVID-19): a clinical update. Front. Med. 2020 Apr 2; 14(2): 126–135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu B, Guo H, Zhou P, et al.: Characteristics of SARS-CoV-2 and COVID-19. Nat. Rev. Microbiol. 2021 Mar 6; 19(3): 141–154. 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Aging Dis. 2012 Aug; 3(4): 313–329. PubMed Abstract\n\nSabetta JR, DePetrillo P, Cipriani RJ, et al.: Serum 25-Hydroxyvitamin D and the Incidence of Acute Viral Respiratory Tract Infections in Healthy Adults. PLoS One. 2010 Jun 14; 5(6): e11088. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaneshkhah A, Agrawal V, Eshein A, et al.: The Possible Role of Vitamin D in Suppressing Cytokine Storm and Associated Mortality in COVID-19 Patients.2020. Publisher Full Text\n\nTian Y, Rong L: Letter: Covid-19, and vitamin D. Authors’ reply. Aliment. Pharmacol. Ther. 2020 May; 51(10): 995–996. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMansur JL: Letter: low population mortality from COVID-19 in countries south of latitude 35 degrees North supports vitamin D as a factor determining severity. Aliment. Pharmacol. Ther. 2020 Jul; 52(2): 411–412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammad M, Mishra A, Ashraf MZ: Emerging Role of Vitamin D and its Associated Molecules in Pathways Related to Pathogenesis of Thrombosis. Biomolecules. 2019 Oct 24; 9(11): 649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao Z, Wu Y, Faucon E, et al.: SARS-CoV-2 & Covid-19: Key-Roles of the ‘Renin-Angiotensin’ System/Vitamin D Impacting Drug and Vaccine Developments. Infect. Disord. Drug Targets. 2020 Jul 20; 20(3): 348–349. PubMed Abstract \| Publisher Full Text\n\nWellbelove Z, Walsh C, Perinpanathan T, et al.: Comparing the 4C mortality score for COVID-19 to established scores (CURB65, CRB65, qSOFA, NEWS) for respiratory infection patients. J. Infect. 2021 Mar; 82(3): 414–451. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnight SR, Ho A, Pius R, et al.: Risk stratification of patients admitted to hospital with covid-19 using the ISARIC WHO Clinical Characterisation Protocol: development and validation of the 4C Mortality Score. BMJ. 2020 Sep 9; 370: m3339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngelidi AM, Belanger MJ, Lorinsky MK, et al.: Vitamin D Status Is Associated With In-Hospital Mortality and Mechanical Ventilation: A Cohort of COVID-19 Hospitalized Patients. Mayo Clin. Proc. 2021 Apr; 96(4): 875–886. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManson JE, Bassuk SS: Commentary. Eliminating vitamin D deficiency during the COVID-19 pandemic: A call to action. Metabolism. 2020 Nov; 112: 154322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDemer LL, Hsu JJ, Tintut Y: Steroid Hormone Vitamin D. Circ. Res. 2018 May 25; 122(11): 1576–1585. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLemire JM, Adams JS, Kermani-Arab V, et al.: 1,25-Dihydroxyvitamin D3 suppresses human T helper/inducer lymphocyte activity in vitro. J. Immunol. 1985 May; 134(5): 3032–3035. PubMed Abstract \| Publisher Full Text\n\nCantorna M, Snyder L, Lin YD, et al.: Vitamin D and 1,25(OH)2D Regulation of T cells. Nutrients. 2015 Apr 22; 7(4): 3011–3021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeffery LE, Burke F, Mura M, et al.: 1,25-Dihydroxyvitamin D3 and IL-2 Combine to Inhibit T Cell Production of Inflammatory Cytokines and Promote Development of Regulatory T Cells Expressing CTLA-4 and FoxP3. J. Immunol. 2009 Nov 1; 183(9): 5458–5467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu PT, Stenger S, Li H, et al.: Toll-Like Receptor Triggering of a Vitamin D-Mediated Human Antimicrobial Response. Science (1979). 2006 Mar 24; 311(5768): 1770–1773.\n\nAdams JS, Ren S, Liu PT, et al.: Vitamin D-Directed Rheostatic Regulation of Monocyte Antibacterial Responses. J. Immunol. 2009 Apr 1; 182(7): 4289–4295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen H, Mei Y, Zhang K, et al.: The Effect of Vitamin D Supplementation on Clinical Outcomes for Critically Ill Patients: A Systemic Review and Meta-Analysis of Randomized Clinical Trials. Front. Nutr. 2021 May 4; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang TT, Nestel FP, Bourdeau V, et al.: Cutting Edge: 1,25-Dihydroxyvitamin D3 Is a Direct Inducer of Antimicrobial Peptide Gene Expression. J. Immunol. 2004 Sep 1; 173(5): 2909–2912. PubMed Abstract \| Publisher Full Text\n\nQuraishi SA, de Pascale G , Needleman JS, et al.: Effect of Cholecalciferol Supplementation on Vitamin D Status and Cathelicidin Levels in Sepsis. Crit. Care Med. 2015 Sep; 43(9): 1928–1937. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDürr UHN, Sudheendra US, Ramamoorthy A: LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochimica et Biophysica Acta (BBA). Biomembranes. 2006 Sep; 1758(9): 1408–1425. Publisher Full Text\n\nMora JR, Iwata M, von Andrian UH : Vitamin effects on the immune system: vitamins A and D take centre stage. Nat. Rev. Immunol. 2008 Sep; 8(9): 685–698. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKankova M, Luini W, Pedrazzoni M, et al.: Inhibition of cytokine production by vitamin D3 deficiency. Int. J. Immunopharmacol. 1991 Jan; 13(6): 773. Publisher Full Text\n\nMcCullough PJ, Lehrer DS, Amend J: Daily oral dosing of vitamin D3 using 5000 TO 50,000 international units a day in long-term hospitalized patients: Insights from a seven year experience. J. Steroid Biochem. Mol. Biol. 2019 May; 189: 228–239. PubMed Abstract \| Publisher Full Text\n\nPal R, Banerjee M, Bhadada SK, et al.: Vitamin D supplementation and clinical outcomes in COVID-19: a systematic review and meta-analysis. J. Endocrinol. Investig. 2022 Jan 24; 45(1): 53–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffin G, Hewison M, Hopkin J, et al.: Vitamin D and COVID-19: evidence and recommendations for supplementation. R. Soc. Open Sci. 2020 Dec; 7(12): 201912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRachman A: Raw Data - Impact of Vitamin D Deficiency in Relation to the Clinical Outcomes of Hospitalized COVID-19 Patients. figshare. Journal Contribution. 2023. Publisher Full Text" }	[ { "id": "169546", "date": "11 May 2023", "name": "Shaun Sabico", "expertise": [ "Reviewer Expertise Nutrition", "medical sciences", "vitamin D", "metabolism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 prospective study by Rachman and colleagues analyzed the impact of vitamin D deficiency among covid-19 patients and found that those who had more comorbidities and had more severe manifestations of covid-19 were more likely to be vitamin D deficient. Although the data itself is not new, this is one of the very few studies coming from Southeast Asia where vitamin D deficiency is not expected to be as pronounced in other regions. The use of ISARIC-4C mortality score is also an added novelty. Despite this fact, their findings largely echo previous observations. I have several comments:\n\nMajor:\nPlease add more details how covid-19 was diagnosed with reference. Was it through nasopharyngeal swab? And where was vitamin D measured? Was it in a BSL3 facility?\n\nThe statistics could have been expanded to control for confounders such as age and BMI. These were not explicitly mentioned in the data analysis. Furthermore, with the number of variables measured, the p-value should have been Bonferroni adjusted unless the authors prove this wasn’t necessary.\n\nLastly, the study design appears to be cross-sectional and not prospective. The observational design and small sample size have limited the findings to at best, suggestive. The limitation section could have been expanded taking into consideration the points raised.\nMinor\nSeveral studies that support your findings from the Middle East where deficiency is very pronounced are suggested to be added including clinical trials on vitamin D and covid-19. Similar findings from different regions and ethnic groups reinforce the role of vitamin D in severity of covid-19.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9679", "date": "13 Jun 2023", "name": "Andhika Rachman", "role": "Author Response", "response": "Response To The Reviewer: Dear Dr. Shaun Sabico, We would like to thank you for your time and consideration in handling our manuscript. We want to confirm that we have revised the manuscript with the tracked changes system. If you have any suggestions, please don’t hesitate to contact us. Sincerely, Andhika Rachman, PhD Medical staff, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia - Dr. Cipto Mangunkusumo General Hospital Major: Please add more details how covid-19 was diagnosed with reference. Was it through nasopharyngeal swab? And where was vitamin D measured? Was it in a BSL3 facility?  Thank you for your construction feedback. We have added more details on the methods. The SARS-CoV-2 infection was confirmed through positive RT-PCR obtained from nasal and oropharyngeal swabs collected.77 The examination was carried out in the Biosafety Level 3-facility (BSL-3) with Biological Safety Cabinet Class II (BSC-II). During the admission, each patient had 3–5 mL of blood collected in an acid citrate dextrose tube from a cuffed venous sample. The samples were transported to the laboratory in a cold chain for the measurement of vitamin D. Reference: 77. Torretta S, Zuccotti G, Cristofaro V, Ettori J, Solimeno L, Battilocchi L, D'Onghia A, Bonsembiante A, Pignataro L, Marchisio P, Capaccio P. Diagnosis of SARS-CoV-2 by RT-PCR Using Different Sample Sources: Review of the Literature. Ear Nose Throat J. 2021 Apr;100(2_suppl):131S-138S. doi: 10.1177/0145561320953231. The statistics could have been expanded to control for confounders such as age and BMI. These were not explicitly mentioned in the data analysis. Furthermore, with the number of variables measured, the p-value should have been Bonferroni adjusted unless the authors prove this wasn’t necessary. The reviewer did raise a very important concern which will improve our manuscript. We strongly agree that the reviewer’s suggestion should be considered for further researches regarding this issue. The confounders such as age and BMI could not be controlled due to the minimum sample size of the subgroups and the fact that the number of samples is unequally distributed, which has not fulfilled the criteria to conduct the analysis. Thus, we have raised the point that the small sample size has become our limitation. We plan to improve the design and participants selection in the next study. Furthermore, we have added Figure 1 (a bivariate analysis that has been adjusted with the Bonferoni correction). Figure 1 presents the bivariate analysis performed on COVID-19 patients with vitamin D deficiency. Serum 25(OH)D levels were significantly different between patients with mild and severe COVID-19 cases (p-value < 0.001). Serum 25 (OH) D levels in mild and moderate COVID-19 cases were also significantly different (p-value 0.002). Lastly, the study design appears to be cross-sectional and not prospective. The observational design and small sample size have limited the findings to at best, suggestive. The limitation section could have been expanded taking into consideration the points raised. We have added the points of observational design and small sample size as limitations of our study. Additionally, we want to verified that our study's design is cohort prospective. Even if the risk variables (vitamin D levels) are measured upon admission, the outcome is followed and observed until the patient leaves the hospital, becomes worse, requires a ventilator, or passes away. The person had not used a ventilator when first recruited. Minor Several studies that support your findings from the Middle East where deficiency is very pronounced are suggested to be added including clinical trials on vitamin D and covid-19. Similar findings from different regions and ethnic groups reinforce the role of vitamin D in severity of covid-19. Thank you for your advice. We have improved our paper by citing the multi-center randomized clinical trial in the Middle East that conducted by Sabico et al. We have also cited the studies carried out by Al-Daghri et al. and Alguwaihes et al. Please don't be hesitant to contact us if you have any concerns or suggestions about the revised manuscript. If you have any suggestions, we would greatly appreciate hearing them. Thanks for your attention. We eagerly await your response." } ] } ]	1	https://f1000research.com/articles/12-394
https://f1000research.com/articles/11-142/v1	03 Feb 22	{ "type": "Case Report", "title": "Case Report: Glans penile necrosis in a patient with SARS-CoV-2 and leprosy infection", "authors": [ "Jufriady Ismy", "Said Alfin Khalilullah", "Aditya Fajar Bahagianto", "Aditya Fajar Bahagianto" ], "abstract": "Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was firstly identified in China and has been declared a global pandemic. Several serious extrapulmonary manifestations due to SARS-CoV-2 infection have also been reported and associated with hypercoagulability thrombotic vasculopathy. In addition, cases of Mycobacterium-leprae infection have also been known associated with blood coagulation abnormality. Methods: Here, we report a 56-year-old male with coronavirus disease-19 (COVID-19) with concomitant leprosy infection with manifestation of glans penile necrosis, presented to the emergency department with acute penile pain. This case is unique because no occlusion blood flow to the penile was observed in the radiographic imaging. We described the potential pathophysiology in this case through a literature review. Results: The patient received treatment according to the COVID-19 protocol and was given low molecular weight heparin (LMWH) therapy for 4 days. During the follow up, the clinical and functional condition of the penis showed significant improvement. Conclusions: Microthrombus involvement, platelet abnormalities and impaired hemostasis due to SARS-CoV-2 and leprosy co-infection are the hypothesis in this case report.", "keywords": [ "case report", "COVID-19", "gland penile necrosis", "leprosy", "SARS-CoV-2" ], "content": "Introduction\n\nAt the beginning of 2020, the World Health Organization declared novel coronavirus disease-2019 (COVID-19) infection as a pandemic which caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) that has led to a global health crisis due to acute respiratory distress syndrome (ARDS).1 Most cases have mild-to-moderate symptoms, with approximately 15% developing severe pneumonia, while about 5% developing ARDS and organ failure.2 Other serious complications related to SARS-CoV-19 infection include hypercoagulability and thrombotic vasculopathy with clinical manifestations such as coronary syndrome, deep vein thrombosis, ischemic stroke, and pulmonary embolism.3,4 Recently, other serious clinical manifestations associated with genitalia condition were reported in patients with SARS-CoV-2 infection with penile ischemia.5 In patients infected by SARS-CoV-2, penile priapism has also been reported.6,7 Some authors have speculated that these conditions related to COVID-19 may be due to severe hypercoagulability and thrombotic tendency observed in patients with COVID-19.5,8\n\nOn the other hand, penile involvement in leprosy is uncommon.9 Leprosy (also known as Hansen's disease) is an infection caused by Mycobacterium leprae (M. leprae) that can damage the peripheral nerves and bone absorption. Leprosy has two distinct phases: direct infection of macrophages and Schwann cells and reactional episodes. Approximately 50% of patients with leprosy are affected by a reactional episode,10 which occurs because of endothelial inflammation leading to necrotizing pan-vasculitis. This condition, in severe cases, progresses to necrotic hemorrhagic lesions of the extremities and trunk, as well as Lucio's phenomenon.11,12\n\nPenile ischemia or necrosis is rare due to its abundant blood circulations. Usually, penile necrosis is associated with thrombotic phenomena and calcium deposits in patients on dialysis.13 However, no previous cases of penile ischemia in a COVID-19 patient who concomitantly infected with leprosy have ever been reported. Here, we presented a unique case of penile necrosis in acute COVID-19 and leprosy infection following the CARE guidelines.14 To the best of our knowledge, this case is the first case presenting penile glans necrosis in COVID-19 patient concomitant leprosy. We also described the potential pathophysiology of glans penis necrosis in this case through a literature review.\n\n\nCase report\n\nA 56-year-old Indonesian male patient was admitted to Dr. Zainoel Abidin General Hospital in Aceh, Indonesia. He was referred from a regional hospital with SARS-CoV-2 infection and necrotic penile glans. Complaints of changes in the color of the glans penis occur progressively without any past intervention. In addition, he also complained of pain in the penis, dysuria and difficulty urinating. He worked as a farmer, actively smoked since he was an adolescent, and does not consume any drugs or alcohol. Currently, he is undergoing treatment for leprosy disease. Table 1 summarizes the patient's characteristics.\n\nThis patient had a past medical history of hypertension, which is well-controlled with 10 mg/day oral amlodipine. There was no complaint in micturition, meatal discharge, and other genital problems.\n\nOn examination, his condition was stable with blood pressure 134/91 mmHg, pulse 90 bpm, respiratory 22 breaths/minute, and oxygen saturation at 97% on room temperature. The patient was afebrile and fully alert. His localized status found a discoloration involving the penile glans (Figure A).\n\nAn urgent radiologic investigation with Doppler ultrasound of the penile and pelvic computerized tomography (CT) angiography revealed no occlusion nor thrombus on the arteria that supplies the penile (Figure B).\n\nThe patient was admitted to the COVID ward with therapy as follows; 2 L/min nasal oxygen, 1000 mg ceftriaxone injection twice daily, 10 mg amlodipine daily, 50mg clofazimine daily, 100mg dapsone daily, 5000 U vitamin D daily, 1000 mg vitamin C daily and 300 international unit low molecular weight heparin (LMWH) intravenous drip daily for 4 days. No challenges were found during treatment.\n\nAfter 4 days of LMWH administration without any treatment changes, the clinical condition of the gland penile was improved, the necrosis was less extensive and limited to one-third of the superficial of the glans penis (Figure C). The patient then continued to isolate in the COVID-19 ward. On 6 weeks follow-up, the clinical and functional condition of the penis were excellent without any unexpected events. The patient's urinary and erectile functions remained unchanged. The patient was also satisfied with the treatment that he received.\n\n\nDiscussion\n\nWe searched four online databases (the Medline EMBASE, Google Scholar, PubMed, and Scopus) for the literature review. Four terms related to the patient’s condition were combined in the searching strategy; 1) Penile; 2) Penis; 3) COVID-19 and 4) SARS-CoV-2 to investigate the relationship between COVID-19 and leprosy infection in necrosis of glans penis. In the end, we found only one publication by Sarkis et al. that presented the penile ischemic condition in COVID-19 infection.5 This publication reported glans penile discoloration due to ischemia secondary to COVID-19. However, the patient had several comorbidities such as type 2 diabetes, hypertension, and end-stage kidney disease which could also be risk factors for thrombus vasculopathy.\n\nPenile necrosis is linked to thrombotic events and calcium deposits in dialysis patients.13 Another study also reported penile necrosis secondary to purpura fulminans.15 One study in Japan described fifteen patients with penile necrosis due to calciphylaxis and a long history of diabetes.16 In this condition, it is clearly understood that diabetic vasculopathy and calcium deposits can become a thrombus which causes impaired blood flow to penis. In our patient, he had no comorbid precipitating thrombus vasculopathy. Although the patient is an active smoker, we still cannot conclude that this is the cause of penile necrosis. Uniquely, the CT angiography investigation of our patient did not reveal any occlusion or thrombus.\n\nOn the basis of the above finding, we hypothesized that the patient's penis glans necrosis is caused by COVID-19 and leprosy co-infection. Many studies have published the role of microvascular thrombus in SARS-CoV-2 infection.17–20 There is a definite link between inflammation, hypercoagulation, and thrombosis, according to previous research. Part of this interaction is likely mediated by a cytokine storm, which increases the risk of developing disseminated intravascular coagulopathy.21 The stimulation of coagulation pathways throughout the body by mediators produced during a cytokine storm can result in a prothrombotic condition marked by the formation of microthrombi, diffuse capillary blockage, tissue ischemia, and organ damage.18 This pathogenesis indicates the possible tendency for physiopathology of glans penile necrosis secondary to SARS-CoV-2 infection.\n\nSimilar to SARS-CoV-2 infection, leprosy has long been known to trigger hemostasis disorders due to platelet abnormalities, blood coagulation, and fibrinolysis.10 Several studies have previously reported that patients with tuberculoid leprosy who develop erythema nodosum leprosum (ENL) had a longer activated partial thromboplastin time (aPTT) with elevated fibrinogen and platelet activation.12,22,23 One review of related studies conducted by Nery has described thrombophlebitis and pulmonary embolism in leprosy patients who develop ENL.24 Another study also found that leprosy disease was associated with coronary thrombosis and cerebral vascular accidents.25,26 These findings indicate that the severity of leprosy potentially leads to occlusion of blood flow which eventually causes organ damage.\n\nWe assume that patients co-infected with SARS-CoV-2 and leprosy have a greater risk of complications, especially hemostasis disorders and thrombus vasculopathy, as in our patient who showed elevated of D-Dimer and fibrinogen levels. This case report and literature review provide preliminary evidence of the association between SARS-CoV-2 and leprosy infection in glans penile necrosis; however, further studies are warranted.\n\nThis case report and literature review is limited by its short-term follow-up period, however its strength is that as we directly observed the patient rather than taking his information retrospectively, this reduces any potential bias.\n\nFinally, several highlights should be considered in managing glans penile necrosis in patients with COVID-19 infection. As previously described, penile necrosis can occur as a result of a vasculopathy thrombus. On the other hand, COVID-19 and leprosy infection can also induce coagulopathy and microvascular thrombus. Therefore, choosing anticoagulant therapy than immediate aggressive amputation of the organ is still a viable option. Administering anti-thrombus therapy is a less-intrusive treatment approach that physicians can consider, which gave a good result in our patient.\n\n\nConclusions\n\nIn our perspective, microthrombus formation, diffuse capillary occlusion and tissue necrosis are the basis of the etiology penile glans necrosis in our patient. It is likely associated with SARS-CoV-2 and leprosy co-infection. This hypothesis makes this case an interesting report. In addition, from what we know at the time of writing this manuscript, this is the first case report of glans penile necrosis in a patient with SARS-CoV-2 and leprosy co-infection.\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 was obtained from the patient for publication of the patient’s images and this case report.", "appendix": "References\n\nOrganization WH: WHO Director-General’s opening remarks at the media briefing on COVID-19-11 March 2020. Geneva, Switzerland: 2020.\n\nCao X: COVID-19: immunopathology and its implications for therapy. Nat. Rev. Immunol. 2020; 20(5): 269–270. PubMed Abstract \| Publisher Full Text\n\nKlok FA, Kruip M, van der Meer NJM , et al.: Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb. Res. 2020; 191: 145–147. 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Haemost. 2020; 120(12): 1668–1679. PubMed Abstract \| Publisher Full Text\n\nNalugo M, Schulte LJ, Masood MF, et al.: Microvascular Angiopathic Consequences of COVID-19. Front. Cardiovasc. Med. 2021; 8(26). PubMed Abstract \| Publisher Full Text\n\nMcFadyen JD, Stevens H, Peter K: The Emerging Threat of (Micro) Thrombosis in COVID-19 and Its Therapeutic Implications. Circ. Res. 2020; 127(4): 571–587. PubMed Abstract \| Publisher Full Text\n\nGupta A, Madhavan MV, Sehgal K, et al.: Extrapulmonary manifestations of COVID-19. Nat. Med. 2020; 26(7): 1017–1032. Publisher Full Text\n\nLevi M: Pathogenesis and diagnosis of disseminated intravascular coagulation. Int. J. Lab. Hematol. 2018; 40(S1): 15–20. Publisher Full Text\n\nGupta M, Bhargava M, Kumar S, et al.: Platelet function in leprosy. Int. J. Lepr. Other Mycobact. Dis. 1975; 43(4): 327–332. PubMed Abstract\n\nMartinuzzo M, De Larrañaga G, Forastiero R, et al.: Markers of platelet, endothelial cell and blood coagulation activation in leprosy patients with antiphospholipid antibodies. Clin. Exp. Rheumatol. 2002; 20: 477–483. PubMed Abstract\n\nNery JA, Salles S, Malta AM, et al.: Cor pulmonale subacute and recurrent erythema nodosum leprosum. Lepr. Rev. 1993; 64(1): 77–79. PubMed Abstract\n\nRogers JH: Coronary thrombosis, cerebral vascular accident and pulmonary embolism in leprosy. Ann. Intern. Med. 1960; 53: 746–753. PubMed Abstract \| Publisher Full Text\n\nWidasmara D: Eritema Nodusum Lepsosum. Penyakit Kusta, Sebuah Perspektif Klinis. 1st ed.Indonesia: UB Press Publisher (P) Ltd.; 2018; 131–133." }	[ { "id": "122238", "date": "22 Feb 2022", "name": "Harrina Erlianti Rahardjo", "expertise": [ "Reviewer Expertise Urology", "functional urology", "neurourology", "urodynamic" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting case report of penile necrosis in a patient with both a Covid and leprosy infection, perhaps this is the first case reported.\nHowever several details need to be provided.\n\nIn the case report the author has included the baseline data of the patient, however during treatment and follow up there was only a clinical picture after 4 days of treatment. The authors should also add the penile clinical condition on the 6th week follow up. The data is also lacking on the status of the patient's leprosy infection in terms of clinical history and medication. It would also add substantial information if the authors would include the changes in laboratory parameters throughout the course of treatment. Was it inline with the improvement seen clinically? Changes of the hemostasis lab parameters, clinical progress during treatment and perhaps cytokine data (if it was measured) would add up to a nice discussion.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "7945", "date": "15 Mar 2022", "name": "Jufriady Ismy", "role": "Author Response", "response": "We thank to the reviewer for their comments, The clinical picture in this article is the clinical condition of the patient at 2 weeks of follow-up. we will improve the narrative in the article so that there are no misunderstandings. Meanwhile, we apologize for not documenting the patient's clinical condition at 6 weeks of follow-up. Regarding the history and medication of leprosy we have summarized it in table 1, but to add to the clinical history, we also include it in the article We apologize, because we do not have data related to cytokines, but changes in hemostasis laboratory parameters is in line with improving clinical conditions. We also added a revision of this information to the article." } ] } ]	1	https://f1000research.com/articles/11-142
https://f1000research.com/articles/12-1366/v1	18 Oct 23	{ "type": "Research Article", "title": "Carbon footprint of Nepalese healthcare system: A study of Dhulikhel Hospital", "authors": [ "Bikash Adhikari", "Ambika Dangal", "Sushila Pandey", "Bijay Thapa", "Ashim Joshi", "Bivek Baral", "Ambika Dangal", "Sushila Pandey", "Bijay Thapa", "Bivek Baral" ], "abstract": "Background: Though direct greenhouse gas emissions cannot be observed in health care sectors, there can exist indirect emissions contributing to global climate change. This study addresses the concept of the carbon footprint and its significance in understanding the environmental impact of human activities, with a specific emphasis on the healthcare sector through gate-to-gate (GtoG) life cycle assessment. Transportation, energy consumption, and solid waste generated by hospitals are the primary sources of carbon emissions. Methods: Different standards, guidelines and parameters were used to estimate emissions from both the primary and secondary data. All steps and sub-steps involved in GtoG were accessed and analyzed within the standard ISO 14040:44 guideline. An extensive review of existing literature was carried out for the evaluation and verification of secondary data. Results: The total carbon footprint of generators, electricity consumption, transportation activities, LPG cylinders, PV systems was found to be 58,780 kg-CO2-eq/yr, 519,794 kg-CO2-eq/yr, 272,375 kg-CO2-eq/yr, 44,494 kg-CO2-eq/yr, 35,283 kg-CO2-eq/yr respectively and the emissions from non-biodegradable solid waste was found to be 489,835 kg-CO2/yr. Local air pollutants such as PM10, CO, SO2, NOX, and VOCs generated by generators and transportation were also estimated. The CH4 emissions from liquid waste were 1177.344 kg CH4/BOD yr, and those from biodegradables were 3821.6954 kg CH4/yr. Conclusions: Healthcare professionals and policymakers can take action to reduce the sector's carbon footprint by implementing best practices and encouraging sustainable behavior. This study can be taken as foundation for further exploration of indirect emissions from healthcare sectors not only in Nepal but also in south Asian scenario.", "keywords": [ "greenhouse gas", "life cycle assessment", "health care sectors" ], "content": "Introduction\n\nDirect emissions in the healthcare industry are relatively low as compared to other sectors.1 However, a sizable portion of the country's CO2 footprint can be attributed to emissions along the supply chain brought on by the healthcare sector's purchases of goods and services.1,2 In terms of energy consumption, hospitals and other healthcare facilities are major users of energy for heating, cooling, lighting, and the operation of medical equipment. The provision of healthcare and the acquisition of goods, energy use, transportation, services, and technological advancements from a carbon-intensive supply chain all contribute to the direct and indirect emission of greenhouse gasses (GHGs) in every country.3 Although health care services are essential for maintaining and enhancing human wellbeing, they also have an environmental impact that adds to the environmental risks to human health. Recently, concerns regarding how to make medical practices more sustainable due to the carbon footprints linked with healthcare have emerged.4,5 Healthcare produces a variety of waste streams, each with a distinctive method of disposal and thus a different carbon impact. The material composition and method of disposal, as well as the available options, determine the carbon footprint emerging from healthcare facilities. In order to access such emissions, life cycle assessment (LCA) is a suitable form.6–8\n\nDue to the increased GHG emissions brought on by human activity, climate change is a global phenomenon that affects all sectors.9 The carbon footprint of health care has been calculated previously in some of the developed countries, including the UK, the USA, Australia, Canada, China, Japan. International comparisons and the studies estimated the health care GHG footprint as percentage of the national GHG footprint to be 8–10% in the US, 3% in England, 7% in Australia, and 5% in Canada.3 The entire CO2 eq emissions in Australia in 2014–15 was 494,930 kilotonnes, and the health care sector accounted for 35,772 (7%) of those emissions. According to total CO2 eq emissions, the five most significant areas of the healthcare industry were: other medications (3347 kilotonnes [9%]), benefit-paid drugs (3257 kilotonnes [9%]), public hospitals (12,295 [34%] of 35,772 kilo tonnes CO2 eq), and capital costs for buildings (2776 kilotonnes [8%]).2 There were significant standby energy emissions from scanners.10 The majority of the carbon footprints left by MRIs and CTs are driven by their heavy reliance on electricity, especially standby power.10 For coagulation profile testing, CO2-eq emissions were 82 g/test (95% CI, 73–91 g/test), and for whole blood examination tests, they were 116 g/test (95% CI, 101–135 g/test). CO2 -eq emissions for biochemical tests were 49 g/test (95% CI, 45–53 g/test) for arterial blood gas assessment, 99 g/test (95% CI, 84–113 g/test) for urea and electrolyte assessment, and 0.5 g/test CO2 eq (95% CI, 0.4–0.6 g/test) for C-reactive protein (low because it is frequently ordered with urea and electrolyte assessment). Most CO2-eq emissions (ranging from 60% for whole blood examination to 95% for coagulation profile) were related to sample collection; emissions related to laboratory reagents and electricity use were substantially smaller.10 Japanese healthcare services had an overall carbon footprint of 62.5 × 106 metric tons of CO2 equivalent (Mt CO2 -eq) in 2011, which is 4.6% of all domestic GHG emissions.11 The entire carbon footprint increased by nearly 15% in just four years, reaching 72.0 metric tonnes CO2-eq in 2015.11 China invested CNY 2539 billion on healthcare in 2012, which generated 315 (68% CI 267–363) megatonnes of CO2-eq emissions. A total of 27% (68% CI 23–31) of China's GHG emissions were attributable to the health sector.12 Public hospitals (148 megatonnes [47%]), non-hospital purchased medications (56 megatonnes [18%]), and building (46 megatonnes [15%]) were the main contributors to GHG emissions in the healthcare sector.12 Only 16% of the carbon footprint in medical facilities was produced by energy use for buildings and transportation; the other 84% was caused by the goods and services that have been purchased.12 The carbon footprint of waste streams in a UK hospital was assessed in 2022 using activity data from three hospitals in one UK National Health Service organization, in accordance with the Greenhouse Gas Accounting Sector Guidance for Pharmaceutical Products and Medical Devices. In England, the National Health Service (NHS) produces 538,600 tonnes of waste per year. According to the study, recycling has the lowest carbon footprint per tonne of hospital waste (21–65 kg CO2-eq), followed by low temperature incineration with energy from waste (172–249 kg CO2-eq).13\n\nThe primary objective of this study is to estimate the carbon print of hospitals of Nepal, whereas it also aims to recommend majors that can be used to reduce the emissions from such public sectors. It provides a complete and clearer picture of carbon footprint of south Asian scenario and can be set as a foundation for further case studies.\n\n\nMethods\n\nThe field of study for this study is Dhulikhel Hospital. Dhulikhel Hospital, a Kathmandu University hospital has 18 health centers with 475 medical beds. Dhulikhel hospital is one of the largest hospitals of Nepal. The functional unit considered is kg CO2-eq per year, with the consideration of transportation mediums life cycle of 20,000 km per year. A gate-to-gate analysis has been employed considering the simplicity of analysis, mainly due to the involvement of considerable numbers of sectors to be assessed.\n\nThe hospital has the capability to utilize up to 1430 KVA of electricity, although the actual demand stands at 500 KVA. To meet its power requirements, the hospital employs four generators, with capacities of 250 KVA, 160 KVA, and two generators of 100 KVA each.\n\nIn terms of transportation, Dhulikhel Hospital maintains a fleet of 16 vehicles specifically dedicated to hospital services, comprising a mix of diesel and petrol vehicles. Additionally, the hospital possesses 52 private cars, out of which 44 operate on petrol, 5 on diesel, and 3 are electric vehicles.\n\nFor its energy needs, the hospital relies on four solar panels, collectively possessing a capacity of 285 kWh.\n\nRegarding waste management, Dhulikhel Hospital generates a daily average of 503.51 kg of non-risk waste, 258.21 kg of risk waste, and 167.78 kg of biodegradable waste. The generated waste is collected, segregated, and subsequently transported to appropriate disposal sites and recycling centers.\n\nTo address waste treatment requirements, the hospital has implemented a medium-sized system, incorporating three phases of treatment. This system comprises an Anaerobic Baffled Reactor, a Horizontal Wetland, and a Vertical Wetland, with two systems operating simultaneously. The combined treatment capacity of these systems is 100 m3/day. See Figure 1 for the energy and material flow of the system.\n\nISO 14040:44 standard was applied during the primary data collection. Intergovernmental Panel on Climate Change (IPCC)14 guideline and U.S. Environmental Protection Agency (EPA)15 guideline have been used to calculate vehicle emissions along with previous data. National electricity carbon footprint of Nepal has been calculated by modifying the data available from Joshi et al.16 In the case of life cycle emission of solar systems, total capacity of the system was multiplied by the life cycle emission provided by Mehedi et al.17 Furthermore, waste reduction method was applied to conduct waste audit to categorize biodegradable and non-biodegradable components to estimate their respective carbon footprints. An average of 10 solid waste samples from each month (December 2022–March 2023) was taken for consideration. Nepal Electricity Authority (NEA) installed smart meter at the hospital was observed to estimate hospital energy consumption, whereas average electricity outage per day was calculated according to the data provided from the authority itself. Normalization of data was done according to the functional unit. Onsite visits to the landfill were conducted to perform grab sampling of solid waste and of liquid waste. The calculations of emissions from each source are explained in respective sections. All data used in the study are available in the data file.27\n\nAs for secondary data, it involved extensive analysis of existing data from sources such as government reports, academic studies, and industry publications. These includes Dhulikhel Hospital and Nepal Electricity Authority and others as referenced studies. Information includes data on energy consumption, operational consumables and liquid waste generation as well as data on the environmental impact of the production and disposal of medical equipment and supplies. Additionally, data from medical research studies were analyzed to assess the environmental impact of certain treatments and procedures. Secondary data were at least validated, by comparing and contrasting with other two sources of last five years. Greet Model18 provided by Argonne National Laboratory was also used for the calculation of vehicle emission updating the electricity carbon footprint of Nepal.\n\nDhulikhel Hospital has four generators, 250 KVA, 160 KVA and two generators of 100 KVA capacity.\n\nThe equation used for the estimation of the emissions:\n\nEi = Total emission of substance i from a stationary combustion engine for the reporting year (kg/yr)\n\nP = Engine power capacity rating (kW)\n\nOpHrs = Operating hours of engine during the reporting year (h/yr)\n\nEFi = Emission factor of substance i (kg/kWh)\n\nER = Emission reduction efficiency for substance i (%)\n\nI = Substance i (-)\n\nLF = Load Factor 75%\n\nSince the pollution control technology employed in the diesel generator sets is not clearly known, the emission reduction efficiency is zero and thus the emissions results are 'uncontrolled' emission values.19 Table 1 provides the emission factors for the estimation of total carbon dioxide equivalent emission.\n\nHospitals are energy-intensive buildings that require a constant and reliable supply of electricity to operate critical medical equipment. In Nepal, most hospitals rely on the national grid for their energy needs. Dhulikhel Hospital can use electricity up to 1430 KVA but the actual demand is 500 KVA. Table 2 shows the estimation of carbon intensity of Nepali electricity.\n\nDhulikhel hospital has 16 different diesel and petrol vehicles for hospital services and 52 private cars in which 44 are found to be petrol cars, five are diesel and three are electric vehicles, found by random sampling. Table 3 and 4 show the emissions generated from IC vehicles and carbon footprint of electric vehicles respectively.\n\nFor electric vehicles,\n\nDhulikhel hospital uses 1020 cylinders of gas weighing 15.6 kg per cylinder in a year.\n\nFor the conversion factor, the density of LPG at standard conditions, which is approximately 0.54 kg/L. This means that 1 liter of LPG at standard conditions (i.e., a temperature of 15 °C and a pressure of 1 atm) has a weight of 0.54 kg. Table 5 shows the emission generated from LPG.\n\nDhulikhel hospital has four solar panels having 285 kWh capacity in total. Table 6 shows the carbon footprint of PV system.\n\nDhulikhel hospital generates 503.51 kg of non-risk waste per day and generates 258.21 kg of risk waste per day. Table 7 provides the emissions factors for the respective types of waste.\n\nFor biodegradable waste,\n\nDhulikhel Hospital generates 167.78 kg of biodegradable waste per day. Estimation of Global warming potential, from IPCC 2006 guidelines (IPCC, 2006), default method (Tier 1) is used to determine the CH4 emission from landfill.\n\nMSWT = Total MSW generated (Gg/yr) MSWF = Fraction of MSW disposed at SWDS\n\nLo = Methane generation potential = [MCF × DOC × DOCF × F × 16/12 (Gg CH4/Gg waste)] MCF = Methane correction factor (fraction)\n\nDOC = Degradable organic carbon [fraction (Gg C/Gg MSW)] DOCF = Fraction DOC dissimilated\n\nF = Fraction by volume of CH4 in landfill gas R = Recovered CH4\n\nOX = Oxidation factor (fraction)\n\nThe direct carbon dioxide (CO2) emissions from the treatment processes and final disposal of organic waste are considered biogenic and are not included in the carbon footprint accounting (IPCC, 2006). This is because organic waste is made up of carbon that was recently taken from the atmosphere by the plants that the waste originated from. When the waste is treated or disposed of, the carbon in the waste is returned to the atmosphere in the form of CO2, which is then taken up by new plant growth in a continuous cycle of carbon uptake and release.\n\nA medium-sized system having three phases of treatment (Anaerobic Baffled Reactor, Horizontal Wetland, and Vertical Wetland) with two systems operating together. To conclude the wastewater treatment process, the system includes a sludge drying bed. The wastewater treatment plant in Dhulikhel Hospital treats 160 m3/d of wastewater each day. See Table 8 for the emission from wastewater.\n\nCH4 Emission factor for Wastewater Treatment/Discharge Pathway or System,\n\nEFj = emission factor, kg CH4/kg BOD\n\nj = each treatment/discharge pathway or system\n\nBo = maximum CH4 producing capacity, kg CH4/kg BOD = 0.6\n\nMCFj = methane correction factor (fraction) = 0.8\n\n\nResults\n\nTotal carbon emissions from the generators were found to be 58,780 kg-CO2-eq/yr. Along with total CO2-eq, the total local air pollutants; PM10, CO, VOCs, SO2 and NOX were found to be 112 kg/yr, 339 kg/yr, 125 kg/yr, 15 kg/yr and 1569 kg/yr, respectively. Figures 2 and 3 show the emission from the generators highlighting that the 250 kVA generator has the maximum emission. The emissions were found to be comparatively lower than the mentioned study as the generators is not operated in full time condition. The generators in hospital are operated in an average of 30 minutes per day (182.5 hours/yr) to ensure that they are working properly.\n\nAccording to a study by the International Hydropower Association (IHA), the carbon footprint of hydropower is about 24 g-CO2-eq/kWh, which is much lower than the carbon emission of fossil fuels like coal (over 1000 g-CO2-eq/kWh).23 The hospital annually consumes 1,942,845 KWh electricity, and with the imported electricity carbon footprint (see Data File27), the GHG emission of electricity consumption was found to be 519,794 kg-CO2-eq/yr. The carbon emission is found to be highest in the month of June–July as shown in Figure 4, as the electricity consumption rate was highest.\n\nThe total carbon emission of Dhulikhel Hospital is quite high as its emission factor is the weighted average of electricity produced in Nepal and imports from India (coal), with T&D loss of Nepalese grid being 15%.\n\nThe total carbon footprint of 16 hospital vehicles was found to be 127,453 kg-CO2-eq/yr, and the other local polluting agents: PM2.5, NOx, CO and VOC were calculated to be 289 kg/yr, 2115 kg/yr, 2053 kg/yr and 481 kg/yr, respectively. The buses used for staff transportation exhibit the highest levels of carbon emissions with 26,994 kg-CO2-eq and 29,582 kg-CO2-eq annually as shown in Figures 5 and 6. Compared to other vehicles used by the hospital, the buses also emit the highest levels of local air pollutants annually.\n\nThe total carbon emissions from 44 Spark Ignition (SI) vehicles were found to be 124,523 kg-CO2-eq/yr, i.e., one SI vehicle emits 2830 kg-CO2-eq/yr, while used for transportation of hospital activity only, while five Compression Ignition (Diesel) vehicles emit 14,334 kg-CO2-eq/yr, i.e., one CI vehicle emits 2867 kg-CO2-eq/yr while used for transportation of hospital activity only. Diesel vehicles emit a slightly higher carbon footprint compared to Spark ignition vehicles due to their superior fuel efficiency. In comparison to other local polluting agents, the total amount of CO found was the highest, measuring 7039 kg/yr in total, as shown in Figures 7 and 8.\n\nThe carbon emissions from electric cars were found to be relatively lower, measuring 2022 kg- CO2-eq/yr, as compared to those from petrol and diesel cars because they do not have traditional internal combustion, they use battery as power source. So, electric vehicles (EVs) do not emit GHGs like CO2, CO, PM, NOx during their operation. EVs that release no greenhouse gases locally, are considered to be a potential response to the concerns of climate change and environmental pollution.24\n\nLiquified Petroleum Gas (LPG) is a clean alternative to traditional solid fuels that has received widespread attention due to its potential for scale. However, the climate, environmental, and health benefits or impacts of widespread LPG adoption in various low-income countries settings have not been thoroughly studied.25 In our case, it can be observed that the total amount of LPG in 1020 cylinders consumed by hospital would be 15,912 kg. Then, using the carbon emission factor of 1.51 kg CO2 per kg of LPG burned, it is found that the carbon footprint from consumption of LPG would be 44494 kg CO2-eq/yr.\n\nThe hospital uses a PV system as a backup power source, operational during power cut-offs. Using the emission factor 123.8 g-CO2-eq/kWh, carbon emission was found to be 35,283 kg-CO2-eq/yr. Case studies like Bhagat Chandra Hospital and Gundersen Health System have successfully reduced their energy consumption by 20-30% and fully converted from fossil fuels to locally produced energy, resulting in annual operational savings of USD 3.7 million.26 By incorporating 50 kW solar panels that are integrated with the electrical system, the hospital has reduced its energy consumption by 20–30% and avoided 93,000 kg of CO2 emissions since 2016. However, this option isn’t feasible in Nepal due to low carbon footprint of electricity from national grid in Nepal having low carbon intensity.\n\nBased on an assessment of the waste composition at Dhulikhel Hospital, it has been determined that approximately 217.42 kilograms of waste per day may be suitable for autoclaving and subsequent reuse. However, certain types of hospital waste, including sharps such as needles, require additional processing such as shredding or melting to ensure their safe disposal. On the other hand, materials such as paper and plastic can be effectively recycled using anaerobic digestion and autoclaving, which has the potential to significantly reduce the overall emissions associated with waste management at the hospital. The hospital generates 167.78 kg of biodegradable waste per day, emitting 3822 kg CH4 per year. Organic waste from the hospital can be converted into energy through various techniques such as anaerobic digestion and composting. The energy recovery potential from anaerobic digestion of sorted and transported organic waste was calculated to be 2252 kWh.\n\nAccording to the study, Dhulikhel Hospital generates an estimated 489,835 kilograms of carbon dioxide (CO2) annually through the disposal of its inorganic solid waste. Figure 9 depicts that hospital waste plays a significant role for greenhouse gas emissions, and highlights importance of implementing sustainable waste management practices to mitigate its environmental impact.\n\nDhulikhel Hospital generates about 11.8272 Kg CH4/BOD liquid waste daily. Likewise, the emission factor of liquid waste has been determined to be 0.48 kg CH4/kg BOD. Based on these data, the total annual emission was estimated to be 4317 kg CH4/BOD yr for a wastewater treatment plant operating at 70% efficiency and generating 160 m3/d of wastewater.\n\nA gas collection system can be installed to capture and transport the methane gas produced during treatment to a biogas utilization system (a type of renewable energy system that converts organic waste into biogas, which can be used for heating, cooking, and electricity generation) to generate electricity or heat and minimize methane emission.\n\nAccording to the study, electricity consumption of the hospital was found to have high the solid waste produced by hospitals, which is primarily composed of inorganic materials, has been found to contribute significantly to greenhouse gas emissions, with an estimated total of 489,835 kg-CO2/yr. Electricity consumption has been found to have the highest carbon footprint among all the activities considered in the study as shown in Figure 10.\n\nAdditionally, the biodegradable waste generated by hospitals, such as food and organic matter, emits approximately 3822 kg-CH4/yr. Moreover, the liquid waste produced by hospitals, which contains significant amounts of organic matter, was found to emit 4316 kg-CH4/BOD/yr. These emissions of methane, a potent greenhouse gas, from hospital waste have a significant impact on the environment and contribute to climate change.\n\nThese findings highlight the importance of proper waste management practices in the healthcare industry, as reducing the amount of waste generated and effectively managing the waste that is produced can help mitigate the environmental impact of hospital operations.\n\n\nDiscussion\n\nFrom this study, it can be recapitulated that electricity consumption leads to CO2-eq emissions. The emission is accounted from the carbon intensity of national grid electricity of Nepal, which in turn uses carbon intensive electricity from Indian grid. Likewise, the environmental impact of transportation is more than that of photovoltaic system (PV system), Generator and liquefied petroleum gas (LPG) due to addition of use of conventional Spark Ignition (Petrol) and Compression Ignition (Diesel) vehicles for hospital transportation purposes.\n\nThis GtoG LCA study only performs static analysis and does not consider any dynamic variable changes, which may be crucial for future scenario analysis. However, no future scenarios have been studied in this study, a potential intervention for reducing emissions has been discussed. Furthermore, only GHG emission has been considered as an environmental impact category whereas freshwater eco-toxicity level (FETP), human-toxicity potential (HTP) and mineral depletion potential (MDP) could also be important. Similarly, gate-to-cradle (GtoC) LCA can incorporate recycling associated emissions as well as potential GHG avoid. This study sets as a foundation for further extending impact analysis.\n\nDhulikhel Hospital has the potential to significantly reduce its carbon emissions by approximately 21,571 kg-CO2-eq/yr through the transition from diesel-powered buses to electric-powered ones. The terms “emission from the real scenario (A)” and “emission after intervention (B)” refer to two distinct measurements of emissions, as mentioned in Table 8. Specifically, “emission from the real scenario (A)” represents the actual emissions generated by staff transportation buses, while “emission after intervention (B)” represents the emissions resulting from the replacement of petrol and diesel buses with electric buses. The hospital's existing photovoltaic (PV) system can be used to substitute the conventional Nepal Electricity Authority (NEA) grid, which is powered primarily by electricity imports from India (coal sourced), for up to eight hours per day. The two sets of emissions data in Table 8 are labeled “Emission from real scenario (A)” and “Emission after Intervention.” The initial category relates to emissions produced when the PV system is not in operation, whereas the other refers to emissions measured after the PV system has been installed. This substitution can play a vital role in reducing the hospital's carbon footprint.\n\nThe actual scenario indicates that the maximum emissions are from transportation, electricity consumption, and solid waste generated by the hospital. After implementing interventions to reduce the amount of carbon dioxide and other greenhouse gases emitted into the atmosphere, the hospital can reduce its carbon footprint. See Table 9 for the emission generated in actual scenario and interventive scenario.\n\nBy relying solely on hydropower-generated electricity, the hospital could reduce its carbon footprint by an estimated 467871 kg-CO2-eq/yr. The two terms, “Emission from real scenario (A)” and “Emission after Intervention (B),” refer to two different measures of total emissions. Specifically, “Emission from real scenario (A)” denotes the total emissions resulting from energy consumption sourced from the NEA grid, which is supplied by coal from India. On the other hand, “Emission after Intervention (B)” represents the total emissions generated after energy consumption from hydropower, which is anticipated to be used in the near future. This reduction is primarily due to the fact that hydropower has substantially lower emissions in comparison to coal.\n\nIntervention for reducing the carbon footprint in the waste sector of hospitals can involve implementing a “reduce, reuse, recycle, and compost” approach. This can be achieved by adopting a waste reduction and recycling program that involves segregating waste into different categories such as recyclable, organic, and hazardous waste. A system could be developed to ensure that each type of waste is managed appropriately.\n\nBy implementing this intervention, hospitals can significantly reduce their carbon footprint by diverting waste from landfills, which are major sources of greenhouse gas emissions. Recycling and composting can also reduce the need for new raw materials, which further reduces the carbon footprint of hospitals.\n\nThe study highlights the urgent need for the Nepalese healthcare system to adopt sustainable practices that reduce carbon emissions and mitigate their impact on the environment. The results of the study suggest that the Nepalese healthcare system, like many others around the world, is a significant contributor to climate change and its associated risks.\n\n\nConclusions\n\nBased on the findings of the study, several conclusions can be drawn. The total carbon emissions from the generators of the hospital were found to be 58,780 kg- CO2-eq/yr. In addition to the CO2-eq emissions, other local air pollutants were also measured, including PM10, CO, VOCs, SO2 and NOx. It is not possible to completely eliminate emissions generated by the operation of generators, as regular operation is necessary to maintain proper lubrication of its engine components and fuel circulation through the system. The hospital's electricity consumption from NEA grid was found to have a carbon footprint of 519,794 kg-CO2-eq/yr. The utilization of a PV system as a substitute for the conventional Nepal Electricity Authority (NEA) grid, which relies mainly on coal sourced from India, for a maximum of eight hours daily can have a significant impact on reducing the carbon footprint of the hospital. The combined carbon footprint of 52 private cars and 16 hospital vehicles was found to be estimated to be 272,374 kg-CO2-eq/yr. In addition to the carbon footprint, the study also measured the emission of other local pollutants such as PM2.5, NOx, CO, and VOC. Replacing only the petrol and diesel buses used for staff transportation with electric buses can result in a significant reduction in carbon footprint and also co-benefit local pollution. The hospital's use of LPG and PV system also contributed to the overall carbon footprint of the hospital. The study highlights the significant impact of Dhulikhel Hospital's inorganic waste on greenhouse gas emissions, emphasizing the need for sustainable waste management practices. Adopting appropriate waste management strategies that are suited to the specific waste stream characteristics, such as autoclaving and subsequent recycling or energy recovery, can greatly reduce the overall emissions associated with waste management. By implementing such practices, hospitals like Dhulikhel can contribute to mitigating the environmental impact of their operations and pave the way towards a more sustainable future. The hospital emitted 4317 kg CH4/BOD per year from its wastewater treatment plant. A gas collection system can be installed to capture and transport the methane gas produced during treatment to a biogas utilization system to generate electricity or heat and minimize methane emission.", "appendix": "Data availability\n\nZenodo: CARBON FOOTPRINT OF NEPALESE HEALTHCARE SYSTEM-A STUDY OF DHULIKHEL HOSPITAL. https://doi.org/10.5281/zenodo.8229778. 27\n\nThe project contains the following underlying data:\n\n- Data File.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nPichler P-P, Jaccard IS, Weisz U, et al.: International comparison of health care carbon footprints. Environ. Res. Lett. May 2019; 14(6): 064004. Publisher Full Text\n\nMalik A, Lenzen M, McAlister S, et al.: The carbon footprint of Australian health care. Lancet Planet. Health. Jan. 2018; 2(1): e27–e35. PubMed Abstract \| Publisher Full Text\n\nLenzen M, et al.: The environmental footprint of health care: a global assessment. Lancet Planet. Health. Jul. 2020; 4(7): e271–e279. Publisher Full Text\n\nTsagkaris C, et al.: Using telemedicine for a lower carbon footprint in healthcare: A twofold tale of healing. J. Clim. Change Health. Mar. 2021; 1: 100006. Publisher Full Text\n\nMolero A, Calabrò M, Vignes M, et al.: Sustainability in Healthcare: Perspectives and Reflections Regarding Laboratory Medicine. Ann. Lab. Med. Mar. 2021; 41(2): 139–144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarancha V, Vitale K, Oreskovic S, et al.: LIFE CYCLE ASSESSMENT IN HEALTHCARE SYSTEM OPTIMIZATION. INTRODUCTION. Bull. Taras Shevchenko Natl. Univ. Kyiv Econ. Oct. 2015; 39–44. Accessed: Jun. 17, 2023. Publisher Full Text Reference Source\n\nMcGinnis S, Johnson-Privitera C, Nunziato JD, et al.: Environmental Life Cycle Assessment in Medical Practice: A User’s Guide. Obstet. Gynecol. Surv. Jul. 2021; 76(7): 417–428. PubMed Abstract \| Publisher Full Text\n\nBotejara-Antúnez M, Garrido-Píriz P, Sánchez-Barroso G, et al.: Life Cycle Assessment (LCA) in the construction of healthcare buildings. Analysis of environmental impact. IOP Conf. Ser. Earth Environ. Sci. May 2021; 664(1): 012053. Publisher Full Text\n\nVan Aalst MK: The impacts of climate change on the risk of natural disasters. Disasters. 2006; 30(1): 5–18. Publisher Full Text\n\nMcAlister S, et al.: The carbon footprint of hospital diagnostic imaging in Australia. Lancet Reg. Health – West. Pac. Jul. 2022; 24: 100459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNansai K, Fry J, Malik A, et al.: Carbon footprint of Japanese health care services from 2011 to 2015. Resour. Conserv. Recycl. Jan. 2020; 152: 104525. Publisher Full Text\n\nWu R: The carbon footprint of the Chinese health-care system: an environmentally extended input–output and structural path analysis study. Lancet Planet. Health. Oct. 2019; 3(10): e413–e419. PubMed Abstract \| Publisher Full Text\n\nRizan C, Bhutta MF, Reed M, et al.: The carbon footprint of waste streams in a UK hospital. J. Clean. Prod. Mar. 2021; 286: 125446. Publisher Full Text\n\nEggleston HS, Buendia L, Miwa K, et al.; 2006 IPCC Guidelines for National Greenhouse Gas Inventories.Jul. 2006. Accessed: Jun. 17, 2023. Reference Source\n\nO. US EPA: Regulations for Emissions from Heavy Equipment with Compression-Ignition (Diesel) Engines.Dec. 28, 2016. (accessed Jun. 17, 2023). Reference Source\n\nJoshi A, Sharma R, Baral B: Comparative life cycle assessment of conventional combustion engine vehicle, battery electric vehicle and fuel cell electric vehicle in Nepal. J. Clean. Prod. Dec. 2022; 379: 134407. Publisher Full Text\n\nMehedi TH, Gemechu E, Kumar A: Life cycle greenhouse gas emissions and energy footprints of utility-scale solar energy systems. Appl. Energy. May 2022; 314: 118918. Publisher Full Text\n\nArgonne GREET Publication: Summary of Expansions and Updates in GREET® 2021. (accessed Apr. 21, 2022). Reference Source\n\nNational Pollutant Inventory - DCCEEW. (accessed Jun. 17, 2023). Reference Source\n\nGoel R, Guttikunda SK: Evolution of on-road vehicle exhaust emissions in Delhi. Atmos. Environ. Mar. 2015; 105: 78–90. Publisher Full Text\n\nHait A, Powers SE: The value of reusable feminine hygiene products evaluated by comparative environmental life cycle assessment. Resour. Conserv. Recycl. Nov. 2019; 150: 104422. Publisher Full Text\n\nGautam A, et al.: Hospital wastewater treatment in Nepal: Status, challenges and future perspectives (case studies of effluent management in Nepal).Jul. 2022. Publisher Full Text\n\nKumar A, Sharma MP, Kumar A: Green House Gas emissions from Hydropower Reservoirs: Policy and Challenges. Int. J. Renew. Energy Res. IJRER. Jun. 2016; 6(2). Art. no. 2.\n\nGhosh A: Possibilities and Challenges for the Inclusion of the Electric Vehicle (EV) to Reduce the Carbon Footprint in the Transport Sector: A Review. Energies. Jan. 2020; 13(10). Art. no. 10. Publisher Full Text\n\nFloess E, et al.: Global Climate, Environmental, and Health Benefits of Replacing Traditional Solid Fuels with Liquified Petroleum Gas.2021 Dec; 2021: pp. GC13C-07.\n\nSalas RN, Maibach E, Pencheon D, et al.: A pathway to net zero emissions for healthcare. BMJ. Oct. 2020; 371: m3785. PubMed Abstract \| Publisher Full Text\n\nAdhikari B, Dangal A, Pandey S, et al.: CARBON FOOTPRINT OF NEPALESE HEALTHCARE SYSTEM-A STUDY OF DHULIKHEL HOSPITAL. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "222216", "date": "16 Nov 2023", "name": "Khem Gyanwali", "expertise": [ "Reviewer Expertise Energy", "Environment and Economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study holds contemporaneous and consequential significance within the domain of the Nepalese healthcare sector, presenting a prospective alignment with the governmental pursuit of achieving net-zero emissions. Nevertheless, several inquiries necessitate clarification. Primarily, the nature of the life cycle assessment (LCA) employed in this research warrants elucidation, specifically discerning whether it adopts a static or dynamic LCA methodology. The relevance of this distinction emerges notably in the evaluation and formulation of prospective scenarios, where a static LCA may lack meaningful applicability. It is imperative to ascertain the nature of the LCA utilized. Additionally, the importation of electricity from India by Nepal introduces a pertinent consideration. A comprehensive exploration of the authors' contemplation of this condition is warranted. Furthermore, an enhanced explication regarding the emissions stemming from vehicular sources is sought, with a particular focus on the potential ramifications in the event of alterations in material flow. Clarification on the envisaged trajectory of vehicular emissions amid shifts in material flow patterns is essential for a comprehensive understanding of the study's implications.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10587", "date": "16 Nov 2023", "name": "Ashim Joshi", "role": "Author Response", "response": "1) Though the LCA is not fully dynamic in nature the parameters considered for the analysis of future scenarios supports the dynamicity of the LCA within the considered boundary. 2)The electricity emission factor of Nepal used in the study is in consideration with the importation of electricity from India as well. 3) Definitely, the emission generated from vehicles changes as the material flow changes, especially during manufacturing phase. However, it was beyond the scope of the study to examine the future emission scenario of vehicles within the GtoG approach." } ] }, { "id": "219318", "date": "28 Nov 2023", "name": "Kestutis Venslauskas", "expertise": [ "Reviewer Expertise Environmental engineering", "sustainability", "LCA", "renewable energy", "bioenergy" ], "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 has many serious limitations:\nAbstract lacks the size of the hospital, there is no functional unit explained, no methodology given. Aim of study in abstract is wrong \"... emissions from healthcare sectors\" - you analyse only one small hospital, but not whole sector.\n\nText contains many typing errors, different measuring units, unidentified abbreviations (MRT, CT, CI), various indexes and subscripts errors.\n\nSentence \"According to the study, recycling has the lowest carbon footprint per tonne of hospital waste (21–65 kg CO2-eq), followed by low temperature incineration with energy from waste (172–249 kg CO2-eq).\" has wrong interpretation. If waste is incinerated, then it generates energy, and CO2 emissions should be negative.\n\nSentence \"The primary objective of this study is to estimate the carbon print of hospitals\" should be \"footprint\".\n\nElectricity is not measured by KVA. kVA is power, but not electricity. As well \"For its energy needs, the hospital relies on four solar panels, collectively possessing a capacity of 285 kWh\" - PV capacity is measured by kWp of installed power, but surely not by kWh.\n\nIt is not clear how waste with Risk and no-Risk are separated or mixed together at collection stage and transportation?\n\nFunctional unit of LCA is not provided. LCA methodology is not clearly presented in internationally acceptable way.\n\nIndicate emissions for \"In the case of life cycle emission of solar systems, total capacity of the system was multiplied by the life cycle emission provided by Mehedi et al\".\n\n\"Normalization of data was done according to the functional unit.\" - denote and describe your functional unit.\n\nFig 1 is completely wrong. a) diesel or petrol must go to Generator as well; b) arrow from Hospital should not go to Generator and PV system. Arrows must go from mentioned systems to Hospital. c) what is Generation (close to liquid waste)? d) why arrow goes to Operational consumables? e) Arrow must go from Supply from NEA to Electricity, but not wise-versa. f) Title of Fig 1 \"Energy and material flow\" - where is energy and materials? It is not indicated where is what.\n\n\"Additionally, data from medical research studies were analyzed to assess the environmental impact of certain treatments and procedures\" - indicate what are \"certain procedures\".\n\nFurther from page 5, the methodology is not readable. It is not clear where are the explanations, where are the equations, there are no references for equations. Equations are wrong (e.g. The equation used for the estimation of the emissions - has P = Engine power capacity rating (kW), but you indicate in kVA). Some text is repeated in several places the same.\n\n\"Dhulikhel hospital has 16 different diesel and petrol vehicles for hospital services and 52 private cars in which 44 are found to be petrol cars, five are diesel and three are electric vehicles, found by random sampling\" - how was it found by random sampling? Please give a table with parameters of vehicles.\n\nSome tables: 2; 4; 5; 6; 8 can be explained in text by words and numbers.\n\nIn many cases not SI system units are used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "219322", "date": "23 Jan 2024", "name": "Nawraj Bhattarai", "expertise": [ "Reviewer Expertise Energy Policy", "Environment", "Renewable and Climate Change" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 carried out Life Cycle Assessment LCA to determine carbon footprint from healthcare facilities. However, the type of LCA carried out is unclear. Is the LCA conducted a Static LCA or Dynamic LCA? As the GtoG LCA of only one hospital has been carried out in this study, how do the findings generalize for Nepalese healthcare? It is mentioned that the emission reduction efficiency is zero and emissions are “Uncontrolled Emissions”. Clarification on what has been termed as “Uncontrolled Emission Values”. IC Vehicles have different fuel consumption on different load conditions/terrain situations. In the study single value has been used for fuel efficiency, why? As the Generators are used on average 30 minutes per day only to ensure proper functioning, has it been included in overall emissions due to having a significant footprint or considering that in case of power outage, its usage then would increase the overall carbon footprint of the Hospital?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10975", "date": "22 Mar 2024", "name": "Ashim Joshi", "role": "Author Response", "response": "This GtoG LCA study only performs static analysis and does not consider any dynamic variable changes, which may be crucial for future scenario analysis. It has also been stated clearly in the paper. The hospital taken for the case study is one of the largest hospitals in Nepal. And all other hospitals are of similar capacity. Since the pollution control technology employed in the diesel generator sets is not known, the emission reduction efficiency is considered zero and thus the emissions results are 'uncontrolled' emission values. The emission of IC vehicles is an average of emissions on different terrain conditions. Emissions of generators have been included." } ] }, { "id": "222215", "date": "24 Jan 2024", "name": "Sanjaynath Khanal", "expertise": [ "Reviewer Expertise Environmental 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\nThe article adequately puts the content and details of analysis and findings. However, I have the following observations for upgrading the article:\nJustify the study in Nepalese context with adequate background information. Method: Explain Functional unit/GtoG/mention about current and intervention analysis in methodology. Provide source of information on details of DH emission sources. Fig 1: Kathmandu not necessary. Mention specifics of collection, segregation and disposal. Fig 2: Should be in results. Table 9: Intervention analysis not mentioned above. Mention as appropriate. Fig 10: Does not include waste but mentioned in text. Update appropriately. Discuss why electricity consumption has the highest emission. Edit the text to make it more concise and focused.\n\nIs the work 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": "10976", "date": "22 Mar 2024", "name": "Ashim Joshi", "role": "Author Response", "response": "Concerns have been addressed in the revised paper." } ] } ]	1	https://f1000research.com/articles/12-1366
https://f1000research.com/articles/13-92/v1	05 Feb 24	{ "type": "Research Article", "title": "Salutogenesis: A sense of coherence and health among British military veterans exposed to impactful life challenges - the emergence of post-traumatic growth and positive deviance within a life-story approach", "authors": [ "Richard Mottershead", "Muhammad Arsyad Subu", "Nabeel Al-Yateem", "Wegdan Bani-Issa", "Fatma Refaat Ahmed", "Mini Sara Abraham", "Jacqueline Maria Dias", "Mustafa Muhamad Habeb", "Nafi Alonaizi", "Muhammad Arsyad Subu", "Nabeel Al-Yateem", "Wegdan Bani-Issa", "Fatma Refaat Ahmed", "Mini Sara Abraham", "Jacqueline Maria Dias", "Mustafa Muhamad Habeb", "Nafi Alonaizi" ], "abstract": "Background This study sought to capture the perspectives and experiences of two sub-groups of military veterans, namely those who had previously been identified as offenders and those employed as practitioners within the criminal justice system in the United Kingdom.\n\nMethods The lead author undertook narrative inquiry in the form of life story research. The life stories of 17 in-depth interviews were conducted across England and Wales, allowing for insight into the lived experiences of two life story trajectories of the health of military veterans. Existing literature on salutogenesis, health, post-traumatic growth, and positive deviance has also been investigated.\n\nResults Life story interviews were transcribed verbatim and analyzed concurrently using thematic analysis to identify emergent themes. The researchers used thematic analysis as an analytical framework to allow descriptive themes from the literature on salutogenesis, health, post-traumatic, and positive deviance to be compared with those of life-story interviews.\n\nConclusion The study adopts a salutogenic approach, which suggests that an important indicator for a sense of coherence is enabled through the positive utilization of resilience through the reconstruction of military identity and experience. Notably, the concept of salutogenesis demonstrates the ability to draw from internal and external resources as circumstances require, to survive, and because of this, the participants’ health is maintained or improved. Hence, adaptation was notable through the coherence identified by the identity of being a veteran noted between the two subgroups and represents a continuous and dynamic process. The study suggests that the development of a sense of coherence is not restricted to the early decades of life but is a continuous process as contexts, personal circumstances, and opportunities arise. This later point is realized through the presence of post-traumatic growth and positive deviance, as they aid in the nurturing and development of a renewed sense of coherence via the known identity of military service and life.", "keywords": [ "Salutogenesis", "Veteran", "Health", "Sense of Coherence", "Post-Traumatic Growth", "Positive Deviance." ], "content": "Introduction\n\nThe authors begin this paper by highlighting that most individuals (86%) leaving the Armed Forces transition into civilian life without difficulty and obtain employment which consequently allows for a positive contribution to society (Lord Ashcroft, 2014; Mottershead, 2019; Mottershead & Alonaizi, 2021). This paper explores the lived experiences of military veterans and themes that have become prominent features within the landscape of their life stories. The authors explored the concept of salutogenesis on the health care needs of these subgroups and their sense of coherence (SOC). The life stories voiced the struggles that the veterans encountered to maintain an identity within the presence of the stigma and associated shame of being identified as offenders. This study is reminiscent of the seminal work of Goffman (1963), who defined stigma as an attribute that is deeply discrediting to the individual. The life stories initially collected by the lead author provide insight into the veteran’s personal accounts of the impact of stigma and shame and the harm that is caused by the transitional friction encountered by the veteran and their family in being labelled as an offender that holds negative connotations to self-image and contrasts to having previously been identified as soldiers, sailors, or aircrews. In the study by Mottershead (2019), the life stories of the participants indicated that there was evidence that for some veterans, their life story trajectory found a sub-group to become segregated and isolated from a familiar veteran identity with few resources to survive the transition to a civilian life and new civilian identity. This exploratory qualitative study provides emancipatory evidence of the health and self of coherence and the impact of entering the criminal justice system (CJS) as offenders. According to McMurran, Khalifa, and Gibbon (2009), in England and Wales, the criminal justice process is delivered by a number of agencies that work collectively under the umbrella of the CJS. These include the Police, the Crown Prosecution Service, Her Majesty’s Court Service, the Youth Justice Board, Probation Service and the Prison Service. McMurran et al. (2009, p.2) further states that the ‘overall aims of the CJS is to detect and prevent crime, to rehabilitate and punish offenders and to support victims and witnesses of crime.\n\nThe aim of this study was to highlight evidence of post-traumatic growth and positive deviance. These two concepts provide inspiration and insight into how comradeship between military veterans is a cherished bond that appears to have been a source of motivation to address the health and social care needs of others. The participants also provided informed insights into the issues relating to veterans and sought to develop new systems needed to expand the knowledge base on the identification, diversion, and management of veterans and how to support those with mental health needs. The authors adopted a life story approach due to the evidence base that it constituted a practical way of gathering information about the veterans’ lives, which enabled others to step inside the personal world of the storyteller and experience other aspects of human experience (Josselson and Lieblich, 1999; Ochberg, 1994). Braun and Clarke (2006, 2019) recommended that thematic analysis be adopted as part of multiple qualitative methods, as utilized within the methodology of this study. These researchers described thematic analysis as a process of visualizing and encoding qualitative research material through the formation of codes and themes, which will be discussed in the next subsection.\n\nHealth and social care research has long aligned itself with narrative approaches, with some notable seminal examples of life-story methods that will be highlighted in this section. In terms of studying veterans’ pathway into the CJS and specific health needs, life stories go beyond diagnosis, where a question-and-answer style is used to gather information (O’Connor and Coleman, 1995). Gadd and Farrall (2004) emphasize that life stories also have a focus that is not associated with statistical risk-based studies on behavior. In relation to this study, this method of data collection enabled the veterans (participants) to position themselves in relation to discussing their health, how they deal with stress, stay well and improve their health, and maintain or develop resilience. Goodey (2000) advocates the use of narrative methods as a way of exploring how and why the individual has become dependent and, in relation to this study, how the individual has overcome and adapted to social structural forces that provided constraints or opportunities in the veterans’ life. Goodey (2000) explains that this approach is beneficial in that it supports the participant in identifying the relevant life choices that are significant in their story and therefore allows the authors to provide insight into the presence of post-traumatic growth and positive deviance within the narratives.\n\nThis approach is invaluable for comprehending veterans’ perspectives on their health and life trajectories. If the veteran’s life became absorbed with maladaptive behaviors unable to cope with the stresses of life, their social network diminished, and it was often left to statutory or charitable services to assist. This study included some veterans who had been incarcerated within the CJS due to offending behavior. According to McKendy (2006), the CJS exerts considerable powers of enforcement, but this study demonstrated that confinement went beyond physical realization as well as through discursive confinement as the participants experienced a diminished sense of coherence. The lead author sought to access these life stories to prevent restrictions on understanding and to ascertain meaning. When looking at the work of Maruna (2001), there is clear evidence that using life story methods with those identified as disenfranchised provides an opportunity for participants to reclaim their voice and develop a deeper understanding and awareness of themselves and their relationships with others. McKendy (2006) supports the approach of the authors, who ensure that this method is adopted so that the veterans can structure and interact with the interviewer and speak at length about their experiences. Leichtentritt and Arad (2005) explained that by using life stories, there is the potential to explore the social, emotional, interpersonal, health, accommodation, educational, and employment factors that have affected the life choices of veterans. In exploring the human element in the veterans account, the life story may prove invaluable in limiting the “us versus them” dichotomy between veterans having a dependency as opposed to them being perceived as positive contributors and protectors of national interests (Williams, 2006; Mottershead, 2019). This is supported by Bochner (2015) and directed this study to include life story research to allow this methodology to empower participants to make sense of their lives. This approach was crucial to this study as placing the veterans’ experience of health and post-traumatic growth in a tangible form, which could then be transcribed and analyzed for meaning by the research team. Atkinson (2007) attests to the credibility of this approach, as it can create in-depth subjective meanings and enables the storyteller to impart life events in a way that they choose and want others to comprehend. This was important, as it facilitated the veterans to feel that they had ownership of their narratives and were able to articulate their life challenges so that meaning and understanding could be conveyed. Plummer (2001) stresses that although the researcher provides guidance, it is the participants’ voice that determines the frame of reference for the story, and the method captures the interactions between the individual and social world. According to Plummer (2001), this may include moments of indecision, turning points, confusion, and ambiguities common to everyday experience. At the commencement of this study, it was anticipated that there would be a wide variety and degree of complexity within the veterans’ life stories, specifically when exploring the impact of stigma, self-image, and evidence of post-traumatic growth. A comprehensive and adaptable methodological approach was required to support the meaning-making process of life stories; therefore, thematic analysis was selected.\n\nAs advised by Holloway and Todres (2003), this framework would need to be relevant to the research question of this study and was necessary to create an efficient method of analyzing the life stories of individual veterans. The life-story interviews were therefore analyzed using thematic analysis, as utilized by Boyatzis (1998) and Braun and Clarke (2006). This process of analyzing life stories was selected because of its flexible and straightforward technique that created an evidenced theoretical framework, which could provide insight into the lived experiences of veterans and their health and well-being. In this study, the lead author explored and revisited the data within the life story transcripts to produce pertinent health-related codes, then cross-referencing the whole data again to analyze coded extracts. Braun and Clarke (2006) stipulate that this constant moving back and forth is a necessity to establish the themes that establish meaning within the research. Table 1 illustrates the phases of analysis adapted from Braun and Clarke (2006, 2019).\n\nThis systematic approach adhered to the recommendations of Braun and Clarke (2006) and Savin-Baden and Major (2013), as this is a crucial procedure for identifying, analyzing, and producing patterns or themes found within the data. Thematic analysis was adopted because of its merits as an analytical device for constructing order and developing the main arguments of the topic for this study (Guest et al., 2012). Braun and Clarke (2006) stated that one of the main benefits of thematic analysis is its flexibility, which is attractive when trying to identify themes with this sub-group of veterans.\n\nEtherington (2008) emphasized the need to be systematic when organizing life-story themes. Therefore, the lead author provided a chronological order of all the veterans’ stories within the life story trajectories of life before military service, during military service, and the period after discharge, thereby creating an aggregated story of all veterans’ stories. Etherington (2008) recommends that the focus on these stages of the life story trajectory allows the researcher to undertake numerous cycles of questioning, reflecting, rephrasing, theorizing, analyzing, and verifying the data, a quality assurance process that is evidenced to enhance the research findings. The researcher created a thematic map, as advocated by Braun and Clarke (2006), which created an image of the produced codes that galvanized the creation of the themes focused on within this study, as illustrated in Figure 1.\n\n\nMethods\n\nThis research study was submitted to the University of Chester’s Faculty of Health and Social Care Research Ethics Committee and was judged to be ethically robust and permission was granted to commence the data collection phase by a team of independent academics (RESC0513-413 – approved: 30.7.2013). All participants provided written informed consent to participate and for their anonymized data to be used for the purposes of this study.\n\nBoth subgroups were interviewed through the use of semi-structured interviews, but given the difference in their contact with the CJS, there was a need to create two separate sets of questions within an interview schedule (Table 2).\n\n\n\n• Please describe your life prior to entering the Armed Forces\n\n- Had you any involvement in the CJS prior to joining the Armed Forces?\n\n• What caused you to join the Armed Forces?\n\n• Please describe your career within the Armed Forces\n\n• What caused you to leave the Armed Forces?\n\n• What where your initial experiences when you re-entered ‘civvie street?’\n\n• Do you believe you handled the transition well? What would your family and friends comments be on this question?\n\n• What support did you get from the Armed Forces during or leading up to this transitional phase?\n\n• What led you to becoming an offender within the criminal justice system?\n\n• Are there any parallels to your time within the Armed Forces and your time within the criminal justice system?\n\n• Do you maintain any links with your old Regiment/Corp?\n\n• Have there been any drastic changes to your life since leaving the Armed Forces? E.g. divorce\n\n• What support do you think could have made a difference in stopping you from becoming an offender since leaving the Armed Forces?\n\n• Do you know of any comrades who left the Armed Forces and became practitioners/professionals within the CJS? If so why do you think this happened?\n\n- How would you describe this individual?\n\n- How do they differ from you?\n\n\n\n• Please describe your life prior to entering the Armed Forces\n\n- Had you any involvement in the CJS prior to joining the Armed Forces?\n\n• What caused you to join the Armed Forces?\n\n• Please describe your career within the Armed Forces\n\n• What caused you to leave the Armed Forces?\n\n• What where your initial experiences when you re-entered ‘civvie street?’\n\n• Do you believe you handled the transition well? What would your family and friends comments be on this question?\n\n• What support did you get from the Armed Forces during or leading up to this transitional phase?\n\n• What led you to a career in the criminal justice system?\n\n• Are there any parallels to your time within the Armed Forces and in working within the Criminal Justice System?\n\n• Do you maintain any links with your old Regiment/Corp?\n\n• Have there been any drastic changes to your life since leaving the Armed Forces? E.g. divorce\n\n• What has been the greatest support to you in allowing you to make the successful transition from the Armed Forces to becoming a practitioner within the criminal justice system?\n\n• Do you know of any comrades who left the Armed Forces and became offenders? If so why do you think this happened?\n\n- How would you describe this individual?\n\n- How do they differ from you?\n\nThe process of collecting the stories from across the country took five months to complete. Interviews were conducted using a semi-structured format incorporating an interview schedule devised to incorporate themes identified from the literature review. Bachman and Schutt (2013) recommend this approach as it ensures that all dominant themes and main areas of interest are covered, guaranteeing consistency between interviews, whilst also maintaining sufficient flexibility to allow for unanticipated themes to be identified and discussed.\n\nA pilot interview before formal data collection took place where one veteran was interviewed using the life story method. Mason (2010) advises that a pilot study or interview should be included within a study’s research design, as this will create a crucial opportunity to analyse and review initial findings and to make forward decisions about whether there is a need to reassess the research question. As recommended by Bryman (2012), a digital recorder was used to record interviews, assisting greatly in the interview process and allowing both the participant and researcher to relax and converse more freely without the need for extensive notetaking, which can cause distraction and inhibit communication flow. One participant withdrew from the interview process due to on-going health issues. The interviews were undertaken in a variety of settings i.e., participant’s home, police station, university campus, probation offices, job centre, participant’s place of work, and hostel. At the time of the interviews only the lead author and the participants were present.\n\nWithin the study, seventeen individuals participated and gave their consent to allow their life stories to be shared. They all held the identity of a veteran, however eight of these veterans had since leaving the Armed Forces acquired roles within the CJS as practitioners. The remaining nine were defined within the CJS as offenders. This study defines the two sub-groups as follows:\n\n1. Veteran practitioner, an individual who has performed military service for at least one day and drawn a day’s pay is identified as a veteran (Royal College of Defence Studies, 2009). They had been an employee within one of the recognised institutions of the CJS.\n\n2. Veteran offender, an individual who has performed military service for at least one day and drawn a day’s pay is identified as a veteran (Royal College of Defence Studies, 2009). In relation to the label of ex-offender, the same individual had been charged with a crime, managed by the CJS and all sentences were spent.\n\nAs recommended by Silverman (2011), the recruitment of participants was based on snowball sampling. This study was supported by the Westminster Parliamentary Group on veterans in the CJS and the All Wales cross-party group on veterans and cadets. These groups were used as a starting point for identifying potential participants and other contacts emerged from initial contact with these organisations. Where necessary, access to participants was granted via internal organisational approval.\n\nThe various statutory and voluntary service providers who engaged with the researcher and study made initial contact with potential participants; this led to a number of practitioners within these services agreeing to be interviewed, or they knew of colleagues with a former military background who could meet the inclusion criteria.\n\nThe following sections within the results and discussion section, are formed from the emergent main and subthemes as identified within Figure 1 by the research team’s collective analysis of the data.\n\n\nResults and Discussion\n\nThe researcher is a health professional and is aware that, in relation to the two separate life story trajectories, health cannot be understood in its narrowest sense. Health underpins ways of understanding how it may be achieved and maintained, even in the adverse circumstances of military transition. Such understandings include the concept of Salutogenesis, developed by Antonovsky (1979, 1987), to explain how some individuals utilize resources available to them in order to survive in adverse social conditions, and the associated construction ‘Sense of Coherence’ (Antonovsky, 1979, 1987; Lindstrom & Eriksson, 2005), which informs ‘Asset based’ approaches to health and wellbeing (Morgan & Ziglio, 2007). These constructions are important as they relate to uniformed personnel and how health is interpreted, understood, and impacts the ability to have resilience to life challenges.\n\nWithin the UK, it is worth acknowledging that around the time of the introduction of the National Health Service, health was highlighted by the United Nations as a subjective state of well-being within a psychosocial context, as well as an absence of illness (UN Department of Public Info., 1948). The idea of a subjective health experience linked holistically to the physical, psychological, spiritual, and social well-being of individuals and populations has gained momentum since that time (Mahler 1987; Lindstrom & Eriksson, 2006). These concepts are elaborated in the following section to explore the issues contributing to resilience. In addition, a review of the literature and exploration of dominant theoretical frameworks on the impact of health on veterans from life stories through the concept of salutogenesis was conducted.\n\nSalutogenesis, originally developed by Antonovsky, explains how some individuals utilize resources available to them to survive and thrive effectively in adverse social conditions (Antonovsky, 1979, 1987). This concept also challenges the traditional understanding of health and illness, as represented by the medical model. Research that initially informed the concept of salutogenesis focused on how Israeli women maintained a good quality of health during incarceration in concentration camps of the Second World War (Lindstrom & Eriksson, 2006). To reiterate, the ‘medical model,’ which emerged as a key concept for medical sociologists (Conrad & Schneider 1992; van Teijlingen, 2005) has been challenged within contemporary medicine where a more holistic understanding has made inroads into the way in which we respond to some illness conditions. Engel (1977) argued that while the medical model constitutes a sound framework within which to understand and treat disease, it is not an appropriate framework within which to understand behavioral and psychological problems, as is clearly being exhibited with members of uniformed services. The acknowledgement that illness has psychological and social dimensions presents a challenge to Cartesian dualism, which underpins this model. For example, Merleau-Ponty (1964) argued that because human consciousness cannot be separated from the physical body, illness cannot be understood as a purely biological phenomenon. Instead, he stressed the importance of a holistic human being, in which the mind and body cannot be divorced. In privileging the centrality of the body in which the subject resides, he argued that the body has a dialogue with the world or, in other words, a reflective, absorbed engagement with the environment. When the body is ill, he argues that individuals must rethink their ability to engage with the world and provide movement, freedom, and creativity. Whereas the healthy body may be understood as transparent and taken for granted when it is ill, the harmony between the biological and lived body is disrupted and the difference between the two becomes apparent. Carel (2008) argued that health and illness can coexist in the same body, but that individuals do not always appreciate health until a body part ceases working, and then the focus of human consciousness becomes a lack of functionality on that part of the body. These may seem far removed from this article’s focus, yet reflexive review supports justification and linkage to causes of feelings of anger, frustration, and irrationality observed within the healthcare needs of uniformed services. For those making a transition between new identities, adaptation to an alternative construction of well-being must occur within the context of functionality. Moreover, this construction of health in its broadest sense is situated within a wider social context, which itself places constraints upon adaptation for veterans’ life post-conflict and those emergency service personnel whose working lives bring them to the front-line of conflict daily.\n\nThe study findings demonstrate how health, social and psychological aspects of the causality of ill-health for military veterans and emergency service personnel, mean it cannot easily be reduced to a few ‘bad eggs’ phenomena alone. This approach acknowledges how social and personal phenomena of a condition cannot be easily reduced to overarching dominant theory, and thus is apt to use as a framework within which to respond. In this study, an inclusive and holistic review of health in understanding emergent themes from the participants’ life stories offered insight into the structures of experience and consciousness of the participants’ physical, psychological, and social/practical support from military and emergency services. This resonates with Engel’s (1977) recommendation that an alternative model should consider not only the individual, but also the social contexts within which they live. He argued that a new biopsychosocial model which took into account all the factors which contribute to illness and patient-hood, (rather than giving primacy to biological factors alone) would make it possible to understand the individual’s ‘experience’ of disrupted well-being (Carrió et al., 2004). This understanding, while not previously applied to the research undertaken on the health of uniformed services, would necessarily be situated within the context of an individual, their family, community, culture, and possibly their religious beliefs (Gawinski et al., 2002). From the life stories, a positive transition from the former military identity could be viewed as achieving health, and the authors suggest that arguably it is not about having perfect physical health as much as coping and living well within current physical capabilities. Research findings would indicate that in relation to the subgroup of military veterans, they appear to have had health issues linked to maladaptive behavior, psychological trauma, and alcohol abuse, which has led to a deterioration in health and well-being, resulting in imprisonment (Howard League of Penal Reform, 2011; Napo, 2009). The findings from the lead authors’ original study (Mottershead, 2019) clearly highlight the importance of non-biological factors in participant well-being, leading some to ill-health and impoverished well-being. Indeed, interview discussions from that qualitative study highlighted issues around managing the transition and struggling to identify transferable skills, with some participants wishing they had never served in the Armed Forces. Likewise, frustrations were also evident for those within the emergency services who struggled with management who had no comprehension for the realities of the professional life and what constituted ‘normality.’ There was an emergent theme around the lack of understanding of what many participants felt the general public and the wider society had no comprehension of the lived experiences of those who had served their communities.\n\nThe authors believe that health and well-being cannot be conceptualized in the narrowest sense as a function solely of biology, which provides much scope for exploring how the quality of life may be achieved and maintained, even in adverse circumstances. It is in the context of this understanding that this study emphasizes that salutogenesis has an important role in creating insight into the health and uniformed services. A range of different theoretical concepts contribute to our understanding of salutogenesis (Strumpfer 1990). These include Sense of Coherence (Antonovsky, 1979, 1987; Lindstrom & Eriksson, 2005); Hardiness (Kobasa, 1979; Kobasa, Maddi & Kahn, 1982); Learned Resourcefulness (Rosenbaum & Palman, 1984); Potency (Ben-Sira, 1985); and Stamina (Colerick, 1985). These have been used to explore the concept of health as a resource for thriving and increasing quality of life (Lindstrom, 1994). Specifically, Sense of Coherence SOC, which is integral to salutogenesis and thus the understanding of why some individuals are able to remain in relatively good health in conditions of adversity, while others are not (Antonovsky, 1979, 1987). This concept relates to how individuals integrate into their society to utilize the resources available to them effectively to create and promote a good state of health, as can be seen within the veteran and emergency service sub-groups. Three concepts which underpin Sense of Coherence (SOC) are; ‘comprehensibility’ (where individuals can make sense of events), ‘manageability’ (where they feel they can take care of things) and ‘meaningfulness’ (where they really care about what happens) (Antonovosky, 1979). These three concepts lie at the heart of Antonovosky’s salutogenic orientation, which represents a global orientation that can help understand how individuals respond to everyday external stressors and overcome stressful and challenging situations (Antonovsky, 1996). Stressors include chronic stressors (such as disposable income), major life events (such as divorce), and acute daily hassles (time or support) (Antonovsky, 1987).\n\nAntonovsky (1979, 1987) argues that people create and build a sense of coherence over the first three decades of life. During this period, skills that operate flexibly and dynamically in any given environment were acquired. Given that the recruitment process into the military can commence at 16 years of age, it could suggest that immersion into a total institution (Goffman,1961) may impact the development of a sense of coherence, evidencing how military identity and culture impact development. This observation of development is significant as coherence creates skills to assist individuals in identifying the resources needed to address needs, related challenges, and source effective solutions to these, which promote positive well-being over the lifetime, as those within the emergency services can have extended working lives, whereas the military is predominantly constrained to 22 years.\n\nIn order to understand how Sense of Coherence might assist in promoting these individuals’ health-related behaviors, Johnson (2004) investigated its relevance in relation to health promotion with respect to self-esteem and locus of control. Johnson (2004) argued that a Sense of Coherence appears to have a strong and unique relationship with the pursuit of general health and a more tentative link to self-esteem or self-worth. A more stable locus of control was associated with an optimistic perspective towards coping with health challenges. Antonovsky (1993) had previously noted that Sense of Coherence, as a part of Salutogenesis, differed from ‘coping’ in the traditional sense. Because humans are continually surrounded by disease and associated stress, it is important to understand why certain people survive every day despite this situation (Eriksson & Lindström, 2005). Hence, Sense of Coherence requires support from other theoretical frameworks such as ‘General Resistance Resources’ (Griffiths et al., 2011) which purported that individuals may have instinctual drives to utilize financial, social, and cultural resources in order to create greater health stability for improved well-being (Antonovsky, 1993). Antonovsky (1993) argued that greater General Resistance Resources (GRRs) means a more effective sense of coherence with respect to health promotion.\n\nHaving these resources increases the individual’s ability to deal with adversity and appears to be a feature lacking or present within the two sub-groups of uniformed services. Furthermore, Lindstrom and Eriksson (2006) identified a range of GRRs at the individual level, including physical (such as genetic resistance), material (such as money), cognitive (such as knowledge), attitudinal (such as self-esteem), and interpersonal (such as social capital) resources. At the social/structural level, they highlight welfare provisions and cultural traditions. While SOC is more of an orientation towards life, GRRs are biological, material, and psychosocial factors that enable individuals to perceive their lives as consistent, structured, and understandable (Lindstrom & Eriksson, 2006).\n\nSense of Coherence has received considerable attention in the health care literature (Langius-Eklof, Lidman & Wredling, 2009). For example, it has been explored in nursing interventions for cancer patients (Delbar & Benor, 2001) and the treatment of young people at risk of developing mental disorders (Ristkari et al., 2009). Delbar and Benor (2001) investigated whether the ability to cope depends on internal resources or can be increased through a structured nursing intervention–the Self-Care Approach. They assessed the impact of the Self-Care Approach on 48 cancer patients living at home, undergoing cancer treatment, including radiotherapy, chemotherapy, or both. These patients were compared with the matched control group. Measurement of coping ability with responsibility for symptom control through the SOC and Multidimensional Health Locus of Control Scales showed that the intervention group had a significant improvement in their SOC scores in three subcategories (comprehensibility, manageability, and meaningfulness). This means that the Self-Care Approach is associated with enhanced coping ability. Ristkari et al. (2009) examined self-reported psychopathology, adaptive functioning, and sense of coherence among adolescent boys in Finland. This quantitative study, using the Young Adult Self-Report (YASR) and Orientation of Life Questionnaire (SOC-13), showed a non-specific association between poor SOC and somatic complaints and a range of diagnosed psychiatric conditions.\n\nA systematic review by Lindstrom & Eriksson (2006) reported that a sense of coherence emerges as a health-promoting resource that strengthens resilience and contributes to the development of a positive subjective state of health. Griffiths et al.’s (2011) qualitative study investigated the usefulness of a sense of coherence in understanding how (n=20) mental health service users dealt with problems they faced in their lives. The study identified various resistance resources for both concrete and relationship-oriented problem solving, suggesting that SOC may play an important role in coping with stressors in the rehabilitation/recovery process and thus contribute to mental health and psychosocial functioning within the general population (Griffiths, 2009) and could have wider implications for uniform services.\n\nOther social scientific theories have explored the process by which individuals, groups or societies use SOC to promote effective health management (Klein, 2013). Research by Ben-Sira (1985) demonstrated that, for the participants of this study, a sense of individual potency increases the ability to buffer stressful issues and manage them effectively, as ordered, predictable, and meaningful within the context of health and well-being. It has been argued that high individual potency weakens the association between stress, coping, and illness, helping individuals thrive in the face of health-related adversity. Kobasa, Maddi and Kahn (1982) focused on individual ‘hardiness’ as an aspect of personality type, suggesting that individuals respond to stressful life events, exhibiting different degrees of commitment, control and challenge. They argued that individuals displaying more hardiness tend to involve themselves more readily in specific encounters and attempt to influence their outcome favorably rather than being helpless (Seligman, 1972). Those with greater hardiness are also argued to be more able (and expected) to embrace change in circumstances as normal rather than static coping. It has been argued that in supporting the sense of coherence approach, hardiness increases the ability to expect and embrace change as the norm (Kobasa, Maddi & Kahn, 1982).\n\nIt should be noted, however, that evidence for the hardiness theory was generated with respect to research on efficacy among business executives rather than on related behaviors associated with participants linked to this study, which means that its applicability to a sense of coherence is unproven (Blaney & Ganellon, 1990). The emergence of adopting a bio-psychosocial model to explore and understand the life stories of military veterans’ exposure to life challenges has merit based on the success attributed to this approach, from simply not adhering to a dominant theoretical model. Mottershead and Ghisoni (2021), Mottershead (2022) and Mottershead and Alonaizi (2023) demonstrate variations in dominant treatment models for specific groups and the benefits of seeking theoretical models that are innovative and holistic. Similarly, focusing on veterans’ presence in the CJS via criminology and penological research alludes to the inclusion of other fields that can bring much-needed insight into this area of study.\n\nIt is essential to acknowledge that Salutogenic theory and SOC underpin health asset approaches (Morgan & Ziglio, 2007). The notion of assets, or resources, as antecedents of health has been increasingly cited in the health promotion literature to explain social differences in health and wellbeing, but never to explore the meaning and understanding of military veterans’ involvement in the CJS. Charlton and White (1995) explain inequalities in health, including differences in health-related risk behaviors (as seen within the veteran community), in terms of the ‘margin of resources’ differentially available to individuals. Hence, an individual’s margin of resources, which is equal to their available resources less their essential needs, constitutes salutogenic factors associated with the optimum opportunity for improving health and well-being. Consequently, when analyzing the two veteran sub-groups of life stories, we can see that a broad definition of health and well-being allows for an understanding of the causality of one life story trajectory as a practitioner and the other as an offender.\n\nThe assets- or strengths-based approach is underpinned by understanding health as a resource for everyday life, rather than the object of living. From this perspective, health denotes social and personal resources as well as physical capabilities. In an asset- or strength-based approach, the strengths and resources of the individual and community are emphasized. Fundamental to this perspective is how the capabilities and characteristics of individuals and communities contribute to health and well-being, and as identified by Ashcroft (2014), these can be significant for a select portion of ex-service personnel entering civilian life. Health assets, defined as resources at the disposal of individuals and communities that protect against negative health outcomes and/or promote health status, can be social, financial, physical, environmental, or human resources (Morgan & Ziglio, 2007). All prominent features within life stories that represent a submerged reef are ready to render those making the transition rudderless and ready for submersion into the CJS as an offender.\n\nAlthough salutogenesis and its applications have received increasing attention from the research community, it should be noted that there is no evidence for the long-term sustainability of the Sense of Coherence and other applications of salutogenic theory with respect to public health (Eriksson & Lindström, 2005). Outside the confines of this study, there is a lack of published research that further identifies the foundation of salutogenesis in the context of thriving and surviving for military veterans exposed to the CJS.\n\nThe 17 veteran participants involved in the study presented here drew on a range of resources to maximize personal well-being. Central to the identification and deployment of these resources was the objective of reconstructing a new civilian identity, allowing for a version of normality within their new lives post-discharge from the Armed Forces. In this reconstructed identity, they attended to the physical, psychological, and social aspects of their new civilian experience; for those unable to adapt, there was evidence of involvement in the CJS as an offender rather than as a practitioner.\n\nThe findings suggest that an important indicator for a Sense of Coherence is enabled through the utilization of resilience resources through which the reconstruction of a version of normality, mapping on to previous lives, is facilitated. Notably, the concept of salutogenesis concerns the ability to draw from internal and external resources as circumstances require, in order to survive, and because of this, it is not about having the static ability to move to a positive place and remain there (Eriksson & Lindström, 2005). Hence, adaptation was notable through the coherence identified by the shared identity of being a veteran noted between the two subgroups and represents a continuous and dynamic process. As Eriksson and Lindström (2005) suggest, the development of a sense of coherence is not restricted to the early decades of life, but is a continuous process as contexts, personal circumstances, and opportunities arise. Given the age of recruitment into the Armed Forces, as highlighted in this study, there may be a need to further explore the impact of adolescents spent within a total institution on the development of a sense of coherence. Based on these points, the following section will highlight the studies and the researcher’s positionality and connect this epistemology simultaneously to acknowledge empowering and disempowering practices and policies towards and possible for veterans exposed to the CJS. The next section seeks to establish an understanding from the theoretical concept of salutogenesis and continues with the need to further investigate veteran identity and culture.\n\nBoth subgroups of veterans described clear duality of identity. Among the practitioners, this was voiced within positive narratives of having achieved, and now in various guises, they reap the rewards of having been trained and served within the Armed Forces. There was a clear indication from this subgroup of participants (veterans) that having belonged to a larger community with a clear focus and purpose had created a lasting positive transferable skill set that had a positive influence, as evidenced within the life stories. As previously identified, most individuals (86%) leave the Armed Forces and enter civilian life without difficulty and obtain employment, which consequently makes a positive contribution to society (Lord Ashcroft, 2014; Mottershead, 2019). However, the same could not be said of the veteran offender subgroup. This group spoke about the shame and stigma of having been a serving member of the Armed Forces and is now being identified as an offender. There appeared to be a wish to have the first identity take priority, as this was discussed with pride and gave substance to their self-image, which was indefatigable and permanent, while the identity of the offender was spoken about in the hope of being transient and keen to have this label resigned to the past. Both subgroups spoke about belonging to a larger veteran community, a group within a society that they perceived as not fully comprehended by those outside of the group. Their life stories alluded to the belief that this was due, in part, to society not having shared attributes, a common value system, a unique culture, and a shared history. Cohen and Taylor (1990) and Clemmer (1958) explain that the replacement of a previous identity with one conducive to life within an institution can force the individual to become institutionalized and adopt traits associated with that environment.\n\nBoth sub-groups perceived that there was still a shared comradeship in belonging to the veteran community, and this bond was evident as there was a bond between the two sub-groups who would speak about their support and, at times, symbiotic relationships when the two would come into contact with each other within the criminal justice system. This feature has been explored and identified within research from the United States, first by Hollingshead (1946), who highlighted the struggles of returning G. I. in making the transmission back into their civilian lives, and later by Wortzel et al. (2009), who saw the support offered to veterans serving custodial sentences in the US by veterans employed as prison staff. This study highlighted the benefits of a shared lived experience in assessing and preventing suicide associated with shame and challenges linked to incarceration.\n\nWithin the life stories of the participants, there was a common thread of how military service led to the process of the creation of a reconstituted identity, formed through training, socialization, and contact with military culture. This reconstituted identity lends itself to allowing the individual to be assimilated into the entire institution of military life. This process is described by Goffman (1961), who described it as ‘disculturation’ involving the internalization of military culture with inculcation of habits, mannerisms and language as well as the breaking down of previously held moral boundaries and the assertion of an ‘adapt and overcome’ ethos. Goffman (1961) noted that this ethos was evident in the presence of confidence, courage, organization, and the suppression of fear. The life stories indicated that these traits were present within the veterans from both subgroups, but it was not clear whether these traits were created as a bi-product of military service or whether the participants already possessed these traits and were attracted to the Armed Forces as a career choice because of a lack of prospects. Research undertaken by McCulloch (2005) and Raffo et al. (2011) indicate that many recruits entering military service from socially and economically deprived areas struggle to meet basic eligibility requirements, have low academic achievement levels, poor health and fitness, histories of drug use, and criminal records. This may indicate that military service had a positive impact on traits, supporting the development of resilience, sense of coherence, and improved health during military service. This innovative ethos was a theme within the life stories, as they bore testimony to the development of schemes that had been born out of a need to improvise, adapt, and overcome a presenting challenge that it was felt had not been addressed by current policies and procedures. Indeed, this necessity to ‘overcome’ held within its discourse a concept of a need to work outside of previously held norms and to grasp upon a method to improve quality, safety, and access to healthcare inside and outside judicial provision.\n\nThe military veterans within this study were a unique population with previous exposure to various potentially traumatic and stressful life events, such as military training, deployment, combat, and the rigors of transition into the CJS as offenders as well as practitioners. A theme emerging from the life stories was the ability of these two subgroups to demonstrate post-traumatic growth. This was realized through meaningful psychological change after stressful and traumatic life events, as discussed by Zoellner and Maercker (2006), and was evident within the participants’ narratives as improved functioning and greater resilience. A relevant study by Feder et al. (2008) showed evidence of post-traumatic growth within a prison population, although this was during the war. A previous study by Boerner et al. (2017) provided some insight into the cautionary view that whilst post-traumatic growth may lead to positive psychological changes, it is also feasible to consider that it may be a dysfunctional attempt to manage and avoid the coping mechanisms that allow individuals to deal with stressful life events. There are two known studies from the UK that have focused on post-traumatic growth with veterans: Murphy et al. (2017) and Dyball et al. (2022), which have had a health focus with the latter study concluding that a moderate or large degree of post-traumatic growth among UK serving and veterans is associated with mostly positive health experiences, except for those with a diagnosis of post-traumatic stress disorder.\n\nThe life stories attested to evidence of post-traumatic growth for the veteran offender sub-group of psychological change resulting from the stigma and shame the individual initially experienced. The participants demonstrated the ability to relate to their veteran peers (offenders), a new understanding of their own personal strength, perceiving new possibilities in their lives that they focused on creating and establishing new innovative initiatives to support veterans now within the CJS as offenders or ex-offenders. The research team believes that this process not only demonstrates the presence of post-traumatic, but also demonstrates how this concept is focused on the action of positive deviance.\n\nThe participants demonstrated an effort to create and promote systems which surpassed conventional expectations outside of conventional norms; which indicated the presence of positive deviance. This concept is the observation that in most settings a few at risk individuals follow uncommon beneficial practices and consequently experience better outcomes than their neighbours who share similar risk (Marsh et al., 2004).\n\nLife stories contest the presence of deviant behavior and positive deviance. The latter appeared as attempts to establish new ways of working to support veteran ex-offenders and echo the military ethos of adapt and overcome. Positive deviance is defined as\n\n“Positive deviance is the observation that in most settings a few at risk individuals follow uncommon beneficial practices and consequently experience better outcomes than their neighbours who share similar risk”.\n\nMarsh, Schroeder, Dearden, Sternin, and Sternin, (2004 p.1177)\n\nA point articulated by the participants was that some behaviors encouraged within the military would be seen as actions that would classify them as deviant within civilian life. However, while this observation was made by the participants, there was a unilateral belief that the assessment and classification of deviant behavior was viewed as a necessity for this society, as it reinforced the social cohesiveness of its population by establishing rules to punish those that infringe and abuse these rules. Even ex-offenders appeared to appreciate the need for custodial boundaries. To this end, the veteran practitioners did not excuse the veteran ex-offenders’ actions but instead sought to establish systems that the research was based on the concept of positive deviance. This process within the life stories demonstrated the ability to stretch a norm by creating a morally correct action for the veterans and the community concerned. The shared veteran identity appeared to resonate between the two subgroups, and there was evidence of an ability to create effective veteran peer-support schemes that built on belonging to this veteran identity. This process appeared to aid in the elevation of frustrations for the participants while allowing for the progression of new improvised systems, adapted to overcome outdated practices towards veterans in the CJS, and demonstrating the presence of positive deviance.\n\nThis concept, not fully explored in military research, explains that positive deviance has started to be identified within the field of international public healthcare and has been identified as a method to improve the quality and safety of healthcare (Marra, 2010). This concept was present within both subgroups but was strongest within the veteran practitioners who appeared to adapt, overcome, and improve current processes with the explicit intention of supporting fellow veterans. Adamsky (2010) and Bassingthwaighte (2011) demonstrated that this scheme of working appeared to have a resonance enshrined within military training, which promoted the individual’s ability to improvise, adapt, and overcome. Dodge (1985) emphasized the need to progress past the overly negative conceptualization of deviance and instill a realization that individuals and/or their actions can be deemed as superior by virtue of surpassing conventional expectations. He argued that these positive deviants had surpassed their counterparts in sports, science, and politics because they operated outside of the perceived normal parameters (Dodge, 1985). This ideological shift within sociological perspectives on the concept of deviance is significant in this study’s findings and demonstrates new knowledge when applied to military culture. With regard to the veterans within both subgroups, there appeared to be evidence of this approach as their actions sought to prevent and reduce harm by striving to negate possible harmful outcomes. Tuhus-Dubrow (2009) identifies positive deviants as exemplary in practice, either as individuals or part of a collective group. The presence of evolving strategies outside of normal practices was evident and demonstrated the presence of positive deviance, as the participants created multiple schemes to help aid veterans within the CJS, including those with specific health and social care needs.\n\nOne such initiative was the Veterans in Custody Support Scheme (VICS). One participant led the development of this scheme to identify offenders who have served at the earliest possible opportunity, with identification of having served in the Armed Forces occurring within police custody or within the prison service (Phillips, 2014).\n\nThe participants’ attempts to establish and disperse the scheme were admirable, but they often faced hardships in their attempts to provide support. Fossey et al. (2017) explain that although the scheme is not mandatory for prisons and has no designated funding, it continues to spread across the prison service. Indeed, participants in the study were actively involved in supporting the scheme by establishing it within police custody suites, and prisons were employed. Once veterans have been identified, the scheme refers to them as community ex-service organizations for resettlement assistance (Phillips, 2014).\n\nIn relation to well-being and mental health, this process of transition into the CJS meant a realization that, for some innovative schemes, focusing on peer support through a shared ex-military service identity aided in the start of rehabilitation. This unexpected benefit through forced capitulation with the CJS created the opportunity for some within this study to identify unmet social and health needs and commence rehabilitation and redemption for mistakes. The thematic analysis of the life stories provided evidence of mental fall-out through stigmatization and shame of being identified as an offender, which then had consequences on the participants’ sense of coherence. However, while the process of progressing through the CJS was traumatic, there was acknowledgement that this environment could at times be a reprieve from the chaos that had led them to become offenders through their maladaptive behaviors.\n\nThe findings of this study do not claim generalizability, having emerged from one country and one homogeneous sample (albeit two sub-groups) providing their subjective lived experiences, together with the researcher’s own interpretation of them. Undoubtedly, the researcher’s own subjectivity, lived experiences, and research interests may have influenced the interpretation of these findings, as highlighted by Blaikie (2007). However, precautions were taken to limit the impact of this bias, where possible, by adhering to a clear and robust methodological framework and review of the original research by a team of researchers. Given the modest deficit of knowledge in this area, this study provides insight and contributes to new knowledge.\n\n\nConclusion\n\nThis study can be utilized to establish a way of knowing within two realities, which has created knowledge about the experiences of individual veterans, to represent the position of stakeholders, and to go beyond the generalization of policies and practices relating to this group’s experiences. This developing issue has been overlooked, perhaps due to financial shortcomings, finite resources, and issues relating to stigma and shame forcing a camouflaged subgroup with specific health and social care needs. This study suggests that there is a need to establish a new perspective and understanding of the impact of being a veteran with challenges around the specific needs of this group, which merit further expansion of bespoke health and well-being services and treatments.\n\nJust over ten years ago, practitioners began to express concern that a problem existed as they recognized fellow veterans entering the CJS as offenders. This realization allowed for synergy between post-traumatic growth that illuminated opportunities for paradigm shifts in individuals’ thinking and the establishment of new practices and policy alignment. Undeterred by budgetary confines and resources, the participants began to engage in positive deviance to use the innovation and knowledge of total institutions to engineer solutions in the establishment of systems to identify this vulnerable group. The authors have utilized the veteran’s own life stories and experiences to ensure that their voices were heard. It is hoped that this act will aid in understanding the stigma and shame associated with many with the eroded veteran identity due to a custodial sentence, as well as health needs that may affect the individual’s sense of coherence, as understood through the study’s salutogenic approach. The presence of post-traumatic growth is testimony to the resilience of this subgroup. Likewise, the establishment of innovative practices and procedures realized through the concept of positive deviance is further evidence of this subgroup’s ability to overcome adversity, demonstrate compassion through comradeship, and attempt to prevent cyclical problems related to a diminished sense of coherence and health through the capacity to cope with the stresses of life, to stay well, and to maintain good physical and mental health.", "appendix": "Data availability\n\nData cannot be shared publicly due to potentially identifiable information in the transcripts. Data will be made available to reviewers and researchers via the corresponding author following approval from participants when and if possible.\n\n\nAcknowledgements\n\nThis research is based on the first author’s PhD thesis (Mottershead, 2019). The authors wish to acknowledge the participants contributions which breathed life into this article. Their resilience and determination to face life’s challenges and to continue to adapt and overcome was inspirational.\n\n\nReferences\n\nAdamsky D: The Culture of Military Innovation: The Impact of Cultural Factors on the Revolution in Military Affairs in Russia, the US, and Israel. Stanford University Press; 2010.\n\nAntonovsky A: Health, stress and coping. San Francisco: Jossey-Bass; 1979.\n\nAntonovsky A: Unravelling the mystery of health. How people manage stress and stay well. San Francisco: Jossey-Bass; 1987.\n\nAntonovsky A: The structure and properties of the sense of coherence scale. Social Science Medicine. 1993; 36: 725–733. 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Publisher Full Text PubMed Abstract \|" }	[ { "id": "244666", "date": "06 Mar 2024", "name": "David Jackson", "expertise": [ "Reviewer Expertise Veteran research", "Creative Methods", "Life story Narrative", "autoethnography", "Multi modal representations of narrative" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 looked at the life experiences and the health of two subgroups of veterans. There were 17 participants 8 who were practitioners within the criminal justice system and 7 who were defined as offenders. They all identified as military veterans. The study explored health issues within the trajectory of their lives using life story methods. It found that there was a process of adaptation through the identity of being a veteran. This was found to be an ongoing and dynamic process. The study suggests that the development of a sense of coherence is ongoing through life trajectories. It is achieved through post-traumatic growth, positive deviance and an identification with their past lives of military service and life. As a researcher who is also a veteran and whose research has extensively used life story methods and thematic analysis, I think this paper is excellent. It contributes to the growth in research that offers a voice and gives agency to the veteran community. For example, the Military Afterlives (Military Afterlives Project) and Stories in Transition Project (Stories in Transition). The reasons for using life story and thematic analysis were very clear and well-argued citing some important published work in this area. The method section showed rigour and clarity. I really liked the contextualisation of salutogenesis and the importance placed upon the sense of coherence and general resistance resources theoretical frameworks. The important point being that these veterans’ life experiences are not lived out within a vacuum. The authors findings from his interviews raise some very important points in relation to the notion of veteran/military identity and a connection with past military experience. It is an important part of having a positive veteran self-identity. It was alluded to by the participants that society did not fully comprehend them and their experiences. I would add to this point that society does comprehend veterans but are informed by historical and cultural assumed knowledge (Armour, C. et al, 2018 (Ref 1), Ashcroft, M., & KCMG 20149 (68364 The Armed Forces and Society.pdf (lordashcroftpolls.com), You.Gov 2018). Secondly, there is potential in the future for further discussions to consider insider/outsider research. There is a growing cohort of researchers who are veterans researching the veteran community. Whilst the author acknowledged his military identity at the end of the paper and recognised possible bias. I think there is an opportunity to explore the benefits of ‘insiders’ conducting veteran research more especially in relation to the comradeship and commonality of experience felt within the veteran community. How does \"the space between\" insider and outsider researchers differ when participating in veteran research? (Kukutai, T. et.al, 2011 (Ref 2) I really enjoyed reading 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? Yes", "responses": [ { "c_id": "11209", "date": "21 Mar 2024", "name": "Dr. Richard Mottershead", "role": "Author Response", "response": "Dear Dr. David Jackson, My sincere gratitude for the insightful and encouraging review. I have found it to be both thought provoking and an engaging read. As you may imagine, it was important to me to provide a voice for the participants and having the article reviewed by a researcher with a military veteran identity (Royal Marine) and embedded within military themed research is not only highly appropriate, but I believe to be lorded. The life stories provided both a practical and holistic approach to collect information about the participants lives, their individual challenges and their unconquerable ability to strive to adapt and overcome. This ethos was not only realised within the presence of post-traumatic growth but also to the wider community via positive deviance. I hope that the article allows the reader to glimpse inside a perceived reality and widen their own understanding of human existence. I would like to invite the reviewer to join me in a collaboration to explore the insider/outside perspectives of veteran research. This emerging presence within the research landscape offers important insight into the realities of lived and meaningful reality. Thank you for your review - Per Mare, Per Terram. Dr. Richard Mottershead" } ] }, { "id": "244671", "date": "06 Mar 2024", "name": "Adam Mahmoud Salameh Khraisat", "expertise": [ "Reviewer Expertise Emotional intelligence", "Qualitative data analysis", "mixed methods research", "Nursing 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\nThis article delves into the experiences and perspectives of two distinct groups within the UK military veteran community: those who have had encounters with the criminal justice system and those working within the criminal justice system. The study utilizes narrative inquiry and life story research, conducting 17 in-depth interviews across England and Wales. The aim is to explore the concept of salutogenesis, which focuses on how individuals maintain health and well-being despite adverse conditions, particularly in the context of military transition. The findings suggest that health cannot be narrowly defined and that various factors, including social, psychological, and spiritual well-being, contribute to resilience and overall health outcomes.\n\nMajor Points:\n\nStrengths:\nComprehensive Exploration: The article thoroughly explores the concept of salutogenesis and its application to understanding the health of military veterans. It delves into various dimensions of health, including physical, psychological, spiritual, and social well-being, providing a holistic perspective.\n\nInclusive Methodology: The use of narrative inquiry and life story research allows for a deep understanding of participants' lived experiences, capturing diverse perspectives within the veteran community.\n\nTheoretical Integration: The article effectively integrates theoretical frameworks such as salutogenesis, Sense of Coherence (SOC), and biopsychosocial models to analyze health outcomes, offering a nuanced understanding of factors contributing to resilience and well-being.\n\nWeaknesses:\n\nLimited Generalizability: The study's sample size is relatively small, comprising only 17 participants, which may limit the generalizability of findings to the broader veteran population. Additionally, the focus on UK military veterans and practitioners within the criminal justice system may restrict the applicability of findings to other contexts.\n\nPotential Bias: As the lead author is a health professional, there might be a bias towards health-centric perspectives, potentially overlooking other important factors influencing veterans' experiences and well-being. and as I mentioned previously, the results should be given to another Qualitative data analyzer to check the codes and themes building process and maintain the trustworthiness of this article's findings.\n\nLack of Discussion on Methodological Limitations: The article lacks a discussion on potential limitations of the methodology employed, such as selection bias, interviewer bias, or the influence of social desirability on participants' responses.\n\nMinor Points:\n\nClarity and Organization: The article could benefit from clearer organization and structure, particularly in presenting the findings and discussing their implications. Clear subsections or headings could enhance readability and comprehension.\n\nIntegration of Additional Literature: While the article extensively discusses salutogenesis and related concepts, it could further enrich the discussion by integrating findings from other relevant literature, particularly studies focusing on veterans' experiences and transition challenges.\n\nOverall, the article offers valuable insights into the complex interplay between health, resilience, and lived experiences among military veterans and practitioners within the criminal justice system. However, addressing the identified weaknesses could strengthen the robustness and applicability of the study's 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? 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? Yes", "responses": [ { "c_id": "11214", "date": "21 Mar 2024", "name": "Dr. Richard Mottershead", "role": "Author Response", "response": "Dear Dr. Adam Mahmoud Salameh Khraisat, Thank you for the time and energy that you have spent on the review. The team have found your review to be insightful and helpful. I myself have reflected on how I might structure future publications to enunciate pertinent features of the research and when to elaborate further to inform the reader. If I may respond to a selection of your points. 1. Within qualitative research and within the life story research approach, 17 participants is seen as more than adequate. It may have been useful if I suggested that data saturation was met in the future. 2. The health approach was adopted to address the balance of distributive justice of those researching in this field. Therefore, this study provides a valuable contribution to the field of inclusive and emancipatory criminology and penological research through a health perspective which forms an element of the criminal justice system (forensic). The data was analyzed by multiple researchers to ensure the validity this was to ensure that the identification of codes could be cross-referenced with the researcher’s own coding process. This validation process was useful in ensuring that relevant material was being identified and created a confidence that there was an effective focus on the relevant subject matter to produce the identified themes and sub-themes. 3. Thank you for highlighting these points and this will be a focus of further exploration within a subsequent article. As discussed the focus of the article was to highlight the emergence of the data on the participants sense of coherence and health for life challenges and the subsequent response of post-traumatic growth and positive deviance. Indeed, this is the first article to capture this evidence and we believe demonstrates a crucial insight into transition challenges of military service personnel. Again, thank you for your time and diligence. Dr. Richard Mottershead" } ] }, { "id": "244664", "date": "06 Mar 2024", "name": "John Williams", "expertise": [ "Reviewer Expertise Vulnerable people and the law. Human Rights." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 explores the sense of coherence and health among British military veterans who have experienced significant life challenges, focusing on the emergence of post-traumatic growth and positive deviance within a life-story approach.\nIt provides valuable insight into two groups of military veterans in the United Kingdom, namely those identified as offenders and those working within the criminal justice system. It adopts a salutogenic approach that emphasises resilience and reconstructing military experience to enable a sense of coherence. The research was conducted based on the first author's PhD thesis and thus has a robust academic approach; it relies on a variety of sources, including the appropriate literature in monographs, journal articles, and reports. Qualitative research methodologies such as narrative inquiry and thematic analysis are employed in the study, delving into issues such as military innovation, health and coping, post-traumatic growth, and the criminal justice system's treatment of veterans.\nThe study's findings are valuable as they present evidence of post-traumatic growth and positive deviation within the veterans' life stories, highlighting the significance of veteran identity and culture and the bond and support within the veteran community. The participants in the research displayed improvisation, adaptation, and problem-solving skills, establishing new systems and initiatives to support fellow veterans within the criminal justice system. Internal, social and community resources all played a part in improving their lives and the lives of other veterans. Overall, the research highlights the resilience and determination of the participants who contributed to the study. The research acknowledges the adversities veterans have faced and are still facing and the need to continue providing them with the support and resources they need to ensure their well-being. An interesting piece of research, the findings of which will help the academic community and others to better understand the complexities experienced by veterans and it provides a good foundation for ongoing work with this 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?\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": "11215", "date": "21 Mar 2024", "name": "Dr. Richard Mottershead", "role": "Author Response", "response": "Dear Professor John Williams, On behalf of the research team, I would like to thank you for your invaluable time and the consideration that you have given to our study. We feel exceptional fortunate to have had a review from such an eminent academic who is so engrained within criminology, law and human rights. The team will look to further disseminate the findings of the study to highlight the insight into human existence and mean making process of the military veterans, highlighted through the salutogenic approach. In so doing so, we hope that this approach acknowledges how social and personal phenomena of a condition (life challenge) cannot be easily reduced to overarching dominant theory (criminology and penological research) and therefore is apt to use as a framework within which we, the research team have responded. Consequently, this holistic review of health creates understanding of emergent themes from the life stories and offered insight into structures of experience and consciousness of the participant’s (veterans) physical, psychological and social support post-discharge during their transition into civilian life. We thank you collectively - Diolch yn fawr. Dr. Richard Mottershead" } ] }, { "id": "244667", "date": "19 Mar 2024", "name": "Mark Russell", "expertise": [ "Reviewer Expertise Educational research." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research (and associated dissemination) through this paper), presents an interesting and useful exploration of two (sub-)groups of veterans using qualitative methods. The research team set out some useful definitions (Salutogenesis, veteran practitioner / offender) and their research methodology.\nThe paper is also usefully located in the literature.\nConducting seventeen interviews, coding and identifying themes from such interviews is a non-trivial activity.\n\nIt is noted, and useful, that the paper includes the question set for the two variants of the semi-structured interviews. Such will prove helpful for other researchers wishing to explore similar areas, (and triangulate the findings). Although some questions appear to be ‘closed questions’, I am sure the researcher(s) opened these closed questions during the semi-structured format. I think it would be useful if the authors elaborated on this point.\nIn terms of some opportunities to enhance the paper, unless the current format is a journal requirement, I think the Abstract could / should be crisper and ‘un-sectioned’.\nIt would be useful to read more about the process of coding and especially hearing how the researcher’s own life experiences / perception did not bias the codes and themes. I note the paper mentions ‘…research team’s collective analysis of the data.’. It would be useful to read more about this ‘collective analysis’ process.\nLinked to the above, it would be useful to read more about the elimination of (recognition of) potential bias and how the research team maintained 'arms length' from the findings. i.e. offer some observations relating to reflexivity.\nGiven the veterans' reported shared experience, I would be interested to read if the researchers felt their non-veteran experiences skewed (in any way) the discussions. Simply to avoid any explicit (or implicit) assertions such as, ‘you wont understand, you haven’t served’.\nI note the paper uses... sense of coherence, Sense of Coherence and SOC. I think a single format would be better.\nTo note: The paper does not include the interview transcripts (source data) hence I cannot comment on predictability of 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-92
https://f1000research.com/articles/12-297/v1	17 Mar 23	{ "type": "Study Protocol", "title": "A protocol for cloning, expression, and purification of Lysine exporter (LysE) gene of Mycobacterium tuberculosis", "authors": [ "Shilpa Upadhyay", "Archana Dhok", "Vinod Agarkar", "Supriya Kashikar", "Zahiruddin Syed Quazi", "Archana Dhok", "Vinod Agarkar", "Supriya Kashikar", "Zahiruddin Syed Quazi" ], "abstract": "Background: Tuberculosis (TB) is among the deadliest diseases and a significant cause of illness across the globe. Several studies on mycobacterial proteins, such as proteases and transporters that are essential for survival and pathogenesis have aimed to develop an efficient anti-tubercular agent. In mycobacterium, lysine exporter (LysE) is an amino acid transporter and a probable target for an anti-tubercular agent as it is responsible for bacterial growth inhibition and is also absent in the widely used Bacillus Calmette-Guérin (BCG) vaccine. Methods: Some studies have purified LysE using different protocols. This study describes a protocol for purifying different constructs of LysE, focusing on its hydrophobic region using immobilized metal affinity chromatography (IMAC) after expressing LysE gene in a bacterial expression system. pET vector (pET28a) is used as an expression vector. Amplied LysE gene is ligated with the pET28a vector, and the resultant plasmid is then transformed into E. coli cells. The vector has a histidine tag that makes the purification process convenient. After IMAC, the samples will be subjected to size-exclusion chromatography for further purification. Results: Cloning and amplification findings will be analyzed using 1% agarose gel, and protein expression and purification outcomes will be examined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Domain-specific constructs of LysE can be further analyzed as an anti-tubercular agent. Conclusions: Despite being a potential anti-tubercular target, research is quite limited on this protein. Therefore, we aim to purify LysE protein for further analysis. Similar protocols have already been implemented to purify several other bacterial proteins with >95% purity.", "keywords": [ "Lysine exporter (LysE)", "Amino acid transporter", "Anti-tubercular agent", "Expression and purification", "Immobilized Metal-Affinity Chromatography (IMAC)", "Histidine-tag", "Bacterial proteins", "Tuberculosis" ], "content": "Introduction\n\nTuberculosis (TB) is caused by Mycobacterium tuberculosis (M.tb), a slow-growing bacteria and a very successful pathogen that acclimates to staying alive inside the host (Chai et al., 2018). According to the World Health Organisation (WHO), 1.5 million people globally died from TB in 2020 (WHO key facts, 2021). In the current scenario, the Bacillus Calmette-Guérin (BCG) vaccine is the only available TB vaccination that is widely used and protects newborns and children from TB meningitis. However, BCG's significance in reducing tuberculosis transmission is restricted since it is considered less efficient in preventing pulmonary TB (Pereira et al., 2007). As a result, it is essential to investigate the M. TB proteins lacking in BCG and capable of eliciting particular humoral responses, cell-mediated responses, and appropriate innate responses in the host (Chen, 2018).\n\nIn bacteria, L-Lysine exporters are transmembrane proteins that remove the surplus of metabolically produced L-lysine and L-arginine from the cytosol. Its deficiency causes high L-lysine cellular levels, inhibiting bacterial growth (Georgieva et al., 2020a). In this protocol, we intend to clone, express, and purify LysE using immobilized metal affinity chromatography (IMAC) to yield protein with high concentration and >95% purity. If the protein is secretory, it is secreted to the cell supernatant and purified by IMAC chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) agarose column. If the protein is present in inclusion bodies (insoluble form), then an extraction buffer containing urea will be used, followed by Ni-NTA purification. Similar protocols have already been adapted to purify other mycobacterial proteins in our collaborative lab with >95% purity.\n\n\nMethods\n\n\n\n1. For cloning: Thermal cycler PCR machine (Eppendorf), electrophoretic assembly (Bio-Rad), gel doc (Vilber), incubator shaker (Labbyscopes), plasmid isolation kit and Gel/PCR extraction kit ((BR Biochem), laminar airflow, water bath (REMI), UV illuminator (Biotech), -80°C and -20°C refrigerator, PCR tubes, Milli-Q Reference Water Purification System (Merck Millipore)\n\n2. For expression and purification: Sonicator (Lobalite), weighing balance (Contech), spectrophotometer (Elico), Cooling centrifuge (REMI), pH meter (BR Biochem), autoclave, dialysis tube, Hot air oven (Sentwin), protein concentrator (Millipore), syringe filter, vortex, falcons, Petri plates, pipettes, vials, tips, beakers, conical flasks, ice flaker, electrophoretic assembly (Bio-Rad), Superdex S200 16/60 columns, AKTA purifier 100.\n\nM.tb LJ slants, E. coli strains ((DH5α, BL-21 (DE3)), Primers (forward and reverse), restriction enzymes for cloning and amplification from New England Biolabs, Plasmids and bacterial strains from Novagen, and Ni-NTA resin for purification from Qiagen (Valencia, CA) will be purchased. Buffers and other chemicals from SIGMA, MP Biomedicals, Invitrogen, Amresco, Himedia, BR-Biochem etc.\n\n1. For genomic DNA isolation: Sodium glutamate, Tris-HCl, EDTA, Lysozyme, RNAse A, Proteinase K, phenol, chloroform, Isoamyl alcohol, sodium acetate, isopropanol, ethanol, agarose, boric acid, tween-80, NaCl, Ethidium Bromide (EtBr).\n\n2. For competent cells preparation: E. coli strain (DH5α), Luria-Bertani (LB) broth, Polyethylene glycol (PEG) 3350, Dimethyl sulfoxide (DMSO), MgSO4, transformation and storage (TSS) buffer, ice, liquid nitrogen.\n\n3. For cloning into pET expression vector: Restriction enzymes, pET vectors, T4 DNA ligase, Taq DNA polymerase, forward and reverse primers, agarose, competent cells, antibiotics, nuclease-free water, MgCl2, Deoxynucleotide triphosphates (dNTPs), Bovine Serum Albumin (BSA), Sodium borate or tris borate buffer, Ethidium Bromide (EtBr), Glycerol, Agar-agar, ice, DNA ladder.\n\n4. For transformation: LB agar media and broth, antibiotics, comp cells, 5X KCM (KCl, MgCl2, CaCl2), nuclease-free water, agar plates, and ice.\n\n5. For protein expression and purification: LB broth, antibiotics, glycerol, Isopropyl β-d-1-thiogalactopyranoside (IPTG), ice, Urea, Sodium Dodecyl Sulphate (SDS), Nickel- Nitrilotriacetic acid (Ni-NTA) resin, Tris, Sodium Chloride (NaCl), β-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), imidazole, Mili Q water, HCl, ethanol, acrylamide, bis-acrylamide, ammonium persulphate, tetramethylethylenediamine (TEMED), dithiothreitol (DTT), glycine, bromophenol blue, protein marker, coomassie brilliant blue, methanol, glacial acetic acid, Bradford's reagent, bovine serum albumin (BSA), phosphoric acid, DNA and protein ladder, Coomassie Brilliant Blue R-250.\n\nGenomic DNA isolation\n\nIsolation and purification of genomic DNA using mycobacterium tuberculosis protocols (Van Helden et al., 2001). Briefly, heat the sealed culture tube of M. tuberculosis H37Rv at 80°C for one hour. Perform all steps in the Class III biosafety cabinet for safety measures. Add 3 ml of extraction buffer (50 mM Tris-HCl, 25 mM EDTA, 5% monosodium glutamate, pH 7.4) in LJ slant and scrape the bacterial colonies using a disposable loop. Collect the buffer and bacterial colonies in a 50 mL falcon tube containing 30 glass beads of 5mm diameter. Again add 3 ml extraction buffer in LJ slant to collect the remaining colonies. Disrupt the suspension by vortexing the tightly sealed falcon at full speed for 2 to 3 minutes to ensure no clumps are left. Lyse the cells using 400 μL of 50 mg/mL lysozyme and add 10 μL of 10 mg/mL RNAase for RNA degradation, followed by incubation for 2 hours at 37°C. Add 600 μL of 10× proteinase K buffer (100 mM Tris-HCl, 50 mM EDTA, 5% SDS, pH 7.8). Add 150 μL of 10 mg/mL proteinase K to remove protein contamination, mix and incubate at 45°C for 16 h. Perform DNA purification by adding 5 ml of phenol, chloroform, and isoamyl alcohol (25:24:1) and slowly mixing by inverting tubes four to five times. Throughout 30 minutes, redo the inversion steps four times. Gently remove the supernatant after centrifuging at 3000g for 20 minutes at room temperature. Add 5 mL of chloroform/isoamyl alcohol (24:1) again, gently mix, and centrifuge at 3000g for 20 minutes at room temperature. Add 700 μl of 3 M sodium acetate, pH 5.5, to the supernatant that has been collected. Add the same amount of isopropanol and mix gently by inverting the tube two to four times. Use a glass rod or sealed pipet tip to collect the DNA, or incubate at -20°C for 30 minutes and then centrifuge it for 30 minutes at 3000g. Wash the pellet in 5 mL 70% ethanol and air dry at 55°C until dry. Finally, suspend the alcohol-precipitated dried M. tuberculosis genomic DNA in 500 μL of lukewarm 1× TE buffer and store it at -80°C. Check the isolated genomic DNA in 1% agarose gel (Van Helden et al., 2001).\n\nComposition of 1× TE buffer\n\n10 mM Tris-HCl, 1 mM EDTA, pH 8.0.\n\nInoculate 10 μl glycerol stock of E. coli strain in 10 ml LB broth (without antibiotics for DH5α), and incubate overnight at 37°C. The next day inoculate 1 ml overnight culture in 100 ml LB broth (without antibiotic for DH5α). Allow it to grow at 37°C until O.D. comes to 0.35-0.4. Swirl the culture flask on ice for 1-2 minutes to pre-chill the cells. Pellet down the cells at 5000 rpm for 10 min at 4°C. Resuspend in 10 ml ice-cold TSS buffer (10 ml TSS for 100 ml culture pellet). Aliquots 50 μl in prechilled vials and snap freeze in liquid nitrogen and store at -80°C.\n\nPreparation of LB broth:\n\nWeigh 25 g LB media and dissolve it in 1 liter of distilled water.\n\nComposition of TSS buffer:\n\nTSS buffer: 100 ml\n\nLB: 73 ml\n\n10% PEG 3350: 20 ml (from 50% stock)\n\n5% DMSO: 5 ml\n\n20 mM MgSO4: 2 ml (from 1 M stock)\n\nConstruction of recombinant plasmid\n\nAfter retrieving the gene sequence of LysE (Rv1986) from the NCBI nucleotide database, design gene-specific forward primer 5′-ATGAACTCACCACTGGTCGTCGG-3′ and reverse primer 5′-CTAGGTCACGGTCAGCGAGATTCC-3′ to amplify the gene of interest isolated from the genomic DNA of Mycobacterium tuberculosis using PCR. Design four different constructs from the LysE gene sequence (Full length, V6, A11, and A64). Perform second PCR using Nde1 (CATATG) and BamH1 (GGATCC) restriction site inserted in the four different forward primers\n\n1. N2_Nde1 FP1: 5′-TAGTGCTACATATGAACTCACCACTGGTCGTCGG-3′\n\n2. V6_BamH1 FP1: 5′-AATTTTGGATCCGTCGTCGGCTTCCTGGCCTGC-3′\n\n3. A11_BamH1 FP1: 5′-TTAATTGGATCCGCCTGCTTCACGCTGATCG-3′\n\n4. A64_Nde1 FP1: 5′-TAATTTACATATGGCACATCCGCGTGCGCTCAATGTCG-3′\n\nand Xho1 (CTCGAG) restriction site inserted in reverse primer 5′-ATTTTCTCGAGCTAGGTCACGGTCAGCGAGATTCC-3′, respectively. The reverse primer remains the same for all four constructs. Analyze the amplified product on 1% agarose gel electrophoresis. Cut and elute from the gel, clean up using the Gel/PCR recovery kit (Zymoclean), and proceed for digestion. See Table 1 for the PCR mixture.\n\nPCR conditions:\n\nDenaturation: 95°C\n\nAnnealing: 64-66°C\n\nExtension: 72°C\n\nNumber of cycles: 30\n\nExpression vector (pET28a) preparation:\n\nThe expression vector (pET 28a) will be provided by our collaborative lab (GeNext Genomics Pvt. Ltd.). Grow 10 ml culture of pET28a vector overnight at 37°C. The next day, isolate the plasmid using a plasmid isolation kit.\n\nDigestion\n\nDouble digestion of vector: 100 μl reaction volume\n\nDigest the expression vectors using Nde1, Xho1 enzymes, and BamH1 and Xho 1 restriction enzymes. Incubate overnight at 37°C. Analyze the digested product on 1% agarose gel electrophoresis. Cut and elute from the gel and clean up using the Gel/PCR recovery kit (Zymoclean). See Table 2 for double digestion mixture for vector.\n\nDouble digestion of amplified LysE: 50 μl reaction volume\n\nDigest the amplified LysE using Nde1/BamH1 and Xho1 enzymes. Incubate overnight at 37°C. The next day, analyze and purify from 1% agarose gel electrophoresis using Gel/PCR recovery kit. See Table 3 for amplified LysE double digestion mixture.\n\nLigation\n\nLigate the digested gene and vector using T4 DNA ligase and transform it into chemically competent E. coli DH5α cells. See Table 4 for ligation reaction mixture.\n\nTransformation\n\nPrepare LB agar plates with antibiotic selection. Take 50 μl DH5α competent cells + 20 μl 5X KCM + 5 μl ligation mixture + 25 μl H2O. Add all the above components, mix well, and keep it on ice for 20 mins. Heat shock at 37°C for 5 mins. Add 500 μl LB media and incubate at 37°C on the shaker for one hour. Centrifuge at 8000 rpm for 3 mins. Discard 550 μl supernatant and resuspend the pellet in the remaining 50 μl supernatant.\n\nPlate the transformed cells on Luria–Bertani (LB) agar plates with antibiotics (kanamycin 50 μg/ml) which act as a selection marker for the pET 28a vector, and incubate overnight at 37°C. The next day, screen the positive clones with gene-specific primers and restriction digestions.\n\nComposition of 5× KCM buffer (10 ml)\n\n2M KCl = 2.5 ml\n\n1M MgCl2 = 1.5 ml\n\n1M CaCl2 = 2.5 ml\n\nMili Q water = 3.5 ml\n\nColony PCR:\n\nInitiate independent PCR reactions for each different primer pair. Mark colonies on a plate, take 20 μl of water in separate tubes, touch colonies from the tip, and dissolve them in 20 μl of water in respective tubes. Prepare glycerol stock by adding 10 μl of the above cells in 100 μl of LB broth with the required antibiotic before disruption. Vortex it properly to disrupt the cells. Take PCR tubes, mark accordingly, and transfer 1 μl from 20 μl dissolved cells in a PCR tube. Add 500 μl LB to the remaining 19 μl cells, and allow it to grow for 1 hr at 37°C, 200 rpm. Check on 1% agarose gel after PCR. Following confirmation of the positive clone, take 10 ml LB + kanamycin add 100 ul of selected clone (grown for 1 hr), and allow it to grow overnight at 37°C, 200 rpm. The next day, isolate the plasmid using a plasmid isolation kit and transform it into BL21 (DE3) competent cells. See Table 5 for colony PCR reaction mixture.\n\nProtein expression and purification\n\nTransform LysEpET28a plasmid in BL21 (DE3) competent cells and inoculate in LB growth media supplemented with kanamycin (50μg/ml) and allow it to grow overnight at 37oC. Re-inoculate the overnight grown culture in flasks containing LB media with antibiotics and allow it to grow for three hours at 37°C at 220 rpm until the O.D. comes to 0.6 to 0.8. Transfer half of the culture at 18°C and after 30 minutes, induce both (18°C and 37°C) cultures using (0.1 to 1 mM) isopropyl β-D-1-thiogalactopyranoside (IPTG) (take out uninduced sample). Harvest the 37°C culture after 3 to 4 hours and the 18°C culture after 18-20 hours. Sonicate the harvested cells on ice in cold extraction buffer A containing 50 mM Tris-HCl pH 8.0, 300-600 mM NaCl, 20% glycerol, 10 mM βME, and (0.1 to 1 mM) PMSF. Centrifuge the sonicated cells for 30 minutes at 4°C at 16000 rpm. Collect the supernatant and store it at -80°C. Collect samples from each step.\n\nFor insoluble inclusion bodies, resuspend the remaining cell pellet at 37°C culture in an extraction buffer containing 0.1% Triton X-100. Shake gently at room temperature for 30-40 minutes and centrifuge at 16000 rpm for 20 minutes. Wash the resultant pellet twice with 50 mM Tris-HCl pH 8.0 and 500 mM NaCl buffer. Finally, separate the inclusion bodies from suspension by centrifugation at 16000 rpm and suspend them by mixing in extraction buffer B containing 50 mM Tris-HCl pH 8.0, 100-300 mM NaCl, 10-20% glycerol, 10 mM βME, and 6 M Urea. Analyze the presence of expressed protein on an SDS-PAGE gel.\n\nProtein purification\n\nFor soluble protein\n\nPerform complete purification steps in cold conditions. Centrifuge the solubilized cell pellet at 16,000 rpm for 30 minutes to remove cell debris. Meanwhile, wash the resin with water and equilibrate with wash buffers containing 50 mM Tris-HCl pH 8.0, 100-300 mM NaCl, and 10-20% glycerol and 5 mM. Wash again with the same buffer containing 20 mM imidazole. Allow the clear supernatant (that shows induction on SDS-PAGE gel) to bind with equilibrated Ni-NTA resin (for overnight rotation). Pack protein-bound resin in the column. Wash the protein-bound resin with wash buffers containing 5 mM and 20 mM imidazole. Finally, elute the bound protein with an elution buffer containing 300 mM imidazole. Collect the fractions at each step and run on an SDS-PAGE gel. Subject the eluted fractions for refolding (dialysis) to yield active protein in buffer containing 30 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol.\n\nFor insoluble protein\n\nPerform complete purification steps in cold conditions. Centrifuge the solubilized cell pellet (inclusion bodies) at 16,000 rpm for 30 minutes to remove cell debris. Meanwhile, wash the resin with water and equilibrate with wash buffers containing 50 mM Tris-HCl pH 8.0, 100-300 mM NaCl, 10-20% glycerol, 5 mM and 6M urea. Wash again with same buffer containing 20 mM imidazole and 6M urea. Allow the clear supernatant (that shows induction on SDS-PAGE gel) to bind with equilibrated Ni-NTA resin (for overnight rotation). Pack protein-bound resin in the column. Wash the protein-bound resin with wash buffers containing 5 mM and 20 mM imidazole and Urea. Last wash will be of 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol, 10 mM βME, and 6 M Urea and then followed by elution with the same buffer containing 300 mM Imidazole. Collect the fractions at each step and run on an SDS-PAGE gel. Subject the eluted fractions for refolding (dialysis) to yield active protein in buffer containing 30 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol and 0.1 to 1 mM CaCl2 or MgCl2.\n\nProceed with the refolded protein for size exclusion chromatography (Superdex S200 16/60 GE Healthcare) attached with AKTA purifier 100. Equilibration and elution buffer consisting of 30 mM Tris-HCl pH 8.0, 300 mM NaCl, 2% glyxcerol, and 1 mM DTT. Collect the fractions and analyze them by SDS-PAGE (Umbarkar et al., 2019). Concentrate the purified protein using a molecular weight cut-off concentrator and measure the concentration by Bradford’s assay.\n\nProtocol may change according to protein behavior during purification and stability.\n\nSize exclusion chromatography\n\nProceed with the refolded protein for size exclusion chromatography (SEC). Superdex S200 16/60 column from GE Healthcare attached with AKTA purifier 100 assembly is used for the purification process. Equilibrate the column and loop before loading the sample in the column. Arrange vials in the collector assembly for fractions collection. Set the chromatographic conditions and inject the sample. Elute the fractions using elution a buffer consisting of 30 mM Tris-HCl pH 8.0, 300 mM NaCl, 2% glycerol, 1 mM DTT and 0.1 to 1 mM CaCl2 or MgCl2. The same buffer is used for equilibration. Collect the fractions (that shows peak) and analyze them by SDS-PAGE (Umbarkar et al., 2019). Concentrate the purified protein using a molecular weight cut-off concentrator and measure the concentration by Bradford’s assay.\n\nChromatographic conditions:\n\n1. Flow rate: 1 to 1.2 ml/min\n\n2. 0.1 to 0.3 MPa\n\n3. Wavelength: 280 and 254 nm\n\n4. Injection volume: 0.5 to 5 ml (depending on loop’s length)\n\nProtocol may change according to protein behavior during purification and stability.\n\nBuffer A for expression check and sonication: 50 mM Tris-HCl pH 8.0, 300-600 mM NaCl, 20% glycerol, 10 mM βME, and 0.1 to 1 mM PMSF.\n\nExtraction buffer (for native protein) Buffer C: 50 mM Tris-HCl pH 8.0, 100-300 mM NaCl, 10-20% glycerol, 10 mM βME.\n\nExtraction buffer (for insoluble protein) Buffer B: 50 mM Tris-HCl pH 8.0, 100-300 mM NaCl, 10-20 % glycerol, 10 mM βME, 6M Urea.\n\nWash buffer A: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol, 5 mM imidazole (6 M Urea for insoluble protein).\n\nWash buffer B: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol, 20 mM imidazole, (6 M Urea for insoluble protein).\n\nElution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20% glycerol, 300 mM imidazole, (6 M Urea for insoluble protein).\n\nDialysis buffer: 30 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol.\n\nBuffer for size exclusion chromatography: 30 mM Tris-HCl pH 8.0, 300 mM NaCl, 2% Glycerol, 1 mM DTT and 0.1 to 1 mM CaCl2 or MgCl2.\n\nRelated studies will be published in indexed journals, and papers will be presented at relevant conferences.\n\nThe study is ongoing. We are validating the protocol using previously purified genomic DNA. Primers, enzymes, reagents and instruments have been arranged.\n\n\nDiscussion\n\nL-lysine exporter (LysE) is a small transmembrane protein of 20-25 kDa that functions as a dimer and has five to six transmembrane hydrophobic spanning helices (Eggeling and Sahm, 2001; Krämer, 2002). In bacteria, L-lysine exporters (transmembrane lysine exporters) remove the surplus of metabolically produced L-lysine and L arginine from the cytosol. Its deficiency causes high L-lysine cellular levels, inhibiting bacterial growth (Georgieva et al., 2020a). To remove the excess lysine from the cytosol, lysine exporters are present on the inner membrane of the bacterial cell. LysE is a target for bacterial inhibition since its deficiency leads to the accumulation of toxic levels or toxic analogs of L-lysine and then the inhibition of bacterial growth (Georgieva et al., 2020b).\n\nLysE was identified in a few studies as a vaccine candidate, drug target, and a potential diagnostic marker (Chen, 2018; Cockle et al., 2002; Georgieva et al., 2020a; Gideon et al., 2010). LysE, also known as the Rv1986 gene in mycobacterium tuberculosis, stimulates innate and adaptive immune cell proliferation and cytokine release. These properties of mycobacterial LysE attract our attention to work and explore more on this protein.\n\nSome studies have purified LysE protein, such as Georgieva et al. (2020a) purified LysE protein on lipodiscs made of native E. coli membranes and in detergent and further characterized it biophysically. Another study (Chen, 2018) claimed to purify LysE as a full-length protein. However, the characterization of this protein is still a new area to explore. This protocol has been designed to purify the LysE gene in four different constructs considering its hydrophobic region using IMAC to unveil new knowledge about this protein. IMAC is a widely adopted method for purifying recombinant proteins containing a 6X histidine tag. In this protocol, we used Ni-NTA resin. Since the protein has a histidine tag, the imidazole ring of histidine readily forms coordination bonds with the immobilized nickel metal. Proteins with polyhistidine sequences can be easily eluted from the column by adding free imidazole to the elution buffer. This method has been developed with discussion with protein experts. Also, the procedures mentioned here have already been validated and implemented in purifying other mycobacterial proteins with >95% purity in our collaborative lab (Umbarkar et al., 2019).\n\n\n\n1. While performing experiments, the buffer’s composition and experimental conditions may slightly differ from the composition and conditions mentioned above depending on the protein's behavior and stability (secretory or insoluble).\n\n2. The concentration of IPTG, urea, PMSF, glycerol, and imidazole may differ after the analysis of expressed protein on SDS-PAGE.\n\n3. To purify protein with high purity (>95%), buffers should be prepared in autoclaved water and stored after filtration.\n\n4. This protocol follows the principles of Good Laboratory Practice (GLP).\n\nThis work has been approved by the Institutional Ethics Committee (IEC), Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nWe would like to acknowledge GeNext Genomics Pvt. Ltd for their support in providing reagents and equipment. We would also like to thank Central Research Laboratory (CRL) DMIHER, Microbiology Lab (JNMC), Biochemistry Lab (JNMC) and COVID lab for their technical support.\n\n\nReferences\n\nChai Q, Zhang Y, Liu CH: Mycobacterium tuberculosis: An Adaptable Pathogen Associated With Multiple Human Diseases. Front. Cell. Infect. Microbiol. 2018; 8: 158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen F: The Role of Mycobacterium tuberculosis Rv1986 and Rv3823c in Stimulating Humoral and Cell-Mediated Immune Responses.2018; 1: 6.\n\nCockle PJ, Gordon SV, Lalvani A, et al.: Identification of Novel Mycobacterium tuberculosis Antigens with Potential as Diagnostic Reagents or Subunit Vaccine Candidates by Comparative Genomics. Infect. Immun. 2002; 70: 6996–7003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEggeling L, Sahm H: The cell wall barrier of Corynebacterium glutamicum and amino acid efflux. J. Biosci. Bioeng. 2001; 92: 201–213. Publisher Full Text\n\nGeorgieva ER, Fanouraki C, Borbat PP: Purification and Biophysical Characterization of LysE Membrane Exporter From Mycobacterium tuberculosis in Lipodiscs Made of Native E. coli Membranes and Detergent. Biophys. J. 2020a; 118: 503a–504a. Publisher Full Text\n\nGeorgieva ER, Fanouraki C, Borbat PP: Expression, purification and initial characterization of LysE membrane exporter from Mycobacterium tuberculosis: Towards comprehensive functional and structural study. FASEB J. 2020b; 34: 1–1. Publisher Full Text\n\nGideon HP, Wilkinson KA, Rustad TR, et al.: Hypoxia Induces an Immunodominant Target of Tuberculosis Specific T Cells Absent from Common BCG Vaccines. PLoS Pathog. 2010; 6: e1001237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrämer R, Burkovski A: Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications. Appl. Microbiol. Biotechnol. 2002; 58: 265–274. PubMed Abstract \| Publisher Full Text\n\nPereira SM, Dantas OMS, Ximenes R, et al.: Vacina BCG contra tuberculose: efeito protetor e políticas de vacinação. Rev. Saúde Pública. 2007; 41: 59–66. Publisher Full Text\n\nUmbarkar CP, Thakre PS, Kashikar SA, et al.: Cloning, expression, and refolding of Nsuccinyl-l-l diaminopimelic acid desuccinylase (DapE) of Mycobacterium tuberculosis. IJLSR. 2019; 7: 110–115.\n\nVan Helden PD, Victor TC, Warren RM, et al.: Isolation of DNA from Mycobacterium tubercolosis. Methods Mol. Med. 2001; 54: 19–30. PubMed Abstract \| Publisher Full Text\n\nWHO key facts: Tuberculosis: Key facts 2021.2021." }	[ { "id": "167046", "date": "26 May 2023", "name": "Seamus J. Martin", "expertise": [ "Reviewer Expertise Immunology", "cell biology", "inflammation", "protein expression", "protein purification" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol, the authors report a protocol for the purification of lysine exporter (LysE) protein, which is a potential drug target, vaccine candidate and diagnostic marker for bacterial infections. To study and utilize the protein effectively, it is necessary to obtain a pure and high concentration sample. This protocol provides a method for purifying LysE protein with >95% purity using immobilized metal affinity chromatography (IMAC). The protocol can serve as a useful resource for researchers who study LysE and other transmembrane protein using IMAC.\nThe authors are in line with the requirements for study protocol. However, I have few suggestions that can further improve the clarity and reproducibility of the protocol.\n\nInclude information on the quality control of the purified protein: I recommend including more information on the expected yield and purity of the final protein. The article mentions that the purified protein has >95% purity but does not provide any information on yield (how much protein was purified from what volume of bacterial culture?) and the purified protein workflow should be demonstrated on SDS-PAGE gels.\n\nThe authors should consider adding some subheadings to break up the text into more manageable sections. This would make it easier for the reader to follow the protocol and locate specific steps if they need to refer back to these later.\n\nThe authors should consider providing some troubleshooting tips for common issues that may occur during the purifications process, especially while purifying the insoluble protein.\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": "9721", "date": "29 Nov 2023", "name": "Shilpa Upadhyay", "role": "Author Response", "response": "Comment 1: In this study protocol, the authors report a protocol for the purification of lysine exporter (LysE) protein, which is a potential drug target, vaccine candidate and diagnostic marker for bacterial infections. To study and utilize the protein effectively, it is necessary to obtain a pure and high concentration sample. This protocol provides a method for purifying LysE protein with >95% purity using immobilized metal affinity chromatography (IMAC). The protocol can serve as a useful resource for researchers who study LysE and other transmembrane protein using IMAC. Response 1: Thank you for appreciating our efforts. Comment 2: Include information on the quality control of the purified protein: I recommend including more information on the expected yield and purity of the final protein. The article mentions that the purified protein has >95% purity but does not provide any information on yield (how much protein was purified from what volume of bacterial culture?) and the purified protein workflow should be demonstrated on SDS-PAGE gels. Response 2: Thank you for your comments. We will definitely include this suggestion in the future publications of this work because this study is still ongoing and we are now validating the cloning part. We will definitely include the SDS-PAGE gels picture in further publications. In this protocol, we had mentioned that “Similar protocols have already been adapted to purify other mycobacterial proteins in our collaborative lab with >95% purity” under the supervision of protein experts. Although, from 1 L culture, we were able to purify 10 -15 mg of similar type of mycobacterial protein. This yield depends upon: 1. The expression of protein depending on type of construct, amino acids sequence, extra tags, folding nature of protein, expression time, temperature and concentration of IPTG. However, only not limited to mention. 2. To purify protein with high purity (>95%), we are adopting the following steps. Which we will publish in details later on getting recombinant protein. First step: As we made the construct in such a way so that protein yield would get as much as at increased level. Second step: The interacting proteins from protein expressing strain with target protein should be as low as possible. However, placing 6 His tag with target protein minimized the problem of protein binding column. Third step: buffers and their stock should be prepared in autoclaved water and stored after filtration. 3. High yield of protein also depends upon the lab conditions. In our Lab, we are following the Good Laboratory Practice (GLP). Comment 3: The authors should consider adding some subheadings to break up the text into more manageable sections. This would make it easier for the reader to follow the protocol and locate specific steps if they need to refer back to these later. Response 3: Thank you for your comments. We will definitely incorporate your suggestion. Comment 4: The authors should consider providing some troubleshooting tips for common issues that may occur during the purifications process, especially while purifying the insoluble protein. Response 4: Thank you for your comments. We will definitely incorporate your suggestion Troubleshooting tips: 1. During Ni-NTA purification, sometimes, while adding the supernatant containing extraction buffer (50 m MTris-HCl pH 8.0, 100-300 mM NaCl, 10-20% glycerol, 10 mM βME), the color of the resin may change from blue to brown. This is due to the presence of β-mercaptoethanol (reducing agent) in extraction buffer. This brown color of resin may disappear after 3-4 washing with wash buffer A containing no βME. This color change will not affect the purification step. 2. During dialysis or refolding of insoluble protein, sometimes protein may get precipitate. To avoid the precipitation, step wise dialysis is required. Since the elution buffer for insoluble protein contains 6M or 8M urea. Step wise removal of urea is recommended. For e.g., use dialysis buffer 1 with 4M urea, dialysis buffer 2 with 2 M urea, dialysis buffer 3 with 1M urea and then the final dialysis buffer with no urea. 3. Concentration of Urea (6M to 8M) may change depending of the nature of protein. This is a protocol and I have added in Notes that “While performing experiments, the buffer’s composition and experimental conditions may slightly differ from the composition and conditions mentioned above depending on the protein's behavior and stability (secretory or insoluble)”." } ] } ]	1	https://f1000research.com/articles/12-297
https://f1000research.com/articles/11-1540/v1	20 Dec 22	{ "type": "Research Article", "title": "Analysis of a self-sufficient photovoltaic system for a remote, off-grid community", "authors": [ "A. S. Aljuboury", "W.K. Al-Azzawi", "L. M. Shakier", "Ahmed Al-Amiery", "A. H. Kadhum", "A. S. Aljuboury", "W.K. Al-Azzawi", "L. M. Shakier", "A. H. Kadhum" ], "abstract": "Background A photovoltaic (PV) system, especially the stand-alone type, along with a battery storage system, plays a significant role in providing the needed electrical energy to remote, off-grid communities. Methods In this study, a stand-alone PV solar cell was enhanced to better understand the system operation. Modeling and simulation were performed using MATLAB software. Results This work has shown that the I-V (current-voltage) and P-V (power-voltage) characteristics of the modelled system have a profound effect on energy when a pulse width modulation controller is used as a charger. The modelled PV system generated the required energy under different conditions, considering temperature and PV battery voltage mismatches. Conclusion\nIn summary, the simulated model in this study can be considered a system capable of detecting different conditions with the presence or absence of light at different temperatures. Finally, monitoring the temperatures, adjusting the voltage with a suitable charger controller, and selecting solar modules made of optimal materials can increase the efficiency of the solar system and allow it to be used in more applications.", "keywords": [ "Stand-alone PV", "Photovoltaic system", "Solar cell", "MATLAB simulation" ], "content": "Introduction\n\nThe world’s population continues to grow, passing the seven billion mark in 2012.1 With seven billion people on the planet, existing resources for food, housing, universal health care, and carbon-based energy will be severely stretched.2 Developing economically and environmentally viable renewable energy technologies to meet the energy needs of the growing population is perhaps the most important of all the challenges we face.3 Photovoltaic (PV) solar modules generate electrical energy that is highly desirable due to their natural advantages such as environmentally friendly and silent operation, long-term performance, and reliability.4 PV solar modules must be affordable, highly efficient, and made from readily available, abundant resources to be economically feasible.5 Stand-alone operation PVislanding system relies heavily on batteries to supply the required amount of electricity.6 Normally, the batteries in these systems are charged during peak solar hours, and the stored energy is used when needed.7 The solar irradiance changes throughout the day, and this irregular pattern affects the PV output.8 Various forecasting techniques are developed in the literature to calculate solar irradiance.9–13 Recently, Javed et al. applied the stand-alone PV systems to a hybrid home storage system with a battery and a supercapacitor.6 Since irradiation is irregular during the day, the charge and discharge patterns of a battery change rapidly. This circumstance degrades battery performance, shortens battery life, and increases battery replacement costs.14–16 As shown in Figure 1, a typical stand-alone PVsystem includes a PVgenerator, a storage battery, a DC/DC converter, a charge controller, an inverter, alternating current (AC) and/or direct current (DC) loads, and an attenuation load.17 There is no connection between a stand-alone PV system and the power grid. A PVgenerator usually consists of a PVgenerator array composed of multiple PV modules, each of which consists of numerous solar cells. When the output of the PV generator exceeds the required demand, the battery stores the excess energy and releases it when the output of the PV generator is insufficient. The load demand of a PV islanding system can take various forms, including DCand/or AC.18–20 The power conditioning unit serves as an interface between all components of the PVsystem, providing control and protection. The DC/DCconverter, the charge controller, and the inverter are the most commonly used components in the power conditioning unit. Additionally, the reducing load is required to absorb any excess energy created when the PV generator’s output exceeds the load requirement and the storage batteries are simultaneously completely charged.\n\n\nMethods\n\nIn this study, a stand-alone PV solar cell was enhanced to better understand the system operation. modeling and simulation were performed using MATLAB software (Version: R2017a, RRID: SCR_001622). In order to determine a regional action plan that can manage different renewable energy techniques, Beccali et al.21 used ELECTRE. Goletsis et al.22 used an energy planning approach to evaluate energy projects. Topcu and Ulengin23 developed energy scenarios based on environmental, economic, physical, political, and other uncontrollable factors. Ribeiro et al.24 developed a multi-criteria decision analysis ranking tool for different scenarios. This classification system is based on technical, economic, environmental, quality of life and labor market factors. The purpose of this research is to model, simulate, and evaluate PV islanding systems in order to increase the operational efficiency of such systems in different large and simple projects.\n\nOne of the most important tasks to achieve the best PV design is to decide on the evaluation criteria for building a stand-alone PV system for a given site.25 In this text, the steps to model a stand-alone PV system using the MATLAB software of the Simulink portal are explained. At an irradiance of 1000 W/m2, a multi-crystalline PV system, namely the Kyocera KC130GT, was used with 36 individual cells connected with two bypass diodes to prevent the breakdown and reverse voltage of the PV system, thus avoiding the generation of a negative voltage (see Figure 2). The terminal voltages of the panel varied, and the P-V and I-V characteristics were measured as in the model in Figure 3. The temperature was set to 25 or 40 °C for the simulation process. The battery is a necessary part of the modelling of the PV islanding system. Therefore, it is connected to a parallel resistor of 8.64 Ω and a 12 V voltage source. The battery is controlled by a charge controller with pulse width modulation (PWM). The PWM simulation was performed at 5 kHz, an 8.02 A saturation current, and a 21.7 volt PV voltage at a duty cycle of 0.5 for 1 second. To make the simulation more accurate, solar irradiance data for the city of Baghdad was used for a sunny day and a rainy day on February 2, 2022 and February 5, 2022, respectively. This in turn incorporated the weather effect into the experimental work to be studied by the model system.\n\n\nResults and discussion\n\nIn this paper, the electrical energy generated from the used PV panels was determined, taking into account the losses in power through the entire system. According to the design and simulation in this research, two different weather conditions during February 2, 2022 and February 5, 2022 were used as minimum data to find out the optimal parameters of the PV system as a home stand alone PV solar system. This system facilitates the process of calculating the amount of power expected to be supplied to the beneficiary throughout the year under different environmental conditions. In order to achieve the load requirements, the current, voltage, and power characteristics must be studied. The overall I-V and P-V characteristics of the modelled PV solar array are plotted in Figure 4. The highest values of the resulting power were about 119 and 125 Watts at 25 °C and 40 °C, respectively, as shown in Figure 5. From the characteristics of the I-V and P-V plots, it is possible to understand the dependence of the cell temperature on the material from which the PV cells are made, including multi-crystalline materials, and how much it is affected mainly by temperature, which in turn is stimulated by the presence of a potential difference applied to its ends. The voltage and current characteristics of the PV panels have a clear impact on the amount of power produced when using a PWM charge controller. Whereas stand-alone PV systems operate at 11-14 V, when a PWM charge controller is connected and the battery provides a 12 V operating voltage, hybrid PV systems operate at 12 V.\n\nBy simulating the main components of the stand-alone PV system, the solar irradiance data were used, as shown in Figure 6. The state of charge of the battery increased during the period 10:00 am–14:00 pm, as shown in Figure 7. The photovoltaic model was equipped with an 8.02 A saturation current and 21.7 volts. During the period of sunny hours, the load consumes part of the power, and the excess is stored in the battery (charging). When the solar radiation was low, it was noticed that the load removed power from the battery. The load would be disconnected from the battery as soon as the charge rate was below 30%. On February 2, 2022 (the sunny day), the state of charge of the applied battery dropped from 75% to 55% as it supplied power to the load. Then the PV panels were able to supply the loading power with the required energy. In the case of February 5, 2022 (the rainy day), the battery’s state of charge gave the same indication. But the PV panels were not able to supply the same quantity of the required power because of the lower irradiation amount. As a result, the simulated system performed significantly better during the sunny day conditions. Whereas the battery reached 95% as a maximum value of state of charge at the end of a sunny day, it ended the day at 82%. This was much better than the other day, which reached 58% at the end of the day.\n\n\nConclusion\n\nStand-alone modeling and simulation PV system with its components were presented using MATLAB and the SIMULINK portal. The simulation proved the effect of the amount of radiation projected and the weather condition on the amount of power generated by the system. The voltage and current characteristics of the PV panels had a clear impact on the amount of power produced when using a PWM charge controller. The simulated model in this research can be considered a system capable of sensing different conditions with the presence or absence of light at different temperatures. Also, it can be concluded from this research that the optimum size of the PV system corresponding to the capacity of the battery is a matter that must be taken into account carefully because of its impact on improving the efficiency of the solar system. It is also worth noting that the cost of the stand-alone PV system modelled in this research is very low. But if this system is implemented in larger projects, the cost will be five times what it was in this research due to the high prices of photovoltaic panels, batteries, and fuel. Finally, monitoring the temperatures, matching the voltage with the appropriate charger controller, and selecting solar panels made of optimal materials can raise the efficiency of the solar system and make it useful in more applications.\n\n\nEthical approval and consent\n\nNo humans or animals were included in this study.", "appendix": "Data availability\n\nNo data are associated with this article\n\n\nReferences\n\nCarbone M, Ly BH, Dodson RF, et al.: Malignant mesothelioma: facts, myths, and hypotheses. J. Cell. Physiol. 2012 Jan; 227(1): 44–58. 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Publisher Full Text\n\nMaharjan L, Inoue S, Akagi H: A transformerless energy storage system based on a cascade multilevel PWM converter with star configuration. IEEE Trans. Ind. Appl. 2008 Sep 19; 44(5): 1621–1630. Publisher Full Text\n\nNehrir MH, Wang C, Strunz K, et al.: A review of hybrid renewable/alternative energy systems for electric power generation: Configurations, control, and applications. IEEE transactions on sustainable energy. 2011 May 27; 2(4): 392–403.\n\nBeccali M, Cellura M, Mistretta M: Decision-making in energy planning. Application of the Electre method at regional level for the diffusion of renewable energy technology. Renew. Energy. 2003 Oct 1; 28(13): 2063–2087. Publisher Full Text\n\nGoletsis Y, Psarras J, Samouilidis JE: Project ranking in the Armenian energy sector using a multicriteria method for groups. Ann. Oper. Res. 2003 Apr; 120(1): 135–157. 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Publisher Full Text" }	[ { "id": "182142", "date": "05 Jul 2023", "name": "Erkata Yandri", "expertise": [ "Reviewer Expertise Solar photovoltaic", "PV performance analysis", "hybrid photovoltaic and thermal collector", "solar thermal collector" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nIn principle, the title is OK. Basically, the title is OK, but the title needs to be strengthened by adding the method used. I suggest that the title consists of purpose + method. However, the title must be adjusted to the keywords chosen. To optimize for search engines, it is better if the words in the title are no longer used for keywords, or vice versa. Please adjust accordingly.\nAbstract:\n\nAbstract is still too general and short. To make your abstract more interesting, I suggest to add some other application from this method and show the important results.\nKeywords:\nAs suggested for title, some keywords are already in the title, so they are less effective in optimizing search engines. I suggest replacing some of the current keywords with some that aren't already in the title. To further optimize search engines, make sure the keywords are free from the words in the title.\nINTRODUCTION:\n\nIn general, INTRODUCTION is too short, too simple, uninteresting and boring., but there are some notes for improvement;\nFrom the existing Introduction, please break it down and develop it into 4 paragraphs, as follows:\nParagraph 1: Focus on the background, starting from the energy situation and ending with the importance of utilizing renewable energy sources, one of which is solar energy.\n\nParagraph 2: This is a transitional paragraph from the background in paragraph 1 to the literature review that will be discussed. You can start right away from connecting the solar energy that was alluded to at the end of paragraph 1 to solar photovoltaic. Then, develop it with developmental phenomena and interesting issues to discuss according to your topic as shown in the title.\n\nParagraph 3: Focus on literature reviews according to the topic of discussion by prioritizing research papers that are highly related; with that title. Make sure the references used are more recent, or the year of publication is no more than 5 years.\n\nParagraph 4: Summary of the results of the literature, explain the gaps and explain the novelties of this study. Finally, state the purpose of this study.\n\nEnsure that Figure 1. Benefits of DC, AC, and hybrid DC-AC coupled configurations of stand-alone PV systems is no longer in the Introduction section. If you still need it to develop your manuscript, then move this Figure to the Methods section.\n\nThere are several references published more than 5 years ago. I suggest updating them with a more recent one.\nMATERIALS AND METHODS\nThe method section is not written with a good writing structure and flow so it is unclear and difficult to understand. I suggest to reconstruct the Method section to be more structured and interesting to understand. Please improve your Method section with the following suggestions;\nYou can split the current 2. Methods section or Materials and Methods section into sub-sections, such as;\n2.1. Materials: please focus here on explaining all the materials or tools used, including software, etc.\n2.2. Modeling and Simulation: please focus here on explaining everything about the modeling and simulation being done, including parameters, coefficients, or other assumptions or scenarios used. If there are basic calculations or theories, or modelling formula, etc., you can develop them as needed here. You can still use Figure 2 and Figure 3 with appropriate explanations, to link with the methods you developed and describe in this section.\nYou can use pictures or tables as appropriate to make it easier for readers to understand your explanation. You should pay attention to writing briefly and concisely, but still understandable.\n\nNo more mini literature reviews in the Material and Methods section, as you are currently doing. Literature review should be in the Introduction section, as previously suggested. Here, if you really have to include references based on pictures, figures, theories, makers, etc., then please include them as references.\n\nThe important thing, please make sure that the Material and Methods section that you develop is based on the objectives that you explained earlier in the last sentence in the 4th paragraph in the Introduction section.\nRESULTS and DISCUSSION:\nThe information presented in RESULT and DISCUSSION is understandable, although less interesting in delivery. However, there are some notes for improvement;\nThere are 4 figures presented, but there are only 2 paragraphs to explain them. For that, I suggest explaining each figure with 1 paragraph. If the two figures are related, you can combine these figures into figures a and b, so that you can explain them in 1 paragraph.\n\nMake sure the numbers or terms used in the Results and Discussion section have been displayed and explained in the Materials and Methods section.\n\nPlease provide and develop some more results based on some of the simulation results that you get, so that the topics you describe become clearer and more interesting and clearer.\n\nFor Discussion, please explain to readers at least 2-3 items for discussion, related to the results you have obtained. Please remember, here do not repeat what has been explained in the Result. However, what is important here is the implications of your results for a more interesting discussion material for the reader.\n\nThen, in the last paragraph of the Discussion section, please briefly explain, such as; the practical application and other contributions of this research, and finish with suggestions for further research directions. Relevant and related references should be cited as necessary.\nCONCLUSIONS:\n\nThe conclusion you give is still out of focus. For that, there are some suggestions;\nWrite a Discussion with a focus on 3-5 key findings.\n\nDo not rewrite what was written in the previous section.\n\nWrite a brief summary of the discussion points that you have written in the discussion section\nREFERENCES:\nCurrent references can still be developed and updated. As suggested in the Introduction section, make sure the references used are more recent, or the year of publication is no more than 5 years.\nENGLISH:\nI suggest that you re-check the English.\n\nThank you for allowing me to review this manuscript. This manuscript purposes to model, simulate, and evaluate PV islanding systems in order to increase the operational efficiency of such systems in different large and simple projects. I think this study is interesting for F1000Research readers. However, this manuscript cannot be indexed in its current condition because many things need to be improved, in addition with a fairly short, look like a draft manuscript (please refer to the comments above). So, I recommend “Rewrite Manuscript for the next review”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10849", "date": "05 Feb 2024", "name": "Ahmed Al-Amiery", "role": "Author Response", "response": "Dear Dr. Erkata Yandri, Thank you for your valuable feedback on our manuscript entitled \"Analysis of a self-sufficient photovoltaic system for a remote, off-grid community.\" We appreciate the detailed suggestions and comments provided, and we have carefully addressed each point in the revision. Below, we outline the changes made to enhance the manuscript: Title: We have revised the title to incorporate the method used, making it more informative and aligning with the paper's content. Enhancing Operational Efficiency of Stand-Alone Photovoltaic Systems: A Simulation-Based Analysis of Self-Sufficient Solar Power for Remote, Off-Grid Communities Abstract: We have expanded the abstract to include additional applications of the proposed method and highlight important results, addressing the need for more specificity and interest. Keywords: Adjusted and replaced some keywords to avoid redundancy with the title and further optimize search engine results. Photovoltaic systems, Off-grid communities, Solar energy simulation, Stand-alone PV, Battery storage, Pulse width modulation, I-V characteristics, P-V characteristics, Solar system efficiency Introduction: The Introduction has been expanded into four paragraphs, as suggested. We have provided more details on the background, connected solar energy to photovoltaic systems, and added a transitional paragraph leading into the literature review. The literature review now focuses on more recent research papers related to the topic. Additionally, Figure 1, \"Benefits of DC, AC, and hybrid DC-AC coupled configurations of stand-alone PV systems,\" has been moved to the Methods section. Materials and Methods: The Methods section has been restructured to improve clarity and flow. It now includes sub-sections (Materials and Modeling/Simulation) to provide a more organized presentation. We have removed mini literature reviews from this section and focused on presenting materials, tools, and the modeling/simulation process. Results and Discussion: Each figure is now explained in a separate paragraph, and the numbers or terms used are properly referenced to the Materials and Methods section. We have added more simulation results to provide a clearer and more comprehensive understanding of the topic. The discussion now includes 2-3 items related to the results, avoiding repetition from the Results section. Conclusion: The conclusion has been revised to focus on 3-5 key findings, avoiding redundant information from previous sections. References: We have updated and refined the references, ensuring that they are more recent and relevant to the topic. English: The manuscript has been carefully reviewed for language, grammar, and clarity. We believe that these revisions have significantly improved the manuscript's quality, addressing the concerns raised. We appreciate the opportunity to resubmit the revised manuscript for your consideration. Thank you for your time and valuable feedback. Sincerely," } ] }, { "id": "175415", "date": "16 Aug 2023", "name": "Hussein Mohammed Ridha", "expertise": [ "Reviewer Expertise Optimal design and of the standalone PV system", "Parameter extraction of the PV models", "MOO", "and MCDM." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors of the presented paper try to simulate the behavior of the standalone PV-Battery system, which is considered an interesting topic. However, there are several suggestions are required to be completed to be suitable for publications:\n\nFigure 1 should be replaced with a higher resolution quality.\n\nMore papers need to be included in the literature.\n\nHence, the authors mentioned the cost analysis in the conclusion. However, I have not seen any table lists the cost of the components of the system. Then, how the cost analysis is performed?\n\nThe load demand of the system should be indicated and described.\n\nMore descriptions on the output PV modules should be included in the result section.\n\nFinally, how the authors describe the reliability of the system? Which criteria are used.\n\nOnce the authors consider the previous comments, the submitted paper can be appropriate for publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10848", "date": "05 Feb 2024", "name": "Ahmed Al-Amiery", "role": "Author Response", "response": "Dear Dr. Hussein Mohammed Ridha, Thank you for your thoughtful and constructive feedback on our manuscript. We appreciate your positive acknowledgment of the paper's interesting topic and potential suitability for publication. We have carefully considered your comments and suggestions, and we will address each of them below: Figure 1 Resolution: We apologize for the low resolution of Figure 1. We will replace it with a higher resolution version to enhance its clarity and readability. Literature Review: We acknowledge the importance of a comprehensive literature review. We will enrich the literature review section by incorporating more relevant papers, particularly recent ones, to strengthen the background and relevance of our study. Cost Analysis: Your observation about the absence of a cost breakdown for the system components is valid. We will include a detailed table listing the costs of individual components in the conclusion section to provide transparency on the cost analysis process. Load Demand Description: We appreciate your suggestion to provide more details on the load demand of the system. In the revised manuscript, we will include a section that describes the load demand characteristics, ensuring a more comprehensive understanding of the system's behavior. Output PV Module Descriptions: We agree with your recommendation to provide more detailed descriptions of the output PV modules in the Results section. We will include additional information on the specifications and characteristics of the PV modules used in the simulation. Reliability Criteria: Your question regarding the reliability of the system and the criteria used is crucial. In the revised manuscript, we will explicitly discuss the criteria and metrics employed to assess the reliability of the stand-alone PV system. We appreciate your thorough evaluation, and we are committed to addressing these suggestions promptly. We believe that incorporating these changes will significantly enhance the quality and completeness of the manuscript. Thank you for your valuable feedback and consideration. Best regards," } ] } ]	1	https://f1000research.com/articles/11-1540
https://f1000research.com/articles/12-340/v1	27 Mar 23	{ "type": "Review", "title": "Technological tools for the measurement of sensory characteristics in food: A review", "authors": [ "José D Martinez-Velasco", "Annamaria Filomena-Ambrosio", "Claudia L Garzón-Castro", "José D Martinez-Velasco", "Annamaria Filomena-Ambrosio" ], "abstract": "The use of technological tools, in the food industry, has allowed a quick and reliable identification and measurement of the sensory characteristics of food matrices is of great importance, since they emulate the functioning of the five senses (smell, taste, sight, touch, and hearing). Therefore, industry and academia have been conducting research focused on developing and using these instruments. In this review, several of these technological tools are documented, such as the e-nose, e-tongue, artificial vision systems, and instruments that allow texture measurement (texture analyzer, electromyography, others). These allow us to carry out processes of analysis, review, and evaluation of food to determine essential characteristics such as quality, composition, maturity, authenticity, and origin. The determination of these characteristics allows the standardization of food matrices, achieving the improvement of existing foods and encouraging the development of new products that satisfy the sensory experiences of the consumer, driving growth in the food sector.", "keywords": [ "Sensorial characteristic", "technological tools", "electronic nose", "electronic tongue", "artificial vision", "texture analyzer", "acoustic analysis", "food sector" ], "content": "1. Introduction\n\nThe world of the food industry search to ensure satisfactory multisensory experiences for consumers through the consolidation of quality standards for food products (Blissett & Fogel, 2013; Tuorila & Hartmann, 2020). The first approach to each food matrix allows the consumer to identify attributes related to size, shape, color, and brightness. A second approach allows more direct interactions related to the perception of smell, aroma, taste, temperature, and texture of the product (Fine & Riera, 2019; Isogai & Wise, 2016; Moding et al., 2020; Nederkoorn et al., 2018). Recognizing these sensory characteristics determines the acceptance or rejection of the food (Costell et al., 2009; Torres Gonzalez et al., 2015; Wadhera & Capaldi-Phillips, 2014). One of the disciplines that study the sensory characteristics of food is sensory analysis. This term became a field of study in the 17th century when Jean Anthelme Brillat-Savarin, in 1825, wrote his first book entitled Philosophy of Taste, in which he established the basis for the analysis of food and how it is perceived (Chong, 2012). The constant evolution of the concept and applicability of sensory analysis has consolidated its study using trained panelists or instrumental methods. Although the analyses carried out by these panelists constitute an essential source of information for the acceptance or rejection of a food product, this can be subjective due to biological, social, and other external factors surrounding the subject (Buratti et al., 2018; Loutfi et al., 2015; Tan & Xu, 2020).\n\nOne of the main limitations when implementing sensory tests is the number of required panelists, ranging from 7 to 100 depending on the test type (Lawless & Heymann, 2010; O’Mahony, 2017). This implies an investment of human and economic resources, raw materials, and/or time. This limitation has motivated researchers to generate technologies to identify and quantify some sensory characteristics of foods with greater precision (Akimoto et al., 2017; Kusumi et al., 2020; Pascual et al., 2018).\n\nSuch developments search to mimic the functioning of the five senses, such is the case of electronic noses (e-noses) and tongues (e-tongues), which upon contact with food, generate an electronic response from a chemical interaction, which is interpreted by a digital information processing system (Banerjee et al., 2019; Bonah et al., 2020). Similarly, image analysis through devices such as cameras seek to simulate the sense of eyesight (Ansari et al., 2021; Barbon et al., 2017; Kakani et al., 2020; Khojastehnazhand & Ramezani, 2020); concerning touch and hearing, some reports show various technological tools that measure force and sound, seeking to imitate the behavior of these senses (Akimoto et al., 2019; Kato et al., 2017; Kusumi et al., 2020).\n\nEach of the technological tools mentioned above contributes a description of the primary sensory characteristics of the food matrix to be evaluated. This article consolidates information on some technological tools reported in the literature for sensory analysis in various food matrices.\n\n\n2. Electronic nose (e-nose)\n\nOdor is one of the most representative attributes of food. This can be expressed as one of the qualities of Volatile Organic Compounds (VOCs), so unique and distinctive that they are considered fingerprints (Bonah et al., 2020; Tan & Xu, 2020).\n\nGenerally, the sensory analysis method to identify such components is performed by panelists who rate and classify on different scales the odor perceived in the sample (Barbieri et al., 2021; Giungato et al., 2018; Niu et al., 2019; Świąder & Marczewska, 2021). On the other hand, different methods have been developed for the identification of VOCs, which are characterized by high accuracy and reliability, such as: Gas Chromatography-Olfactometry (GC-O), Gas Chromatography-Mass Spectrometry (GC-MS), Headspace Solid Phase Microextraction (HS-SPME), as some of the most used methods (Attchelouwa et al., 2020; Chen et al., 2021). However, these methods usually require sample conditioning, which involves investing many different types of resources (Shi et al., 2018). Considering the above, devices such as the e-nose have been developed, consisting of an array of electrochemical sensors articulated with a pattern recognition system that identifies, groups, and discriminates the VOCs (Gliszczyńska-Świgło & Chmielewski, 2017; Loutfi et al., 2015). This has become an alternative to generating fast and reliable results in the food industry (Barbosa-Pereira et al., 2019; Conti et al., 2021; Wasilewski et al., 2019).\n\nFor the articulation of three fundamental systems characterizes E-nose: sensing, electrical conditioning, and pattern recognition; see Figure 1.\n\nThe sensing system is composed of a matrix of sensors that can be of different types such as: conductivity, polymers, intrinsic conductive polymers, metal oxide, surface acoustic waves, and quartz crystal balance, which allow the detection of VOCs through absorption, adsorption, or chemical reaction methods. Depending on the characteristics of the food matrix to be evaluated, the sensors that make up the e-nose must be carefully considered, as they will react more efficiently to certain particles (Tan & Xu, 2020; Wilson & Baietto, 2009). This detection produces an electronic signal, from which it is possible to characterize the VOCs.\n\nThe electrical conditioning system is responsible for matching the signal emitted by each of the sensors. Signal matching consists of amplification and filtering to identify the analyzed food matrix sample (Shi et al., 2018).\n\nFinally, the pattern recognition system receives the already conditioned electrical signal and is in charge of processing it. For this procedure, extraction methods are used, which aim to obtain reliable and robust information from the electrical signal, guaranteeing greater measurement efficiency. Some extraction methods are: Principal Component Analysis (PCA), Support Vector Machines (SVM), Artificial Neural Networks (ANN), Linear Discrimination Analysis (LDA), Discriminant Function Analysis (DFA), decision trees, and other machine learning classifiers (Tan & Xu, 2020; Yan et al., 2015).\n\nE-nose is used in several food matrices to identify their authenticity due to the growing number of counterfeit products that represent a significant risk to the health of consumers (Gliszczyńska-Świgło & Chmielewski, 2017). Additionally, this device also allows users to identify and group according to their specifications some food matrices such as: alcoholic beverages, dairy products, and juices (Sanaeifar et al., 2017); the ripeness of fruits and vegetables; quality of meats; shelf life of grains, among others (Du et al., 2019; Tan & Xu, 2020; Wang et al., 2019).\n\nFor example, the e-nose of the Alpha MOS FOX family has been used to identify possible adulteration of olive oil with hazelnut and sunflower oils (Mildner-Szkudlarz & Jeleń, 2008). Also, in the analysis of flaxseed oil detecting adulteration with other similar components (Wei et al., 2015).\n\nIn research conducted by Nurjuliana (2011), the volatile compounds in pork, beef, lamb, and chicken sausages were analyzed. The samples taken from each of the sausages were analyzed by mass spectrometry, gas chromatography, and zNose™ electronic nose, which allowed the identification of the type of meat from which the sausages were made. Although the results of the tests carried out by all the instruments were highly efficient, the speed and low cost of using the zNose™ e-nose were highlighted.\n\nAdditionally, in the research by Ghasemi-Varnamkhasti et al. (2019), an e-nose was custom designed using five types of MOS sensors to classify two pieces of cheese: Roquefort and Camembert. This classification was carried out by taking into account the milk (sheep, goat, or cow) with which it was made, the degree of pasteurization, and the maturity of these cheeses.\n\nOther reports show the use of e-noses to analyze fish. Güney and Atasoy (2015), used a low-cost e-nose developed at Karadeniz University, composed of 8 metal oxide gas sensors, to classify three fish species (Horse mackerel (Trachurus murphyi), Anchovy (Engraulidae) and Whiting (Merlangius merlangus). In addition, Zhang et al. (2012), analyzed VOCs during the storage and freezing process of sawfish (Scomberomorus niphonius), finding a linear relationship between a volatile nitrogen base with triethylamine. A separate investigation reports the use of the commercial e-nose Alpha MOS FOX 3000, composed of 18 MOS-type sensors, to establish the sensory profile of the active aromatic compounds of cumin (Cuminum cyminum L.) (Ravi et al., 2013).\n\nTable 1 shows some relevant studies using e-nose in the food, specifying: product, purpose of the analysis, e-nose model, type of sensor, extraction method, and main result obtained.\n\n\n3. Electronic tongue (e-tongue)\n\nThe human tongue can identify five basic tastes: sour, salty, sweet, bitter, and umami (Beauchamp, 2019). Usually, the evaluation and classification of the basic flavors of a product are done through trained panelists and sometimes consumers (Jiang et al., 2018). However, these measurements can be subjective, which can be reduced by using technological tools such as the e-tongue, thus ensuring repeatability and reproducibility of the results (Schlossareck & Ross, 2019). Ross (2021) showed that combining different electrodes makes it possible to identify different flavors, such as fatty, metallic, and others. Different investigations have shown that by using the e-tongue, it is possible to determine the quality, adulteration, classification, or origin of food (de Morais et al., 2019; Elamine et al., 2019; Jiang et al., 2018; Sobrino-Gregorio et al., 2018). The previously mentioned characteristics have allowed the e-tongue to become a fast, economical and impartial detection alternative (Titova & Nachev, 2018); this is because it allows the characterization of the flavor of the food matrix (di Rosa et al., 2017). Additionally, the e-tongue has a matrix of electrodes that, according to their combination and characteristics, produce potentiometric, voltammetric, and impedimetric signals (Jiang et al., 2018).\n\nE-tongue is characterized for articulating three fundamental systems: sensing, electrical conditioning, and pattern recognition (di Rosa et al., 2017) (see Figure 2).\n\nE-tongue sensing system is composed of two or more electrodes, each electrode has a membrane that upon contact with the analyte generates a chemical interaction causing a reversible change in the electronic properties, which allows the characterization of the food matrix (Tan & Xu, 2020).\n\nPotentiometric-type electrodes measure the voltage differences between the working and the reference electrodes (Wasilewski et al., 2019). The voltage change in the measurement given by the working electrode will have a proportional relationship to the concentration of the analyte (Jiang et al., 2018; W. Wang & Liu, 2019). Some of the membranes used in potentiometric electrodes can be multi-channel lipid with a reference electrode made of a silver/silver carbon alloy (Ag/AgC), chalcogenide glass with a polyvinyl chloride (PVC) film, liquid or polymeric, which allow the detection of the voltage generated when in contact with the food matrix (Tan & Xu, 2020).\n\nRegarding voltammetric electrodes, these are used in conjunction with a minimum electrode configuration in which one must have a working, a reference, and an auxiliary electrode (Jiang et al., 2018; Wasilewski et al., 2019). Generally, these working electrodes are constituted by a bare or modified metal, which contemplates any of the following compounds: copper (Cu), nickel (Ni), palladium (Pd), silver (Ag), tin (Sn), titanium (Ti), zirconium (Zr), gold (Au), platinum (Pt) and radium (Ra) (Jiang et al., 2018). Its operation encourages the transfer of electrons through the food matrix, measuring the resulting polarization current, which has a direct relationship with the concentration of certain components present in the food (Wei et al., 2018).\n\nAnother group of electrodes is those of impedimetric type, characterized by being coated with different polymeric materials, which, upon receiving an alternating signal of variable frequency and constant amplitude, produce an alteration in the impedance value (Garcia-Hernandez et al., 2018). This impedance change allows for characterizing, detecting, and discriminating different components such as: sucrose (C12 H22 O11), sodium chloride (NaCl), potassium chloride (KCl), and hydrochloric acid (HCl) (Podrazka et al., 2017). According to the literature, the most commonly used electrodes on the market are potentiometric and voltammetric electrodes due to advanced development (Wang & Liu, 2019).\n\nTan and Xu (2020) indicated that electrodes in the development phase incorporate biomaterials such as enzymes, whole cells, tissues, receptors, or antibodies, whose chemical interaction with the food generates a transfer of electrons, ions, or molecules. This transfer modifies the characteristics of the electronic signal, like those produced by potentiometric and voltammetric electrodes. It is expected that these biosensors will be a technology that will contribute to improving results in the future.\n\nThe electrical conditioning and pattern recognition systems of the e-tongue present particularities closely like those of the e-nose. The only substantial difference between these two technological tools is presented in the sensing system in terms of the characteristics specific to the internal and structural design of the sensors (Tan & Xu, 2020; Wasilewski et al., 2019).\n\nThe use of the e-tongue in the food industry encompasses a wide range of applications, including discrimination by type and place of origin, verification of authenticity, adulteration or counterfeiting, and quantification of food matrix components (Titova & Nachev, 2018; Wasilewski et al., 2019).\n\nA clear example of the use of such technology for classifying products by type and place of origin is evidenced in the research developed by Souayah (2017), where a potentiometric e-tongue was used to classify 60 samples of olive oil. Moreover, Elamine et al. (2019) discriminated 31 samples of honey from Portugal by botanical origin using an impedimetric e-tongue.\n\nCetó and Pérez (2020) used an inset voltammetric e-tongue from Bas Inc. configured with three electrodes of gold (Au), platinum (Pt), and glassy carbon (C), to carry out the process of identification of authenticity and classification of 44 samples of six different varieties of vinegar. The measurement results of the equipment were subjected to the PCA and LDA extraction methods, which allowed the discriminating and categorizing of the total of the analyzed samples with 100% accuracy. This research allowed it to generate records of the electrochemical fingerprints of the vinegar.\n\nFurthermore, a voltammetric-type e-tongue was custom-developed to identify adulteration in roasted ground coffee (de Morais et al., 2019). This research analyzed 90 cups of coffee (60 unadulterated and 30 adulterated). LDA, SPA, and PLS-DA identification methods were applied to the measurements obtained; as a result, the adulterated beverages were identified and the purity percentage in each sample was quantified.\n\nAnother example is the investigation of the evolution process of taste compounds in the chicken stew at different cooking times, which focused on detecting nucleotides and free amino acids using a commercial e-tongue (TS-5000Z, Insent). As a result, the proportion of the components detected in each cooking stage and the identification of inosine monophosphate (IMP), glutamic acid (Glu), lysine (Lys), and sodium chloride (NaCl) as the main compounds highlighted the final flavor attributes of the chicken were evidenced (Liu et al., 2017). Table 2 shows some relevant studies in which e-tongues in different food matrices.\n\n\n4. Artificial Vision System\n\nComputer Vision System (CVS) also known as Artificial Vision System (AVS), is an image analysis tool used to obtain information about objects through them (Bhargava & Bansal, 2018; Wu & Sun, 2013). This is due to its ability to characterize: shape, size, color, and other particularities of the object, which can be static or moving (Zhu et al., 2021). Therefore, the CVS can be used in both continuous and static production lines, achieving a real-time analysis, as it allows fast, accurate, and non-invasive captures, with reliable and reproducible results (Barbon et al., 2017; Patrício & Rieder, 2018). Due to its flexibility and technological development, a CVS can store information about an object to perform further analysis using new images (Taheri-Garavand et al., 2019; Wu & Sun, 2013). Thus, the CVS becomes an alternative to avoid the possible errors of quality inspection of the objects which the human eye can incur (Patrício & Rieder, 2018).\n\nA CVS is composed of three fundamental stages: illumination, image detection, and pattern recognition (Kakani et al., 2020), see Figure 3. The first stage plays an important role in image acquisition, since light has a direct impact on the clarity and color of the images and its improper use can generate shadows and unwanted reflections, cataloged as noise in the images (Vithu & Moses, 2016). Therefore, depending on the application of the system, an appropriate selection of the light-generating elements must be made, considering characteristics such as wavelength, intensity, and direction. These light-generating elements can be light bulbs (incandescent, fluorescent, halogen), lasers, light emitting diodes (LEDs), X-ray tubes, and infrared lamps (Naik & Patel, 2017; Sun et al., 2019; Zhu et al., 2021). These ensure clarity, repeatability, and reliability of the image (Barbon et al., 2017).\n\nTwo of the most commonly used technologies in the second stage are cameras or scanners, which are responsible for taking an image of the object to be analyzed. Cameras capture a two-dimensional image instantaneously, while scanners take a line of pixels in an instant of time, so it requires a mechanism that performs a displacement of the scanner or the object to capture a succession of data and thus obtain the two-dimensional image (Patrício & Rieder, 2018). Internally, these devices have specialized sensors that can capture color, monochromatic, thermal, or ultraviolet images depending on their characteristics (Patrício & Rieder, 2018; Sun, 2016; Vithu & Moses, 2016; Zhang et al., 2014). Other technologies used in this stage are: Hyperspectral, Magnetic Resonance, and X-Ray (Sun, 2016; Zhang et al., 2014).\n\nFinally, the third stage aims to extract quantitative and qualitative information from the image using an analysis algorithm usually run on a processor (Zhu et al., 2021). Depending on the application and the complexity of the system, image processing is divided into three different levels: low, medium, and high. At the first level, operations such as cleaning of noise caused by shadows or external elements, quality enhancement, or correction of image illumination errors are performed (Patrício & Rieder, 2018). Then, at the medium level, segmentation, description, classification of shapes, and image dimensions are performed (Sun et al., 2019; Taheri-Garavand et al., 2019). Finally, at the third level, more complex operations are performed, including classification, comparison, and discrimination of the characteristics of the object in the image. These operations can be applied to the area or regions of interest using analysis methods such as statistical tools or computational models such as neural networks, which are some of the most used extraction methods (Kakani et al., 2020; Patrício & Rieder, 2018).\n\nGiven the versatility and advantages presented by a CVS, the food industry has been implementing these systems to identify properties such as: morphology, color, texture, freshness, and quality (Bhargava & Bansal, 2018; Patrício & Rieder, 2018; Taheri-Garavand et al., 2019; Vithu & Moses, 2016). In general, the information collected is fed into databases to train learning algorithms and establish patterns to build a knowledge base, with which a system for autonomous decision-making can be implemented to provide an agile and flexible solution (Zareiforoush et al., 2015).\n\nThe applications that recurrently use CVS are focused on the classification and prediction of the characteristics of a food matrix, whether it is an individual analysis, a production batch, or harvesting (Arsalane et al., 2020; Kakani et al., 2020; Velesaca et al., 2021). Research such as the one carried out by Arselane et al. (2020) in which they were able to successfully evaluate and determine the freshness of beef based on color and texture obtained by a portable custom-designed CVS. The system comprises fluorescent lighting, a GigEPRO camera, and an EVM6678 processing system in which PCA, SVN, PNN, and LDA algorithms were evaluated using Matlab®. In a similar investigation carried out by Barbin (2016) to find the relationship between color and quality of chicken meat, a CVS was used with a Doc L-Pix camera.\n\nResearchers such as Ghyar & Birajdar (2017), implemented a CVS, with which the state of pests in the rice plants was identified, to determine and discriminate anomalies or disease traits using leaf texture and color as reference parameters. The system developed consists of a Sony F470 camera, LED illumination, and computer analysis where ANN and SVM algorithms were run. Similarly, Koklu and Ozkan (2020) carried out the classification of seven different bean varieties to ensure the uniformity and quality of the seeds, identifying the characteristics of each bean species such as: area, perimeter, length of major and minor axes, aspect ratio, roundness, equivalent diameter, among others. The CVS was equipped with a Prosilica GT2000C camera, LED lighting, and a processor where an ANN algorithm was implemented in Matlab®.\n\nThe research performed by Shrestha et al. (2016) reported a morphological analysis of wheat kernels to segment and classify them into three groups: healthy, damaged, and very damaged, as a consequence of premature germination. The result obtained was the segmentation and classification of the three groups of grains with an accuracy of 95% and 72.8%, respectively. The custom-designed system has two RL04C-OC cameras (Ximea GmbH, Germany), LED lighting system, and ANN implemented in Matlab®.\n\nOther applications of CVS systems are in fruits and vegetables, such as the one carried out by Santos Pereira (2018), where he classified the ripeness level of harvested papayas through the identification of color, length, diameter, and weight with an accuracy of 94.3% compared to manual classification. The CVS developed in-house, incorporates a Sony camera (Japan) located in an environment illuminated with white LED light. The pictures of each fruit were analyzed in Matlab® using a decision tree algorithm. Table 3 shows some relevant investigation where CVS has been used.\n\n\n5. Texture analyzer\n\nThe texture of a food is perceived through the response to the contact between the body part and the food. It is a determining characteristic in the acceptance of the product by the consumer (Civille, 2011; Liu et al., 2019; Muthukumarappan & Karunanithy, 2021). Texture is a quality attribute used in the food industry (Torres Gonzalez et al., 2015), allowing the parameterization and standardization of food products (Liu et al., 2019). For example, freshness, a determining characteristic in selecting a vegetable or fruit, can be described by its hardness (Liu & Zhang, 2021). The latter is one of the primary properties of texture, as well as cohesiveness, viscosity, elasticity, and adhesiveness (Foegeding et al., 2011).\n\nTo determine some of the main textural characteristics mentioned, Friedman in 1963 established a method called Texture Profile Testing (TPA) (Nishinari et al., 2019). This method generates characteristic curves from the force measurement performed by the jaw to realize a change in the geometrical property of the product, generating deformation or fracture (Kohyama, 2020; Peleg, 2019). The study of these curves allows for establishing and quantifying texture characteristics such as: brittleness, hardness, adhesiveness, cohesiveness, elasticity, gumminess, and chewiness (Nishinari et al., 2019).\n\nFor the measurement of texture characteristics, different methodologies and instruments have been developed, the most widely used technology is centered on texture analyzers or texturometers (Torres Gonzalez et al., 2015), which are based on the TPA principle, this device simulates the bite of the jaw in two cycles (compression and decompression), through a controlled mechanism that vertically displaces a uniaxial compression cell (Peleg, 2019). When the cell comes into contact with the product, it generates an electrical signal conditioned by a transducer and sent to a computer to be read by operating software (Taniwaki & Kohyama, 2012). The displacement is carried out until it reaches either a distance threshold or a force level defined by the operator. When this limit is exceeded, the cell moves back and repeats the cycle (Liu et al., 2019).\n\nThe texture analyzer usually has three fundamental parts: a moving beam, a load cell, and a control panel (Schmidt, 2018). The first part has a mechanical system that performs the precise vertical displacement of the beam where the load cell is supported; these mechanisms work with a spindle-type system, which has a motor coupled to it that transmits the controlled circular motion (Sussex, 2013). The load cells are electrical elements that generate a voltage signal when they come into contact with a surface (Liu et al., 2019). The cells used are in a range of operation from 100 g to 500 kg (Schmidt, 2018; Sussex, 2013), which will depend on the design of each manufacturer's analyzer.\n\nWith the basic structure of the texture analyzer already mentioned, a variety of probes can be incorporated, which, coupled with the load cell, make it possible to measure a large part of the common texture parameters in foodstuffs (Liu et al., 2019). Among which are the cylindrical probe, which was used to determine the firming kinetics of breadcrumbs (Jekle et al., 2018). The conical probe that allowed me to measure the texture for deep-fried and air-fried French fries (Gouyo et al., 2020), The Spherical probe with which they analyzed the texture of the surface of cured ham (Fulladosa et al., 2021). Also, there are gel and cut probe, each with properties to perform certain texture tests.\n\nSome applications in which the texture analyzer is used are evidenced in investigations such as the one conducted by Aguirre et al. (2018), where texture attributes were validated in the “woody breast” and “cooking methods on the marination” (marinated breast), for which a texture analyzer (TA. XT plus, Texture Technologies, Hamilton, MA) was used. The results were compared with a descriptive test, finding a significant difference in 9 of the 11 texture attributes. Another application is shown in the research conducted by Jiménez et al. (2017), where two lionfish surimi patties were studied to validate the efficiency of high-power ultrasound on textural properties. The measurement was performed with a texture analyzer (TA. XT plus, Texture Technologies, Hamilton, MA) correlated with trained panelists.\n\nOther relevant studies, such as those mentioned above, where the aim is to characterize products and correlate them with sensory tests using a texture analyzer, are shown in Table 4.\n\n\n6. Electromyographic analysis\n\nAlthough TPA is a method that simulates the chewing process, its shear rate is low compared to that of the human bite (Nishinari & Fang, 2018). Therefore, some researchers have focused on finding other mechanisms that allow an understanding of the bite processes of people in a real environment. One of the alternatives is the study of Electromyographic (EMG) signals, which are produced by the nervous system so that the muscles involved during the chewing process react in a certain way producing electrical signals that can be measured (Besomi et al., 2020; Pereira de Caxias et al., 2021). These signals are captured with an electromyograph, which integrates an instrumentation amplifier that captures and amplifies the EMG signal with the help of three reference electrodes (Fang et al., 2020). This signal is sent through a data acquisition board (DAQ), to a processing system where it is processed and sent to a data acquisition system (DAS) (Gohel & Mehendale, 2020) to a processing system where it is subjected to extraction methods that perform the analysis of the signal (Ahsan et al., 2009; Zabala et al., 2019).\n\nSodhi et al. (2019) correlated bite EMG signals with texture variables (instrumental and sensory) of seven Indian sweets, identifying EMG parameters that distinguish the different textured foods. In addition, the PCA determined the significant correlation between hardness (instrumental and sensory) and sensory stickiness. Similarly, Shimada et al. (2012) established intraoral force recordings to analyze the mechanics of human chewing by measuring the force (using strain gauges located on the molars) and the EMG signals (using electrodes located on the masseter muscle) during the biting process of five different products (rice, bread, almonds, banana, and apple). Other relevant studies where the effectiveness of the analysis of EMG signals to determine the texture of a food matrix is sought to be validated are shown in Table 5.\n\n\n7. Acoustic analysis\n\nFood products have the characteristic that when consumed they generate sounds that allow identifying or relating some textural properties such as hardness, crispness, and crunchiness to it (Dias-Faceto et al., 2020). Some of the equipment to perform these measurements use devices such as microphones connected to computers, texture analyzers integrated with microphones (Dias-Faceto & Conti-Silva, 2022), and alternative designs with oscillating tips and piezoelectric sensors (Taniwaki et al., 2006). All these devices allow capturing the acoustic waves produced by the deformation of the product.\n\nResearchers such as Błońska et al. (2014), showed that adding inulin with reduced fat content significantly affected the acoustic parameters of Short-Dough Biscuits. Eight Short-Dough Biscuits with different percentages of inulin addition were compared, determining the impact on the acoustic properties and the decrease in the breaking workforce. For example, the biscuit with 74.1% fat and 18.5% inulin, showed a low acoustic energy level of 1.134 a. u. this compared to a biscuit with 55.6% fat and 9.3% inulin, in which a high acoustic energy level of 17.373 a. u. was found, the former being less brittle and hard compared to the latter. This was achieved using a Zwick 1445 measuring system (Zwick GmbH & Co. KG, Ulm, Germany). Separately, Jakubczyk et al. (2017) studied the acoustic signals generated during puncture tests on some coextruded cereal products with different fillings (toffee, milk, fruit jelly, coconut, and chocolate creams), to perform the analysis of hardness, crunchiness, and texture sound attributes for each product. The results showed that the snacks with jelly filling were perceived as less crunchy and soft, compared to the snack with milk cream filling, which showed high acoustic and mechanical values that link it to crunchiness. The variables were measured with a BC45 cooking extruder (Clextral, Firminy, France). Other relevant investigation, such as those mentioned above, where acoustic analysis was performed to determine some textural properties of certain foods, can be seen in Table 6.\n\n\n8. General conclusions\n\nAs evidenced in this review, some technological tools have been developed to emulate the functioning of the five senses (smell, taste, sight, touch, and hearing), seeking to quantify and characterize some sensory properties of different food matrices, to compare, parameterize and standardize a product. These investigations show that the use of technological tools guarantees the repeatability and reproducibility of the process, compared to the results obtained when working with trained panelists. Therefore, the use of this type of device reduces the number of samples required to perform the analysis, in addition to dispensing with the need for a team of trained panelists, which generates a reduction in costs. In addition, another advantage of these tools is the wider measurement capacity compared to that of human beings. However, most of the tools analyzed only have the property of measuring a single characteristic in a food matrix, this becomes an inconvenience when it comes to characterizing an entire product, for which many tools must be available, samples required and therefore an increase in the time of the analysis and availability of personnel to carry out the process. This is why both the scientific community and the industry, increasing the development of research that seeks to create new technological tools that allow the measurement of two or more sensory characteristics in a food matrix. All the above, seeking to develop new food products and improve existing ones to satisfy the sensory experiences of the consumer, driving growth in the food sector.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgments\n\nThe authors would like to thank Universidad de La Sabana. Mr. Martinez thanks the Universidad de La Sabana for the “Graduated Assistant Program” Scholarship awarded, and to the master's Process Design and Management Program of Universidad de La Sabana.\n\n\nReferences\n\nAguirre ME, Owens CM, Miller RK, et al.: Descriptive sensory and instrumental texture profile analysis of woody breast in marinated chicken. Poultry Science. 2018; 97(4): 1456–1461. 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Publisher Full Text\n\nZhu L, Spachos P, Pensini E, et al.: Deep learning and machine vision for food processing: A survey. Current Research in Food Science. 2021; 4: 233–249. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "194983", "date": "07 Sep 2023", "name": "Ítala M. G. Marx", "expertise": [ "Reviewer Expertise Olive oil", "Bioactive compounds", "Phenolic compounds", "Emerging technologies of phenols extraction", "electrochemical sensor devices", "e-tongue", "e-nose", "chromatography." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Technological Tools for Sensory Analysis in the Food Industry\" provides an overview of various technological tools used in the food industry to measure and analyze sensory characteristics of food products. The paper covers electronic nose, electronic tongue, artificial vision systems, texture analyzers, electromyographic analysis, and acoustic analysis.\nThis present manuscript provides a valuable overview of technological tools used for sensory analysis in the food industry. It offers practical insights into how these tools can be applied to assess various sensory attributes of food products. However, it would benefit from a more critical evaluation of the limitations and challenges associated with these tools and a discussion of recent advancements in the field. Additionally, a deeper exploration of data analysis methods would enhance the paper's utility for researchers and practitioners in the food industry.\nThis article does not demonstrate an innovative character. There are several reviews already published in this area. This manuscript should draw attention to real innovations and aspects that have not yet been addressed in the literature.\nBelow I detail some positive aspects of the review, but also some points that must be improved so that this manuscript can be accepted.\nThe paper provides a comprehensive overview of various technological tools used in sensory analysis in the food industry. It covers a wide range of devices and techniques, including e-nose, e-tongue, artificial vision systems, texture analyzers, electromyographic analysis, and acoustic analysis. This comprehensive coverage is valuable for readers interested in understanding the diversity of tools available for sensory analysis.\nThis manuscript is well-structured, with each section dedicated to a specific technological tool. This organization makes it easy for readers to navigate and find information on each tool separately. Additionally, the internal structure and applications of each tool are described in detail, providing a clear understanding of their operation and potential uses.\nThe authors emphasize the practical applications of these technological tools in the food industry. It highlights how these tools can be used to assess various sensory characteristics such as flavor, texture, and appearance. The practical examples and applications mentioned in the paper demonstrate the real-world utility of these tools for quality control and product development.\nHowever, the authors should provide a wealth of information on the various tools, it lacks critical evaluation and discussion of their limitations and challenges. A review should not only highlight the strengths but also address potential weaknesses and constraints associated with the use of these tools. For instance, the paper does not discuss the cost, maintenance requirements, and calibration challenges that may arise when implementing these tools in food production settings. The manuscript does not discuss recent advancements and developments in the field of sensory analysis technology. Given the rapid pace of technological innovation, it would be beneficial to include information on any emerging tools or techniques that have been developed since the paper's publication.\nFinally, the authors briefly mention some data analysis methods used in conjunction with these tools, such as Principal Component Analysis (PCA) and machine learning classifiers. However, it would be helpful to provide more in-depth discussions on data analysis techniques and how they are applied to interpret the results obtained from these tools.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "10630", "date": "01 Feb 2024", "name": "Claudia Garzón", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: 1. However, the authors should provide a wealth of information on the various tools, it lacks critical evaluation and discussion of their limitations and challenges. A review should not only highlight the strengths but also address potential weaknesses and constraints associated with the use of these tools. For instance, the paper does not discuss the cost, maintenance requirements, and calibration challenges that may arise when implementing these tools in food production settings. Answer. To give scope to this recommendation, we have added section 8. 2. The manuscript does not discuss recent advancements and developments in the field of sensory analysis technology. Given the rapid pace of technological innovation, it would be beneficial to include information on any emerging tools or techniques that have been developed since the paper's publication. Answer. Thanks for the recommendation. We have added a new table that contains works where the colorimeter is used to characterize food matrices (table 3). Additionally, we add works developed between 2022 to date in tables 1, 2, 4, 5 and 6. 3. Finally, the authors briefly mention some data analysis methods used in conjunction with these tools, such as Principal Component Analysis (PCA) and machine learning classifiers. However, it would be helpful to provide more in-depth discussions on data analysis techniques and how they are applied to interpret the results obtained from these tools. Answer. We appreciate the suggestion made; however, this review is focused on presenting some of the technological tools used for the analysis of sensory characteristics in food matrices. Therefore, data analysis methods used in conjunction with these tools, such as Principal Component Analysis (PCA) and machine learning classifiers, were not discussed in this review. However, we are working with a master's student on a review article which includes the compilation of research studies related to existing data analysis methods that have been used in the study of food matrices." }, { "c_id": "10631", "date": "01 Feb 2024", "name": "Jose martinez", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: 1. However, the authors should provide a wealth of information on the various tools, it lacks critical evaluation and discussion of their limitations and challenges. A review should not only highlight the strengths but also address potential weaknesses and constraints associated with the use of these tools. For instance, the paper does not discuss the cost, maintenance requirements, and calibration challenges that may arise when implementing these tools in food production settings. Answer. To give scope to this recommendation, we have added section 8. 2. The manuscript does not discuss recent advancements and developments in the field of sensory analysis technology. Given the rapid pace of technological innovation, it would be beneficial to include information on any emerging tools or techniques that have been developed since the paper's publication. Answer. Thanks for the recommendation. We have added a new table that contains works where the colorimeter is used to characterize food matrices (table 3). Additionally, we add works developed between 2022 to date in tables 1, 2, 4, 5 and 6. 3. Finally, the authors briefly mention some data analysis methods used in conjunction with these tools, such as Principal Component Analysis (PCA) and machine learning classifiers. However, it would be helpful to provide more in-depth discussions on data analysis techniques and how they are applied to interpret the results obtained from these tools. Answer. We appreciate the suggestion made; however, this review is focused on presenting some of the technological tools used for the analysis of sensory characteristics in food matrices. Therefore, data analysis methods used in conjunction with these tools, such as Principal Component Analysis (PCA) and machine learning classifiers, were not discussed in this review. However, we are working with a master's student on a review article which includes the compilation of research studies related to existing data analysis methods that have been used in the study of food matrices." } ] }, { "id": "200385", "date": "11 Sep 2023", "name": "Zahir Al-Attabi", "expertise": [ "Reviewer Expertise Food sensory", "food analysis", "gas chromatography", "E-nose" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 described several technologies applied in the field of food sensory. The flow of the manuscript is good and well-structured. The information provided is useful. However, some of these technologies are extensively reviewed like e-nose and e-tongue.\nThe following would improve the manuscript:\nGC and GC/O were mentioned in the introduction but were not discussed.\n\nHighlighting the advances in these technologies.\n\nThe colorimeter was not discussed.\n\nDiscuss the correlation between human sensory evaluation and these technologies.\n\nDisadvantages of these technologies.\nOther minor comments are amended in the manuscript PDF file linked.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\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": "10456", "date": "01 Feb 2024", "name": "Claudia Garzón", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: GC and GC/O were mentioned in the introduction but were not discussed. Answer. In session 2 the following technologies were discussed: Gas Chromatography-Olfactometry (GC-O), Gas Chromatography-Mass Spectrometry (GC-MS) and Headspace Solid Phase Microextraction (HS-SPME). 2. Highlighting the advances in these technologies.  Answer. In session 2 the following technologies were discussed: Gas Chromatography-Olfactometry (GC-O), Gas Chromatography-Mass Spectrometry (GC-MS) and Headspace Solid Phase Microextraction (HS-SPME). 3. The colorimeter was not discussed. Answer. We have added sub-section 4.1 which talks about the colorimeter. 4. Discuss the correlation between human sensory evaluation and these technologies. Answer. We have added in sections 2.1, 3.1, 4.2.1, 5, and 7 information indicating the correlation between human sensory evaluation and the technological tools mentioned in the article.  5. Disadvantages of these technologies. Answer. To give scope to this recommendation, we have added section 8." }, { "c_id": "10632", "date": "01 Feb 2024", "name": "Jose martinez", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: 1. GC and GC/O were mentioned in the introduction but were not discussed. Answer. In session 2 the following technologies were discussed: Gas Chromatography-Olfactometry (GC-O), Gas Chromatography-Mass Spectrometry (GC-MS) and Headspace Solid Phase Microextraction (HS-SPME). 2. Highlighting the advances in these technologies.  Answer. In session 2 the following technologies were discussed: Gas Chromatography-Olfactometry (GC-O), Gas Chromatography-Mass Spectrometry (GC-MS) and Headspace Solid Phase Microextraction (HS-SPME). 3. The colorimeter was not discussed.  Answer. We have added sub-section 4.1 which talks about the colorimeter. 4. Discuss the correlation between human sensory evaluation and these technologies. Answer. We have added in sections 2.1, 3.1, 4.2.1, 5, and 7 information indicating the correlation between human sensory evaluation and the technological tools mentioned in the article.   5. Disadvantages of these technologies. Answer. To give scope to this recommendation, we have added section 8." } ] }, { "id": "200391", "date": "19 Sep 2023", "name": "Mustapha Muhammad Nasiru", "expertise": [ "Reviewer Expertise Food Processing and Preservation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 explores various technological tools, including e-nose, e-tongue, artificial vision systems, and texture analysis instruments used to analyse and assess food properties like quality, composition, maturity, authenticity, and origin. By standardising these characteristics, they enhance existing food products and create new ones that cater to consumers' sensory preferences. This advancement supports growth in the food sector by delivering satisfying sensory experiences to consumers.\nThe paper exhibits a well-organised structure, with coherent headings and subheadings that contribute to the overall clarity of the manuscript. The write-up is satisfactory and presented in good English. The figures and tables provided are clear and comprehensible, aiding in understanding the findings.\nHowever, there are some suggestions as follows:\nThe abstract should be rewritten to include the findings of the study.\n\nColour measurement devices were not discussed sufficiently, so more discussion is needed.\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": "10455", "date": "01 Feb 2024", "name": "Claudia Garzón", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: The abstract should be rewritten to include the findings of the study. Answer. The abstract was adjusted. Colour measurement devices were not discussed sufficiently, so more discussion is needed. Answer. We have added sub-section 4.1which talks about the colorimeter." }, { "c_id": "10633", "date": "01 Feb 2024", "name": "Jose martinez", "role": "Author Response", "response": "We thank the reviewer for the valuable comments made on version 1 of the manuscript. We answer the reviewers’ comments as follows: 1. The abstract should be rewritten to include the findings of the study. Answer. The abstract was adjusted.  2. Colour measurement devices were not discussed sufficiently, so more discussion is needed. Answer. We have added sub-section 4.1which talks about the colorimeter." } ] } ]	1	https://f1000research.com/articles/12-340
https://f1000research.com/articles/11-714/v1	28 Jun 22	{ "type": "Research Article", "title": "Search engine optimisation (SEO) strategy as determinants to enhance the online brand positioning", "authors": [ "Umar Faruq Ahmad", "Junainah Mahdee", "Normazalila Abu Bakar", "Umar Faruq Ahmad", "Normazalila Abu Bakar" ], "abstract": "Background - Marketers face evolution of online brand positioning marketing strategy due to changes of search engine algorithm that affects the reaching out of brands to potential internet users. Brand owners realise that to be relevant in modern market, they need to transition and focus more into online market. However, many brand owners have ignored the power of search engine optimisation (SEO) strategy for attracting the online market, which is highly competitive and faces rapid changes. A brand can be considered as old fashioned if it does not utilise the SEO as their marketing strategy, in penetrating the online marketplace. Various studies have analysed factors that can enhance the persistency of using the SEO strategy, however gaps remain regarding the relationship of this strategy with the online brand positioning. The main aim of this study was to identify the persistency of using the SEO strategy including the niche point of differentiation, valuable content, targeted keyword and scalable link building, as the determinants that enhance the success of online brand positioning. Methods – This study applies quantitative design using online survey to gather information from the online business entrepreneurs. The survey questionnaire was arranged to focus on the use of SEO as a new way to strategies online business. Results – Based on the results of this study, most online entrepreneurs have somewhat realised the effects of using the SEO strategy to enhance effectiveness of online brand positioning. Conclusion – This research provides insights on the importance of SEO strategy in online business positioning. It is hoped that the online entrepreneurs will take the SEO strategy into consideration in the positioning of their brand in the marketplace.", "keywords": [ "SEO", "online", "brand", "positioning", "determinants", "marketing", "strategy" ], "content": "Introduction\n\nSearch Engine Optimisation (SEO) incorporates technicality and creativity elements that enable online entrepreneurs to increase users’ traffic, thus upgrade its rankings in search engines utilisation.1 Online entrepreneurs are knowledgeable with up-to-date technologies in marketing and selling products via online platforms. Online entrepreneurs must adapt with the online evolution especially search engines because any changes of search engine algorithm will affect brands’ reach out to potential buyers. To stay relevant in the modern market, brand owners must be ready to migrate from conventional to online business, with more focus towards online brand strategy.1\n\nThe aim for any marketing campaign is finding the right online brand marketing strategy, e.g., defining the right marketing message to influence consumers on their brands. The technical and creativity aspects of digital marketing will enable online entrepreneurs to improve their brand position in online search engine platforms.2 Online entrepreneurs must also have the ability to quickly adapt with SEO strategies to position their brands effectively.\n\nDue to the rapid changes of technology and tough competition from rivals, many brand owners have opted to ignore the power of SEO in their online brand positioning strategy. This has caused a big loss to the brand owners for not taking advantage of the “limitless world” of internet for their marketing strategies. The SEO saves a lot of money to reach the highest numbers of customers compared to other marketing methods that has limited reach.\n\nVarious studies related to the factors that enhance the persistency of using a SEO strategy have been performed, however the use of this strategy as determinants to enhance online brand positioning has not been investigated. As such this study analysed the online brand positioning for entrepreneurs using the SEO strategy for longevity in the modern market. Specifically, the objective was to investigate the influence of being persistent by using niche point of differentiation, valuable content, targeted keyword, and scalable link building, in SEO strategy among the online entrepreneurs.\n\n\nLiterature review\n\nOnline business has been referred to as “as the process of buying, selling, or exchanging products, services, and or information via computer network, including the internet”.3 Online business consists of business transactions via online whereby sellers can advertise, promote, and sell their items via internet. In comparison to the traditional brick-and-mortar, online business provides 24-hours services that are operating throughout the year. Online platforms enhance business market position and its presence to the target audience.4 The most crucial part for an online business success is the effective information technology and communication system in the World Wide Web for serving customers.5,6\n\nOnline business attracts new markets, thus enhancing the productivity and performance of firms.7–11 This type of business was not only adopted by huge firms but also small businesses globally.12 Conventional off-line businesses that are perceived as a growing new market are also attracted to move to online platforms.13 Since online business is relatively new, entrepreneurs must consider several barriers such as security and privacy, dependability on technology, maintenance cost, hectic online price and competition; especially for small sizes online entrepreneurs.14–17 Despite all obstacles and challenges facing online business, there are many innovative opportunities that online business entrepreneurs can venture into, to ensure their success18; inclusive of trust, cyberlaw and cybersecurity, mobile commerce, e-warranty, effective services deliveries, prompt and easy payment schemes and many others which are better than conventional business transactions.19\n\nAdditionally, online business can take advantage from the rapid increase of Internet user base, positive mindset, especially among youngsters towards easier online shopping regardless of time and location.20\n\nThe global increase of Internet penetration and enhancement of legal aspects of online business can grow online businesses in any country.21 Search engines support online business strategies; however they are not designed for online business and consumers to search anything by browsing just one web page. There are many search engines available with main key players dominated by well-known names such as Google, Yahoo, and Bing. The search engines have been used by consumers to find anything online, therefore they have become a new platform for businesses opportunities.22 For a brand to be perceived as popular, the SEO strategy targets for the brand name to be listed on the first page when a keyword is entered in the search engine. Normally, customers will not scroll down to the bottom of the first page, or they will not visit too many pages due to time constraint.23 Online entrepreneurs must acknowledge the importance of dependency on the SEO strategy to launch brand campaigns, so that target audience can choose their brand despite the competition. The review of the relevant literature has indicated that there are four major components of the SEO strategy: niche point of differentiation, valuable content, targeted keyword, and scalable link building to enhance online brand positioning.24\n\nNiche marketing with the use of SEO strategy means the utilisation of the online advertising strategy that focuses on specific group of customers as the niche market; to enable the full benefits to the target markets at the lowest costs and fastest coverage as possible.25 The differentiation of niche could give advantage to a brand that has particular strategies in online marketing and branding. Not only that, the niche can also be determined by where the brand is positioned in the search engine competition.25\n\nA fresh page with quality content is more valuable than old contents. Many scholars agreed that ‘Content is King’, whereby any good content will link with other content automatically. High rated content results in high quality search engine optimisation.26 Relative positioning of the content and the quality of the content are the main elements of the valuable content factors. In addition, the frequency of the content will give ‘bonus mark’ to the search engine.27\n\nSpecific placement of targeted keywords for the brand will enhance the effectiveness of the SEO in online brand positioning.28 An organic branded keywords traffic naturally comes from the search engine. Therefore, discovering certain related keywords to the brand that are always being searched is crucial in online branding. Another plus point is that integrating keywords can create a competitive advantage to the online brand.29 Keywords can bring direct competition for the higher-ranked slots. Site visibility in Google search can be significantly improved by using targeted keywords.30\n\nScalable link building is a process whereby consumers start with a generic keyword when searching for any products, and later become more specific to certain brand’s uniqueness.31 Navigational link with other online retailers will associate the brand positioning in the online market. Increasing number of links to the online brand placement and link architecture can give some additional information to the search engine web crawlers. Anchor links are recognised as important factors in Google’s algorithm.32\n\nOnline brand positioning via SEO is affected by the numbers of key contextual factors.33 Online marketers recognises that ranking of the brand is effectively enhanced by brand positioning in the online market.34 By associating with the big brands or market leaders of the industry in the same platform using a search engine will also increase the brand value for the consumers.35\n\nBased on the deliberations of the SEO components and the online brand positioning, the research theoretical framework is illustrated in Figure 1.\n\nFive hypotheses were developed based on this research theoretical framework:\n\nH1: There is a significant positive relationship between the persistency of using SEO strategy with the success of online brand positioning.\n\nH2: There is a significant positive relationship between the persistency of using niche point of differentiation in SEO strategy with the success of online brand positioning.\n\nH3: There is a significant positive relationship between the persistency of using valuable content in SEO strategy with the success of online brand positioning.\n\nH4: There is a significant positive relationship between the persistency of using targeted keyword in SEO strategy with the success of online brand positioning.\n\nH5: There is a significant positive relationship between the persistency of using scalable link building in SEO strategy with the success of online brand positioning.\n\n\nMethods\n\nThe research design is quantitative whereby a set of online survey questionnaires (See Underlying data)36 was used to collect data randomly from online entrepreneurs who offer various products to customers. The respondents were contacted via Internet, websites, Facebook, and other social media sites and convenience sampling was used to selected 275 online entrepreneurs for this study. All the participant gave informed written consent to take part in this study.\n\nThe hypotheses were tested using Pearson Correlation analysis to determine causal relationships between variables, i.e., the positive or negative direction and strength of relationship based on the R-value, and at the significant of the relationship based on p-value.\n\n\nResults\n\nThe results shows that all the five hypotheses were accepted with the Pearson Correlation (R) values of more than 0.70 at significant values of less than 0.05.\n\nAll the relationships are positive and significantly strong. The Pearson Correlation results for relationships between persistency of using SEO strategy with the efficiency of online brand positioning are shown in Table 1 and Figure 2.\n\n\nDiscussions\n\nThe results show that the online entrepreneurs realise the effects of using the SEO strategy to enhance effectiveness of online brand positioning. Niche point of differentiation allows the online brand to appear distinctive and unique by making the brand appear more valuable and desirable to the customers.37 Niche differentiation strategy gains competitive advantage by classifying the customers into smaller target, and later distinguish products for its excellent design, high awareness, easy accessibility, or other aspects.38\n\nContent can increase the SEO strategy components effectiveness, whereby niche consumers will be attracted to online brands with up-to-date content.39 Content must have detailed brand information suitable for the target niche market, including a picture of the product/brand, ingredients, product usage and usefulness, warranty, returnable policies, companies’ details and other information; since without valuable content consumers may not trust the brands.\n\nIntegrating keywords can create a competitive advantage to online brand.40 Good keywords provide direct natural traffics from the SEO strategy.41 Keyword’s popularity can be determined by examining the search engine users’ behaviour. Focusing on how branded keywords across different markets, languages and search engines can also improve on international branding.41,42\n\nScalable link building influences the effectiveness of online brand positioning by laddering technique. In searching for a brand information, consumers will start searching using general keywords that are not very popular, and later laddering the search to specific keyword.43 Therefore in the SEO strategy, it is crucial for online entrepreneurs to ensure that the brand keywords appear on the first page of search engine and enhance the generic list of keywords to the specific ones gradually.\n\n\nConclusions\n\nThis research has achieved its objectives in investigating the persistency of using the SEO strategy which includes niche point of differentiation, valuable content, targeted keyword, and scalable link building among online entrepreneurs. It contributes to new information on the online brand positioning, which can be utilized by online entrepreneurs. Nevertheless, the limitation of this study was that it only focused on the online entrepreneurs in Malaysia, therefore, future studies should investigate the real time usage of SEO platforms by the online entrepreneurs. Similarly, researchers can also investigate further appropriate training models to enhance online entrepreneurs’ knowledge and skills in using the SEO strategy as enablers in the branding market.\n\n\nData availability\n\nData Archiving and Networked Services (DANS): Search Engine Optimisation (SEO) strategy as determinants to enhance the online brand positioning. DOI: 10.17026/dans-xc5-jx74.\n\nThis project contains the following underlying data:\n\nData set information _SEO_Paper ID_ TIM21173_DIFCON2021 15102021.pdf. This is the data information sheet for this study QUESTIONNAIRE UMAR_SEO Paper ID TIM21173.pdf. This file contains the questionnaire used.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” Attribution 4.0 International (CC BY 4.0)\n\n\nAuthor contributions\n\nU.F.A. worked on the idea and conceptualisation of the research topic, data curation and data analysis. J.M. was responsible on supervision of the research project, reviewing and editing work. N.A.B. worked on software and validation of findings.\n\n\nThe ethical approval was obtained from the Research Ethics Committee of the DIFCON conference\n\nEthical Approval Number: EA1172021.", "appendix": "Acknowledgements\n\nWe acknowledge the support from the Multimedia University (mmu) for providing opportunity to publish this article.\n\n\nReferences\n\nYuvaraj S, Indumathi R: Influence of Digital Marketing on Brand Building. International Journal of Mechanical Engineering and Technology. 2018; 9(7): 235–243.\n\nSchwarz S, Grabowska M: Online Marketing Strategies: The Future is Here. 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MIS Q. 2020; 17(6): 261–279.\n\nMakrides A, Vrontis D, Christofi M: The Gold Rush of Digital Marketing: Assessing Prospects of Building Brand Awareness Overseas. Business Perspectives and Research. 2020; 8(1): 4–20. Publisher Full Text\n\nHo CH, Chiu KH, Chen H, et al.: Can Internet Blogs Be Used as an Effective Advertising Tool? The Role of Product Blog Type and Brand Awareness. Journal of Enterprise Information Management. 2015; 28(3): 346–362. Publisher Full Text\n\nAhmad UF, Mahdee J, Bakar NA: Search Engine Optimisation (SEO) Strategy as Determinants to Enhance the Online Brand Positioning. DANS. 2021. Publisher Full Text\n\nSilvia SE, Basariya R: Role of Niche Marketing on Search Engine with Reference to Pay Per Click Advertisement. International Journal of Recent Technology and Engineering. 2019; 7(5C): 258–260.\n\nOrtiz-Cordova A, Jansen BJ: Classifying Web Search Queries in Order to Identify High Revenue Generating Customers. Journal of the American Society for Information Sciences and Technology. 2012; 63(7): 1426–1441. Publisher Full Text\n\nTyagi G, Sharma M, Kaushik, et al.: Using Search Engine Optimization Technique Increasing Website Traffic and Online Visibility, 2015.2015.\n\nChen CY, Shih BY, Chen ZS, et al.: The Exploration of Internet Marketing Strategy by Search Engine Optimization: A Critical Review and Comparison. African Journal of Business Management. 2011; 5(12): 4644–4649.\n\nVignesh J, Deepa V: Search Engine Optimization to Increase Website. International Journal of Science and Research. 2014; 3(2): 425–430.\n\nJerath K, Ma L, Park YH: Consumer Click Behaviour at a Search Engine: The Role of Keyword Popularity. Journal of Marketing Research. 2014; 51(4): 480–486. Publisher Full Text\n\nAhmad M, Faris M, Suhel P: Search Engine Optimization (SEO). International Research Journal of Engineering and Technology. 2019; 6(4): 4113–4117." }	[ { "id": "142588", "date": "30 Aug 2022", "name": "Umi Kartini Rashid", "expertise": [ "Reviewer Expertise Entrepreneurship", "Business Management", "Marketing", "E-Commerce" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFor the discussion and conclusion part, H1 tested the four (4) major components of SEO as unidimensional, while H2~H5 tested them independently/multidimensional. So, what can you say about the differences between the results of unidimensional (combination of four components of SEO) and multidimensional? This paper would be more interesting if you could elaborate and justify further on these parts.\nI have included further comments by annotating the article, which you can find 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?\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": "148925", "date": "04 Oct 2022", "name": "Rizwan Raheem Ahmed", "expertise": [ "Reviewer Expertise Management", "marketing", "advertising", "digital marketing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 tried their best to execute this paper, however, I have still the following reservations and suggestions for the sake of improvement of the undertaken study:\nThe logical sequence of the abstract should be 1) objective, 2) methodology, 3) findings, 4) conclusion, and 5) implication. Thus, the authors should rewrite the abstract in this manner.\n\nThe authors did not establish the motivation, significance, and novelty of the undertaken study. The authors are suggested to improve this important factor in the \"Introduction\" section. The introduction needs further details regarding the background, objectives, structure of the paper, etc. There is a lack of relevant and current citations in the introduction, the scientific paper needs appropriate, current, and relevant citations.\n\nThe literature should be presented in a separate section, it should be presented in an audit form, and it should be linked with the paper’s objectives, moreover, further relevant and current citations should be added to the introduction and previous literature.\n\nThe material and methods should be presented in a comprehensive manner. The conclusion should be elaborated in a comprehensive manner. The conclusion is always one step ahead of the findings.\n\nThe practical, theoretical and societal implications should be discussed in a detailed manner after the conclusion, and in the light of the conclusion and results.\n\nThe limitations and areas of future studies should be written at the end of the paper.\n\nThe spelling and grammatical mistakes should be corrected throughout 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10921", "date": "13 Apr 2024", "name": "Junainah Mahdee", "role": "Author Response", "response": "The abstract sequence has been adjusted according to the F1000 format. We established the significance of research, added recent citations, elaborated the conclusion in a comprehensive manner with limitations/future research, and amended typo errors throughout the paper." } ] } ]	1	https://f1000research.com/articles/11-714
https://f1000research.com/articles/11-982/v1	25 Aug 22	{ "type": "Case Study", "title": "Advancing South-South cooperation in education: Indonesian experience with South Africa", "authors": [ "R. Dudy Heryadi", "Shanti Darmastuti", "Ayu Anastasya Rachman", "R. Dudy Heryadi", "Shanti Darmastuti" ], "abstract": "Education collaboration is important to increase the quality of human capital. South-South cooperation is conducted to exchange resources, technology, skills, and knowledge between southern countries. The purpose of this research is to investigate the collaboration between Indonesia and South Africa toward advancing South-South Cooperation in education. The data collection used in this qualitative research is an interview and literature study with 9 purposive informants from the Indonesian Ministry of Foreign Affairs, the Indonesia Ministry of Education and Culture, and the Embassy of South Africa. The data were obtained within six months. The interview was primarily used to explore the roles and practices of government institutions and universities in fostering education collaboration and human resources development. In this study, the South-South Cooperation helps Indonesia address its educational needs. The result showed that most international education cooperation between both countries is agreed upon at the University-to-University level through a Memorandum of Understanding (MoU), facing less sustainable challenges and low impact. Therefore, an investment-led model, multi-stakeholder collaboration, and a tool to monitor and evaluate this process are needed to improve South-South Cooperation to achieve its goals.", "keywords": [ "South-South Cooperation", "Middle-Income Countries", "Indonesia", "and South Africa Education Cooperation" ], "content": "Introduction\n\nThe international development landscape has shifted and allowed new forms of cooperation to flourish. International cooperation has always promoted sharing information and best practices as significant features in education. Compared to the North-South Cooperation, the South-South is perceived as more economical, effective, and favorable. Therefore, this research seeks to fill the gap in the education cooperation model between Indonesia and South Africa to maintain diplomatic relations between the two countries.\n\nThe South-South Cooperation (SSC) establishes a collaborative scheme among the least or Middle-Income Countries, most of which are located in the southern part of the globe. In practice, it incorporates the basic principles of relationships between sovereign states, such as respect for sovereignty, non-interference in internal affairs, and equal rights. This process promotes the exchange of successful experiences between countries that share the same historical realities with similar challenges. Indonesia has been working closely with southern countries since the Asia-Africa Conference, the Non-aligned Movement (NAM) formation, and the G-77 in 1955, 1961, and 1964. Today, it is committed to enhancing its role and contribution to the development of South-South Cooperation, especially in South Africa.\n\nEducation is important in most developing countries because it aids in the formation of human capital for long-term investment and nation-building. Countries with a focus on human capital become more attractive to domestic and foreign private investment (Becker et al., 2001). Through the Ministry of Education and Culture, Indonesia has carried out systemic reforms by revising various regulations, including the national education law on curriculum, research, expertise, and accreditation. Indonesia’s education system endorses the philosophy of “Merdeka Belajar,” translated as freedom of learning. This concept focuses on revising the curriculum of higher institutions towards greater quality, competence, and expertise of teachers and students. It is also aimed to advocate the academic mobility of students and lecturers outside their study programs, including cross-university and continents.\n\nThe presence of various forums such as the Indonesia-Africa Infrastructure Dialogue (IAID) and the Indonesia Africa Forum (IAF) help the two countries to meet each other’s needs. South Africa is Indonesia’s largest trading partner in Africa therefore, the increase in capital goods procurement from this country indicates productivity, which is mainly related to the need for infrastructure development. Indonesia has significant potential to increase the economic balance of South Africa. Both countries share scientific expertise and scholarly exchanges, which are largely unprompted by the government. Although this collaboration has been mentioned multiple times in forums, no cooperation framework has been specifically designed, implemented, or evaluated for educational-related programs such as scholarship and exchanges. The progress seems sporadic and unmonitored, which led to the inception of this research.\n\nAccording to the Indonesian Ministry of Foreign Affairs hosting “Diplomatic Training/Education “for African diplomats, the educational activity at the state level is technical vocation and training. Meanwhile, at the non-state level, it is carried out through different forms, including the signing of a Memorandum of Understanding (MoU) in universities from both countries, the establishment of the African Indonesian Center of Studies in Indonesian University, scholarship scheme for African students and by sending speakers to international seminars. A study conducted by McLaughlin (1996) provides a framework for global education by stating that it builds cross-cultural understanding and develops the cooperative attitude needed to solve world problems.\n\nThe existing SSC between Indonesia and South Africa is mostly in the agriculture sector. However, satisfaction would still be lagging, without the sustainability of education cooperation, especially in the important agenda, such as teacher’s, girls and women’s education, technical vocation and training, as well as education on technology and halal products. Therefore, this study aims to analyze the existing model of education funding and cooperation between southern countries and later on construct the revised model of SSC in education between Indonesia and South Africa\n\n\nLiterature review\n\nAccording to the literature, education can generate human capital and contribute to economic growth and development by creating human capital. The notion of education as a form of human capital was first used in the study of Schultz (1963), sometime after he discussed whether or not knowledge and skills become a sort of capital (Schultz, 1961). A focused study also conducted by Becker between 1960 and 1964 theorized the linkage between economic development meaning high-income revenue and education. Similarly, earlier studies had emphasized the role of education as an intangible capital which means “improvement in basic science, technology, business administration, education and training” to enhance the productivity of a nation (Mincer, 1958; Fabricant, 1959; Schultz, 1961), even further they have found the direct relationship between education and knowledge of the population and national growth, estimated “that knowledge and education accounted for at least 43% of national income growth” and suggested “education contribute to health and nutritional improvements” (Denison, 1974; Schultz, 1963; Denison, 1962). The human capital theorized the type of education and knowledge obtained from both formal education, informal education at home and work, vocational education, and on-the-job training (Cohn & Geske, 1990; Schultz, 1981; Corazzini, 1967; Mincer, 1974). Despite the breakthrough, education as human capital receives backlash by criticism of the quality of education, impact of learning on experience, the role of nutrition and health, and unemployment among high credentials graduates (Wößmann, 2003; Gundlach, 1997; Livingstone, 1997)\n\nIn contemporary studies, more attention was paid to adult education, especially the knowledge and competence required in the labor market. Countries particularly developing have included human capital development in their national planning (Devadas, 2015). A study by Miyamoto (2003) found that some developing countries have not yet achieved access to education, quality of education, and adult literacy. The difficulties had prompted the policy initiative namely ‘education for all’ in collaboration with international donors, inter-government, and NGOs. Miyamoto (2003) also found that human capital can contribute to civil liberties, political stability, health, and reduced crime and corruption which are determinants of Foreign Direct Investment (FDI). Muhr (2016) explains that not only security and economy, but human development is also a shared responsibility of countries, “commitment to mutual support and joint efforts to achieve sustainable and integral human development, and the appropriate care of developing countries”. The position of education in developing major countries is then adapted to various global sustainable development agendas. The MDGs and SDGs emphasize achieving universal, free, and compulsory primary education through Education for All (EFA). Improving and enhancing the quality of education and gender equality to support long-life learning is the collective responsibility of countries. McLaughlin (1996) provides a framework for global education, “global education seeks to teach people how to live in an increasingly interconnected and interdependent world”. This educational method serves to build cross-cultural understanding and develop the cooperative attitude needed to solve world problems. The emergence of developing or middle-class countries as new donors and providers of technical assistance in the 2000s has reshaped the landscape of international development cooperation. This cooperation allows countries to respect each other’s cultures and develop relations and friendships. While building various ideas and strategies to deal with global issues and various perspectives.\n\nThe concept of SSC is understood as \"the best practice transfer and the exchange of resources, such as but not limited to technology, skills and knowledge among the members of southern countries, as well as promoting the development of social, economic, cultural, political by building coalitions.\" This cooperation model combines funding, knowledge, expertise, and technology to transfer between Southern countries, either bilaterally or multilaterally. A previous study on SSC between Ethiopia, Kenya, and Uganda shows that South-South Cooperation maintains domestic stability covering various fields, such as Agriculture, Trade, and Education. Siahaan (2017) also found that SSC is an effective form of activity to increase “friends,” known as diplomatic relations. Important to note is evidence which support the output of the best practices transfer and programs is infrequent and it is required to pay more attention to evaluate these practices and programs (du Toit et al., 2017)\n\nThe emergence of developing or middle-class countries as prospectus donors and providers of technical assistance in the 2000s has reshaped the landscape of international development cooperation. This process allows countries to respect each other’s cultures and develop friendships while building ideas and strategies to deal with international issues. International educational cooperation has monetary and non-monetary significant effects on countries. In monetary terms, the higher a person’s education level, the lower their unemployment and poverty rates with better income returns, used to build the country through taxes. Meanwhile, in non-monetary terms, education affects a person’s health and nutrition practices, childcare, and participation in social activities.\n\nRegarding the form of educational cooperation, one form of educational cooperation can be found in the case of cooperation between China and Kenya. In this matter, China provides a form of training to strengthen the capacity of Kenyan human resources. In addition, the cooperation between the two sides appears in the form of providing scholarships from both the Kenyan and Chinese governments to Chinese students studying in Kenya and vice versa. The educational cooperation carried out between the two parties is carried out in a reciprocal South-South mode of cooperation. The interesting thing here is that China also provides this form of training through Chinese companies operating in Kenya, and the trainers are not only from China but also from Kenyans themselves (King, 2010). The scholarship form is also one of the programs used by the United States in conducting educational cooperation with Africa. Scholarships are awarded in both technical and social sciences at US educational institutions (Amanor 2013).\n\nCheru (2016) explained the cooperation between Ethiopia and India in the field of education. The form of educational partnership carried out by the Indian government provides 50 scholarships for students from Ethiopia to study at universities in India every year. This exchange is monitored by a shared working group which was founded in 2007. Education is one of India’s grant priority sectors. The grant program is given a priority on capacity building in the field of education. Several education programs can be seen in the construction of schools (in the Maldives) and assistance in transforming the South African education system (Tilak, 2016). From several forms of educational cooperation within the framework of SSC, it can be seen that the provision of scholarships and the provision of training in the context of capacity building are the main programs in educational cooperation.\n\nSome studies have discussed international aid to education, which also helped the exploration of the SSC model. In general, there are two kinds of funding models in international education: the traditional development-aid model and the southern model. The traditional development aid was given by INGOs, such as but not limited to, Development Assistance Committee (DAC), OPEC and OECD countries. Whereas the southern model is given by Non-DAC, Non-OPEC, or “emerging donors” (du Toit et al., 2017; Zimmerman & Smith, 2011; Manning 2006). The latter one is therefore in line with the SSC model comprising the collaboration between partners in the global south which pay a greater focus on partnerships and cooperation. In terms of cooperative education programs based on SSC, there are two kinds of relationships. The first one is vertical intervention by the foreign government in local society. The second one is a twinning relationship or triangular model, which also means best practice transfer (Walensky & Kuritzkes, 2010; Sa e Silva, 2009). Other than funding, Dr. Diplomat in The Economist (2007) mentioned a study about the education cooperation model between Cuba and Africa which was implemented as capacity building and the provision of infrastructure.\n\nThere is a discussion surrounding the effectiveness of various implementation models of the aid, as well as the measurement of impact in the recipient country and population. There is so much theory about the intended outcomes of development, rather than what aid micro-aims to achieve. The focus of the conversation is on how aid is utilized to enhance the quality of life rather than economic return alone (UNDP, 2010). Some suggest the need to comprehend the motivation of all the actors involved in the aid relationship, from the political desires and to the consequence of the relationship of donor and recipient that were formed historically (Schraeder PJ, et al., 1998). Furthermore, Rosseel et al. (2009) express the social role of higher education in development. University has a prominent role in promoting and conducting capacity building such as training and workshops that are beneficial to communities, both in which they are based and beyond. As the co-creators of knowledge, universities promote society participation and produce types of science that address the needs of society.\n\nPrevious models may have not been focused on the process of monitoring and evaluating the programs, and rather on theorizing so much of the intended outcomes of the development (Du Toit, 2017). Scholars that discussed SSC have just focused on limited types of education cooperation such as scholarship, training, and workshop, and less attention has been paid to more progressive models, like investment-led mode. Therefore, our model these two factors have been emphasized to fill the gaps.\n\n\nMethods\n\nThis research is qualitative research, specifically case study. According to Creswell & Cresswell (2018), it is explained that a case study form is a research design in which the researcher develops an in-depth analysis of a topic. The cases taken are limited by time and activity, and the researcher collects detailed information using various data collection procedures over a continuous-time. Analysis of the data used in this study is divided into three stages, namely data reduction, data display, and verification. As explained by Miles and Huberman (1994), data reduction focuses on the process of selecting, focusing, abstracting, and transforming the data that appears in the transcription. In this case, data reduction is based on the framework of thought and research questions that have been determined. Next, the data display stage will be carried out. The results of the data reduction carried out will be made in the form of tables, or matrices to organize the reduced data. The last is the data verification process and making conclusions on the data that has been obtained.\n\nThis research uses qualitative methods to unravel the SSC in Education between Indonesia and South Africa. The qualitative method was selected because the topic requires an understanding of how the education cooperation process is carried out and developed in the future. The primary concepts in this study are Education as Human Capital and South-South Cooperation Model in Education.\n\nThe purposive sampling method was used during the interviewing process to acquire information-rich cases related to education activity between Indonesia and South Africa. The total number of informants is nine people, three per institution. The informants’ criterion is based on two points. First, having the expertise and the task to coordinate foreign cooperation specifically with southern countries. Second, his/her position is exclusive as the director and/or in charge of the public relations in the relevant institution as follows: the Indonesia Directorate General of Higher Education, the Indonesia Ministry of Foreign Affairs, and the South Africa Embassy in Indonesia.\n\nData were obtained from primary and secondary sources with the duration of data collection being six months covering the data of SSC between Indonesia and South Africa from 2015 to 2020. The primary data were collected through an interview process with selected informants, and the secondary data were from documentation studies, such as ministerial regulations, technical guidelines, scientific journals, and mass/electronic media, in the forms of research reports, documents, archives, or videos. The data set was established to collect the intended information about SSC in education. Firstly, the information regarding the SSC Indonesia with South Africa which consist of: a) programs; b) agreement; c) implementation, was collected from the Indonesia Ministry of Foreign Affairs (code of Informant 2) and The South Africa Embassy in Indonesia (code of Informant 3). Secondly, the data about Indonesian scholarship, exchange programs, and capacity building with South Africa were obtained from the Indonesia Directorate General of Higher Education (code of informant 1)\n\nThis research is deductive, focusing on identifying existing educational cooperation between Indonesia and South Africa and analyzing the gap using the South-South Cooperation Framework. In this study, Taguette open-source was used to help with qualitative data analysis in different stages. In maintaining validity, a triangulation method was formed using various available data sources, which were juxtaposed to select the valid ones. Triangulation is a technique which uses multiple methods and/or data sources in a qualitative approach to construct an holistic understanding of phenomena (Patton, 2002). Meanwhile, this research triangulation method matches the data obtained through the interview and literature study methods.\n\nAll informants completed the consent form. It was made clear that their identities and response would remain confidential and only used for the data analysis of this study. Participants were voluntary, meaning that they could decide to withdraw from any questions they felt not comfortable with. Permission to record the data was obtained from informants through email correspondence to ensure the accuracy of the analysis. This study protocol was approved by the ethics committee from the research, education, and society outreach department of UPN Jakarta.\n\nThe results of the interview show that increasing the competence and capacity of human resources is one of the main objectives in the implementation of south-south cooperation. This capacity building can be carried out by sending human resources to internship or training activities. As stated by an informant from the Ministry of Education and Culture of the Republic of Indonesia who explained that:\n\n\"In creating quality human resources under the direction of President Joko Widodo, the position of international cooperation in the field of Indonesian education is very diverse. One of these things can be seen through bilateral cooperation, among others: the existence of cooperation to improve the quality of learning in terms of encouraging and facilitating the exchange of information, and scientific publications on early, basic, secondary, vocational, and technical education through formal, non-formal and informal education\" [Informant 1.2, Table 1]\n\nBoth countries’ education and skills development are critical because they share similar challenges such as poverty, teacher’s education, girls’ education, adaptive curriculum, and technological disruption. In 2063 African Union Agenda conveyed the need for a revolution in education and skills, which was emphasized in the Common African Position (CAP) of the Sustainable Development Goals (SDGs). According to CAP, there is a need to promote and support research, technology, and innovation, to build human resources in Africa. This includes establishing education centers, an African accreditation board to monitor the educational standards, and strengthening the relationship of Pan African University.\n\nMeanwhile, the educational philosophy of “Merdeka Belajar,” applied at the tertiary level as Strategic Planning of Indonesia Education Development to accelerate the quality, is categorized into three different streams. The first is the provision of scholarship programs for lectures, followed by organizing research collaboration programs, visiting professors, student exchanges, joint laboratories, and others. The last is the 3) fulfillment and utilization of university infrastructure.\n\nVarious forums, such as the Indonesia-Africa Infrastructure Dialogue (IAID) and the Indonesia Africa Forum (IAF), help the two countries meet their needs, especially trading partners. Despite the significance, the education agenda has not been discussed much bilaterally between Indonesia and South Africa. This is because the investment-led model is a suitable cooperation mechanism between the two countries. Therefore, to develop mutual respect toward freedom and democracy, a business-oriented approach to cooperation is needed, which allows to achieve win-win and avoid interference in internal affairs. On the contrary, the unconditional cooperation of the SSC may entrench the unaccountable political elites as a trade-off of democratic reforms and jeopardize the protection of human rights.\n\nIndonesia’s role as a contributor under the framework of SSC has been initiated since 1950, and it is increasingly significant, especially in education.\n\nThe Indonesian government realizes that higher education plays a role to help the country achieve socio-economic goals and have practical impact on society. Higher education promotes responsible citizens, which hold ethical behavior, educational desires, professionalism in various fields, and cross-border engagement. Therefore, as a part of our commitment in helping the international public to survive the global challenges, sharing our experience and resources by nurturing a highly skilled and educated society has also become our priority. [Informant 2.2, Table 2]\n\nAccording to Aikins (2008), the socio-political and economic crisis in the African region in the 1970s and 1980s has reduced funding for research and academics, scholarships, decline in student enrollment, and problems in academic-related tasks. Other challenges are related to shortages of a qualified labor force and an institution’s capacity. The lack of skills translates into a poor labor market and problematic transition from school to work, affecting the country’s productivity (Garcia & Fares, 2008). Varghese (2015) stated that educational cooperation is better directed to support higher education in implementing national policies and institutional enhancement.\n\nIndonesia’s Ministry of Education and Culture increased the Darmasiswa scholarship quota from 250 to 750 students between 2015 and 2020. Darmasiswa Scholarship is a program initiated in 1974 to offer scholarships to international students from countries with diplomatic relations with Indonesia. It is a non-degree scholarship program given to international students at 51 Higher Education Institutions spread across Indonesia for 12 months. Therefore, through this scholarship program, international students can study the Indonesian language and culture at various universities in Indonesia.\n\n“Participants can select their subjects based on their preference, such as Indonesian language, traditional instruments (gamelan), ethnomusicology, shadow puppetry, traditional dance, crafts, the art of making batik, culinary and photography and et cetera from any of Indonesian university and college” [Informant 2.3, Table 2]\n\nDarmasiswa Scholarship is a quota based on the Ministry of Foreign Affairs’ country’s priority, the state secretariat, and alumni (Figure 1). The total budget allocated is IDR 40,024,025,000 covering tuition fees, living expenses, international round tickets, health insurance, and visa fees. Currently, there are 22 South African alumni of the scholarship program, and 4 are studying in different universities under the Darmasiswa Scholarship Program for the present academic year.\n\nSource: Ministry of Education and Culture Indonesia, 2018.\n\nAnother program is Kemendikbud Excellence Scholarships, a degree funded by the Indonesian government for international students. These scholarships are accessible to international students outside Indonesia with no limitations. It is offered to various areas of study, such as management, administration, information technology, and food security. Currently, 10 South Africans are benefitting from the Kemendikbud Excellence Scholarship. The last educational aid provided is Kemitraan Negara Berkembang (KNB), known as the Developing Countries Partnership Scholarship offered by the Indonesian government to prospective international students from MICs to pursue their master’s degrees. The regional exclusiveness of the scholarship was revoked in 2002 since it had developed a global interest.\n\nDarmasiswa, Kemendikbud Excellence, and KNB Scholarship are infamous for Indonesia’s educational aid given to international students. This scholarship aims to promote Indonesian culture and language among the younger generation and strengthen cultural ties and enhance skills under the current curriculum of “Merdeka belajar,” which requires international students to carry out internship programs in Indonesian industries at the end of their study. Among the three, KNB was created specifically to enhance a south-south cooperation mission in which Indonesia contributes to international public goods of education as indicated by the yearly budget allocation. However, from Indonesia’s standpoint, the scholarship is also considered charity rather than an investment for development, which is caused by the lack of framework between its SSC in education and South Africa.\n\nBased on information from several informants, it was explained that educational cooperation between Indonesia and South Africa has so far been emphasized more on the provision of scholarships and cooperation in the field of culture. The scholarship quota has also increased from year to year. The cooperation can also be seen in the form of the MoU that has been agreed upon by the two countries. In connection with this MoU, an informant (2.2) Table 2, from the Ministry of Foreign Affairs of the Republic of Indonesia explained that:\n\n\"A Memorandum of Understanding between the Government of the Republic of South Africa was signed on March 17, 2008, in Pretoria. This agreement is valid for 5 years and will automatically be extended for another 5 years\"\n\nAccording to the Indonesia Ministry of Foreign Affairs, there is no document on the education cooperation between Indonesia and South Africa. However, universities in both countries have accidentally collaborated with South Africa’s universities, with non-state actors becoming more influential in international relations. Therefore, a proper discussion is needed to advocate bilateral relations in education at the state level, especially when honoring the SSC between MICs.\n\nGovernment plays an important role in orchestrating the SSC effort between Indonesia and South Africa, from setting up funding, infrastructure, and human resources, to monitoring and evaluating the results for both sides. Most Memoranda of Understanding between Indonesia and overseas universities are ineffective due to the limited resources and the lack of framework. Moreover, President Jokowi mandated the Indonesia Ministry of Education and Culture to ensure all forms of an international agreement signed by Indonesian universities are registered and monitored by the state.\n\nRegional exclusiveness of the Indonesian scholarship shows unexpected results and is therefore revoked. Meanwhile, inter-regional cooperation plays an integral role in advancing SSC in education and skills development. The NAASP (New Asia-Africa Strategic Partnership), a strategic inter-regional partnership between Asia and Africa, established the education cooperation that connects NAASP member countries, called the Asia-Africa Development Universities Network (AADUN), consisting of 21 universities with a few from Indonesia and South Africa.\n\nDespite the increasing mobility of South African students to Indonesia, there is still an underlying challenge due to the state’s inadequate involvement in granting visas. While South Africans can travel to Indonesia without a visa, Indonesians do not enjoy the same benefits. They need a minimum of 30 days to get a travel visa, which is sometimes difficult to obtain. Deregulation of immigration rules between both countries can promote students, researchers, and lecturers’ relationships.\n\n\nDiscussion\n\nPrevious studies have shown that there is an approach of the “triangular model” in SSC (Abdenur & Da Fonseca, 2013; Walz & Ramachandran, 2011; Sa e Silva, 2009) and the nature of the triangular model allows an effective planning by focusing on participation and long-term involvement for all parties involved. However, in this study, we argue that there is also a need to involve collaboration with civil society.\n\nSSC is not a well-defined debate due to its inability to determine the role of civil society and its exclusive engagement with bilateral or state-state affairs. Interviews conducted with leaders of universities in Indonesia indicate that not many have detailed knowledge regarding the concept and practices of SSC and civil society. Rather, they mostly depend on information from the internet and social media. Civil society plays a significant role in ensuring that Indonesia’s SSC education agenda with South Africa is aligned with the needs and interests of Indonesians. Besides, as the ‘watchdogs’, the people can help identify some obstacles to meeting their various ambitions. Currently, the coordination of the civil society, NGOs, and foundation at the national level is not enough to push the agenda, as there are no concrete plans to scale up the involvement of civil society in SSC-related topics.\n\nThe process of increasing state involvement tends to develop an incentive mechanism for actors involved in South-South Cooperation, thereby promoting positive progress and outcomes. Indonesia and South Africa need to cooperate in education and skills development, especially regarding teacher training, girls’ education, halal products, and education technology by implementing the SDGs. Simultaneously, identifying requires actions for monitoring and evaluating SSC to incorporate lesson-learned into the future initiatives in education development. Therefore, with the help of civil society, NGOs, and foundations, the state can reflect on how investment-led policy platforms for SSC can further advance the implementation of the SDGs in education for both countries.\n\nAt the inter-regional level, a multilateral organization for civil society networks such as the global network of Asian African Scholars is used to promote good governance and education capable of protecting and maintaining human capacity, dignity, rights, and values. Asian-African relations are becoming increasingly important globally, in the political and economic spheres and social and cultural fields. This is because it opens up new economic development options, political solidarity, intellectual solidity, and the emergence of a transnational civil society.\n\nThere is a crucial need to engage with the academic and scientific community and leverage technical knowledge in the private sector and civil society to develop and implement practical solutions. The private sector plays a significant role in sharing information about their capacities and resources, thereby opening up opportunities to include each institution’s comparative advantages in the two countries. For example, in the current pandemic, cooperation forums with the private sector play an important role in facilitating medical equipment and medicines.\n\nIn 2014, joint university projects, which focus on poverty reduction and human development were established through a collaboration between the African-Asian Development University Network (AADUN), the Center for Civilizational Dialogue, the University of Malaya, the Asia Africa Option research project (AFRASO), and Goethe University Frankfurt, Germany.\n\nAcademics and higher education are significant in strengthening educational cooperation between Asia and Africa, especially in Indonesia and South Africa. Universities are becoming the front-row in international cooperation in education through the Memorandum of Understanding (MoU), which contains programs including student exchanges, research, joint scientific publications, double-degree, and training.\n\nSince 2008, Indonesia and South Africa have been strategic partners with halal certification and education based on the Joint Declaration on Strategic Partnership. Since this partnership, several universities in South Africa have collaborated with those in Indonesia. South Africa has 26 public universities with nearly one million students in various areas, such as agriculture, mining, mineral engineering, and public health.\n\nOn 28 Jan 2020, the Chancellor of the Muhammadiyah University of Yogyakarta (UMY) and the Judicial Council (MCJ) Cape Town, South Africa, signed the MoU agreement to cooperate with the education sector through full scholarships. This program is given to South African students willing to complete undergraduate, postgraduate, and doctoral degrees. Establishing international cooperation between tertiary institutions can create superior human resources.\n\nA study conducted by Liao et al. (2019) reported a positive correlation between education investment and sustainable economic growth, with a 1% increase leading to a gross domestic product of 0.14% on average. Bhusnurmath (2016) stated that the investment-led model relies on creating new capacity, which provides more employment opportunities with higher demand and production. Therefore, any investment linked to innovation increases supply, demand, and growth. However, the influence of educational investment on economic growth is a long-run process and not immediate.\n\nRather than equal benefits in economic growth, a win-win in SSC means both parties have access to the output, such as a positive country’s image, transfer of knowledge, improvement in education through the investment in each other’s learning infrastructure. Others include developing the information and communications technology (ICT) and improving the quality and service of educators and facilitators. MICs often viewed science as a luxury affordable only by developed countries. Emerging countries, such as South Africa, have also managed to catch up by being Africa’s strongest scientific capacity and world-class infrastructures. The SSC promotes more mutual respect while offering substantial experience in dealing with the developing nation related-issues. Therefore, we construct a comparison between the existing model and the new model as appears in Table 3.\n\n\n\n1. Government to Government\n\n2. Government to Southern Public\n\n3. University to University\n\n\n\n1. International students through scholarship and internship\n\n2. Diplomat training\n\nIn order to achieve gradual benefit towards the contributor of SSC in Indonesia, the government needs to take adequate measures by monitoring the international student scholarship closely, adding quotas, or designing a certain priority in requirements when necessary. It rejects the concept of charity and rather emphasizes the principles of equality, respect for national sovereignty, ownership, and mutual goals.\n\nMeanwhile, the characteristics of SSC do not require the imposition of conditions because they can strengthen the power of the irresponsible political elite at the expense of democratic reform and respect for human rights. A business-oriented approach can end in unfair trade-offs; therefore, commitment needs to be ensured when designing the cooperation. There are several factors responsible for successful cooperative activities when achieving objectives. According to McKenzie et al. (2008), some of the key success factors of international cooperation include identifying the genuine needs of all parties, using a two-way process to share strengths to help others, and providing strong links, including central governments like ministries of education. Others include providing a program framework approved by both parties, a realistic time frame, a resourceful secretariat to maintain the implementation, including support from civil society, sharing good practices, and developing plans. The agreement must be tailored to cover both needs, and when implementing the SSC, strong political support from multi-stakeholder is required.\n\n\nConclusion\n\nIn conclusion, the growing need and efforts to solve today’s critical problems require international cooperation and multi-stakeholder support. The cooperation agenda between Indonesia and South Africa focuses on economic balance through trading with education cooperation limited to demand-based vocational training. Furthermore, the current SSC in education between both countries is still unfathomable with the absence of resources focused on developing and implementing a south-south education cooperation model. This incidental agenda is mostly initiated and implemented by non-state actors, such as the university. This research suggests using the investment-led model involving multi-stakeholders to advance SSC in education in both countries. We recognize there were a number of limitations in this study, we sought to follow the recommendation of Du Toit (2017) to emphasize the importance of time-bound monitoring and evaluation in SSC cooperation. Nevertheless, we acknowledge that we were also not able to measure the effectiveness of the existing model of cooperation between Indonesia and South Africa. Therefore, this study suggests for future research to conduct a quantitative approach to measure the effectiveness of the model, particularly in the context of university-to-university cooperation.\n\n\nDeclaration\n\nDudy Heryadi: head of research, analyzed\n\nShanti Darmastuti: conceived, designed interview, analyzed\n\nAyu Anastasya Rachman: analyzed, interpreted data, wrote the paper\n\n\nData availability\n\nFigshare: Advancing South-South Cooperation in Education: Indonesia Experience with South Africa, https://doi.org/10.6084/m9.figshare.20113892.v2 (Heryadi et al., 2022).\n\nThe transcripts of all participants will be made available in English upon request.", "appendix": "References\n\nAASGON (Asia Africa Scholars Global Network): Strategic Partnership.Reference Source\n\nAbdenur AE, Da Fonseca JMEM: The North’s Growing Role in South–South Cooperation: keeping the foothold. Third World Q. 2013; 34(8): 1475–1491. 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Publisher Full Text" }	[ { "id": "162006", "date": "06 Feb 2023", "name": "Indra Yohanes Kiling", "expertise": [ "Reviewer Expertise Community psychology", "qualitative studies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written article. It has the potential to advance current literature and practice in the field of education. I have some suggestions to the authors to help improve the article.\nThe introduction needs more work especially in citing the more recent literature. I would like to suggest the authors to find some relevant systematic or scoping reviews to help in highlighting the current evidence and research gap. I think the authors could also work on making the objectives of the study more explicit.\n\nThe authors should discuss their positioning as a research team in this study. This kind of reflexivity will help to improve trustworthiness of the study.\n\nMoreover, I would suggest the authors use other efforts to improve trustworthiness of the study besides using triangulation. I would like to recommend the authors to at least use audit trail and diary which is vital to any qualitative study. If they have these documents, they should attach them as appendixes.\n\nSome more quotes can be used to help explain themes.\n\nThe discussion is strong in the contextual and implications but I think is still lacking in terms of conceptually. Some analysis on the limitations and strengths of the study might benefit the readers as well.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [ { "c_id": "9337", "date": "20 Feb 2023", "name": "Dudy Heryadi", "role": "Author Response", "response": "Dear Dr. Indra, thank you for your review. We have looked through it and agree to make changes accordingly to improve the article in order to advance current literature and practice in the field of education." }, { "c_id": "9338", "date": "20 Feb 2023", "name": "Dudy Heryadi", "role": "Author Response", "response": "Dear reviewer, here are point-by-point respond made according to the given comments. Comment: \"The introduction needs more work especially in citing the more recent literature. I would like to suggest the authors to find some relevant systematic or scoping reviews to help in highlighting the current evidence and research gap. The authors should discuss their positioning as a research team in this study. This kind of reflexivity will help to improve trustworthiness of the study.\" Respond: The new version have cited and discussed more recent literature by Nguyen et al. (2015), Hanada (2021), and Zhu & Chikwa (2021) Comment : \"the authors could also work on making the objectives of the study more explicit.\" Respond: The new version have highlighted research gap and study the objectives based on the research gap founded Comment: \"to improve trustworthiness, recommend the authors to at least use audit trail and diary, they should attach them as appendixes.\" Respond: Based on data disclose policy from F1000 is already made available through Figshare and it is stated in the appendix. The reader may refer to the following link https://doi.org/10.6084/m9.figshare.20113892.v2 ( Heryadi et al., 2022). Comment: \"More quotes can be used to help explain themes.\" Respond: The new version have added more quotes to explain the themes of international education cooperation (role and the rationale of the government) Comment: \"Lacking in terms of conceptually. Some analysis on the limitations and strengths of the study might benefit the readers as well.\" Respond: The new version have added and highlighted the limitation and strength in terms of conceptually. Thank you so much for your valuable inputs." } ] } ]	1	https://f1000research.com/articles/11-982
https://f1000research.com/articles/11-1402/v1	29 Nov 22	{ "type": "Research Article", "title": "Measuring the psychosocial wellbeing of Rohingya mothers in Cox’s Bazar, Bangladesh: Psychometric properties of an MHPSS assessment battery", "authors": [ "Amanda Nguyen", "Anvita Bhardwaj", "Kh Shafiur Rahaman", "Suzit Barua", "Karine Le Roch", "Molly Lasater", "Matthew Schojan", "Catherine Lee", "Kim Berg", "Sarah M Murray", "Anvita Bhardwaj", "Kh Shafiur Rahaman", "Suzit Barua", "Karine Le Roch", "Molly Lasater", "Matthew Schojan", "Catherine Lee", "Kim Berg", "Sarah M Murray" ], "abstract": "Background: Psychosocial research in humanitarian settings has been historically dominated by a focus on distress and disorder. As such, there is a need to establish the validity of instruments for a broad range of psychosocial outcomes, particularly among highly affected and under-represented populations. The current study describes the adaptation and testing of multiple psychosocial instruments among displaced Rohingya mothers in Bangladesh. Methods: We used baseline data from 600 mothers of malnourished children aged 2 and under enrolled in an intervention study in Cox’s Bazar, Bangladesh. Instruments assessed distress (International Depression Symptom Scale [IDSS] and Kessler-6 [K-6]); functional impairment (World Health Organization Disability Assessment Schedule [WHODAS]); subjective wellbeing (global Satisfaction With Life [SWL] and Personal Wellbeing Index [PWI]; and, coping (Brief COPE and locally developed coping items). Instruments were piloted and refined, then used for data collection by Bangladeshi-Rohingya interviewer pairs. We conducted exploratory factor analysis, evaluated internal consistency, examined construct validity through correlation with other scales, and used regression models to explore demographic factors associated with psychosocial health. Results: Both the WHODAS and coping items fit 2-factor models; other scales were unidimensional. Cronbach’s alphas ranged from .76 to .90 for the refined scales. With the exception of coping, scale correlations supported construct validity; separate measures of the same construct were highly correlated, distress and impairment were moderately correlated, and both were inversely correlated with wellbeing. Correlates of poorer psychosocial health included relative socioeconomic disadvantage, current pregnancy, and being unmarried.\n\nConclusions: Most of the standard psychosocial assessment tools performed adequately, but they did not appear to fully capture local experiences and included items of little local relevance. Findings highlight the need for further mixed methods research to develop a rich battery of instruments with cross-cultural validity, particularly for positive outcomes such as coping which was particularly challenging to assess.", "keywords": [ "MHPSS", "psychosocial", "psychometrics", "measurement", "humanitarian", "Rohingya", "Bangladesh" ], "content": "Introduction\n\nThe myriad ways in which culture shapes both experiences and expressions of mental wellbeing and distress have been increasingly recognized, leading to cautions against an assumption that psychosocial measures are equally valid across a range of cultures and contexts.1 While there has been increased attention to culturally and contextually specific measurements of distress within global mental health research,2–4 recent reviews have highlighted that there is less consensus on how to best measure other key mental health and psychosocial domains (e.g., wellbeing, coping, social behavior, and social connectedness).5,6 This discrepancy in the focus of psychosocial measurement has also been raised in calls for greater conceptual clarity when considering both clinical and social-environmental intervention models,7 while efforts such as the Inter-Agency Standing Committee (IASC)’s Common Monitoring and Evaluation Framework have catalyzed new progress in evaluation.8 To improve the evidence-base for prevention and promotion programming targeting a broader array of psychosocial outcomes among highly impacted populations, increased attention to their measurement is needed.6 Even as the IASC has recently released updates to the Monitoring and Evaluation (M&E) Framework that include guidance on means of verification,9 there is a continued need to disseminate findings on the validity, reliability, feasibility, and relevance of the most commonly used measurement tools in under-studied populations to advance both programming and research.10\n\nRefugees often reside in a complex environment that makes measurement even more difficult.11 Spurred by ethnic violence in 2017, nearly a million Rohingya forcibly displaced from Myanmar have taken refuge in Cox’s Bazar Bangladesh - dubbed the world’s largest refugee settlement – where they remain highly dependent on humanitarian aid and are living in overcrowded, difficult conditions with serious impacts on health and wellbeing.12 Both prior exposure to traumatic events as well as current stressors related to food insecurity, mobility, and safety concerns are highly prevalent and associated with poor mental health in this population.13 A previous needs assessment within Northern Rakhine State, Myanmar, found that approximately a third of all Rohingya adults screened by the humanitarian organization Action contre La Faim, reported extreme levels of stress, and half of adults who underwent a full psychosocial evaluation reported suicidal ideation.14,15\n\nGiven the magnitude of need for mental health and psychosocial support (MHPSS) for displaced Rohingya people, there is an urgent need to grow our inventory of valid tools for needs assessment and program evaluation. This is particularly needed for sub-groups who are at higher risk of poor mental health outcomes or for whom standardized instruments may perform differently, such as pregnant and parenting mothers of young children.16 Yet, at present, only limited information is available on the psychosocial wellbeing of the Rohingya population in Cox’s Bazar.17 Language barriers that already complicate or delay provision of critical services18 could also complicate attempts to adapt or develop new measures. The Rohingya dialect, which is unwritten, has a vast vocabulary for emotions, behaviors, and idioms of distress, yet they are often not translated well, could vary between established migrants and more recent arrivals,17 and few MHPSS personnel in Bangladesh know the Rohingya dialect.17,18 These challenges point to a need to explore the psychometrics of common measures of mental wellbeing in this population.\n\nThe purpose of the current study was to adapt, translate, pilot, and assess the psychometric properties of a collection of instruments for use with displaced Rohingya mothers enrolled in an intervention study in Cox’s Bazar, Bangladesh. A second aim of this study was to explore demographic characteristics associated with psychosocial health among a sample of mothers accessing nutrition treatment services for their children (i.e., women not necessarily seeking MHPSS support).\n\n\nMethods\n\nThis study was carried out in Rohingya settlements in Cox’s Bazar, Bangladesh, where the study’s operational partner, Action Against Hunger (Action contre la Faim; ACF), has provided critical humanitarian services through a number of Integrated Nutrition Centers (INCs) since 2017. As a core feature of their holistic approach to supporting child wellbeing, ACF also offered maternal psychosocial support at these INCs, including the Baby Friendly Spaces (BFS) program. BFS is a low-intensity psychosocial support intervention aimed at promoting maternal and child wellbeing and improving care practices among mothers of children under age two who are receiving nutritional treatment for acute malnutrition. During the study period, the BFS program completed approximately 50-100 intakes of mother-child dyads per month at each of the 10 included INCs. Resident populations across INC catchment areas were generally comparable, with the exception of length of time in displacement across camps; some camps primarily hosted people who were displaced in the 1990s and others from the more recent 2017 influx. For this study, we prioritized INCs located in camps hosting more recent arrivals. Data were collected from November, 2021 through January, 2022.\n\nThis paper uses baseline data from a clustered randomized controlled trial (RCT) of the BFS program collected among a sample of 600 Rohingya mothers who were newly enrolling in BFS services at 10 participating INCs. RCT eligibility criteria included being a mother, age 18 or older, of a child under two years of age identified as suffering from moderate or severe acute malnutrition without complication by ACF as a part of their routine programming. Mothers who had cognitive impairment or psychosis that would preclude participation in program activities, as well as mothers of children with severe developmental disabilities or severe malnutrition with complications, were excluded. Additionally, while mothers who received individual counseling from a counselor or psychologist within ACF remained eligible, anyone receiving referral care outside of ACF for more severe mental health or protection needs (in accordance with standard ACF protocols) were excluded.\n\nMeasures were chosen to assess multiple indicators of person-focused mental health and psychosocial wellbeing as outlined in the IASC Common M&E Framework for MHPSS in emergencies.8 As this study was undertaken as one of five projects involved in the USAID-funded Health Evaluation and Applied Research Development (HEARD) learning collaborative of related MHPSS studies,19 key research domains and assessment instruments were aligned for consistent use across projects. Thus, selection prioritized existing, standardized instruments with demonstrated prior utility in trauma-affected populations across multiple cultures and contexts.\n\nPrior to launching the study, all instruments were translated into Bengali language with independent back-translation (English-Bengali). Following reconciliation, the Bengali versions were further translated and back-translated to create a version in the Chittagonian Bengali dialect that is widely spoken in Cox’s Bazar and has a substantial degree of mutual intelligibility with the Rohingya dialect (which has no standard written form). Following these initial translations and back translations, ten Chittagonian Bengali-speaking interviewers from the Cox’s Bazar district who had conversational fluency in the Rohingya dialect were trained in the full assessment battery. In partnership with Rohingya volunteers, the team engaged in collaborative decision-making about standardized phrasing in the Rohingya dialect for any items in which the Chittagonian dialect did not properly reflect Rohingya terms. Ten data collector pairs (comprised of a Bangladeshi interviewer and Rohingya volunteer; see procedures below) each subsequently piloted the instruments with 1-2 Rohingya mothers recruited from the BFS program at the same INCs that would be involved in study recruitment. After noting all feedback, the Bangladeshi interviewers met together with the research team to make collective recommendations on instrument revisions to clarify wording, reduce redundancy, remove problematic items, align response options, and shorten the overall survey.\n\nPsychological distress was measured using two separate scales. To align across the HEARD learning collaborative, we used the Kessler 6-item scale (K-6), a standardized measure of general distress that has been used in diverse settings.20 Responses are based on frequency of experience over a 2-week recall period, with a 5-point Likert type response option of “none”, “a little’, “some”, “most”, and “all” of the time. We supplemented this primary instrument with the International Depression Symptom Scale (IDSS), a 29-item measure of depression developed to reflect symptoms that were commonly reported in a global review of qualitative studies on depression and showed reliable cross-cultural performance in quantitative analyses.21 The IDSS has subsequently been validated, refined, and tested among four distinct groups of people from Myanmar,21–24 though not with Rohingya people. Responses are typically provided on a 4-point response scale, but for this administration we revised the response scale to match the K-6. After piloting, we reduced the IDSS from 29 to 12 items by removing items that were redundant to the K-6 and ones that were removed during a prior validation of a 15-item Myanmar-wide short version (e.g., items related to weight, appetite, and sleep, as well as a number of items describing heart-related symptoms).23,24 Three somatic items from the IDSS (headache, stomach ache, and other bodily aches) that were not included in the Myanmar-wide short version were retained on an exploratory basis due to the potential relevance in this population. Both the IDSS and K-6 were scored by taking the average of all contributing items to generate scale scores ranging from 0-4, with higher scores indicating greater distress. Given the initial redundancy between K-6 and IDSS items, we also explored the psychometric properties of a combined score using items from both scales, again calculated on the 0-4 score range.\n\nFunctional impairment was measured using the 12-item version of the WHO Disability Assessment Schedule 2.0 (WHODAS), a widely used assessment of health and disability developed to be applicable across a range of cultures and contexts25 and used previously with displaced Rohingya people.26 The WHODAS assesses six domains of functioning: cognition, mobility, self-care, getting along/social interactions, life activities, and participation. The WHODAS is typically administered by asking how much difficulty the respondent has had carrying out various activities in the past month, but we adjusted the recall period to two weeks to align with other scales on the assessment battery. Response options included “none”, “mild”, “moderate”, “severe”, and “extreme or cannot do”. Scores were calculated by taking the average of all contributing items for a range of 0-4, with higher scores indicating greater functional impairment.\n\nSubjective wellbeing was measured using items recommended by the Organization for Economic Co-operation and Development (OECD),27 including a single-item rating of global Satisfaction With Life (SWL) that is widely used internationally and the 8-item Personal Wellbeing Index (PWI).28 Frequently administered with the PWI, the SWL item asks “Thinking about your own life and personal circumstances, how satisfied are you with your life as a whole these days?” The PWI has one item for each of seven core life domains (e.g., health, safety, and personal relationships) and an optional item on religion, which we included. Previous research with Rohingya people has used similar question stems, i.e., asking “how satisfied” the respondent is with personal relationships and support from friends.13 Both the SWL item and the PWI used the same 0-10 response scale, accompanied by the image of a ladder with ten rungs that correspond to the ratings with 0 at the bottom (i.e., “no satisfaction at all”) and ten at the top (i.e. “completely satisfied”). After piloting, one item “what you are achieving in life” was removed, as the question was not clear or meaningful to respondents. The PWI was scored by taking the average of all contributing items for a range of 0-10, with higher scores indicating greater subjective wellbeing.\n\nCoping strategies were assessed using the Brief COPE,29 a 28-item instrument that has been translated and validated in numerous languages,30–32 and which is suggested as a means of verification in the IASC M&E framework.9 The Brief COPE assesses a broad range of coping strategies, such as denial, humor, active coping, and use of instrumental and emotional support. Responses were provided on a 4-point scale regarding how frequently each strategy has been used, ranging from 0 “I haven’t been doing this at all” to 3 “I’ve been doing this a lot”, with a 2-week recall period. The Brief COPE has previously been used to generate three overarching coping styles of problem-focused, emotion-focused, and avoidant coping. However, studies have shown the factor structure of the Brief COPE to vary across samples; for example, in a previous maternal sample the instrument consisted of two separate sub-scales: active coping and disengaged coping.33 For the current study, the items on the original Brief COPE were supplemented with several items depicting additional coping strategies found to be relevant locally through qualitative research with Rohingya adults (e.g., “thinking about a better future for the children”).13 After piloting, feedback regarding redundancy, item irrelevance, and respondent fatigue led us to remove some items resulting in a 24-item scale that retained 18 items from the Brief COPE and six locally developed coping items. Scores were generated by taking the average of contributing items for a range of 0-3, with higher scores indicating more frequent use of the included coping strategies.\n\nAs part of their programmatic intake, women enrolling in the BFS program were screened for study eligibility by BFS providers using the inclusion/exclusion criteria listed above. Eligible participants were informed of the study, and those who indicated an interest in study participation were referred to a trained data collector who obtained oral informed consent and administered the assessment interview. We used an oral consent process due to low literacy levels in the target population. As described above, these interviewers were native Chittagonian Bengali speakers who were also proficient in Rohingya, and who were additionally accompanied by a Rohingya volunteer to facilitate comfort and understanding. As such, consent was obtained by the interviewer with the volunteer present as a witness, and assessment interviews were conducted in Rohingya, the participants’ native language. Baseline data collection lasted between 1-2 hours and was completed prior to the participant engaging in BFS program activities. Data were recorded in KoBo Toolbox34 using a handheld tablet. As consent was obtained at the same time as the interview, consent was also documented in Kobo Toolbox.\n\nThis study was approved by the Institutional Review Board of the Institute of Health Economics (approval number IHE-IRB/DU/2021/33/Final) at the University of Dhaka.\n\nAnalyses were conducted in Stata 17.0.35 Descriptive analyses included cross-tabulations of demographic variables and histograms of individual item distributions. We then examined each scale’s factor structure using exploratory factor analysis (EFA). An exploratory rather than confirmatory approach was used due the dearth of prior instrument validation research with this population, the substantial revisions made to some of the instruments prior to data collection that could impact factor structure, and prior evidence for different factor structures for some instruments.33,36 Because item responses were categorical, we first generated and examined polychoric correlation matrices for the items on each instrument. We determined the number of factors to extract based on number of eigenvalues over 1 and Horn’s parallel analysis for principal components. Factor models used iterated principal factor estimation with promax rotation. Items were retained based on high factor loadings (>.4) and low uniqueness. Where there was ambiguity in factor structure, decisions were made based on parsimony and meaningful interpretation. Once refined, scale scores were calculated and summarized to examine sample distributions, and each scale’s internal consistency was assessed using Cronbach’s alpha (α).\n\nWe then evaluated construct validity by examining correlations between scales, hypothesizing that different indicators of similar domains (e.g., K-6 and IDSS, both measures of distress) would be highly correlated; indicators of unique but related domains (e.g., IDSS and WHODAS, measuring distress and functional impairment) would be moderately correlated; and, indicators of positive wellbeing (e.g., PWI) would be negatively correlated with distress but positively correlated with each other. Lastly, we examined correlates of psychosocial health by regressing each scale (as separate models) onto a consistent set of demographic characteristics in multiple linear regression models with cluster-robust standard errors to account for clustering. Due to low missingness (e.g., <1%), listwise deletion was used.\n\n\nResults\n\nA total of 600 Rohingya mothers, ranging in age from 18 to 46, were enrolled in the study and completed baseline assessments. Sample characteristics are reported in Table 1. Women had been living in the camp anywhere from 1-34 years (mean = 5.7, SD = 4.2). The vast majority of women were married (95.8%) and did not work outside the home (96.3%). Most also had no (74.3%) or limited (some primary; 20.5%) formal education. Women reported having anywhere from 1 to 9 children (mean = 3.2, SD = 1.7). Their index child (the child for whom they were receiving ACF supports and attending the BFS program) ranged in age from 6-24 months (mean = 11.4, SD = 4.5) and was more often female (65.5%) than male. Eleven percent (n = 66) of women were currently pregnant. Only 21% of women reported that their family ate meat on at least a weekly basis; 55% reported monthly meat consumption, and 24% less than once per month. The full raw data can be found under Underlying data.48\n\nInternational Depression Symptom Scale. Parallel analysis identified two eigenvalues above 1, although the second was marginal (adjusted: 1.03) and fell below that of the simulated value in Horn’s analysis. To explore potential multidimensionality, we fit a 2-factor EFA model. Factor loadings and uniqueness are provided in Table 2. Most items loaded highly onto a single factor that accounted for 84% of the variance, with only the three exploratory somatic items loading onto the second factor. As these items were excluded from prior short versions of the IDSS in Myanmar, we exclude these items. Additionally, the item assessing recent thoughts of self-harm had high uniqueness (.82) with no substantial loading onto either factor. In prior use of the Myanmar short instrument this item has been retained for clinical purposes but not factored into scale scores; we opted to do the same for the current study. Thus, the final IDSS scale score was comprised of eight items (IDSS-8) with high internal consistency (α = .87).\n\n\n\n1. feel sad\n\n\n\n2. feel no interest in things/less interest in daily activities\n\n\n\n3. feel lonely\n\n\n\n4. feel tired, low in energy or slowed down\n\n\n\n5. worry too much about things\n\n\n\n6. experience headachesa,b\n\n\n\n7. experience stomach achesa,b\n\n\n\n8. feel other bodily aches and painsa,b\n\n\n\n9. feel you were thinking too much\n\n\n\n10. have difficulty concentrating\n\n\n\n11. have difficulty doing your usual activities at home or work\n\n\n\n12. have thoughts of wanting to kill yourselfb\n\na Items not included in Myanmar-Wide Short Instrument.\n\nb Items removed from final scale scores.\n\nKessler 6-item Scale. Parallel analysis of the K-6 identified only one eigenvalue above 1. In a single-factor model, all six items had satisfactory factor loadings (.57-.69) and satisfactory internal consistency (α = .78).\n\nCombined IDSS/K6. Using the items retained from the separate IDSS and K-6 EFAs, parallel analysis of the combined items supported a single factor structure that accounted for nearly 94% of the variance. Factor loadings for contributing items ranged from .55 to .79, and internal consistency for the 14-item combined scale was high (α = .90).\n\nWHO Disability Assessment Schedule 2.0. Parallel analysis identified three adjusted eigenvalues over 1, although the 3rd was marginal (1.01) and fell below the simulated value. The first two factors together accounted for 95% of variance. As such we fit a 2-factor EFA (Table 3). Six unique items loaded on each factor sufficiently, and internal consistency of the two subscales were .73 and .78, respectively. Factor 1 was comprised of items reflecting aspects of self-care (e.g., washing your whole body) and social interaction (e.g., joining in community activities), and appeared to present less difficulty (mean = .37, SD = .44), whereas Factor 2 was comprised of more task-oriented items (e.g., standing, walking, household activities) for which a higher level of impairment was reported (mean = .78, SD = .63). However, the factors were moderately correlated (r = .49), and internal consistency was improved for the full scale (α = .81); as such, we opted to explore the relative contribution of each factor to the overall scale score and construct validity, but retained the single full-scale score for regression analysis.\n\n\n\n1. Standing for long periods such as 30 minutes\n\n\n\n2. Taking care of your household responsibilities\n\n\n\n3. Learning a new task, for example, learning how to get to a new place\n\n\n\n4. Joining in community activities (for example, festivities, religious or other activities) in the same way as anyone else can\n\n\n\n5. How much have you been emotionally affected by your health problems\n\n\n\n6. Concentrating on doing something for ten minutes\n\n\n\n7. Walking a long distance such as a kilometer [or equivalent]\n\n\n\n8. Washing your whole body\n\n\n\n9. Getting dressed\n\n\n\n10. Dealing with people you do not know\n\n\n\n11. Maintaining a friendship\n\n\n\n12. Your day-to-day work/school\n\nPersonal Wellbeing Index. Parallel analysis clearly supported a single factor model with only one eigenvalue over 1 and X% of the variance explained. The six standard items all loaded highly, with factor loadings ranging from .61 to .78. The optional item assessing satisfaction with religion did not load at or above .4 and had high uniqueness (.96) and was thus excluded. Cronbach’s alpha with that item excluded was .85.\n\nBrief COPE and Local Coping Items. Parallel analysis for the adapted Brief COPE (inclusive of locally developed items) indicated four adjusted eigenvalues greater than one, although two of these fell at or below those of the randomly generated set. We first fit 4- and 3-factor models, yet each of these models produced one factor with only a single sufficiently loading item. Thus, we fit a more parsimonious 2-factor (shown in Table 4) in which standard COPE items loaded on Factor 1 and locally developed items on Factor 2, with the exception that finding comfort in religion (a standard item) loaded with the locally developed items. A number of items did not load on either factor (e.g., substance use, making jokes). The internal consistency of Factor 1 items (standard COPE) was satisfactory (α = .84) and all contributing items had positive item-rest correlations; this was unexpected, given that the items reflected a blend of presumably adaptive (e.g., getting comfort, accepting reality) and maladaptive (e.g., criticizing yourself, giving up) strategies which would make a cumulative score not hold utility. Given this, we further examined scale psychometrics using theoretical or previously demonstrated subscales (e.g., emotion-focused, problem-focused, avoidant, adaptive, maladaptive), none of which produced satisfactory scale properties (results not shown). We then examined the internal consistency of only those theoretically adaptive items loading onto Factor 1, and were able to identify a theoretically useful 10-item subscale of adaptive Brief COPE items (i.e., “B-COPE”, α = 81). A similar effort to identify a maladaptive subset was unsuccessful (e.g., 3-items, α = .47). Using Factor 2, we were able to generate a second 4-item subscale of locally derived items (i.e., “L-Cope”) after excluding “solving disputes”, which had low factor loading and high uniqueness. Removal of that item increased the subscale’s internal consistency from α = .74 to α = .76. We then re-ran the parallel analysis and EFA using only those items retained across the two scales, and items continued to clearly organize themselves across the two distinct factors. As the factors were only minimally correlated, (r = .24), we opted to treat these as two separate constructs for analysis.\n\n\n\n1. concentrating your efforts on doing something about the situation you are ina\n\n\n\n2. taking action to try to make the situation bettera\n\n\n\n3. refusing to believe that it has happened\n\n\n\n4. saying things to let your unpleasant feeling escapea\n\n\n\n5. getting help and advice from other peoplea\n\n\n\n6. criticizing yourself.\n\n\n\n7. trying to come up with a strategy about what to doa\n\n\n\n8. getting comfort and understanding from someonea\n\n\n\n9. giving up the attempt to cope\n\n\n\n10. looking for something good in what is happeninga\n\n\n\n11. making jokes about it\n\n\n\n12. doing something to think about it less (Take your mind off of it)a\n\n\n\n13. accepting the reality of the fact that it has happeneda\n\n\n\n14. trying to find comfort in your religion or spiritual beliefsb\n\n\n\n15. learning to live with ita\n\n\n\n16. blaming yourself for things that happened\n\n\n\n17. praying or meditating; participating in religious practices,b\n\n\n\n18. Doing acts of service\n\n\n\n19. Helping to solve disputes/arguments\n\n\n\n20. Working or earning money (e.g., household chores, daily tasks, or job/occupation)\n\n\n\n21. Thinking about a better future for the children,b\n\n\n\n22. Practicing good hygiene (washing, cutting nails, etc) ,b\n\n\n\n23. Participating in physical activities (e.g., sports, exercise, walking, etc)\n\n Taken from locally developed items reported in Riley et al. (2017).\n\na Contributes to final originally sourced scale composition.\n\nb Contributes to final locally developed scale composition.\n\nDescriptions of each of the final scales, including the number of contributing items, internal consistency, possible and observed score ranges, and sample distributions are reported in Table 5. Distress and functional impairment were relatively low, with moderate subjective wellbeing ratings. Comparing score distributions among like constructs, the Myanmar-specific IDSS items had a slightly higher mean relative to the K-6 (1.03 vs. .77). The two subjective wellbeing scores performed relatively similarly, as evidenced by similar means (6.89 vs. 6.67). There was a larger variation between the two measures of coping, with scores indicating more frequent use of strategies included in the local coping scale (mean = 2.3) relative to the Brief COPE items (mean =1.06).\n\nCorrelations between scores, as shown in Table 6, aligned with our hypotheses aside from coping. For example, the IDSS and K-6 (i.e., measures of distress) were highly correlated with each other (r = .71), and both moderately correlated with total functional impairment (IDSS: r = .65; K-6: r = .58), although these correlations appeared to be driven largely by WHODAS Factor 2 (r range .61 to .70) rather than Factor 1 (r range .31 to .34). PWI scores, reflecting a composite score of satisfaction with various life domains, were likewise positively correlated with a global rating of life satisfaction (r = .68). Both measures of subjective wellbeing were negatively correlated with both measures of distress and functional impairment (r range: -.35 to -.44). However, the two coping measures were not highly correlated with each other (r = .27) and had seemingly divergent relationships with other scales. For example, the Brief COPE items appeared to be more highly – and positively – correlated with functional impairment and distress (r range from.26-.38), with very low – but negative – correlations with subjective wellbeing (r = -.09 and -.17). The local coping scale had lower positive correlations with distress, but also slight positive correlations with subjective wellbeing (r = .09 and .17).\n\nAdjusted associations between demographic characteristics and scale scores are reported in Table 7. Controlling for other factors, demographic characteristics most strongly and consistently associated with psychosocial outcomes included household meat consumption, pregnancy status, and marital status. Relative to mothers from households with the least access to meat, mothers from households eating meat on either a monthly or more frequent basis reported significantly lower distress (β range from -.18 to -.30, most p < .05) and significantly higher subjective wellbeing (β range from .75 to 1.85, all p < .05). Mothers who were currently pregnant also reported significantly higher distress (β range from .18 to .22, most p < .05) and lower wellbeing (SWL: β = -.47; PWI: -.32), though not all estimates were not statistically significant at p < .05. Likewise, the small number of unmarried mothers in the sample reported marginally significant but substantially higher distress (β range from.51 to.53) and functional impairment (β = .36), and lower subjective wellbeing (SWL: β = -1.42; PWI: -1.09). Other characteristics were less associated with distress and wellbeing, but meaningfully associated with other outcomes. For example, mothers with higher educational attainment reported lower functional impairment (completed primary: β = -.23, p < .01) and higher use of coping strategies (any primary: β = .31; completed primary: β = .44; both p < .01). Length of time in displacement had a small but positive association both with indicators of distress (all β = .02, p < .05) and coping (β = .01 and.03, p < .05). Other correlates of coping strategies were less consistent and largely not significant.\n\n** p < .01.\n\n* p < .05.\n\n† p < .10.\n\n\nDiscussion\n\nMHPSS research in humanitarian settings has historically been dominated by a narrow focus on distress-oriented outcomes, particularly depression and post-traumatic stress. This focus has facilitated the availability of well-tested assessment tools for these outcomes, with demonstrated validity across a range of cultures and contexts,37 that have helped create a relatively strong evidence base for the treatment of distress with psychotherapy. To build a similar evidence base for a broader range of prevention and promotion interventions, there is a great need for progress in establishing instrument validity and utility for the psychosocial outcomes that should more often be directly targeted by those interventions, such as subjective wellbeing, functioning, and coping. Further, there is a critical need to test these instruments that are underrepresented in psychometric studies to determine where and how to balance use of standardized tools that support comparability across studies with the need for capturing meaningful experiences that are highly contextual.\n\nThis study, conducted with a community-based, non-clinical sample of displaced Rohingya mothers, provides a valuable illustration of this tension. The included distress and wellbeing instruments generally showed remarkable consistency with both theoretical expectations and prior performance. For example, our finding that the three exploratory somatic items (headache, stomach ache, and other bodily aches) did not load together with the core IDSS items from the Myanmar-Wide Short Version aligns with prior research among different ethnic groups in Myanmar,23,24 demonstrating the consistency of this instrument across populations. The IDSS and K6 were also highly correlated, items had good internal consistency as a single scale, and both measures aligned with hypothesized expectations about their relationships with functional impairment and subjective wellbeing. This consistency in performance likely reflects the long history of research that has informed the development of these distress-based measures;21 we note that in fact the IDSS typically does include items similar to those on the K-6, and the use of both in this study – and resulting removal of redundant IDSS items – was due to outcome alignment across the broader learning collaborative. However, the items retained on the IDSS did appear to be more sensitive to maternal distress than the K-6 as evidenced by the higher mean scores.\n\nThe subjective wellbeing measures, likewise, were well correlated with one another and findings support their construct validity. Additionally, that the magnitude of correlation between measures of subjective wellbeing and distress was relatively moderate, supporting conceptualization of subjective wellbeing as a distinct construct rather than simply the inverse of distress.38 Yet, the higher variation on the global SWL rating may indicate that the domain-based PWI is failing to capture certain aspects of life among the Rohingya in Cox’s Bazar that they are factoring into their perceptions of wellbeing. Moreover, the PWI item on satisfaction with what achievements in life seemed so irrelevant and difficult to understand during piloting that it was not recommended for use. It is thus plausible that a scale developed based on locally sourced domains of relevance would hold notable variations from standard instruments; such work would be valuable in the future.\n\nSimilar to the PWI, the WHODAS also performed satisfactorily but not without some questions regarding the overall utility and optimal scale composition in this population. The WHODAS is one of the most common assessments of functional impairment worldwide,39 with good evidence for reliability and validity across a range of cultures, supporting use of full and comparable scale scores when possible. Yet there is also evidence that it is a multidimensional instrument for which the factor structure may vary, suggesting that different factors may be driving overall scores across different groups.36 In this study, it appeared that a subset of role or task-oriented items (walking, standing, household tasks, daily school, or work) for which impairment was higher loaded onto a separate factor and drove the relationships between distress and functional impairment, whereas basic self-care and social engagement were less impaired and less closely related to distress. This may reflect the influence of a community-based sample with relatively low rather than clinical levels of distress and impairment. It is noteworthy that the women in this study were recruited from a program seeking to improve child’s health and development by improving caregiver functioning; that role or task-oriented items were more likely to be impaired aligns with this theoretical approach. That tasks of daily living are particularly context- and role-driven, locally developed tools that value contextual relevance over cross-cultural comparability are likely to be particularly useful.40\n\nThe most problematic assessment we encountered was that of coping. Coping strategies, like expectations for functioning, are likely to be highly dependent on cultural beliefs and practices, as well as what is feasible and accessible in a given setting.41 Whereas we sought to include standard items from the Brief COPE, multiple items were found to be irrelevant or simply not endorsed and thus removed, while the remaining standard items did not cohere together in meaningful ways. While we pragmatically identified a subset of “adaptive” items that appeared to have good internal consistency, its relationship with other constructs, particularly temporal relationships that we could not account for in cross-sectional data, was unclear. For example, do observed positive correlations between coping and distress potentially support validity as the presence of distress necessitates leveraging coping strategies, or would construct validity be supported if coping and distress were inversely related because coping protects from distress? How are similar positive relationships between coping and both distress and subjective wellbeing accounted for? The endorsement of strategies included on the COPE was relatively limited compared to items added from local qualitative research13 these latter items reflected a combination of physical, mental, and spiritual self-care strategies that were highly endorsed, but seemingly irrespective of other characteristics and thus potentially less sensitive to change. Moreover, some locally reported coping strategies also seemed to conflate coping and functioning (e.g., solving disputes, practicing good hygiene); as improved coping is likely to be a key intermediate outcome for many psychosocial interventions.7 As such, there is a critical need to improve the conceptualization and measurement of coping in humanitarian settings, which our findings suggest will require in-depth qualitative research that seeks to understand how participants conceptualize the relationship between coping and distress and the types of strategies with the most relevance and utility in a given context.\n\nBeyond scale psychometrics, the current study also identified factors associated with the measured psychosocial outcomes. The Rohingya community in Bangladesh faces substantial political and societal opposition to integration.41–43 This reception, paired with a rapid influx of newly displaced Rohingya that have further strained relations and resources in recent years, has contributed to stressful very stressful living conditions.12 Within this context, among food-insecure mothers with limited education or employment opportunities, key risk factors associated with poorer psychosocial health included relative socioeconomic disadvantage, current pregnancy, and being unmarried. These findings align with previous research indicating that displaced Rohingya women are especially vulnerable and even more so if they are separated, widowed or divorced due to numerous reasons ranging from poorer economic opportunities to increased gender-based violence.44,45 Recalling that these women were all mothers to children aged 6-24 months, that current pregnancy emerged as a clear risk factor may indicate a lack of women’s empowerment in reproductive health and planning. Also related to female empowerment, higher educational attainment was associated with less functional impairment and greater use of coping strategies, consistent with previous studies amongst other refugee groups.41,46 While employment may have been expected to follow a similar pattern, this was not shown in our data potentially due to restrictions on employment in the camps such that very few women reported employment, and it is possible that women seeking such opportunities do so in the absence of other supports. Gender preferences favoring male children may further explain why this sample – presenting for child malnutrition – reflected a higher proportion of female children and why having a female child in some cases appeared to be associated with poorer maternal psychosocial health.47\n\nA key limitation of this study is a lack of formative qualitative work that would have helped to better refine instruments and potentially explain difficult findings. That said, instruments were piloted and refined with careful consideration to the interview process and, for the most part, did show good psychometric properties when used. This study also relied on a convenience sample of mothers enrolled in a longer intervention study, which imposed some inclusion restrictions that would not have otherwise been included (e.g., excluding women referred to outside support for severe distress or suicidality). Thus, this study likely failed to capture the full range of distress that would be observed in a representative community-based sample which could alter the observed factor structures, but does represent a population that could be targeted with prevention and promotion interventions. Lastly, we took an exploratory approach to factor analysis given the changes made to the instruments and the limited prior research with this population; future research can build upon this work to take a more confirmatory approach.\n\n\nConclusions\n\nAmong a group of displaced Rohingya mothers seeking nutrition treatment for their malnourished young children, a set of standard assessment tools performed adequately to assess a range of psychosocial outcomes but did not appear to fully capture local experiences. Some items included on standard measures were seldom endorsed, suggesting less relevance. Most critically, both standard and locally developed coping items showed weak evidence of construct validity and utility. Testing and reporting on the validity of outcome instruments intended for use in program evaluation and intervention research is a critical step in addressing current knowledge gaps regarding the effectiveness of many commonly implemented, broadly supportive psychosocial programs that do not necessarily target mental illness. Findings highlight the need for further mixed methods research to develop a rich battery of instruments with cross-cultural validity, particularly for positive psychosocial wellness outcomes.\n\n\nData availability\n\nOSF: Measuring the psychosocial wellbeing of Rohingya mothers in Cox’s Bazar, Bangladesh: Psychometric properties of an MHPSS assessment battery, https://doi.org/10.17605/OSF.IO/AMDF2.48\n\nThis project contains the following underlying data:\n\n- HEARD BFS Baseline Psychometrics Limited Dataset.csv\n\nOSF: Measuring the psychosocial wellbeing of Rohingya mothers in Cox’s Bazar, Bangladesh: Psychometric properties of an MHPSS assessment battery, https://doi.org/10.17605/OSF.IO/AMDF2.48\n\nThis project contains the following extended data:\n\n- HEARD BFS Baseline Questionnaire.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBass JK, Bolton PA, Murray LK: Do not forget culture when studying mental health. 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Publisher Full Text\n\nNelson EL, Saade DR, Gregg GP: Gender-based vulnerability: combining Pareto ranking and spatial statistics to model gender-based vulnerability in Rohingya refugee settlements in Bangladesh. Int. J. Health Geogr. 2020; 19(1): 1–14.\n\nCrabtree K: Economic challenges and coping mechanisms in protracted displacement: A case study of the Rohingya refugees in Bangladesh. J. Muslim Ment. Health. 2010; 5(1): 41–58. Publisher Full Text\n\nGladden J: The coping skills of East African refugees: A literature review. Refug. Surv. Q. 2012; 31(3): 177–196. Publisher Full Text\n\nChowdhury SA, Mostafa AT: Rohingya Refugees: Risks and Safety in Bangladesh. Chowdhory N, Mohanty B, editors.Citizenship, Nationalism and Refugeehood of Rohingyas in Southern Asia.Singapore:Springer;2020;pp. 155–170.\n\nNguyen A, Le Roch K, Murray S: Measuring the psychosocial wellbeing of Rohingya mothers in Cox’s Bazar, Bangladesh: Psychometric properties of an MHPSS assessment battery. [dataset].2022, September 8. Publisher Full Text" }	[ { "id": "157099", "date": "09 Jan 2023", "name": "Pieter Ventevogel", "expertise": [ "Reviewer Expertise Refugee mental health", "global mental health", "humanitarian mental health and psychosocial support" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 impression\nThis paper describes an exercise to validate a series of mental health questionnaires that were used in an intervention research among Rohingya refugee mothers in Cox’s Bazar in Bangladesh. The rationale of the research is that much humanitarian mental health research (and its instruments) focusses on ‘negative outcomes’ (symptoms of mental disorders) with less attention to measures of well-being. The authors looked at a range of measures in a sample of 600 Rohingya women and analyze their psychometric properties. This is by itself a valuable exercise and I agree with the authors that much mental health research in humanitarian settings is done with questionnaires that have not been validated for the contexts. The authors are to be lauded for their work.\nThe manuscript is well written and logically set up.\nHowever, I have various suggestions to strengthen the manuscript which I will describe below.\nIntroduction\nThe second sentence and its references for (“While there has been increased attention to culturally and contextually specific measurements of distress within global mental health research2–4)” seem to be a bit haphazard. References 2 and 3 are not even about low-income settings, let alone about humanitarian settings. Can this be tailored to humanitarian settings? There is by now a substantive body of research in such contexts.\n\nThe second and third paragraphs that describe what is known about mental health and psychosocial wellbeing of Rohingya is incomplete. The essential reference to Riley et al 20171 needs to be completed with more recent data such as Riley et al 20202. I also find it remarkable that the authors cite two papers by authors from Action Against Hunger (ACF) among Rohingya in Myanmar, but do no not mention the research work by ACF in Cox’s Bazar 3,4 which is even more relevant.\n\nThe authors cite the overview paper by Tay et al to state that there is a lack of information on Rohingya MHPSS, but seem to have overlooked the dozens of papers on this topic in a special issue of Intervention several of which address more in detail several of the issues the authors mention such as challenges with the language of the Rohingya and community perceptions around mental health 5,6.\n\nWhile I generally agree that the use of unvalidated questionnaires is a major impediment for mental health research among Rohingya, there have been some (minor) efforts to explore the psychometric properties of instruments, see for example the work of Mahmuda et al 2019 which contains a section on the adaptation of psychometric tools in the Rohingya dialect 7.\n\nRohingya in Bangladesh fulfill all definitions of refugees and the humanitarian documents such as the Refugee Response Plan clearly acknowledge this. The manuscript however avoids referring to Rohingya as ‘refugees’, instead resorting to euphemisms such as ‘Rohingya forcibly displaced from Myanmar’ or ‘displaced Rohingya people’ or even worse ‘migrants’. I strongly recommend the authors to correct this and use the term refugees.\nMethods\nIn the description of the setting, the authors suggest that ACF has provided critcal humanitarian services in Cox’s Bazar since 2017. This is not correct. In fact, ACF is one of the longest serving humanitarian agencies in Cox’s Bazar with a track record that starts long before 2017.\n\nMeasure selection and adaption. I find the process of instrument adaptation quite confusing. I understand that the English version were first translated into standard Bangla, which was subsequently adapted to the Chittagonian dialect and then somehow adapted into Rohingya. I am aware of the challenges around writing the Rohingya language but I do not understand why these procedure was chosen. Could the authors not better have worked directly with Rohingya refugees instead of taking all these tedious steps of translation and then adapting and then again adapting?\n\nThe chosen instruments are an acceptable battery of questionnaires. But I find it questionable how these questionnaires were modified. One of the instrument, the IDSS contains 29 items in the original English version. I do not quite understand how and why the authors ended up with a 12-item version, deleting 17 items. They also talk about a 15-item version that was used in Myanmar, but the Rohingya version was not merely a further reduction because it also added items that had been deleted in the Myanmar version. And the authors apparently also deleted items that were similar to those in the K-6. I find this confusing and wonder whether perhaps all this acrobatics leads to a questionnaire that is so far removed from the original that it has become another scale. Are the authors sure this is still the IDSS? I doubt it and believe this needs serious reflection in the discussion section.\n\nSimilar concern apply to the Brief Cope which was originally a 28-item questionnaire. Ten items were removed and six new ones were added, leading to a 24-item news scale. Can we call this the Brief Cope? I think not and it could better be given a different name, which somehow the authors do by considering the scale as consisting of two independent subscales.\nResults\nThis seems generally sound but I am not an expert in these matters. Some minor things: can the authors make sure the language around significance is correct. They write in the section ‘demographic correlates’ that that in a subgroup the distress is ‘marginally significant but substantially higher’. That seems not correct. If the result is not significant it cannot be a substantial difference.\nDiscussion\nIs to the point and clear. I would doubt however their opening sentence ‘MHPSS research in humanitarian settings has historically been dominated by a narrow focus on distress-oriented outcomes, particularly depression and post-traumatic stress. This focus has facilitated the availability of well-tested assessment tools for these outcomes, with demonstrated validity across a range of cultures and contexts.’ This may be true for some settings, it is certainly not the case for the Rohingya context, where there has been quite some research with pathology-focussed questionnaires (such as the Patient Health Questionnaire for depression) while these have not been validated 1,2,8,9,10,11,12,13,14 which makes it hard to distinguish frank disorder from adaptive distress as I and colleagues have argued for the Afghan context 15 as have others elsewhere 16. In my view there is a major need to do a clinical validation of such symptom-focussed tools to gauge how adequately they measure depression and other mental disorders. That would require validation against a clinical gold standard. So I would suggest the authors adapt their discussion and conclusion by arguing for the development of ‘grounded’ tools that are derived with bottom-up qualitative approaches, as well as clinical validation of tools that are meant to measure clinical constructs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10944", "date": "22 Mar 2024", "name": "Amanda Nguyen", "role": "Author Response", "response": "We greatly appreciate the opportunity to receive and respond to reviewers’ comments on our manuscript. In the following, please find point by point responses. We believe these changes have substantially strengthened out manuscript. Reviewer 1 Critique: This paper describes an exercise to validate a series of mental health questionnaires that were used in an intervention research among Rohingya refugee mothers in Cox’s Bazar in Bangladesh. The rationale of the research is that much humanitarian mental health research (and its instruments) focusses on ‘negative outcomes’ (symptoms of mental disorders) with less attention to measures of well-being. The authors looked at a range of measures in a sample of 600 Rohingya women and analyze their psychometric properties. This is by itself a valuable exercise and I agree with the authors that much mental health research in humanitarian settings is done with questionnaires that have not been validated for the contexts. The authors are to be lauded for their work. The manuscript is well written and logically set up. Response: Thank you, we are very appreciative of your thorough and thoughtful review. Introduction Critique: The second sentence and its references for (“While there has been increased attention to culturally and contextually specific measurements of distress within global mental health research2–4)” seem to be a bit haphazard. References 2 and 3 are not even about low-income settings, let alone about humanitarian settings. Can this be tailored to humanitarian settings? There is by now a substantive body of research in such contexts. Response: This is well noted, and we have now refined this paragraph to give greater detail and provide additional references from studies based in low- and middle-income countries Critique: The second and third paragraphs that describe what is known about mental health and psychosocial wellbeing of Rohingya is incomplete. The essential reference to Riley et al 2017 needs to be completed with more recent data such as Riley et al 2020. I also find it remarkable that the authors cite two papers by authors from Action Against Hunger (ACF) among Rohingya in Myanmar, but do no not mention the research work by ACF in Cox’s Bazar 3,4 which is even more relevant. Response: Thank you for noting this omission. We agree these are critical to include and have updated these paragraphs to reference and discuss this body of work. Critique: The authors cite the overview paper by Tay et al to state that there is a lack of information on Rohingya MHPSS, but seem to have overlooked the dozens of papers on this topic in a special issue of Intervention several of which address more in detail several of the issues the authors mention such as challenges with the language of the Rohingya and community perceptions around mental health 5,6. Response: We appreciate the direction to this special issue. We’ve added content from and cited relevant papers from the special issue to this paragraph Critique: While I generally agree that the use of unvalidated questionnaires is a major impediment for mental health research among Rohingya, there have been some (minor) efforts to explore the psychometric properties of instruments, see for example the work of Mahmuda et al 2019 which contains a section on the adaptation of psychometric tools in the Rohingya dialect 7. Response: We have noted some effort in this area and cited Mahmuda 2019 while advocating for continued advancement in this area Critique: Rohingya in Bangladesh fulfill all definitions of refugees and the humanitarian documents such as the Refugee Response Plan clearly acknowledge this. The manuscript however avoids referring to Rohingya as ‘refugees’, instead resorting to euphemisms such as ‘Rohingya forcibly displaced from Myanmar’ or ‘displaced Rohingya people’ or even worse ‘migrants’. I strongly recommend the authors to correct this and use the term refugees. Response: We very much appreciate this point and agree that accurate language here is critical given the legal and political context. We have made revisions throughout the manuscript to ensure we are referring to our participants and the population in Cox’s Bazar as refugees Methods Critique: In the description of the setting, the authors suggest that ACF has provided critical humanitarian services in Cox’s Bazar since 2017. This is not correct. In fact, ACF is one of the longest serving humanitarian agencies in Cox’s Bazar with a track record that starts long before 2017. Response: We have clarified that ACF has been working in Cox’s Bazar since 2008, which enabled it to be responsive to the 2017 influx. Critique: Measure selection and adaption. I find the process of instrument adaptation quite confusing. I understand that the English version were first translated into standard Bangla, which was subsequently adapted to the Chittagonian dialect and then somehow adapted into Rohingya. I am aware of the challenges around writing the Rohingya language but I do not understand why these procedure was chosen. Could the authors not better have worked directly with Rohingya refugees instead of taking all these tedious steps of translation and then adapting and then again adapting? Response: We appreciate this point, as the process was complex. We have added clarifications in the text that the purpose of first translating to standard Bangla was to facilitate IRB review, that our reason for creating the Chittagonian translation was to have a written document in a local dialect to work from, and that the Rohingya volunteers available to work with us had limited literacy. It is plausible that even with these constraints, we could have worked to create a written form directly in Rohingya while bypassing Chittagonian; as such, we have further acknowledged in the limitations that the approach taken was complex and may have introduced inconsistencies from the original. Critique: The chosen instruments are an acceptable battery of questionnaires. But I find it questionable how these questionnaires were modified. One of the instrument, the IDSS contains 29 items in the original English version. I do not quite understand how and why the authors ended up with a 12-item version, deleting 17 items. They also talk about a 15-item version that was used in Myanmar, but the Rohingya version was not merely a further reduction because it also added items that had been deleted in the Myanmar version. And the authors apparently also deleted items that were similar to those in the K-6. I find this confusing and wonder whether perhaps all this acrobatics leads to a questionnaire that is so far removed from the original that it has become another scale. Are the authors sure this is still the IDSS? I doubt it and believe this needs serious reflection in the discussion section. Response: This is a fair point. We have reworked this paragraph to hopefully clarify the description of the scale development for the IDSS items, have revised throughout to refer to an “IDSS-derived scale” rather than calling it the IDSS, and have added to the limitations the point that it cannot be presumed to reflect the original. Given the burdensome length and that the IDSS was not a required cross-study outcome measure, it was necessary to shorten this scale. With time constraints precluding formative qualitative research, we relied on two authors’ (CL, AN) previous experience developing the Myanmar-wide short version, though we acknowledge Reviewer 2’s point that cultural differences between Rohingya and the previously involved ethnic groups is substantial. We also now state clearly the two items redundant to the K6 that were removed. Additionally, the decision to retain items on concentration and somatic complaints were at the recommendation of the implementing partner given their potential additional relevance due to the program’s focus on families affected by malnutrition. Critique: Similar concern apply to the Brief Cope which was originally a 28-item questionnaire. Ten items were removed and six new ones were added, leading to a 24-item new scale. Can we call this the Brief Cope? I think not and it could better be given a different name, which somehow the authors do by considering the scale as consisting of two independent subscales. Response: We agree and have similarly mentioned this in the limitations. Results Critique: This seems generally sound but I am not an expert in these matters. Some minor things: can the authors make sure the language around significance is correct. They write in the section ‘demographic correlates’ that that in a subgroup the distress is ‘marginally significant but substantially higher’. That seems not correct. If the result is not significant it cannot be a substantial difference. Response: This wording has been clarified to state that there were seeming differences, but that the differences did not reach statistical significance. Discussion Critique: Is to the point and clear. I would doubt however their opening sentence ‘MHPSS research in humanitarian settings has historically been dominated by a narrow focus on distress-oriented outcomes, particularly depression and post-traumatic stress. This focus has facilitated the availability of well-tested assessment tools for these outcomes, with demonstrated validity across a range of cultures and contexts.’ This may be true for some settings, it is certainly not the case for the Rohingya context, where there has been quite some research with pathology-focussed questionnaires (such as the Patient Health Questionnaire for depression) while these have not been validated 1,2,8,9,10,11,12,13,14 which makes it hard to distinguish frank disorder from adaptive distress as I and colleagues have argued for the Afghan context 15 as have others elsewhere 16. In my view there is a major need to do a clinical validation of such symptom-focussed tools to gauge how adequately they measure depression and other mental disorders. That would require validation against a clinical gold standard. So I would suggest the authors adapt their discussion and conclusion by arguing for the development of ‘grounded’ tools that are derived with bottom-up qualitative approaches, as well as clinical validation of tools that are meant to measure clinical constructs. Response: Thank you for this clear and thoughtful comment. We have clarified in this paragraph that while distress-oriented measures have been more frequently tested in global mental health broadly, they have not been validated when used with Rohingya people and there is a lack of case identification tools. We revisit this in the limitations, noting that our sample would not have supported clinical validation because of the exclusion criteria, but advocating for future efforts toward improving clinical decision-making tools as well as ground-up psychosocial assessments." } ] }, { "id": "183364", "date": "16 Aug 2023", "name": "Yubaraj Adhikari", "expertise": [ "Reviewer Expertise Public Mental Health in Humanitarian settings", "Cultural Validation and Adaptation of Psychological Measures", "Task shifting - Training and Supervision", "Moral Injury", "and Mental Health at Work" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Impression\nI found this research paper on contextualization and validation of psychological and mental health screening tools in the Rohingya population extremely relevant. I agreed with the authors that mental health in a humanitarian context was more focused on addressing the distress and consequences of ill health rather than investing in a deeper understanding of socio-cultural, anthropological, and spiritual aspects and contextualization/validation of psychological tools. Nevertheless, the situation is now different than a few decades ago. The research in the humanitarian context and validation of measures are still dominated by researchers representing the global north with the inclusion of a few interpreters without any role in the designing and implementation process. Minimally, the contributions of such processes have enriched the access to the tools and knowledge of scholars from the global south.\n\nIn my work with the Rohingya population (mostly with detainees) in Myanmar, I learned that they had challenges communicating with their families in writing due to not having their script. Many of them had difficulties understanding Bengali. This situation gives enormous challenges for the researchers. I admire the efforts of the authors in working on the relevant topic.\nThe manuscript is presented in a logical order, the information provided is substantial, and is reader friendly. However, I have some critical observations and a few recommendations, which are presented below:\nIntroduction\nThe introductory paragraph highlights the ISAC body guiding the applied research in the humanitarian context. There are many other examples and agreed consensus before the IASC’s Common Monitoring and Evaluation Framework for emergency settings on the contextualization and validation of tools in local contexts.\n\nMethods and measures\nNotably, the study with the Rohingya population in Cox Bazar is a post-emergency situation rather than an ongoing emergency, so the researchers could have paid more attention to the detailed processes of the adaptation and cultural validation of the measures. The process of translation and adaptation is not straightforward. The reasons behind the initial translation of the measures from English to Bengali and then to the Chittagonian Dialect are confusing. The process of cultural adaptation and validation was also not thoroughly followed.\nAs per the International Test Commission (2017) and other relevant recommendations from the researchers (Van Ommeren et al., 1999; Borsa et al., 2012; Fenn et al., 2020)), the process should follow two independent translations from the original language to intended language, then consensus on the translated version, after that lexical back translation by two experts followed by adjustments through the FGDs/face to face meeting with 6-8 experts on the subject, and pilot testing with intended research population with a sample size of 30 or above to look at the psychometric properties and face validity of the measures. The research instruments were only piloted with 1-2 Rohingya mothers and no psychometric properties from the pilot test were presented. Often the researchers use a convenience sampling technique and one category of population based on the needs of the program. Such studies conducted in a rush often provide inadequate baselines for future researchers, which could have been avoided.\nAnother important drawback of this research is to play with established measures. Researchers took out many items from the measures to align with the HEARD project funded by USAID (Psychological distress) and added new items without proper theoretical background (Coping). The author did not mention whether any permissions were taken from the test developers to add new items to the established measure or not. While eliminating the items, as a general practice, no more than 20% of items from each measure can be eliminated from an established measure (Field, 2013), during the validation process. I am surprised to read the comparison of the Myanmar study that was done with Karen and Kachin populations and researchers assumed the potential relevance with the Rohingya population. Based on my experience of staying and working in Myanmar for some years, the culture, language, religion, geography, and way of living of those studied population in Myanmar with the Rohingya population from Rakhine state is incomparable.\n\nResults/discussion/conclusion:\nThe findings from the study and statistical analysis outcomes of all measures are adequately presented. It seems that measures related to distress and coping mostly impacted the statistical analysis process. Researchers have explained the potential reasons in the discussion section. The conclusion clearly states the limited relevancy of these measures in the chosen population and the need for mixed methods for proper validation of the measures. This paper provides ample knowledge and potential areas to be strengthened for cultural adaptation and validation of psychological measures in the Rohingya population. Still having some methodological caveats, this study is relevant and useful for future readers.\n\nLastly, I checked under the data availability section but could not find the final version of the battery (retained after the analysis). I would suggest to make this available (if not provisioned yet) for future researchers as a reference.\nReferences:\nBorsa, J. C., Damásio, B. F., & Bandeira, D. R. (2012). Cross-cultural adaptation and validation of psychological instruments : Some considerations. Paidéia (Ribeirão Preto), 22(53), 423-432. https://doi.org/10.1590/S0103-863X2012000300014\nFenn, J., Tan, C. S., & George, S. (2020). Development, validation and translation of psychological tests. BJPsych Advances, 26(5), 306-315.\nField, A. (2013). Discovering statistics using IBM SPSS statistics. Sage.\nInternational Test Commission. (2017). The ITC Guidelines for Translating and Adapting Tests (Second edition). [www.InTestCom.org]\nVan Ommeren, M., Sharma, B., Thapa, S., Makaju, R., Prasain, D., Bhattarai, R., & de Jong, J. (1999). Preparing instruments for transcultural research: use of the translation monitoring form with Nepali-speaking Bhutanese refugees. Transcultural Psychiatry, 36(3), 285-301. doi: 10.1177/136346159903600304\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10945", "date": "22 Mar 2024", "name": "Amanda Nguyen", "role": "Author Response", "response": "We greatly appreciate the opportunity to receive and respond to reviewers’ comments on our manuscript. In the following, please find point by point responses to the critique. We believe these changes have substantially strengthened out manuscript. Reviewer 2 Critique: I found this research paper on contextualization and validation of psychological and mental health screening tools in the Rohingya population extremely relevant. I agreed with the authors that mental health in a humanitarian context was more focused on addressing the distress and consequences of ill health rather than investing in a deeper understanding of socio-cultural, anthropological, and spiritual aspects and contextualization/validation of psychological tools. Nevertheless, the situation is now different than a few decades ago. The research in the humanitarian context and validation of measures are still dominated by researchers representing the global north with the inclusion of a few interpreters without any role in the designing and implementation process. Minimally, the contributions of such processes have enriched the access to the tools and knowledge of scholars from the global south. In my work with the Rohingya population (mostly with detainees) in Myanmar, I learned that they had challenges communicating with their families in writing due to not having their script. Many of them had difficulties understanding Bengali. This situation gives enormous challenges for the researchers. I admire the efforts of the authors in working on the relevant topic. The manuscript is presented in a logical order, the information provided is substantial, and is reader friendly. I have some critical observations and a few recommendations, which are presented below: Response: Again, we very much appreciate the reviewer sharing their expertise and providing a thoughtful review of our work. We respond to each of these points below in turn. Introduction Critique: The introductory paragraph highlights the ISAC body guiding the applied research in the humanitarian context. There are many other examples and agreed consensus before the IASC’s Common Monitoring and Evaluation Framework for emergency settings on the contextualization and validation of tools in local contexts. Response: Thank you for this comment- we agree and have added broader citations to the academic literature and guides for practitioners on instrument adaptation and validation for mental health, particularly in emergency settings, in the first paragraph, and then re-refer to them here as well Methods and measures Critique: Notably, the study with the Rohingya population in Cox Bazar is a post-emergency situation rather than an ongoing emergency, so the researchers could have paid more attention to the detailed processes of the adaptation and cultural validation of the measures. The process of translation and adaptation is not straightforward. The reasons behind the initial translation of the measures from English to Bengali and then to the Chittagonian Dialect are confusing. The process of cultural adaptation and validation was also not thoroughly followed.As per the International Test Commission (2017) and other relevant recommendations from the researchers (Van Ommeren et al., 1999; Borsa et al., 2012; Fenn et al., 2020)), the process should follow two independent translations from the original language to intended language, then consensus on the translated version, after that lexical back translation by two experts followed by adjustments through the FGDs/face to face meeting with 6-8 experts on the subject, and pilot testing with intended research population with a sample size of 30 or above to look at the psychometric properties and face validity of the measures. The research instruments were only piloted with 1-2 Rohingya mothers and no psychometric properties from the pilot test were presented. Often the researchers use a convenience sampling technique and one category of population based on the needs of the program. Such studies conducted in a rush often provide inadequate baselines for future researchers, which could have been avoided. Response: We acknowledge that both Reviewers raised questions about the instrument adaptation and translation process, and refer to our response to Reviewer 1 to clarify why we undertook the approach we did as it relates to IRB review and interviewer characteristics. We agree a longer preparatory phase – including foundational qualitative work – would have been optimal to produce high quality instruments. However, we faced time constraints related to our funding timeline, launch delays due to pandemic restrictions, and participation in the multi-site study collaborative that made such preparatory work unfeasible. We have also clarified that each of the ten teams piloted the battery with 1-2 separate mothers (not 1-2 mothers total across the ten teams). Critique: Another important drawback of this research is to play with established measures. Researchers took out many items from the measures to align with the HEARD project funded by USAID (Psychological distress) and added new items without proper theoretical background (Coping). The author did not mention whether any permissions were taken from the test developers to add new items to the established measure or not. While eliminating the items, as a general practice, no more than 20% of items from each measure can be eliminated from an established measure (Field, 2013), during the validation process. I am surprised to read the comparison of the Myanmar study that was done with Karen and Kachin populations and researchers assumed the potential relevance with the Rohingya population. Based on my experience of staying and working in Myanmar for some years, the culture, language, religion, geography, and way of living of those studied population in Myanmar with the Rohingya population from Rakhine state is incomparable. Response: We appreciate and agree with the reasonable concerns both reviewers raised about removing items from established instruments and how that can impact interpretation of findings. Again, these decisions ended up being pragmatic in trying to balance our priority outcome measures with the cross-study alignment while avoiding excessive burden on participants. As described in our response to Review 1, we have tried to clarify the rationale for the process and decisions made regarding the IDSS items, including that two members of our team were involved in the previous testing and so drew on that experience, though we also note and agree with Reviewer 2’s point that cultural differences between Rohingya and the previously involved ethnic groups is substantial. We have also discussed in the limitations that the adapted scales cannot be presumed to reflect the original. Results/discussion/conclusion: Critique: The findings from the study and statistical analysis outcomes of all measures are adequately presented. It seems that measures related to distress and coping mostly impacted the statistical analysis process. Researchers have explained the potential reasons in the discussion section. The conclusion clearly states the limited relevancy of these measures in the chosen population and the need for mixed methods for proper validation of the measures. This paper provides ample knowledge and potential areas to be strengthened for cultural adaptation and validation of psychological measures in the Rohingya population. Still having some methodological caveats, this study is relevant and useful for future readers. Response: Thank you for these points. We are glad these sections were clear. Critique: Lastly, I checked under the data availability section but could not find the final version of the battery (retained after the analysis). I would suggest to make this available (if not provisioned yet) for future researchers as a reference. Response: We’ve uploaded a new file called “Final scales after psychometric analysis” to the OSF Page Registry" } ] } ]	1	https://f1000research.com/articles/11-1402
https://f1000research.com/articles/11-184/v1	14 Feb 22	{ "type": "Systematic Review", "title": "The benefit of tissue sealant on urethroplasty in hypospadias patients – A systematic review and meta-analysis", "authors": [ "Safendra Siregar", "Steven Steven", "Akhmad Mustafa", "Steven Steven", "Akhmad Mustafa" ], "abstract": "Background: Hypospadias was ranked second after undescended testis as the most prevalent congenital abnormality in newborn males. Hypospadias can be successfully repaired through multiple surgeries in the majority of children. Postoperative complications were not rarely seen after surgeries, such as urethrocutaneous fistula (UCF), meatal stenosis, and glans breakdown. Tissue sealant application in hypospadias repair serves as additional suture line coverage and reduces the post surgery complications5.However, the effects of sealants usage during urethroplasty are still uncertain. This review aimed to know the effects of tissue sealant usage on patients with hypospadias who undergo urethroplasty. Methods: The study was reported based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Literature was searched on PubMed, Embase, and Scopus following PRISMA guidelines. The search was conducted on October 12th, 2021, using the search term (\"glue\" OR \"sealants\" OR \"tissue glue\" OR \"tissue sealant\" OR \"tissue adhesive\") AND (\"hypospadias\" OR \"urethrocutaneous fistula\" OR \"urethral repair\" OR \"urethroplasty\" OR \"hypospadiology\"). Result: Systematic searching from all databases resulted in 160 potential articles. After a full-text review, eight articles were included in this study. UCF complication was reported in all studies. The occurrence of complication reported by all studies was urethrocutaneous fistula. Several studies also reported tissue edema and flap-related complications. Tissue sealant had no significant effect in reducing meatal stenosis.\n\nConclusions: This systematic review revealed additional benefits from several types of tissue sealant in hypospadias repair surgery. Fibrin sealant application over the urethroplasty suture line in hypospadias repair offers a water-proof coverage and may enhance the outcome from the surgery.", "keywords": [ "Hypospadias", "Urethroplasty", "Tissue Sealant", "Benefit" ], "content": "Introduction\n\nHypospadias was ranked second after undescended testis as the most prevalent congenital abnormality in newborn males.1 Hypospadias is a displaced urethral opening at the penis ventral side because of incomplete penile structures closure during embryogenesis.1At present, there are more than 300 known methods of surgical management of hypospadias however, hypospadias repair remains a complex procedure, even for sophisticated urologiest and paediatrics surgeons.2\n\nHypospadias can be successfully repaired through multiple surgeries in the majority of children.3 The overall success rate for first surgery was 55.9%. Postoperative complications were not rarely seen after surgeries, such as urethrocutaneous fistula (UCF), meatal stenosis, and glans breakdown.4\n\nIn the recent past, the role of tissue sealants has been taken into consideration.5 Sealants are agents that can prevent the leakage of fluids by providing a physical barrier, which also aids in hemostasis.6 Tissue sealant application in hypospadias repair serves as additional suture line coverage and reduces the post surgery complications.5 However, the effects of sealants usage during urethroplasty are still uncertain. This review aimed to know the effects of tissue sealant usage on patients with hypospadias who undergo urethroplasty.\n\n\nMethods\n\nCriteria for inclusion:\n\n1. The study was published in full-text and written in English.\n\n2. Published between 2002 and 2021.\n\n3. Randomized controlled trials (RCTs) or prospective cohort studies.\n\n4. Participants aged up to 18 years, who required operative repair for hypospadias.\n\n5. The intervention included tissue sealants usage.\n\n6. The comparison was made with patients without tissue sealant (Control).\n\n7. The outcome of the studies was the number of children with complication after sealant use.\n\nThe Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was used in reporting this study.7 The flow diagram can be found in Figure 1.\n\nThe literature search was performed on PubMed, Embase, and Scopus following PRISMA guidelines (See reporting guidelines). The search was conducted on October 12th, 2021, using the search term (“glue” OR “sealants” OR “tissue glue” OR “tissue sealant” OR “tissue adhesive”) AND (“hypospadias” OR “urethrocutaneous fistula” OR “urethral repair” OR “urethroplasty” OR “hypospadiology”).\n\nOne reviewer (SS) performed literature selection and data extraction to an Excel database. Titles and abstracts were screened by two reviewers (SFS and AM) to determine the qualified articles. Next, the full text was reviewed to gather detailed information. Data were extracted by two reviewers (DK and SS) independently. Authors, study design, publication year, population, sample size, age, hypospadias severity, type of procedure performed, and characteristics of sealant information were extracted to Microsoft Excel. Any discrepancies among the observers were discussed until consensus was reached.\n\n\nResult\n\nSystematic searching from all databases resulted in 160 potential articles (Figure 1). Duplicates were checked and excluded, leaving 79 articles. A total of 13 articles were qualified in this study. After a full-text review, eight articles were selected for review. These studies used various tissue sealant types. All the studies reported the procedure.\n\nThe general characteristics of reviewed articles are listed in Table 1. All studies are prospective in study design. A total of 863 subjects with hypospadias were included. They were divided into the sealant and non-sealant groups, with 494 and 369 participants.\n\nShenoy et al.,8 Gopal et al.,9 and Ambriz-González et al.,10 used fibrin sealant in their study. There were several distinctions regarding the application of fibrin sealant. Shenoy et al.8 applied the sealant on the neo-urethra's suture line, and no vascular cover was used. Then, Gopal et al.9 made a thin layer of the sealant in the urethral and dartos suture line. Whereas Ambriz Gonzalez et al.10 applied 2 ml of fibrin glue over the surgical site and suture line.\n\nIn another study, Guinot et al.11 applied autologous platelet-rich fibrin (PRF) over urethroplasty. Meanwhile, Kajbafzadeh et al.12 aimed to determine fibrin sealant efficacy for UCF repair after multiple hypospadias and epispadias surgery. Over the closure area, a fibrin glue cover was made, and the dartos fascia layer was placed over this area.\n\nOther sealants used in the included study were cryocalcium glue, BioGlue, and cyanoacrylate. Hosseinpour et al.13 used cryocalcium glue as a thin urethral layer and dartos suture lines. Kocherov et al.14 applied BioGlue as a thin cover on the the neo-urethra second suture line. Lastly, Hosseini et al.15 poured cyanoacrylate over the glans. A rubbery consistency was formed in 60 seconds, then reapplied 4-6 times.\n\nKey findings of reviewed articles are listed in Table 2. Postoperative follow-up ranges between 7 days to 2 weeks to evaluate immediate complications, and between 1 month to 5 years to evaluate late complications. Hypospadias location differs from coronal, subcoronal, distal penile, etc. Some studies reported statistically significant differences of complication between the sealant and non-sealant groups. A study by Shenoy et al. reported distinction in early postoperative ooze, skin flap-related complications, and tension between the sealant and non-sealant groups (p<0.05).8 Hosseinpour et al. revealed differences between edema and fistula (p=0.002).13 Kocherov et al. reported differences in poor cosmetic outcomes (p=0.007).14\n\n\n\n• Complications in 5 patients\n\n• Complications: coronal fistula (3), poor cosmetic outcome (3)\n\n\n\n• Complications in 9 patients\n\n• Complications: early postoperative ooze (2), skin flap-related complications (3), UCF (7), poor cosmetic outcome (7), penile torsion (5)\n\n\n\n• Differences in early postoperative ooze, skin flap-related complications and torsion were significant (p<0.05)\n\n• There were fewer patients with UCF and poor cosmetics in the fibrin sealant group\n\n• The overall improvement in outcome was found to be significantly different among the two groups, with a higher number in group 1 (p<0.05)\n\n\n\n• Complications: edema (24), fistula (5), meatal stenosis (10)\n\n\n\n• Complications: edema (15), fistula (6). Diverticulum (1), meatal stenosis (4)\n\n\n\n• Statistically significant differences in edema (p=0.02)\n\n• Statistically significant differences in fistula (p=0.02)\n\n• No case of allergic in the case group\n\n\n\n• Coronal fistula (2)\n\n\n\n• Urethral fistula (3)\n\n\n\n• No statistically significant difference between both groups (p=0.65)\n\n\n\n• Complications: UCF (4), breakdown of the suture line (4), meatal stenosis (1), poor cosmetic (n=12.6%)\n\n\n\n• Complications: UCF (3), breakdown of the suture line (1), meatal stenosis (1), poor cosmetic (n=19.95%)\n\n\n\n• Statistically significant differences in poor cosmetic (p=0.007)\n\n\n\n• Complications: hematoma (1), skin necrosis (1)\n\n\n\n• Complications: hematomas (7), painful removal of the dressing (12), repeat dressing (10)\n\n\n\n• Wound dehiscence (1), subcutaneous hematoma (1)\n\n\n\n• A patient with the largest fistula (7 mm) had partial wound dehiscence.\n\n\n\n• Complications in 12 patients\n\n• Complications: postoperative edema (10), fistula (6), infection (4), meatal stenosis (3), proximal stricture (3).\n\n\n\n• Complications in 34 patients\n\n• Complications: postoperative edema (21), fistula (19), diverticulum (3), infection (2), meatal stenosis (3), proximal stricture (10)\n\n\n\n• Statistically significant differences in postoperative edema (p=0.039), fistula formation (p=0.027), and overall complication (p=0.003)\n\n\n\n• Complications: urethrocutaneous fistula (3), flap dehiscence (4), flap necrosis (2)\n\n\n\n• Statistically significant differences in urethrocutaneous fistula (p=0.002), flap dehiscence (p=0.001), and flap necrosis (p=0.01)\n\nShenoy et al.8 and Kajbafzadeh et al.12 reported less complication in the fibrin glue than in the non-sealant group. In the non-sealant group, the complications included early postoperative ooze, skin flap-related complications, UCF, poor cosmetic outcome, penile torsion, postoperative edema, diverticulum, meatal stenosis, proximal stricture, flap dehiscence, and flap necrosis. Early postoperative ooze, skin flap-related complications, and torsion were significantly different in the sealant and non-sealant groups (p<0.05). However, in a study by Kajbafzadeh et al.,12 the difference of complications between both groups was not statistically significant.\n\nHosseinpour et al.,13 in their study about the application of Cryocalcium glue, revealed that there was no allergy found in the sealant group. Then, Kocherov et al.14 reported poorer cosmetic outcomes in the non-sealant group compared to the BioGlue group (p=0.007). Subsequently, Hosseini et al.15 found poorer results in the non-sealant group than the cyanoacrylate group. His study revealed some complications, including hematomas, painful dressing removal, and repeat dressing.\n\nIn addition, Ambriz-González et al. compared the use of fibrin sealant on proximal, medial, and distal hypospadias.10 No statistically significant differences were found in the use of fibrin glue in proximal hypospadias to prevent complications. For medial hypospadias, fibrin glue was found to significantly reduce the occurrence of urethrocutaneus fistula (p=0.03) and flap dehiscence (p=0.001). Then, for distal hypospadias, fibrin glue was also found to reduce the occurrence of urethrocutaneus fistula (p=0.04) and flap dehiscence (p=0.04).10\n\nThe analysis of clinical and statistical findings were shown in Figure 2. We compared the occurance of complication after hypospadias repair. The occurrence of UCF was reported in all studies. UCF with sealant complication was the occurrence of complication reported by all studies was urethrocutaneous fistula. The pool risk ration (RR) for UCF was 0.47 (95% CI 0.30 – 0.73 p=0.001) compared to non-sealant group. Tissue edema and flap related complication also reported by several studies with pooled RR for tissue edema and flap related complication was 0.45 (95% CI0.29 – 0.71 p=0.0004) and 0.44 (95% CI 0.31 – 0.62 p<0.0001). Tissue sealant gave no significant effect in reducing meatal stenosis with Pool RR 0.9 (95% CI 0.38 – 2.15 p=0.8).\n\n\nDiscussion\n\nHypospadias is a congenital anomaly discovered in 1 of every 300 live births.9 About 2-15% of infants with hypospadias experienced complications following repair procedures.16 The most common complications found, in order, are fistula, stricture, and dan wound breakdown.16 The need for hypospadias repairing technique, especially for cosmetic issues, is very demanding. The most popular technique is 'tabularized incised plate urethroplasty' or TIP. Although superior in cosmetic issues, it is reported that the complication is as high as 20-30%.17 Various tissue sealants were offered to gain a better seal of neo-urethra formation in the urologic procedures.14\n\nThis systematic review revealed five different sealant types were used in hypospadias repair. The sealants are classified into natural and synthetic. The natural sealants are made of albumin, fibrin, and cryocalcium, while the synthetic sealant is made of cyanoacrylate.\n\nFibrin glue contains fibrinogen-rich coagulation cascade, factor XII, dan thrombin.5 According to the coagulation cascade, scavenging macrophages will reabsorb the fibrin glue in 14 days so that it has a weak mechanical strength.5 Other complications are anaphylactic reaction and infection.18\n\nFibrin sealant has been used in many surgical procedures for hemostasis (topical agent) and tissue approximation (as an adhesive). Its usage has expanded through numerous procedures, including urologic surgery. Urologic procedures need the fibrin glue's sealing power to improve wound healing in penile urethroplasty.19\n\nTisseel, a fibrin glue preparation, was first utilized by Kinahan et al. to enhance hypospadias repair.20 In addition, Hick et al. stated that fibrin glue could be beneficial in promoting early catheter removal and accelerating the process of wound healing.21 Study from Barbagli et al. shared their experience using fibrin glue in the buccal mucosa graft urethroplasty for bulbar urethral stricture.22\n\nBetter postoperative outcomes were found in patients with fibrin sealant usage during surgery. There were no flap necrosis or penile torsion found as early postoperative complications.8 Havez et al. stated this benefit might be influenced by the regeneration of the cellular and angiogenic of the tunica albuginea defect.23 Urethra-cutaneous fistula was found less in the sealant group. They also had a better cosmetic outcome. There is a statistically significant improvement in the overall outcomes.24\n\nAnother complication assessed in the study was postoperative edema. A larger number of early postoperative edema incidences in the non-sealant group might be due to undiscovered microscopic leakage in the tissue spaces between the suture lines. This could lead to a fistula formation.10 The application of fibrin glue forms an additional layer over the urethral tube, seals minute cracks between sutures, and decreases post-surgery edema. Therefore, its application might accelerate the wound healing process.10\n\nCommercial fibrin sealants had a higher risk of allergic reactions compared to the homologous, blood bank product, fibrin sealants, which are prepared from single unit plasma. Risk of infection could be decreased by using autologous blood as the fibrinogen source. The setback is that the production took longer and created non-uniform fibrinogen concentration.25,26 Kajbafzadeh et al.12 described fibrin glue's benefits preventing further recurrence in patients with recurrent fistula after hypospadias or epispadias repair.12\n\nChoukroun et al.27 developed platelet-rich fibrin (PRF) in the early 2000s that has been used to augment the process of tissue healing. The procedure is simple and less expensive than fibrin glue: the PRF patch is collected by centrifuging the autologous blood sample without any adjuvant. The patch creation could be done amid the urethroplasty procedure and used promptly.11,28\n\nA study by Guinot et al.11 found no negative effect of PRF as there were no adverse effects or skin infections discovered. Unfortunately, PRF superiority over conventional covering technique has not been proven.\n\nThe use of cryocalcium glue has been limited in underdeveloped or developing countries. This is due to its higher risk of infection transmission than fibrin glue, despite having been through donor screening, heat-treating, and the use of solvent and detergent suspension.13 The preparation of cyrocalcium glue is very simple and can be done during the urethroplasty procedure. It can be used shortly after its prepatation.13 It is formed from combining a patient's cryoprecipitate and calcium gluconate.13\n\nThis cryocalcium glue could be a safe and less expensive alternative than commercial fibrin glue to reduce fistula formation. The case group showed a minimal incidence of fistula by using cryocalcium glue.13 It is also safe from allergic complications due to its autologous origin.13\n\nIn a prospective study, Kocherov et al. reported the efficacy of BioGlue in hypospadias repair.14 BioGlue does not carry a risk of infection transmissions, such as HIV, HBV, or HCV, different from natural human fibrin tissue sealant. Therefore, it appears to be an ideal sealant.29 BioGlue consists of two purified bovine serum albumin and glutaraldehyde.30 Glutaraldehyde acts to bridge the bovine serum albumin amine groups to the target tissue's extracellular matrix, forming a covalent bond between tissue and adhesive.30 However, the advantages of BioGlue in reducing fistula formation and reconstruction breakdowns were not shown in the Kocherov et al. study.14 No difference in fistula formation and surgical breakdown were seen in both groups.14 In addition, the BioGlue group tends to have a more prominent severe fibrotic skin reaction, therefore inferior in cosmetics than the control group. Other publicity stated some discouraged effects of BioGlue, such as toxicity to tissue, local inflammation, and postoperative wound complications.31,32 It is hypothesized due to glutaraldehyde toxicity.14\n\nIn a study by Hosseini et al., cyanoacrylate was used as a dressing in urethroplasty.15 This dressing has many purposes with others in edema restriction, hematoma formation, and wound stabilization if treated in several layers and lacks urine and feces permeability.15 There were no regular changes or pain upon removal. This type of dressing was more convenient for patients and the wound was kept hygienic at home. The occurrence of complications is less, 10% versus 30%; but late complications indirectly related to the dressing have been excluded.15\n\nPrestipino et al. used cyanoacrylate in four patients with UCF. Three of them had fistula healing.33 In another study, cyanoacrylate was used for simple sharp wounds in various locations, including the face and extremities, inguinal and umbilical hernia, cleft lip, hypospadias, and post-hypospadias fistula repair. The results showed improvement in efficacy, cosmetics, procedure duration, and patient comfort.34\n\nBased on our meta-analysis, tissue sealant can reduce urethroplasty complications during hypospadias repair. In our analysis, tissue sealant can reduce the occurrence of fistula urethocutan, tissue edema, and flap-related complication. This result is in line with the previous systematic review.5\n\n\nLimitation\n\nThere are several limitations to this study. The included studies reported non-uniform complications. Information on the sealant and surgery cost of hypospadias repairment was limited. Moreover, several hypospadias types and various repair techniques used might serve as confounding factors as not all studies report on the complication and success rate based on the type of hypospadias.\n\n\nConclusion\n\nThis systematic review revealed additional benefits from several types of tissue sealant in hypospadias repair surgery. Fibrin sealant applied over the urethroplasty suture lines forms an adequate water-proof cover and improves outcomes in hypospadias repair. Fibrin glue reduces the incidence of complications in medial and distal hypospadias but not in proximal hypospadias. The application of PRF had no harmful effects, but it did not demonstrate superiority over conventional covering techniques. Cryocalcium glue could be used as an alternative to fibrin glue to reduce fistula formation. It is also safe and less expensive than fibrin glue. The use of cyanoacrylate dressing lessens the immediate complication of hypospadias repair surgery. Meanwhile, BioGlue does not show any additional advantages in hypospadias repair in pediatric patients than the standard procedures. A conclusion cannot be made due to studies diversity. Therefore, randomized controlled trials with a significant sample size are necessary to precisely compare the sealant and non-sealant groups, types of sealants, and hypospadias location.\n\n\nData availability\n\nAll data used in the research are available as part of articles and no additional source are needed to disclose\n\n\nReporting guidelines\n\nFigshare: PRISMA checklist for ‘The Benefit of Tissue Sealant on Urethroplasty in Hypospadias Patients – A Systematic Review and Meta-analysis’.\n\nDOI: 10.6084/m9.figshare.19102184\n\nData are available under the terms of the Attribution 4.0 International license (CC BY 4.0)\n\n\nAuthor contributions\n\nSFS: Conceptualization, Methodology, Project administration, Supervision\n\nSS: Data curation, Software, Writing original draft\n\nAM: Conceptualization, Supervision, Validation, Wiriting review and editing", "appendix": "References\n\nvan der Horst HJR , de Wall LL : Hypospadias, all there is to know. Eur. J. Pediatr. 2017; 176(4): 435–441. Publisher Full Text\n\nAkramov NR, Kagantsov IM, Sizonov VV, et al.: Advancement urethroplasty for distal hypospadias repair without dismembering urethra spongy body and glans penis. Urology Herald. 2020; 8(3): 5–12. Publisher Full Text\n\nLu YC, Huang WY, Chen YF, et al.: Factors associated with reoperation in hypospadias surgery — A nationwide, population-based study. Asian J. Surg. 2017; 40(2): 116–122. PubMed Abstract \| Publisher Full Text\n\nMammo TN, Negash SA, Negussie T, et al.: Hypospadias Repair in Ethiopia: A Five Year Review. Ethiop. J. Health Sci. 2018; 28(6): 735–740. PubMed Abstract \| Publisher Full Text\n\nSingh A, Anand S, Goel P, et al.: Can sealing promote healing?: A systematic review and meta-analysis highlighting the adjunctive role of tissue sealant application during urethroplasty for hypospadias. J. Pediatr. Urol. 2021; 17: 805–812. PubMed Abstract \| Publisher Full Text\n\nKommu SS, McArthur R, Emara AM, et al.: Current Status of Hemostatic Agents and Sealants in Urologic Surgical Practice. Rev. Urol. 2015; 17(3): 150–159.\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement.2009; 6(7).\n\nShenoy NS, Tiwari C, Gandhi S, et al.: Efficacy of fibrin sealant as waterproof cover in improving outcome in hypospadias surgery. Afr. J. Paediatr. Surg. 2021; 18(4): 215–218. PubMed Abstract \| Publisher Full Text\n\nGopal SC, Gangopadhyay AN, Mohan TV, et al.: Use of fibrin glue in preventing urethrocutaneous fistula after hypospadias repair. J. Pediatr. Surg. 2008 Oct; 43(10): 1869–1872. Publisher Full Text\n\nAmbriz-González G, Velázquez-Ramírez GA, García-González JL, et al.: Use of fibrin sealant in hypospadias surgical repair reduces the frequency of postoperative complications. Urol. Int. 2007; 78(1): 37–41. Publisher Full Text\n\nGuinot A, Arnaud A, Azzis O, et al.: Preliminary experience with the use of an autologous platelet-rich fibrin membrane for urethroplasty coverage in distal hypospadias surgery. J. Pediatr. Urol. 2014; 10(2): 300–305. PubMed Abstract \| Publisher Full Text\n\nKajbafzadeh AM, Abolghasemi H, Eshghi P, et al.: Single-donor fibrin sealant for repair of urethrocutaneous fistulae following multiple hypospadias and epispadias repairs. J. Pediatr. Urol. 2011; 7(4): 422–427. PubMed Abstract \| Publisher Full Text\n\nHosseinpour M, Etezazian S, Hamsaieh M: Cryocalcium Glue in Hypospadias Surgery. J. Indian Assoc. Pediatr. Surg. 2019; 24(3): 226–227. PubMed Abstract \| Publisher Full Text\n\nKocherov S, Lev G, Chertin B: Use of BioGlue surgical adhesive in hypospadias repair. Current Urology. 2014; 7(3): 132–135. PubMed Abstract \| Publisher Full Text\n\nHosseini SMV, Rasekhi AR, Zarenezhad M, et al.: Cyanoacrylate glue dressing for hypospadias surgery. N. Am. J. Med. Sci. 2012; 4(7): 320–322. PubMed Abstract \| Publisher Full Text\n\nSnodgrass WT: Tubularized incised plate hypospadias repair: indications, technique, and complications. Urology. 1999 Jul; 54(1): 6–11. PubMed Abstract \| Publisher Full Text\n\nCook A, Khoury AE, Neville C, et al.: A multicenter evaluation of technical preferences for primary hypospadias repair. J. Urol. 2005 Dec; 174(6): 2354–2357. discussion 2357. Publisher Full Text\n\nBao Z, Gao M, Sun Y, et al.: The recent progress of tissue adhesives in design strategies, adhesive mechanism and applications. Mater. Sci. Eng. C 2020; 111(March): 110796. PubMed Abstract \| Publisher Full Text\n\nYang H, Xuan X, Hu D, et al.: Comparison of effect between dartos fascia and tunica vaginalis fascia in TIP urethroplasty: a meta-analysis of comparative studies. BMC Urol. 2020; 20(1): 161. PubMed Abstract \| Publisher Full Text\n\nKinahan TJ, Johnson HW: Tisseel in hypospadias repair. Canadian journal of surgery Journal canadien de chirurgie. 1992 Feb; 35(1): 75–77. PubMed Abstract\n\nHick EJ, Morey AF: Initial experience with fibrin sealant in pendulous urethral reconstruction. Is early catheter removal possible? J. Urol. 2004 Apr; 171(4): 1547–1549. PubMed Abstract \| Publisher Full Text\n\nBarbagli G, De Stefani S, Sighinolfi MC, et al.: Experience with fibrin glue in bulbar urethral reconstruction using dorsal buccal mucosa graft. Urology 2006 Apr; 67(4): 830–832. PubMed Abstract \| Publisher Full Text\n\nHafez AT, El-Assmy A, El-Hamid MA: Fibrin glue for the suture-less correction of penile chordee: a pilot study in a rabbit model. BJU Int. 2004 Aug; 94(3): 433–436. PubMed Abstract \| Publisher Full Text\n\nAmbriz-González G, Aguirre-Ramirez P, García-De León JM, et al.: 2-octyl cyanoacrylate versus reintervention for closure of urethrocutaneous fistulae after urethroplasty for hypospadias: A randomized controlled trial. BMC Urol. 2014; 14(1): 2–7. Publisher Full Text\n\nMoser C, Opitz I, Zhai W, et al.: Autologous fibrin sealant reduces the incidence of prolonged air leak and duration of chest tube drainage after lung volume reduction surgery: a prospective randomized blinded study. J. Thorac. Cardiovasc. Surg. 2008 Oct; 136(4): 843–849. PubMed Abstract \| Publisher Full Text\n\nde Hingh IHJT , Nienhuijs SW, Overdevest EP, et al.: Mesh fixation with autologous platelet-rich fibrin sealant in inguinal hernia repair. European surgical research Europaische chirurgische Forschung Recherches chirurgicales europeennes. 2009; 43(3): 306–309. PubMed Abstract \| Publisher Full Text\n\nLi Q, Pan S, Dangaria SJ, et al.: Platelet-rich fibrin promotes periodontal regeneration and enhances alveolar bone augmentation. Biomed. Res. Int. 2013; 2013: 638043.\n\nDohan DM, Choukroun J, Diss A, et al.: Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 2006 Mar; 101(3): e37–e44. PubMed Abstract \| Publisher Full Text\n\nJoch C: The safety of fibrin sealants. Cardiovasc. Surg. (London, England). 2003 Aug; 11(Suppl 1): 23–28. PubMed Abstract \| Publisher Full Text\n\nBahouth Z, Moskovitz B, Halachmi S, et al.: Bovine serum albumin-glutaraldehyde (BioGlue(®)) tissue adhesive versus standard renorrhaphy following renal mass enucleation: a retrospective comparison. Ther. Adv. Urol. 2017; 9(3–4): 67–72. PubMed Abstract \| Publisher Full Text\n\nFürst W, Banerjee A: Release of glutaraldehyde from an albumin-glutaraldehyde tissue adhesive causes significant in vitro and in vivo toxicity. Ann. Thorac. Surg. 2005 May; 79(5): 1522–1528; discussion 1529. PubMed Abstract \| Publisher Full Text\n\nKlimo PJ, Khalil A, Slotkin JR, et al.: Wound complications associated with the use of bovine serum albumin-glutaraldehyde surgical adhesive in pediatric patients. Neurosurgery. 2007 Apr; 60(4 Suppl 2): 305–309. discussion 309. PubMed Abstract \| Publisher Full Text\n\nPrestipino M, Bertozzi M, Nardi N, et al.: Outpatient department repair of urethrocutaneous fistulae using n-butyl-cyanoacrylate (NBCA): a single-centre experience. BJU Int. 2011 Nov; 108(9): 1514–1517. PubMed Abstract \| Publisher Full Text\n\nCastañón García-Alix M, Sancho Vendrell MA, Parri Ferrandis FJ, et al.: Utility of octyl 2-cyanoacrylate in pediatric surgery. Anales de Pediatria (Barcelona, Spain: 2003) 2003 Dec; 59(6): 548–551. Publisher Full Text" }	[ { "id": "123542", "date": "28 Feb 2022", "name": "Borko Stojanovic", "expertise": [ "Reviewer Expertise Paediatric urology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 deals with a very interesting and up-to-date topic, since successful hypospadias repair still presents a great challenge. Literature data on the use of tissue sealant in urethroplasty show quite heterogeneous results, and authors made a very good choice to assess this by a systematic review. The methodology is based on PRISMA guidelines, with detailed explanation on search strategy, as well as good illustration of results. However, I have to note that the rest of the manuscript is poorly written, in several points:\nThe manuscript should be copy-edited to improve the quality of the writing.\n\nRationale and objectives are poorly explained, and introduction section needs more details and corrections, especially about the use of tissue sealant in hypospadias repair. Is it indicated in every case and form? What type of tissue sealant and when...?\n\nIntroduction Section: The sentence \"Hypospadias can be successfully repaired through multiple surgeries in the majority of children\" has to be corrected. Today, most of hypospadias cases are repaired by a one-stage surgery.\n\nIntroduction Section: \"The overall success rate for first surgery was 55.9%.\". Please provide reference. The rate of success is much higher today.\n\nAbstract Section: \"UCF complication was reported in all studies.\" Do not start a sentence with an acronym.\n\nSome sentences within the Results and Discussion sections are unclear and require copy-editing.\nSection Discussion: \"Although superior in cosmetic issues, it is reported that the complication is as high as 20-30%.17\" I don't think this data is true, based on provided reference.\n\nDiversity of the studies is large, and it should be primarily denoted in Conclusion section.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "10826", "date": "14 Jun 2024", "name": "Steven Steven", "role": "Author Response", "response": "Thank you for the review. We tried to revise the article in line with the recommendation you made. we also update several data according to newer articles." } ] }, { "id": "185863", "date": "07 Sep 2023", "name": "V V S Chandrasekharam", "expertise": [ "Reviewer Expertise PEDIATRIC UROLOGY & Hypospadias" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a systematic review & meta-analysis (SR & MA) on an interesting topic. However, a similar review as published recently1, which found no difference in the overall complication rate with the use of tissue sealants. The present article by Siregar et al has several drawbacks, some of which the authors themselves have observed and mentioned. Since an SR & MA represents a high level of evidence, it is important to apply certain stringent criteria to define the quality of the included studies.\n\nIn the methodology of SR, it is important to calculate the heterogeneity of the included papers by the use of I2 statistics and Funnel plot. The publication bias of each included paper needs to be assessed and reported using methods such as Doi plot and LFK index. These statistics provide the reader with information regarding the comparability of the included papers and thus is a measure of the reliability of the results of the SR & MA. Without these data, it is impossible for the reader to arrive at any conclusions.\n\nThe authors themselves have acknowledged that the included studies have several shortcomings in their methodology, reporting of complications, type of sealant used and follow-up. This makes the results of the SR & MA unreliable.\n\nIn the discussion, the authors should compare their results with other similar studies1 and explain the differences in the outcomes.\n\nThe single most important criteria for the success of hypospadias repair is the expertise and experience of the surgeon. This factor has not been discussed in the studies not in the SR & MA.\nThus, although this SR & MA is a good effort, the results must be interpreted with caution.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/11-184
https://f1000research.com/articles/12-279/v1	14 Mar 23	{ "type": "Research Article", "title": "Validation of the Dominican system for measuring early childhood development", "authors": [ "Laura V. Sánchez-Vincitore", "María Angélica Alonso Pellerano", "María Elena Valdez", "Angie Sabrina Jiménez", "Carlos B Ruiz-Matuk", "Arachu Castro", "Felipe Díaz", "Daniel Cubilla-Bonnetier", "María Angélica Alonso Pellerano", "María Elena Valdez", "Angie Sabrina Jiménez", "Carlos B Ruiz-Matuk", "Arachu Castro", "Felipe Díaz", "Daniel Cubilla-Bonnetier" ], "abstract": "Background: The purpose of the study was to determine the psychometric properties of the Dominican System for Measuring Early Childhood Development (SIMEDID, for its Spanish acronym), to adjust the sequence of item presentation, and to provide age-standardized norms for each item, to enable policy and program managers to make decisions based on specific and structured data. Methods: After approval from an ethics committee, a total of 948 children from 0 to 60 months participated in this study. Participants were evaluated on four early childhood development domains (gross motor, fine motor, language development, and socio-emotional development). The data were collected from November 2021 to February 2022, either at early childhood care centers or at home, using mobile devices that guided the evaluators through the screener. Data were later synced to a global database. Psychometric properties were calculated using Cronbach’s alpha and split-half parallel reliability. For reorganizing item presentation and to obtain age-standardized norms, we conducted a logistic regression analysis for each item on dependent variable item success, and independent variable age. Results: The instrument showed excellent reliability and additional evidence of validity. The item presentation order was rearranged according to the probability of item success progression. In addition, the study characterized the expected evolution of item success probability across participants' age. Conclusions: SIMEDID is a valid and reliable instrument for depicting childhood development in national evaluations. Its integration with electronic platforms for national monitoring represents a cost-effective, time-efficient screening tool adapted to the Dominican sociocultural context. This represents a promising tool to strengthen strategies that support early childhood development.", "keywords": [ "Early childhood development", "Dominican Republic", "Monitoring", "Screening" ], "content": "Introduction\n\nThe Dominican Republic confirmed its commitment to achieving the United Nation’s sustainable development goal 4.2 for 2030 to guarantee that children are ready for primary education by offering quality early childhood services, including care and early education (United Nations, 2015). One of this goal’s indicators is “[the] proportion of children aged 24-59 months who are developmentally on track in health, learning, and psychosocial well-being, by sex” (UNESCO UIS, n.d., para. 1). Although this indicator is conceptually straightforward, and numerous efforts have been conducted to establish a global methodology for measuring it, some challenges associated with determining cut-off points for early childhood development remain (Daelmans et al., 2017; Richter et al., 2017).\n\nFirst, high-income countries' screening tools might translate poorly to low- and middle-income countries. This poses the risk of either underestimating or overestimating childhood development, which in turn precludes making accurate, evidence-based decisions regarding early childhood interventions and resource allocation (Gladstone et al., 2008; Sabanathan et al., 2015). The second challenge is the need for more funding for monitoring systems in low- and middle-income countries, which translates into needing more sufficiently qualified personnel to conduct periodic childhood development screenings (Lokuketagoda et al., 2016). And third, there is a critical need to obtain a large pool of data from developing children to identify those at risk for developmental delay (Lokuketagoda et al., 2016), which is particularly challenging in low- and middle-income countries (Richter et al., 2017).\n\nIn Latin America and the Caribbean, childhood development measurement has attracted attention, evidenced by the creation of the Regional Network for Measuring Childhood Development (REMDI) (Interamerican Dialogue, 2020). This international network of specialists is dedicated to promoting national measurements of childhood development to obtain data for decision-making and comparison between and within countries. Since the year 2000, the Dominican Republic has collected data on childhood development by participating in a series of Multiple Indicator Cluster Surveys (MICS), a household survey methodology designed by UNICEF that analyzes the situation of women and children across the world. The instrument collects data on children’s health, education, protection, and environment (such as sanitation), among other variables. The Dominican Republic participated in MICS2 (Molina Achécar and Polanco, 2001), MICS5 (ONE and UNICEF, 2016), and MICS6 (ONE and UNICEF, 2021) survey rounds with an evolving early childhood development measurement (Loizillon et al., 2017).\n\nThe latest data from 2019 reported that 87.1% of Dominican children meet the minimum development indicators. Data generated by MICS have been useful for guiding the advocacy and system-strengthening plans of early childhood development and children’s rights institutions, including sustainable investments in the multi-year governmental planning for 2020-2024. In addition, the MICS data have been used to create predictive models that quantify the impact of multiple sociodemographic and psychosocial factors in childhood development (Sánchez-Vincitore and Castro, 2022).\n\nHousehold surveys provide useful information on general trends in childhood development, but they are not comprehensive enough to assess development in its various dimensions or sensitive enough to generate alerts to detect developmental delays. These limitations highlight the need for specific child development screening tools that, although quick and cost-effective in the application, have adequate psychometric properties.\n\nMany private, informal, and some public initiatives have been conducted in the Dominican Republic to provide early childhood services. However, until 2019 there were no standardized instruments to measure the impact of such efforts. In 2019, Sánchez-Vincitore et al. (2019) initiated the validation of the Dominican adaptation of the Malawi Developmental Assessment Tool (MDAT) (Gladstone et al., 2008, 2010). It is a childhood development screener in which an evaluator observes the behavior of a child in four different domains: gross motor, fine motor, language, and socio-emotional development. One of the advantages of this screener is that it optimizes test application time by only presenting items that correspond to the child’s expected evolutionary stage according to the child’s age. Therefore, providing a more precise item order is crucial to prevent bias in obtaining total scores. Although the instrument presented good psychometric properties, some limitations had to be considered before upscaling it as a national surveillance tool. First, as a preliminary pilot implementation within the academic context, research assistants with vast experience and training in data collection administered the instrument, which is unlikely in naturalistic environments. Second, the study had a small sample size (N = 42), meaning that there was no national representativeness. Therefore, age-standardized norms were not obtained for each item, threatening item presentation order. Finally, the study evaluated children up to 24 months, limiting the age range for which data were available.\n\nTo overcome these limitations, the National Institute for Early Childhood Comprehensive Care (INAIPI, for its acronym in Spanish) –the state institution responsible for providing quality comprehensive care services to children from 0 to five years and their families–, the Universidad Iberoamericana (UNIBE), and the United Nations Children’s Fund (UNICEF) developed the Dominican System for Measuring Early Childhood Development (SIMEDID, for its acronym in Spanish). SIMEDID was created by re-analyzing the content and structure of the Dominican version of the MDAT (Sánchez-Vincitore et al., 2019) and integrating other items from international and national instruments (Alonso et al., 2022).\n\nSIMEDID is an electronic platform that hosts an early childhood development screener. This platform allows data collection through mobile devices and connects to INAIPI’s servers as part of its monitoring and evaluation system. The mobile application extracts sociodemographic information from the server, configuring individual evaluations according to each child’s age. As a result, INAIPI personnel already in the field can administer the early childhood development screener time-efficiently with little training. Once the assessment is over and the device connects to the internet, the data sync to the server—reducing the risk of losing the data.\n\nGiven the current stage of development of SIMEDID, the aims of this study were: (1) To determine the psychometric properties of SIMEDID; (2) To adjust the sequence of item presentation according to developmental milestones obtained from data from a large sample; (3) To provide age-standardized norms for each item.\n\n\nMethods\n\nThe Universidad Iberoamericana’s ethics committee approved this study (CEI2021-3). Written informed consent was obtained from participant’s parents or guardians before participation in the study.\n\nThis is a cross-sectional, non-experimental, and descriptive study that evaluated children who receive services at INAIPI.\n\nData collection occurred in Santo Domingo, Dominican Republic, from November 1st, 2021, to February 17th, 2022. INAIPI participants who attended Comprehensive Care Centers for Early Childhood (CAIPIs, for its Spanish acronym) were assessed at their centers. In contrast, those who participated in Comprehensive Care Centers for Children and the Family (CAFI, for its Spanish acronym) received community and family-based services at their home, which is where children were assessed. The instrument was applied during regular service hours. The evaluation personnel consisted of 20 educational agents (who work at CAIPIs) and 20 community agents (who work at CAFIs).\n\nAn intentional sample of 948 children who live in Santo Domingo was selected from the INAIPI’s System of Information and Management for Early Childhood (SIGEPI, for its Spanish acronym) according to their age and type of service received (CAIPI or CAFI, which were kept proportional to the actual service: 36% and 64%, respectively). The inclusion criterion was to be beneficiaries of INAIPI. Participants were 428 girls (45.1%) and 520 boys (54.9%).\n\nSociodemographic variables: This set of questions addressed general demographic variables: sex assigned at birth (male and female), age (in days at the moment of evaluation), and type of service (CAIPI vs CAFI). These variables, obtained directly from SIGEPI, determine the starting item of SIMEDID 's subscales.\n\nSIMEDID: This electronic instrument assesses childhood development in four development areas: gross motor, fine motor, language development, and social development. Each subtest has 33 items (except for language development, which has 34 items) in ascending difficulty levels. The presentation of the first item depends on the child’s age. Once the first item is completed on each dimension, the instrument is presented backward until the participant passes three items. Then the presentation continues forward until the participant misses three items. This allows evaluators to know the actual state of child development, in addition to detecting alerts, either delays in childhood development (backward presentation) or above the average development (forward presentation). Since the purpose of the study was to determine the age range in which each item is accomplished, we used the Malawi MDAT norms for this first trial. Subsequent data collection will use the sequence based on the findings from this study.\n\nThe evaluation is conducted using a mobile device with the SIMEDID app that connects to the INAIPI server and instructs the evaluator to assess a specific child. The app calculates the first item in each development area and presents items backward and forward. Passed items were scored 1, while missed items scored 0. Items not shown (for not corresponding to the participant's age range) were automatically completed: items before the three first achieved items were scored 1 (since it is assumed that the child has already passed these), and items after three consecutive misses scored 0 since the child is not ready to perform these.\n\nWe obtained an expert panel's consensus before collecting data to guarantee the instrument's construct definition (Sireci and Sukin 2013). The expert panel consisted of a group of professionals who represent Dominican institutions that provide early childhood services, including the Early Childhood Education Department and the Special Education Department from the Ministry of Education; the Ministry of Health; the National Health Services (SNS for its Spanish acronym); the National Council for Childhood and Adolescence (CONANI for its Spanish acronym); and the National Council for Disabilities (CONADIS for its Spanish acronym). The experts had the opportunity to revise each item and their definitions. The experts suggested to modify the definition of the item “[They] walk on tiptoes”. They included a statement that differentiates walking on tiptoes as a possible disability indicator vs. the intentional action of walking on tiptoes. The experts unanimously approved all changes.\n\nThe items were presented in Spanish and were translated to English by the research team for this publication.\n\nThe variables included in this study are listed in Table 1.\n\nA total of 40 evaluators received a six-hour training session and conducted two practice evaluations. During the practice evaluations, the research team coached and supervised the evaluators. Recruitment was conducted by an institutional message indicating that either a CAIPI or CAFI was selected to participate. Children from CAIPI attending services during the data collection day were evaluated after a parent signed the consent form when dropping off their children at the centers. For CAFI participants, evaluation was conducted at home, and the assigned in-field INAIPI personnel contacted their families. Parents signed the informed consent before the interview took place at home. Each evaluation had a duration of 25-30 minutes.\n\nTo determine the instrument's psychometric properties, which corresponds to the first aim, we calculated Cronbach’s alpha and split half-parallel reliability. Then, for additional evidence of content validity, we conducted descriptive analyses (means and standard deviations) of each sub-scale score for each age group to confirm alignment of the instrument with development by age.\n\nTo determine the most appropriate item presentation order according to these data (second aim) and age-standardized norms (third aim), we conducted a logistic regression analysis on each item with item success (0 and 1) as the dependent variable and age in days as the independent variable. Following the methodology used by Gladstone et al. (2008, 2010), after ensuring a good model fit, the alpha and beta coefficients were used to calculate the cut-off age associated with a.9 probability of success following the formula in Equation 1:\n\nThe formula was also used to calculate the .75, .50, and .25 probabilities of success for each item. We used predictive probabilities from the regressions to calculate ages corresponding to 25%, 50%, 75%, and 90% of children passing each item, as proposed by Gladstone et al. (2008, 2010). For the statistical analyses, we used the IBM SPSS Statistics 25 program (https://www.ibm.com/products/spss-statistics). An open-source alternative to SPSS that can conduct the same processes is JASP (https://jasp-stats.org/).\n\n\nResults\n\nTo confirm the content validity of SIMEDID, we obtained descriptive statistics on each age group for the evaluated developmental areas. Table 2 shows that mean growth is progressive through age groups.\n\n* Sample size for this group was insufficient for an accurate representation.\n\nTwo internal consistency indices were calculated for each developmental area to confirm the instrument reliability, Cronbach's alpha, and split-half Spearman-Brown’s correlation (see Table 3).\n\nTo complete the second and third aims, we conducted a logistic regression analysis on each item with independent variable age and dependent variable item success. The results show a good fit (p < .05), except for the first item in the gross motor area and three of the first four items in the language development area.\n\nThe item presentation order was determined by sorting the age at which each item had a .9 probability of success from the results of logistic regression analyses on each item. To describe the expected evolution of item success probability across age, we also determined the .75, .50, and .25 probability of success. We thus provided the range amplitude for each item's success predicted by age. Figures 1 to 4 contain a visual representation of sorted items and corresponding probabilities of success. Note that results are presented in two scales for the X axis: for the first two years, the X axis corresponds to age in months, and for the following age ranges, in years.\n\nNotes: MG1 Hold their head when carried; MG2 Lift their chin off the floor; MG3 From the prone position, they can lift their head to 90 degrees; MG4 Support head when lifted by hands; MG5 Flip over; MG6 Raise head, shoulders, and chest from a prone position; MG7 Start creeping; MG8 Start crawling position; MG9 Stand up with support; MG10 Sits up unassisted; MG11 Crawl with displacement alternating knees and hands; MG12 Take steps with help; MG13 Stand up unassisted; MG14 Walk without help; MG15 They crouch and stand up; MG16 Walk well with cross-scroll; MG17 Run, they may fall; MG18 Throw ball; MG19 Kick the ball; MG20 Run showing coordination in their movements; MG21 Run well, stops and start again without falling; MG22 Jump with feet together; MG23 Jump moving with both feet; MG24 Stand on one foot for 3 seconds; MG25 Stand on tiptoe with both feet; MG26 Walk on tiptoe; MG27 Walk in a straight line keeping balance; MG28 Jump on one foot without support; MG29 Can catch a ball with both hands; MG30 Bounce and catch the ball; MG31 Stand on one foot for 5 seconds; MG32 Jump moving with one foot; MG33 Jump alternating feet.\n\nNotes: MF1 Palmar grasp reflex; MF2 Stare the midline; MF3 Visually focuses on an object and tracks it horizontally; MF4 Keep hands open when awake; MF5 Hold an object in hand; MF6 Show interest in putting an object in their mouth; MF7 Visually focus on an object and follow it from top to bottom; MF8 Grasp large objects voluntarily; MF9 Hold an object in each hand; MF10 Pass an object from one hand to another; MF11 Pick up small objects as if their fingers were a rake; MF12 Find the object under a blanket; MF13 Put and take out objects from the container; MF14 Grasp with thumb and forefinger (tweezers); MF15 Pick up a spoon and brings it to their mouth; MF16 Scribble; MF17 Push a car; MF18 Turn pages of a book; MF19 Make a tower of two cubes; MF20 Put nails on a board; MF21 Make a tower of six cubes; MF22 Make a ball of paper; MF23 Tear paper with both hands; MF24 Make shapes with putty; MF25 Rotate hand to unscrew; MF26 String; MF27 Copy a horizontal and vertical line; MF28 Copy a circle; MF29 Copy a cross; MF30 Know how to button and unbutton; MF31 Color without leaving the outline of the drawing; MF32 Draw a human figure; MF33 Cut paper with scissors.\n\nNotes: DL1 Calm down when speaking to them; DL2 Startle or jump in response to sounds; DL3 Cry to express needs; DL4 They laugh; DL5 Make sounds with the throat; DL6 Turn their head when they search for a sound; DL7 React when called by their name; DL8 Pronounce syllables like Ma, Pa, Ba, Ta; DL9 Point with their finger when they want something; DL10 Repeat the same syllable twice “Dada, Mama, Mimi, Tata, Papa, Yaya, Baba”; DL11 Understand the meaning of the word no; DL12 Follow one-step commands; DL13 Follow two-step instructions; DL14 Answer with yes or no; DL15 Pronounce their first words with communicative intention; DL16 Recognize at least 6 objects; DL17 Point to 5 parts of their body; DL18 Use a two-word phrase; DL19 Say 6 words; DL20 Say their name; DL21 Know the use of three or more objects; DL22 Can identify 10 objects by name; DL23 Pronounce sentences of three words; DL24 Use more than 15 words; DL25 Use long sentences; DL26 Know the qualities or characteristics of an object; DL27 Pronounce the sounds of words correctly; DL28 Describe the drawing; DL29 Name at least three things in a category; DL30 Recognize opposites; DL31 Can count up to 5 or more objects; DL32 Answer two comprehension questions; DL33 Compare objects; DL34 Tell a story from a sequence of images.\n\nNotes: DS1 Calm down with family members or caregivers; DS2 Smile spontaneously; DS3 Smile in response to a person; DS4 Recognize the voice of the main caregiver; DS5 Make eye contact; DS6 Touch the examiner's hands; DS7 They are aware of their hands (body); DS8 Try to hold a cup when being fed; DS9 Respond to a conversation; DS10 Raise their arms or indicate that they want to be carried; DS11 Laugh out loud; DS12 Explore their face when they are in front of the mirror; DS13 Show interest or intention to feed themselves; DS14 Look for continuing the game; DS15 Explore the environment; DS16 Participate in games; DS17 Wave or verbally greet; DS18 Express their satisfaction when they achieve something; DS19 Take a glass without spilling; DS20 Imitate adult actions; DS21 Recognize their belongings; DS22 Express interest in playing with other children; DS23 Symbolic game; DS24 Refer to themselves as “I”; DS25 Say the names of the people with whom they live; DS26 They urinate or defecate independently without dirtying their clothes; DS27 Indicate in some way that they need to urinate or defecate; DS28 Identify basic emotions in images; DS29 Come up with games; DS30 Share their belongings; DS31 Recognize basic emotions in themselves and express them verbally; DS32 Recognize and express basic emotions in others; DS33 Participate in games respecting rules and turns.\n\n\nDiscussion\n\nThis paper collects evidence for the validity of SIMEDID, an electronic early childhood development screening tool adapted to the Dominican context conducted by INAIPI’s personnel through an electronic application. Regarding the first objective, we found that the instrument has adequate psychometric properties: the instrument’s subscales showed high internal consistency scores, evidencing excellent reliability. Furthermore, total scores for each sub-scale increased progressively across age, which evidenced alignment with standards already provided by an expert panel (Alonso et al., 2022) and criterion validity with a previous version of this instrument on a small sample size (Sánchez-Vincitore et al., 2019).\n\nRegarding the second aim, we found that age predicted item success in most items, which supports similar results in children from Malawi Gladstone et al. (2008, 2010). However, age did not predict four initial items from the gross motor and language development subscales. We attribute these null findings to the fact that our study did not include an acceptable sample size for the age group for which these items were relevant. This is because INAIPI’s services to very young children were scarce at the time of this study, which will be considered for future studies. To comply with the third aim, age-standardized norms for each item were established, obtaining correspondences between ages and different probabilities of success for each item, which will allow comparing the achievement of participants with what is expected at their age.\n\nAdapting this instrument to the Dominican context guarantees that cultural aspects of childrearing do not overshadow developmental scores (Suchdev et al., 2017) and that development is not under or overestimated. Having the instrument in an electronic platform solves two main challenges. First, personnel training is kept to the minimum since the platform guides the evaluator throughout the evaluation, presenting the items that only pertain to the child according to their age, with suggested videos and additional testing resources. Second, having SIMEDID connected to INAIPI servers and incorporating the data on national services provided by INAIPI creates a continuous stream that otherwise would be costly and logistically convolute data. This data stream will allow the development of new research agendas that include correlational modeling, intervention studies, and longitudinal studies to understand better the factors associated with childhood development in the Dominican Republic in a timely and cost-efficient way.\n\nThe study findings demonstrate that SIMEDID passes three elements of a checklist of critical methodological elements to consider when appraising a childhood development assessment tool: (1) the instrument measures domains affected by the risk factor or intervention; (2) reliability and validity of the instrument in the population of interest; (3) sensitivity of the instrument to identify changes; (4) logistics and methodology is suitable for evaluating the outcome; and (5) consideration of control group (Sabanathan et al., 2015). SIMEDID passes the first two elements from this checklist, as it measures specific domains of early childhood development previously identified as risk factors in the Dominican Republic, such as the sociodemographic and psychosocial factors that predict childhood development (Sánchez-Vincitore and Castro, 2022) and low levels of oral language comprehension in school-aged children that should be addressed during early childhood development before children enter primary school (Sánchez-Vincitore et al., 2020, 2022) among other risk factors. In addition, it passes the fourth element, given that the logistic and methodology was specifically designed to assess the outcome in the Dominican context. Future studies will address the third and fifth items when SIMEDID is used as a monitoring tool on a population basis.\n\nThis study has some limitations that should be considered before its interpretation. Children from the sample for which these age standards were obtained receive services at INAIPI. Socio-economic vulnerability is one of the main inclusion criteria for receiving such services. This means that the results of this study can only be generalizable for this population. Future studies should consider the whole socio-economic position spectrum to obtain national norms. The data generated in the Dominican context using SIMEDID has limited comparability to data from other countries.\n\nAnother important limitation is that the experience of creating this platform in the Dominican Republic was cost-effective due to the already existing infrastructure within INAIPI, which should be considered when transferring it to other countries. The institution is the national administrator of early childhood services, which gives them access to the population of interest and trained personnel already working with children. In addition, INAIPI has the Division of Early Childhood Development Measurement, with dedicated personnel to designing, creating, supervising, and training the personnel in childhood development measurement. And finally, INAIPI has a dedicated Information and Communications Technology Department, which developed the online platform and made it to synchronize with SIGEPI, the national database for managing data from early childhood services. Further studies should conduct a cost-per-user analysis to evaluate its efficiency.\n\nEven with these limitations, these results will allow the pertinent institutions of the Dominican Republic to implement and report more accurate early childhood development indicators. They will also contribute to creating a robust monitoring system with a high-quality data collection process that allows evidence-based and timely decision-making. Furthermore, such a system will contribute to generating longitudinal data that can establish the association between childhood development and sociodemographic and psychosocial variables and determine the impact of initiatives and interventions (Richter et al., 2017), which is not sufficiently evaluated in most countries (Daelmans et al., 2017).\n\nAlthough SIMEDID was created to be integrated into the services provided by INAIPI, and the instrument so far has only been administered to INAIPI beneficiaries, efforts to make a paper version of SIMEDID are on the way under the name of TADID (Tamizaje de Desarrollo Infantil Dominicano). This will allow other institutions, clinicians, schools, and pediatricians to use this validated tool at no cost.\n\n\nConclusion\n\nIn conclusion, this study provides evidence for the validity of SIMEDID, an electronic early childhood development screening tool adapted to the Dominican context, with adequate psychometric properties and age-standardized norms for each item. Adapting this instrument to the Dominican context ensures that cultural aspects of childrearing do not overshadow developmental scores. While SIMEDID is a screening tool and not intended for diagnosis, it offers valuable insights for caregivers and stakeholders, both at a group level for program design and decision-making, as well as at the individual level to monitor each child's progress.", "appendix": "Data availability\n\nOpen Science Framework: Database for Validation of the Dominican System for Measuring Early Childhood Development. https://doi.org/10.17605/OSF.IO/KW3B8 (Sánchez-Vincitore et al., 2023b).\n\nThe project contains the following underlying data:\n\n• Codebook SIMEDID.docx (names and values of each variable).\n\n• Database – Validation study – SIMEDID.csv (database).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nOpen Science Framework: Questionnaire and item presentation for SIMEDID validation study. https://doi.org/10.17605/OSF.IO/SWN8C (Sánchez-Vincitore et al., 2023a).\n\nThis project contains the following extended data:\n\n• SIMEDID – Presentation order.xlsx. (Order of item presentation before and after data collection. Spanish and English translations)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors sincerely thank Besaida Manola Santana and Penélope Melo Ballesteros for their exemplary leadership throughout the project. We also extend our heartfelt appreciation to the dedicated team of data collectors whose hard work and commitment to quality ensured the success of this study.\n\n\nReferences\n\nAlonso MA, Valdez ME, Jiménez A, et al.: Creación del Sistema de Medición de Desarrollo Infantil Dominicano (SIMEDID). Ciencia y Educación. 2022; 6(3): 71–77. Publisher Full Text\n\nDaelmans B, Darmstadt GL, Lombardi J, et al.: Early childhood development: The foundation of sustainable development. Lancet. 2017; 389(10064): 9–11. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSánchez-Vincitore LV, Castro A: The role of sociodemographic and psychosocial variables in early childhood development: A secondary data analysis of the 2014 and 2019 Multiple Indicator Cluster Surveys in the Dominican Republic. PLOS Global Public Health. 2022; 2(7): e0000465. Publisher Full Text\n\nSánchez-Vincitore LV, Mencía-Ripley A, Veras-Díaz C, et al.: Efectos de una intervención de alfabetización en las habilidades lectoras de estudiantes de primaria: Proyecto USAID Leer. Revista Caribeña de Investigación Educativa. 2020; 4(2): 78–95. Publisher Full Text\n\nSánchez-Vincitore LV, Schaettle P, Castro A: Validation of the Malawi Developmental Assessment Tool for children in the Dominican Republic: Preliminary results. PLoS One. 2019; 14(8): e0221162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSánchez-Vincitore LV, Veras C, Mencía-Ripley A, et al.: Reading comprehension precursors: Evidence of the simple view of reading in a transparent orthography. Front. Educ. 2022; 7: 914414. Publisher Full Text\n\nSánchez-Vincitore LV, Alonso Pellerano MA, Valdez ME, et al.: Questionnaire and item presentation for SIMEDID validation study. Dataset. 2023a, February 21. Publisher Full Text\n\nSánchez-Vincitore LV, Alonso Pellerano MA, Valdez ME, et al.: Database for Validation of the Dominican System for Measuring Early Childhood Development. Dataset. 2023b, February 7. Publisher Full Text\n\nSireci SG, Sukin T: Test validity.Geisinger KF, Bracken BA, Carlson JF, et al., editors. APA handbook of testing and assessment in psychology, Vol. 1. Test theory and testing and assessment in industrial and organizational psychology. American Psychological Association; 2013; pp. 61–84. Publisher Full Text\n\nSuchdev PS, Boivin MJ, Forsyth BW, et al.: Assessment of neurodevelopment, nutrition, and inflammation from fetal life to adolescence in low-resource settings. Pediatrics. 2017; 139: S23–S37. PubMed Abstract \| Publisher Full Text\n\nUNESCO Institute of Statistics (UIS): Proportion of children aged 24-59 months who are developmentally on track in health, learning and psychosocial well-being, by sex.n.d.Reference Source\n\nUnited Nations: Transforming our world: The 2030 agenda for sustainable development (A/RES/70/1).2015. Reference Source" }	[ { "id": "166639", "date": "11 Apr 2023", "name": "Melissa J. Gladstone", "expertise": [ "Reviewer Expertise Neurodevelopmental Paediatrics and International Child Health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides information about the recent validation of a system for measuring child development in Dominican Republic. This is quite a large study which provides some initial information as to how a new tool for measuring child development in the Dominican Republic is working. This is extremely laudable and very important. Tools such as this are required to ensure that better developmental surveillance can be done across countries globally.\n\nI do have some reservations that could be addressed but presently the article is not in a fit state to publish. I outline these below.\n\nMethods:\nIt is not clear if the tool has been adapted from the mentioned MDAT tool and whether it is parent report or directly observed or a mixture of the two. It is not clear if it needs a kit to support assessment or not. It is also not clear if there are any pictures or materials that have been created to support assessment. It is also not entirely clear if it has been piloted prior to this use.\nThe authors describe how the tool was reviewed by an expert pane to provide information on construct validity but also to revise the items and their definitions. The authors provide examples of items they modified but it is not clear what the initial item was. It might be helpful to have a list of items and what was and was not changed. It also might be helpful to use the COSMIN framework to clarify the various validation procedures to ensure they were done as would be recommended and if not, this could be discussed more in the discussion part of the paper.\nDo the items have to be passed in succession? Can they move from one area to another within the app?\nMethods\nThe authors describe that a Normative sample of children were assessed. Can the authors be clearer about how this is known. It looks like the children who were assessed were those who attended Comprehensive Care Centers for Early Childhood but it is not clear how much or little these represent the general population of children in the Dominican Republic. The authors do not make it clear if any other demographics were taken into account e.g. born prematurely? Birth weight? Adversities and sociodemographic status (maternal education), health status and frequency of admissions to hospital, nutritional status etc?\nThe authors describe the use of Cronbach’s alpha internal reliability and “split half parallel reliability” but it is not clear if any reliability was done at the time of training and whether any quality control was done between examiners?\nWhat did the 6 hour session for training consist of? And could it be clearer who did this and what is needed for the future? Were there online sessions? In person sessions? Any materials that were created? Certification of assessors?\nFurthermore, could the authors provide more information about whether children were assessed with a disability and if not, how did they decide to exclude them? Can the present tool go down further to assess a child who is not performing? How did the team decide if they had a child that they felt was below the norm?\nDiscussion\nThe authors describe that age did not predict certain items. Is this because these items are not acceptable or work in the setting or is it because there were not enough children to assess these items? It seems the latter.\nThe authors conclude that the study passes three elements which include that it is reliable and valid. The authors may want to check the COSMIN criteria to make decisions on what reliability and validity they are describing as having definitively concluded on through this study. It is not clear that the authors have definitively shown that the tool can sensitively identify changes? Could they make that clearer and that they have demonstrated its use in another population who are known to have a problem through doing a Gold Standard assessment of these children.\nCould the authors provide a little more information on what they plan to do with the info from this study? At the moment, it seems like it goes into a data collection system but what and how will they create “cut offs” decided for referral? These next steps are very important and could be provided more clearly for the reader.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10946", "date": "22 Mar 2024", "name": "Laura V. Sánchez-Vincitore", "role": "Author Response", "response": "Dear Dr. Gladstone, We are thankful for your constructive feedback, which has contributed to improving our manuscript. We have addressed all your questions and comments, as explained below and included in the manuscript’s new version. 1. It is not clear if the tool has been adapted from the mentioned MDAT tool. The SIMEDID tool has been adapted from the MDAT tool and other instruments, as reported by Alonso et al. 2022, which provides more details on the process of adaptation and validation. How was this addressed in the manuscript? We described the process of adapting the instrument in more detail, summarizing Alonso et al. 2022. 2. It is not clear if the assessment tool requires direct observation or is based on parent report, or a combination of both. The SIMEDID tool combines direct observation of the child's behavior by educational agents and reports from individuals who have spent enough time with the child. That person must have observed the desired behavior. For example, in INAIPI’s case, the evaluator is the person in contact with the child, the educational agent, the community agent, and/or the parent. How was this addressed in the manuscript? We included the following statement in the methods section: “The assessment tool requires an observation of the child’s behavior, either by the evaluator, parent, or the person in charge of the child at a given time. In case the child does not show the expected behavior at the time of testing, the informant’s report is considered a valid response as long as they have witnessed the expected behavior from the child.” 3. It is not clear if the assessment tool requires a kit to support the assessment or not. Yes, there is a kit available to support the assessment with the SIMEDID tool. How was this addressed in the manuscript? We included the following statement in the methods section: “The INAIPI team created a kit to support the assessment tool (which includes props, pictures, and everyday objects). Materials are easily found in the Dominican Republic as a strategy for upscaling data collection.” 4. It is not entirely clear if there are any pictures or materials that have been created to support the assessment. Yes, the SIMEDID tool includes pictures and materials to support the assessment. How was this addressed in the manuscript? See comment #3. 5. It is not entirely clear if the assessment tool has been piloted before this use. The SIMEDID assessment tool underwent an initial pilot study involving 100 children before its use in this study. The pilot study, which is unpublished, provided valuable insights into the tool's performance and informed the validation process. How was this addressed in the manuscript? The following statement has been included “The pilot study included 100 children aged 45 days to 5 years who were receiving INAIPI services. The study confirmed the platform’s offline functionality, speed, and user-friendly interface, along with the willingness of educators and animators to participate. In addition, the instrument showed good internal consistency.” 6. It might be helpful to have a list of items and what was and was not changed. The extended data now include a list of the items in the SIMEDID tool and details on what was and was not changed during the adaptation process. How was this addressed in the manuscript? The extended data are referenced in the manuscript. 7. It might be helpful to use the COSMIN framework to clarify the various validation procedures to ensure they were done as would be recommended. A new supplementary table that follows the COSMIN framework has been included in the extended data. This table will serve as a live document to track data collection activities and ensure that the instrument continues to improve over time. How was this addressed in the manuscript? The extended data are referenced in the manuscript. 8. Do the items have to be passed in succession? Can they move from one area to another within the app? Items must be completed in sequence as they are organized based on increasing difficulty and follow an algorithm that assesses children's development from minimum to maximum levels. Evaluators can easily switch between different developmental areas by clicking on tabs with corresponding names. However, to progress to the next item, the current one must be answered. Additionally, to finish the test and submit results, all age-specific items must be filled out. Any missing responses will trigger an alert from the platform. How was this addressed in the manuscript? We included the following statement in the instruments section: “Each subtest comprises 33 items, except for language development, which has 34 items; all are arranged in ascending difficulty levels. The initial presentation of the first item is age-dependent. Once the first item on each dimension is completed, the instrument presents items in reverse until the participant successfully completes three items. Subsequently, the tool proceeds forward until the participant misses three items. This approach enables evaluators to ascertain the child's current developmental state, identifying potential delays (backward presentation) or advanced development (forward presentation). Items must be completed in sequence as they are organized based on increasing difficulty and follow an algorithm that assesses children's development from minimum to maximum levels. Evaluators can easily switch between different developmental areas by clicking on tabs with corresponding names. The study's primary aim was to determine the age range at which each item is accomplished, utilizing the Malawi MDAT norms for the initial trial. Subsequent data collection will adopt the sequence based on the findings from the present validation study.” 9. Can the authors be clearer about how they obtained the sample of children for the study and if these children are representative of the general population in the Dominican Republic? The children in this study attended CAIPIs and CAFIs in the metropolitan area. While this may not be a fully representative sample of the general population of children in the Dominican Republic, we are currently obtaining norms from a representative sample of INAIPI beneficiaries from all regions. Future data collection activities will also aim to include a sample of children outside of INAIPI to ensure greater representativeness. How was this addressed in the manuscript? We have included the following statement in the discussion section: “One limitation of this study is that the sample may not accurately reflect all children in the Dominican Republic since it comprises only those involved in INAIPI programs within the metropolitan region. During the study period, 77,000 children enrolled in INAIPI from the metropolitan region accounted for 1.23 percent of the total population of children ages 0 to 5. The limited sample size was influenced by the challenges posed by the COVID-19 pandemic, including restrictions and prevention measures.” 10. Were other demographic factors taken into account, such as birth weight, adversities, sociodemographic status, health status, frequency of admissions to hospital, nutritional status, etc.? For the the current validation study, no other demographic factors were considered since we were looking for a progression in item order. However, future data collection activities, which will involve a larger sample of children from INAIPI, will take into account variables such as birth weight, prematurity, and other health factors. How was this addressed in the manuscript? The following statement has been included in the method section. “No other demographic factors were considered for this analysis.” In addition, the following statement was included in the discussion section: “Another limitation is that the study did not account for other relevant factors in childhood development such as low birth weight and other health factors. However, future data collection activities, which will involve a larger sample of children from INAIPI, will consider variables such as birth weight, prematurity, and other health factors to determine cutoff points that better identify children at risk for developmental delay and to further understand the sensitivity of SIMEDID.” 11. The authors describe the use of Cronbach’s alpha for internal reliability and “split half parallel reliability”, but it is unclear if any reliability was done at the time of training and whether any quality control was done between examiners? During the pilot (unpublished), Cronbach’s alpha and split-half parallel reliability were conducted to ensure the reliability of the instrument. Additionally, we conducted a quality control analysis to check for consistency between examiners. This involved comparing the mean scores of participants per evaluator. How was this addressed in the manuscript? The following statement has been included in the method section: “To guarantee uniformity in assessments among evaluators, we conducted an analysis of variance (ANOVA), comparing the mean scores of participants within each age group per evaluator. The results revealed no significant differences between evaluators within each age group across all dimensions.” 12. What did the 6-hour session for training consist of? Could it be clearer who did this and what is needed for the future? Were there online sessions? In-person sessions? Any materials that were created? Certification of assessors? The 6-hour training session, conducted by INAIPI personnel, encompassed practical and theoretical components based on a module designed by experts in training and child development. The training, mainly conducted face-to-face, included virtual follow-up by the Child Development Evaluation Division. While there was no initial certification process for assessors, facilitators of the training received certification as official SIMEDID facilitators. The module covered essential topics such as the concept of child development, the Dominican Child Development Measurement System (SIMEDID), its structure, and dimensions. It explained who applies SIMEDID in INAIPI's service modalities and provided guidelines on application rules. Evaluators learned the assessment process in homes, including guidance for parents, the periodicity of the system, and the specifications of the tool. The practical section involved the hands-on use of evaluation kit materials in simulated assessments, emphasizing adherence to instructions for standardized data. The training concluded with a presentation on the app's functionality, using visual aids and videos to reinforce the covered content. How was this addressed in the manuscript? The following statement has been included in the method section: “A total of 40 evaluators received a six-hour training session and conducted two practice evaluations. The training session comprised both theoretical and practical components. The training, facilitated by experts in training and child development, covered essential topics such as the concept of child development and SIMEDID. It included virtual follow-ups by the Child Development Evaluation Division and certification for facilitators. The module provided insights into SIMEDID's structure, dimensions, and its application in INAIPI's service modalities. Evaluators learned about assessment processes in homes, including guidance for parents, system periodicity, and tool specifications. The practical section involved hands-on use of evaluation kit materials in simulated assessments, emphasizing adherence to standardized data collection. The training concluded with a presentation on the SIMEDID app's functionality, utilizing visual aids and videos to reinforce the covered content. At the end of the training, evaluators were certified as official SIMEDID evaluators.” 13. Furthermore, could the authors provide more information about whether children were assessed with a disability, and if not, how did they decide to exclude them? Can the present tool go down further to assess a child who is not performing? How did the team decide if they had a child that they felt was below the norm? For this specific data collection activity, we did not exclude children with disabilities because such data were not available. However, for the normalization study, in which we are setting the cutoff points to detect developmental delays, we have a mechanism to exclude them from the analysis, not from the evaluation. This way, we will be able to determine SIMEDID's sensitivity to disability. The tool is designed to assess children from ages 0 to 5, and there are items that can be used to assess children who are not performing at their age level. How was this addressed in the manuscript? The following statement has been included in the discussion “Another limitation is that the study did not account for other relevant factors in childhood development, such as low birth weight and other health factors or disabilities. However, future data collection activities, which will involve a larger sample of children from INAIPI, will consider variables such as birth weight, prematurity, and other health factors to determine cutoff points that better identify children at risk for developmental delays and to further understand the sensitivity of SIMEDID.” 14. The authors describe that age did not predict certain items. Is this because these items are not acceptable or work in the setting, or is it because there were not enough children to assess these items? It seems to be the latter. We did not have enough children to assess these items, and we are currently working on obtaining a sample of children within the age range to evaluate these items. We are meeting with other governmental institutions that work with infants younger than 45 days to expand the age range of the study. How was this addressed in the manuscript? The following statement has been included in the discussion section: “We could not validate initial items of SIMEDID given that INAIPI does not provide services to infants younger than 45 days, given that mothers are on maternity leave during that period. However, the research team is meeting other governmental institutions that work with infants that young to expand the age range of the study. “ 15. It is not clear that the authors have definitively shown that the tool can sensitively identify changes. Could they make that clearer and that they have demonstrated its use in another population who are known to have a problem through doing a Gold Standard assessment of these children? We have yet to demonstrate the tool's ability to sensitively identify changes, as this is the first large-scale data collection using this instrument. Our current focus is to establish the reliability and validity of the SIMEDID tool. However, we are designing a study to compare the SIMEDID tool with another widely used instrument by UNICEF that is applied during household surveys. How was this addressed in the manuscript? The following statement has been included in the discussion section: “We have yet to demonstrate the tool's ability to sensitively identify changes, as this is the first large-scale data collection using this instrument. However, since SIMEDID is already part of the monitoring strategy, with constant data collection, it would be relatively simple to demonstrate its sensitivity to changes.” 16. Could the authors provide a little more information on what they plan to do with the information from this study? At the moment, it seems like it goes into a data collection system, but what and how will they create “cut-offs” decided for referral? These next steps are very important and could be provided more clearly for the reader. The information collected from this study will be used to establish cut-off scores for referral to early intervention services. We will use statistical methods to determine the optimal cut-offs. A subsequent standardization study allowed us to establish correction parameters and normative scores, with which it will be possible to establish when alerts occur in the development of a child. How was this addressed in the manuscript? This was addressed in the Discussion section: “These results will allow the pertinent institutions of the Dominican Republic to implement and report more accurate early childhood development indicators. Still, they will also contribute to creating a robust monitoring system with a high-quality data collection process that allows evidence-based and timely decision-making. Furthermore, such a system will contribute to generating longitudinal data that establish the association of childhood development with sociodemographic and psychosocial variables and determine the impact of initiatives and interventions (Richter et al., 2017), which is not sufficiently evaluated in most countries (Daelmans et al., 2017).”" } ] }, { "id": "194708", "date": "31 Aug 2023", "name": "Mphelekedzeni Caroline Mulaudzi", "expertise": [ "Reviewer Expertise Child health and Early Childhood 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\nI commend the researchers for doing research in the area of instruments for early child development in low/middle-income countries.\nLiterature review\nThe researchers should have shared more on their initial work of adapting MDAT to the Dominican instrument and how it differs.\nMethodology and statistical analysis\nThere is no description of the sampling technique used and no information on how they determined the population was normal\nAs far as whether the correct statistical analysis was done to meet the aims of the study.\nThere is clear information and a report on reliability but not validity.\nResults\nFigures should be labeled appropriately with an explanation of the range of colors' meanings.\nThe Y axis of the item should not only mention the domain but also what analysis\nDiscussion\nInterpretation of the result is poor. The researchers gave results of the Chronbach alpha and split-half correlation but did not elaborate on the relevance\nThey did not comment on how the instrument should be used in the future, especially with items that had a standard deviation that was higher than 3 and even some items that had a standard deviation greater or equal to 6.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10947", "date": "22 Mar 2024", "name": "Laura V. Sánchez-Vincitore", "role": "Author Response", "response": "Dear Dr. Mulaudzi, We thank you for your comments and questions, which have helped us improve the quality of our manuscript. We address your questions below and in the manuscript: 1. The researchers should have shared more on their initial work of adapting MDAT to the Dominican instrument and how it differs. How was this addressed in the manuscript? We have described the process of adapting the instrument in more detail, summarizing Alonso et al. 2022. 2. There is no description of the sampling technique used and no information on how they determined the population was normal How was this addressed in the manuscript? We used an intentional sampling with an N=948. We have clarified that the sample came from INAIPI beneficiaries who lived in the capital city of Santo Domingo and provided additional details. 3. There is clear information and a report on reliability but not validity. How was this addressed in the manuscript? We have included additional details on the creation of SIMEDID that includes more validity information, such as collaboration with experts, content analysis, and ongoing data collection activities. As SIMEDID transitions into the national surveillance system, continued data collection will contribute further evidence of its validity. 4. Figures should be labeled appropriately with an explanation of the range of colors' meanings. How was this addressed in the manuscript? We have added a note to each figure explaining the color range. 5. The Y axis of the item should not only mention the domain but also what analysis How was this addressed in the manuscript? The Y represents the distribution of the probability of success, as explained by the color ranges. We have added this information to each figure. 6. Interpretation of the result is poor. The researchers gave results of the Chronbach alpha and split-half correlation but did not elaborate on the relevance How was this addressed in the manuscript? We have expanded the conclusion with an additional interpretation of the results. 7. They did not comment on how the instrument should be used in the future, especially with items that had a standard deviation that was higher than 3 and even some items that had a standard deviation greater or equal to 6. How was this addressed in the manuscript? In the future, SIMEDID will identify children at risk for developmental delays. The current study aimed to enhance item presentation order. The observed large standard deviations may result from presenting items in an order that doesn't fully adapt to the Dominican context. The upcoming standardization study, which includes a more fitting presentation order of items obtained in the present validation study, seeks to establish national norms by calculating cut-off points based on standard deviations once the presentation order is appropriately configured. We have explained these plans in the discussion." } ] } ]	1	https://f1000research.com/articles/12-279
https://f1000research.com/articles/12-1337/v2	29 Jan 24	{ "type": "Research Article", "title": "On solving system of differential-algebraic equations using adomian decomposition method", "authors": [ "Srinivasarao Thota", "Shanmugasundaram P", "Srinivasarao Thota" ], "abstract": "Background In this paper, we focus on an efficient and easy method for solving the given system of differential-algebraic equations (DAEs) of second order.\n\nMethods The approximate solutions are computed rapidly and efficiently with the help of a semi-analytical method known as Adomian decomposition method (ADM). The logic of this method is simple and straightforward to understand.\n\nResults To demonstrate the proposed method, we presented several examples and the computations are compared with the exact solutions to show the efficient. One can employ this logic to different mathematical software tools such as Maple, SCILab, Mathematica, NCAlgebra, Matlab etc. for the problems in real life applications.\n\nConclusions In this paper, we offered a method for solving the given system of secondorder nonlinear DAEs with aid of the ADM. We shown that the proposed method is simple and efficient, also one can obtain the approximate solutions quickly using this method. A couple of examples are discussed for illustrating this method and graphical and mathematical assessments are discussed with the analytical solutions of the given problems.", "keywords": [ "Differential-algebraic equations", "Adomian decomposition method", "Approximate solutions." ], "content": "Introduction\n\nThe applications of system of differential-algebraic equations (DAEs) occur in many branches of engineering, scientific and real life applications. For example, these equations arise in circuit analysis, electrical networks, computer aided design (CAD), optimal control, real-time simulation of mechanical (multi-body) systems, incompressible fluids dynamics, power system and chemical process simulations. DAEs are a combination of algebraic equations and differential operations, and many mathematical models in different fields are expressed in terms of DAEs. The system of DAEs is a combination of algebraic and differential equations. In the recent years, several algorithms or methods are introduced by various researchers, engineers and scientists to solve the linear/nonlinear system of DAEs and many of them are focused on the numerical solution.7,13,14 In the literature, there are many numerical methods available and these are developed using various existing classical methods. For example, in the literature, there are numerical methods with help of Padé approximation method,4,5 there are methods created using implicit Runge-Kutta methods,36 also there are methods developed using back difference formula (BDF)3,13,35 and etc. Many existing methods are working for low indexed problems or functions. However, using these methods, many real life applications can be solved. There are many other algorithms or methods for solving DAEs and also for differential equations available in the literature.20–34 In this paper, we propose a general numerical method to solve the second-order system of DAEs using Adomian decomposition method (ADM). There are some general approaches methods available in the literature,18,19,37,38 and these are developed for solving the first order DAEs.\n\nThe main aim of this manuscript is to develop a method that gives us quick approximate solutions of a given system of second order DAEs. In order to develop the proposed method, we use a powerful technique, namely ADM, to get the solution of DAEs system. Since 1980, the ADM has been used widely to solve the nonlinear or linear problems in various fields. For example, recently, ADM is widely used as a straightforward powerful tool for solving a large class of nonlinear equations1,2,8–12,15 such as functional equations, integro-differential equations (IDEs), partial differential equations (PDEs), algebraic equations, differential equations (DEs), differential-delay equations and different kind of equations arise in chemical reactions, physics and biology. We use the ADM to obtain a rapid approximation solution of a given DAEs systems.\n\nThis paper is planned as follows: in the next section we recall the ADM to solve the ODEs. The method proposed in this paper for DAEs systems is presented in the following section. Then a number of numerical examples are presented to illustrate the method, followed by concluding remarks.\n\n\nAdomian Decomposition Method: An Overview\n\nIn this section, we recall ADM briefly to solve ODEs. More details about the ADM can be found in.2,9,15,17 Consider the nonlinear DE of the following type\n\nWe can solve (1) for y by applying the inverse operator L−1. Indeed, we have the following solution by solving (1) for Ly and then apply the inverse operator L−1 on to both sides,\n\nTo apply the ADM to (3), let y be the solution of (1), and it can be expressed in the form of infinite series as follows,\n\nNow, choose y0 as\n\nOn comparing the general terms of (7), we obtain the following equation for the ADM\n\nWe have y0 from (6), and using (8) we can generate the components yn for an approximate solution. Further, we can obtain the exact solution of (1) if the series (4) converges. The K-order approximation solution is obtained as\n\nThe next section presents a method for DAEs systems using the ADM.\n\n\nProposed Method using ADM\n\nConsider a system of second-order DEs as follows\n\nWe can rewrite the system (10), as follows:\n\nFrom replacement lemma,16 we have the following equation. The replacement lemma helps us to convert the double integral into a single integral as given below,\n\nThus, (12) can be expressed in terms of integral operator I as follows\n\nUsing the inverse operator on (11), we get\n\nApplying ADM, we have the solution of (13) in the series sum,\n\nfrom (8) we define, for n=0,1,…,\n\nSince fi,0 are known, we can use fi,n+1 to generate the approximate solution components.\n\n\nNumerical Examples\n\nExample 1. Let us consider the following system of second order DAEs with initial conditions to illustrate the proposed method.39\n\nIn order to apply the proposed method, we rewrite the given system (18) as follows\n\nOn simplifying above equations, we have y1″=2y1+x+1ex. Following procedure as given in (13), we get\n\nUse the alternate algorithm to find the Adomian polynomials as given in,6,10–12 the Adomian method is as following:\n\nWe have iterations (approximate solutions components) from above equations as follows\n\nNow we have the approximate solution after three steps\n\nAfter nine steps, we have the solution\n\nGraphical assessment of the analytic solution with the approximate solution after three steps is visualized in Figure 1 and the comparison of the exact solution with approximate solution after nine steps is shown in Figure 2. From these figures, we can observe that the approximate solutions are near to the analytic solution. A greater number of steps gives us a more accurate solution (the graphs are drawn using Maple 16.0).\n\nNumerical results of the exact solution, approximate solution yapx3 after three steps, approximate solution yapx9 after nine steps and absolute error are given in Table 1. From the numerical values in Table 1, one can observe that the solution yapx9 is closer to the exact solution y1. To get more appropriate solution of the given system, we increase the number of iterations.\n\nExample 2. Consider a DAEs system of second order.39\n\nFollowing the procedure of the proposed method, similar to Example 1, we get\n\nUsing the alternate algorithm for computing the Adomian polynomials, we have\n\nNow, we can get iterations from above equations as follows\n\nAfter five steps, we have the solution\n\nIn Figure 3 and Figure 4, we show the graphical comparisons of the exact solutions y1x,y2x with the approximate solution after five steps respectively. From the graphs in Figure 3 and Figure 4, one can observe that the approximate solutions are very close to the exact solution. Higher number of iterations give us more accurate solution (one can use Microsoft Excel to draw the graphs).\n\nIn Table 2 and Table 3, mathematical results of the analytical solution and approximate solutions after five steps y1,apx5,y2,apx5 with absolute errors are given respectively. From these numerical results, one can observe that the approximate solutions y1,apx5 and y2,apx5 are closer to the exact solution y1 and y2 respectively. For more appropriate solution of the given system, we increase the number of iterations.\n\n\nConclusions\n\nIn this paper, we offered/presented a numerical method for solving the given system of second-order nonlinear DAEs with aid of the ADM. We illustrated and shown that the proposed method is simple and efficient, also one can obtain the approximate solutions quickly using this method. Logic of the method in this paper is straightforward and simple.", "appendix": "Data availability\n\nMendeley data: On solving system of DAEs using ADM https://doi.org/10.17632/r89zy3y657.1. 39\n\nThis project contains the following underlying data:\n\n• Paper_Example_1.mw\n\n• Paper_Example_2.mw\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nThe author is thankful to the reviewers and editor for providing valuable inputs to improve the quality and present format of this manuscript.\n\n\nReferences\n\nWazwaz AM: A new approach to the nonlinear advection problem: An application of the decomposition technique. Appl. Math. Comput. 1995; 72: 175–181. Publisher Full Text\n\nBenhammouda B: A novel technique to solve nonlinear higher-index Hessenberg differential-algebraic equations by Adomian decomposition method. Springerplus. 2016; 5: 590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGear CW, Petzold LR: ODE systems for the solution of differential algebraic systems. SIAM J. Numer. Anal. 1984; 21: 716–728. Publisher Full Text\n\nÇelik E, Karaduman E, Bayram M: Numerical Method to Solve Chemical Differential-Algebraic Equations. Int. J. Quantum Chem. 2002; 89: 447–451. Publisher Full Text\n\nÇelik E, Bayram M: On the numerical solution of differential-algebraic equations by Padé series. Appl. Math. Comput. 2003; 137: 151–160. Publisher Full Text\n\nÇelik E, Bayram M, Yeloğlu T: Solution of Differential-Algebraic Equations (DAEs) by Adomian Decomposition Method. International Journal Pure & Applied Mathematical Sciences. 2006; 3(1): 93–100.\n\nHairer E, Norsett SP, Wanner G: Solving Ordinary Differential Equations II. Stiff and Differential-Algebraic Problems. New York: Springer; 1992. 978-3-642-05221-7.\n\nAomian G: Nonlinear Stochastic Systems Theory and Applications to Physics. Dordrecht/Norwell, MA: Kluwer Acaemic; 1989.\n\nAdomian G, Rach R: On the solution of algebraic equations by the decomposition method. J. Math. Anal. Appl. 1985; 105(1): 141–166. Publisher Full Text\n\nDuan JS: Recurrence triangle for Adomian polynomials. Appl. Math. Comput. 2010; 216: 1235–1241. Publisher Full Text\n\nDuan JS: An efficient algorithm for the multivariable Adomian polynomials. Appl. Math. Comput. 2010; 217: 2456–2467. Publisher Full Text\n\nDuan JS: Convenient analytic recurrence algorithms for the Adomian polynomials. Appl. Math. Comput. 2011; 217: 6337–6348. Publisher Full Text\n\nBrenan KE, Campbell SL, Petzold LR: Numerical Solution of Initial Value Problems in Differential-Algebraic Equations. SIAM Philadelphia. 1989; 26: 976–996. 978-0-89871-353-4. Publisher Full Text\n\nPetzold LR: Numerical Solution of Differential-Algebraic Equations. Advances in Numerical Analysis IV. 1995.\n\nAlmazmumy M, Hendi FA, Bakodah HO, et al.: Recent modifications of Adomian decomposition method for initial value problem in ordinary differential equations. Am. J. Comput. Math. 2012; 02: 228–234. Publisher Full Text\n\nPeter J: Collins: Differential and integral equations. Oxford University Press; 2006.\n\nRamana PV, Raghu Prasad BK: Modified Adomian decomposition method for Van der Pol equations. Int. J. Non Linear Mech. 2014; 65: 121–132. Publisher Full Text\n\nCampbell SL: A Computational method for general higher index singular systems of differential equations. IMACS Trans. Sci. Comput. 1989; 89: 555–560.\n\nCampbell SL, Moore E, Zhong Y: Utilization of automatic differentiation in control algorithms. IEEE Trans. Automat. Control. 1994; 39: 1047–1052. Publisher Full Text\n\nThota S, Kumar SD: Solving system of higher-order linear differential equations on the level of operators. International journal of pure and applied mathematics. 2016; 106(1): 11–21. Publisher Full Text\n\nThota S, Kumar SD: On a mixed interpolation with integral conditions at arbitrary nodes. Cogent Mathematics. 2016; 3(1): 1–10. Publisher Full Text\n\nThota S, Kumar SD: Symbolic algorithm for a system of differential-algebraic equations. Kyungpook Mathematical Journal. 2016; 56(4): 1141–1160. Publisher Full Text\n\nThota S, Kumar SD: A new method for general solution of system of higher-order linear differential equations. International Conference on Inter Disciplinary Research in Engineering and Technology. 2015; 1: 240–243.\n\nThota S, Kumar SD: Symbolic method for polynomial interpolation with Stieltjes conditions. International Conference on Frontiers in Mathematics. 2015; 225–228.\n\nThota S, Rosenkranz M, Kumar SD: Solving systems of linear differential equations over integro-differential algebras. International Conference on Applications of Computer Algebra, June 24–29, 2012, Sofia, Bulgaria.\n\nThota S: A Study on Symbolic Algorithms for Solving Ordinary and Partial Linear Differential Equations, Ph.D. thesis.2017.\n\nThota S, Kumar SD: Maple Implementation of Symbolic Methods for Initial Value Problems. Research for Resurgence-An Edited Multidisciplinary Research Book. 2017; I: 240–243.\n\nThota S: On a Symbolic Method for Fully Inhomogeneous Boundary Value Problems. Kyungpook Mathematical Journal. 2019; 59(1): 13–22.\n\nThota S: On A New Symbolic Method for Initial Value Problems for Systems of Higher-order Linear Differential Equations, International Journal of. Mathematical Models and Methods in Applied Sciences. 2018; 12: 194–202.\n\nThota S: A Symbolic Algorithm for Polynomial Interpolation with Integral Conditions. Applied Mathematics & Information Sciences. 2018; 12(5): 995–1001. Publisher Full Text\n\nThota S: Initial value problems for system of differential-algebraic equations in Maple. BMC. Res. Notes. 2018; 11: 651. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThota S: On A Symbolic Method for Error Estimation of a Mixed Interpolation. Kyungpook Mathematical Journal. 2018; 58(3): 453–462.\n\nThota S: A Symbolic Algorithm for Polynomial Interpolation with Stieltjes Conditions in Maple. Proceedings of the Institute of Applied mathematics. 2019; 8(2): 112–120.\n\nThota S: On A New Symbolic Method for Solving Two-point Boundary Value Problems with Variable Coefficients, International Journal of. Math. Comput. Simul. 2019; 13: 160–164.\n\nAscher UM: On symmetric schemes and differential-algebraic equations. SIAM J. Sci. Stat. Comput. 1989; 10: 937–949. Publisher Full Text\n\nAscher UM, Petzold LR: Projected implicit Runge Kutta methods for differential-algebraic equations. SIAM J. Numer. Anal. 1991; 28: 1097–1120. Publisher Full Text\n\nAscher UM, Lin P: Sequential regularization methods for higher index differential-algebraic equations with constant singularities: the linear index-2 case. SIAM J. Numer. Anal. 1996; 33: 1921–1940. Publisher Full Text\n\nAscher UM, Lin P: Sequential regularization methods for non-linear higher index differential-algebraic equations. SIAM J. Sci. Comput. 1997; 18: 160–181. Publisher Full Text\n\nPalanisamy S, Thota S: On solving system of DAEs using ADM. [Data]. Mendeley Data. 2023; V1. Publisher Full Text" }	[ { "id": "243837", "date": "05 Mar 2024", "name": "Mohammad Faisal Khan", "expertise": [ "Reviewer Expertise Optimisation", "Special functions" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors focused on an efficient and easy method for solving the given system of differential-algebraic equations (DAEs) of second order. The method includes computing the approximate solutions with rapidly and efficiently with the help of a semi-analytical method known as Adomian decomposition method (ADM). The logic of this method is simple and straightforward to understand. To demonstrate the proposed method, they presented several examples and the computations are compared with the exact solutions to show the efficient. They also claimed that one can employ this logic to different mathematical software tools such as Maple, SCILab, Mathematica, NCAlgebra, Matlab etc. for the problems in real life applications.\nGeneral Comments: The paper is organized in well-order and interesting to the general readers. The main text is to propose a method for DAEs using ADM which produces quick approximate solution which is novel as far as my knowledge. This paper can be accepted. Minor comments:\nIn the paper, authors are focused on only second order DAEs. Is this technique applicable for higher order DAEs? In the integral operator I, authors have taken lower value of the integral zero. What is the reason for fixed lower value zero? If the lower value is not fixed, say c (constant), that are changes occur in the process?\n\nIs the work 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": "11206", "date": "13 Apr 2024", "name": "Shanmugasundaram P", "role": "Author Response", "response": "The authors of this paper are very thankful to the reviewer for giving valuable comments on this paper. The response to the minor comments are as follows: 1. In the paper, authors are focused on only second order DAEs. Is this technique applicable for higher order DAEs? Response: The proposed technique in this paper is applicable to the higher-order DAEs. For the sake of simplicity, we have taken 2-order DAEs. 2. In the integral operator I, authors have taken lower value of the integral zero. What is the reason for a fixed lower value of zero? If the lower value is not fixed, say c (constant), are changes occur in the process? Response: The lower value of the integral can be any number. Based on this, we compute integrals in the proposed algorithm. For simplicity, we took the lower value to be zero." } ] }, { "id": "243839", "date": "14 May 2024", "name": "Arun Prasath GM", "expertise": [ "Reviewer Expertise Inventory Model", "Parking lot problems", "Analytic Hierarchy Process", "Optimization" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 focused on an efficient and easy method to solve the second-order systems of differential-algebraic equations (DAEs). They computed the approximate solutions with the help of a semi-analytical method known as “Adomian decomposition method” (ADM). They claimed that the logic of the proposed method is simple and straightforward to understand. Authors presented few examples to illustrate the method. The paper is attractive and planned fine. The key of this paper is to solve the given system of DAEs with the help of ADM to give speedy approximate solution. The design of the work in this paper is constructive and novel. I recommend, this paper can be acceptable. After review, the authors can include the following comments. Comments: This paper solves second order DAEs. Can we apply the same logic for higher order DAEs, if so; put it as NOTE point in the conclusion section. Integral operator is fixed with lower-limit as zero or it can be any constant? Give explanation about 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?\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": "11579", "date": "29 Jun 2024", "name": "Shanmugasundaram P", "role": "Author Response", "response": "* This paper solves second order DAEs. Can we apply the same logic for higher order DAEs, if so; put it as NOTE point in the conclusion section. Response: The proposed technique in this paper is applicable to the higher-order DAEs. For the sake of simplicity, we have taken 2-order DAEs. This information is given conclusion section. * Integral operator is fixed with lower-limit as zero or it can be any constant? Give explanation about it. Response: The lower value of the integral can be any number. Based on this, we compute integrals in the proposed algorithm. For simplicity, we took the lower value to be zero. There is no restriction on lower bound of the integral." } ] } ]	2	https://f1000research.com/articles/12-1337
https://f1000research.com/articles/12-141/v1	07 Feb 23	{ "type": "Research Article", "title": "Exploring potential strategies to enhance memory and cognition in aging mice", "authors": [ "Shreevatsa Bhat M", "Ramesh Babu M G", "Anandh Dhanushkodi", "Prof Kiranmai S Rai", "Shreevatsa Bhat M", "Ramesh Babu M G", "Anandh Dhanushkodi" ], "abstract": "Background: Aging population is rapidly expanding worldwide, and age-related cognitive impairments prove detrimental for achieving a better productive and quality of life. Lack of effective therapies for age-related cognitive impairment focuses attention on developing preventive strategies, such as nutritional interventions, cell therapies and environmental manipulations. The objective of the present study was to explore the comparative benefits of potential memory-enhancing strategies like supplementation of choline (Ch) and docosahexaenoic acid (DHA) or administration of human embryonic kidney stem cell conditioned media (HEK-CM) or exposure to environmental enrichment (EE), that attenuates cognitive impairments in aging mice. Methods: 12-month-old CF1 male mice were subdivided [n=6/group] into normal aging control (NAC), saline vehicle control (SVC), Ch-DHA, EE, heat-inactivated HEK-CM (HIHEK-CM) and HEK-CM mice. Spatial working and reference memory were assessed using an eight-arm radial maze test and cognition using a novel object recognition test (NORT). Results: Spatial memory and cognition were decreased in normal aging mice. Aged mice exposed to dietary Ch-DHA or HEK-CM showed significant enhancement in spatial learning tasks, memory and cognition compared to the same in age-matched NAC mice. Ch-DHA and HEK-CM treated mice committed significantly lesser reference memory errors and attained a higher percentage of correct choices in spatial learning and memory tasks. Moreover, on testing for cognition in NORT, significantly higher number of visits to the novel object was observed in Ch-DHA supplemented and HEK-CM administered aging mice whereas HEK-CM and EE mice groups showed significantly greater number of visits to familiar object, when compared to same in age-matched NAC and HIHEK-CM groups, respectively. Conclusion: Supplementation of Ch-DHA and HEK-CM treatment strategies have a higher potential [~ 20—30%] for enhancing spatial learning, memory and cognition in normal aged mice, whereas exposure to EE seems to enhance only their short-term memory.", "keywords": [ "Aging", "cognition", "spatial memory", "choline", "and DHA", "enriched environment", "and HEK cell-conditioned media" ], "content": "Introduction\n\nNormal aging is associated with a decline or functional deficits in multiple physiological domains, including cognitive functions. Cognitive abilities crucially determine successful/healthy aging and their impairment during aging specifically affects the quality of life and reduces productivity in elderly individuals. The growing burden of cognitive loss in rapidly increasing aging populations is shouldered not only by elderly patients and their family members but also by country/worldwide health care organizations. Although some mental functions such as general knowledge, numerical and verbal abilities are less affected during aging, other mental abilities like short-term memory, working memory, reasoning, processing speed, and executive functions weaken from middle age onwards or before.1,2 Age-related learning and memory impairments are due to neuronal deficits that are associated with hippocampal damage. Age-related changes in cognitive ability are also attributed to altered hippocampal function.3 All cognitive functions are not equally affected by aging, and it is also reported that functions like delayed recall of verbal information,4 short-term recall, working memory5 and spatial memory6 decline with aging. Lack of effective disease-modifying therapies for age-related cognitive impairment seeks increasing attention on preventive strategies, such as nutritional interventions, cell therapies and environmental manipulations. Nutritional intervention has a major role in preventing/delaying deficits observed on neuronal function in aging such as cognitive ability and behavior. Important nutrients like choline (Ch) and docosahexaenoic acid (DHA) are required for normal brain function in humans and animals. Ch is derived from diet and by de novo synthesis in liver.7 It is essential for membrane structural integrity, methyl group metabolism and neurotransmitter synthesis.8 In rodents, maternal intake of Ch during gestation increases nerve cell proliferation and decreases apoptosis of fetal hippocampal neural progenitor cells,9 enhances long-term potentiation in the hippocampus, and improves the auditory and visuospatial memory throughout their life. In adult rats, Ch supplementation improves cognitive abilities10 and enhances the temporal memory.11 DHA supplementation increases the level of synaptic proteins and membrane phospholipid in the hippocampal neurons12 and enhances cognitive activities.13 Several studies report that DHA deficiency may have an increased risk of developing cognitive disorders such as dementia and Alzheimer’s disease.14–16 Cellular senescence is the restricted proliferation of cells, which contributes to aging of tissue and organism.17 Further, currently, cell and cell-derived therapies are emerging as potential modes for treating various brain diseases/neurological disorders. Significant findings indicate that stem cell-based therapies potentiate functional recovery of neurological injury or neural disorders in animals.18–21 Embryonic stem cells provide great potential for cell-based therapy in the field of regenerative medicine.22,23 Alternatively, embryonic stem cells derived conditioned medium has been shown to be beneficial mainly through their chemical factors, for cell proliferation and tissue regeneration.24,25 Moreover, studies also show that environmental enrichment (EE) impacts the brain positively by exposure to varying and stimulating physical and social surroundings. Increased rates of synaptic formation in the brain and improved activity have been observed when exposed to richer and stimulating surroundings. Studies in aged rats show that EE exposure results in permanent neuronal plasticity in the hippocampus and prevents age-associated impairments of spatial learning.26 Daily exposure to EE in old rodents revealed its stimulating effect on neurogenesis possibly by increasing the survival rate of new neurons till their maturity.27,28 Long-term exposure to EE in aged mice results in overall enhancement in cognitive ability. This proposes that long-term EE could provide cognitive stabilization.29\n\nAlthough these studies indicate the potential of each of the aforementioned therapies for improving cognitive functions and memory, no studies have explored comparing these beneficial strategies to identify the best in preventing normal age-associated decline in cognition and memory functions. Thus, the objective of the present study was to explore and identify the comparative potential memory-enhancing benefits of supplementation of Ch-DHA or administration of human embryonic kidney stem cell conditioned media (HEK-CM) or exposure to EE, in preventing normal age-associated decline in cognition and memory functions of normal aging mice.\n\n\nMethods\n\nApproval for the study was obtained from Institutional Animal Care and Use Committee [No. IAEC/KMC/107/2014], MAHE, Manipal. All experiments were carried out in accordance with guidelines provided by the IAEC and CPCSEA, Government of India. Total number of 66 middle-aged (12 to 15-months-old) CF1 male mice housed in Central animal house research facility, MAHE, Manipal, were used for the study. CF1 mouse species is ideal for general multipurpose model, safety and efficacy testing and infectious disease model, and also have been used in various neuroscience research studies worldwide. During 44 days of experimental period [09/03/2019 to 22/04/2019], 4 to 6 mice were housed and bedded in standard polypropylene cages containing paddy husk as bedding material that was replaced every two days. Standard lab conditions, with temperatures ranging between 23±20C, humidity (50±5%), and 12 hrs light-dark cycle were maintained for all the mice. Pellet feed with Ch content of 1 mg/kg, procured from VRK Nutritional Solutions [VRK’s “Scientist’s Choice” Laboratory Animal Diets, Pune] and water ad libitum were freely accessible to these mice. As expected, 10 to 15% of mortality was observed in the mice due to ageing.\n\nMiddle-aged male CF1 mice were randomly grouped [n=6/group] into normal aging control (NAC), saline vehicle control (SVC), Ch-DHA, HEK-CM, heat inactivated HEK-CM (HIHEK-CM) and EE groups. The NAC mice group remained undisturbed in the home cage throughout the 30 days interventional period of the experiment. The SVC group mice (included only for eight arm radial maze test) were fed equi-volume of saline for 30 days, Ch-DHA group mice were fed 45mg/kg body weight of Ch and 300mg/kg body weight of DHA for 30 days, HEK-CM group of mice were injected 100 μL of HEK-CM and HIHEK-CM group mice were injected 100 μL of heat inactivated HEK-CM through (tail vein) intravenous injections for 5 days in a 30-day period and EE group of mice were exposed to enriched environment for 30 days.\n\nCryopreserved 293T cells, which are derived from human embryonic kidney (HEK), were rapidly thawed at 37°C. HEK cells from a cryovial which contained dimethyl sulfoxide were transferred to a centrifuge tube (15 mL) and 5 mL of HEK media (Dulbecco’s modified Eagle’s medium-high glucose) was added along with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids and 1% of 200 mmol/L L-glutamate and centrifuged at 1800 rpm for 5 min. After discarding the supernatant, the pellets were re-suspended in 3 mL of HEK media and the cell suspension was then transferred into 25-cm2 culture flasks to incubate with 5% CO2 at 37°C for 24 hrs. The HEK cells were grown until they attained a confluency of 70 to 80%. HEK-CM, the conditioned media in which the HEK cells were grown was stored at -80°C for further use.\n\nThe eight-arm radial maze is an elevated plexiform maze placed 80 cm above the floor. It consists of an octagonal central platform from which equally spaced eight arms (each arm is 42 cm long, 11.4 cm high, and 11.4 cm wide) radiate and has a video monitor attached to a computer. After 30 days of treatment, mice were semi-starved for 2 days to reduce their body weight up to 85% and then subjected to eight arm radial maze test for 10 days, which consisted of 2 days of habituation phase followed by 4 days each of acquisition and retention phases.\n\nDuring the habituation/orientation phase, all the eight arms were baited with food pellets and mice were allowed to orient and get habituated to the maze during two trials per day carried out for 2 days.\n\nAcquisition phase of a spatial task is conducted following habituation in the radial maze. During this phase, bait of food pellets was placed only in four arms. Prior to each trial and each session, the maze was wiped with 50% ethanol to avoid any olfactory cues. The mice were placed in the centre of the maze and allowed to explore the maze freely. The mice were trained to take the food from baited arm without making a re-entry into the already visited arm. The trial was ended when the mice have taken the food from all four baited arms or after 5 min if mice did not visit the baited arms. During the trial, the animal’s performance was monitored and the number of entries into the arms were noted. Each mouse was given two trials per day for 4 days. The performance of the animal was scored by calculating the percentage of correct responses (a correct entry is the animal’s number of first visits to the baited arms) divided by the total number of entries made by the animal. Re-entries into previously visited baited arms were counted as working memory errors and entries into the unbaited arms were taken as reference memory errors.\n\nRetention phase of spatial task in the radial maze: Subsequent to learning/acquisition phase, mice were retained in their individual cages for 4 days without any training. In order to assess the retention of the learned/acquired task, the performance of mice in the radial maze was again assessed for a single trial on the 4th day. The experimental protocol remained same as that for the acquisition test.\n\nThe cognitive functions of mice were assessed by using NORT. Rodents have an innate tendency to visit the novel objects repeatedly than to the familiar objects. Two round plastic container boxes filled with sand were used as familiar objects, and a wooden cube box with different shape and color was used as novel object. Number of visits to a novel object gives a behavioral measure of retention of memory and discriminating ability between familiar and novel objects, thus revealing their cognitive and hippocampal function. The test was done as a new one-trial NORT method. In the habituation phase, animals were allowed to orient and habituate in an open field for 30 min. Mice were retained in their home-cage for 5 min after the 30 min habituation phase. Subsequently, during the acquisition phase, mice were exposed to two identical objects and allowed to explore and get familiarized with the objects for 5 min. Subsequently, mice were placed back in their respective cages. During the test phase [one day after the acquisition phase], mice were again exposed to the open-field arena with one familiar object and a novel object, for 5 min. The test phase was video monitored, and the number of visits by each animal to the familiar and novel objects were marked manually from the monitor and then counted. Only the active contact of animal with its nose, mouth or paws to the objects was considered as the number of visits. The accidental touch, such as if animal was backing into the object or bumping the object accidentally as it passed was not included for scoring. To remove any olfactory clues, the test arena and the objects were cleaned with 70% alcohol before placing a successive mouse for the test.\n\nData were presented as mean ±SEM and one-way analysis of variance [ANOVA] with Bonferroni's post-hoc test were used to compare the treatment effects between the groups, and a value of p< 0.05 was considered as statistically significant. SPSS (RRID: SCR_002865) was used for statistical analysis.\n\n\nResults\n\nAging mice supplemented Ch-DHA, or administered HEK-CM showed significantly increased mean % of correct choices (p< 0.001) indicating enhanced spatial learning during both the 3rd and 4th day of training whereas those mice exposed to EE showed significantly enhanced spatial learning (p<0.001) only on fourth day of training relative to same in age-matched SVC, HIHEK-CM, and NAC mice (Figure 1, Table 1). During the initial days of training, mice from all groups made random re-entries into arms already visited. As the training continued, although aging mice exposed to Ch-DHA or HEK-CM or EE, showed progressive decrease in working memory errors, they were significantly lower (p<0.01) only in Ch-DHA supplemented mice compared to the same in age-matched SVC (Figure 2, Table 2). However, when compared with aging mice exposed to Ch-DHA, HEK-CM and EE group of mice, NAC mice and HIHEK-CM mice groups committed more working memory errors throughout training, indicating poor learning. Moreover, although the mean number of reference memory errors committed by Ch-DHA and HEK-CM mice were lesser during the 3rd and 4th day of training, no significant difference in reference memory errors was observed when compared to the same in age-matched NAC mice groups (Figure 3, Table 3). However, Ch-DHA and HEK-CM mice attained a significantly higher percentage (p<0.001 and p<0.05, respectively) of correct choices during the retention test. In contrast, NAC, HIHEK-CM mice showed significant impairment in the retention of spatial tasks (Figure 4, Table 4).\n\nValues represent mean ±SEM percentage of correct choices along with Bonferroni post-hoc test p-values.\n\nHuman embryonic kidney stem cell conditioned media (HEK-CM) and choline-docosahexaenoic acid (Ch-DHA) mice vs heat inactivated HEK-CM (HIHEK-CM) or saline vehicle control (SVC) or normal ageing control (NAC) mice made significantly more correct choices on 3rd and 4th day of trials @@@ and ###p<0.001 respectively, whereas environmental enrichment (EE) exposed mice vs NAC mice made significantly more correct choices on 4th day of trial. **p<0.001.\n\nValues represent mean ±SEM numbers of working memory errors, along with Bonferroni post-hoc test p-values.\n\nCholine-docosahexaenoic acid (Ch-DHA) mice vs saline vehicle control (SVC) mice made significantly less numbers of working memory errors on 4th day of trial. ##p<0.01.\n\nHuman embryonic kidney stem cell conditioned media (HEK-CM) and Choline-docosahexaenoic acid (Ch-DHA) mice made slightly fewer mean numbers of reference memory errors on 3rd and 4th day of trials when compared with all other groups.\n\nCholine-docosahexaenoic acid (Ch-DHA) and human embryonic kidney stem cell conditioned media (HEK-CM) mice vs saline vehicle control (SVC) or normal ageing control (NAC) and heat inactivated HEK-CM (HIHEK-CM) mice respectively made significantly more percentage of correct choices during retention phase. ###p< 0.001 and @p< 0.05.\n\nWhen aging mice supplemented Ch-DHA, or exposed to HEK-CM or EE were assessed for cognition in NORT, significant differences in visits to the familiar object, were observed in HEK-CM and EE groups when compared to NAC and HIHEK-CM groups (p<0.05). Moreover, aging mice supplemented Ch-DHA also showed slightly higher number of visits to familiar object compared to NAC mice but it was not significant (Figure 5, Table 5). When the number of visits to novel objects was compared between all groups, HEKCM aging mice showed significantly higher preference (p<0.001) with higher number of visits to the novel object as compared to the same in age-matched NAC and HI-HEKCM mice groups. Whereas, Ch-DHA supplemented aging mice showed higher preference at lower significant levels (p< 0.05) to the novel object as compared to the same in age-matched NAC mice group (Figure 6, Table 6).\n\nEnvironmental enrichment (EE) and human embryonic kidney stem cell conditioned media (HEK-CM) mice vs normal ageing control (NAC) and heat inactivated HEK-CM (HIHEK-CM) mice respectively made significantly greater number of visits to familiar object and @ p<0.05.\n\nHuman embryonic kidney stem cell conditioned media (HEK-CM) and choline-docosahexaenoic acid (Ch-DHA) mice vs heat inactivated HEK-CM (HIHEK-CM) or normal ageing control (NAC) mice respectively made significantly greater number of visits to novel object. @@@p<0.001 and #p<0.05\n\n\nDiscussion\n\nThe present study shows that normal aging in mice is associated with impairment in cognition, learning and memory capabilities. Comparison of potential strategies to attenuate age-associated cognitive and memory impairments during aging in this study has shown interesting findings. In the radial arm maze task during the 3rd and 4th trial days of acquisition phase of the test, it was observed that aged mice supplemented with Ch-DHA performed significantly better in spatial learning with reduced numbers of working and reference memory errors and higher ability to correctly choose the baited arms in comparison to same in age-matched normal aging mice. Comparatively, HEK-CM treated aged mice also showed similar findings except that on the 4th trial day of acquisition phase they performed significantly better compared to the same in age-matched Ch-DHA supplemented aged mice as well as aged mice exposed to EE. Whereas aged mice exposed to EE performed significantly better compared to same in age-matched NAC mice only on the 4th trial day of acquisition phase. Additionally, it was observed that during the retention phase of the radial arm maze test too, aged mice supplemented with Ch-DHA and those treated with HEK-CM performed significantly better than the same in age-matched NAC mice, indicating better memory of the learnt correct choices. These findings indicate that all three strategies help to some extent in attenuating spatial memory deficit in aging mice. High consumption of Ch during the perinatal period showed neuroprotective effect in animal models including during age related neuronal dysfunction. Perinatal Ch supplemented rats showed improvement in spatial memory performance and showed less errors relative to the untreated group when subjected to a 12-arm radial maze task.30–32 Rats which were supplemented with Ch as infants were able to retain a larger number of items in working memory during their adulthood.33 Studies also report that, dietary DHA supplementation enhances spatial memory in DHA deficient rats.34 Recent studies observed that, combined supplementation of both Ch and DHA was more efficacious in enhancing hippocampal neurodevelopment. Prenatal supplementation of Ch and DHA showed significant improvement in spatial learning and other cognitive functions during adolescence.35,36 Supplementation of Ch and DHA reduced memory errors in obese rats when subjected to eight arm radial maze test.37 The hippocampus plays a major role in memory function. Hippocampal damage disturbs the memory consolidation.38 Many studies have also shown the beneficial effects of cell therapies in restoring spatial learning in animal models of hippocampal degeneration.39,40 Conditioned media which are derived from cell cultures are known to have excellent sources of neurotrophic factors and cytokines.41,42 Furthermore, HEK-CM could potentiate megakaryopoiesis due to the presence of erythropoietin and other cytokines.43 Erythropoietin is a known neuroprotectant.44,45 In kainic acid induced hippocampal damaged mice, HEK-CM treatment showed significant increase in neuro-protection and improvement of hippocampal cognitive function.46 Further, previous studies in aged animals also showed that environmental enrichment prevents age-associated impairments in spatial learning and cognition.26,47\n\nIn the present study, behavioral testing for novel object recognition showed that, aging mice treated with HEK-CM had significantly higher preference for familiar object as well as novel object when compared to the same in age-matched NAC as well as HIHEK-CM mice groups. Additionally, aging mice supplemented Ch and DHA although had higher visits to familiar object, they were not significant as compared to the same in age-matched NAC, whereas visits to novel object were significantly higher when compared to the same in age-matched NAC mice. Whereas aging mice exposed to EE had significantly higher preference for familiar object but not for novel object when compared to the same in age-matched NAC mice.\n\nStudies show that kainic acid (KA) lesioned mice that received HEK-CM showed significantly greater number of visits towards the novel object as compared with KA alone treated mice. Normal mice that received HEK-CM also showed higher preference for novel object as compared with familiar objects.46 Combined supplementation of nutrients like Ch and DHA after perinatal brain injury in mice showed a substantial preference for the novel object when subjected to NORT, indicating that recognition memory after brain injury was improved by the supplementation of DHA and Ch.48 Aged mice, exposed to standard environment rather than EE, were able to discriminate between familiar and novel objects. Animals exposed to EE were able to distinguish stationary from displaced objects with different preferences independent of age. It was observed that young mice spent more time with displaced objects and aged mice with stationary objects.49\n\n\nConclusion\n\nDuring normal aging in mice, supplementations of Ch-DHA and HEK-CM treatment strategies have a higher potential [~ 20—30%] for enhancing spatial learning, memory and cognition, whereas exposure to EE seems to enhance only short-term memory. Further studies need to be done in order to analyse the underlying mechanisms for the cognitive changes.\n\n\nAuthor contributions\n\nKiranmai S Rai and Anandh Dhanushkodi conceptualized and designed the methodology for this study. They have worked as project administrators and mentors/supervisors. The study was carried out in detail and investigated by Shreevatsa Bhat M. Data collection, curation and formal analysis of the data were carried out by Shreevatsa Bhat M. Resources were provided by Manipal Academy of Higher Education and partly by Anandh Dhanushkodi and Kiranmai S Rai. Shreevatsa Bhat M and Kiranmai S Rai have analysed and validated data. Shreevatsa Bhat M has written original draft. It has been critically revised and suggestions for editing were given by Kiranmai S Rai, Anandh Dhanushkodi and Ramesh Babu MG. Shreevatsa Bhat M, Ramesh Babu MG, Anandh Dhanushkodi and Kiranmai S Rai read the final version of article and have approved this version to be published.", "appendix": "Data availability\n\nDryad: Data for Radial arm maze tests and Novel object recognition test, https://doi.org/10.5061/dryad.xpnvx0kj3. 50\n\nThis project contains the following underlying data:\n\nFile 1. Percentage_of_correct_choices-Learning_phase.csv (Percentage of correct choices made by mice during learning phase of eight arm radial maze test)\n\nFile 2. Working_memory_error.csv (Number of working memory errors made by mice during trial phase of eight arm radial maze test)\n\nFile 3. Reference_memory_error.csv (Number of reference memory errors made by mice during trial phase of eight arm radial maze test)\n\nFile 4. Percentage_of_correct_choices-Retention_phase (Percentage of correct choices made by mice during retention phase of eight arm radial maze test)\n\nFile 5. Visits_to_familiar_object.csv (Number of visits to familiar object made by mice during the test phase of novel object recognition test)\n\nFile 6. Visits_to_novel_object.csv (Number of visits to novel object made by mice during the test phase of novel object recognition test)\n\nDryad: ARRIVE checklist for ‘Exploring potential strategies to enhance memory and cognition in aging mice’. https://doi.org/10.5061/dryad.xpnvx0kj3. 50\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\nAcknowledgments\n\nThe authors would like to thank Manipal Academy of Higher Education for providing all infrastructure and support needed for this study.\n\n\nReferences\n\nHedden T, Gabrieli JDE: Insights into the ageing mind: A view from cognitive neuroscience. Nat. Rev. Neurosci. 2004; 5: 87–96. PubMed Abstract \| Publisher Full Text\n\nPark DC, Reuter-Lorenz P: The adaptive brain: Aging and neurocognitive scaffolding. Annu. Rev. Psychol. 2009; 60: 173–196. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiniz DG, et al.: Age, environment, object recognition and morphological diversity of GFAP-immunolabeled astrocytes. Behav. Brain Funct. 2016; 12: 28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhat Shreevatsa M, Rai KS, Ramesh Babu MG, Dhanushkodi A: Data for Radial arm maze tests and Novel object recognition test. Dryad Dataset. 2022. Publisher Full Text" }	[ { "id": "164703", "date": "09 Mar 2023", "name": "Kumar M.R. Bhat", "expertise": [ "Reviewer Expertise Human gross anatomy", "Neuroanatomy", "neurobehaviour", "Natural medicine and their mechanism of actions in human diseases", "cancer biology", "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 is very well conceptualized and objectives are designed. All sections of the article are well written. Results are encouraging further research in this area of age and cognition.\n\nAbstract - sufficient\nIntroduction - covers all necessary information to understand the objectives and its importance\nMethodology - overall content are good enough. However, few details about need to use of CF1 mouse would be important in this study.\nResult - good descriptions\nDiscussion - can be enriched if the explain about:\nmolecular mechanism of Ch and DHA in relation to cognition microconstituents of HEK media and its effect on cognition possible mechanism about how EE works on cognition\nWas there any signs of immune reactions due to HEK media? What are the difference between HEK-CM AND HIHEK-CM in terms of constituents and its effect on immune system and cognition?\nThe author needs to make an attempt to explain the possible reasons for differences in the results after exposure to different enhancing agents.\n\nIs the work 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": "9646", "date": "13 Jun 2023", "name": "Prof Kiranmai S Rai", "role": "Author Response", "response": "Methodology - However, few details about need to use of CF1 mouse would be important in this study. Answer: CF1mice were used as they genetically resembles human gene and also since these mice are smaller in size the total cost for the experiments was lesser. Discussion - can be enriched if the explain about: molecular mechanism of Ch and DHA in relation to cognition: Choline is known to enhance cholinergic function and DHA is an essential nutrient that helps development of neurons / neurogenesis, synaptogenesis / synaptic plasticity in hippocampus which enhances cognitive abilities microconstituents of HEK media and its effect on cognition- HEK conditioned media are excellent sources of neurotrophic factors and cytokines. Furthermore, the conditioned medium contains known neuroprotectants such as erythropoietin (EPO) and other cytokines. Previous studies show that these neurotrophic factors enhance cognition. possible mechanism about how EE works on cognition: Previous studies revealed that exposure to EE has stimulating effect on neurogenesis, enhances granule cell layer and increases number of dentate gyrus granule cells in the hippocampus. Further, long-term EE exposure prevents age-associated cognitive impairment by increasing cAMP response element-binding protein (CREB) level in the hippocampus which potentially contributes to improved cognitive abilities such as learning and memory in aged animals.[Ref. -in revised version] Was there any signs of immune reactions due to HEK media? - No What are the difference between HEK-CM AND HIHEK-CM in terms of constituents and its effect on immune system and cognition? HIHEK-CM- used as vehicle control for HEK CM, has all the protein constituents inactivated by heat, hence has no effect on immune system and cognition" } ] } ]	1	https://f1000research.com/articles/12-141
https://f1000research.com/articles/11-763/v1	08 Jul 22	{ "type": "Research Article", "title": "Macro-economic determinants of the IPO waves: An assessment of G-7 countries", "authors": [ "MUSTAFA ÖZYEŞİL", "Esin Benhür Aktürk", "Esin Benhür Aktürk" ], "abstract": "Background: The purpose of this study was to reveal the macroeconomic determinants that companies take into account when scheduling initial public offerings (IPOs). Methods: For this purpose, the relationship between the frequency of the IPOs and the selected macroeconomic indicators in the stock market of the G-7 countries for the period 1999-2020 was examined by panel data analysis method. A random effect model was used as econometric model in the analysis, and the Driscoll-Kraay resistant standard estimator developed against deviations was used in the application of the model. Results: According to the results of the analysis, it was determined that stock market returns have a positive and significant effect on the volume of public offerings. Conclusions: Based on our findings, it can be concluded that companies take into account the periods when stock market returns are relatively high for timing the IPO.", "keywords": [ "IPO Wave", "Stock Returns", "Panel Data", "Random Effect Model", "Driscoll-Kraay Standard Error", "New Product Development", "Operations Management", "Organization Development Change", "Continuous Improvement" ], "content": "Introduction\n\nInitial public offerings (IPOs) are one of the equity financing methods that companies apply in capital markets. As a strategic financing process, public offerings play a critical role in the financial success of companies. The advantages and disadvantages of IPOs for companies are a matter of debate in the literature. The company that makes the IPO gains the status of a publicly traded company, is listed on the stock exchange, and thus the stocks are traded among the investors in the secondary market, that is, a liquid market is formed for the investors. In addition to this, after issuance process, the firms, as a publicly traded firm, have the opportunity to raise capital under much more favorable terms in the coming years (Ibbotson and Ritter, 1995). On the other hand, public offerings have many direct and indirect costs, such as the intermediary costs paid to the financial institutions and the listing fee paid to the regulatory institutions; these are accepted as one-time costs, while the obligation of the firms to provide information to the regulatory institutions on a regular basis is accepted as a continuous cost (Ibbotson and Ritter, 1995). Considering other factors such as company owners are sharing the management with others, companies should carefully evaluate advantages and disadvantages during the public offering process.\n\nPublic offerings are one of the most important issues in corporate finance. There have been many studies on initial public offerings in the literature. While some of the studies have examined the macro- and micro-determinants that lead companies to go public, some have examined the short and long-term price performance of stocks after the public offering. For example, Chemmanur and He (2011), Rydqvist and Högholm (1995) and Draho (2004) have studied the reasons for companies to go public and the frequency of public offerings. Loughran and Ritter (2004), Booth and Chua (1996), Ljungqvist (2007), Ritter and Welch (2002), Ritter (1984) et al. focused on price movements and their determinants. In studies on the causes and frequency of public offerings, hypotheses such as hot and cold public offerings have been developed. Many hypotheses such as Winner’s Curse by Rock (1986) have been put forward regarding the post-IPO price performance.\n\nIn this study, macroeconomic determinants that affect the frequency of IPOs was examined. In other words, we tried to determine which indicator companies take into account for timing of the public offering. The study is carried out on the G-7 countries for the period 1999-2020 using the panel data analysis method.\n\nThe number, volume and returns of IPOs are influenced by both the firm-related and macroeconomic environment and intermediary institution behaviors. For example, investment banks have a very high public offering experience, as they constantly manage public offerings. For this reason, the information produced by investment banks for market participants in the public offering processes provides significant guidance both companies and investors. Particularly during periods of heavy issuance and high initial returns, the information produced by investment banks improves the quality of IPOs, enabling previously low-quality firms to go public and increasing secondary market prices, synchronizing high IPO volumes and high first-day returns (He, 2007).\n\nIn the following part of the study, research in the international literature on this subject were examined. The relationship between the frequency of public offerings and the macroeconomic indicators selected for this study were then analyzed. In the last part of the study, the findings were interpreted and evaluated.\n\nThe sample for the study consisted only of G-7 countries, which are developed markets.\n\nIn this respect, a comparative analysis and interpretation between country groups cannot be made, since the results of emerging or underdeveloped markets were not included in the study. However, it is recommended to conduct similar analyzes on other country groups in order to obtain more comprehensive and comparative results.\n\nIn addition, in this study, macroeconomic factors that are thought to be effective on the number of IPOs were included, and micro factors related to the company were ignored. It will be possible to obtain more meaningful results by including micro factors in the model.\n\nStudies on the number of initial public offerings were examined. The majority of the studies in the literature have analyzed the trend of IPOs in terms of number and volume and have created the concepts known as IPO Wave and Hot and Cold Issue markets in the literature. In most of the studies in the literature, the markets in which the number and volume of IPOs increase gradually are defined as “hot issue markets”, while the markets where the number and volume of IPOs decrease are defined as “cold issue markets”. In hot issue markets, many companies can plan their IPOs for this period, as the investor’s demand for the stock market is increasing. This way, the company makes a successful public offering and the investors have the opportunity to obtain abnormal returns in the short term thanks to the high discounts they obtain.\n\nPástor and Veronesh (2005) developed an optimal IPO timing model for firms. They emphasized that many studies in the literature explained the change in IPO volume over time with the concept of market inefficiency, where IPOs are made when stocks are overvalued. Contrary to previous literature, they created a model in which the fluctuation in the volume of initial public offerings occurs without any mispricing mechanism and the volume of the IPO is more closely related to recent changes in stock prices rather than the level of market stock prices. In the model, the variables that companies take into account during the public offering are, respectively: expected market return, expected aggregate profitability and prior uncertainty about the average future profitability of IPOs. According to the results of the analysis, they determined that there were high market returns before the IPO waves and low market returns after the IPO waves.\n\nButtimer, Hyland and Sanders (2005), in their study, comparatively examined the waves of IPOs in the real estate investment trust (REIT) sector and the waves of general initial public offerings. In terms of initial returns, it was observed that the REIT sector provided lower returns than the general market average after the IPO waves. In addition, the long-term underperformance anomaly, which is frequently observed in stock markets according to many studies such as Ritter (1991), Loughran and Ritter (1997), Levis (1993), Schultz (2003) among others, has not been observed in the REIT sector. Based on the findings of their study, authors stated that the Capital Demand Hypothesis best describes the REIT public offering market.\n\nYung, Colak and Wang (2008) created a model in which real investment opportunities that change over time cause adverse selection in the market for IPOs. As a result of the analysis, it has been determined that economic expansions are highly correlated with the number of companies that go public, which is positively correlated with underpricing. In line with the model they created, greater cross-sectional return variance and higher delisting rates were detected for hot market public offerings.\n\nChristoffersen, Nain and Tang (2010) examined the waves and the quality of IPOs. They stated that the quality of the public offering will not be the same in the first and later periods of the IPO wave. They noted that in the early stages of the IPO wave, the average quality of IPO stock is low when initial returns and IPO demand are low. However, later in the IPO wave, when initial returns and IPO demand are low, IPOs have better operating performance, higher market share, and consequently higher long-term abnormal returns. According to the results of the study, they found that institutional investors benefit from short-term high returns in early public offerings, but they prefer IPOs that are done in later stages of the IPO wave thanks to their higher quality in the long run.\n\nIn their study, Tran and Jeon (2011) analyzed the macroeconomic factors that were effective on the initial public offerings in the US capital market during the 1970-2005 period. By applying time series econometric methods in the analysis, they determined the long-term balance between macroeconomic variables and public offerings. As in many studies, Tran and Jeon also found that stock market return performance and volatility have the most significant impact on the timing of the IPO. In their study, they also examined the effects of interest rates and bond market returns. They found that there are not only long-term but also short-term statistically significant relationships between macroeconomic variables and IPO activities.\n\nAngelini and Foglia (2018) examined the short- and long-term relationships between initial public offerings and macroeconomic factors in the UK stock market for the period 1996-2016. In the analysis, they sought answers to the questions of how macroeconomic conditions affect the initial public offerings and how long the recent shock effect lasted. Business cycle, volatility, interest rates and stock returns were used as macroeconomic variables. According to the results of the correlation analysis made in the study, it was determined that the business cycle, volatility and interest rate variables could explain the change in the number of IPOs. However, unlike many studies in the literature, no significant effect of stock market performance on the number and timing of IPOs could be detected.\n\nThanh (2020) used macroeconometric models to examine the cycles of initial public offerings in US stock markets. According to the results of the time series analysis they applied in their studies, they determined the strong and negative effect of macroeconomic uncertainty on the public offering activity. It has been determined that a one-unit standard deviation increase in macroeconomic uncertainty reduces the monthly number of initial public offerings by approximately four units in the long run. In the analysis, they also found that both the decrease in the number of IPO applications and the increase in withdrawn IPOs contributed to the decrease in the number of IPOs in response to an uncertainty shock.\n\nCarosi and Mengoli (2021) analyzed local IPO waves in their studies. They analyzed public offerings on the basis of region and sector. They found that the waves of public offerings overlapped on a sectoral and regional basis. In other words, public offerings on a sector basis are similar to public offerings on a regional basis. They observed that IPOs at the beginning of the IPO wave were equally priced lower than IPOs at the end of the wave. According to the results of the analysis, it was determined that the IPO decision is sensitive not only to the high valuations of the companies in the same sector, but also to the high valuations of the companies in the same region but in different sectors.\n\n\nMethods\n\nIn the study, we examined whether macroeconomic variables have an effect on the frequency of public offerings. Since the aim is to determine the factors affecting the IPO wave through developed capital markets, the sample consists of G-7 countries. The analysis period covered the period 1999-2020, and annual data were used in the analysis.\n\nMacroeconomic indicators such as annual consumer inflation, stock market returns and economic growth over the years (GDP growth) were used as independent variables. Explanations regarding on variables included in analysis are provided in Table 1 as follows.\n\nOur preliminary expectations were a negative relationship between consumer inflation and the frequency of public offerings, and a positive relationship between stock returns and economic growth and frequency of public offerings.\n\nIn the study, the relationship between the IPO waves (frequency) and macroeconomic variables were analyzed in terms of country sections. For this reason, panel data analysis method was used. In the analysis, it was deemed appropriate to apply the random effect model as the econometric model. While macroeconomic variables were included in the analysis as independent variables, the number of IPOs was included in the analysis as a dependent variable.\n\nSince there was a cross-section dependency between the series in the analysis, the stationarity of the data was measured with the Peseran (2007) unit root test, which is one of the second-generation unit root tests. Afterwards, the Hausman test was applied to determine the econometric model to be applied. According to the results of the test, the application of the random effect model was deemed appropriate and the Driscoll-Kraay resistant standard estimator developed against deviations was used in the application of the model.\n\n\nResults\n\nDescriptive statistics for the sample used in the study are shown in Table 2 below.\n\nCorrelation analysis is one of the most typical analyzes used to examine the relationship between variables. Pearson’s correlation analysis was used in this study, as the data showed normal distribution.\n\nThe correlation relationship between the variables is shown in Table 3 below.\n\nAccording to the results of the correlation test, since the degree of correlation between the variables varies between 0.05 and 0.49, there was a positive but weak relationship. The degree of correlation differed according to the variables used. For example, there was a very weak correlation of 0.05 between stock returns and inflation, while a weak correlation of 0.49 between the GDP growth and stock returns. A strong and very strong relationship between the variables in the correlation analysis should have a degree of correlation between 0.70-0.89 and 0.90-1.00, respectively. As a result, according to the results of the analysis, there is a weak relationship between the variables in this sample.\n\nIn order not to encounter the spurious regression problem in the analysis, it is necessary to measure the stationarity of the series before the regression analysis. However, in order to determine which stationarity test will be used, a cross-section dependency test should be applied. According to the result of the cross-sectional dependence, the appropriate unit root test was applied. Cross-section dependence was tested with Pesaran (2004) cross-section dependence method.\n\nThe hypotheses of this test were as follows:\n\nH0: There is no cross-section dependence between the series.\n\nH1: There is a cross-section dependence between the series.\n\nThe results of the test are shown in Table 4 below:\n\nAccording to the results of the test, there was a cross-sectional dependence between the series. As a consequence, Pesaran (2007) second generation unit root test was applied, taking this stiuation into account.\n\nThe hypotheses of these tests were as follows:\n\nH0: Series are not stationary.\n\nH1: Series are stationary.\n\nThe results of the test are shown in Table 5 below.\n\nAccording to the unit root test results, IPOs and Inflation were found to be stationary after taking the first difference, while the GDP growth and stock returns variables were found to be stationary at level values. Therefore, non-stationary IPOs and inflation variables were included in the regression analysis after taking their first difference. For this reason, a cointegration test was not be applied in the study.\n\nA Hausman test was used to determine the econometric model to be established. The Hausman test results are summarized in Table 6 below.\n\nAccording to this result, the random effect model was used as the econometric model.\n\nTests related to autocorrelation and heteroscedasticity problems in the model were performed in Table 7 and Table 8 below.\n\nIn the model, macroeconomic variables were used as independent variables, and IPO numbers were used as dependent variables, and a random effect regression analysis with the Driscoll-Kraay resistant standard estimator was applied.\n\nRegression analysis results are shown in Table 9 as follows.\n\nGLS: generalised least squares.\n\nAccording to the results of the regression analysis, stock returns is the only variable that has a positive and significant effect on the number of IPOs. The effects of all other variables on the frequency of IPOs were not found significant. Based on the analysis findings, it can be stated that companies take into account the stock returns variable in timing the public offering. The correlation between stock returns variable and IPOs was found to be positive. In other words, as the stock market index returns increase, companies may choose to go public in order to enter the hot public offering market and maximize their issuance proceed (or reduce the underpricing degree of public offerings). The findings are compatible with many studies in the previous literature, especially Pástor and Veronesh (2005), and Tran and Jeon (2011).\n\n\nDiscussion and conclusions\n\nIPOs are one of the most important methods used by companies as long-term equity financing in the capital market. In an IPO, the issuer receives issuance income in exchange for shares sold to investors. Investors, on the other hand, become a shareholder of a newly publicly traded company, often taking advantage of the discounted issue price, and as long as they hold the stock in the future, they will receive dividend income or can get capital gain by selling the discounted shares at a higher price. Considering the above-mentioned advantages and long-term results of public offerings, it was found that this strategic financing process has an important place in the financial success of companies. For this reason, the success of the IPOs is critical for all parties in the IPO process as well as for the general market functioning. Due to the importance of IPOs, there have been numerous studies on IPOs in the literature. The majority of the studies focused on the short- and long-term price performances and determining factors of IPOs, as well as the cycle of IPOs.\n\nIn this study, macroeconomic factors that were thought to be effective on the cycle of IPOs were examined, and especially whether economic growth, inflation and stock market returns are determinants on the number of IPOs. The analysis was applied to the G-7 countries representing the developed world economies, for the period 1999-2020.\n\nIn the analysis, first of all, the stationarity of the series was tested with the Pesaran (2007) unit root test, which takes into account the cross-section dependence. The series that were not stationary at the values level values were included in the regression analysis by considering their primary difference. Before the regression analysis, we tested whether the problems of autocorrelation and heteroscedasticity existed in the model, and it was determined that there were no such problems in the model. A Hausmann test was applied in the selection of the econometric model to be established, and according to the results obtained, it was deemed appropriate to use the random effect model. Regression analysis was performed using the Driscoll-Kraay resistant standard estimator, which was resistant to deviations and errors.\n\nAccording to the results of the analysis, it was determined that only the stock returns variable, among the independent variables in the model, had a significant effect on the number of initial public offerings. The stock returns variable has a positive effect on the number of IPOs. In other words, as stock return increases, the number of IPOs also increases. Based on this finding, it can be stated that stock returns is one of the macroeconomic factors that affect companies’ public offering decision. In other words, companies prefer to go to public offerings during periods when stock market returns increase. The inflation and GDP growth variables, which were the other variables investigated in the analysis, could not have a significant effect on the frequency of the initial public offering.\n\nIn this analysis, only the macroeconomic determinants, which are thought to be effective on the IPO waves, were examined. In addition, more comprehensive and meaningful results can be obtained by including microeconomic factors that may affect the number of IPOs in the analysis. A study on developing economies in addition to developed world economies will be extremely useful in terms of showing comparatively how macro- and microeconomic factors change according to the level of development of countries.\n\n\nData availability\n\nInflation and GDP growth rate data were retrieved from WorldBank databank and filtered for the G-7 countries for the period of interest (https://data.worldbank.org/indicator/FP.CPI.TOTL.ZG). In the menu on the right of the home page, the indicators for sample countries can be selected. No account creation is needed.\n\nIPO numbers were retrieved from Statista. Scholars don’t have to create account and make subscription on Statista when they are downloading IPO numbers for each country seperately. For multiple countries and periods, the system requires to create an account.\n\nStock returns were retrieved from Investing.com. In thesearch box located at the top of the home page of investing, equity code is written and profile page of related stock wil be opened. The historical data section is located just above the price chart. After determining start and end dates all information regarding on closing price, opening price, highest and lowest will be visible.\n\nAll data used are open access.", "appendix": "References\n\nAngelini E, Foglia M: The relationship between IPO and macroeconomics factors: An empirical analysis from UK market [J]. Ann. Econ. Financ. 2018; 19(1): 319–336.\n\nBooth JR, Chua L: Ownership dispersion, costly information, and IPO underpricing. J. Financ. Econ. 1996; 41(2): 291–310. Publisher Full Text\n\nButtimer RJ, Hyland DC, Sanders AB: REITs, IPO Waves and Long-Run Performance. Real Estate Econ. 2005; 33(1): 51–87. Publisher Full Text\n\nCarosi A, Mengoli S: Local IPO waves, local shocks, and the going public decision. Int. J. Finance Econ. 2021. Early View.\n\nChemmanur TJ, He J: IPO waves, product market competition, and the going public decision: Theory and evidence. J. Financ. Econ. 2011; 101(2): 382–412. Publisher Full Text\n\nChristoffersen SK, Nain A, Tang Y: IPO Cycles, Firm Characteristics, and the Role of Underwriters.2010. Publisher Full Text\n\nDraho J: The IPO Decision. Why and How Companies Go Public. Cheltenham, UK:Edward Elgar Publishing;2004.\n\nHe P: A theory of IPO waves. The Review of Financial Studies.2007; 20(4): 983–1020.\n\nIbbotson RG, Ritter JR: Initial Public Offerings. Handbooks in Operations Research and Management Science. 1995; 9(1): 993–1016. Publisher Full Text\n\nLevis M: The Long-Run Performance of Initial Public Offerings : The UK Experience 1980 – 1988. J. Int. Fin. Manag. Acc. 1993; 22(1): 28–41.\n\nLjungqvist A; IPO Underpricing, Handbook of Empirical Corporate Finance, Handbooks in Finance.2007; 375–422.\n\nLoughran T, Ritter J: Why Has IPO Underpricing Changed over Time? Financ. Manag. 2004; 33(3): 5–37.\n\nLoughran T, Ritter JR: The Operating Performance of Firms Conducting Seasoned Equity Offerings. J. Financ. 1997; 52(5): 1823–1850.\n\nPástor Ľ, Veronesi P: Rational IPO waves. J. Financ. 2005; 60(4): 1713–1757. Publisher Full Text\n\nPesaran MH: General Diagnostic Tests for Cross Section Dependence in Panels. Cambridge Working Papers in Economics 0435, Faculty of Economics. University of Cambridge;2004.\n\nPesaran MH: A Simple Panel Unit Root Test in The Presence of Cross-Section Dependence. J. Appl. Econ. 2007; 22: 265–312. Publisher Full Text\n\nRock K: Why New Issues Are Underpriced. J. Financ. Econ. 1986; 15: 187–212. Publisher Full Text\n\nRitter JR: The Long - Run Performance of Initial Public Offerings. J. Financ. 1991; 46(1): 3–27.\n\nRitter JR: The Hot Issue Market of 1980. J. Bus. 1984; 57(2): 215–240. Publisher Full Text\n\nRitter J, Welch I: A Review of IPO Activity, Pricing, and Allocations. J. Financ. 2002; 57(4): 1795–1828. Publisher Full Text\n\nRydqvist K, Högholm K: Going public in the 1980s: Evidence from Sweden.1995; 1(3): 287–315.\n\nSchultz P: Pseudo Market Timing and the Long-Run Underperformance of IPOs. J. Financ. 2003; 58(2): 483–517.\n\nThanh BN: Macroeconomic uncertainty, the option to wait and IPO issue cycles. Financ. Res. Lett. 2020; 32: 101100. Publisher Full Text\n\nTran AL, Jeon BN: The dynamic impact of macroeconomic factors on initial public offerings: evidence from time-series analysis. Appl. Econ. 2011; 43(23): 3187–3201. Publisher Full Text\n\nYung C, Çolak G, Wang W: Cycles in the IPO market [J]. J. Financ. Econ. 2008; 89(1): 192–208. Publisher Full Text" }	[ { "id": "177271", "date": "28 Jun 2023", "name": "Prof.Dr.Cemal Zehir", "expertise": [ "Reviewer Expertise Strategic management", "business administration", "and economist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 peer-reviewed article, it has been examined whether the macroeconomic factors that are thought to be effective on the public offering cycle, and especially economic growth, inflation and stock market returns, are determinants of the number of public offerings. From this point of view, it will contribute to the literature.\nThe analysis employs a random effect model and applies the Driscoll-Kraay resistant standard estimator to account for deviations.\nThe findings reveal that stock market returns have a positive and significant effect on the volume of public offerings. This suggests that companies tend to time their IPOs during periods when stock market returns are relatively high.\nThis research provides insights into the factors influencing companies' decision-making processes regarding IPO timing, specifically emphasizing the role of macroeconomic indicators. By understanding the relationship between stock market performance and IPO frequency, policymakers and investors can gain valuable insights for strategic decision-making.\nThe analysis has been applied to the G-7 countries representing the developed world economies for the period 1999-2020. The countries analyzed and the fact that it covers a period of 20 years will be useful information for the readers.\nResearch results are also a guide for practitioners. This article is suitable for publication.\n\nIs the work 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": "182871", "date": "14 Jul 2023", "name": "Daniel Penido L. Amorim", "expertise": [ "Reviewer Expertise Financial Economics", "Econometrics" ], "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 \"Macro-economic determinants of the IPO waves: An assessment of G-7 countries\" aimed to analyze which macroeconomic factors affect the number of IPOs in the G7 countries. The authors estimated a static model using a conventional panel data econometric method (random effects model). The results suggest that stock market returns influence the number of IPOs.\nThe results are not new. Many articles on the topic have also found positive effects of stock market returns (or prices) on IPOs. However, the study contributes to the literature because it considers the countries that compose the G7. Adding this contribution and others in the introduction would be relevant to improving the article. Except for this, the introduction is good.\nRegarding the literature review, some citations are missing in the first paragraph of this section. The way the authors wrote the paragraphs, dedicating each to a single study, is not convenient. The authors should discuss the articles together, according to some questions they have in common. Furthermore, the authors need to add some articles on IPOs that are more recent. I list these articles at the end of this referee report.\nIt is necessary to add at least the variables of interest rate and economic policy uncertainty (Backer et al., 2016) to the analysis. In several previous studies, the authors considered the interest rate in the models. Effects of economic policy uncertainty have appeared in recent studies such as Thanh (2020) and Amorim et al. (2021).\nThe authors stated that the data used had a normal distribution, but this is not what the Jarque-Bera test suggests for some variables in the table of summary statistics. Moreover, the authors can remove the less common summary statistics from this table. IPO number data from Statista does not appear to be openly available, even after a basic registration.\nThe more critical problems in the article arise from the econometric methods employed. The Hausman test is an asymptotic method. Therefore, it is not convenient to choose between fixed effects and random effects models based on it when the panel has only seven countries.\nStill on the models, we know that the process of deciding to go public (IPO) is dynamic. Agents can make these decisions with information from past periods. Therefore, it is common that the empirical models include lags. Moreover, stock prices affect IPOs because of the choice to go public in periods that favor IPO proceeds. However, IPOs also affect stock prices because these events cause stock market fluctuations. Thus, endogeneity is probable. Finally, there is also cross-sectional dependence. This requires the use of more advanced econometric methods than conventional panel data estimators with robust standard errors.\nI recommend that the authors estimate a dynamic model for long panel data, specifically the cross-sectionally augmented ARDL (CS-ARDL) approach (Chudik & Pesaran, 2015). The lags included in this model will overcome endogeneity and autocorrelation. The author can apply this method with variables I(0) (stationary) and I(1) (with a unit root). In addition, the method deals with cross-sectional dependence. In this approach, it has been common to use some tests jointly (two of them even the authors have used), which the authors can apply using the Stata software:\nPesaran's (2007) CIPS unit root test [command xtcips]; Pesaran’s (2021) cross-sectional dependence CD test [command xtcd]; Westerlund's (2007) cointegration test [command xtwest]; Chudik and Pesaran's (2015) CS-ARDL estimator [command xtdcce2]\nThese methods appear in studies such as Herzer (2019), Vo et al. (2022), and Uddin et al. (2023). Other dynamic models (for example, DOLS and FMOLS estimators) can be useful as robustness tests.\nFinally, the article needs to have the text revised. The authors need to improve their writing. Firstly, they need to use accurate vocabulary. For example, it is better to write \"evidence indicates\" or \"evidence suggests\" when referring to the results (no single study has a result \"determinant\"). There are well-known concepts that the authors miswrite, such as \"resistant standard estimator\" (I believe they meant \"robust standard error\" or \"robust estimator\") and \"macro-economics\" (do not use a hyphen). Furthermore, authors should avoid adopting passive voice in English and writing very long sentences. Some tools, such as Grammarly and Quillbot, contribute to the improvement of texts written by non-native English speakers.\nI hope the authors will continue to seek publication of the article.\nReferences\nAmeer, R. (2012). Macroeconomic factors and initial public offerings (IPOs) in Malaysia. Asian Academy of Management Journal of Accounting and Finance, 8(1), 41–67. https://ejournal.usm.my/aamjaf/article/view/aamjaf_vol8-no1-2012_3\nAmorim, D. P. L., Camargos, M. A., & Ferreira, C. M. B. L. (2021). Macroeconomic Factors and Initial Public Offerings in Brazil. Global Business Review. https://doi.org/10.1177/09721509211003696\nBaker, S. R., Bloom, N., & Davis, S. J. (2016). Measuring economic policy uncertainty. The Quarterly Journal of Economics, 131(4), 1593–1636. https://doi.org/10.1093/qje/qjw024\nChudik, A., & Pesaran, M. H. (2015). Common correlated effects estimation of heterogeneous dynamic panel data models with weakly exogenous regressors. Journal of Econometrics, 188(2), 393–420. https://doi.org/10.1016/j.jeconom.2015.03.007\nDeloof, M., de Jong, A., & Legierse, W. (2022). Going public: evidence from stock and bond IPOs in Belgium, 1839–1935. Cliometrica. https://doi.org/10.1007/s11698-022-00258-5\nHerzer, D. (2018). The long-run effect of aid on Health: Evidence from panel cointegration analysis. Applied Economics, 51(12), 1319–1338. https://doi.org/10.1080/00036846.2018.1527449\nMehmood, W., Mohd-Rashid, R., & Ahmad, A. H. (2020). The variability of IPO issuance: evidence from Pakistan stock exchange. Global Business Review. https://doi.org/10.1177/0972150920929198\nMehmood, W., Mohd-Rashid, R., Abdullah, Y. et al. (2023). Inclusive mapping of initial public offerings: a bibliometric and literature review study. Quality & Quantity, 57, 655–700. https://doi.org/10.1007/s11135-022-01387-9\nMehmood, W., Mohd-Rashid, R., Ong, C.Z., & Abbas, Y.A. (2021). Factors driving IPO variability: evidence from Pakistan stock exchange. Journal of Economics, Finance and Administrative Science, 26(52), 300-316. https://doi.org/10.1108/JEFAS-04-2021-0036\nPesaran, M. H. (2007). A simple panel unit root test in the presence of cross-section dependence. Journal of Applied Econometrics, 22(2), 265–312. https://doi.org/10.1002/jae.951\nPesaran, M. H. (2020). General diagnostic tests for cross-sectional dependence in panels. Empirical Economics, 60(1), 13–50. https://doi.org/10.1007/s00181-020-01875-7\nUddin, I., Ahmad, M., Ismailov, D., Balbaa, M. E., Akhmedov, A., Khasanov, S., & Haq, M. U. (2023). Enhancing institutional quality to boost economic development in developing nations: New insights from CS-ARDL approach. Research in Globalization, 7, 100137. https://doi.org/10.1016/j.resglo.2023.100137\nVo, D. H., Ho, C. M., Le, Q. T., & Vo, A. T. (2022). Revisiting the energy-growth-environment nexus in the OECD countries: An application of the CS-ARDL approach. Energy, Sustainability and Society, 12(1). https://doi.org/10.1186/s13705-022-00375-z\nWesterlund, J. (2007). Testing for error correction in panel data. Oxford Bulletin of Economics and Statistics, 69(6), 709–748. https://doi.org/10.1111/j.1468-0084.2007.00477.x\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": "185960", "date": "15 Aug 2023", "name": "Sanjib Biswas", "expertise": [ "Reviewer Expertise Multicriteria Decision Making", "Multivariate analysis", "Logistics & Supply Chain Management", "Sustainability" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper addresses a relevant issue which is however not very new. I find the following concerns\nThe paper suffers from flow and connectivity issues\n\nWhy will a researcher be interested to read this article? What is the value addition?\n\nWhy have the authors undertaken this work?\n\nLiterature review is outdated. It is required to study the papers published in 2020-2023.\n\nThe selection of methodology needs justification\n\nThe results need to be aligned with the key research questions and limitations need to be elaborated\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10859", "date": "29 Jan 2024", "name": "Esin Benhür aktürk", "role": "Author Response", "response": "First of all, we’d like to thank the respected professor reviewer for invaluable comments. We consider all these comments as crucial to improve our study. You can kindly find responses to your comment as follows: 1. The paper suffers from flow and connectivity issues We improved this issue. Thank you very much. 2. Why will a researcher be interested to read this article? What is the value addition? The study aims to assist investors in the stock market with market timing. By examining the relationship between macroeconomic indicators and the hot and cold issue market, it is intended to provide higher returns to investors. Investors can use macroeconomic indicators as a signal to gain insight into the trend of initial public offerings (IPOs) and, accordingly, time their investments to achieve higher short-term returns. 3. Why have the authors undertaken this work? The reason for our study is the relatively limited presence of research in the literature examining the relationship between stock returns and IPO trends. Through this study, we aim to contribute to the literature. We believe that the findings of this analysis, particularly focusing on the developed G-7 countries' capital markets, could serve as guiding insights for researchers delving into this subject. 4. Literature review is outdated. It is required to study the papers published in 2020-2023. As mentioned earlier, there exists a significant subject area in the literature on this matter. Consequently, the number of studies available is limited. Furthermore, due to its publication in 2021, this study naturally does not encompass research from the years 2022 and 2023. Nevertheless, three relevant studies published between 2021 and 2023 have been included in the literature. 5. The selection of methodology needs justification 6.The results need to be aligned with the key research questions and limitations need to be elaborated. In panel data analysis, the dataset encapsulates information from different cross-sections (e.g., various firms or countries) and across different time dimensions (e.g., different time periods). The Discroll-Kraay regression analysis is a method utilized for analyzing data within a panel data structure. This method specifically provides robust predictions against common issues such as autocorrelation and heteroscedasticity present in panel datasets. The Discroll-Kraay method is recommended to address concerns like autocorrelation and heteroscedasticity in panel data analysis. Autocorrelation denotes the situation where relationships between observations remain similar over time, whereas heteroscedasticity indicates varying variance among observations. By addressing such scenarios, this method ensures more reliable and robust regression outcomes. Moreover, the Discroll-Kraay method is widely recognized in the literature and is acknowledged for its reliability and effectiveness in panel data analysis. Hence, this method has been chosen for panel data analysis and forms part of the reasons behind its selection in this study." } ] } ]	1	https://f1000research.com/articles/11-763
https://f1000research.com/articles/12-54/v1	13 Jan 23	{ "type": "Systematic Review", "title": "The refusal of COVID-19 vaccination and its associated factors: a systematic review", "authors": [ "Fredo Tamara", "Jonny K. Fajar", "Gatot Soegiarto", "Laksmi Wulandari", "Andy P. Kusuma", "Erwin A. Pasaribu", "Reza P. Putra", "Muhammad Rizky", "Tajul Anshor", "Maya Novariza", "Surya Wijaya", "Guruh Prasetyo", "Adelia Pradita", "Qurrata Aini", "Mario V.P.H. Mete", "Rahmat Yusni", "Yama S. Putri", "Chiranjib Chakraborty", "Kuldeep Dhama", "Harapan Harapan", "Laksmi Wulandari", "Andy P. Kusuma", "Erwin A. Pasaribu", "Reza P. Putra", "Muhammad Rizky", "Tajul Anshor", "Maya Novariza", "Surya Wijaya", "Guruh Prasetyo", "Adelia Pradita", "Qurrata Aini", "Mario V.P.H. Mete", "Rahmat Yusni", "Yama S. Putri", "Chiranjib Chakraborty", "Kuldeep Dhama", "Harapan Harapan" ], "abstract": "Background: To date, more than 10% of the global population is unvaccinated against the coronavirus disease 2019 (COVID-19), and the reasons why this population is not vaccinated are not well identified. Objectives: We investigated the prevalence of COVID-19 vaccine refusal and to assess the factors associated with COVID-19 vaccine refusal. Methods: A meta-analysis was conducted during August to November 2022 (PROSPERO: CRD42022384562). We searched articles investigating the refusal of COVID-19 vaccination and its potential associated factors in PubMed, Scopus, and the Web of Sciences. The quality of the articles was assessed using the Newcastle–Ottawa scale, and the data were collected using a pilot form. The cumulative prevalence of the refusal to vaccinate against COVID-19 was identified using a single-arm meta-analysis. The factors associated with COVID-19 vaccine refusals were determined using the Mantel-Haenszel method. Results: A total of 24 articles were included in the analysis. Our findings showed that the global prevalence of COVID-19 vaccine refusal was 12%, and the highest prevalence was in the general population, and the lowest prevalence was in the healthcare worker subgroup. Furthermore, individuals with a high socioeconomic status, history of previous vaccination, and medical background had a lower rate of COVID-19 vaccination refusal. Subsequently, the following factors were associated with an increased risk of COVID-19 vaccine refusal: being female, educational attainment lower than an undergraduate degree, and living in a rural area. Conclusion: Our study identified the prevalence of and factors associated with COVID-19 vaccine refusal. This study may serve as an initial reference to achieve global coverage of COVID-19 vaccination by influencing the population of COVID-19 vaccine refusal.", "keywords": [ "COVID-19", "vaccination", "refusal", "acceptance", "risk factors." ], "content": "Introduction\n\nAt the beginning of 2021, the coronavirus disease 2019 (COVID-19) vaccination program involving several designs including protein subunit, vector, inactivated, and mRNA, was started.1 Currently, referring to data presented on Our World in Data, this vaccination program has included 84.6% of the global population, and the reason the rest of the population (15.4%) did not receive vaccination is still unknown.2 The high number of vaccinated country populations is the result of the hard work of various parties, and this may be associated with factors such as the seriousness of governments in promoting vaccination programs, equitable distributions of vaccines, hard work of healthcare workers, good public awareness about the importance of vaccination, and good promotion of vaccines to populations who have the power to hesitate about vaccines.3 Contrarily, in the unvaccinated population, several factors may contribute to hesitation, including fear of harmful ingredients in vaccines, distrust of pharmaceutical companies, lack of knowledge about COVID-19, belief that a healthy lifestyle and a good diet are enough to fight against COVID-19, preference for natural immunity, lack of seriousness from the government in promoting vaccination programs, religious rules suggesting not to vaccinate, and limited information regarding the safety of vaccination. These factors have been reported to trigger hesitancy and refusal of the COVID-19 vaccination.4–7 In our previous study, we had explored the global prevalence of COVID-19 vaccination hesitancy and its potential associated factors.8 However, because the hesitancy population consists of hesitancy and refusal populations, and the refusal population can influence people in their circle to become hesitant or refuse the COVID-19 vaccine, the prevalence of the COVID-19 vaccine refusal should also be investigated.\n\nIt is widely known that new vaccines or vaccine candidates are commonly hesitated against or rejected by the public. Before the COVID-19 pandemic, this phenomenon has been widely reported in several cases, such as: dengue,9 malaria,10 Ebola,11 chikungunya,12 and monkeypox.13 This might be caused by poor public knowledge regarding the vaccine, including the poor knowledge of vaccine efficacy and side effects. In the case of COVID-19, this phenomenon might be affected by multiple factors, and theoretically, the factors had been contextualized into three major categories, including poor knowledge of vaccination programs, socioeconomic status, and social interaction.14 Moreover, recently, influencers in their podcasts discussed the rejection of the COVID-19 vaccine, which is a dilemma that can govern people in society to reject COVID-19 vaccinations, thereby threatening the success of the COVID-19 vaccination program.15 However, to date, there are no precise data on the prevalence of COVID-19 vaccination refusal and its potential associated factors. Several previous studies have investigated the refusal of COVID-19 vaccines; however, the results of these studies have been inconclusive. In the present study, we seek to explore the global prevalence of COVID-19 vaccination refusal and identify the associated factors using a meta-analysis approach.\n\n\nMethods\n\nA meta-analysis following the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) protocol was carried out from August to November 2022 (PROSPERO: CRD42022384562). The PRISMA checklist in our study is provided in Figshare.16 A systematic search was implemented in PubMed, Scopus, and Web of Science; and the information was collected from each relevant article to determine the prevalence and associated factors of COVID-19 vaccines refusal.\n\nPre-defined eligibility criteria were assigned prior to performing the search strategy. The inclusion criteria were: (1) assessment of the prevalence of COVID-19 vaccination refusal, and (2) investigation of the factors associated with COVID-19 vaccination refusal. Articles with double publications, letters to the editor, commentaries, and reviews were excluded.\n\nPubMed, Scopus, and Web of Science were searched up to November 5th, 2022. Before conducting a search for the primary outcome, we identified the factors that might have an impact on the incidence of refusal of COVID-19 vaccines. The potential keywords adapted from medical subject headings were applied: “vaccine,” “vaccination,” or “immunization;” “COVID-19” or “coronavirus disease 2019;” “refusal” or “rejection” or “acceptance.” The search strategy used only English words. In case of duplication, articles with a lower sample size used in the study were excluded. Moreover, to acquire additional references, a search on the reference list of related articles was also carried out. A pilot form was used to collect data from each study and consisted of the following items: (1) first author name, (2) time of publication, (3) design of study, (4) study period, (5) Newcastle–Ottawa scale (NOS), (6) the event rate of COVID-19 vaccination refusal, and (7) factors associated with COVID-19 vaccination refusal. Data were collected by FT, JKF, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, and YSP.\n\nThe NOS was used to assess the quality of potential articles. We included articles of moderate and high quality, and low quality articles were excluded. A score of 0–3, 4–6, and 7–9 indicated low, moderate, and high quality articles, respectively. The NOS assessment was performed by JKF, APK, and EAP using the NOS pilot form. Disagreements were resolved through discussion.\n\nThe primary endpoints of our study were the global prevalence and factors associated with COVID-19 vaccination refusal. The potential factors associated with the refusal of COVID-19 vaccination were: age group, gender, marital status, educational attainment, employment status, healthcare-related job, socioeconomic status (SES), urbanity, presence of children and elderly people at home, individual with medical background, history of testing for COVID-19, family member/friend ever diagnosed with COVID-19, personal history of COVID-19 diagnosis, history of hospitalization due to COVID-19 among people in a social circle, and history of previous vaccination.\n\nData are presented as n (%). The statistical analysis consisted of the following parameters: publication bias among studies, heterogeneity among studies, event rate, and odds ratio with a 95% confidence interval (OR95%CI). Publication bias was assessed using Egger’s test. A p-value of less than 0.05 indicated that publication bias existed among studies. The heterogeneity in our study was determined using the Q test. Evidence of heterogeneity was found if the p-value was less than 0.10. If we found heterogeneity among studies, we applied the random effects model, and in cases where no heterogeneity was found, we used a fixed effects model. The cumulative event rate of COVID-19 vaccine refusal was calculated using a single-arm meta-analysis with a dichotomous model, and the pooled OR95%CIs of factors associated with the refusal of COVID-19 vaccination were calculated using the Mantel-Haenszel method. The analysis was performed using the R package (RStudio version 4.1.1, R Studio, California, MA, USA).\n\n\nResults\n\nA total of 3,422 papers and 4 papers were assessed from the databases and reference lists of related articles, respectively. In the initial evaluation, we excluded 33 papers because of duplication, and 3,318 papers because of irrelevant topics. Subsequently, 75 articles were included in further review. We further excluded 17 articles as they were reviews and 34 articles because of insufficient data. Finally, the data retrieved from 24 articles were analyzed to estimate the cumulative prevalence and factors associated with COVID-19 vaccination refusal.17–40 The flow diagram of article selection in our study is outlined in Figure 1, and the characteristics of the articles included in our study are listed in Table 1.\n\nOur analysis identified that the cumulative prevalence of the refusal to COVID-19 vaccination was 12% (event rate: 0.12; 95%CI: 0.10, 0.15; p Egger: 0.5290; p Heterogeneity<0.0001; p<0.0001) (Figure 2A). Subsequently, sub – group analysis found that the prevalence of the refusal to COVID-19 in general population was 20% (Figure 2B), healthcare workers 10% (Figure 2C), and students 11% (Figure 2D).\n\nA). All prevalence of COVID-19 refusal (Event rate: 0.12; 95%CI: 0.10, 0.15; p Egger: 0.5290; p Heterogeneity<0.0001; p<0.0001).\n\nB). The prevalence in general population subgroup (Event rate: 0.20; 95%CI: 0.15, 0.26; p Egger: 0.4100; p Heterogeneity<0.0001; p<0.0001).\n\nC). The prevalence in healthcare workers subgroup (Event rate: 0.10; 95%CI: 0.07, 0.14; p Egger: 0.6760; p Heterogeneity<0.0001; p<0.0001).\n\nD). The prevalence in student subgroup (Event rate: 0.11; 95%CI: 0.06, 0.20; p Egger: 0.5830; p Heterogeneity<0.0001; p<0.0001).\n\nTable 2 and Figures 3–5 summarize the factors associated with refusal of COVID-19 vaccination. Our calculation revealed that six of the 15 factors had a significant impact on COVID-19 vaccine refusal. We found that an increased risk of COVID-19 vaccine refusal was observed in the following covariates: female (Figure 3A), educational attainment lower than an undergraduate degree (Figure 4A) and living in rural areas (Figure 5B).\n\nIn contrast, the decreased risk of refusal of COVID-19 vaccination was affected by the following factors: high socioeconomic status (Figure 5A), history of previous vaccination (Figure 3B), and individuals with medical background (Figure 4B).\n\nOur analysis using the Q test revealed that two variables (single marital status and history of testing for COVID-19) had no evidence of heterogeneity; thereafter, we applied a fixed-effects model. In contrast, a random-effects model was applied to the other covariates (Table 2). Subsequently, our analysis using Egger’s test revealed that the marital status and ever tested for COVID-19 covariates had a risk of publication bias (Table 2).\n\n\nDiscussion\n\nOur meta-analysis revealed that the prevalence of refusal to undergo the COVID-19 vaccination was 12%. Our findings were lower than those reported by Cenat et al. and Robinson et al.41,42 In our study, we had a larger sample size than those reported by in these studies. Moreover, studies by Cenat et al. and Robinson et al. also involved articles that reported COVID-19 vaccination hesitancy.41,42 It is well known that the terminologies of refusal and hesitancy to vaccinate are different, and not everyone is hesitant to vaccinate. Thus, it can be assumed that the context of previous studies has a gap in the definition of vaccine refusal. Therefore, our study may provide better data on the prevalence rate of COVID-19 vaccination refusal. Moreover, we also reported the prevalence of COVID-19 vaccination refusal in some subgroup populations: general population, healthcare workers, and students. We found that healthcare workers had the lowest prevalence of COVID-19 vaccination refusal, followed by students, and the general population. Our current findings indicate that vaccination knowledge might affect our findings. We assumed that healthcare workers and students may have a better knowledge of vaccination programs than the general population. This assumption is supported by the results of previous studies, which found that healthcare workers and students had better knowledge of COVID-19 vaccination than the general population,43,44 and this factor was also shown to contribute to the acceptance of vaccination programs.45\n\nOur study found that the increased risk of COVID-19 vaccination refusal was higher in females and individuals with educational levels below an undergraduate degree (BSc). In contrast, lower risk of COVID-19 vaccination refusal was found in individuals with a history of previous vaccination and medical background. Our current findings suggest that the factors related to knowledge of COVID-19 vaccination had the potential to affect the refusal to vaccinate against COVID-19. As previously reported, a study revealed that females lacked literacy regarding COVID-19 vaccination than males.46 This may be due to the fact that the majority of females are housewives, and therefore, may have less social interaction than males, as they are based at home rather than going out to work.47 This possibility might contribute to the lack of knowledge on COVID-19 vaccination in the female population. Furthermore, one study found that the majority of the side effects of COVID-19 vaccination were found among female individuals48 Taken together, those factors may affect the decision to accept or refuse the vaccines. Moreover, individuals with educational level below the undergraduate (BSc) degree might have an inadequate source of literature regarding COVID-19 vaccination compared to those with an educational level higher than an undergraduate (BSc) degree. In the context of vaccination knowledge, a study found that educational attainment was one of the predictors of vaccination knowledge, where lower educational attainment was associated with poorer knowledge of vaccination.49 Therefore, the population with an educational level below the undergraduate (BSc) degree might have insufficient consideration for COVID-19 vaccination compared to those with an educational level higher than the undergraduate (BSc) degree. Further, individuals with a history of previous vaccination and a medical background may have adequate information regarding the importance of COVID-19 vaccination, therefore, may have sufficient awareness regarding COVID-19 vaccination. Previous studies found that individuals with a medical background had better knowledge of COVID-19 vaccination than the general population.43,44 Likewise, a study revealed that individuals with a history of previous annual vaccination had good awareness and knowledge of the importance of vaccination programs.50 Prior to the COVID-19 pandemic, studies have widely reported that knowledge on disease prevention and the good practice of health behavior had a significant impact on the acceptance rate of vaccination in the case of Monkeypox, Ebola, and Dengue.51–53 Thus, this might imply that this population (individuals with a history of previous vaccination and medical background) has a low rate of refusal to vaccinate against COVID-19, as reported in our meta-analysis.\n\nOur study also found that a higher risk of COVID-19 vaccination refusal was observed in rural than urban populations, and a lower risk of COVID-19 vaccination refusal was observed in individuals with high SES than in those with low SES. Currently, the precise explanations regarding the reasons underlying our findings might be difficult to explain and might have differences between each region. However, we can propose the following reasons: social privileges, administrative requirements, and social circles. First, in the social privilege aspect, individuals with high SES might have certain pride if they have been vaccinated, while this pride might not belong to the rural population. Studies found that COVID-19 vaccination was considered a socioeconomic privilege and political ideology,54 while the rural population did not consider that the COVID-19 vaccine was a privilege and tended to have poor perception toward vaccine safety.55 The second reason is administrative requirements. Individuals with high SES might need COVID-19 vaccination as an administrative requirement for their activities; for example, business, travelling, and carrier requirements as the World Health Organization (WHO) has implemented a COVID-19 vaccine certificate as an administrative requirement for travel or business.56 However, these administrative requirements were not necessary for rural individuals, as the majority of rural individual jobs are in private and traditional sectors, such as farmers, fishermen, and manual laborers.57,58 The third factor is social circle. Individuals with high SES might have social circles that interact with high intellectual content, whereas in rural populations, their social circle might be limited to their neighbors with similar intellectual contents. This factor might also indirectly contribute to the understanding of COVID-19 vaccinations, and consequently, affect their decision to accept or refuse the COVID-19 vaccine. This is supported by previous studies that revealed that SES was associated with the level of knowledge of vaccination programs and physical health status.59,60 Moreover, our previous study on dengue also revealed that SES was one of the predictive indicators for the acceptance of vaccination.53\n\nOur meta-analysis is one of the first to report the prevalence of COVID-19 vaccination refusal and the potential factors associated with refusal of COVID-19 vaccination. Our study also had a larger sample size compared to previous meta-analyses in a similar context.41,42 The findings of our study might serve as the initial step to prevent the failure of COVID-19 vaccination programs. By identifying the potential factors associated with refusal to vaccinate against COVID-19, we expect that governments may provide advanced intervention to those populations. As previously reported, the main concern to obtain public trust regarding the COVID-19 vaccines was the lack of adequate evidence from long-term and large-scale studies on the effectiveness and safety of COVID-19 vaccination.61 However, several studies have provided suggestions on how to provide interventions to the refusal population: providing reliable information regarding the COVID-19 pandemic and the COVID-19 vaccination, providing customized, effective, ethical, and evidence-based communication; and the information should be provided by community leaders and healthcare practitioners.62–64\n\nOur meta-analysis has several limitations. First, several potential confounding factors, such as the level of knowledge about COVID-19 vaccination, attitude toward COVID-19 prevention, government regulation, types of vaccine, environmental factors, and the source of literature regarding COVID-19 vaccination, were not included in the analysis due to the lack of available data. Second, the sample size in our present study was limited; therefore, further studies involving larger sample sizes are needed. Third, our meta-analysis could not reflect the prevalence of the global numbers because the proportion of sample sizes in each region was unequal. Fourth, all articles included in our analysis were cross-sectional studies. Further meta-analyses involving only randomized controlled trials are required.\n\n\nConclusion\n\nIn conclusion, we revealed that the cumulative prevalence of the refusal to COVID-19 vaccination was 12%, the highest in the general population, and the lowest in the healthcare worker subgroup. The individuals with the following characteristics are found increased risk to refuse COVID-19 vaccination: female, having an educational attainment lower than an undergraduate degree, and living in a rural area, while the following factors are associated with reduced risk to refuse COVID-19 vaccination: high socioeconomic status, history of previous vaccination, and individuals with a medical background.\n\n\nAuthor contribution\n\nConceptualization: FT, JKF, GS; Data Curation: FT, JKF, GS, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Formal Analysis: JKF, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Investigation: FT, JKF, GS, LW, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Project Administration: APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Resources: APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Methodology: FT, JKF, GS, LW, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Software: FT, JKF, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Visualization: APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP, CC, KD, HH; Supervision: FT, JKF, GS, LW, CC, KD, HH; Validation: FT, JKF, LW, CC, KD, HH; Writing – Original Draft Preparation: FT, JKF, LW, APK, EAP, RPP, MR, TA, MN, SW, GP, AP, QA, MVPHM, RY, YSP; Writing – Review & Editing: FT, JKF, GS, LW, CC, KD, HH. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional sources of data are required.\n\nFigshare: PRISMA checklist for ‘The refusal of COVID-19 vaccination and its associated factors: A meta-analysis’. https://doi.org/10.6084/m9.figshare.21617979.v1. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Lembaga Pengelola Dana Pendidikan (LPDP) and Kementerian Pendidikan, Kebudayaan, Riset, dan Teknologi (Kemendikbudristek) Republic of Indonesia for supporting this project.\n\n\nReferences\n\nCarneiro DC, Sousa JD, Monteiro-Cunha JP: The COVID-19 vaccine development: A pandemic paradigm. Virus Res. 2021; 301: 198454. Epub 2021/05/21. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKose S, Mandiracioglu A, Sahin S, et al.: Vaccine hesitancy of the COVID-19 by health care personnel. Int. J. Clin. Pract. 2021; 75(5): e13917. Publisher Full Text\n\nKozak A, Nienhaus A: COVID-19 Vaccination: Status and Willingness to Be Vaccinated among Employees in Health and Welfare Care in Germany. Int. J. Environ. Res. Public Health. 2021; 18(13). Epub 2021/07/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManning ML, Gerolamo AM, Marino MA, et al.: COVID-19 vaccination readiness among nurse faculty and student nurses. Nurs. Outlook. 2021; 69(4): 565–573. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMena G, Blanco B, Casas I, et al.: Attitudes of Spanish hospital staff towards COVID-19 vaccination and vaccination rates. PLoS One. 2021; 16(9): e0257002. Epub 2021/09/11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOusseine YM, Vaux S, Vandentorren S, et al.: Predictors of Uncertainty and Unwillingness to Receive the COVID-19 Vaccine in Men Who Have Sex with Men in France. Int. J. Environ. Res. Public Health. 2022; 19(9). Epub 2022/05/15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParis C, Benezit F, Geslin M, et al.: COVID-19 vaccine hesitancy among healthcare workers. Infect. Dis. Now. 2021; 51(5): 484–487. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPataka A, Kotoulas S, Stefanidou E, et al.: Acceptability of Healthcare Professionals to Get Vaccinated against COVID-19 Two Weeks before Initiation of National Vaccination. Medicina (Kaunas). 2021; 57(6). Epub 2021/07/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodriguez-Blanco N, Montero-Navarro S, Botella-Rico JM, et al.: Willingness to Be Vaccinated against COVID-19 in Spain before the Start of Vaccination: A Cross-Sectional Study. Int. J. Environ. Res. Public Health. 2021; 18(10). Epub 2021/06/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaied SM, Saied EM, Kabbash IA, et al.: Vaccine hesitancy: Beliefs and barriers associated with COVID-19 vaccination among Egyptian medical students. J. Med. Virol. 2021; 93(7): 4280–4291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwarzinger M, Watson V, Arwidson P, et al.: COVID-19 vaccine hesitancy in a representative working-age population in France: a survey experiment based on vaccine characteristics. Lancet Public Health. 2021; 6(4): e210–e221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaw J, Stewart T, Anderson KB, et al.: Assessment of US Healthcare Personnel Attitudes Towards Coronavirus Disease 2019 (COVID-19) Vaccination in a Large University Healthcare System. Clin. Infect. Dis. 2021; 73(10): 1776–1783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpinewine A, Petein C, Evrard P, et al.: Attitudes towards COVID-19 Vaccination among Hospital Staff-Understanding What Matters to Hesitant People. Vaccines (Basel). 2021; 9(5). Epub 2021/06/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVignier N, Brureau K, Granier S, et al.: Attitudes towards the COVID-19 Vaccine and Willingness to Get Vaccinated among Healthcare Workers in French Guiana: The Influence of Geographical Origin. Vaccines (Basel). 2021; 9(6). Epub 2021/07/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang K, Wong ELY, Ho KF, et al.: Intention of nurses to accept coronavirus disease 2019 vaccination and change of intention to accept seasonal influenza vaccination during the coronavirus disease 2019 pandemic: A cross-sectional survey. Vaccine. 2020; 38(45): 7049–7056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson E, Jones A, Lesser I, et al.: International estimates of intended uptake and refusal of COVID-19 vaccines: A rapid systematic review and meta-analysis of large nationally representative samples. Vaccine. 2021; 39(15): 2024–2034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenat JM, Noorishad PG, Moshirian Farahi SMM, et al.: Prevalence and factors related to COVID-19 vaccine hesitancy and unwillingness in Canada: A systematic review and meta-analysis. J. Med. Virol. 2022. Epub 2022/09/17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJi W, Camara MA, Xia J, et al.: Knowledge, Attitude and Practice Toward COVID-19: A Cross-Sectional Study of Staff in China-Guinea Friendship Hospital. Guinea. Front Public Health. 2022; 10: 889227. Epub 2022/06/17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarapan H, Wagner AL, Yufika A, et al.: Acceptance of a COVID-19 Vaccine in Southeast Asia: A Cross-Sectional Study in Indonesia. Front. Public Health. 2020; 8: 381. Epub 2020/08/08. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalle F, Sabella EA, Roma P, et al.: Knowledge and Acceptance of COVID-19 Vaccination among Undergraduate Students from Central and Southern Italy. Vaccines (Basel). 2021; 9(6). Epub 2021/07/03. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIshimaru T, Okawara M, Ando H, et al.: Gender differences in the determinants of willingness to get the COVID-19 vaccine among the working-age population in Japan. Hum. Vaccin. Immunother. 2021; 17(11): 3975–3981. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeong S, Cho SI: Concordance in the Health Behaviors of Couples by Age: A Cross-sectional Study. J. Prev. Med. Public Health. 2018; 51(1): 6–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Qazaz HK, Al-Obaidy LM, Attash HM: COVID-19 vaccination, do women suffer from more side effects than men? A retrospective cross-sectional study. Pharm. Pract (Granada). 2022; 20(2): 2610–2678. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVoo JYH, Lean QY, Ming LC, et al.: Vaccine Knowledge, Awareness and Hesitancy: A Cross Sectional Survey among Parents Residing at Sandakan District, Sabah. Vaccines (Basel). 2021; 9(11). Epub 2021/11/28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarry MA, Aljammaz KI, Alrashed AA: Knowledge, Attitude, and Barriers Influencing Seasonal Influenza Vaccination Uptake. Can. J. Infect. Dis. Med. Microbiol. 2020; 2020: 7653745. Epub 2020/10/31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarapan H, Wagner AL, Yufika A, et al.: Acceptance and willingness to pay for a hypothetical vaccine against monkeypox viral infection among frontline physicians: A cross-sectional study in Indonesia. Vaccine. 2020; 38(43): 6800–6806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMudatsir M, Anwar S, Fajar JK, et al.: Willingness-to-pay for a hypothetical Ebola vaccine in Indonesia: A cross-sectional study in Aceh. F1000Res. 2019; 8: 1441. Epub 2020/07/29. 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. Archives of. Clin. Infect. Dis. 2017; 12(3). Publisher Full Text\n\nAgarwal R, Dugas M, Ramaprasad J, et al.: Socioeconomic privilege and political ideology are associated with racial disparity in COVID-19 vaccination. Proc. Natl. Acad. Sci. U. S. A. 2021; 118(33). Epub 2021/07/31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarter J, Rutherford S, Borkoles E: COVID-19 Vaccine Uptake among Younger Women in Rural Australia. Vaccines (Basel). 2021; 10(1). Epub 2022/01/23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCascini F, Causio FA, Failla G, et al.: Emerging Issues From a Global Overview of Digital Covid-19 Certificate Initiatives. Front. Public Health. 2021; 9: 744356. Epub 2021/12/07. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPattayat SS, Parida JK, Awasthi IC: Reducing Rural Poverty Through Non-farm Job Creation in India. Indian J. Labour Econ. 2022; 65(1): 137–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar A, Kumar S, Singh DK: Rural employment diversification in India: trends, determinants and implications on poverty. Agric. Econ. Res. Rev. 2011; 24(conf): 361–372.\n\nWang Y, Yang C, Zhang Y, et al.: Socioeconomic Status and Prosocial Behavior: The Mediating Roles of Community Identity and Perceived Control. Int. J. Environ. Res. Public Health. 2021; 18(19). Epub 2021/10/14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWirawan GBS, Harjana NPA, Nugrahani NW, et al.: Health Beliefs and Socioeconomic Determinants of COVID-19 Booster Vaccine Acceptance: An Indonesian Cross-Sectional Study. Vaccines (Basel). 2022; 10(5). Epub 2022/05/29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodriguez-Morales AJ, Franco OH: Public trust, misinformation and COVID-19 vaccination willingness in Latin America and the Caribbean: today's key challenges. Lancet Reg. Health Am. 2021; 3: 100073. Epub 2021/09/16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRatzan SC: “Vaccine Communication in a Pandemic: Improving Vaccine Literacy to Reduce Hesitancy, Restore Trust and Immunize Communities”: Editor’s Introduction. J. Health Commun. 2020; 25(10): 745–746. Publisher Full Text\n\nHussain B, Latif A, Timmons S, et al.: Overcoming COVID-19 vaccine hesitancy among ethnic minorities: A systematic review of UK studies. Vaccine. 2022; 40(25): 3413–3432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGendler Y, Ofri L: Investigating the Influence of Vaccine Literacy, Vaccine Perception and Vaccine Hesitancy on Israeli Parents' Acceptance of the COVID-19 Vaccine for Their Children: A Cross-Sectional Study. Vaccines (Basel). 2021; 9(12). Epub 2021/12/29. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "190442", "date": "09 Aug 2023", "name": "Amy Morrison", "expertise": [ "Reviewer Expertise Dengue epidemiology", "arbovirus epidemiology", "systematic review" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents results from a systematic review and meta-analysis on the global prevalence of COVID-19 vaccine refusal factors that increase or decrease the risk of refusal. This manuscript follows the publication from the same group titled “Global Prevalence and Potential Influencing Factors of COVID-19 Vaccination Hesitance: A Meta-Analysis” which includes considerable overlap with the current article. The important distinction is summarized by the following difference in the search strategy: search term HESITANCY (original article) replaced by terms REFUSAL or REJECTION. Interestingly, this subtle distinction is important, and should be a key message and be the focus of the introduction, and the contrasts between this manuscript highlighted throughout the methods and discussion sections. For example, vaccine hesitancy represents a continuum from complete acceptance to refusal to be vaccinate under any circumstances. The authors need to clearly describe how they are making this distinction in their article assessment and how actual vaccination rates equate to refusal. That is, in the included studies was the survey methodology able to distinguish between individuals who will never allow vaccination versus those who might opt be able to overcome their hesitancy. I agree that there is a distinction between hesitancy and refusal, but how to clearly measure that distinction requires more explanation. This is clearly an important issue that merits publication, the analysis appears rigorous and appropriate, but could be better contextualized for readers. It is also important to contrast this study with the previous study by this group. Below I provide general and specific comment I hope the authors will consider.\nGeneral Comments:\nAs mentioned above the distinction between vaccination refusal and hesitancy needs to be clearly described, in the introduction of the manuscript. The authors do point out why the distinction is important, but they could elaborate more. But most importantly, how can we make this distinction within the evaluated studies. Your search strategy was slightly different and despite this search extending to November 2022 in comparison to your previously published study extending through May 2022, a total of 56 and 24 manuscripts were evaluated, respectively. I want to know if some of those manuscripts from the previous review, that were not included in the review would have been appropriate for the current analysis. Again, contrasting this information with your previous publication would be helpful.\nOne co-variate which is mentioned but not included in your analysis is country. This seems like it could be an important factor. One objective for systematic reviews is the identification of clear knowledge gaps, which deserves a strong place in your discussion. The absence of studies in LMICs is concerning. I would also argue that the issue of refusal/hesitancy and how we really identify these hard-core refusers is important and the follow up to that is how many of these people are sources of misinformation, another potential gap.\nContext for your meta-analysis statistics: Although you report Egger and heterogeneity (Q) scores it is no clear readers unfamiliar with them what they are telling us.\nThe manuscript needs further editorial review, there are numerous grammatical errors, missing words, etc. Overall, the article was understandable, but the errors did reduce the quality of the article.\nConsider providing a discussion of the range of prevalence values in addition to the model output, but there is considerable variability shown in the figures.\nI’m not an expert in Meta-Analysis, but I’m curious is a multivariate model is possible since many of the factors you are analyzing could be confounders.\n\nHigh socioeconomic status is significant, but marginally, that deserves a bit more emphasis.\nAgain, more discussion on the continuum of hesitancy to refusal is needed, more context about the included studies, what the key questions were and how populations were selected.\nSpecific Comments\nTitle: consider including Meta-analysis is the title, you are doing both.\nStatistical analysis: How is the Egger and Q statistics interpreted beyond the p-value. Do they range from 0-1 with…\nIntroduction: See my general comments. Another model which is not mentioned in your manuscript which I think could be helpful is Larson et al. 20141 that describes the 3 C’s model (Complacency, convenience, and confidence). Your article focuses on confidence rather than the other factors.\nTable 1. Consider including a column with a summary of the study findings. Also, abbreviations should be specified in the Table legend. You could eliminate the study design column as you state in the text that all the studies were cross-sectional.\nDiscussion, last paragraph: “Fourth, all articles included in our analysis were cross-sectional studies, further meta-analyses involving only randomized controlled trials are required.” Please expand, having a hard time imagining this design for this question. What intervention would be randomized here. Sample selection is an important issue here. That is, how was a representative sample achieved with in individual studies, but beyond cross-sectional study we are give no additional information on sampling strategy, if the study was properly powered etc. This seems like an important issue, that may be reflected in the NOS score but not shared with the reader.\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": [ { "c_id": "10942", "date": "20 Mar 2024", "name": "Jonny Fajar", "role": "Author Response", "response": "This manuscript presents results from a systematic review and meta-analysis on the global prevalence of COVID-19 vaccine refusal factors that increase or decrease the risk of refusal. This manuscript follows the publication from the same group titled “Global Prevalence and Potential Influencing Factors of COVID-19 Vaccination Hesitance: A Meta-Analysis” which includes considerable overlap with the current article. The important distinction is summarized by the following difference in the search strategy: search term HESITANCY (original article) replaced by terms REFUSAL or REJECTION. Interestingly, this subtle distinction is important, and should be a key message and be the focus of the introduction, and the contrasts between this manuscript highlighted throughout the methods and discussion sections. For example, vaccine hesitancy represents a continuum from complete acceptance to refusal to be vaccinate under any circumstances. The authors need to clearly describe how they are making this distinction in their article assessment and how actual vaccination rates equate to refusal. That is, in the included studies was the survey methodology able to distinguish between individuals who will never allow vaccination versus those who might opt be able to overcome their hesitancy. I agree that there is a distinction between hesitancy and refusal, but how to clearly measure that distinction requires more explanation. This is clearly an important issue that merits publication, the analysis appears rigorous and appropriate, but could be better contextualized for readers. It is also important to contrast this study with the previous study by this group. Below I provide general and specific comment I hope the authors will consider. General Comments: As mentioned above the distinction between vaccination refusal and hesitancy needs to be clearly described, in the introduction of the manuscript. The authors do point out why the distinction is important, but they could elaborate more. But most importantly, how can we make this distinction within the evaluated studies. Response: We sincerely appreciate the insightful guidance given. The difference between vaccine hesitancy and refusal has been clarified in our introduction. Hesitancy signifies uncertainty about accepting the vaccine, whereas refusal pertains to outright rejection. As a meta-analysis, our study involves extracting data from prior publications within the same context, and details regarding vaccine refusal are sourced from studies explicitly stating results using terms such as refusal or rejection. However, we have also included additional explanations about the distinction between vaccine hesitancy and refusal in the introduction section. Your search strategy was slightly different and despite this search extending to November 2022 in comparison to your previously published study extending through May 2022, a total of 56 and 24 manuscripts were evaluated, respectively. I want to know if some of those manuscripts from the previous review, that were not included in the review would have been appropriate for the current analysis. Again, contrasting this information with your previous publication would be helpful. Response: We appreciate the invaluable advice. Indeed, our study is a continuation of our previous study on vaccine hesitancy. From the papers included in our earlier study, we have also incorporated several papers into our current study. These papers contain data specifically related to vaccine rejection or refusal. One co-variate which is mentioned but not included in your analysis is country. This seems like it could be an important factor. One objective for systematic reviews is the identification of clear knowledge gaps, which deserves a strong place in your discussion. The absence of studies in LMICs is concerning. I would also argue that the issue of refusal/hesitancy and how we really identify these hard-core refusers is important and the follow up to that is how many of these people are sources of misinformation, another potential gap. Response: We appreciate the highly valuable advice. We wholeheartedly agree with your suggestion. The country is a crucial variable in the context of vaccine refusal. Factors influencing vaccine rejection behavior may vary across different countries. Initially, we aimed to create sub-groups based on countries; however, this was not feasible due to data limitations. Forcing this could potentially introduce a high level of data bias. Nevertheless, we have included the country factor as one of the limitations in our study. Context for your meta-analysis statistics: Although you report Egger and heterogeneity (Q) scores it is no clear readers unfamiliar with them what they are telling us. Response: We sincerely appreciate your advice. The Egger test for assessing potential publication bias and the Q test for evaluating heterogeneity among studies are actually quite familiar in the context of meta-analysis. Most meta-analysis software includes these features (Egger and Q test). The manuscript needs further editorial review, there are numerous grammatical errors, missing words, etc. Overall, the article was understandable, but the errors did reduce the quality of the article. Response: We appreciate the very important advice. We have re-evaluated the grammatical aspects of our article. Consider providing a discussion of the range of prevalence values in addition to the model output, but there is considerable variability shown in the figures. Response: We are very grateful for the highly valuable advice. Indeed, the prevalence data for vaccine refusal exhibits variability across each study. However, here we report the cumulative prevalence of vaccine refusal, in accordance with our study method, which is meta-analysis. I’m not an expert in Meta-Analysis, but I’m curious is a multivariate model is possible since many of the factors you are analyzing could be confounders. Response: We fully agree with your suggestion. Due to the multitude of factors analyzed, the likelihood of confounding factors is also quite high. Unfortunately, in meta-analysis, we cannot eliminate confounding factors that we consider to have an impact if the data is not present in the studies we include. Nevertheless, we have outlined the potential confounding factors in the limitations section of our study. High socioeconomic status is significant, but marginally, that deserves a bit more emphasis. Response: Thank you for your highly valuable advice. We have added emphasis on socioeconomic status in the discussion. Again, more discussion on the continuum of hesitancy to refusal is needed, more context about the included studies, what the key questions were and how populations were selected. Response: We appreciate your very important advice. We have added a discussion on the contextual differences between vaccine refusal and hesitancy. Additionally, we have included key questions in the search strategy, and we have also added the study population in the data extraction. Specific Comments Title: consider including Meta-analysis is the title, you are doing both. Response: Thank you for your highly valuable advice. We have revised our title according to your suggestion. Statistical analysis: How is the Egger and Q statistics interpreted beyond the p-value. Do they range from 0-1 with… Response: Thank you for your question. The interpretation of the Egger test is based on the p-value, where a p-value below 0.05 is considered to indicate potential publication bias. Regarding the Q test for evaluating heterogeneity among studies, the interpretation relies on the p-value, with a value above 0.10 indicating no heterogeneity, while a value below 0.10 suggests the presence of heterogeneity. In addition to assessing heterogeneity, we have included I-squared. If the I-squared value is above 50%, it indicates heterogeneity, whereas if it's less than 50%, it suggests no heterogeneity. If there is heterogeneity, the analysis model uses a random-effects model, while if there is no heterogeneity, the analysis model employs a fixed-effects model. The explanation regarding the interpretation of the assessment of potential publication bias and heterogeneity has been elaborated in the sub-heading \"statistical analysis.\" Introduction: See my general comments. Another model which is not mentioned in your manuscript which I think could be helpful is Larson et al. 20141 that describes the 3 C’s model (Complacency, convenience, and confidence). Your article focuses on confidence rather than the other factors. Response: Thank you for your suggestion. We have added an additional discussion regarding the 3Cs model. Table 1. Consider including a column with a summary of the study findings. Also, abbreviations should be specified in the Table legend. You could eliminate the study design column as you state in the text that all the studies were cross-sectional. Response: We appreciate your crucial advice. We have revised Table 1 by adding study findings and eliminating study designs since all studies have a cross-sectional design. Instead, we have explained in the text that all studies have a cross-sectional design. Additionally, we have also added abbreviations explanations in the table legend. Discussion, last paragraph: “Fourth, all articles included in our analysis were cross-sectional studies, further meta-analyses involving only randomized controlled trials are required.” Please expand, having a hard time imagining this design for this question. What intervention would be randomized here. Sample selection is an important issue here. That is, how was a representative sample achieved with in individual studies, but beyond cross-sectional study we are give no additional information on sampling strategy, if the study was properly powered etc. This seems like an important issue, that may be reflected in the NOS score but not shared with the reader. Response: Thank you for your valuable advice. We wholeheartedly agree with your suggestion. The study limitations regarding the design of included studies have been revised. Furthermore, the sample selection method of included studies has also been added to Table 1. As for the NOS assessment results, we have included this information in the Table 1." } ] }, { "id": "201174", "date": "30 Aug 2023", "name": "Angelo Capodici", "expertise": [ "Reviewer Expertise Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRegarding the question of whether ample methodological and analytical details are furnished to facilitate replication by fellow researchers, my response leans toward the negative. The omission of the search string and the absence of a reported PRISMA checklist, despite claims of adhering to PRISMA guidelines, hinder the transparency and reproducibility of the study. It's worth noting that when delving into the assessment of vaccine hesitancy, a well-defined framework should ideally be employed. Notably, the widely recognized BESD framework by WHO stands out as a prominent choice. Disappointingly, the authors of the study failed to acknowledge this framework through proper citation, consequently weakening the robustness of their conclusions.\nFinally, since they cite studies about willingness regarding COVID-19 vaccination, I should mention that no studies updated to 2023 are cited, which is a shame. I would recommend improving this. Here's a few ideas: Gori et al. (2023)1; Morgan et al. (2023)2; Huang et al. (2023)3.\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10943", "date": "20 Mar 2024", "name": "Jonny Fajar", "role": "Author Response", "response": "Regarding the question of whether ample methodological and analytical details are furnished to facilitate replication by fellow researchers, my response leans toward the negative. The omission of the search string and the absence of a reported PRISMA checklist, despite claims of adhering to PRISMA guidelines, hinder the transparency and reproducibility of the study. It's worth noting that when delving into the assessment of vaccine hesitancy, a well-defined framework should ideally be employed. Notably, the widely recognized BESD framework by WHO stands out as a prominent choice. Disappointingly, the authors of the study failed to acknowledge this framework through proper citation, consequently weakening the robustness of their conclusions. Response: We appreciate your comments and suggestions. Regarding the PRISMA guidance, we have included the PRISMA checklist in the supplementary files. We assure that all points from the PRISMA checklist for meta-analysis studies are documented in the supplementary files. Additionally, concerning the WHO's BESD framework, initially, we aimed to incorporate all its variables. However, in the context of meta-analysis, our ability to analyze specific variables depends on the data available from supporting studies. Nevertheless, we have added a discussion on the limitations related to the BESD framework in our study. Finally, since they cite studies about willingness regarding COVID-19 vaccination, I should mention that no studies updated to 2023 are cited, which is a shame. I would recommend improving this. Here's a few ideas: Gori et al. (2023)1; Morgan et al. (2023)2; Huang et al. (2023)3. Response: We sincerely appreciate your advice regarding additional studies that could serve as supplementary data. However, we have previously registered our study protocol in PROSPERO, and the three additional studies were published after the timeframe specified in our protocol. Our study protocol has received approval from PROSPERO, and adding these additional studies would result in our departure from the registered study protocol in PROSPERO." } ] } ]	1	https://f1000research.com/articles/12-54
https://f1000research.com/articles/11-1560/v1	22 Dec 22	{ "type": "Research Article", "title": "Until the dawn: everyday experiences of people living with COVID-19 during the pandemic in Thailand", "authors": [ "Ek-uma Imkome", "Kamonchanok Moonchai", "Kamonchanok Moonchai" ], "abstract": "Background: This study aims to understand and analyze the complex experiences of people living with COVID-19 to support future cases. Methods: Purposive and snowball sampling techniques were employed to collect data through in-depth interviews; the data were combined with non-aggregated behavioral observations. Lived experiences of 15 individuals were collected through semi-structured interviews and analyzed using a phenomenological-hermeneutic approach. We used the thematic analysis technique to analyze the data. Results: Through analysis, themes of stress, economic impact, social stigma, social support, and unexpected benefits were identified. Participants further expressed economic and social concerns during the interviews. Conclusion: The identified themes can help develop multidisciplinary treatment strategies that would be useful during a healthcare crisis, establish comprehensive support systems that could address economic and social problems, and provide training for employees for post-COVID-19 assistance.", "keywords": [ "COVID-19", "Mental health", "lived experiences", "phenomenological-hermeneutic approach", "stress", "economic impact", "social stigma", "social support", "multidisciplinary treatment strategies", "multidisciplinary treatment teams" ], "content": "Introduction\n\nOver 219 million cases of COVID-19 were reported globally until October 10, 2021. With 4.55 million fatalities and threats of new variants, the virus continues to evolve. To control COVID-19 outbreaks, governments and health experts worldwide have been experimenting with various vaccines, treatment modalities, new measures, and preventative strategies within a short timeframe. Lockdowns and lenient curbs have caused turmoil worldwide since the beginning of the pandemic. People have been trying to use several new coping strategies to deal with market turbulence (Nurunnabi et al., 2020). After its rapid spread worldwide, the number of people contracting COVID-19 and succumbing to it reached 512,963,517 and 6,259,611, respectively, by May 25, 2020 (World Meter, https://www.worldometers.info/coronavirus/). COVID-19 outbreaks affect the population’s immediate morbidity and mortality from COVID-19, disrupt the emergency care for non-COVID-19 conditions, change chronic illness care modality, and rapidly escalate the incidence of mental health problems such as stress, depression, and anxiety (Sing, et al., 2021). Approximately 20% of all COVID-19 cases may experience severe symptoms, and require oxygen therapy or other inpatient interventions, and only 5% require hospitalization in the intensive care unit (Wu & McGoogan, 2020). However, studies have reported that COVID-19 has a broader physical and psychosocial impact beyond acute symptoms. Patients with COVID-19 demonstrate various symptoms such as fever, dyspnea, muscle ache, headache, fear, diarrhea, nausea, vomiting, increased systolic blood pressure, and hemoptysis, all of which require invasive and non-invasive therapeutic support during the acute course of the disease (Davis et al., 2019; Huang et al., 2020). The mortality rate of COVID-19 has been estimated to range from 1% to 5%, but this varies depending on patients' age group and the presence or absence of underlying diseases. The epidemiological distribution of mental health problems and associated factors are heterogeneous among COVID-19 patients. The current evidence suggests that a psychiatric epidemic is co-occurring with the COVID-19 pandemic, which necessitates the attention of the global health community (Hossain, 2020). During the COVID-19 pandemic, patients have faced problems such as fear, loneliness, boredom, anger, anxiety, insomnia, and a feeling of taboo. Patients have also been concerned about the effect of being quarantined on their psychological well-being and the risk of infecting family members and community members.\n\nIn the initial crisis response to the pandemic, researchers primarily conducted epidemiological studies and clinical trials. However, qualitative research is needed to provide the nuance and detail of COVID-19 patients' lived reality and contextualized experiences. Such rich data can help develop interventions and policies alongside obtaining quantitative data (Tremblay et al., 2021). Besides, a few studies have explored the needs of COVID-19 cases, their stress and adaptation to the disease, and health support. Explaining the perspectives of people living with COVID-19 on their needs and the impact can help improve the quality of care and their quality of life. The study findings can guide policymakers in making public policy decisions and strategic plans for public health organizations, and provide accessibility of assessment and treatment services, thereby improving the quality of care provided to this population and ensuring research subsidy priorities at state, regional, and national levels. The findings can guide public mental health initiatives across multiple ecological levels that can mitigate the toll of COVID-19.\n\nThe theoretical framework of this study will help deepen our understanding of the lived experiences and the impact of COVID-19 on patients. The Roy adaptation model (Roy, 2001, 2009, 2011) states that the three elements are coping processes, adaptive modes, and adaptive levels. Coping processes are primary elements of understanding individual COVID-19 cases, their adaptation, and their life strategies for enhancing adaptation. Furthermore, the adaptive modes classify COVID-19 case data and help us understand this population's daily life and experiences of people living with COVID-19.\n\n\nMethods\n\nThis research applies narrative-based data collection procedures (NbDC). NbDC (Kirkpatrick, 2008), underpinning the phenomenological-hermeneutic approach, allows researchers to listen to and understand human experiences and contribute to planning quality of care in the future.\n\nFifteen participants were selected through purposeful sampling. As per the inclusion criteria, participants included COVID-19 survivors who had been discharged from the hospital and provided written consent for participation. COVID-19 cases that experienced stress or mental health problems were prevented from giving informed consent, and their participation was thus excluded.\n\nThe phenomenological-hermeneutic approach was applied through discussions after examining and interpreting lived experiences and perceptions related to the COVID-19 pandemic (Al Kalaldeh et al., 2018; Graor & Knapik, 2013; Kvale & Brinkmann, 2014). Dialogical conversations were conducted with participants who were in a vulnerable state. During the interviews, the interviewer used a checklist to report essential aspects of the research team, methods, context, findings, analysis, and interpretations (Tong et al., 2007).\n\nWe recruited participants through the nursing offices of a hospital in a different location in Thailand. First, participants' willingness to participate was sought through phone calls. Second, the nurse apprised the participants of the interview. Third, semi-structured individual in-depth video interviews were conducted via Microsoft Teams in a time slot of the participants’ preference after explaining the research goals and acquiring their verbal and written consent. The sampling process continued until data saturation, and the researcher, a psychiatric nurse, took on the role of interviewer to maintain a professional relationship with the participants. The duration of the interviews was between 25 and 60 minutes. Data were anonymized. In the final step, the transcripts were returned to participants for comment and/or correction.\n\nEthical approval was obtained from the Ethics Review Committee for Research Involving Human Research Participants (COA No. 119/2563) on 20 October 2020.\n\nThe research scope, risks, and benefits were explained to the participants; they were assured that anonymity and confidentiality would be maintained. They were informed that their participation in the research was voluntary (Imkome & Moonchai, 2022). We confirm that we obtained written and verbal consent to use data from the participants included in this study. The interview duration was determined based on the participants' preferences, patience, and experiences. All interviews were recorded on video.\n\nInterviews and data analysis were conducted simultaneously using the phenomenological-hermeneutic method (Kvale & Brinkmann, 2014). The three levels of description and interpretation were processed. The first level was self-understanding, wherein in the first readings, the authors created the narrative, summarized all the participants’ experiences, and protected data anonymity. The second level was structural understanding, as several readings of the transcription constructed general themes, and we named and interpreted the central theme. We analyzed the data using SPSS Modeler software (RRID:SCR_002865) for qualitative data analysis. The third level was a structural understanding that was extended through theoretical interpretation. The Roy adaptation model (Roy, 2011) was applied.\n\n\nResults\n\nThis section describes the participants' understanding through a structural account of the findings, followed by a theoretical interpretation of the results.\n\nOut of the 15 participants, 11.33% were unemployed, and 62.5% had an insufficient family income per month (Table 1). The data analysis led to four themes (Figure 1).\n\nThese early findings highlight how the comprehensive theme shaped by the participants' structural understanding brings attention to their perceptions of experiences and the impact of COVID-19. Major themes derived from content analysis were evident across interviews and included stress, economic and social impact, social stigma, and the concept that bad luck occasionally brings good luck, which was described in all interviews. Positive experiences of \"feeling good\" and \"social support\" were also emphasized throughout the transcripts (Figure 1).\n\nStress\n\nIn this context, stress refers to how people with COVID-19 perceive the infection and related circumstances as threatening and challenging to manage.\n\n\"It is stressful to be alone in the room. It was not easy to go out and find food. The city was locked down; what would happen next? Will our family be okay? Will the people who live in the community hate us? A disease spreading heavily in Thailand is stressful.\" (Cl. 1)\n\n\"Going to the hospital, I coughed a lot the first day and felt stressed, and I did not feel better on the third day. The doctor from the hospital called to check my signs and symptoms, and I felt like I would not survive. After being admitted to the hospital, I was completely unconscious for about 40 days. What is the difference in oxygen? What is bulking? My family cannot visit, but they can look from outside.\" (Cl. 9)\n\n\"Covid infection affects health and causes stress.\" (Cl. 15)\n\nEconomic and social impact\n\nThe economic and social impact here refers to the physical, mental, social, spiritual, and economic situations that people living with COVID-19 experience that affect their daily lives.\n\n\"After being infected with COVID-19 and locked down, I had no job. When there was no job, there was no money. I kept saving money until I could not manage it. Shops were closed, so it was not easy to buy food. My salary was reduced when I returned to work because we were expected to help the company financially. Our expenditures remained the same, but our incomes were smaller, which had a huge impact. But other matters are not affected as much as the finances.\" (Cl. 1)\n\n\"An essential part of people today is the economy. If the economy is good, mental health is good. I do not need anything; trade does nothing; I do not feel upset and offended; I am already happy. It is a good environment; it is good, I think. Everyone has their duties. Why are there so many robbers and thieves? Maybe because they do not have a choice. The whole problem lies in economic and mental health.\" (Cl. 3)\n\n\"A stupid economy. If we get infected, we cannot work; it affects our family.\" (Cl. 5)\n\n\"In health, stress, or unemployment, I have no money to spend like this.\" (Cl. 6)\n\n\"COVID-19 is affecting work. I closed shop; cannot open now; no money.\" (Cl. 13)\n\n\"I am unemployed, and I have no money.\" (Cl. 15)\n\nSocial stigma\n\nIn this study, social stigma refers to how people with COVID-19 are stereotyped and treated with discrimination, dissociation, and loss of certain status due to association with a perceived disease.\n\n\"It is stressful that people around you have to be poorly judged by society, such as when someone was evicted from their condo when it was learned that he had infected a friend with COVID-19 even though he had a young child. Some people know that it is my friend in the condo. So many people push them away from their homes. I told him to go elsewhere for 14 days and then return.\" (Cl. 5)\n\n\"An infected friend posted a picture on Facebook. There will be a team to hunt him. This is a true story.\" (Cl. 5)\n\n\"Do you not understand? Are you disgusted or not? My mother told a group friend that her daughter was infected with the coronavirus. My mother gave them a gift and souvenir, and they returned it as if afraid it was infected with COVID-19.\" (Cl. 8)\n\n\"I had to stop work. I had to take a break from work first. It was too bad. My friend is a football coach, and he has COVID-19. Parents do not let their children learn football from him. The room was locked when he returned to his condo; the owner had kicked him out.\" (Cl. 11)\n\n“You will be stigmatized by society as an infected person. You have to go somewhere else. Those who are infected or have a friend who is infected are disrespected and bullied. Let us go together and not live in a normal society.\" (Cl. 13).\n\nSocial support\n\nIn various fields, social support involves purposeful interactions that bring about help. For those infected with COVID-19, cultivating concrete and abstract assistance, such as recognizing, understanding, and responding to emotions, providing information, giving objects, and being accepted as part of the group, affects how a person perceives social support. Individuals are perceived as being loved, cared for, valued, and positively affected by their physical and mental health. This enables a person to face life-threatening events more effectively.\n\n\"I call home every day, and I jog to my room to watch television.\" (Cl. 2)\n\n\"During stressful times, listening to music, reciting the prayers, doing housework, and watching TV with my girlfriend can relieve stress.\" (Cl. 4)\n\n\"I was infected, and therefore [my] friends were exposed. They only have sympathy for us. We called to apologize. He said hey, it is okay. You can take care of yourself. It is not a problem. He wanted to encourage us and not make us more stressed.\" (Cl. 5)\n\n\"I have a neighbor who acts as if she is his older sister and consults about dealing with COVID-19.\" (Cl. 6)\n\n\"Luckily, most people are encouraging, and those around sick people are worried. They will never bully or scold.\" (Cl. 1)\n\n\"When stressed, I listen to music and watch TV.\" (Cl. 4)\n\n\"Doctor and his teams were highly qualified, and people received good treatment; they felt confident, and their health status gradually improved.\" (Cl. 1)\n\n\"The health team from the hospital that has taken care of me since I was discharged still contacts me to ask about my symptoms, invite me to work, become a lecturer, and sometimes get a job. Recently, the agency made an appointment for an interview. If anything could help, such as providing information about COVID-19 or others, I would be happy to help. The hospital produces an application called Clinical App to track the symptoms of COVID-19 patients. So we do not have to go to the hospital. You can ask for advice in the app. There will be a team that takes care of coordinating.\" (Cl. 1)\n\n\"Bang Khae Health Center contacted and coordinated care such as home visits and consults. They gave good help.\" (Cl. 2)\n\n\"During the treatment, the medical team was excellent and did not show disgust. The doctors and nurses took good care of the patients, spoke well, cared for them, and encouraged them. They gave us medicines, checked the temperature and pressure, and always called to tell us. They provided detailed treatment care. The medical team is perfect.\" (Cl. 4)\n\n\"At that time, I was in an intensive care unit. The healthcare team provided a PPE gown. All the care and treatment procedure was done quickly so that I could get out of the intensive care unit faster.\" (Cl. 5)\n\n\"I was very well taken care of. The doctor would video call and ask about my condition. Then, a doctor would check the fever in the morning and evening. Everything about the food was good. I got lung x-rays, and they took blood samples. When I came out of the hospital, I called to ask, but I did not come to visit because it was difficult. After all, I was staying at work—I called to ask.\" (Cl. 6)\n\n\"When my symptoms improved and I woke up in the hospital, the doctor took good care and was friendly.\" (Cl. 9)\n\n\"The healthcare team at the Tambon health-promoting hospital providing care is outstanding, so I call and ask about health issues when I get any problem. They support me when I get a problem by providing information and medicine.\" (Cl. 10)\n\n\"During the hospital stay, we would talk on the phone. He would call to check the fever, check here and there, and check every time there was a headache and fever. Ask how we were feeling. Check in the morning and at noon. If we had a fever, we called via Line and told him. There was a blood test and heart rate measurement. Since I coughed a lot, I had many medicines but no side effects. As soon as it got into my lungs, I suffered for 4–5 days, but now my lungs are normal, and I only had medication.\" (Cl. 14)\n\n\"I wish there were a cure for the disease. It is essential for life because it is not only in Thailand; it is worldwide, and I want it to be researched quickly.\" (Cl. 11)\n\n\"At first, I was worried because it was a new case. I do not know what the symptoms of COVID-19 are. Believing in the craftsmanship of Thai doctors and their teams, I felt that it was not scary, and I was fortunate to be taken care of by a hospital that felt that it was a leading hospital, so I was not worried.\" (Cl. 1)\n\nSometimes, bad luck brings good luck\n\nGood luck in unfortunate situations means someone with COVID-19 has symptoms and needs to be treated in a hospital. There are various consequences, but one can get through that period.\n\n\"It was good luck in bad luck. Our bad luck is being infected with COVID-19, but good luck is learning things that we did not know before, such as knowledge of the disease and, most importantly, society. Encouragement and being a spokesperson. We felt lucky because we could not find an experience like this. Advertising for life insurance companies and interviews with other agencies, including this interview and conducted research, is something that other people cannot do because they have not been infected. I am fortunate in the misfortunes that I have to face in this life.\" (Cl. 1)\n\n\"When I found out that I was infected, I felt unlucky. There was a feeling of 'why so unlucky.' But I was lucky enough to enter the treatment process until finally recovering and going out to live a normal life. I think that COVID-19 also brings good things, such as getting to know the medical system in Thailand. There is a line between the patient group and doctors in the medical personnel and equipment system. I will ask a lot because I want to know and check it out on Facebook. We will ask and check with the doctor. After getting the information, try to share it on Facebook with some friends. This infection was helpful. It is not just a punishment. It allows us to spread good things and benefit others after we are infected with COVID-19. We are lucky to be able to benefit society.\" (Cl. 5)\n\nAdaptation is a concept that has been used to understand participants' experiences, and further illumination can be gained through the Roy adaptation model (Roy, 2001, 2009). Based on this model, the participants' stress, economic and social impact, social stigma, social support, and a sense that sometimes bad luck brings good luck can be interpreted.\n\nThis model is based on the four dimensions as the person submitted to a scheme, including the stimulus, which generates the coping mechanisms and results that make up the individual, family, and community response. This scheme focuses on three stimulus types: 1) focal stimuli, which require stimulation, including fatigue, dyspnea, high fever, and cough, i.e., the signs and symptoms of COVID-19; 2) contextual stimuli, which can be defined as comorbidities; and 3) residual stimuli, which are described as internal and external factors, such as stress from unemployment, viral infodemic, financial problems, social stigma, and lack of appropriate Personal Protective Equipment (PPE).\n\nAdaptative behavior is assessed in four modes: physiological, self-concept, role function, and interdependence. Additionally, the subdivided models are regulators, including the physiological mode, which we describe as the situation and function of people infected by COVID-19. This is pertinent since the body's homeostasis is directly related to the lower probability of worsening symptoms. Secondly, the cognate coping mechanisms are self-concept, role function, and interdependence. The self-concept mode defines coping and highlights psychological and spiritual aspects. Indeed, considering the context of stress generated by this pandemic, asking for emotional support in chaotic times eases anguish and favors psychological well-being. The coping mechanism that complements the role function mode refers to the individual's ability to understand their role in the world and the self-knowledge of their role in society. During the pandemic, this acknowledgment is necessary because the population does not participate in essential services; instead, it supports control measures when it fulfills social isolation measures such as visiting the hospital after an appointment.\n\nIn contrast, in the scope of essential services, health professionals, for example, legitimize their functional importance when they perform their duty with technical skills and humanity. The coping mechanism of the interdependence mode includes the affective demands of everyone. In fact, with a social distancing policy, it is common to observe anguish in the community, which has a particular need related to complete well-being. However, information and communication technologies can be alternatives to increasing physical distancing and its repercussions on the population's biopsychosocial health. For the interdependence mode, social support from family and multidisciplinary treatment teams can decrease the experience of fear around the transmission and conditions related to COVID-19. Social stigma increases the participants' stress, which increases the severity of COVID-19 (Sing et al., 2021).\n\nIn conclusion, the theoretical interpretation of COVID-19 case perceptions showed that participants experienced stress, economic and social impact, stigma, and social support during the global COVID-19 pandemic. The participants' adaptive process promoted their mental integrity and positively affected their health. Nonetheless, they discussed their existential perceptions during the interviews, raising questions about work and the future during crises. Knowledge gathered from this point of view will help plan to fight such crises in the future.\n\n\nDiscussion\n\nAn interest driving this study was to 1) describe the stressful experiences of life during the COVID-19 pandemic and 2) describe the impact caused by the COVID-19 infection on those infected during the global COVID-19 pandemic.\n\nPeople with COVID-19 were stressed by being in lockdown and quarantined. Difficulty finding food sources and disgust from the community exacerbated the symptoms of the disease.\n\nSocial stigma toward people with COVID-19 existed among people such as relatives or neighbors who feared infection. People with COVID-19 were severely judged by society and experienced situations such as being hunted by a group of citizens who came to find them, who then publicized infection information, meaning that there was no safe place in society. The owners locked rooms and stopped renting to patients. Patients were kicked out of their place (Tsai & Wilson, 2020) and were insulted and bullied. Anyone who had an infected friend was stigmatized by society, meaning they could not stay together and not live in a typical community. Shreyaswi & Shashwath (2020) reported that reducing stigma and providing mental health services is a necessary public health response to COVID-19. Challenges related to the spread of COVID-19, stigma, and discrimination can affect patients diagnosed with COVID-19 and those who are quarantined. Effective communication is both accurate and timely. It positively affects coordination and community involvement and is a cornerstone for reducing stigma and promoting mental health. Besides, an integrated psychosocial rehabilitation program to reduce social stigma and improve the resilience of COVID-19 patients is needed (Son et al., 2021). While several steps are required to address stigma and promote mental health, a clear strategy to integrate mental health services into meeting public healthcare needs becomes necessary during the pandemic; for example, elaborating on possibilities to deliver mental health care through technology.\n\nAdditionally, participants mentioned the impact of stigmatization and discriminatory experiences on physical and psychological health during the pandemic. This situation has increased discrimination against East and Southeast Asians, with reports of anti-Asian harassment and attacks rising globally (Lee & Waters, 2021; Chen et al., 2020; Dhanani & Franz, 2020; Hahm et al., 2021; Noel, 2020).\n\nAmong those infected with COVID-19, social support was received during the COVID-19 outbreak, including treatment and assistance, information, and material provisions. For example, the landlord and neighbors showed no disgust when accepted as a part of society. Receiving love, care, appreciation, and acceptance from friends and family, such as encouraging calls during treatment, friends that did not mind doing activities with family members, such as exercising, listening to music, or watching TV, and consulting trusted people, such as a family member, and a health team that provided good care was also experienced. Hospitals produced an app called Clinical App to track the symptoms of COVID-19 patients, coordinate care, ask questions, help people receive care at home, and give advice; on this app, the medical team was beneficial and did not show disgust. Nurses took good care of the patient, unconditioned positive regards of care, encouraged (Galehdar et al., 2020a), (Galehdar et al., 2020b) and made video calls to ask about patients’ symptoms. Nurses checked their temperature in the morning and evening. Lung X-rays were obtained. A study by Rathnayake et al. (2021) examined nurses' perspectives on caring for COVID-19 patients; their phenomenological study found that nurses were willing to provide care for COVID-19 patients because it was their duty and responsibility as well as a humanitarian issue. In the beginning, infected people always thought they were unlucky because of the infection's severe economic, social, health, and mental impacts. However, after entering a good treatment regimen, symptoms improved, and they found themselves fortunate to be cured of this disease. Many established friendships with the health team. They acquired good morale and the ability to educate and share experiences during treatment to benefit society.\n\nPeople were unhappy during the COVID-19 lockdown. The physical, mental, social, spiritual, and economic impacts experienced by people with COVID-19 affected their daily lives, such as having no job and using their savings instead of their salary. Unbalanced trade led to more robbers and thieves and increased stress and depression (Joo et al., 2021). this is consistent with a study by Hertz-Palmor et al. (2021) that examined the relationship between income loss, financial strain, and depressive symptoms during COVID-19. Moreover, according to research by Lei et al. (2020), stress during COVID-19 impacted the participants, and individuals with no psychosocial support were highly vulnerable to anxiety and depression during this pandemic (Hossain et al, 2020). Moreover, Nicola and research teams (Nicola et al., 2020a) (Nicola et al., 2020b) reported that social support was associated with a lower risk of mental health problems and correlated with another study of emotion regulation growth during the pandemic (Cuan-Baltazar et al. 2020; Dhanani, & Franz, 2020). Post-traumatic growth, psychological conditions, and both hopeless and post-stress growth were reported during the COVID-19 pandemic (Qi & Sheng, 2022; Gutiérrez-Cobo et al., 2021; Hu et al., 2021).\n\nThe interview was conducted during community quarantine and after the participant was discharged from the hospital. The participants may have had stress and anxiety about their health status, long COVID, work, and economic problems; this may have prevented the authors from fully understanding the studied phenomena. In addition, the research consisted of a homogenous population of only 15 participants. Thus, a similar study that included these participants might have revealed different perceptions of experiences. The small sample size also increased the risk of bias.\n\n\nConclusions\n\nThis research explores the impact of COVID-19 on patients by analyzing their lived experiences. The data show that COVID-19 patients were psychologically, physically, socially, economically, and spiritually affected by the disease. Therefore, healthcare staff and other support systems should be comprehensively maintained by offering economical, employee-oriented, or occupational therapy and establishing a new career that includes providing training for post-COVID-19 treatment.\n\nPolicy implications\n\n• The research shows that stress, economic and social aspects, social stigma, social support, and the concept that sometimes bad luck brings good luck have impacted COVID-19 cases. We recommend that the government provide a policy to support the incomes, employment, and training of this new era's career in the new generation.\n\n• Understanding the challenges that the COVID-19 cases faced in these outbreaks (stress, economic and social aspects, social stigma, social support, and the concept that sometimes bad luck brings good luck) will advance hospitals and nurses to prepare better for the future.\n\nNursing interventions\n\n• Social stigma is the factor that influences COVID-19 cases to worsen and leads to mental health problems. Media can efficiently decrease social stigma in the community via social media and gain more cooperation from the healthcare unit in the community.\n\nNursing research\n\n• There is a need for a more exhaustive holistic assessment, including return-to-work strategies for future research studies.\n\n• This research depicts the impact of stigmatization and discriminatory experiences. This increase in discrimination and harassment has important implications for health care/nursing care.\n\n• Considering the limitations of this study, other researchers should use larger sample sizes or conduct research in other regions of the country.\n\n\nEthical approval\n\nEthical approval was obtained from the Ethics Review Committee for Research Involving Human Research Participants (COA No. 119/2563) on 20 October 2020.\n\n\nConsent\n\nThe authors obtained written and verbal informed consent from all participants to use their data in this study.\n\n\nORCID IDs\n\nEk-Uma Imkome https://orcid.org/0000-0001-5714-4249", "appendix": "Data availability\n\nFigshare: Until the Dawn: The Impact of COVID-19, https://doi.org/10.6084/m9.figshare.21369810 (Imkome E-u, & Moonchai K, 2022).\n\nThe project contains the following underlying data:\n\n• Demographic questions\n\n• Field notes\n\n• Participant information\n\n• Semi-structured interview questions\n\n• Raw data\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThis research was supported by Thammasat University Research Unit in the innovation of Mental Health and Behavioral Healthcare and funded by the Faculty of Nursing, Thammasat University.\n\n\nReferences\n\nAl Kalaldeh M, Shosha GA, Saiah N, et al.: Dimensions of phenomenology in exploring patient's suffering in long-life illnesses: Qualitative evidence synthesis. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNurunnabi M, Hossain SFAH, Chinna K, et al.: Coping strategies of students for anxiety during the COVID-19 pandemic in China: a cross-sectional study. F1000Res. 2020; 9: 1115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQi L, Sheng HJ: Posttraumatic growth and psychological resilience during the COVID-19 pandemic: A serial mediation model. Front. Psych. 2022; 361.\n\nRathnayake S, Dasanayake D, Maithreepala SD, et al.: Nurses' perspectives of taking care of patients with Coronavirus disease 2019: A phenomenological study. PLoS One. 2021; 16(9): 1–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoy C:Alterations in cognitive processing. Stewart-Amidei C, Kunkle J, Bornstein K, editors. American Association of Neuroscience Nurses' Neuroscience nursing: human responses to neurologic dysfunction. Philadelphia: 2nd ed.Saunders;2001; (pp. 275–323)\n\nRoy C: The Roy adaptation model. Upper Saddle River, NJ:Prentice-Hall Health;3rd ed2009.\n\nRoy SC: Research-based on the Roy adaptation model: Last 25 years. Nurs. Sci. Q. 2011; 24(4): 312–320. PubMed Abstract \| Publisher Full Text\n\nShreyaswi Sathyanath M, Shashwath Sathyanath M: Stigma Reduction and Provision of Mental Health Services in the PublicHealth Response to COVID-19. Indian J. Community Health. 2020; 32: 269–272. Publisher Full Text\n\nSing Joo G, Owen Devan DM, Shao Qi C, et al.: Association between depression, anxiety, stress and perceived quality of life in a Malaysian B40 urban community during the COVID-19 lockdown: A cross-sectional study [version 1; peer review: 1 approved]. F1000Res. 2021; 10: 693. Publisher Full Text\n\nSon H-M, Choi W-H, Hwang Y-H, et al.: The Lived Experiences of COVID-19 Patients in South Korea: A Qualitative Study. Int. J. Environ. Res. Public Health. 2021; 18(14): 7419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTong A, Sainsbury P, Craig J: Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int. J. Qual. Health Care. 2007; 19(6): 349–357. PubMed Abstract \| Publisher Full Text\n\nTremblay S, Castiglione S, Audet LA, et al.: Conducting qualitative research to respond to COVID-19 challenges: Reflections for the present and beyond. Int J Qual Methods. 2021; 20: 160940692110096. Publisher Full Text\n\nTsai J, Wilson M: COVID-19: a potential public health problem for homeless populations. Lancet Public Health. 2020; 5(4): e186–e187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu Z, McGoogan JM: Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese center for disease control and prevention. JAMA. 2020; 323(13): 1239–1242. PubMed Abstract \| Publisher Full Text" }	[ { "id": "175113", "date": "20 Jun 2023", "name": "Chomphunut Srichannil", "expertise": [ "Reviewer Expertise Counseling Psychology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 used online interviews to explore the lived experiences of 15 COVID-19 survivors, focusing on the impact of COVID-19.\n\nData Collection\nData saturation was mentioned, but it is not clear how saturation was reached. Indeed, the question should also be extended to “Is data saturation required for using phenomenological-hermeneutic approach?”\nIn terms of recording online video interviews, there should be more information regarding how the issues of confidentiality, data storage, and data safety have been addressed.\n\nData Analysis\n\nThere is an inconsistency in mentioning the use of the methods for analyzing the data in the abstract and other sections. Specifically, in the abstract, the authors stated that “using a phenomenological-hermeneutic approach. We used the thematic analysis technique to analyze the data”. (The use of “thematic analysis technique” could also be confused with Thematic Analysis, a range of methods for qualitative data analysis). In the data analysis section, only the phenomenological-hermeneutic approach was stated (Here a citation to “Kvale & Brinkmann, 2014” was claimed, but this was not on the reference list). However, content analysis was also mentioned in the result section when the authors wrote “Major themes derived from content analysis were evident across interviews”. The question is thus what is the data analysis method used in the study? Additionally, the word “structural understanding” appeared to be redundant in the second and third levels. Please check for accuracy.\n\nResults It appears that the results were fairly descriptive, mostly done by presenting (translated) quotes from the participants. With the use of the phenomenological-hermeneutic approach, interpretation should be more widely incorporated in the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10142", "date": "25 Sep 2023", "name": "Ek-uma Imkome", "role": "Author Response", "response": "To respond to the reviewer's comments on Data Collection. We added the sentence “the saturation was reached when no additional data were found” on the part of data collection. Additionally, we had to add more information regarding how the issues of confidentiality, data storage, and data safety have been addressed on the part of consent. We have updated Kvale & Brinkmann, 2014 as the reference, and The IPA analysis methods were added. We removed the redundant “structural understanding” in the second and third levels for the results and corrected it for the data analysis part." } ] }, { "id": "193387", "date": "07 Aug 2023", "name": "Cheah Phaik Kin", "expertise": [ "Reviewer Expertise Public Health", "education", "policing" ], "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\nBecause this study was done in Thailand, it is helpful to describe the situation there during the pandemic to help readers understand the context in which this study was based.\n\nThe knowledge gap needs to be further established and supported to justify the need for this investigation and the significance of this study.\nIt is important that the researchers provide support for this statement - \"Besides, a few studies have explored the needs of COVID-19 cases, their stress and adaptation to the disease, and health support.\"\n\nIn this regard, it would also be appropriate to refer to past studies that have been done in Thailand, as well as in other countries to establish the need to conduct this investigation and fill this knowledge gap.\n\nTheoretical framework - While it is helpful to use a theoretical framework to guide this work, the researchers need to explain how this model is not stifling this study that uses qualitative methods, ensuring that it does not limit creativity or the exploration of alternative perspectives, but rather enhances understanding and provides a structured approach to the research.\n\nMethods\nIn the same vein (ie. No. 3), please explain how the interview questions were developed.\n\nThis statement can be rephrased to make it clearer. Does it also mean that the authors have designed the interview questions by drawing upon the instrument/findings from these studies? - \"The phenomenological-hermeneutic approach was applied through discussions after examining and interpreting lived experiences and perceptions related to the COVID-19 pandemic (Al Kalaldeh et al., 2018; Graor & Knapik, 2013; Kvale & Brinkmann, 2014).\"\n\n\"Ethical approval was obtained from the Ethics Review Committee for Research Involving Human Research Participants (COA No. 119/2563)\" - Please state more specifically which committee this refers to e.g. the University of ??? Ethics Review Committee for Research Involving Human Research Participants\n\nPlease state when these interviews were carried out as this is a cross-sectional study.\n\nPlease describe how the authors ensured data trustworthiness.\n\nPlease provide support and justification for the data analysis methods employed.\n\nResults\nTable 1 - The marital status \"Couple\" means living with a partner or married?\n\nLevel 1 - It would be helpful to describe the results found about the respondents Self-Understanding.\n\nIs Figure 1 showing the results of this study? What does this statement mean - \"Apply from Roy, 2011\"? There may be a typo or grammatical mistake here.\n\nIt is not clear why the Results of the analysis are labelled as \"Level\". This is not a conventional way to report the results of a qualitative study. Labelling each theme/category as a \"level\" indicates a hierarchy. Is this the intention, if so, the hierarchy is not clear.\n\nThe sub-themes in Leval 2 appears confusing. From the formatting of the paper, it appears that within \"Level 2: Structural understanding\", there are Stress; Economic and Social Impact; Social Stigma; Social Support; Sometimes, bad luck brings good luck. Are these sub-themes all of the same level sub-themes under \"Level 2\"? Or are some of them of a third level sub-theme?\n\nOverall, while the purpose of this paper is rationale and very necessary, this paper lacks the scientific rigour. I would recommend a resubmission.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10141", "date": "25 Sep 2023", "name": "Ek-uma Imkome", "role": "Author Response", "response": "This version has addressed reviewer 2's comments by: Adding the statement of the situation during the COVID-19 pandemic in Thailand, the gap of knowledge was address and citation Adding an explanation of how the interview questions were developed. rephrased the statement to make it clearer on the part of procedure Added the state of the Ethics Review Committee’s institution Trustworthiness was describing The IPA analysis methods were employed Table 1 - The marital status \"Couple\" means living with a partner and/or married." } ] } ]	1	https://f1000research.com/articles/11-1560
https://f1000research.com/articles/12-1327/v1	12 Oct 23	{ "type": "Research Article", "title": "Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.", "authors": [ "Angel Fernando Huamán-Pilco", "Tito Ademir Ramos-Carrasco", "Mario Emilio Ernesto Franco", "Daniel Tineo-Flores", "Richard Estrada-Cañari", "Pedro Eduardo Romero", "Vilma Aguilar-Rafael", "Lourdes Adriana Ramírez-Orrego", "Rosalina Tincopa-Marca", "Fanny-Rosario Márquez", "Manuel Oliva-Cruz", "Jorge Ronny Díaz-Valderrama", "Angel Fernando Huamán-Pilco", "Tito Ademir Ramos-Carrasco", "Mario Emilio Ernesto Franco", "Daniel Tineo-Flores", "Richard Estrada-Cañari", "Pedro Eduardo Romero", "Vilma Aguilar-Rafael", "Lourdes Adriana Ramírez-Orrego", "Rosalina Tincopa-Marca", "Fanny-Rosario Márquez", "Manuel Oliva-Cruz" ], "abstract": "The thread blight disease (TBD) of cacao (Theobroma cacao) in the department of Amazonas, Peru was recently reported to be caused by Marasmius tenuissimus (Sect. Neosessiles). This same species is known to be the main causal agent of TBD in West Africa. However, some morphological characteristics, such as the presence of rhizomorphs, the almost exclusively white color, and pileus sizes less than 5 mm, among others, differ to the description of M. tenuissimus. Therefore, we aimed to conduct a taxonomic revision of the cacao-TBD causal agent in Peru, by using thorough micro and macro morphological, phylogenetic, and nuclear and mitochondrial genomic approaches. We showed that the causal agent of TBD of cacao in Amazonas, Peru, belongs to a new species, Marasmius infestans sp. nov. This study enriches our knowledge of species in the Sect. Neosessiles, and strongly suggests that the M. tenuissimus species complex is highly diverse.", "keywords": [ "Agaricales", "cocoa", "Marasmiaceae", "tropical phytopathogens" ], "content": "Introduction\n\nSpecies of Marasmius are mainly decomposers of leaves and twigs in natural ecosystems, but some species can be pathogenic in agricultural settings, such as the cacao (Theobroma cacao) agroecosystem.1–3 Cacao can get infected by several species of Marasmius, including M. crinis-equi and some species within the section Neosessiles, causing thread-blight disease (TBD).4 The Sect. Neosessiles is a paraphyletic group mainly characterized by the pleurotoid habit of growth and the absence or rudimentarity of the stipe.5,6 Among the TBD-causing Neosessiles species, M. tenuissimus seems to be the most frequent in West Africa,3 and in native Awajun and Wampis communities from Northern Peru.7 Marasmius tenuissimus is characterized by pilei between 7 and 22 mm in diameter, with rusty brown, light grayish fusco or greyish orange color; and basidiospore dimensions of about 9-10 × 4-6 μm.1,8 Even though, nuclear rDNA sequence similarities over 99% point to M. tenuissimus type strains, the morphological characteristics of specimens from West Africa and Northern Peru do not quite match the original description.2,3 Pileus sizes less than 5 mm, the white color, and the presence of rhizomorphs, as in the West African and Peruvian specimens, were never reported before for M. tenuissimus,2,3 suggesting they may not be of that species. Moreover, the mitochondrial genome of six strains and the genome of one strain of M. tenuissimus from West Africa have been recently published,4,9 which opens up the door to perform mitochondrial and nuclear genomic comparisons between the Neosessiles TBD agents from both continents. Therefore, in this study, we aimed to conduct a taxonomic and phylogenetic revision of the status of the cacao TBD-causal agent in Northern Peru, including nuclear and mitogenomic evidence.\n\n\nMethods\n\nSpecimens were obtained during an expedition into the Imaza District (4°47′09.4″S 78°16′51.6″W) in the department of Amazonas, Peru, during August–September 2022 (Table 1). Collections were performed under the authorization N° AUT-IFL-2021-052 granted by the Peruvian National Forestry and Wildlife Service Agency - SERFOR. Morphological studies were conducted in the Plant Health Laboratory of the National University Toribio Rodriguez de Mendoza de Amazonas, Peru (UNTRM-A). The macroscopic characterization was made from fresh pilei. We described the color, shape, and size using a stereomicroscope SMZ18 (NIKON, Tokyo, Japan). The microscopic description was made from fresh and dried material. First, a tiny piece of dried material was carefully sectioned and placed onto a microscope slide. We applied 95% ethanol to the sample for 30 seconds, and then a drop of distilled water was added. The slides were dried out and 5% KOH was applied. We placed a cover slip onto the sample and observed it under an OLYMPUS DP74 (Tokyo, Japan) microscope. The dimensions of basidia and basidiospores were measured with at least thirty individual structures. Finally, pure cultures were obtained from rhizomorph tissues by the hyphal tip technique. The holotype specimen (TAIM04) was deposited in the KUELAP herbarium of UNTRM-A under voucher number KUELAP-2940.\n\n* Taxa marked with an asterisk are type specimens of the corresponding species.\n\n§ These accession numbers correspond to the translated coding sequences of RPB1 and TEF. The DNA sequence of RPB1 sequence is located from position 29369 to 32406 of JANHQD010001077.1; TEF sequence from positions 10248 to 12091 of JANHQD010000042.1.\n\n¥ These accession number correspond to the accession number of the mitochondrial genome. COX1 sequence is located from position 43,391 to 44,992.\n\nDNA extractions were performed from pure cultures using the Wizard® genomic DNA purification kit (Catalogue number A1120; Promega, Wisconsin, USA). DNA was quantified with the BioSpectrometer® Basic (Eppendorf, New Jersey, USA), and diluted to 0.5 ng/μl for PCR reactions. The internal transcribed spacer region (ITS1-5.8S-ITS2, or simply ITS), the large subunit (LSU) rDNA gene, and partial fragments of the genes Translation Elongation Factor 1-α (EF1α) and the largest subunit of RNA polymerase II gene (RPB1) were amplified following.10 The amplified PCR products were Sanger sequenced at MACROGEN (Seoul, South Korea). LSU, EF1α, and RPB1 sequences from the West African TBD-causing specimen GHA37 were retrieved from the recently published genome.9 In addition, we included the mitochondrial cytochrome oxidase I gene (COX1) from isolate INDES-AFHP31, which was retrieved from the mitogenome generated in this study, and from other phylogenetic-related species.4 The introns of COX1 sequences were removed as in.11 Most other sequences used in phylogenetic analyses were obtained from other relevant literature.3,6–8,12–18 We also included unpublished sequences available in NCBI from M. tenuissimus-phylogenetically related isolates C2/06 and C2/33 (accession numbers KM246261 and KM246277 for ITS, and KM246066 and KM246082 for LSU, respectively).\n\nGenerated sequences were edited and assembled with Sequencher v.5.4 (Gene Codes, USA). Once all sequences were gathered, they were aligned with MUSCLE19 implemented in MEGA-X,20 and concatenated with SeaView 4.7.21 We used jModelTest v222 to identify the most appropriate nucleotide evolution model under the Akaike information criterion, for the five locus concatenated data set. Phylogenetic analysis was performed with IQ-TREE v223 which implements the Maximum Likelihood algorithm,24 in the CIPRES Science Gateway v3.3 portal.25 The phylogenetic trees were visualized and edited with FigTree.26\n\nDNA extraction, sequencing, and assembly\n\nTotal DNA extraction was performed using the Wizard Purification Kit (Promega Corp., Madison, Winsconsin) following the manufacturer’s instructions. For nuclear and mitogenome sequencing, we used the strain INDES-AFHP31 from Northern Peru, previously reported.7 A paired-end sequencing library was constructed using the TruSeq Nano DNA Kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina NovaSeq 6000 platform in paired-end, 2 × 150 format.\n\nThe raw reads were checked by FastQC v.0.11.9. Also, quality trimming (Phred Q > 25) and remotion of adapters were conducted with Trimmomatic v0.3627 and TrimGalore software,28 respectively. Jellyfish v.2.29 was used for k-mer counting, and Genome Scope v1.0.030 for assessing genome size, repeat content, and heterozygosity rate. This analysis involved utilizing the output of Jellyfish and the count of 17-mers for k-mer analysis. Additionally, k-depth estimation was performed to identify a predominant single-peak pattern in the frequency distribution analysis of k-mers.\n\nDe novo assembly was performed with two assembly algorithms: SOAPdenovo2 v.2.04,31 and Masurca v.4.0.6.32 Next, we used QUAST v.5.2.033 to evaluate the statistics of assemblies. The assembly validation process employed two distinct methods. First, the filtered paired-end Illumina reads were realigned to identify any errors in the assembly. This was accomplished using Bowtie2 v.2.4.234 and SamTools v.1.735 software. Second, the completeness of the assembly was evaluated using the Agaricales-specific profile of the BUSCO strategy.36 To identify vector contamination, we employed VecScreen JCVI (https://github.com/tanghaibao/jcvi), which utilizes the Univecdatabase (https://ftp.ncbi.nlm.nih.gov/pub/UniVec/). We also performed a BLAST v.2.2.2637 analysis, mapping the scaffolds against the nt/nr NCBI database (found at https://www.ncbi.nlm.nih.gov/). Following this, any contaminated scaffolds and vectors were eliminated before submitting the remaining data to the NCBI database. This assembly has been deposited at DDBJ/ENA/GenBank under the accession: JANHQD01.\n\nNuclear genome annotation\n\nGenome annotation was performed using Funannotate v1.8.12.38 Repetitive elements were soft-masked by TANTAN.39 Ab initio gene prediction was carried out using AUGUSTUS v.3.3.3,40 GlimmerHMM v.3.0.4,41 and SNAP42 trained with alignments of the BUSCO agaricales_odb10 dataset,36 and with GeneMark-ES v.4.6943 self-trained on the repeat-masked genome sequence. Protein evidence from the UniProt/SwissProt database44 was mapped to the genome using DIAMOND v.2.0.1545 and Exonerate v.2.4.0.46 Finally, EVidenceModeler v.1.1.147 was used to generate consensus gene models based on all the above ab initio and evidence-based gene models. The tRNA genes were identified with tRNAscan-SE v.2.0.9.48 Functional annotations were assigned by similarity to UniProtKB (2021_02),44 InterPro v89.0,49 Pfam v.34.0,50 EggNOG v.5.0,51 BUSCO (agaricales_odb10 dataset),36 dbCAN v.10.0,52 and MEROPS v.12.0.53 Phobius54 and SignalP v.5b55 were used to predict transmembrane topology and signal peptides, respectively. DeepLoc 2.056 was used to determine protein localization. Effectors were predicted by EffectorP 3.057 based on the subset of extracellular proteins. AntiSMASH v.6.058 was used to predict secondary metabolite Clusters of Orthologous Groups (smCOGs).\n\nMitochondrial genome annotation\n\nThe mitochondrial genome was confirmed using the default Geneious Prime 2023.1 setup (Biomatters Ltd., Auckland, New Zealand) and GetOrganelle v1.7.6.1.59 Genes were annotated with MITOS,60 MFannot61 and manually confirmed with ORFfinder available in NCBI, and tRNAscan-SE 1.21,62 finally adjusted in Geneious. A physical map of the mitogenome was created with OGDRAW v 1.2.63 Our results were compared to the ones reported for another published M. aff. tenuissimus mitogenomes.4\n\n\nResults\n\nWe collected new cacao tissues infected by TBD-bearing fruiting bodies of the pathogen in Imaza province, department of Amazonas, in Northern Peru. Besides AFHP-31 from a previous study,7 and for which no basidiocarps and only pure agar culture were preserved, two additional specimens were included in this study: TAIM-04 and AFHP-101. The presence of white rhizomorphs, and white pilei less than 5 mm in diameter with no reticulation are macro morphology hallmarks of the TBD- causing specimens in Amazonas, Peru (Figure 1). In terms of micromorphological features, these specimens produce ellipsoid and smooth basidiospores with dimensions of 7.6-8.1 × 4.3-4.8 μm; they also produce Siccus-type cheilocystidia and pileipellis broom cells; cheilocystidia are cylindrical slightly narrower at the base, and pileipellis broom cells are ovoid to globose shape (Figure 2). All these morphological features provides the first point of evidence that the cacao TBD-causal agent in Peru is a new species.\n\na) Small basidiocarps growing on decomposing cacao leaves, b) Mature basidiomata, c) Young basidiomata, d) White-rhizomorphs colonizing cacao stem. Photographed by A. F, Huaman-Pilco. Scale bar in b) and c) = 1 mm.\n\nScale bar = 20 μm. Drawings by T. A. Ramos-Carrasco.\n\nWe used 49 taxa (holotype and other reference specimens) of Maramius spp. in the Neosessiles and other closely related sections. The best model of evolution on the aligned concatenated dataset was the transversion model with unequal base frequency, with a discrete Gamma model for the rate of heterogeneity (TVM + G). As expected for paraphyletic groups, the phylogenetic analysis grouped Sect. Neosessiles taxa in two different parts of the tree (Figure 3).6,18 The Peruvian specimens were grouped together in a highly supported clade (100% bootstrap support; Figure 3). The closest related clade was composed of the unpublished Marasmius isolates (C2/33, C2/06), putatively soybean endophytes according to their NCBI passport information. These two clades were phylogenetically distinct to the M. tenuissimus sensu stricto clade conformed by the type strains NW192, and NW199, and other reference specimens (Figure 3). Therefore, these results, combined with the unique morphological characteristics of the Peruvian specimens, support they belong to a new species within the Sect. Neosessiles, herein after called Marasmius infestans sp. nov.\n\nValues above branches = Maximum likelihood bootstrap values. Taxa in blue are the specimens causing thread-blight disease of cacao in Amazonas, Peru, and conform a new species clade, Marasmius infestans sp. nov. Type specimens are marked with an asterisk.\n\nWest African specimens were phylogenetically distinct to both, M. tenuissimus sensu stricto, and the Peruvian-specimen clade (Figure 3). They were grouped in at least four phylogenetic clades. The informally described M. neosessiliformis nom. prov. was grouped together with isolate GHA64.\n\nThe nuclear genome assembly of Marasmius infestans AFH-31 reveals a size of 84.7 Mb, organized in 3,213 contigs (≥1,000 bp) with a GC content of 49.32% and a N50 value of 42,194 kb. We predicted 21,762 protein-coding genes and 656 tRNA genes in the nuclear genome of M. infestans strain AFHP31. Functional annotation resulted in the identification of 15,414 Pfam domains; 29,349 InterPro protein families; 31,346 Clusters of Orthologous Groups of proteins (COGs) and EggNog proteins; 606 proteases/protease inhibitors, and 679 carbohydrate-active enzymes (CAZymes). Moreover, we predicted 1,823 signal peptides and 4,010 transmembrane regions. About 8.9% of the proteome (2,152) are extracellular proteins, of which 25.6% (550) and 16.5% (356) were predicted to be apoplastic and cytoplasmic effectors, respectively. The genome contained 11 smGCs, 18 biosynthetic enzymes, and 27 secondary metabolite Clusters of Orthologous Groups (smCOGs). The draft nuclear genome of M. infestans AFHP-31 is larger in size than isolate GHA-37 genome size and, despite having fewer proteins, has more effectors. (Table 2).\n\nThe mitochondrial genome of Marasmius infestans is circular, 47,389 bp long, and contains 42 genes. It is A + T rich (72.83%) and includes 25 tRNA (trnR, trnL and trnS occur in duplicate, while trnM in triplicate), 14 ribosomal proteins, two rRNA (rnl, rns), and one orf (orf868) (Figure 4). A comparison with the mitochondrial genome of TBD-causal agents from West Africa reveals M. infestans differs to them in size, and gene number, and has lost all their introns in COX1I (Table 3).\n\nThe genes inside and outside the circle are transcribed in clockwise and counterclockwise directions, respectively. Genes belonging to different functional groups are shown in different colors. The dark gray area in the inner circle denotes GC content.\n\nTaxonomy:\n\nMarasmius infestans Huamán, Ramos C. & Díaz Val., sp. nov. IF 901138 (Figures 1-2)\n\nEtymology: ‘infestans’, in reference to the capacity to infect branches and leaves of cacao trees.\n\nDiagnosis: Similar to Marasmius tenuissimus but it has white pilei less than 5 mm, lamella not reticulated, and presence of ovoid to globose pileipellis broom cells.\n\nType: PERU: Amazonas department, Bagua province, Imaza district, Pumpu native community; -4.785944, -78.281000. Holotype specimen TAIM-04 (voucher KUELAP-2940).\n\nOther examined specimens: AFHP-101 and dried culture of AFHP-31 (voucher KUELAP-2251).\n\nPileus: (0.7-)1.8-3.1(-4.9) mm diam., convex, smooth, glabrous, dull to shiny, white to white-cream (n = 84). Context white, thin. Lamellae adnate with 1-5 series of lamellulae, narrow, non-reticulate, intervenose, white, non-marginate. Stipe absent or extremely rudimentary, eccentric, cylindrical. Odor not distinctive.\n\nBasidiospores: (6.2-)7.6-8.1(-8.8) × (3.7-)4.3-4.8(-5.2) μm, ellipsoid, smooth, thin-walled (n = 44). Basidia length (17.3-)18.9-21.1(-22.2) μm, thicker part (7.0-)8.0-9.4(-10.2) μm, thinner part (2.6-)4.2-5.8(-6.3) μm, cylindrical to clavate (n = 30). Cheilocystidia Siccus-type broom cells; main body, cylindrical, slightly narrower at the base, thin-walled; apical setulae length 2.2-4.1 μm, obtuse, thin-walled. Pleurocystidia rarely present, main body length 66.1-75.4 μm, thicker part 8.9-11.9 μm, thinner part 4.1-5.1 μm (n = 3). Pileipellis composed of Siccus-type broom cells; main body (9.6-)10.1-11.9(-12.2) × (8.5-)9.9-14.5(-17.9), ovoid to globose, thin- to thick-walled. Pileus trama interwoven. Lamellar trama hyphae 3.4-4.6 μm diam. μm diam., hyaline. Stipitipellis hyphae diam 3.4-4.6 μm., hyaline. Clamp connections present in all tissues.\n\nHabit and habitat: Rhizomorphic colonizing leaf and twigs of cacao, which subsequently causes thread blight disease. Rhizoids are white; fruiting bodies develop on dead tissue. The fungus occurs on poorly managed cacao farms with excessive shade and high relative humidity, typical characteristics of the tropical rainforest.\n\nGeographic distribution Imaza province, Amazonas department, Peru.\n\nNotes: Marasmius infestans differs from M. tenuissimus in the color and size of the pilei, and lamellae appearance. Marasmius infestans pilei are white to white-cream, while M. tenuissimus is between light grayish fuscous to rusty brown when fresh,1 or greyish or pale orange to golden brown, light brown or orangish white.1,8,14 Pilei in M. infestans can reach up to 5 mm broad, while pilei in M. tenuissimus can have diameters typically of 7–40 mm.8,14 Lamellae in M. infestans lacks reticulation while in M. tenuissimus lamellae is heavily reticulate. Additionally, West African species within the M. tenuissimus species complex can produce pure-white to brown-colored rhizomorphs,3 while M. infestans produces pure-white rhizomorphs. At the genomic level, M. infestans is 13,636,061 pb larger than M. aff. tenuissimus GHA37. Marasmius infestans also has 3,229 fewer genes, and 231 more effectors (Table 2).\n\n\nDiscussion\n\nThe causal agent of cacao TBD had been previously analyzed based on nuclear rDNA comparison, macro morphology of the fruiting body and rhizomorphic structures, and mycelial culture.7 However, other important characteristics, such as the micro morphological features of lamella and pileus, were not considered in reports from both, West Africa and Peru.3,7 In this study, we present morphological, phylogenetic, and genomic evidence that the species of Marasmius causing TBD in the Amazonian areas of Northern Peru is a new species in the Sect. Neosessiles, namely M. infestans.\n\nIn this study we revealed morphological differences with M. tenuissimus sensu stricto in color and size of pilei, lamellae appearance, and growth habit. Marasmius infestans differs from M. tenuissimus mainly because of their smaller basidiocarps. With respect to color, M. infestans can be easily distinguished by its white to white-cream pilei. Also, M. tenuissimus has never been reported to colonize the leaves and stems of plants with abundant white rhizomorphs as M. infestans.1,64\n\nIn West Africa, four different species were reported to cause TBD of cacao: M. crinis-equi, M. aff. tenuissimus, M. scandens, and Paramarasmius palmivorus (reported as Marasmius palmivorus).3 Besides the molecular differences, these TBD causal agents presented five rhizomorph morphotypes.3 The morphotype A of rhizomorphs is characterized by abundant thin, black, “horsehair”-type rhizomorphs, and was only found on M. crinis-equi. The morphotype B is characterized by its brown coloration; and the type C, by its intense white color. Both morphotypes B and C, were found on M. aff. tennuisimus. The morphotype D is characterized by a faint cream or dull white rhizomorph, with the presence of smooth or cream-pruinose pilei, with a diameter up to 8 mm; and the morphotype E is characterized by its aggregation of shiny or silky white hyphae and white or pale yellow basidiocarps, with smooth and convex pilei, with diameter from 10 to 50 mm, observed on P. palmivorus.3 If we followed this classification in our study, we find M. infestans has rhizomorphs of morphotype C. Unfortunately, in the West African study, no fruiting bodies from this type of rhizomorphs were reported, so we cannot make a macro morphological comparison.\n\nIn the Marasmius genus, ITS was the main locus for phylogenetic studies but did not provide sufficient support for resolving species in Sect. Neosessiles.8 In this study, we found M. infestans is a sister species of M. tennuisimus sensu stricto, both species forming individual clades with 100 % and 98 % bootstrap support, respectively. We also found that the M. tenuissimus species complex has at least five other species that will need proper description, including the previously reported yet not formally described M. neosessiliformis nom. prov.,12 provided the discovery of corresponding basidiocarps. One of these species (Marasmius sp. 5) corresponds to the endophytic strains C2/33 and C2/06 from soybean in Brazil forming a distinct phylogenetic clade, with 97% bootstrap support and closely related to M. infestans and M. tennuisimus. Additionally, TBD-causal strains from West Africa form separate and well-supported clades. Isolate GHA64 groups together with the informally described species M. neosessiliformis nom. prov. specimen Buyck 97.615, suggesting it may be another member of this species requiring formal description.\n\nOn top of morphological and phylogenetic evidence that M. infestans distinguishes from other species within the Sect. Neosessiles, in this study we present its nuclear and mitochondrial genome sequences. The nuclear genome is about 13 Mb longer than M. aff. tennuisimus GHA37. Marasmius infestans also has 231 more effectors than M. aff. tennuisimus GHA37. Pathogenic effectors are secreted by pathogenic fungi during infection and play an important role in silencing plant defenses response,57,65,66 which may help M. infestans during cacao infection. Moreover, mitochondrial genomes are known for different sizes and rearrangements despite their similar gene function.4,67 Marasmius infestans mitogenome has 47,389 bp and has undergone the loss of introns in the COX1 gene, as opposed to M. cf. tenuissimus causing TBD in Africa (Table 3). The gain and loss of introns are common in fungal mitogenomes and are related to their evolution .67 Moreover, we found that M. cf. tennuisimus specimens GHA74, GHA37, MS2, and GHA07, which group together in the phylogenetic analyses, have very similar mitochondrial genome sizes, ranging from 44,399 to 44,859 bp, supporting they all belong to another species (Marasmius sp. 1; Figure 3). Additionally, specimens GHA63, and GHA79, which also formed individual specific lineages, have different mitochondrial genome sizes (51,210 and 48,952 bp, respectively).3 Therefore, they also represent other Neosessiles species in need of formal description (Marasmius sp. 3 and Marasmius sp. 4, respectively).", "appendix": "Data availability\n\nBioProject: The Genome Shotgun project of specimen AFHP-31. Accession number PRJNA860982; https://identifiers.org/NCBI/bioproject:PRJNA860982\n\nThe NCBI accession number of the version of the assembled genome of specimen AFHP-31 described in this paper is JANHQD010000000.\n\nNucleotide: The mitochondrial genome assembly. Accession number OQ343345; https://www.ncbi.nlm.nih.gov/nuccore/OQ343345.1/\n\nSRA: The raw reads for the nuclear and mitochondrial genome under accession number SRR20354643. https://identifiers.org/insdc.sra:SRR20354643\n\nSequences obtained through the Sanger method used are deposited at NCBI through accession numbers OR359411, OR364495, OR420729 and OR420730 for specimen TAIM-04; OM720123 and OM720135 for specimen AFHP-31; and OR359410 and OR364494 for specimen AFHP-101.\n\n\nReferences\n\nSinger R: Marasmieae (Basidiomycetes-Tricholomataceae). Flora Neotropica. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "214961", "date": "31 Oct 2023", "name": "Atik Retnowati", "expertise": [ "Reviewer Expertise Macro-fungal taxonomy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 that aims to describe a new species of Marasmius infestans from Peru. Combination molecular and morphological study is the best approach to describe these new species. Overall, the work is good, but here are some suggestions:\nA mycobank registration number must be provided for a valid description.\n\nThe previous report of Marasmius from Peru must be added to the introduction.\n\nMap of locality where the species was collected must be added. It will help of the ordinary people to see where the exact type locality.\n\nScale bar at the Figure 2 can clearly be seen, and it probably white color scale bar would be better.\n\nDescription: Lamellae adnate with 1-5 series of lamellulae. It should be “Lamellae adnate, distant (1-5) with 0 series of lamellulae”.\n\nIs the work 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": "10917", "date": "11 Apr 2024", "name": "Jorge Ronny Díaz-Valderrama", "role": "Author Response", "response": "This is a study that aims to describe a new species of Marasmius infestans from Peru. Combination molecular and morphological study is the best approach to describe these new species. Overall, the work is good, but here are some suggestions: A mycobank registration number must be provided for a valid description. To register a new fungal name, you need to do it in only one recognized repository (Aime et al., 2021). The recognized repositories for fungal names are: Fungal Names, Index Fungorum, and MycoBank (Aime et al., 2021). We have registered “Marasmius infestans” in Index Fungorum (IF 901138), so there is no need for a MycoBank registration number. - Aime, M. C., Miller, A. N., Aoki, T., Bensch, K., Cai, L., Crous, P. W., ... & Schoch, C. L. (2021). How to publish a new fungal species, or name, version 3.0. IMA fungus, 12, 1-15. The previous report of Marasmius from Peru must be added to the introduction. The report of the thread-blight disease-causing Marasmius species in Peru (Huamán et al., 2023; reference number 7 in the manuscript) is in the introduction as follows: “… in native Awajun and Wampis communities from Northern Peru7” Map of locality where the species was collected must be added. It will help of the ordinary people to see where the exact type locality. A map is now included in version 2 of the manuscript as Figure 1. Because of this, all other figure numbers have been updated. Scale bar at the Figure 2 can clearly be seen, and it probably white color scale bar would be better. We have made the scale bars wider and of white color, so they are more visible to the reader. Description: Lamellae adnate with 1-5 series of lamellulae. It should be “Lamellae adnate, distant (1-5) with 0 series of lamellulae”. We agree with this comment and the statement was updated as indicated." } ] }, { "id": "214959", "date": "31 Oct 2023", "name": "Nopparat Wannathes", "expertise": [ "Reviewer Expertise Taxonomy and systematics on agaric" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.\" described a new species of Marasmius from Peru. This manuscript is interesting, with new data and nice photographs and drawings. However, some points need to be considered.\nThis research reported a new species of Marasmius, which is a taxonomic report with some molecular evidence. So a clear morphological description of a new species is needed. The key characteristics of Marasmius species have to be informed, such as Melzer's reagent reactions of spores, and the trama tissues. A comparison of a new species with phenetically similar taxa is needed, especially for the species reported from the related geographical area --the Amazon rainforest, not scope to the Marasmius species that caused the thread blight disease (TBD) only. A comprehensive dataset for phylogenetics analyses is required, ITS sequences of related species from the Amazon rainforest (Oliveira et al 2014--the sixth reference of this manuscript) have to be included. The phylogenetic analyses of a single gene of ITS and LSU and a combined gene of ITS and LSU are required at least as supplemented data. Due to the dataset used in this study being mainly based on both genes, Only a new species and a single specimen of undescribed Marasmius sp. have other genes.\n\nThere are some suggestions and comments, please look carefully at the following:\nIntroduction: the current classifications of Marasmius should be informed. Since, this research aimed to conduct a taxonomic and phylogenetic revision of the fungi in this genus.\n\nMethods: The spore Quotient (Q) should be determined, and the statistical analysis of spore sizes should show the arithmetic mean with standard deviations. The type specimens (holotype, lectotype, or neotype) are designated to a single specimen. So please correct the data in Table 1 and Figure 3. Please correct the abbreviation of the translation elongation factor 1-alpha gene.\n\nResults: Please make the scale bar in Figure 1 more clearly. In the Taxonomy subsection, the description that the author provided is not concordant with the specimen photo shown in Figure 1. Please correct the spacing, and number of the lamellae and clarify whether lamellae are intervernose or not. Please indicate the attachment of the stipe base and substrate. The Melzer's reaction is a taxonomic chemical reaction used in Marasmiaceae. Please inform the results of the test in the spores and all kinds of tissue. The color of spores, cells, and hypha are required to be stated in a description. The type of pileipellis, and all kinds of trama have to be informed. In Notes, a comprehensive comparison of related taxa is required especially for the Amazonia species.\n\nDiscussion: Since a new species reported in this study caused the TBD. The comparison of new species and other TBD causal species is valuable data. However, a species is described based on the sexual stage and the basidiocarp is mainly characterized. Other characters like the rhizomorph or culture morphology could be additional data. So please keep in mind that the TBD causal strains from West Africa are identified without the morphological characteristics of basidiocarp. The author cites that ITS did not provide sufficient support for resolving species in the section Neosessiles refers to the work of Wannathes et al 2009. I disagree with this information, please check this carefully. According to the number of available sequences in GenBank and the recent data on the barcode gaps in ITS of Marasmius (Wilson et al., 20231), it would be better if the author could investigate the phylogenetic analysis on combined sequences of ITS and LSU.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10918", "date": "11 Apr 2024", "name": "Jorge Ronny Díaz-Valderrama", "role": "Author Response", "response": "The manuscript entitled \"Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.\" described a new species of Marasmius from Peru. This manuscript is interesting, with new data and nice photographs and drawings. However, some points need to be considered. This research reported a new species of Marasmius, which is a taxonomic report with some molecular evidence. So a clear morphological description of a new species is needed. The key characteristics of Marasmius species have to be informed, such as Melzer's reagent reactions of spores, and the trama tissues. A comparison of a new species with phenetically similar taxa is needed, especially for the species reported from the related geographical area --the Amazon rainforest, not scope to the Marasmius species that caused the thread blight disease (TBD) only. A comprehensive dataset for phylogenetics analyses is required, ITS sequences of related species from the Amazon rainforest (Oliveira et al 2014--the sixth reference of this manuscript) have to be included. The phylogenetic analyses of a single gene of ITS and LSU and a combined gene of ITS and LSU are required at least as supplemented data. Due to the dataset used in this study being mainly based on both genes, Only a new species and a single specimen of undescribed Marasmius sp. have other genes. In this new version we have examined our specimens with Melzer’s reagent as suggested. Besides comparing the new species with TBD-causing Marasmius species, we have now included a discussion highlighting the main differences between the new species and phenetically similar taxa, such as M. neosessilis, M. griseoroseus, M. conchiformis, and M. jasingensis. In the phylogenetic analyses, we had included related species from Oliveira et al. (2014), such as Marasmius conchiformis and M. griseoroseus. In this new version, we are adding the other two species described in Oliveira et al. (2014), M. linderioides and M. longisetosus. In this regard, we are also including separate phylogenies with ITS, LSU, and concatenated data sets to accommodate reviewer suggestions. In all cases, phylogenies support that M. infestans is a new species. There are some suggestions and comments, please look carefully at the following: Introduction: the current classifications of Marasmius should be informed. Since, this research aimed to conduct a taxonomic and phylogenetic revision of the fungi in this genus. The current classification of Marasmius is now informed in the first paragraph of the introduction. Methods: The spore Quotient (Q) should be determined, and the statistical analysis of spore sizes should show the arithmetic mean with standard deviations. The type specimens (holotype, lectotype, or neotype) are designated to a single specimen. So please correct the data in Table 1 and Figure 3. Please correct the abbreviation of the translation elongation factor 1-alpha gene. In this new version we have calculated the Quotient values of spores along with other important statistical metrics such as the arithmetic means and standard deviations, as suggested by the reviewer. We recognized the mistakes in the original submission regarding the designation of type specimens, as pointed out by the reviewer. Therefore, we have updated Table 1 and Figure 3 accordingly. Regarding the abbreviation of the translation elongation factor 1-alpha gene, we have kept the abbreviation as “EF1α” following the nomenclature of Aime and Phillips-Mora (2005). We have also reviewed for consistency the abbreviations of other genes such as COX1 and updated the manuscript throughout. Results: Please make the scale bar in Figure 1 more clearly. In the Taxonomy subsection, the description that the author provided is not concordant with the specimen photo shown in Figure 1. Please correct the spacing, and number of the lamellae and clarify whether lamellae are intervernose or not. Please indicate the attachment of the stipe base and substrate. The Melzer's reaction is a taxonomic chemical reaction used in Marasmiaceae. Please inform the results of the test in the spores and all kinds of tissue. The color of spores, cells, and hypha are required to be stated in a description. The type of pileipellis, and all kinds of trama have to be informed. In Notes, a comprehensive comparison of related taxa is required especially for the Amazonia species. Scale bars have been adjusted for better clarity. As pointed out by the reviewer, there were several inconsistencies and lack of details in the description section. We have made a thorough revision in this regard. We corrected the lamellae characteristics as follows: “Lamellae adnate, distant (1-5) with 0 series of lamellulae, narrow, non-reticulate, non-intervenose, white, non-marginate”. We have updated information about the stipe as follows: “Stipe insititious not arising from white rhizomorphs”. All microstructures were examined with Melzer’s reagent, finding that only Caulocystidia were strongly dextrinoid, while all other structures and tissues were inamyloid. We have included a new figure to show these findings. The color of spores, cells, and other tissues was included in the description (hyaline, except for the Siccus-type broom cells that in some cases were light brown) The type of pileipellis is hymeniderm, and other characteristics of trama tissues were added. We have added a discussion comparing the new species with related taxa, with emphasis on Amazonian species from Oliveira et al. (2014). Discussion: Since a new species reported in this study caused the TBD. The comparison of new species and other TBD causal species is valuable data. However, a species is described based on the sexual stage and the basidiocarp is mainly characterized. Other characters like the rhizomorph or culture morphology could be additional data. So please keep in mind that the TBD causal strains from West Africa are identified without the morphological characteristics of basidiocarp. The author cites that ITS did not provide sufficient support for resolving species in the section Neosessiles refers to the work of Wannathes et al 2009. I disagree with this information, please check this carefully. According to the number of available sequences in GenBank and the recent data on the barcode gaps in ITS of Marasmius (Wilson et al., 20231), it would be better if the author could investigate the phylogenetic analysis on combined sequences of ITS and LSU. We have included a paragraph in which we have added a comparison of the new species with other related basidiocarp-forming species in the Neosessiles section. We agree with the reviewer, so we have clarified the statement in which we indicated that ITS did not provide sufficient phylogenetic resolution power within the Neosessiles. We have also added new phylogenies with ITS alone, LSU alone, and concatenated, to provide extra evidence for the new species." } ] } ]	1	https://f1000research.com/articles/12-1327
https://f1000research.com/articles/11-94/v1	26 Jan 22	{ "type": "Research Article", "title": "Personality typologies of smokers and excessive drinkers: a cross-sectional survey of respondents in the BBC Lab UK Study", "authors": [ "Olga Perski", "Astrid Nikiel", "Jamie Brown", "Lion Shahab", "Astrid Nikiel", "Jamie Brown", "Lion Shahab" ], "abstract": "Background: Several personality traits have been linked to addictive behaviours, including smoking and excessive drinking. We hypothesised that the combination of low conscientiousness, high extraversion and high neuroticism would be synergistically associated with smoking, excessive drinking and both behaviours combined. Methods: Respondents aged 16+ years (N=363,454) were surveyed between 2009-2013 as part of the BBC Lab UK Study, with no restrictions on geographical location. Respondents provided information about sociodemographic characteristics, personality traits, and smoking and alcohol consumption. A series of multivariable logistic regression analyses were conducted.\nResults: No significant three-way but significant two-way interactive effects were observed. The association of high extraversion with smoking was more pronounced in those with high (vs. low) conscientiousness (ORadj=1.51, 95% CI=1.46, 1.56, p<.001; ORadj=1.38, 95% CI=1.35, 1.42, p<.001). The association of high extraversion with excessive drinking was more pronounced in those with low (vs. high) conscientiousness (ORadj=1.70, 95% CI=1.67, 1.74, p<.001; ORadj=1.60, 95% CI=1.56, 1.63, p<.001). The association of high extraversion with both behaviours combined was more pronounced in those with high (vs. low) conscientiousness (ORadj=1.74, 95% CI=1.65, 1.83, p<.001; ORadj=1.62, 95% CI= 1.56, 1.68, p<.001). Results remained largely robust in sensitivity analyses. Conclusions: In a large international survey, we identified two-way ‘personality typologies’ that are associated with greater odds of smoking, excessive drinking and both behaviours combined. The results may be useful for the tailoring of behaviour change interventions to at-risk individuals.", "keywords": [ "tobacco smoking", "excessive alcohol consumption", "personality typologies", "cross-sectional survey" ], "content": "Introduction\n\nCigarette smoking and excessive alcohol consumption are two of the most serious public health problems globally (Stanaway et al., 2018). Each year, up to eight million people die of a smoking-related disease (World Health Organisation, 2021). Excessive alcohol consumption resulted in three million global deaths in 2016, the majority of which were due to injuries or digestive diseases (World Health Organisation, 2018a). Excessive alcohol consumption is implicated in substantial costs to the economy through lost productivity, crime and healthcare costs (Gowing et al., 2015; Lim et al., 2012). Differences in human personality can be accounted for by a limited number of dimensions or traits (Cattell, 1973). The “Big Five” model is a widely applied taxonomy which proposes that personality is underpinned by five major (and intentionally broad) dimensions: openness to experience (i.e. the tendency to be curious and excitable), conscientiousness (i.e. the tendency to be organised and deliberative), extraversion (i.e. the tendency to be sociable and outgoing), agreeableness (i.e. the tendency to be sympathetic and warm) and neuroticism (i.e. the tendency to be self-conscious and moody) (Costa Jr & McCrae, 1992). Several personality traits have been linked to addictive behaviours in general, and with smoking and excessive alcohol consumption in particular (outlined in detail below). To the authors’ knowledge, no study to date has explored whether particular evidence-informed combinations of personality traits (i.e. ‘personality typologies’) are synergistically associated with greater odds of being a current smoker, excessive drinker or both. We aimed to explore this in a large, cross-sectional sample of respondents from the international British Broadcasting Corporation (BBC) Lab UK Study.\n\nEvidence for associations between openness to experience and smoking and excessive drinking is mixed: while some studies have found a positive association of openness with excessive drinking (Martin et al., 2015), others have found a negative or non-significant association with smoking (Jokela et al., 2018; McCann, 2010) or excessive drinking (Luchetti et al., 2018; Scaife & Duka, 2009).\n\nAs conscientiousness comprises facets of self-discipline and deliberation, it is plausible that the association between this trait and smoking and drinking is mediated by behavioural mechanisms associated with self-discipline (e.g. reduced exposure to others who smoke or drink, increased ability to inhibit prepotent responses to smoking or alcohol-related environmental cues). Several cross-sectional and longitudinal studies of representative and non0pt-representative samples indicate that low conscientiousness is associated with increased risk of smoking (Bogg & Roberts, 2004; Hagger-Johnson et al., 2012; Hampson et al., 2006; Hong & Paunonen, 2009; Jokela et al., 2018; Raynor & Levine, 2010; Terracciano & Costa Jr, 2004; Welch & Poulton, 2009) and excessive drinking (Adan et al., 2017; Bogg & Roberts, 2004; Hagger-Johnson et al., 2012; Hakulinen et al., 2015; Ibáñez et al., 2015; Jokela et al., 2018; Kotov et al., 2010; Luchetti et al., 2018; Malouff et al., 2007; Raynor & Levine, 2010; Ruiz et al., 2010).\n\nAs extraversion comprises facets of sociability and sensation seeking, it has been suggested that the association between this trait and smoking and drinking might be mediated by behavioural or biological mechanisms associated with sociability (e.g. greater exposure to others who smoke or drink) or sensation seeking (e.g. experiencing a proportionately greater reinforcing, as opposed to aversive, effect of nicotine or ethanol) (Munafò et al., 2007). In cross-sectional and prospective studies, high extraversion is associated with increased odds of smoking (Jokela et al., 2018; Munafò et al., 2007; Raynor & Levine, 2010) and excessive drinking (Adan et al., 2017; Cheng & Furnham, 2013; Hakulinen et al., 2015; Ibáñez et al., 2015; Jokela et al., 2018; Luchetti et al., 2018; Raynor & Levine, 2010).\n\nEvidence for an association between agreeableness and smoking and excessive drinking is mixed: while some studies have found a positive association of agreeableness with excessive drinking (Whelan et al., 2014), others have found a negative association with smoking (Hampson et al., 2006; Hong & Paunonen, 2009; Terracciano & Costa Jr, 2004) or excessive drinking (Cheng & Furnham, 2013; Hong & Paunonen, 2009; Ibáñez et al., 2015; Jokela et al., 2018; Luchetti et al., 2018; Malouff et al., 2007).\n\nAs neuroticism comprises facets of emotional instability and negative affect, it has been proposed that individuals who score highly on neuroticism may self-medicate with cigarettes or alcohol to reduce anxiety and enhance low mood (Munafò et al., 2007). Results from cross-sectional and longitudinal studies indicate that individuals scoring high on neuroticism have increased odds of being a smoker (Munafò et al., 2007; Terracciano & Costa Jr, 2004; Welch & Poulton, 2009) and excessive drinker (Adan et al., 2017; Jokela et al., 2018; Kotov et al., 2010; Luchetti et al., 2018; Malouff et al., 2007; Ruiz et al., 2010). However, in a sample of university students, low neuroticism was associated with being categorised as a heavy drinker (Lac & Donaldson, 2016).\n\nAlthough several studies have examined the independent associations between the Big Five personality traits and smoking/excessive drinking, few studies have assessed their synergistic (or interactive) effects, henceforth referred to as ‘personality typologies’. Previous research has found an interactive effect of low conscientiousness and low agreeableness (Hong & Paunonen, 2009) and of low conscientiousness and high neuroticism (Terracciano & Costa Jr, 2004) on smoking. Based on the evidence reviewed above, we hypothesised that the combination of (i.e. the three-way interaction between) low conscientiousness, high extraversion and high neuroticism would be synergistically associated with smoking, excessive drinking and the combination of both behaviours.\n\nEvidence suggests that personality traits (Feingold, 1994), smoking (Peters et al., 2014) and alcohol consumption (Wilsnack et al., 2000) vary by sex/gender. There is also evidence to suggest that personality may change over time, with average declines in neuroticism, extraversion and openness, and increases in agreeableness and conscientiousness, over the life span (McCrae et al., 1999). Moreover, low socioeconomic status is associated with both smoking (Casetta et al., 2017) and excessive drinking (Beard et al., 2019) and the prevalence of both behaviours vary by country (World Health Organisation, 2018b, 2019). We therefore included sex, age, socioeconomic status (measured by education) and country of residence as covariates in our analyses.\n\nSpecifically, this study aimed to address the following research questions in a large, cross-sectional sample:\n\n1. Is the combination of low conscientiousness, high extraversion and high neuroticism synergistically associated with current smoking, without and with adjustment for sex, age, education and country of residence?\n\n2. Is the combination of low conscientiousness, high extraversion and high neuroticism synergistically associated with excessive drinking, without and with adjustment for sex, age, education and country of residence?\n\n3. Is the combination of low conscientiousness, high extraversion and high neuroticism synergistically associated with the combination of current smoking and excessive drinking, without and with adjustment for sex, age, education and country of residence?\n\n\nMethods\n\nThis was a correlational study involving cross-sectional data. The STROBE guidelines were used in the design and reporting of this study (Elm et al., 2007). The study protocol and analysis plan were pre-registered on the Open Science Framework (OSF; osf.io/5h9sj). A series of open access online surveys were hosted by the BBC Lab UK Study website between 2009 and 2013. Anyone able to access the website could take part. The pre-registered protocol specified that only respondents from the UK would be included; however, we subsequently decided to include respondents irrespective of their country of residence.\n\nRespondents who were aged 16+ years were included.\n\nInterested respondents were invited to take part in open access experiments and surveys via the BBC Lab UK website. The survey was advertised and promoted via the BBC website, radio programmes and television shows. This was a citizen science project with data being collected by members of the general public in collaboration with scientists. As such, participants were not reimbursed for their time.\n\nThis study involved secondary analyses of fully anonymised data obtained from the BBC Lab UK Study. Hence, ethical approval was not sought. Respondents were told that by clicking on the link to proceed to the survey, they were providing consent to participate. Initiating the survey was used as a record of consent.\n\nOutcome variables\n\nThe outcome variables were current smoking (no vs. yes), excessive drinking (no vs. yes) and the combination of both behaviours (no vs. yes). Current smoking was assessed by the following two items: “Have you ever smoked cigarettes daily, that is, at least one cigarette every day for 30 days?” and “During the past 30 days, on average how many cigarettes did you smoke per day?”. Respondents indicating that they had ever smoked cigarettes daily and any cigarettes in the past 30 days were coded as a ‘current smoker’. Excessive drinking was assessed with the following item: “During the past 30 days, on how many days did you have 5 or more drinks of alcohol in a row, that is, within a couple of hours?” Response options were: 1) 0 days, 2) 1 day, 3) 2 days, 4) 3 to 5 days, 5) 6 to 9 days, 6) 10 to 19 days, 7) 20 to 29 days and 8) all 30 days. Respondents were dichotomised into ‘low/moderate drinkers’ (response options 1-2) and ‘excessive drinkers’ (response options 3-8); this deviated from the pre-registered analysis plan, in which we had specified that response options 2-8 would be categorised as ‘excessive drinkers’. This item broadly corresponds to the third item on the validated Alcohol Use Disorders Identification Test-Consumption (AUDIT-C) scale (Frank et al., 2008) and single-item measures of binge drinking (Wechsler et al., 1995). Respondents categorised as both a ‘current smoker’ and ‘excessive drinker’ received a score of 1 on the clustering variable, with those categorised as either or neither receiving a score of 0.\n\nExplanatory variables\n\nThe explanatory variables were extraversion, conscientiousness and neuroticism, measured with the Big Five Inventory (John et al., 1991). Raw scores were transformed by the BBC Lab UK Study team into a percentage of maximum possible (POMP) score ranging from 0 to 100. As population norms for the Big Five Inventory are lacking, we relied on local norms to categorise responses (low vs. high) using the median split. In a planned sensitivity analysis (pre-specified on the OSF; osf.io/5h9sj), we categorised those scoring 1 standard deviation (SD) above the mean POMP score for each trait as ‘high’ (or 1 SD below the mean as ‘low’ for conscientiousness) vs. all others. In an unplanned sensitivity analysis, we categorised those scoring 1 SD above the mean POMP score for each trait as ‘high’ and those scoring 1 SD below the mean as ‘low’, excluding participants falling within 1 SD either side. This alternative cut-off was selected based on the assumption that any effect is more likely to be detected at the extreme ends of the personality scales and that such a cut-off may also be more clinically useful.\n\nCovariates\n\nCovariates were sex (male, female), age (continuous) and education, measured by combining responses to the following two items: “What is your highest level of formal schooling?” and “If you are still in education, what is the highest level of education you expect to obtain?”. For respondents indicating that they were still in education, responses to the second item were used. Response options were: 1) Did not complete GCSE/CSE/O-Levels, 2) Completed GCSE/CSE/O-Levels, 3) Completed post-16 vocational course, 4) A-Levels, 5) Undergraduate degree, 6) Postgraduate degree. Responses were then categorised into ‘no post-16 qualifications’ (response options 1-2), ‘post-16 qualifications’ (response options 3-4) and ‘higher education’ (response options 5-6). The age variable was capped at 100 years, with responses >100 coded as missing. Respondents were also asked to indicate their country of residence.\n\nData were analysed in RStudio v.3.5.2. Respondents with missing data on any of the variables of interest were excluded from the analyses.\n\nTo address the research questions, multivariable logistic regression analyses were conducted for each of the three outcome variables, including each personality trait, the component two-way interaction effects of the personality traits and the three-way interaction effect of the personality traits, without and with adjustment for all covariates.\n\nBayes factors\n\nPlanned further analyses involved the calculation of Bayes Factors (BFs) using an online calculator to examine whether non-significant associations could best be characterised as evidence of no effect or whether the data were insensitive to detect an effect. In the limited research on synergistic associations between two personality traits and smoking/excessive drinking, small effects have been identified (Terracciano & Costa Jr, 2004). At the same time, meta-analyses and syntheses of data from multiple cohort studies suggest that independent associations between personality traits and smoking/excessive drinking also tend to be small at approximately OR = 1.36 (extraversion and smoking) and OR = 1.2 (neuroticism and excessive drinking) (Hakulinen et al., 2015; Munafò et al., 2007). For a synergistic association (i.e. a three-way interaction) to be considered meaningful, we proposed that the effect needed to be at least similar to those observed for the independent associations between the Big Five traits and smoking/excessive drinking. We therefore set the expected effect sizes to OR = 1.4. The alternative hypothesis was conservatively represented by a half-normal distribution. BFs of ≥3 can be interpreted as substantial evidence for the alternative hypothesis (and against the null), while BFs of ≤1/3 can be interpreted as evidence for the null hypothesis. BFs between 1/3 and 3 suggest that the data are insensitive to distinguish the alternative hypothesis from the null (Dienes, 2011).\n\n\nResults\n\nA total of 588,014 respondents completed the survey, with 363,454 (61.8%) respondents included in the analytic sample (see Table 1). Compared with the overall sample, a nominally greater proportion of those included in the analytic sample had a higher education, resided in the UK, were a current smoker, were an excessive drinker and were a combined smoker and excessive drinker.\n\nNo significant three-way interactions were observed (see Table 2). However, significant two-way interactive effects of high extraversion and low conscientiousness on smoking, excessive drinking and both behaviours combined were observed, with associations remaining robust in the covariate adjusted model. These interactions were further explored in stratified analyses. In addition, significant two-way interactive effects of low conscientiousness and high neuroticism on smoking and excessive drinking were observed, but these associations were no longer significant following adjustment for covariates.\n\nIn stratified analyses to explore the significant two-way interactions between high extraversion and low conscientiousness (including all covariates but none of the interaction terms from the previous models), the association of high extraversion with smoking was more pronounced in those with high (ORadj = 1.51, 95% CI = 1.46, 1.56, p < .001) compared with low conscientiousness (ORadj = 1.38, 95% CI = 1.35, 1.42, p < .001). In contrast, the association of high extraversion with excessive drinking was more pronounced in those with low (ORadj = 1.70, 95% CI = 1.67, 1.74, p < .001) compared with high conscientiousness (ORadj = 1.60, 95% CI = 1.56, 1.63, p < .001). Finally, the association of high extraversion with both behaviours combined was more pronounced in those with high (ORadj = 1.74, 95% CI = 1.65, 1.83, p < .001) compared with low conscientiousness (ORadj = 1.62, 95% CI = 1.56, 1.68, p < .001).\n\nThe calculation of Bayes Factors (BFs) indicated that the data on the three-way interactive effect of extraversion, conscientiousness and neuroticism on smoking and excessive drinking provided evidence for the null hypothesis of there not being any effect compared with large associations of OR = 1.4 (BF = 0.07 and BF = 0.03, respectively).\n\nThe results remained largely robust in the planned sensitivity analysis in which respondents who scored 1 SD above the mean POMP score for each trait were categorised as ‘high’ (or 1 SD below the mean as ‘low’ for conscientiousness), with the remaining respondents categorised as ‘low’ (or ‘high’ for conscientiousness). As in the primary analysis, no significant three-way interactions were observed. In the covariate adjusted model, there were similar main effects and a significant two-way interactive effect of high extraversion and low conscientiousness on excessive drinking (see the Supplementary Online Information, Supplementary Table 1; https://osf.io/c6vmr/). However, unlike the primary analysis, no such two-way interactive effect was observed for smoking, while a two-way interactive effect of low conscientiousness with high neuroticism reached significance, as did a two-way interactive effect of high extraversion with high neuroticism on excessive drinking.\n\nIn the unplanned sensitivity analysis, excluding participants falling within 1 SD of the mean on the selected personality traits, there were fewer significant associations, but in the covariate adjusted model, the three-way interactive effect of high extraversion, low conscientiousness and high neuroticism on excessive drinking reached significance (see the Supplementary Online Information, Supplementary Table 2). In stratified analyses to explore the significant three-way interaction, the association of low conscientiousness with excessive drinking was more pronounced in those with low neuroticism and high extraversion (ORadj = 2.25, 95% CI = 2.02, 2.51, p < .001) compared with those with high neuroticism and high extraversion (ORadj = 1.87, 95% CI = 1.50, 2.32, p < .001), high neuroticism and low extraversion (ORadj = 1.87, 95% CI = 1.65, 2.14, p < .001) and low neuroticism and low extraversion (ORadj = 1.41, 95% CI = 1.10, 1.80, p = .006).\n\n\nDiscussion\n\nThis study identified two-way combinations of personality traits (‘personality typologies’) that were associated with greater odds of being a smoker, excessive drinker or both. Specifically, we observed a two-way interactive effect of extraversion and conscientiousness on smoking, excessive drinking and both behaviours combined. The magnitude (but not direction) of the effects differed depending on the level of conscientiousness. However, as results were only somewhat robust in two sensitivity analyses with different methods of categorising respondents into the levels of the exposure variables, these results must be interpreted with caution. Contrary to expectations, no significant three-way interactions were observed in the primary analysis. The calculation of Bayes Factors indicated that data provided evidence for the null compared with large associations for smoking and excessive drinking. Our findings are somewhat at odds with those reported in the literature: for example, the combination of low conscientiousness and high neuroticism has previously been associated with increased odds of being a smoker (Terracciano & Costa Jr, 2004). Indeed, this was replicated in the first (but not the second) sensitivity analysis with a slightly different method of categorising respondents into the levels of the exposure variables.\n\nTo our knowledge, this was the first study to examine whether ‘personality typologies’ are synergistically associated with greater odds of being a smoker, excessive drinker or both. This study was further strengthened by the large sample size and international recruitment. However, this study also had several limitations. First, the generalisability of the findings is likely limited due to self-selection bias. Although the UK proportion of the sample has previously been found to be representative of the UK population with regards to local authority districts, age and ethnicity (Rentfrow et al., 2015), our analytic sample was biased towards women, respondents with high education and the proportion of current smokers was substantially lower than what would have been expected from representative population surveys (e.g. 20% of adults aged 16+ years in England in 2012). However, the focus of the study was to examine associations between variables of interest (as opposed to prevalence of specific characteristics) and we deem it unlikely that respondents with a specific combination of personality traits and behaviour(s) have self-selected out of the sample. Second, the BBC Lab UK database does not include validated measures of nicotine dependence or patterns of alcohol consumption, such as the Heaviness of Smoking Index (Heatherton et al., 1989) or the Alcohol Use Disorders Identification Test (Babor et al., 2001). This may limit comparisons of the results from this study with those in the wider literature. However, it should be noted that observed associations of sociodemographic characteristics and smoking/excessive drinking were in line with the extant literature. Third, although contested (Jokela et al., 2018), health behaviours such as smoking and drinking may themselves influence the stability and change of personality over time (Allen et al., 2015; Stephan et al., 2019). Due to the cross-sectional nature of the study, we were unable to explore potential temporal trends in personality. Fourth, data collection for the BBC Lab UK Study ceased in 2013. Although the prevalence of smoking and excessive drinking has changed, we deem it unlikely that the associations under investigation have changed over time.\n\nThe finding that respondents with certain combinations of personality traits have greater odds of being a smoker, excessive drinker or both may be useful for informing the tailoring of behaviour change interventions to better engage individuals with high-risk personality typologies. As the identification of at-risk individuals does not require anything other than a brief self-report instrument, this form of tailoring may be particularly feasible and attractive for practitioners to implement. However, as the results were not completely robust in the two sensitivity analyses (with the exception of high extraversion and low conscientiousness being associated with excessive drinking), additional work is required to establish meaningful cut-offs for the personality traits. A useful approach for future work may be to select an analytic technique that maximises variance explained, automatically selecting different cut-offs in relation to smoking and excessive alcohol consumption (e.g. principal components analysis or estimating a receiver operating characteristic curve).\n\n\nConclusion\n\nWe identified two-way ‘personality typologies’ that are associated with greater odds of smoking, excessive drinking and both behaviours combined. The results may be useful for the tailoring of behaviour change interventions to at-risk individuals.\n\n\nData availability\n\nWe used data from the BBC Lab UK Study for this work. We do not have approval to release the individual-level data underpinning the analyses. Anonymised and de-identified individual-level data are available upon request from the corresponding author to bona fide researchers and following approval from the British Broadcasting Corporation (BBC).\n\nAnalysis code available from: https://github.com/OlgaPerski/Personality-typologies\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.5862139 (Perski, 2022).\n\nThis project contains the following files:\n\n- personality_smoking_drinking_28.04.21.R (source code used for analysis)\n\n- variable_names.R\n\nLicense: MIT", "appendix": "References\n\nAdan A, Forero DA, Navarro JF: Personality Traits Related to Binge Drinking: A Systematic Review. Front. Psych. 2017; 8(134): 1–11. PubMed Abstract \| Publisher Full Text\n\nAllen MS, Vella SA, Laborde S: Health-related behaviour and personality trait development in adulthood. J. Res. Pers. 2015; 59: 104–110. 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PubMed Abstract \| Publisher Full Text\n\nWorld Health Organisation: Global status report on alcohol and health 2018. 2018a.\n\nWorld Health Organisation: Global status report on alcohol and health 2018. 2018b; (pp. 1–478).\n\nWorld Health Organisation: WHO report on the global tobacco epidemic. 2019. Reference Source\n\nWorld Health Organisation: WHO report on the global tobacco epidemic 2021: Addressing new and emerging products. 2021. Reference Source" }	[ { "id": "157918", "date": "27 Jan 2023", "name": "Arielle S. Selya", "expertise": [ "Reviewer Expertise Tobacco use behavior" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 examines the interactive effects of personality dimensions on outcomes of smoking, excessive drinking, and both behaviors together, using data from the BBC Lab UK Study, with the hypothesis being that the combination of low conscientiousness, high extraversion, and high neuroticism would interact to have higher risk of smoking and drinking. Results instead found that the combination of high extraversion and high conscientiousness was associated with two of the outcomes (smoking and both behaviors combined), while the combination of high extraversion and low conscientiousness was associated with excessive drinking.\nMajor comments:\nThe Introduction appropriately reviews the five major dimensions of personality, but the hypothesis focuses only on three of them. Why were these particular aspects chosen?\n\nThe authors explain that there are currently no population norms for personality dimensions. How does this impact the implications that these dimensions might be used to identify at-risk individuals?\nMinor comments:\nTypo in Introduction/conscientiousness: “non0pt-representative”.\n\nWhat was the rationale behind the deviation from the pre-registration with respect to the categorization of “excessive drinking?”\n\nTable 1 lists the number excluded from the sample (N=224,560) but the exclusion criteria do not seem to be explained in the text – please add\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9376", "date": "26 Jan 2024", "name": "Olga Perski", "role": "Author Response", "response": "Thank you for the helpful feedback on our manuscript. Please find point-by-point responses to the comments below. Major comments: The Introduction appropriately reviews the five major dimensions of personality, but the hypothesis focuses only on three of them. Why were these particular aspects chosen? Thank you – we have now added the following explanation to the end of the Introduction: “As limited research has been conducted relating to openness to experience and agreeableness, with available studies providing mixed evidence of the associations between these personality traits and addictive behaviours, we were unable to generate a directional hypothesis for these traits.” The authors explain that there are currently no population norms for personality dimensions. How does this impact the implications that these dimensions might be used to identify at-risk individuals? We now further reflect on this in the Discussion: “As this study involved a very large sample, using the median split is likely to provide initially useful population-level insight as to what cut-off may be useful for identifying at-risk individuals. However, although such a pragmatic approach provides initial utility in the absence of population-level norms, further work is required to establish meaningful cut-offs and analyses should be repeated were population-level norms to become available.” Minor comments: Typo in Introduction/conscientiousness: “non0pt-representative”. This has now been amended. What was the rationale behind the deviation from the pre-registration with respect to the categorization of “excessive drinking?” We now provide additional explanation in the Methods: “This deviation was based on a better fit between our categorisation and a validated measure (AUDIT-C). The original categorisation would have overestimated excessive drinking.” Table 1 lists the number excluded from the sample (N=224,560) but the exclusion criteria do not seem to be explained in the text – please add We already state that respondents with missing data on any of the variables of interest were excluded from the analyses. There were no additional exclusion criteria." } ] } ]	1	https://f1000research.com/articles/11-94
https://f1000research.com/articles/11-1166/v1	12 Oct 22	{ "type": "Research Article", "title": "Effect of linear alkylbenzene sulfonate on the uptake of microcystins by Brassica oleracea and Solanum tuberosum", "authors": [ "Glynn Pindihama", "Mugera Gitari", "Ntakadzeni Madala", "Ntakadzeni Madala" ], "abstract": "Background Hypereutrophic conditions in major water reservoirs used for irrigation purposes in South Africa promote the co-existence of cyanotoxins and other pollutants such as linear alkylbenzene sulfonate (LAS). LAS is known to alter the permeability of membranes and promote the uptake of other pollutants by plants. In light of the potential human health risks and prevailing hypereutrophic conditions in some catchments in South Africa, we investigated the combined effects of LAS and microcystins (MCs) when cyanobacteria infested water is used to irrigate terrestrial crops. Methods To understand the potential risks, pot-culture experiments were conducted to assess the effect of LAS on the accumulation of MCs in Brassica oleracea (cabbage) and Solanum tuberosum (potato) plants. The plants were watered with dam water containing 3.48 mg L-1 of the LAS (sodium dodecyl sulfate) and MCs (MC-LR: 10.47 ± 3.879; 6.158 ± 4.127 for MC-RR and 8.160 ± 2.544 for MC-YR μg L-1) for 20 days. Results The presence of LAS, at environmentally relevant concentrations in the irrigation water, did not enhance the uptake of MCs in the two plants, as demonstrated by statistically insignificant differences in the means of the treatments (with and without LAS). In addition, the presence of LAS, high pH, electrical conductivity (EC), and cyanotoxins in the water did not affect the total chlorophyll or the well-being of the plants. However, in some cases the levels of MCs bioaccumulated by the two plants exceeded the WHO recommended tolerable daily intake (TDI). Conclusions These findings imply that the tested levels of LAS and MCs did not have any synergic effects on the two plant species, but irrigating food crops with such water still poses a human health risk.", "keywords": [ "Linear alkylbenzene sulfonate", "Microcystins", "Brassica oleracea", "Solanum tuberosum", "combined effects" ], "content": "Introduction\n\nCases of harmful algal blooms (HABs) have been on the rise globally in the last few decades. Such increases have been linked to growing urbanization and the resultant increase in nutrients loads into the aquatic environment, higher atmospheric temperatures and salinity, all of which are worsened by human-induced climate change (Zhao et al., 2013; Howard et al., 2017; Díez-Quijada et al., 2018). South Africa is a semi-arid country with limited water resources and thus relies on surface waters for irrigation, domestic and industrial uses. The country mainly relies on man-made reservoirs for potable and irrigation water supplies and unfortunately, such surface water sources are increasingly being contaminated by cyanobacteria and cyanotoxins which can bioaccumulate in plant tissue when such water is used for irrigation. This makes the consumption of crops and vegetables irrigated with the contaminated water a potentially dangerous route for human exposure to different cyanotoxins.\n\nThere are numerous types of cyanotoxins that have been reported and documented, but the liver toxins, microcystins (MCs), are the most commonly occurring toxins in freshwater, hence the most widely studied. Contaminated drinking and recreational water are known to pose immediate human health threats, but recent studies have demonstrated food consumption as another exposure route, since cyanotoxins can accumulate in animal and plant tissues (Manubolu et al., 2018). Thus, drinking water and the consumption of contaminated fish, crops and other food supplements are now recognized as routes for human oral exposure (Díez-Quijada et al., 2018).\n\nDue to the recognition of the potential health threats that could be posed by cyanotoxins, the World Health Organization (WHO) has set thresholds for MCs in both drinking water and food (Howard et al., 2017). WHO has set a provisional tolerable daily intake (TDI) of 0.04 μg kg-1 of body weight (b.w.) for MCs and 1 μg L-1 was set as the upper limit for MC-LR in drinking water (Díez-Quijada et al., 2018; Yao et al., 2019). In plants, MCs are also known to have numerous damaging effects such as reducing photosynthesis, causing necrosis of tissues, inducing oxidative stress, reducing productivity of crops, and causing economic losses (Campos et al., 2021).\n\nLinear alkylbenzene sulfonates (LAS) on the other hand, belong to a group of anionic surfactants commonly used in domestic and industrial processes (Wang et al., 2015). Anionic surfactants are a common ingredient in detergents due to their simple synthesis and low cost (Pierattini et al., 2018). LAS is known to alter membrane permeability and in turn affect the toxicity and accumulation of other toxins such as cyanotoxins in organisms (Wang et al., 2012). LAS find their way into the aquatic environment through the discharge of untreated and treated wastewater. LAS elimination in the aquatic environment occurs via adsorption and biodegradation, but their degradation is very slow in anaerobic and anoxic environments and this leads to their accumulation under such conditions in water (Wang et al., 2012).\n\nThis makes hypereutrophic lakes and reservoirs ideal environments for the co-existence of toxic cyanobacteria since the excessive growth of cyanobacteria in eutrophic lakes consumes oxygen and their eventual death and degradation makes water bodies anoxic and anaerobic (Wang et al., 2012). Previous studies have reported enhanced MCs uptake by plants in the presence of LAS (Wang et al., 2012). Furthermore, Wang et al. (2015) also found increased production of MCs by Microcystis aeruginosa in the presence of LAS.\n\nIn South Africa, the Crocodile (West) Marico Water Management Area (WMA) which covers parts of Gauteng and Northwest Provinces, houses dams such as Hartbeespoort, Rietvlei, Roodeplaat, and Bospoort. For many decades these dams have been classified as hypertrophic (van Ginkel, 2004). Thus, the co-existence of pollutants such as LAS, cyanotoxins, and other pollutants is likely in these reservoirs and the ecotoxicological risk of cyanotoxins, such as MCs in combination with other stressors including LAS, on terrestrial food plants is of importance. It is particularly important because the water derived from these hypertrophic dams is mainly used for irrigation.\n\nThe aim of this study was to assess the effect of anionic surfactants (LAS) on the accumulation of three MC congeners (MC-LR, MC-RR and MC-YR) in Brassica oleracea (cabbage) and Solanum tuberosum (potato) plants using environmentally realistic concentrations of the pollutants. Water used in the study was collected from reservoirs in Crocodile (West) and Marico catchment, considering the prevailing hypertrophic conditions in the catchment.\n\n\nMethods\n\nA field survey was conducted from the 23rd to the 25th of June 2019 and again from the 14th to the 16th of September 2019 to collect field water to be used for the experiments. The water was collected from canals and farm dams from the Roodeplaat and Hartbeespoort dam sites. Total dissolved solids (TDS), EC, pH, and turbidity of the water were monitored in-situ and anionic surfactants, chlorophyll-a, microcystins (MCs), and cations were measured ex-situ. The water was kept frozen at −20oC until required. The LAS used in this study was sodium dodecylbenzene sulfonate (SDS) (CAS No. 25155-30-0; molecular weight 348.48 g mol-1; and chemical formula C18H29NaO3S acquired from BYMAZ Pty Ltd, Johannesburg, South Africa).\n\nThe B. oleracea seeds were purchased from NTK Agricultural Products & Services (S.A) and the S. tuberosum seeds were purchased from Livingseeds Heirloom Seeds (Pty) Ltd Midvaal, Gauteng (S.A). The S. tuberosum seeds were first washed with distilled water before being planted in 200 mm plant pots filled with non-contaminated soil. The B. oleracea seedlings were produced and pre-grown in plastic trays with non-contaminated soil. The soil used in this study was collected from the agricultural farm at the University of Venda. The farm lies in the low veld climate and has well-drained deep red soils mostly dominated by clay and falls in the Hutton classification, which is the same as the Rhodic Ferralsol (Mabasa, 2019). Regarding the main nutrients, phosphorus (P), potassium (K), total nitrogen (N) and organic matter, the soil contained 25.86 (mg kg-1); 184 (mg kg-1); 0.079% and 2.07%, respectively. All of which indicated healthy soils for plant growth. The soil was collected from a depth of 0-50 cm, and approximately 15 kg of the soil was placed into 350 mm plastic pots for the experiments and treated with half a cup of Protek General Fertilizer with N:P:K (%) 2:3:2 (14) before introducing the plants.\n\nTo investigate the effect of LAS on MCs uptake and accumulation in B. oleracea and S. tuberosum, plants were watered daily with dam water with a mean concentration ± standard deviation of MC-LR: 10.47 ± 3.879; 6.158 ± 4.127 for MC-RR, and 8.160 ± 2.544 for MC-YR. In order to maintain approximately constant concentrations of the LAS (3.4 mg L-1), the media were tested daily and refreshed accordingly. After 5 days and 20 days of exposure, the accumulations of MCs in edible parts of B. oleracea (leaves) were measured (Table 1). For S. tuberosum, the accumulated MCs in the tubers were assessed only after 20 days of exposure, since the seeds take longer to sprout. Leaves were also harvested from both plant species on the 20th day of exposure for total chlorophyll determination.\n\nChlorophyll content was measured according to Baskar et al. (2015). 50 mg of the leaves were crushed using a mortar and pestle, and then soaked in 10 mL of 95 % (v/v) ethanol, followed by incubation in the dark for 72 hours. This was then followed by centrifuging for 30 minutes at 2264 × g, then collection of the supernatant and reading of the absorbance at 664.2 and 648.6 nm using a UV-vis spectrophotometer (SPECTROstar Nano, BMG LABTECH, Germany). Equations 1, 2 and 3 (below) were then used to calculate the chlorophyll-a and b, and the total chlorophyll content.\n\nTotal chlorophyll content was expressed as milligram per gram per fresh matter (FM).\n\nMicrocystins (MC-LR, MC-RR, and MC-YR) in plant tissues were determined using a triple quadrupole LC–MS/MS system (model 8045, Shimadzu Corporation, Japan). Toxin extraction was conducted using a modification of the method used by Manubolu et al. (2018). Accurately weighed, 100 g of plant material (leaves for B. oleracea and tubers for S. tuberosum) were freeze-dried for 48 hours at −48oC under a constant vacuum of 44 μmHg (Telstar Lyoquest Freeze Dryer, Terrassa, Spain). The freeze-dried material was then ground to powder using a mortar and pestle.\n\n10 mL of 50% methanol solution was added to 1 g of each freeze-dried sample and sonicated for 5 minutes in a water bath (SCIENTEC Ultrasonic Cleaner, Model 705, South Africa) for 5 minutes. Upon sonication, the plant extracts were then centrifuged for 30 minutes at 2264 × g. The whole process of sonication, centrifugation, and collecting the supernatant was repeated thrice and the supernatant was pooled to give approximately a 30 mL extract of each sample. The 30 mL extract was then cleaned up using solid phase extraction (SPE) with HLB (3 cc, 60 mg, Waters Oasis).\n\nFor SPE with HLB, the cartridges were first conditioned with methanol (6 mL), followed by ultrapure water (6 mL). The samples (± 30 mL) were slowly loaded onto the cartridges, followed by rinsing with 20% methanol. The cartridges were then eluted with 10 mL of 80% methanol. Lastly, the eluent was then dried at 50oC under a stream of nitrogen (N2) gas. The dried samples were reconstituted in 1 mL of 80% methanol prior to LC-MS/MS analysis.\n\nChromatographic conditions\n\nLevels of microcystins (-LR, -RR and -YR) in the plant extracts were determined on a triple quadrupole LC–MS/MS system (model LCMS-8045, Shimadzu Corporation, Japan) with a Shim-pack Velox SP-C18, 2.7 μm, with dimensions 2.1 × 100 mm (Shimadzu, Japan). The injection volume was set at 10 μL and the mobile phases used were 0.1% formic acid (FA) in water (A) and 0.1% FA in acetonitrile (B). A flow rate of 0.4 mL min-1 and a 5-minute binary gradient was used with an elution profile of: 2% B (0.4 min), linear gradient to 70% B (3.1 min), 100% B (0.5 min), and, finally, 2% B (1 min).\n\nThe LC-MS/MS interface conditions were: 300oC interface temperature, 3 L min-1 for the nebulizing gas flow, 235oC DL temperature, 10 L min-1 for both drying gas and heating gas flow and interface voltage of 3.0 kV for electrospray in the positive (ES+) mode.\n\nThe final concentration of toxins in each sample was determined using equation 4:\n\nWhere Co = the concentration of the sample determined from the calibration curve (μg L-1).\n\nEquation 5 was utilized to estimate the daily intake of cyanotoxins for an average size human (Bartos, 2020):\n\nWhere:\n\nT = the concentration of toxins in the edible fractions of the cabbage (T, μg kg-1 fresh weight).\n\nC = the daily consumption amounts of cabbage (C, kilograms per day).\n\nW = weight of an average-sized human (W, 60 kg adult).\n\nWe assumed that the consumption of cabbage is similar to that of lettuce and used 85 g dry weight (DW) of cabbage and 148 g dry weight (DW) of potatoes based on the U.S. FDA (2017) suggested serving size (Bartos, 2020). An EDI of >0.04 exceeds the total daily intake limit set by WHO.\n\nTo compare the levels of accumulated MCs and the total chlorophyll of the various plant treatments, analysis of variance (ANOVA) and/or the Kruskal-Wallis tests were used at 95% confidence intervals (CI) using GraphPad InStat 3 (GraphPad Software, California, United States; RRID:SCR_000306). Levels of MCs are presented by their means ± the standard deviation (SD). Kolmogorov–Smirnov and Bartlett tests were used to test for normality and variance homogeneity at 95% CI. Data which passed this test was compared using ANOVA while data which did not pass the test was compared using Kruskal–Wallis at 95% CI. The Tukey-Kramer multiple comparisons test and Dunn’s multiple comparisons test were used as post-hoc assays for data which passed the normality tests and data which did not pass the normality test, respectively.\n\n\nResults & discussion\n\nThe dam water used to water the plants was alkaline, with a mean pH of 9.02 ± 0.29, had high EC and TDS levels (380 ± 16.52 μS cm-1 and 228 ± 7.51 mg L-1, respectively). The water also had a high cyanobacterial biomass (Chlorophyll-a 440.24 ± 328.147 μg L-1). The raw dam water had the following mean concentration ± standard deviation of MC-LR: 10.47 ± 3.879 μg L-1; 6.158 ± 4.127 μg L-1 for MC-RR, and 8.160 ± 2.544 μg L-1 for MC-YR. The pH of the dam water used for irrigation was above the 6.5–8.4 threshold for water intended for irrigation in S. A (DWAF, 1996). Even though the EC of the dam water was quite high, it was within the S. A (DWAF, 1996) guideline and the FAO (1985) (Ayers and Westcot, 1985) limits for irrigation water, which are set at ≤400 μS cm-1 and 700 μS cm-1, respectively. The levels of anionic surfactants in the water ranged from 0.13 to 3.4 mg L-1.\n\nTo identify and quantify MCs, two multiple reaction monitoring (MRM) transitions for MC-LR and MC-YR were selected and optimized, with the most abundant ionic product utilized for quantitation and the other for confirmation, whereas for MC-RR only one transition was used. For MC-LR and MC-YR, the single protonated molecular ions [M + H]+ were formed, as a result of the presence of one arginine moiety, which is the most preferred protonation site for these compounds (Zervou et al., 2017). The MRM transitions employed for MC-LR were 996.0078/996.00 and 498.5078/162.90; for MC-RR: 520.0078/134.90; for MC-YR: 1046.5078/1046 and 523.7578/127.00, with the first one being used for quantification and the second one for confirmation (for MC-LR and MC-YR).\n\nWith respect to all the three MCs monitored, the mass-to-charge ratio (m/z) signal 135 was the main ionic product. Like other polypeptides, MCs form sodium replacement ions which results in ion envelopes at each charge state apparent in mass spectra (Draper et al., 2013). For MC-RR the transition with a m/z of 520.0078 corresponds to the double charged protonated molecular ion [M + 2H]2+ precursor ions, as they contain two arginine residues in their molecular structure (Draper et al., 2013; Zervou et al., 2017).\n\nStandard solutions at seven different concentrations (1, 2, 5, 10, 20, 50, and 100 μg L-1) were prepared using cabbage leaves extracts and potato tuber extracts and these were used to quantify the toxins in the plant samples (Figure 1). The MRM chromatograms of the quantification ions for the three MCs at a concentration of 100 μg L-1 are shown in Figure 2.\n\nWhen exposed to the four different treatments for a period of 20 days, Table 2 shows the mean ± SE concentrations of MCs accumulated in the tubers of S. tuberosum. The accumulation patterns resembled the levels of MCs in the raw water samples, with plants exposed to raw dam water (T2 and T4) showing higher levels of MC-LR, followed by MC-RR then MC-YR. Statistically significant differences among the mean levels of accumulated toxins were reported for MC-LR and MC-YR, whereas MC-RR did show any statistically significant differences (ANOVA/Kruskal-Wallis Test, at 95% CI) among the four treatments. Higher levels of the toxins accumulated in plants exposed to raw dam water (T2) compared to the other three treatments. The presence of LAS in the raw dam water in T4 did not result in higher uptake and accumulation as anticipated.\n\nData labelled with a–d differed significantly (ANOVA/Kruskal-Wallis Test) at p < 0.05 in each row (n = 6).\n\n* p < 0.05.\n\n p < 0.01.\n\n* p < 0.001, n.s = not significant.\n\nExcept for MC-LR, the levels of MCs accumulated by the tubers did not reach levels high enough to exceed the TDI of 0.04 mg kg-1 of body weight recommended by WHO. Since MC-LR is normally the dominant congener in many waters dominated by the Microcystis and Aeruginosa genera, the raw dam water used was dominated by MC-LR, hence it was the only congener which exceeded the recommended TDI.\n\nRegarding the accumulation of the toxins in B. oleracea leaves, Table 3 and Table 4 show the mean levels of MCs accumulated in the leaves of the plants after 5 days and 20 days, respectively. Based on the findings, a clear increase in the accumulation of the three MCs in the plants from the 5th day to the 20th day is observed. Statistically significant differences in treatments were observed for MC-YR and MC-LR after 5 days of exposure, with significantly higher accumulations observed in T2 followed by T4, compared to the other treatments (ANOVA/Kruskal-Wallis Test at p = 0.05).\n\nData labelled with a–d differed significantly (ANOVA/Kruskal-Wallis Test) at p < 0.05 in each row (n = 6).\n\n* p < 0.05.\n\n p < 0.01.\n\n* p < 0.001, n.s = not significant.\n\nData labelled with a–d differed significantly (ANOVA/Kruskal-Wallis Test) at p < 0.05 in each row (n = 6).\n\n* p < 0.05.\n\n p < 0.01.\n\n* p < 0.001, n.s = not significant.\n\nFindings in Table 4 show that statistically significant differences were found among the treatments for all of the three congeners of MCs (ANOVA/Kruskal-Wallis Test at p = 0.05) in B. oleracea leaves upon 20 days of exposure to the four treatments. Similar to the patterns observed for S. tuberosum tubers and for B. oleracea leaves after 5-day exposures, higher levels of MCs accumulated in T2, followed by T4, compared to the other treatments. The findings imply that the presence of LAS in T3 did not have any impact on the uptake of MCs from the soil (without exposure to MCs in irrigation water) and that the presence of LAS in raw dam water in T4 did not enhance the uptake and accumulation of MCs by the plants.\n\nMuch of the work on the combined ecotoxicological risks of LAS and MCs has been done by Wang et al. (2011, 2012). According to Wang et al. (2011), LAS affects organisms by altering their membrane permeability, the activity of enzymes, and the structure of tissues in organisms (Wang et al., 2011). Unlike our findings, where the presence of LAS did not impact the accumulation of MC-LR in plants, Wang et al. (2011) reported higher accumulation rates when lettuce seedling were exposed to a combination of MC-LR and LAS compared to MC-LR alone.\n\nSimilar to our findings, where we found higher levels of MCs in potato tubers compared to cabbage leaves, Wang et al. (2011) reported higher levels in roots compared to other parts of the plants. In contrast to our findings, Wang et al. (2012) found enhanced uptake of MC-LR in duckweed even at the lowest concentration of 3 μg mL-1, which was comparable to the 3.4 μg mL-1 used in this study. The major difference between these experiments was the media in which the experiments were conducted, with Wang et al. (2012) having used aquatic plants compared to the terrestrial plants tested in this study.\n\nIn our study, the presence of microbes with the potential to degrade LAS in the soil could have been a major factor. According to Mao et al. (2015) at low concentrations, surfactants build up at the liquid to liquid or at the solid to liquid interface as monomers. Increasing their concentrations, eventually replaces the interfacial solvent, such as water, leading to decreased polarity of the aqueous-phase and a surface tension reduction. In high concentrations of surfactants, dissolved pollutants in the aqueous phase gain more mobility which is conducive for removal and uptake by plants and even degradation by microbes. Also, the properties of the soil and the surfactant itself influence the adsorption of a surfactant (Mao et al., 2015). In addition, previous studies have demonstrated that soils have the potential to temporarily make cyanotoxins unavailable for uptake by plants through chemical and physical modification, though this is dependent on the type of soil (Bartos, 2020).\n\nThe interaction and combination of LAS and other contaminants including microcystins and metal ions has been found to be both synergistic and, in some cases, antagonistic (Chai et al., 2020). Our findings did not suggest any synergistic nor antagonistic effects of LAS in combination with MCs in the water used. Consistent to our findings, Zhang et al. (2008) did not find increased uptake of Cadmium (Cd) by soybeans in the presence of LAS. Jensen and Sverdrup (2002) also did not find any combined effect of LAS and pyrene on the Folsomia fimetaria. According to Chai et al. (2020) synergistic or combined effects are influenced by a number of factors including the types of contaminants tested, the plant species, the concentrations tested, and the duration of exposure. In this study factors such as faster biodegradation of LAS by microbes and the low concentrations of LAS tested could all have affected the activity and toxicity to the plants of LAS, MCs, and other contaminants.\n\nEven though MC-RR was of a lower concentration in the raw dam water compared to MC-LR, the findings of the current study have shown that it can accumulate in cabbage leaves to levels which can exceed the 0.04 mg day-1 kg-1 of body weight when plants are watered with contaminated dam water. This is of concern since this limit was reached after only 5 days of exposure to the dam water. However the fact that the EDI was not exceeded in the cabbage leaves after 20 days of exposure to the same dam water could be a reflection that the plants were finding ways of copying and bio-transforming the toxins as the exposure was prolonged. Xiang et al. (2020) established that the biotransformation and depuration rates of MC-LR in plants can sometimes exceed its uptake and there can also be higher degradation in soils, thus lower bioaccumulation from soil.\n\nIt is also important to mention that WHO has provisional TDI values for MC-LR only and not for other MC congeners and here we calculated EDIs for each of the three MC congeners monitored. Based on that, assuming that all the MCs have similar impacts on human beings, the combined EDIs for the MCs monitored here and those not monitored in this study can easily exceed the set TDI values.\n\nSince exposure to increased levels of MCs in plants is known to induce oxidative stress, hinder the plants’ ability to produce chlorophyll, and inhibit photosynthesis (Campos et al., 2021), total chlorophyll levels in the two plant species were monitored to assess the potential effects of LAS and MCs on the plants. In addition to the two stressors (MCs and LAS), the dam water used in treatments 2 and 4 also had a high EC (380 ±16.52 μs cm-1) and TDS levels (228 ±7.51 mg L-1), indicating contamination with other salts. All these contaminants, for example MCs (Saqrane et al., 2008; Machado et al., 2017), high pH, high EC (Huang et al., 2017), and anionic surfactants (Pandey & Gopal, 2010; Wang et al., 2012) are also known to induce oxidative stress, reduce chlorophyll production, and affect plant growth.\n\nFigure 3 (i) shows higher total chlorophyll content in the leaves of B. oleracea plants exposed to treatment 1 compared to other treatments. There were no statistically significant differences in the mean total chlorophyll content among the plants exposed to the four treatments after 20 days of exposure (p > 0.05). This implied that the levels of MCs, LAS, and other pollutants in the raw dam water used were not high enough to impact the synthesis of chlorophyll and other photosynthetic processes in the plants.\n\n(n = 6). a indicates no significant difference among treatments (p > 0.05).\n\nConcerning the total chlorophyll content of the S. tuberosum leaves, higher total chlorophyll levels were observed in plants exposed to treatments 1 and 4, but there were no statistically significant differences in the mean total chlorophyll levels among the four treatments (p > 0.05). The findings imply that exposure to environmentally relevant levels of MCs and LAS, as applied in this study, and the high EC in the raw dam water did not induce oxidative stress nor inhibit chlorophyll production in the plants. In addition, no significant visual impacts were observed on the plants exposed to the four treatments.\n\n\nConclusions\n\nBased on the findings, the presence of the anionic surfactant (LAS) did not induce or promote the uptake of MCs by the two plant species. In addition, the presence of LAS and MCs in the irrigation water did not affect the total chlorophyll content and well-being of the tested plants. The study demonstrated that irrigation of terrestrial food plants with cyanobacteria-infested water from dams, such as Roodeplaat, can lead to MCs accumulating in the edible parts of the plants to levels that can exceed the set TDI of 0.04 mg kg-1 of body weight, as recommended by WHO. Long-term studies investigating the levels of cyanotoxins in irrigation water in areas such as the Crocodile (West) Marico WMA and their potential impacts on crop productivity and the dietetic acceptability of such plants for human consumption are recommended. Such studies will need to factor in the local climate, soil types, degradation rates of cyanotoxins in the soil, and also consider a variety of cyanotoxin classes for a proper risk assessment.\n\n\nAuthor contributions\n\nPindihama G.K.: Conceptualization, Methodology, Data curation Roles/Writing - original draft; Writing - review & editing, Investigation; Methodology.\n\nGitari W.M.: Conceptualization, Methodology, Data curation Roles, Supervision; Funding acquisition; Investigation; Resources; Roles/Writing - original draft; Writing - review & editing.\n\nMadala N.E.: Data acquisition/sample analysis, Writing - review & editing\n\n\nData availability\n\nThe knowledge network for biocomplexity: Effect of linear alkylbenzene sulfonate on the uptake of microcystins by Brassica Oleracea and Solanum Tuberosum, DOI: 10.5063/F16M3589 (Pindihama, 2022).\n\nThis project contains the following underlying data:\n\n- Data file 1: comparison of MC levels in the two plants\n\n- Data file 2: total chlorophyll data for Brassica oleracea\n\n- Data file 3: total chlorophyll for Solanum tuberosum\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\nThe authors would like to acknowledge the Department of Nutrition (Faculty of Health Sciences) at the University of Venda for the use of the Shimadzu High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometer (LCMS-8045).\n\n\nReferences\n\nAyers RS, Westcot DW: Water Quality for Agricultura. FAO;1985; 29.\n\nBartos AD: Harmful Algal Blooms: Dominance in Lakes and Risk for Cyanotoxin Exposure in Food Crops.2020.Reference Source\n\nBaskar V, Venkatesh J, Park SW: Impact of biologically synthesized silver nanoparticles on the growth and physiological responses in Brassica rapa ssp. pekinensis. Environ. Sci. Pollut. Res. 2015; 22: 17672–17682. PubMed Abstract \| Publisher Full Text\n\nCampos A, Redouane EM, Freitas M, et al.: Impacts of microcystins on morphological and physiological parameters of agricultural plants: A review. Plants. 2021; 10. PubMed Abstract \| Publisher Full Text\n\nChai L, Yang L, Zhang Y, et al.: Antagonism or synergism? Responses of Hydrocharis dubia (Bl.) Backer to linear alkylbenzene sulfonate, naphthalene and their joint exposure. Ecotoxicol. Environ. Saf. 2020; 200: 110747. PubMed Abstract \| Publisher Full Text\n\nDíez-Quijada L, Guzmán-Guillén R, Prieto Ortega AI, et al.: New method for simultaneous determination of microcystins and cylindrospermopsin in vegetable matrices by SPE-UPLC-MS/MS. Toxins (Basel). 2018; 10: 1–16. PubMed Abstract \| Publisher Full Text\n\nDraper WM, Xu D, Behniwal P, et al.: Optimizing LC-MS-MS determination of microcystin toxins in natural water and drinking water supplies. Anal. Methods. 2013; 5: 6796–6806. Publisher Full Text\n\nDWAF: WATER QUALITY GUIDELINES VOLUME 4 AGRICULTURAL USE: IRRIGATION. Department of Water Affairs and Forestry;1996; 4.\n\nHoward MDA, Nagoda C, Kudela RM, et al.: Microcystin prevalence throughout lentic waterbodies in coastal southern California. Toxins (Basel). 2017; 9: 1–21. PubMed Abstract \| Publisher Full Text\n\nHuang L, Liu X, Wang Z, et al.: Interactive effects of pH, EC and nitrogen on yields and nutrient absorption of rice (Oryza sativa L.). Agric. Water Manag. 2017; 194: 48–57. Publisher Full Text\n\nJensen J, Sverdrup LE: Joint toxicity of linear alkylbenzene sulfonates and pyrene on Folsomia fimetaria. Ecotoxicol. Environ. Saf. 2002; 52: 75–81. PubMed Abstract \| Publisher Full Text\n\nMabasa HZ: Spatial variability of aggregate stability, size distribution, erosion and runoff in selected soils in South Africa. University of Venda;2019.Reference Source\n\nMachado J, Campos A, Vasconcelos V, et al.: Effects of microcystin-LR and cylindrospermopsin on plant-soil systems: A review of their relevance for agricultural plant quality and public health. Environ. Res. 2017; 153: 191–204. PubMed Abstract \| Publisher Full Text\n\nManubolu M, Lee J, Riedl KM, et al.: Optimization of extraction methods for quantification of microcystin-LR and microcystin-RR in fish, vegetable, and soil matrices using UPLC–MS/MS. Harmful Algae. 2018; 76: 47–57. PubMed Abstract \| Publisher Full Text\n\nMao X, Jiang R, Xiao W, et al.: Use of surfactants for the remediation of contaminated soils: A review. J. Hazard. Mater. 2015; 285: 419–435. Publisher Full Text\n\nPandey P, Gopal B: Effect of Detergents on the Growth of Two Aquatic Plants: Azolla pinnata and Hydrilla verticillata. Int. J. Sci. Technol. 2010; 5: 107–114.\n\nPierattini EC, Francini A, Raffaelli A, et al.: Surfactant and heavy metal interaction in poplar: A focus on SDS and Zn uptake. Tree Physiol. 2018; 38: 109–118. PubMed Abstract \| Publisher Full Text\n\nPindihama GK: Effect of linear alkylbenzene sulfonate on the uptake of microcystins by Brassica Oleracea and Solanum Tuberosum. 2022. Publisher Full Text\n\nSaqrane S, El Ghazali I , Oudra B, et al.: Effects of cyanobacteria producing microcystins on seed germination and seedling growth of several agricultural plants. J. Environ. Sci. Heal. - Part B Pestic. Food Contam. Agric. Wastes. 2008; 43: 443–451. Publisher Full Text\n\nvan Ginkel CE : A National Survey of the Incidence of Cyanobacterial Blooms and Toxin Production in Major Impoundments. Directorate: Resource Quality Services Department of Water Affairs and Forestry. 2004; 54. (April).\n\nWang Z, Xiao B, Song L, et al.: Responses and toxin bioaccumulation in duckweed (Lemna minor) under microcystin-LR, linear alkybenzene sulfonate and their joint stress. J. Hazard. Mater. 2012; 229-230: 137–144. PubMed Abstract \| Publisher Full Text\n\nWang Z, Xiao B, Song L, et al.: Effects of microcystin-LR, linear alkylbenzene sulfonate and their mixture on lettuce (Lactuca sativa L.) seeds and seedlings. Ecotoxicology. 2011; 20: 803–814. PubMed Abstract \| Publisher Full Text\n\nWang Z, Zhang J, Song L, et al.: Effects of linear alkylbenzene sulfonate on the growth and toxin production of Microcystis aeruginosa isolated from Lake Dianchi. Environ. Sci. Pollut. Res. 2015; 22: 5491–5499. PubMed Abstract \| Publisher Full Text\n\nXiang L, Li YW, Wang ZR, et al.: Bioaccumulation and Phytotoxicity and Human Health Risk from Microcystin-LR under Various Treatments: A Pot Study. Toxins (Basel). 2020; 12: 523. PubMed Abstract \| Publisher Full Text\n\nYao L, Steinman AD, Wan X, et al.: A new method based on diffusive gradients in thin films for in situ monitoring microcystin-LR in waters. Sci. Rep. 2019; 9: 17528–17528. PubMed Abstract \| Publisher Full Text\n\nZervou S, Christophoridis C, Kaloudis T, et al.: New SPE-LC-MS/MS method for simultaneous determination of multi-class cyanobacterial and algal toxins. J. Hazard. Mater. 2017; 323: 56–66. PubMed Abstract \| Publisher Full Text\n\nZhang Y, Liao B-H, Zeng Q-R, et al.: Surfactant Linear Alkylbenzene Sulfonate Effect on Soil Cd Fractions and Cd Distribution in Soybean Plants in a Pot Experiment. Pedosphere. 2008; 18: 242–247. Publisher Full Text\n\nZhao H, Qiu J, Fan H, et al.: Mechanism and application of solid phase adsorption toxin tracking for monitoring microcystins. J. Chromatogr. A. 2013; 1300: 159–164. PubMed Abstract \| Publisher Full Text" }	[ { "id": "153265", "date": "07 Nov 2022", "name": "Rialet Pieters", "expertise": [ "Reviewer Expertise Ecotoxicologist with a good understanding of what is required in a scientific paper containing analytical chemistry data." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The possibility of LAS enhancing uptake of MC from water and soil into 2 vegetables was investigated. Seedlings were treated with uncontaminated water, water with LAS, water with MCs, and water with both MCs and LAS.\n\nIssues to be addressed:\nUse 'mass' in all instances where the unit is ug or g or kg and not 'weight'. The SI unit for weight is Newton. E.g., body mass, dry mass and molecular mass.\n\nElimination of LAS in Intro \"occurs via adsorption and biodegradation\". Elaborate onto what LAS may adsorb with references.\n\n\"This makes hypereutrophic lakes and reservoirs ideal environments for the co-existence of toxic cyanobacteria since\" insert 'and LAS' between 'cyanobacteria' and 'since'.\n\n\"Previous studies have reported enhanced MCs\" Remove 'have'\n\n\"combination with other stressors including LAS, on terrestrial food plants is of importance.\" Insert 'to the humans consuming these plants' after 'importance'.\n\nIssues with methods:\nAnalysis of LAS never described.\n\nOnly nominal concentrations of MCs in treatments, and not measured concentrations which are crucial.\n\nNo internal standards were used in the analysis of plants for MCs. Therefore, no determination of recoveries.\n\n\"The farm lies in the low veld climate...\" Lowveld\n\n\"...respectively. All of which indicated...\" , all\n\n\"...total daily intake limit set by WHO\" Insert 'the' after 'by'.\n\n\"...15 kg of the soil was placed into 350mm plastic pots for...\" This, 350 mm, is only one length. The pots are likely to have volume and this should be reported.\n\n\"...the media were tested daily and refreshed accordingly\". This description is too vague. At which depths were the soil media sampled? And how was it 'refreshed'? By removing the plant from the soil and replacing with newly prepared media containing concentrations of which? Or is the media referring to 'dam water'? If this is the case, what is meant by 'refreshing the media/water?\n\n\"Chlorophyll content was measured...\" What is this a measure of? In other words, why would you want to determine this?\n\n\"...followed by incubation in the dark for 72 hours.\" 'incubation' usually associated with temperature?\n\n\"The water also had a high cyanobacterial biomass (Chlorophyll-a 440.24 +- 328.147 μg L-1).\" Chlorophyll a is a measure of ALL algal mass, not only cyanobacterial (Biogeosciences Discuss., 4, 707–745, 2007).\n\n\"Standard solutions at seven different concentrations (1, 2, 5, 10, 20, 50, and 100 μg L-1) were prepared using cabbage leaves extracts and potato tuber extracts and these were used to quantify the toxins in the plant samples (Figure 1). The MRM chromatograms of the quantification ions for the three MCs at a concentration of 100 μg L-1 are shown in Figure 2.\" Should move to M&M section.\n\n\"...water) and that the presence of LAS in raw dam water in T4 did not enhance...\" The quantification of this was not demonstrated in the preceding sections of the paper\n\nSuggestion: Report data in tables 2-4 in bar graphs. Will make for an easier comparison of results. Explain treatments again, or name them more descriptively.\n\nReport SD and not SE in tables 2-4.\n\nExpress concentration of Table 2-4 in ng/g to reduce zeroes after decimal point.\n\n\"...used. Consistent to our findings, Zhang et al. (2008) did not find increased uptake of Cadmium...\" No metals are reported in Results? And no analysis thereof is described in M&M. Rephrase this so that it becomes clear that you use the Cd as an example of a toxicant that similarly to MC is not taken up more by plants when in the presence of LAS\n\n\"...plants were finding ways of copying and bio-transforming...\" coping rather than copying.\n\n'It is also important to mention that WHO has...' the WHO.\n\nFig 3 legend: \"a indicates no significant difference among treatments (p > 0.05).\" Remove this sentence and the a's from the figure. You should really only indicate when there was stat. sig. difference like in tables 2-4 and not when there NOT stat. sig.\n\n\"...the high EC in the raw dam water did not induce oxidative stress nor inhibit...\" The preceding text in the manuscript does not show any evidence of the quantification of oxidative stress. This claim is therefore not supported by the data in the manuscript and should be removed.\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?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "182592", "date": "12 Jul 2023", "name": "Ioannis-Dimosthenis S Adamakis", "expertise": [ "Reviewer Expertise Plant stress 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 subject that is being addressed is very interesting however there are some concerns which need to be addressed prior acceptance.\nHypereutrophic conditions leading to increased presence of microcystin producing bacteria is a problem not only in South Africa. Some statements in the manuscript makes it of topical interest and should be avoided.\n\nMicrocystins are toxic to plants. Even if you did not find any symptoms of toxicity you should discuss this. Below I have added some references.\n\nThe material and methods section is not adequate. A more detailed description of the methodology is needed. For instance, the authors state that the plants were watered with water for the dam where microcystins were found. How were those concentration measured? From which species of cyanobacteria?\n\nThe well being of plants are not only determined be the chlorophyll content. The determination of more parameters are needed to make such a statement.\n\nThe English needs polishing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/11-1166
https://f1000research.com/articles/13-86/v1	26 Jan 24	{ "type": "Research Article", "title": "Sustainable economic development through strategic planning, environmental sustainability, innovation, and learning in community enterprises: evidence from Ranong Province, Thailand", "authors": [ "Supattra Pranee", "Natnaporn Eaknarajindawat", "Nattachai Aeknarajindawut", "Sodsri Pulphon", "Chutirom Suwannapirom", "Natnaporn Eaknarajindawat", "Nattachai Aeknarajindawut", "Sodsri Pulphon", "Chutirom Suwannapirom" ], "abstract": "Background This study explores the factors influencing the performance of community enterprises in Ranong Province, Thailand. Drawing on a literature review, the research focuses on the role of strategic marketing, green supply chain management, innovation capabilities, organizational learning, social entrepreneurship, and destination vulnerability in shaping the performance and competitiveness of these enterprises. The study aims to provide insights into how community enterprises can enhance their performance and contribute to the sustainable development of the region.\n\nMethods The findings were based on data collected from 460 community enterprise operators in Ranong province. The data was acquired through questionnaires and in-depth interviews using a combination of qualitative and quantitative research techniques.\n\nResults The findings of this study suggest that strategic marketing plays a crucial role in helping community enterprises identify opportunities, develop unique value propositions, and respond to dynamic market trends. Green supply chain management emerges as an important factor for community enterprises to consider, as adopting environmentally friendly practices can improve their reputation, attract loyal customers, and enhance their overall performance. Innovation capabilities are identified as essential for community enterprises to stay competitive and adapt to changing market conditions. Organizational learning and the establishment of communities of practice are found to be critical for fostering knowledge sharing and enhancing employee skills, thus improving organizational performance. Social entrepreneurship can enable community enterprises to leverage their social missions and differentiate themselves from competitors, while destination vulnerability assessment can help them build resilience and ensure long-term sustainability.\n\nConclusions This study contributes to the understanding of the influences on the performance of community enterprises in Ranong Province and offers practical implications for entrepreneurs, policymakers, and local communities. By focusing on these key areas, community enterprises can improve their performance, achieve sustainable growth, and make a positive impact on their communities and the environment.", "keywords": [ "Community enterprises", "strategic marketing", "green supply chain management", "innovation capabilities", "organizational learning", "social entrepreneurship", "destination vulnerability" ], "content": "Introduction\n\nCommunity businesses in the province of Ranong can enhance their performance by becoming learning organizations. A learning organization improves the financial and social performance of community companies positively, according to Chansuchai (2019). Thus, community enterprises in the province of Ranong can improve their performance by emphasizing becoming learning organizations (Kelloway, 1998).\n\nStrategic Orientation, Green Supply Chain Management (GSCM) Processes, Innovation Capacity, Learning Organization and Performance Literature demonstrates that strategic orientation toward sustainability, GSCM practices, innovation capabilities, and learning organization are favorably related to the performance of organizations, particularly community enterprises. Madhavan, Sharafuddin, and Chaichana (2022) discovered, for instance, that GSCM procedures positively influenced innovation capabilities and company performance. Pranee, Kakhai, Pulphon, and Wongjunai (2022) also discovered that GSCM techniques favorably benefited community firms’ creative capability and financial performance. In addition, Chotsuwan (2017) discovered that strategic orientation toward sustainability favorably benefited the implementation of GSCM processes, which in turn positively impacted the financial performance of enterprises. In addition, Chaowarat (2005) discovered that a learning organization favorably impacted innovation capability and financial performance. In addition, Deerod (2018) discovered that a learning organization significantly influenced the application of GSCM practices, which in turn positively impacted the financial performance of enterprises. Thus, community enterprises in the province of Ranong can improve their performance by adopting a strategic orientation toward sustainability, using GSCM principles, cultivating innovation capability, and transforming into learning organizations. Community enterprises can acquire a competitive advantage over larger firms and contribute to regional sustainability in this manner (Kline, 2016).\n\nSimilarly, a learning organization can enhance the performance of community-based organizations. According to Prasunpangsri (2013), the financial performance of community enterprises that are learning organizations is superior. Thus, community enterprises in the province of Ranong might enhance their performance by transforming into learning organizations.\n\nCompetitive advantages refer to a company’s unique advantages over its competitors. It is a crucial aspect in deciding a business’s success or failure. It has been discovered that competitive advantages have a positive effect on the success of businesses. For instance, Vichitdhanabadee (2010) discovered that competitive advantages significantly impacted the financial success of businesses. Similarly, competitive advantages can enhance the effectiveness of community-based organizations. According to Soedarmanto and Kartika Cahya (2018), the financial performance of community enterprises with competitive advantages is superior. Hence, community firms in the province of Ranong can enhance their performance by cultivating competitive advantages.\n\nSeveral research have investigated the connections between strategic orientation, GSCM practices, innovation capabilities, learning organization, competitive advantages, and performance. For instance, Marnarath and Ranong (2022) discovered that a strategic orientation toward sustainability substantially impacted GSCM procedures, which in turn positively impacted innovation capabilities and performance. Similarly, Phuthong, Anuntavoranich, Chandrachai, and Piromsopa (2022) discovered that GSCM procedures positively impacted innovation capabilities, which in turn positively impacted financial performance.\n\nIn addition, Horadal (2016) discovered that a learning organization favorably impacted innovation capability, which in turn positively impacted competitive advantages and financial performance. Similarly, Egbunike and Obi (2018) discovered that a learning organization influences competitive advantages and financial performance positively. The relationship between strategic orientation, GSCM processes, innovation capabilities, learning organization, competitive advantages, and performance is complex. Community enterprises in the province of Ranong can enhance their performance by adopting a strategic orientation toward sustainability, using GSCM principles, increasing innovation capability, becoming learning organizations, and establishing competitive advantages.\n\nThe 20-year National Strategy Framework (2017-2036) consists of six strategies. The workers and development of small and medium-sized enterprises (SMEs) help develop community enterprises and farmers’ institutions internationally. Community enterprises are small businesses essential for the economy’s development. In addition to increasing productivity by using science, research technology, and innovation in all manufacturing and services sectors, the existing and new revenue bases (Burana-at, 2021; Agic, E., Cinjarevic, M., Kurtovic, E., & Cicic, M. 2016). At the same time, it lays a solid foundation to support competitiveness enhancement in both stability and macroeconomic performance. For the development of areas, particular economic areas, geopolitics, and cities, it is possible for Thailand to use the strengths of each area and community to support the overall economic development as well as to support the distribution of wealth to different regions in the country, reducing inequality in all dimensions and strengthening the economy and society at the local level in a sustainable way. Brändle, L., Golla, S., & Kuckertz, A. (2019) said that developing a foundational economy to build a local community economy, be self-reliant, and help each other. It is a virtuous and economic system that facilitates the development of other aspects in the following area. Thailand’s economic development during the 13th development plan accelerates the restructuring of the manufacturing sector economy to transform into economic mobility. By innovating and towards sustainable development. Focus on creating value for qualitative products and services. It also emphasizes distributing benefits to relevant sectors within the country thoroughly and concretely. It conveys the idea of transforming the country into policies and plans at various levels that support upgrading the manufacturing sector to the industries and services of the future. Both to revive the economic conditions from the COVID-19 pandemic and to drive the development of manufacturing fields that will drive Thailand’s economic growth in the next phase.\n\nCommunity enterprises the department of agriculture defines as “small entrepreneurship to creatively manage community capital for self-reliance and adequacy of families and communities (Schumaker and Lomax, 1996). A community enterprise is a business started and run by a group of people who share a common way of life. The purpose of a community enterprise is to generate income and help the families involved to become self-reliant community and inter-community” (Venkatachalam, S., Marshall, A., Ojiako, U., & Chanshi, C. S., 2019). Following the criteria announced by the Board of Promotion of Community Enterprises, the nature of community enterprises has been defined from the definition. In summary, the following are essential characteristics: 1) The community owns the business. However, outsiders may participate, with shareholdings for participation, cooperation, and assistance, but small shares give them the power to make decisions. 2) The produce comes from community processes, which may bring in some raw materials from outside but focus on making the most of local resources. 3) Creative initiatives are innovative for the community to develop the potential of the community, which has knowledge and wisdom. Suppose there is a proper learning process. Born with confidence in yourself, takes the initiative. 4) Innovate without imitating or seeking a recipe for success. There is a local wisdom base combined with universal wisdom, a local wisdom base, and a critical capital base blended with knowledge of universal wisdom or from somewhere else. 5) An integrated operation systematically links various activities, similar to a single crop, not as a single project. However, as agroforestry, i.e., there are many activities, synergy, and support. 6) There is a learning process at its core. Learning is at the heart of the development process. Without learning, there will be only imitation. Without learning, there would be no community enterprises based on knowledge but on a sense-based basis. The demands that the media in society encourage make cravings necessary for life. 7) Self-reliance as a goal Self-sufficiency is community enterprises’ first and foremost goal. If you miss this goal, you miss your target. Community enterprises will become businesses that aim for a profit before they are thought of as surviving. It’s important to develop a step by step process to achieve stability before moving on to business development (Borazon, Huang, & Liu, 2021).\n\nSetting up a community enterprise to accomplish our goals is a great way to bring people together and work toward a common objective. It is necessary to understand the new theory’s three steps. Those members of community enterprises look primarily at income because community enterprises are not solely focused on profit creation Fornell and Larcker (1981). It does not aim for profit as much as possible but fulfills coexistence. Good health has a good quality of life for people in the community. The production of goods by community enterprises is based on an extension of local wisdom. Local ingredients are available. This important aspect has been an essential factor in the sustainability of community enterprises. Many communities will receive government support to establish career promotion groups to increase income and develop them into community enterprises. The entry of various government agencies promotes careers for that community. There needs to be more understanding of local wisdom, and many communities need to have local wisdom to support it. The problem that follows is that the community produces the goods as they are promoted and asks to be registered as a community enterprise for grants, but the goods produced are not due to their wisdom. Make it the same product—inability to compete in markets characterized by industrial products. As a result, community enterprises needed more sustainability and eventually fell away. The nature of local wisdom is often accompanied by folk sages who are knowledgeable in wisdom or, it can be said, own that wisdom. Developing the potential of the sage locals is, therefore, necessary to raise the level of knowledge to a standard. Build knowledge leadership in the community so that the professional integration of the community can be developed into a sustainable community enterprise.\n\nTherefore, this research aims to study strategic orientation, green supply chain management, innovation capability, learning organization, and competitive advantages affecting the performance of community enterprises in Ranong province, Thailand, in transition to driving the economy through innovation and towards sustainable development. Identifying the value of local wisdom is transformative to keeping pace with change and generating sustainable income for the community.\n\nHypothesis 1: There is a significant relationship between Strategic Orientation and Competitive Advantages. H2: There is a significant relationship between Strategic Orientation and Community Enterprises Performance. H3: There is a significant relationship between Green Supply Chain Management and Competitive Advantages. H4: There is a significant relationship between Green Supply Chain Management and Community Enterprises Performance. H5: There is a significant relationship between Innovation Capability and Competitive Advantages. H6: There is a significant relationship between Innovation Capability and Learning Organization. H7: There is a significant relationship between Innovation Capability and Community Enterprises Performance. H8: There is a significant relationship between Learning Organization and Competitive Advantages. H9: There is a significant relationship between Learning Organization and Community Enterprises Performance. H10: There is a significant relationship between Competitive Advantages and Community Enterprises Performance.\n\nH1: There is a significant relationship between Strategic Orientation and Competitive Advantages.\n\nStrategic orientation is a critical component that determines a company’s competitive advantage. A strategic orientation toward sustainability, according to Poapongsakorn (2011), can assist organizations in creating a competitive edge. Firms that prioritize sustainability can distinguish themselves from their competition by implementing sustainable practices that fulfill the needs of their consumers and stakeholders. Furthermore, a strategic focus on sustainability can boost the firm’s reputation, leading to enhanced customer loyalty and improved stakeholder relationships.\n\nH2: There is a significant relationship between Strategic Orientation and Community Enterprises Performance.\n\nAccording to research, a strategic focus on sustainability can improve the success of community enterprises. Community enterprises with a strategic orientation toward sustainability perform better financially and socially, according to Calgaro, Pongponrat, and Naruchaikusol (2009) and Taghipour, Akkalatham, Eaknarajindawat, and Stefanakis (2022). Moreover, a strategic emphasis on sustainability can assist community firms differentiate themselves from rivals, resulting in increased consumer loyalty and strengthened stakeholder relationships.\n\nH3: There is a significant relationship between Green Supply Chain Management and Competitive Advantages.\n\nGreen supply chain management (GSCM) methods can give organizations a competitive advantage. GSCM practices are favorably connected with corporate financial success, according to Poontai (2019) and the Climate Bonds Initiative (2022). GSCM techniques can assist organizations in gaining a competitive edge by increasing environmental performance, lowering expenses, and boosting their reputation. Furthermore, applying GSCM processes can help organizations comply with environmental standards while also lowering their exposure to environmental risks.\n\nH4: There is a significant relationship between Green Supply Chain Management and Community Enterprises Performance.\n\nGSCM practices can also improve the performance of community enterprises. GSCM techniques, according to Wiraphanphong (2020), have a beneficial impact on the financial performance of community companies. Adopting GSCM techniques can assist community enterprises in lowering costs, improving environmental performance, and improving their reputation. Furthermore, employing GSCM techniques can help community enterprises comply with environmental rules and mitigate environmental concerns.\n\nH5: There is a significant relationship between Innovation Capability and Competitive Advantages.\n\nThe ability to innovate is a vital component that determines a company’s competitive edge. According to Hoa (2019), corporate financial performance is favorably related to innovation capability. Businesses can gain a competitive advantage through producing new and improved products, processes, and services. Furthermore, the capacity to innovate can help firms stay ahead of their competition by adjusting to market developments and fulfilling the changing needs of their clients.\n\nH6: There is a significant relationship between Innovation Capability and Learning Organization.\n\nInnovation capabilities can also positively influence the performance of community enterprises. Developing innovation capability can help community enterprises develop new and better products, processes, and services that meet the needs of their customers. Moreover, innovation capability can help community enterprises stay ahead of their competitors by adapting to changes in the market and meeting the changing needs of their customers (Chansuchai, 2019).\n\nH7: There is a significant relationship between Innovation Capability and Community Enterprises Performance.\n\nInnovation capability and being a learning organization are interrelated concepts. Innovation capability is essential for community enterprises to develop new and better ways of doing things. Being a learning organization facilitates continuous learning and improvement, which is essential for community enterprises to adapt to changes in the market and meet the changing needs of their customers (Chotsuwan, 2017).\n\nH8: There is a significant relationship between Learning Organization and Competitive Advantages.\n\nA learning organization can provide a competitive advantage to businesses. Being a learning organization can help businesses develop new and innovative solutions to problems, adapt to changes in the market, and continuously improve their operations. Moreover, a learning organization can facilitate the development of new skills and knowledge, leading to improved productivity and employee satisfaction (Hair et al., 2010).\n\nH9: There is a significant relationship between Learning Organization and Community Enterprises Performance.\n\nInnovation capability is an essential factor that influences the performance of community enterprises. Developing innovation capability can help community enterprises develop new and better products, processes, and services that meet the needs of their customers. Moreover, innovation capability can help community enterprises stay ahead of their competitors by adapting to changes in the market and meeting the changing needs of their customers (Chotsuwan, 2017).\n\nH10: There is a significant relationship between Competitive Advantages and Community Enterprises Performance.\n\nCompetitive advantage is a critical factor that influences the performance of community enterprises. Developing competitive advantages can help community enterprises differentiate themselves from their competitors, leading to increased customer loyalty and improved stakeholder relationships. Moreover, competitive advantages can enable community enterprises to reduce costs and improve their operations, leading to improved financial performance (Climate Bonds Initiative, 2022).\n\nThis literature review examined the relationship between strategic orientation, GSCM practices, innovation capability, learning organization, competitive advantages, and performance of community enterprises in Ranong Province, Thailand. The review found that a strategic orientation towards sustainability, GSCM practices, innovation capability, being a learning organization, and developing competitive advantages can improve the performance of community enterprises. Moreover, the literature review identified the interrelationship between these factors and the critical role they play in enhancing the competitiveness and sustainability of community enterprises.\n\nCommunity enterprises in Ranong Province can improve their performance by integrating environmental considerations into their supply chain process, developing new and better ways of doing things, facilitating continuous learning and improvement, and developing unique advantages that set them apart from their competitors. Furthermore, community enterprises need to adopt a holistic and integrated approach to sustainability to create long-term value for their stakeholders.\n\nHowever, it is essential to note that the literature review is limited to the context of community enterprises in Ranong Province, Thailand. Therefore, caution should be taken when generalizing the findings to other regions or types of enterprises. Further research is needed to confirm the relationships found in this literature review and to explore other factors that may influence the performance of community enterprises.\n\n\nResearch methods\n\nThis paper explores the influence of strategic orientation, green supply chain management, innovation capabilities, and learning organization on the competitive advantage and performance of community companies in the province of Ranong, Thailand. The study analyzed the data acquired through questionnaires and in-depth interviews using a combination of qualitative and quantitative research techniques. Based on a survey of 460 community enterprise operators in Ranong province, this paper was written.\n\nParticipants were recruited over a specific period from March to July 2022. The recruitment phase involved reaching out to community organizations, local authorities, and relevant stakeholders to identify potential participants. Data collection included administering questionnaires and conducting in-depth interviews. This phase took place from April to June 2022 to gather a comprehensive and representative dataset. After data collection, the analysis phase involved processing and analyzing the collected data (Pranee et al., 2023).\n\nThis research has been approved by the Suan Sunandha Rajabhat University and the approval number for the project is COE. 2-474/2022. A detailed explanation of the project was provided to the participants willing to participate in the study, and written consent was obtained from them. The researcher told them that all their information would remain anonymous and would not be used for other purposes.\n\nThe study participants were community enterprise operators with businesses located in Ranong province, Thailand. The researchers collected data using a recruitment process that involved accessing the list of registered community enterprise operators from the Division of Community Enterprise Promotion. The researchers collaborated with local authorities, community organizations, and relevant stakeholders to facilitate the participant recruitment process. To be eligible, participants had to be officially registered with the Division of Community Enterprise Promotion within the Department of Agriculture Promotion. From the larger group of community enterprise participants, the researchers selected a subsample of 460 community enterprise operators for a more in-depth examination, using systematic sampling to ensure that the chosen subset is representative of the broader population of community enterprise operators in Ranong Province.\n\nThe researchers concentrated on analyzing samples of community enterprise operators in the province of Ranong. To assess the Structural Equation Model, the sample size was estimated using multivariate techniques (SEM). The sample size was found to be 20 times the observed variables used to determine samples in the algorithm, or a 1:20 ratio. The minimal sample size necessary to examine the data using the SEM technical equipment was determined to be 460 community enterprise operators in Ranong province.\n\nParticipants\n\nA total of 460 participants completed the questionnaires and were interviewed to enrich the dataset.\n\nQuestionnaire\n\nThe researchers collected data using a combination of qualitative and quantitative research techniques. They created a questionnaire by examining several ideas, theories, and studies pertaining to supply chain management, innovative capabilities, competitive advantage, and performance of community firms.\n\nThe questionnaire was created by the following procedures:\n\nThe process of developing the questionnaire for this study was thoroughly planned. The researchers took a meticulous journey encompassing various steps to ensure the reliability and relevance of the questions (Pranee et al., 2023). We conducted an extensive exploration of relevant concepts, theories, and past studies, distilling each query into a practical definition that served as the foundation for questions included in the questionnaire. Each question was then evaluated and revised to ensure that they were clear, relevant, and unbiased. To enrich the dataset, researchers conducted in-depth interviews to provide a more comprehensive exploration of the research topic. By integrating questionnaire data and insights gleaned from in-depth interviews, we were able to provide a holistic perspective that captured both the quantitative and qualitative dimensions of the research inquiry.\n\nThe researchers conducted quality checks on the questionnaires by consulting an expert to determine their content validity.\n\nThe questionnaire details are as follows: The questionnaires were administered at various locations, including community enterprise offices and online platforms. Participants were instructed to finish tasks within 30 minutes to ensure constancy in response times. To encourage open and reflective answers free from external influence or coercion, the environment was carefully selected.\n\nThe reliability and construct validity of the questionnaires were verified by analyzing affirmative elements. Confirmatory factor analysis revealed that the focus of the research was on green supply chain management strategies, innovation capabilities, learning organization, competitive advantage, and operational effectiveness. The tables present various fit indices for the Default Model. Key findings include a CMIN/DF of 49.770, indicating a poor fit, while the NFI and CFI values of .976 suggest a good fit. Parsimony may be an issue, with PNFI and PCFI values of .130. The RMSEA value of.326 points to a poor fit. The Default Model’s AIC is 149.540, higher than the Saturated Model’s 54.000, and the ECVI is .326, also higher than the Saturated Model’s.118. Hoelter Index values are 28 (.05 significance) and 43 (.01 significance), suggesting limited sample size adequacy. Overall, the model presents a mix of good and poor fit indices, with parsimony being a potential issue.\n\nInterviews\n\nOur approach to gathering information and insights into our series of community enterprise descriptions involves structured interviews with active participants in various community enterprises (Supattra et al., 2023). We carefully designed interview questions to capture the key aspects of each participant’s involvement and experience. These interviews were semi-structured, allowing for flexibility while ensuring consistency in the collected information. We reached out to participants through various communication channels based on their preferences and provided an overview of the purpose of the interviews and the topics that will be covered. Consent to participate was obtained from everyone before proceeding with the interviews. The participants shared their experiences, motivations, and roles within their respective community enterprises during the interviews. We gathered detailed information on the duration of their active participation, specific responsibilities held, and the impact of their contributions. We also gathered insights into their community enterprises’ strategic orientation, including clear visions or long-term plans. Our inquiries into sustainability practices prompted participants to elaborate on how their community enterprises incorporate sustainability into their strategic orientations. We solicit specific examples to illustrate how these practices have given enterprises a competitive advantage and have contributed to their overall success. We also included questions on implementing green supply chain management practices, asking participants to share concrete examples of how these practices have benefited their operations. We assessed innovation capability by asking participants to share examples of innovative initiatives or projects within their community enterprises. The data collected from the interviews were carefully analyzed, and common themes, patterns, and insights were identified. These findings were used to evaluate participants’ experience and their community enterprises’ strategic orientation, sustainability practices, innovation capability, and overall impact. Ethical considerations were upheld throughout the data collection and analysis process, and participants’ privacy and consent were respected.\n\nFrom the questionnaire survey, a subset of 460 community enterprise operators were chosen for the interview process. A specified number of participants were involved in both the questionnaires and interviews. Quantitative and qualitative data collection are what dual engagement provided, resulting in a more robust approach. The interviews were conducted for approximately [30-45 minutes] to fully explore the comments of each participant. A specific environment was used to create a private and peaceful atmosphere for the participants to interact. Only the researcher(s) and the participant were present during the interviews to ensure a focused and secure conversation, which allowed the participants to speak openly. A guided yet flexible discussion was facilitated by the semi-structured interview format, enabling participants to offer intricate insights.\n\nThe study utilized both model fit indices and descriptive statistics to assess the fit of the research model to the data and provide insights into the participants’ perceptions (Figures 1 and 2). The key model fit indices, summarized in Table 1, offer information on the goodness-of-fit and predictive accuracy. In contrast, Table 2 presents the analysis results of variables related to the level of strategic focus on different factors. The descriptive statistics offer an overview of the participant’s responses to each variable, providing insights into their perceptions and viewpoints on strategic orientation, practices, and outcomes in the context of community enterprises.\n\nModel fit index analysis\n\nThe model fit indices provide information on how well the research model fits the data and the predictive accuracy of the model. These indices help evaluate the goodness-of-fit of the proposed model. Structural equation modeling software was used to conduct the model fit analysis is structural equation modeling (SEM). The model fit indices in Table 1 included CMIN/DF, NFI, CFI, PNFI, PCFI, RMSEA, AIC, ECVI, and Hoelter. These indices collectively assess model fit, complexity, and predictive accuracy.\n\nDescriptive statistics analysis\n\nDescriptive statistics were used to summarize and analyze the characteristics of the variables under investigation. The statistics included the mean and standard deviation, providing insights into the central tendency and variability of the data. Data was analyzed using IBM SPSS Statistics, version 27 (IBM Corp., Armonk, NY, USA).\n\n\nResults\n\nThe level of strategic focus on green supply chain management, innovation capabilities, learning organization, competitive advantage, and operational effectiveness are shown in Table 2.\n\nThe table provides insights into the characteristics of the six variables. Strategic Orientation and Performance of Community Enterprises show moderately high mean values, suggesting that respondents generally exhibit strong strategic orientation and community enterprises have a good performance. Green Supply Chain Management, Innovation Capability, and Competitive Advantages present moderate mean values, indicating there is room for improvement in these areas.\n\nThe standard deviations reveal the dispersion in scores for each variable. The small dispersions in Strategic Orientation and Performance of Community Enterprises indicate that the majority of respondents have similar perceptions of these factors. The larger dispersion in Learning Organization scores suggests that respondents may have diverse levels of learning organization practices.\n\nIt is crucial to explore relationships between these variables to understand their impact on one another. For instance, investigating the influence of Strategic Orientation, Green Supply Chain Management, and Innovation Capability on Competitive Advantages and Performance of Community Enterprises could provide valuable insights for improving organizational performance. The equal number of observations for all variables (N=460) ensures a robust analysis.\n\nSkewness values indicate the symmetry of the distributions. Negative skewness for all variables suggests left-skewed distributions, with Competitive Advantages being the most left-skewed. This indicates that there might be more high values in the dataset for these variables.\n\nKurtosis values provide insights into the shape of the distributions. Strategic Orientation and Green Supply Chain Management have negative kurtosis values, indicating lighter tails and flatter distributions (platykurtic). In contrast, Innovation Capability, Competitive Advantages, and Performance of Community Enterprises exhibit positive kurtosis, suggesting heavier tails and more peaked distributions (leptokurtic). This information helps inform the choice of appropriate statistical techniques for further analysis (Tables 3, 5, and 7).\n\nTable 4 shows the association between all factors identified in the research. A strong strategic orientation is positively associated with green supply chain management, innovation capabilities, the adoption of a learning organization culture, competitive advantages, and ultimately, improved performance of community enterprises.\n\n** Correlation is significant at the 0.01 level (2-tailed).\n\nThe adoption of green supply chain management practices is linked to enhanced strategic orientation, innovation, learning organization culture, competitive advantages, and overall performance of community enterprises.\n\nA higher innovation capability is found to contribute to better strategic orientation, green supply chain management, learning organization culture, competitive advantages, and consequently, improved community enterprise performance.\n\nThe presence of a learning organization culture is positively correlated with strategic orientation, green supply chain management, innovation capabilities, competitive advantages, and superior performance of community enterprises.\n\nCompetitive advantages are identified as being interconnected with strategic orientation, green supply chain management, innovation capabilities, and learning organization culture, leading to an overall positive impact on community enterprise performance.\n\nThe performance of community enterprises is strongly influenced by the combination of strategic orientation, green supply chain management, innovation capabilities, learning organization culture, and competitive advantages.\n\nStrategic Orientation positively influences Learning Organization (β = .575, p < .001). Green Supply Chain Management positively influences Learning Organization (β = .241, p = .004). Innovation Capability positively influences Learning Organization (β = .711, p < .001). Strategic Orientation positively influences Competitive Advantages (β = .593, p < .001). Green Supply Chain Management positively influences Competitive Advantages (β = .151, p = .002). Innovation Capability negatively influences Competitive Advantages (β = -.117, p = .027). Learning Organization positively influences Competitive Advantages (β = .373, p < .001). Strategic Orientation negatively influences Performance of Community Enterprises (β = -.145, p = .015). Green Supply Chain Management positively influences Performance of Community Enterprises (β = .238, p < .001). Innovation Capability does not significantly influence Performance of Community Enterprises (β = .082, p = .210). Competitive Advantages positively influence Performance of Community Enterprises (β = .170, p = .003). Learning Organization positively influences Performance of Community Enterprises (β = .275, p < .001).\n\nIn Table 6, hypothetical test results are summarized for the relationships between various factors and their impact on community enterprises performance in Ranong Province, Thailand. Each hypothesis is presented along with the relationship, estimate (beta coefficient), p-value, and the result (supported or not supported).\n\nOverall, most hypotheses are supported, indicating significant relationships between strategic orientation, green supply chain management, innovation capability, learning organization, and competitive advantages on community enterprises performance. Notably, H2 and H5 show negative relationships, suggesting that the impact of strategic orientation and innovation capability on community enterprises performance and competitive advantages, respectively, is inverse. The only hypothesis that is not supported is H7, indicating no significant relationship between innovation capability and community enterprises performance. These findings provide valuable insights for understanding the factors affecting the performance of community companies in Ranong Province, Thailand, and can guide future research and practical initiatives to enhance their performance.\n\nIn the Standardized Direct Effects section, the table shows the direct impact of each independent variable on the dependent variables. For instance, Strategic Orientation has a positive direct effect on Learning Organization (.253) and Competitive Advantages (.389), while having a negative direct effect on the Performance of Community Enterprises (-.101). Green Supply Chain Management has positive direct effects on all three dependent variables.\n\nThe Indirect Effects section represents the influence of the independent variables on the dependent variables mediated through other variables in the model. For example, Innovation Capability has an indirect effect on Competitive Advantages (.265) and Performance of Community Enterprises (.221) through its impact on Learning Organization.\n\nIn the Total Effects section, the table displays the combined impact of both direct and indirect effects. This section reveals the overall influence of each independent variable on the dependent variables. Notably, Strategic Orientation has strong total effects on both Learning Organization (.575) and Competitive Advantages (.808), while Green Supply Chain Management demonstrates substantial total effects on Performance of Community Enterprises (.345).\n\nThis comprehensive analysis of the effects provides a deeper understanding of the relationships between the independent and dependent variables. The findings can inform decision-makers and researchers about the critical factors that influence the performance of community companies in Ranong Province, Thailand, and provide guidance for implementing effective strategies to improve their performance. The results also highlight potential areas for future research, such as examining the reasons behind the negative direct effect of Innovation Capability on Competitive Advantages and the negative direct effect of Strategic Orientation on the Performance of Community Enterprises.\n\nIn conclusion, the results of this study’s hypothesis testing give solid evidence for the linkages between strategic orientation, green supply chain management, innovation capability, learning organization, competitive advantages, and community enterprise performance. These findings have significant ramifications for community business operators, legislators, and researchers who aim to enhance the effectiveness and sustainability of these organizations.\n\nStrong positive links exist between strategic orientation, competitive advantage, and performance, indicating that community companies that prioritize strategic planning and development are more likely to produce superior commercial outcomes. In addition to emphasizing the significance of environmental sustainability and social responsibility in community enterprise operations, the positive impact of green supply chain management on competitive advantage and performance highlights the significance of environmental sustainability and social responsibility in community enterprise operations.\n\nInnovation capability was found to have a strong beneficial effect on both competitive advantage and performance, suggesting that inventive and adaptive community companies are more likely to flourish. The favorable association between a learning organization and competitive advantage and performance highlights the significance of continual learning and information sharing in community enterprise development.\n\nThe findings of this study reveal that community companies in the Thai province of Ranong can improve their performance by adopting strategic orientation, green supply chain management, innovation capabilities, and learning organization practices. These findings can guide future research and policy initiatives aimed at boosting community enterprises and improving the region’s sustainable economic development.\n\nIt is crucial to highlight, however, that these results are exclusive to Ranong Province and may not be applicable to other contexts. There is a need for additional study to investigate the correlations between these variables in various contexts and to uncover additional factors that may affect the performance of community enterprises.\n\nIn conclusion, this study offers valuable insights into the factors influencing the performance of community enterprises in Ranong Province, Thailand, and highlights the significance of strategic planning, environmental sustainability, innovation, and learning in achieving sustainable economic development. This study’s findings can influence future research and policy initiatives intended to boost community enterprise development and promote sustainable economic growth in the region.\n\n\nDiscussion\n\nIn this discussion, we synthesize findings from the literature to provide insights into the factors that influence the performance of community enterprises in Ranong Province, Thailand. Our analysis is based on key references that examine various aspects of strategic marketing, green supply chain management, organizational performance, and social entrepreneurship.\n\nAgic et al. (2016) suggest that strategic marketing patterns are essential for achieving competitive advantages and improving organizational performance. Community enterprises in Ranong Province could benefit from adopting strategic marketing practices to better position themselves in the market and respond to the dynamic needs of consumers. Focusing on the development of unique value propositions, targeted marketing efforts, and continuous market analysis can help these enterprises gain a better understanding of the market trends and respond accordingly. Green supply chain management (GSCM) is another critical factor influencing the performance of community enterprises. Ardakani et al. (2022) emphasize the importance of collaboration between customers and suppliers in achieving a sustainable green supply chain. By incorporating environmentally friendly practices into their operations, community enterprises in Ranong Province can enhance their reputation, increase customer loyalty, and improve their overall performance. Borazon et al. (2021) also highlight the role of green market orientation in driving organizational performance in the context of Taiwan’s electric and electronic industry. A similar approach could be applied to community enterprises in Ranong Province, fostering the adoption of green practices and aligning their operations with sustainable development goals.\n\nInnovation capabilities play a crucial role in organizational performance and competitiveness, as demonstrated by Farzaneh et al. (2020). By investing in innovation, community enterprises in Ranong Province can enhance their capabilities to develop new products, services, and processes, which will allow them to stay competitive in the market. Additionally, innovation can help these enterprises overcome challenges and adapt to changing market conditions, ensuring long-term growth and sustainability.\n\nManuti et al. (2017) highlight the importance of managing social and human capital in organizations through the establishment of communities of practice. By fostering a culture of learning and knowledge sharing, community enterprises in Ranong Province can enhance their organizational performance, develop their employees’ skills, and increase their adaptability to changing market conditions. Venkatachalam et al. (2019) also emphasize the significance of organizational learning in small and medium-sized energy project organizations in South Africa. Community enterprises in Ranong Province can draw lessons from these findings, implementing practices that foster learning and collaboration among employees.\n\nSocial entrepreneurship is another relevant aspect for community enterprises, as it can help them create a positive impact on society while achieving financial success. Brändle et al. (2019) explore how entrepreneurial orientation translates social identities into performance, showing that organizations with a strong social purpose can perform better in the market. Community enterprises in Ranong Province can leverage their social missions to differentiate themselves from competitors and attract customers who share their values, thus enhancing their performance. Calgaro et al. (2009) emphasize the importance of assessing destination vulnerability for sustainable recovery and resilience building in the tsunami-affected region of Khao Lak, Thailand. Their findings highlight the need for community enterprises in Ranong Province to be aware of the potential risks and vulnerabilities in their area, particularly regarding natural disasters and environmental challenges. By incorporating risk assessment and mitigation strategies into their operations, these enterprises can build resilience and ensure their long-term sustainability. Chansuchai (2019) investigates the value-added product of community enterprises following the principles of sufficiency economy, which is geared towards sustainable development for entrepreneurs. This study sheds light on the potential for community enterprises in Ranong Province to adopt practices that promote self-reliance, moderation, and responsible consumption, leading to a more sustainable business model.\n\n\nConclusion\n\nThis study’s findings provide vital insights into the elements that influence the performance of community enterprises in Ranong Province, Thailand. The favorable correlations between strategic orientation, green supply chain management, innovation capability, learning organization, and competitive advantages with the success of community firms emphasize the significance of implementing these approaches to improve business outcomes. These findings are consistent with prior research that underlined the significance of strategic planning, innovation, and sustainability for corporate performance (Msomphora, 2018).\n\nThe substantial positive correlation between strategic orientation and both competitive advantage and performance shows that community firms that prioritize strategic planning and development are more likely to achieve favorable commercial outcomes. This finding is consistent with prior research that has underlined the significance of strategic planning and development to business performance (Burana-at et al., 2021). Community enterprises can better connect their actions with their aims and objectives, hence enhancing their competitive advantage and performance, by adopting a clear and cohesive strategic plan.\n\nIn addition to emphasizing the significance of environmental sustainability and social responsibility in community enterprise operations, the positive impact of green supply chain management on competitive advantage and performance highlights the significance of environmental sustainability and social responsibility in community enterprise operations. This result is consistent with earlier research that has highlighted the significance of sustainability and social responsibility to corporate success (Do, 2019). Community firms can reduce their environmental effect and improve their social responsibility by adopting green supply chain management strategies, while enhancing their competitive advantage and performance.\n\nInnovation capability was found to have a strong beneficial effect on both competitive advantage and performance, suggesting that inventive and adaptive community companies are more likely to flourish. This finding is consistent with prior research that has highlighted the significance of innovation to corporate success (Muneeb, Khong, Ennew, & Avvari, 2019). Community companies may adjust to changing market conditions and produce new products and services, hence enhancing their competitive advantage and performance, if they foster an inventive and adaptable culture.\n\nThe favorable association between a learning organization and competitive advantage and performance highlights the significance of continual learning and information sharing in community enterprise development. This result is consistent with earlier research that has highlighted the significance of organizational learning to corporate performance (Glaveli, & Geormas, K., 2017). By promoting a culture of continuous learning and knowledge-sharing, community firms can increase their innovation capacity and operational efficiency, thereby enhancing their competitive advantage and performance.\n\nIn conclusion, this study’s findings emphasize the significance of strategic planning, environmental sustainability, innovation, and learning for the creation and success of community enterprises. These findings can guide future research and policy initiatives aimed at boosting community enterprises and improving the region’s sustainable economic development. Community enterprises in Ranong Province, Thailand, can improve their performance and contribute to the region’s sustainable economic growth by implementing these strategies.\n\nIn addition, the findings of this study provide practitioners and policymakers with a great resource for promoting the development of community enterprises. By adopting these techniques, community companies can increase their capacity for innovation, enhance their operational effectiveness, and acquire a competitive edge in the marketplace (Ardakani, D. A., Soltanmohammadi, A., and Seuring, S., 2022). This can result in improved regional economic growth, employment creation, and social development.\n\nIt is essential to recognize the study’s limitations. Cross-sectional designs reduce the capacity to demonstrate causal links between variables (Sherani, S., Zhang, J., Riaz, M., Boamah, F. A., and Ali, S., 2022; Farzaneh, Ghasemzadeh, Nazari, and Mehralian, G., 2020). The relatively small sample size of the study may restrict the generalizability of its findings. In addition, the study was conducted in a particular place, and the results may not be applicable to other regions or settings. Future research could address these limitations by using a longitudinal study design, a bigger sample size, and a more diversified variety of geographic locations (Manuti, A., Impedovo, & De Palma, 2017).\n\nDespite these limitations, the findings of this study provide useful insights into the elements that determine the operation of community enterprises in Thailand’s Ranong Province. By adopting strategic orientation, green supply chain management, innovation capability, a learning organization, and competitive advantages, community enterprises can increase their innovation capacity, improve their operational efficacy, and obtain a competitive advantage in the marketplace (Sherani, Zhang, Riaz, Boamah, & Ali, 2022). These strategies can improve regional economic growth, job creation, and social development.\n\nOverall, this study’s findings highlight the significance of sustainability, innovation, and knowledge-sharing for the creation and success of community companies. (Soedarmanto, S., & Kartika Cahya, 2018). Community enterprises can improve their performance and contribute to the region’s sustainable economic growth by implementing these strategies (Venkatachalam, S., Marshall, Ojiako, & Chanshi, 2019). This study contributes significantly to the literature on community enterprise growth and can guide future research and policy interventions targeted at fostering sustainable economic development in the region.\n\nFuture research could expand on the findings of this study by employing a longitudinal study design, using a larger sample size, including a more diverse range of geographic locations, examining the role of social capital and networks, and analyzing the impact of government policies and interventions. Such research would provide a more thorough understanding of the elements that contribute to the sustainable growth of community enterprises and guide future policy interventions targeted at encouraging sustainable economic development in the region.", "appendix": "Data availability\n\nFigshare: Sustainable Economic Development through Strategic Planning, Environmental Sustainability, Innovation, and Learning in Community Enterprises: Evidence from Ranong Province, Thailand. https://doi.org/10.6084/m9. figshare.23003774.v1 (Pranee et al., 2023).\n\nThis project contains the following underlying data:\n\n- Interview Data.xlsx\n\n- Questionnaire Data.xlsx\n\nThis project contains the following extended data:\n\n- Interview – Blank Copy.docx\n\n- Questionnaire – Blank Copy.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAgic E, Cinjarevic M, Kurtovic E, et al.: Strategic marketing patterns and performance implications. 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Publisher Full Text\n\nGlaveli N, Geormas K: Doing well and doing good: Exploring how strategic and market Orientation impacts social enterprise performance. International Journal of Entrepreneurial Behavior & Research. 2017.\n\nHair JF, Black WC, Babin BJ, et al.: Multivariate Data Analysis. 7th ed.Upper Saddle River: Pearson Education; 2010.\n\nHoa AX: Value Chain Analysis and the Potential of Cassava-Based Bioethanol in Dak Lak Province, Vietnam (Doctoral dissertation, Prince of Songkla University).2019.\n\nHoradal K: Drivers and barriers to growth of the Thai rice milling industry (Doctoral dissertation, RMIT University).2016.\n\nKelloway EK: Using LISREL for structural equation modeling: A researcher’s guide. United States of America: SAGE Publications Inc; 1998.\n\nKline RB: Principles and practice of structural equation modeling. 4th ed.New York: The Guilford; 2016.\n\nMadhavan M, Sharafuddin MA, Chaichana T: Impact of Business Model Innovation on Sustainable Performance of Processed Marine Food Product SMEs in Thailand—A PLS-SEM Approach. Sustainability. 2022; 14(15): 9673. Publisher Full Text\n\nManuti A, Impedovo MA, De Palma PD: Managing social and human capital in organizations: Communities of practices as strategic tools for individual and organizational development. Journal of Workplace Learning. 2017; 29(3): 217–234. Publisher Full Text\n\nMarnarath P, Ranong AN: A STUDY OF ROLES OF CIVIL SOCIETY, PRIVATE SECTOR AND PUBLIC SECTOR FOR ORGANIC AGRICULTURAL POLICY IMPLEMENTATION: A CASE STUDY OF ORGANIC RICE IN LOWER NORTHEASTERN REGION (Doctoral dissertation, National Institute of Development Administration).2022.\n\nMsomphora MR: Interactive governance of small-scale fisheries and aquaculture: Transdisciplinary challenges of community involvement.2018.\n\nMuneeb D, Khong KW, Ennew C, et al.: Building an integrated conceptual model of competitive learning capability: A strategic management perspective. Asia-Pacific Journal of Business Administration. 2019; 11: 267–287. Publisher Full Text\n\nObi Egbunike , Laura Purvis , Mohamed. M. Naim , et al.: A systematic review of research into the management of manufacturing capabilities. Production Planning & Control. 2018; 29(16): 1349–1366. Publisher Full Text\n\nPhuthong T, Anuntavoranich P, Chandrachai A, et al.: Developing and Validating an Assessment Scale to Measure the Competitiveness of Wellness Destinations. Sustainability. 2022; 14(7): 4152. Publisher Full Text\n\nPoapongsakorn N: R&D and performance of the Thai agriculture and food processing industry: The role of government, agribusiness firms, and farmers. Agricultural Development, Trade & Regional Cooperation in Developing East Asia. 2011; 401–475.\n\nPoontai Y: The role of port authority in hybrid governance structure: a comparative case study of Laem Chabang Port and Bangkok Port, Thailand.2019.\n\nPranee S, Kakhai K, Pulphon S, et al.: Factors Affecting Marketing and Publicity of Processed Fishery Products in Ranong Province. Journal of Positive School Psychology. 2022; 1381–1392.\n\nPranee S, Eaknarajindawat N, Aeknarajindawut N, et al.: Sustainable Economic Development through Strategic Planning, Environmental Sustainability, Innovation, and Learning in Community Enterprises: Evidence from Ranong Province, Thailand. Dataset. figshare. 2023. Publisher Full Text\n\nPrasunpangsri S: Contribution of university-industry linkages (UILs) to tourism clusters: multiple-case studies in Thailand.2013.\n\nSchumaker RE, Lomax RG: notdef] modeling. Mahwah, NJ: L.1996.\n\nSherani S, Zhang J, Riaz M, et al.: Harnessing technological innovation capabilities by the mediating effect of willingness to share tacit knowledge: a case from Pakistani software SMEs. Kybernetes. 2022. (ahead-of-print). Publisher Full Text\n\nSoedarmanto S, Kartika Cahya K: The Effect of Over Workload and Role Conflicts Behavior of Tax Auditors Dysfunction with Job Stress as Mediation Factor.2018.\n\nTaghipour A, Akkalatham W, Eaknarajindawat N, et al.: The impact of government policies and steel recycling companies’ performance on sustainable management in a circular economy. Resources Policy. 2022; 77: 102663. Publisher Full Text\n\nVenkatachalam S, Marshall A, Ojiako U, et al.: Organizational learning in small and medium-sized South African energy project organizations. Management Research Review. 2019; 43: 595–623. Publisher Full Text\n\nVichitdhanabadee J: Performance measurement and management practices in Thai SMEs (Doctoral dissertation, University of Tasmania).2010.\n\nWiraphanphong A: A study of multidimensional poverty management in Thailand according to the Thai people map and analytics platform (TPMAP) through provincial budget budget allocation and provincial groups, annual budget 2017-2019.2020." }	[ { "id": "278389", "date": "12 Jun 2024", "name": "Marian Oliński", "expertise": [ "Reviewer Expertise social enterprises", "supporting of social enterprises", "Industry 4.0/5.0", "business model" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well-written and structured (it contains all the necessary sections). However, there are some issues that can be easily corrected: -Firstly, formulate the aim (goal). Stating that “the research aims to study strategic orientation, green supply chain management, innovation capability,…” is insufficient. Rather, ‘to study’ is for a purpose, to achieve something, and is not a goal in itself. -After the hypotheses, there is a paragraph: “This literature review examined the relationship between strategic orientation, GSCM practices, innovation capability, learning organization, competitive advantages, and performance of community enterprises in Ranong Province, Thailand.” Indeed, much of the literature directly refers to Thailand—but not exclusively, and certainly not specifically to Ranong Province. I suggest removing the last words of this sentence. -The sample is not as small as indicated in the limitations, although it is strange that it was possible to conduct in-depth interviews exactly in the same group as questionnaires. It is not clearly specified what time interval separated the two studies, or if they were conducted simultaneously. There is only the information that “from the questionnaire survey, a subset of 460 community enterprise operators were chosen for the interview process.” Overall, congratulations on conducting extensive research and writing a good article. Good luck.\n\nIs the work 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": "307897", "date": "20 Sep 2024", "name": "Teerawat Charoenrat", "expertise": [ "Reviewer Expertise 14 - Economics1402 - Applied Economics1499 - Other Economics1503 - Business and Management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n-\n\nIn the abstract, the paper should provide a brief policy recommendation regarding the results of this study. -\n\nIn introduction, it does not explain enough information to guide the reader to understand the research such as why this study is important and which questions in the area of the research are not answered. Hence, it should also it should provide more information and statistic Community enterprises in Thailand during the period 2018-2022. -\n\nIn introduction, the paper needs a descriptive section which will provide a brief overview of Community enterprises in Thailand in the period of 2018-2022. This is useful for interpretation of results and is especially important in these sectors. For example, the reader is likely to be interested to know which industries are more important in the Thai economy. Lastly, the author does not clearly identify research aims, expressing the gap of the research, which question will investigate and what are the precise aims of the study. - For the section of literature review, it is important to note that there are a number of old in-text references in the section of literature review. -\n\nThe authors should consider including a literature review – highlighting the key methods used and empirical findings from the literature. I would recommend that the paper include a section on methodology with the subsection on data coming after a discussion on the empirical model. - Focusing on methodology section, it should also provide a discussion justifying or explaining the model used. This can be linked to the literature (cite). It should highlight the key methods used and empirical findings from the literature. -\n\nIt should strongly emphasize the advantages and disadvantages of methodology (model) used in this study. - In the section of conclusion, the author does not provide a brief overview of the study based on the empirical results. The paper requires a section of policy implications in attempt to have the discussions on policy implications structured along the lines of Community enterprises in Thailand and link these to the results. - Finally, limitations of this study are required, and further research possibilities are also needed in this paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/13-86
https://f1000research.com/articles/13-84/v1	25 Jan 24	{ "type": "Study Protocol", "title": "Hospital-based interventional two-arm parallel comparative study on dydrogesterone vs combined oral contraceptive pills for functional ovarian cysts", "authors": [ "Muneeba Shaikh", "Neema Acharya", "Neema Acharya" ], "abstract": "Background Functional ovarian cysts are common among women of reproductive age, often necessitating medical intervention. This hospital-based interventional study compares the efficacy and safety of combined oral contraceptive pills (COC) and dydrogesterone in managing functional ovarian cysts.\n\nMethods This randomized controlled trial will be conducted over two years at the Department of Obstetrics & Gynecology, AVBRH, Datta Meghe Institute of Medical Sciences. The study population consists of reproductive-age women seeking care at the outpatient unit of Obstetrics and Gynecology at AVBRH hospital. The sample size of 46 participants per group has been calculated based on a 95% confidence interval and the estimated prevalence of functional ovarian cysts. Group A will receive low-dose COC for three menstrual cycles. At the same time, Group B will be administered dydrogesterone (10 mg twice daily) for ten days during the luteal phase, repeated across three cycles.\n\nExpected outcomes The primary outcomes include evaluating the recession of cysts within three months, monitoring alterations in menstrual patterns (frequency, regularity, duration, and volume), assessing the necessary treatment duration, and observing potential side effects (e.g., nausea, vomiting, weight gain, and acne) and complications (e.g., thromboembolism, delayed menstrual cycles post-treatment, and interactions with other drugs). Data analysis will encompass descriptive statistics, comparative tests, and regression models to assess the primary outcomes. The significance level for hypothesis testing will be 0.05 with a two-tailed approach.\n\nRegistration\nCTRI/2023/04/051811.", "keywords": [ "Functional ovarian cysts", "Combined oral contraceptive pills", "Dydrogesterone", "Reproductive-age women", "Efficacy", "Safety" ], "content": "Introduction\n\nFunctional ovarian cysts, a prevalent occurrence among women of reproductive age, frequently require medical intervention due to their potential to disrupt fertility and overall health. These cysts originate from the intricate interplay of normal physiological processes involved in ovulation and follicular development.1 However, in certain instances, deviations from this intricate balance can lead to the development of cysts that warrant medical attention. Such deviations can manifest as variations in size, persistence, or symptomatic expression, prompting clinical intervention to prevent complications and alleviate patient discomfort.\n\nThe management landscape for functional ovarian cysts comprises a spectrum of approaches, encompassing both surgical and medical modalities. Surgical interventions, while effective, often involve inherent risks and potential disruptions to reproductive function. On the other hand, medical options promise to preserve fertility and avoid invasive procedures. Despite the existing armamentarium, pursuing treatments that balance efficacy and safety remains an ongoing endeavor.2,3\n\nThis study seeks to address this need by conducting a hospital-based interventional two-arm parallel comparative investigation to compare the effectiveness and safety of two distinct medical interventions – combined oral contraceptive pills (COC) and dydrogesterone. COC, renowned for their hormonal regulatory effects, have demonstrated potential in mitigating cystic growth. Conversely, dydrogesterone, a synthetic progestin, presents an alternative therapeutic avenue with its luteal phase support capabilities.4\n\nThe primary objective is to evaluate the efficacy of these interventions in terms of cyst regression and their influence on the menstrual pattern. Additionally, the study will focus on assessing the safety profile, including potential side effects, treatment duration, and the occurrence of complications. The outcomes of this research will contribute essential insights to inform clinical decision-making, guide treatment strategies, and optimize patient care. By comparing the outcomes of these interventions in a controlled and systematic manner, this study advances our understanding of medical management approaches for functional ovarian cysts, ultimately enhancing the quality of care for women facing this common medical concern.\n\nTo compare the effectiveness and safety of combined oral contraceptive pills (COC) and dydrogesterone for the medical management of functional ovarian cysts.\n\n\n\n1. To study the effectiveness and safety of combined oral contraceptive pills in treating functional ovarian cysts.\n\n2. To study the effectiveness and safety of dydrogesterone in treating functional ovarian cysts.\n\n3. To compare the effectiveness and safety between combined oral contraceptive pills and dydrogesterone.\n\n\nProtocol\n\nThis research employs a randomized controlled trial methodology, falling within the category of interventional studies. The study is planned to span a duration of 2 years. The study will be conducted at the Department of Obstetrics & Gynecology, AVBRH, Datta Meghe Institute of Medical Sciences, in Sawangi (Meghe), Wardha.\n\nThe study will focus on women of reproductive age seeking medical attention at the outpatient unit of Obstetrics and Gynecology within AVBRH hospital. These participants will be selected based on their fulfillment of the defined inclusion and exclusion criteria.\n\nParticipants within the reproductive age range of 18 to 44 years, possessing ovarian cysts between 3 to 10 centimeters with a single compartment and precise contents, and having a body mass index (BMI) within the range of 18.5 to 29.9 will be considered for inclusion.\n\nProspective participants with dermoid cysts, endometriosis, malignancies, ruptured cysts, cyst torsion, recent use of hormonal medications, history of ovarian cyst removal (cystectomy), and specific medical conditions will be excluded from the study.\n\nThe eligible participants will be assigned randomly to two study groups utilizing a 1:1 allocation ratio. The random allocation will be achieved by applying the sealed envelope technique.\n\nParticipants in Group A will receive a course of ethinyl estradiol (0.03 mg) + Levonorgestrel (0.15 mg) combined oral contraceptive pills over three menstrual cycles. In Group B, participants will be administered dydrogesterone, with 10 mg taken twice daily for ten days during the luteal phase. This protocol will be repeated across three consecutive menstrual cycles.\n\nCriteria for discontinuing or modifying allocated interventions will be clearly defined in our study. These criteria may include, but are not limited to, the development of severe adverse events, non-compliance with the treatment protocols, or the emergence of conditions that contraindicate further participation. In such cases, the participant will be withdrawn from the study, and the reasons for discontinuation or modification will be documented.\n\nTo ensure participant adherence to allocated interventions, we will implement the following strategies:\n\n• Regular follow-up appointments and reminders to participants regarding treatment regimens.\n\n• Monitoring of participant compliance through self-reporting and pill counts for both intervention groups.\n\n• Tracking participant satisfaction and addressing any concerns or issues that may affect adherence.\n\n• Continuous communication between the research team and participants to address any questions or issues related to the interventions.\n\nParticipants will be advised to avoid the use of other hormonal medications or treatments during the study period, as these may confound the results. However, the use of non-hormonal over-the-counter medications will be allowed as needed, and these instances will be recorded.\n\nWe will employ several strategies to achieve adequate participant enrolment:\n\n• Collaboration with healthcare providers in the Department of Obstetrics & Gynecology, AVBRH, to facilitate referrals and patient recruitment.\n\n• Public awareness campaigns and information dissemination about the study in the hospital’s outpatient unit.\n\n• Ensuring that the inclusion and exclusion criteria are clear and well-communicated to potential participants.\n\nThe allocation sequence will be generated by a biostatistician, who is not directly involved in the enrolment process. The enrolment of participants will be conducted by clinical research coordinators, while the assignment of participants to interventions will be performed by a research pharmacist based on the allocation sequence. Due to the nature of the interventions, participants cannot be blinded. However, outcome assessors and data analysts will be blinded to the treatment assignments. Unblinding may be considered in the event of a medical emergency or if knowledge of the treatment assignment is deemed necessary for the safety and well-being of the participant. Unblinding will only occur when the unblinded status is required for clinical management.\n\nWe will implement the following plans to promote participant retention and complete follow-up:\n\n• Regular follow-up appointments and communication to encourage participants to complete the study.\n\n• Collecting contact information and maintaining updated records for all participants.\n\n• Clearly outlining the importance of continued participation and the potential consequences of non-completion at the informed consent stage.\n\n\nMethods: monitoring\n\nA Data Monitoring Committee is not deemed necessary for this study due to its relatively low-risk nature and the absence of interim analyses or stopping guidelines. Interim analyses are not planned for this study, and as such, there are no specific stopping guidelines. The final decision to terminate the trial will be made by the principal investigator in consultation with the research team.\n\nWe will implement systematic procedures for collecting, assessing, reporting, and managing adverse events. Adverse events will be documented by the clinical research team, assessed for causality and severity, and reported to the appropriate regulatory authorities as per regulatory requirements.\n\nAn independent auditing process for trial conduct will not be conducted, as the study is not expected to pose significant risks to participants. However, regular internal quality control checks and monitoring of data integrity will be performed throughout the study to ensure data accuracy and adherence to the protocol.\n\nEffectiveness assessment:\n\n1. Regression of cysts: To monitor the regression of ovarian cysts, we will conduct regular ultrasound evaluations. Participants from both study groups will undergo ultrasound assessments after each menstrual cycle. These assessments will continue until either the cysts dissipate or for a maximum duration of three months.\n\n2. Menstrual pattern changes: To observe shifts in menstrual patterns, we will have participants maintain menstrual diaries. Participants will record information regarding the frequency, regularity, duration, and volume of their menstrual cycles. Data will be collected at each follow-up appointment, allowing us to track any changes over time.\n\nSafety assessment:\n\n1. Duration of treatment: We will determine the duration of treatment necessary for each participant by monitoring their response and progress during follow-up appointments. The treatment duration will be based on individual responses and the predefined treatment protocol.\n\n2. Monitoring side effects: Participants will be encouraged to report any potential side effects during regular follow-up appointments and will also have access to a dedicated contact point for immediate reporting. Adverse events, including nausea, vomiting, weight gain, and acne, will be documented and assessed for severity and causality.\n\n3. Effects on liver function tests: We will conduct regular blood tests to assess liver function. Liver function tests, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, will be measured at specific intervals during the study. Any abnormal results will be monitored closely.\n\n4. Tracking critical complications: We will monitor for critical complications such as thromboembolism, delayed menstrual cycles post-treatment, and potential interactions with other drugs through continuous review of participants’ medical records and regular assessments during follow-up appointments. In the event of a critical complication, immediate actions will be taken following our predefined clinical management plan.\n\nAn ultrasound evaluation will be integrated into the study’s follow-up process. Participants enrolled in both study groups will undergo thorough ultrasound assessments after each menstrual cycle. This evaluation will continue until either the cysts dissipate or for a maximum duration of three months.\n\nFormula using mean difference\n\nDydrogesterone vs combined oral contraceptive pills for functional ovarian cysts (δ) = 0.3\n\nConsidering 30% superiority = (0.3*30)/100 = 0.09\n\nStandard deviation = 1\n\nMinimum sample size N = n1=n2=21.96+0.092120.092=42per group\n\nConsidering 10% drop out = 4\n\nTotal sample size required = 42 + 4 = 46 per group. Reference study.5\n\nThe statistical analysis plan for this study will involve several key steps. Descriptive statistics will be used to summarize baseline characteristics of participants in both treatment groups. Continuous variables will be presented as means with standard deviations or medians with interquartile ranges, while categorical variables will be summarized as frequencies and percentages.\n\nFor the primary outcomes, the effectiveness and safety of the two treatments will be assessed separately. The regression of cysts within the three-month timeframe will be compared between groups using appropriate statistical tests, such as the Chi-square test or Fisher’s exact test. Changes in the menstrual pattern will be analyzed using paired t-tests or non-parametric tests if assumptions are not met.\n\nSafety outcomes, including the duration of treatment needed and the incidence of side effects, will be compared using appropriate statistical tests, depending on the distribution of data. The impact on liver function tests and the occurrence of serious complications will be analyzed using appropriate statistical methods, potentially logistic regression, or survival analysis for time-to-event outcomes.\n\nA comparative analysis between the two treatment groups will be performed using appropriate statistical tests, such as t-tests, Mann-Whitney U tests, or chi-square tests, depending on the nature of the data. Adjustments for potential confounders, such as age, BMI, and baseline cyst characteristics, will be considered using regression models. The significance level for hypothesis testing will be set at 0.05, and all tests will be two-tailed. Confidence intervals will be calculated where relevant.\n\n\nDiscussion\n\nThe present study protocol outlines a comprehensive framework for investigating the efficacy and safety of combined oral contraceptive pills (COC) and dydrogesterone in managing functional ovarian cysts. The study’s design and methodology reflect a systematic approach to addressing the clinical challenges posed by these cysts while contributing to evidence-based treatment decisions.\n\nFunctional ovarian cysts and clinical significance: Functional ovarian cysts are common among reproductive-aged women, often necessitating medical attention due to their potential to disrupt fertility and overall well-being. These cysts arise from the complex interplay of hormonal fluctuations and follicular development, with deviations from the norm occasionally resulting in cysts that require clinical intervention. The clinical significance lies in the potential for these cysts to cause discomfort, pain, and menstrual irregularities, thereby impacting the quality of life and reproductive potential of affected individuals.6\n\nThe rationale for study design: The chosen study design, an interventional two-arm parallel comparative study, is apt for evaluating the relative efficacy and safety of COC and dydrogesterone. This design allows for direct comparison between the two treatments, enabling researchers to draw meaningful conclusions about their merits. By employing random allocation and well-defined inclusion and exclusion criteria, the study minimizes bias and enhances the validity of the results.7\n\nTreatment options and therapeutic landscape: The study’s focus on COC and dydrogesterone is well-founded, as both interventions have demonstrated potential in addressing functional ovarian cysts. With their hormonal regulation properties, COC may modulate the hormonal milieu to suppress cystic growth. Dydrogesterone’s specific luteal phase support properties offer an alternative mechanism of action, potentially influencing cyst resolution. This dual intervention approach enriches the understanding of treatment possibilities.8,9\n\nPrimary outcome measures: The primary outcomes, centered around effectiveness and safety, encompass the regression of cysts, alterations in menstrual patterns, treatment duration, side effects, and complications. These outcomes holistically capture the impact of the interventions on both the physical and overall well-being of participants. Monitoring cyst regression and menstrual pattern changes offers insights into the interventions’ therapeutic effects, while evaluating safety profiles contributes to a comprehensive understanding of potential risks.\n\nImplications and future directions: The findings of this study have far-reaching implications for clinical practice. The data generated from the study will aid clinicians in making informed treatment decisions, balancing effectiveness and safety considerations. Furthermore, this research sets the stage for more targeted investigations into the mechanisms of action of COC and dydrogesterone, potentially uncovering novel insights into the pathophysiology of functional ovarian cysts.\n\nLimitations and considerations: It’s important to acknowledge potential limitations, such as the limited follow-up duration and the exclusive focus on two treatment options. Future studies could explore the effects of alternative interventions and longer-term outcomes, providing a more comprehensive understanding of functional ovarian cyst management.\n\nThe Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted its approval to the study protocol, dated 21/07/22, reference DMIHER (DU)/IEC/2022/115. Prior to commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives. We will prioritize the interviewee’s privacy and comfort during the interview process. This protocol has also been registered with CTRI: CTRI/2023/04/051811.\n\nThis study protocol will be published in an indexed journal.\n\nThe study has not yet started, we will start after publishing the study protocol.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nRosenfield RL, Ehrmann DA: The Pathogenesis of Polycystic Ovary Syndrome (PCOS): The Hypothesis of PCOS as Functional Ovarian Hyperandrogenism Revisited. Endocr. Rev. 2016; 37: 467–520. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMobeen S: Apostol R: Ovarian Cyst. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023.\n\nSinha A, Ewies AAA: Ovarian Mature Cystic Teratoma: Challenges of Surgical Management. Obstet. Gynecol. Int. 2016; 2016: 2390177–2390178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFraison E, Kostova E, Moran LJ, et al.: Metformin versus the combined oral contraceptive pill for hirsutism, acne, and menstrual pattern in polycystic ovary syndrome. Cochrane Database Syst. Rev. 2020; 2020: CD005552. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhattacharya SM, Jha A: Comparative study of the therapeutic effects of oral contraceptive pills containing desogestrel, cyproterone acetate, and drospirenone in patients with polycystic ovary syndrome. Fertil. Steril. 2012; 98: 1053–1059. PubMed Abstract \| Publisher Full Text\n\nMantecon O, George A, DeGeorge C, et al.: A Case of Hemorrhagic Ovarian Cyst Rupture Necessitating Surgical Intervention. Cureus. 14: e29350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThiese MS: Observational and interventional study design types; an overview. Biochem. Med. 2014; 24: 199–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng C, Huang Y, Zhou Y: Dydrogesterone in the treatment of endometriosis: evidence mapping and meta-analysis. Arch. Gynecol. Obstet. 2021; 304: 231–252. Publisher Full Text\n\nSteinkampf MP, Hammond KR, Blackwell RE: Hormonal treatment of functional ovarian cysts: a randomized, prospective study. Fertil. Steril. 1990; 54: 775–777. Publisher Full Text" }	[ { "id": "245739", "date": "13 Feb 2024", "name": "Nilaj Bagde", "expertise": [ "Reviewer Expertise obstetrics and Gynecoloy", "Infertility", "endometriosis", "Gynecology endoscopy", "cancer cervix" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 design is appropriate. The justification and background are adequately presented. The study objectives are clear and in measurable terms. Randomization method, The method of generation of allocation is clear and appropriate. Sample size with statistical formula is appropriate for study design statistical Methodology, Data collection and statistical analysis have been written sufficiently and clearly.\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": "245736", "date": "04 Mar 2024", "name": "Sandhya Gadre", "expertise": [ "Reviewer Expertise Subjects of clinical orientation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 deals with functional ovarian cysts, which is seen in reproductive age group. The objective of the study is to compare the efficacy and safety of combined oral contraceptive pills (COC) and dydrogesterone in managing functional ovarian cysts. It is proposed that a randomized controlled trial will be conducted over a period of two years. In each group 46 subjects would be included. One group will receive low-dose COC ethinyl estradiol (0.03 mg) + Levonorgestrel (0.15 mg) for three menstrual cycles. Other Group will be given dydrogesterone 10 mg twice a day for ten days during the luteal phase, which will be repeated for three cycles. Random allocation of the study participants will be done using 1:1 allocation ratio, using sealed envelopes. The researchers expect the cysts to regress in three months, menstrual pattern would also get altered. The subjects will be monitored for potential side effects & complications of the interventional drugs. The data analysis is planned with the help of descriptive statistics, comparative tests, and regression models for assessment of primary outcomes. The significance level for hypothesis testing will be 0.05. Adverse events and unintended effects will be documented by the clinical research team, assessed for causality and severity, and reported to the appropriate regulatory authorities. The effectiveness will be assessed by the regression of cysts and menstrual pattern change. The safety would be assessed with the help of duration of treatment, monitoring the side effects, effects on liver function tests & tracking critical complications. Monitoring of the cysts is planned by ultrasound on a monthly basis. The statistical analysis involves many key steps. Baseline characteristics of participants in both treatment groups will be summarized using Descriptive statistics. Continuous variables will be presented as means with standard deviations or medians with interquartile ranges and categorical variables will be summarized as frequencies and percentages. The regression of cysts will be compared between groups using Chi-square test or Fisher’s exact test. Changes in the menstrual pattern will be analyzed using paired t-tests or non-parametric tests. Safety outcomes, impact on liver function tests and the occurrence of serious complications will be compared using appropriate statistical tests. A comparative analysis between the two treatment groups will be performed using, such as t-tests, Mann-Whitney U tests, or chi-square tests, depending on the nature of the data. Adjustments for potential confounders, will be considered using regression models. All the ethical considerations will be taken care of. Strengths of the Study :\n\nMajor Points\n\n1.The effectiveness & safety profile, both effects are considered for the intervention planned, with various assessment parameters reflecting the positivity of the study. 2.Sufficient details of the methods are provided in a way it can be replicated by other researchers. 3.For different assessment parameters, different statistical analysis methods are thoughtfully planned. 4.The proposal is worth approving for study.\n\nMinor Points\n\n1.The objectives of the study are clearly described 2.The study design is appropriate for the research question. 3.The study deals with a problem which is faced routinely in clinical practice. The conclusion of the completed study may prove beneficial for practical purposes. I do not advice any changes in the study.\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": [] } ]	1	https://f1000research.com/articles/13-84
https://f1000research.com/articles/11-526/v1	16 May 22	{ "type": "Research Article", "title": "Complementary and alternative system of medicine for type 2 diabetes mellitus and its complications: A scoping review", "authors": [ "Shradha S Parsekar", "Nachiket Gudi", "Sujata Chodankar Walke", "Prabhu Dutta Shaw", "Aditi Hombali", "Anupama V Nayak", "Basavaraj S Hadapad", "Shradha S Parsekar", "Nachiket Gudi", "Sujata Chodankar Walke", "Prabhu Dutta Shaw", "Aditi Hombali", "Anupama V Nayak" ], "abstract": "Background: This review aims to map the evidence on the effectiveness of Complementary and Alternative Medicine (CAM) for Type 2 Diabetes Mellitus (T2DM) and its associated complications and identify research gaps in key outcomes and CAMs. Methods: We followed the Johanna Briggs Institute guidelines to undertake this scoping review. We carried out a comprehensive search from inception to March 2020 in electronic databases and registers. Forward and backward citations of included studies were also searched. The randomized controlled trials (RCTs) and non-randomized studies with intervention and control arm were mapped based on the effectiveness of various CAM (Yoga, Ayurveda, Homeopathy, Siddha, Naturopathy, Unani, and Sowa-rigpa) on outcomes among individuals with prediabetes or metabolic syndrome, T2DM with and without any comorbidities. Results: A total of 249 studies were included after screening 3798 citations. Most of the included studies were conducted in India and Iran. Most frequently studied CAM interventions include Yoga, Ayurveda, and herbal products as compared to other CAM therapies. Conclusion: Although we identified a lot of literature on the effectiveness of CAMs for T2DM, there is a lack of standardization of preparation of these medicines and the interventional modalities. To validate the potential benefits of CAM, robust research with high-quality RCTs and systematic reviews are needed to bridge the gap of evidence in CAM therapies. Future investments for research in CAM may focus on assessing the quality of existing trials and finding out an optimal dose, frequency, and duration of CAM, both as an alternative and complementary approach.", "keywords": [ "Complementary and alternative medicine", "Type II diabetes mellitus", "Scoping review" ], "content": "Introduction\n\nType 2 diabetes mellitus (T2DM) is one of the important public health problems and accounts for 90-95% of all diabetes.1 T2DM is a long-lasting metabolic disease caused as a result of a combination of resistance to insulin action and an insufficient compensatory insulin secretory response.2,3 Globally, the number of people having diabetes mellitus is rising.4,5 In 2017, 6.28% of the world’s population had T2DM, which is equivalent to approximately 462 million individuals. Furthermore, these estimates are expected to rise to 7,079 individuals per 100,000 by the year 2030.4 T2DM ranked seventh and ninth in the year 2017, among the leading causes of disability adjusted life years lost and deaths, respectively, as compared to the nineteenth and eighteenth position, respectively, in 1990.4\n\nDiabetes in general has a significant effect on individuals' functional capacities and quality of life (QoL).4 Individuals with diabetes are vulnerable to many health-related complications such as cardiovascular diseases, kidney failure, delayed wound healing (leading to diabetic foot and ultimately to leg amputation if not treated), vision loss, nerve damage3,6 and microvascular damage.5,7 It is also known to reduce life expectancy.3 Diabetes and its complications bring about significant financial loss to individuals with diabetes and their families through direct medical cost and loss of work and wages, and in some cases catastrophic health spending.3 Increased duration of treatment, presence of complications, hospitalization, surgery and insulin therapy leads to increased expenditure8 affecting the health system and national economy.3\n\nAlternative treatments with potential anti-hyperglycemic effects are commonly used for chronic diseases such as T2DM. “Complementary and Alternative Medicine” (CAM) is defined by the World Health Organization as “broad set of health care practices that are not part of that country’s own tradition or conventional medicine and are not fully integrated into the dominant health-care system”.9 CAM includes various types of therapies such as acupuncture, body work (e.g., massage), energy healing (e.g., reiki), herbal or traditional medicines (e.g., naturopathy, Ayurveda, Chinese medicine), mind body techniques (e.g., meditation, yoga), faith healing and music therapy.10,11\n\nThe global use of CAM varies between 17% and 72.8% of the population.12 Every four out of five individuals living in developing countries still depend on CAMs for treatment of certain health conditions.10 A recently published meta-analysis found a global prevalence of CAMs to be 51% (95% confidence interval 43, 59). Use of CAM as a complementary medicine in addition to conventional therapy was found to be 78%. However, as an alternative to conventional medicine was found to be 21%.9 A review also found that about 67% of the people who use CAMs did not disclose its use to healthcare professionals.9 Furthermore, individuals with diabetes are 1.6 times more likely to use CAMs than non-diabetics as reported in 2014.10 Similarly, diabetes is one of the main diseases, for which patients consult Ayurvedic practitioners and consume Ayurvedic medications.6 In the United States, around 2-3.6 million people depend on CAMs for the treatment of diabetes, whereas in Australia and the United Kingdom about 46% rely on it. India showed a very high use of CAMs among diabetics accounting for 67% in 2014.10 The reasons for high proportion of individuals with diabetes using CAMs are multi-faceted. These reasons could be higher cost, high level of medication adherence and fear of side-effect of modern allopathic medicines, dissatisfaction with healthcare providers, and easy availability of CAMs without doctor’s prescription.10\n\nDiabetes, a complex disease, caused due to pathophysiological changes that affects the whole-body glucose homoeostasis, is a typical example requiring an integrative medicine method for its holistic (complete) management. The pathophysiological web of diabetes along with obesity, inflammation and insulin resistance is a challenge for its management. The current diabetes treatment based on modern medicine focusses on reducing hyperglycemia through targeted molecular medications, which needs long-term use and adherence, along with a continuous increase in dose.13 CAMs may act as complementary to modern medicines and help in contributing to holistic approach. Table 1 provides the list of some of the CAMs and its possible effect in lowering blood glucose levels.\n\nReducing premature mortality from non-communicable diseases including T2DM by one-third, achieving universal health coverage and providing access to affordable essential medicines to all by 2030 are the targets set by member states as a part of the agenda for 2030 Sustainable Development.3 The emergence of T2DM into global epidemic and accessibility and availability issues of modern medicines to many individuals in low resource-settings have encouraged low-cost, easily available alternative therapies. Most of these alternative therapies claim to manage diabetes in no or minimal side-effects, due to which, most of the people using CAMs for T2DM use it along with conventional therapy.18\n\nEvidence on assessing the effectiveness of CAM has increased in recent years. It is recommended by the American Diabetes Association position statement that a CAMs can be used based on evidence from research.30 To make the process of implementation of global partnership stronger, SDG 17 is persuading collection, monitoring and accountability of the data.31 Therefore, timely, good quality and reliable data on CAM therapies for T2DM will help subsequent governments and organizations to take effective steps. Good quality data are also essential in framing the clinical practice guidelines. To inform policymakers, CAMs and the outcomes measured among T2DM and its complications should be known for implementing findings for policy, program development and direct future research. A scoping review can contribute to prioritizing evidence needs as suggested by SDG goal number 17. To the best of our knowledge, till date there is no scoping review in the area of CAMs and T2DM. Therefore, this scoping review intends to map the evidence on effectiveness of CAMs for T2DM and its associated complications. We will also identify research gaps in key outcomes and CAMs.\n\n\nMethods\n\nWe followed the Johanna Briggs Institute guidelines to undertake the scoping review and adhered to “PRISMA-ScR (Preferred Reporting Items for Systematic review and Meta-Analyses extension for Scoping Reviews) Checklist”.32 The protocol was registered with Open Science Framework (DOI 10.17605/OSF.IO/6CNH7).\n\nEvidence source: Publications that were eligible for inclusion were randomized controlled trials (RCTs) and quasi randomized controlled trials (QRT) (i.e., interventional studies having two groups assessed pre and post and might not have adhered to strict randomization procedures) published until March 2020. Single group pre-post studies, systematic reviews and observations studies were excluded.\n\nPopulation: Adults (>/= 18 years of age) with a confirmed diagnosis of T2DM (based on appropriate standards such as American Diabetic Association), metabolic disorder or prediabetic condition (marked by elevated levels of blood glucose) were eligible for inclusion. Additionally, individuals experiencing complications, which are associated with DM were included. We excluded adults living with T2DM with multiple organ dysfunction and failure, and those with life support. Additionally, we excluded studies involving type-1 DM, gestational diabetes or diabetes insipidus. In case of mixed population, we coded information of individuals with population of this review interest, if information on the subgroup was provided. If information on the subgroup was not available or in the case of missing information, the study was excluded.\n\nConcept or interventions: Considering the authors’ experience and knowledge we considered specific types of CAM therapies viz. Ayurveda, herbal medications, homeopathy, yoga, Unani, Siddha, massage therapy and naturopathy involving multiple modalities. Although CAMs included a wide range of strategies, we did not include vitamin and mineral nutritional supplementations and other CAMs such as Chinese traditional medicines, qigong, tai-chi and reiki. Additionally, a combination of CAM or CAM administered along with conventional medications and lifestyle modifications were eligible. However, these interventions had been equally distributed in all the arms of clinical trials. For orally administered therapy, a drug could be mono- or combi-preparation of any dose, frequency or duration, but it should have been restricted to one type of therapy (e.g., Ayurveda). Similarly, for yoga and massage therapy there was no restriction on type, length per session, frequency and duration. Any mode of administration of these therapies were eligible to be included such as oral or topical application.\n\nComparison: No intervention, placebo or lower dose of the same intervention, comparison between different therapy and active medications of conventional medicines (e.g., oral hypoglycemic medications) were eligible for inclusion. In case of co-intervention, one arm should have received at least one CAM.\n\nOutcome measures: We considered the following outcomes after reviewing the literature, after consulting the CAM practitioners (who are known to authors).\n\na. Clinical outcomes:\n\n• Glycemic control: glycosylated hemoglobin (HbA1c), blood sugar levels: fasting blood sugar levels (FBSL) and 2-hour post-prandial blood sugar levels (PPBSL)\n\n• Serum insulin\n\n• Anthropometric measures such as body mass index, body weight, waist circumference\n\n• Adverse effects (e.g., hypoglycemia)\n\n• Lipid profiles\n\n• Diabetic complications and related outcomes: retinopathy, neuropathy, cardio-vascular complications, nephropathy, ketosis, wound healing etc.\n\nb. Quality of life (QoL)\n\nc. Activities of daily living\n\nd. Economic outcomes: Cost-benefit Analysis (e.g., monetary units or QALY)/Cost-effectiveness Analysis/ Cost-utility Analysis/ Cost-identification/ Cost-minimization/Cost-consequence.\n\nContext: We did not impose any geographic or setting restriction. Studies could have been hospital or community-based.\n\nA comprehensive search was conducted to locate relevant records in electronic bibliographic databases, using a comprehensive search strategy. The scoping review included published studies on CAMs. However, due to time and financial constraints we considered only English publications. Search was undertaken in the following list of databases from their inception till March, 2020.\n\n1. Electronic databases- Medline (PubMed, Web of Science), “Cochrane Central”, “Cumulative Index to Nursing and Allied Health Literature”, “SCOPUS”, and “ProQuest”.\n\n2. Database of trial registry.\n\n3. Reference list of included studies was searched for eligible records (backward and forward citations in April 2021).\n\nSearch terms such as “Alternative Medicine”, “Alternative Therapies”, “Complementary Medicine”, “Complementary Therapy”, “Ayurveda”, “Yoga”, “Homoeopathy”, “Massage therapy”, “Diabetes” etc. were used to identify relevant literature. A pre-set search strategy was formed by referring to previous studies and trial search carried out initially in PubMed, which was modified with incorporating more keywords and MESH terms. A search string that was resulted from the process was altered to fit the identifiers of each database separately. The PubMed search strategy as an example can be found in the Extended data - DOI: 10.6084/m9.figshare.19512349.33\n\nSearch results of all databases and records identified from other sources were exported to EndNote X7 software and duplicates were removed. Screening, coding and presentation of data was undertaken in Microsoft Excel.\n\nScreening was undertaken at three stages (title, abstract and full texts) by two review authors, independently. A thorough discussion was held between the review authors until consensus, in the case of any discrepancies on the exclusion of eligible records. We followed an extensive screening protocol. The review team did not have expertise in Naturopathy, Unani and Sowa-Rigpa therefore, once the screening was completed, a list of medicines was prepared to be confirmed by professional working in the field (through our extensive contacts). Based on the experts' recommendations CAMs were categorized.\n\nA pretested coding list was used that consisted of the following variables in details; citation details, study design, country, population (number, age, duration of diabetes, history of oral hypoglycemics and details on the complications), CAM therapies (type, dose, frequency, duration), comparator and outcomes. Data charting was done by six authors. As data charting was not done independently, each study extraction was cross-checked by second author. Any discrepancy on charted data was resolved with discussion until consensus. We did not carry out critical appraisal of included studies.\n\nThe extracted information was mapped descriptively using tables. Frequencies of population, intervention, study details, and country has been reported.\n\n\nResults\n\nOn conducting searches in various databases, registers and reference searches, we retrieved 3245, 105 and 448 citations, respectively. Of which 249 records are included finally. The study selection process is shown in Figure 1 with reasons for exclusion.\n\nAs per Figure 2, the published studies assessing the effect of CAM on diabetes/prediabetes have gradually increased over the years. Additionally, there are three protocols34–36 that we did not consider while summarizing the findings.\n\nTable 2A contains detailed characteristics of included studies and citations and is provided in the Extended data- DOI: 10.6084/m9.figshare.19512349.33 In the below text, we have summarized the characteristics of included studies.\n\nStudy design: Majority of the studies were RCTs, however we also included quasi-randomized/non-randomized studies having an intervention and control group. Twenty-one studies were efficacy, feasibility or pilot interventional studies.\n\nCountry: Studies were carried out in various parts of the world. The geographical distribution of the studies is depicted in Figure 3. As seen in the figure, most of the studies were conducted in India and Iran; both contributing to three-fourths of the identified studies. About four-fifth studies were conducted in Asia compared to other continents.\n\nExtended data: 10.6084/m9.figshare.1951234933 includes PubMed search strategy, characterisitcs of included studies, study IDs of included studies cited in tables 2-5 and bibliography of inluded studies as well as PRISMA-ScR checklist.\n\nPopulation details: Population comprised of prediabetes/metabolic syndrome (n=28 studies), T2DM not on any hypoglycemic drugs (n=46), T2DM with hypoglycemic drugs (n=116) and T2DM with comorbidity (n=59). Most commonly studied comorbidity was diabetic foot ulcer (n=27 studies), followed by peripheral neuropathy (n=11) and other conditions (n=21). Fifteen studies included newly diagnosed T2DM population (within one year of diagnosis) and remaining others (n=113) included chronically diagnosed T2DM, however, ninety-three studies did not report the duration of T2DM diagnosis. Majority of the studies (n=167) included populations with mean ages ranging between 45 to 60 years, however, some studies included older adults or mean ages > 60 years (n=37) and mean ages younger than 45 years (n=16), while remaining others (n=29) included adult population with varying ages. The sample size employed by the studies ranged between 10 and 375. We categorized the studies into sample size of 20 or less (n=15), 21-50 (n=84), 51-100 (n=104), 101-200 (n=31), and greater than 200 (n=15).\n\nIntervention details: Herbal or Ayurveda products were assessed by majority of the studies (n=146), followed by yoga therapies (n=52), other CAM including massage and topical applications of herbal products (n=52), Unani medicines (n=2), Sowa-Rigpa (n=1) and Homeopathy (n=1). Within these broad categories various unique complementary therapies were identified and are listed using botanical or trade names in the Extended data – DOI: 10.6084/m9.figshare.19512349.33\n\nWith the help of tables, we have mapped the studies that measured the effectiveness of various CAMs on outcomes among individuals known to have prediabetes or metabolic syndrome, T2DM without any comorbidities and T2DM with comorbidities.\n\nYoga for prediabetes and T2DM\n\na) Prediabetes or metabolic syndrome: Effectiveness of yoga was measured on following outcomes; FBSL (n=8), anthropometric measures (n=5), HbA1c (n=4), lipid profile (n=4), PPBSL (n=3), insulin (n=3), QoL (n=3).\n\nb) T2DM without any comorbidity: Most measured outcome was FBSLs (n=31), followed by HbA1c (n=21), lipid profile (n=16), QoL (n=16), PPBSL (n=15), anthropometric measures (n=12), insulin or insulin resistance (n=7) and adverse events (n=2). Table 2 depicts the number of studies that measured the effect of yoga on various health outcomes.\n\nAyurveda or herbal products for prediabetes and T2DM\n\na) Prediabetes or metabolic syndrome: Curcuma longa (turmeric) was studied by three studies, followed by Cuminum cyminum (cumin), Ayurveda polyherbal medication and other products.\n\nb) T2DM without any associated comorbidity: T2DM population were further classified as taking hypoglycemic modern medicines or not. Momordica charantia L. (bitter melon), Trigonella foenum-graecum (fenugreek seed), Cuminum cyminum (cumin), as compared to other products were mostly studied among individuals having T2DM who were newly diagnosed or had no history of oral hypoglycemic drugs as standard therapy. Whereas cinnamon, Crocus sativus L. (saffron), Juglans regia (walnut), Momordica Charantia, and Trigonella foenum-graecum were majorly identified as herbal products that assessed glycemic indices, lipid profile, anthropometric measures and other outcomes among individuals with T2DM on hypoglycemic agents.\n\nTable 3 depicts the number of studies that measured the effect of Ayurveda or herbal products on various health outcomes.\n\n1 Ayurveda poly herbal product.\n\n2 Bitter melon fruit, Fenugreek seed, Cinnamon stem bark, & other chemicals.\n\n++ Securigera securidaca, vaccinium arctostaphylos, citrullus colocynthis & coriandrium sativum.\n\n# Poly herbal formulation.\n\n## Terminalia chebula fruit extract, Commiphora mukul, and Commiphora myrrhaoleo-gum-resin.\n\n^ Capparis spinose, Rosa canina, Securidaca securigera, Silybum marianum, Urtica dioica, Trigonella foenum-graecum and Vaccinium arctostaphylos.\n\n^^ Nettle leaf, berry leaf, onion and garlic, fenugreek seed, walnut leaf, and cinnamon bark.\n\nOther CAMs for prediabetes and T2DM\n\nEffect of acupuncture on glycemic indices was mostly studied among those identified and studied (See Table 4).\n\nCAM for T2DM with associated comorbidity\n\nWe identified studies that assessed the effect of various CAMs on comorbidities that are associated with T2DM. These are mapped in Table 5. Massage for diabetic peripheral neuropathy, laser therapy and honey dressing for diabetic foot ulcer were most studied CAMs.\n\nTable 3D in Extended data33 provides the citation details of these studies.\n\n\nDiscussion\n\nThis scoping review provides an up-to-date evidence on studies conducted to identify the effectiveness of CAMs on various health outcomes among T2DM and individuals who had prediabetes. Multiple CAMs were identified, of which yoga and Ayurveda or herbal products were most studied. Majority of these studies were conducted in India and Iran. One of the reasons for identifying higher number of studies from these countries on Ayurveda, herbal products and yoga could be that these products are deeply rooted in the tradition and thus are culturally accepted by the people. Additionally, there might be higher awareness among academic and research communities and promotion of these products by some of the international organization such as the WHO. For instance, one-hundred and seventy WHO members have admitted of promoting and integrating traditional and complementary medicines into their health services.37 Furthermore, yoga has also been promoted by the United Nations, which in its general assembly established, June 21st as International Yoga Day.38\n\nAlthough we identified a lot of literature on the effectiveness of CAMs for T2DM, there is a lack of standardization of preparation of these medicines and interventional modalities. This leads to a great setback in the acceptance of CAM for the management of T2DM among the scientific fraternity. Unlike modern medicines, the concept of most of CAMs’ mode of action over any disease is not solely based on the active principles. Each CAM is, however, based on its own principles and approach through a personalized medicine. For example, in Ayurveda, parameters like Rasa (taste of substance), Guna (properties), Veerya (active principle), Vipaka (final transformation), Prabhava (specific or special power of the drug) are important while deciding the medicines; and each patients gets a tailored medicine, diet and lifestyle advice.6,15 May be because of the tailored approach of many CAMs, it is difficult to design a robust RCT, which might be the reasons for not identifying or limited evidence on some of the CAMs in the current scoping review. We did not identify any studies on the Siddha system of medicine. Also, the evidence on Unani, Homeopathy and Sowa-Rigpa is limited.\n\nAlthough some of the CAMs are found to have used complementary to conventional therapies,39 there are many CAM formulations available in the market that haven't been tested for efficacy or effectiveness.20 Lack of high standard, quality CAM clinical trials with inadequate suitable methodology and systematic documentation and reporting have led to lack of credibility of evidence-based science. Owing to the poor quality of some of the CAM trials, they are often published in journals having lower impact.40 It is one of the reasons we identified almost three-fifth studies through reference searching of included studies. Furthermore, almost two-fifths of identified studies employed a 50 or less sample size and many studies were of short duration, which may be due to lack of adequate funding. Despite this, as discussed previously in the introduction section, vast majority of people (e.g. Asian) use CAMs and, in many cases, do not disclose its use20 with the fear of disapproval by the conventional healthcare professional.41\n\nTo validate the potential benefits of CAM, robust research with suitable methodology such as high quality RCTs and systematic reviews can bridge the gap of CAM therapies in delivering a holistic approach to the wellbeing of humankind. Future investments for research in CAM should be focused on assessing the quality of existing trials and finding out optimal dose, frequency, and duration of CAM, both as an alternative and complementary approach. It is also important to carry out rigorous systematic reviews of CAMs and our scoping review can provide the base for such reviews. Also, researchers should consider carrying out cost-effectives studies on CAM. In this scoping review, we did not identify any of the studies that measured economic outcomes of CAM. In the recent past the “National Health and Medical Research Council, Australia” has banned practice of homeopathy in Australia as there was no evidence to demonstrate the effectiveness of Homeopathy and this was achieved through conducting a robust systematic review.42\n\nOur extensive scoping review provided the groundwork for a systematic review in coming times to investigate the effect of CAMs on important health outcomes. Our intent was to map the evidence hence, we did not critically appraise the included studies. We also did not group the multiple publications arising from same studies. However, future researchers intending to do systematic reviews could overcome these shortcomings. Although we carried out extensive exercise of identifying the literature, important subject specific databases were not freely accessible, and had language barriers. Additionally, while searching we did not use unique names (or botanical names) of the individual drugs. Due to the aforementioned reasons, we do not refute the possibility of missing important publications.\n\nChanges from protocol: We changed the title and throughout manuscript the terminology ‘AYUSH system’, which has restricted usage in India to ‘Complementary and alternative system’ of medicine so as to focus globally. At the protocol stage, we had an intent of mapping systematic reviews, however, due to large volumes of studies we excluded at a full text screening stage.\n\n\nConclusion\n\nThis scoping review summarized Ayurveda and herbal products, yoga, naturopathy and other complementary and alternative therapies for prediabetes and T2DM by including 249 interventional studies. Most studies were conducted in India and Iran. This scoping review also identified gaps in evidence in effectiveness of CAMs and outcomes. Designated ministries and governmental agencies must train CAM practitioners in clinical trial methodologies as it would help in thoroughly appraising the evidence to guide future practice. Research of this magnitude should be collaborative in nature where physicians from allopathic systems of practice, methodologists and practitioners of CAM should work in parallel to systematically synthesize literature on clinical conditions of interest. Although funding for evidence synthesis for CAM is generally low across countries, it is a need of the hour to direct resources to support evidence-based practice.\n\n\nData availability\n\nFigshare. Complementary and alternative system of medicine for type 2 diabetes mellitus and its complications: A scoping review. DOI: https://doi.org/10.6084/m9.figshare.19512349.v233\n\nThis project contains the following underlying data:\n\n- This review aims to map the evidence on the effectiveness of Complementary and Alternative Medicine (CAM) for Type 2 Diabetes Mellitus (T2DM) and its associated complications and identify research gaps in key outcomes and CAMs. The protocol was registered with Open Science Forum with DOI: 10.17605/OSF.IO/6CNH7.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReporting guidelines\n\nFigshare. PRISMA-ScR checklist. DOI: https://doi.org/10.6084/m9.figshare.19512349.v2.33\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors' contributions\n\nSSP, NG and BH conceptualized the research. Search and data management was undertaken by SSP. Title screening was done by SSP and NG, abstract and full text screening was done by SSP, NG, AH, and AN. Reference search and screening of additional studies was done by SSP and SCW. Data extraction was done by SSP, NG, SCW, PDS, AH and AN. Analysis was undertaken by SSP and NG. First draft was written by SSP, SCW and NG. All authors critically read, edited and approved the final manuscript.", "appendix": "Acknowledgement\n\nWe acknowledge Manipal Academy of Higher Education for providing technical support to carry out this scoping review.\n\n\nReferences\n\nSeetaloo AD, Aumeeruddy MZ, Rengasamy Kannan RR, et al.: Potential of traditionally consumed medicinal herbs, spices, and food plants to inhibit key digestive enzymes geared towards diabetes mellitus management — A systematic review. S. Afr. J. Bot. 2019; 120: 3–24. Publisher Full Text\n\nAssociation AD: Diagnosis and classification of diabetes mellitus. Diabetes Care. 2014; 37: S81–S90. Publisher Full Text\n\nGlobal Report on Diabetes. Geneva: World Health Organization; 2016.\n\nKhan MAB, Hashim MJ, King JK, et al.: Epidemiology of Type 2 Diabetes - Global Burden of Disease and Forecasted Trends. J. Epidemiol. Glob. Health. 2020; 10(1): 107–111. PubMed Abstract \| Publisher Full Text\n\nSamarakoon D, Uluwaduge DI, Siriwardhene MA: Mechanisms of action of Sri Lankan herbal medicines used in the treatment of diabetes: A review. J. Integr. Med. 2020; 18(1): 14–20. PubMed Abstract \| Publisher Full Text\n\nGordon A, Buch Z, Baute V, et al.: Use of Ayurveda in the Treatment of Type 2 Diabetes Mellitus. Glob. Adv. Health Med. 2019; 8: 2164956119861094. PubMed Abstract \| Publisher Full Text\n\nRizvi SI, Mishra N: Traditional Indian medicines used for the management of diabetes mellitus. J. Diabetes Res. 2013; 2013: 712092. PubMed Abstract \| Publisher Full Text\n\nOjha A, Ojha U, Ojha H, et al.: Role of AYUSH Therapies in Modern Medicine: A Qualitative Study to Explore the Awareness and Attitudes of Doctors Towards the Utilization of Alternate System of Medicine for Diabetes Mellitus. Int. J. Gen. Med 2020; 13: 1–8. PubMed Abstract \| Publisher Full Text\n\nAlzahrani AS, Price MJ, Greenfield SM, et al.: Global prevalence and types of complementary and alternative medicines use amongst adults with diabetes: systematic review and meta-analysis. Eur. J. Clin. Pharmacol. 2021; 77: 1259–1274. PubMed Abstract \| Publisher Full Text\n\nMedagama AB, Bandara R, Abeysekera RA, et al.: Use of Complementary and Alternative Medicines (CAMs) among type 2 diabetes patients in Sri Lanka: a cross sectional survey. BMC Complement. Altern. Med. 2014; 14: 374. PubMed Abstract \| Publisher Full Text\n\nMiner SA, Robins S, Zhu YJ, et al.: Evidence for the use of complementary and alternative medicines during fertility treatment: a scoping review. BMC Complement. Altern. Med. 2018; 18(1): 158. PubMed Abstract \| Publisher Full Text\n\nVishnu N, Mini GK, Thankappan KR: Complementary and alternative medicine use by diabetes patients in Kerala, India. Glob. Health Epidemiol. Genom. 2017; 2: e6. PubMed Abstract \| Publisher Full Text\n\nThottapillil A, Kouser S, Kukkupuni SK, et al.: An 'Ayurveda-Biology' platform for integrative diabetes management. J. Ethnopharmacol. 2021; 268: 113575. PubMed Abstract \| Publisher Full Text\n\nVasudevan R, Buch Z: Ayurveda for diabetes in India. Lancet Diabetes Endocrinol. 2016; 4(11): 884. Publisher Full Text\n\nChattopadhyay K, Panniyammakal J, Biswas TK, et al.: Effectiveness and safety of Ayurvedic medicines in type 2 diabetes mellitus management: a systematic review protocol. JBI Evid. Synth. 2020; 18(11): 2380–2389. PubMed Abstract \| Publisher Full Text\n\nOoi CP, Yassin Z, Hamid TA: Momordica charantia for type 2 diabetes mellitus. Cochrane Database Syst. Rev. 2012; (8). Publisher Full Text\n\nZhang D-W, Fu M, Gao S-H, et al.: Curcumin and diabetes: a systematic review. Evid. Based Complement. Alternat. Med. 2013; 2013: 636053. PubMed Abstract \| Publisher Full Text\n\nPandey A, Tripathi P, Pandey R, et al.: Alternative therapies useful in the management of diabetes: A systematic review. J. Pharm. Bioallied Sci. 2011; 3(4): 504–512. PubMed Abstract \| Publisher Full Text\n\nRaveendran AV, Deshpandae A, Joshi SR: Therapeutic Role of Yoga in Type 2 Diabetes. Endocrinol. Metab. (Seoul). 2018; 33(3): 307–317. PubMed Abstract \| Publisher Full Text\n\nDham S, Shah V, Hirsch S, et al.: The role of complementary and alternative medicine in diabetes. Curr. Diab. Rep. 2006; 6(3): 251–258. Publisher Full Text\n\nRamamoorthi R, Gahreman D, Skinner T, et al.: The effect of yoga practice on glycemic control and other health parameters in the prediabetic state: A systematic review and meta-analysis. PLoS One. 2019; 14(10): e0221067. PubMed Abstract \| Publisher Full Text\n\nThind H, Lantini R, Balletto BL, et al.: The effects of yoga among adults with type 2 diabetes: A systematic review and meta-analysis. Prev. Med. 2017; 105: 116–126. PubMed Abstract \| Publisher Full Text\n\nEqbal K, Alam MA, Quamri MA, et al.: Efficacy of Qurs-e-Gulnar in Ziabetus (type 2 Diabetes Mellitus): a single blind randomized controlled trial. J. Complement. Integr. Med. 2020; 18(1): 147–153. PubMed Abstract \| Publisher Full Text\n\nSathasivampillai SV, Rajamanoharan PRS, Heinrich M: Siddha Medicine in Eastern Sri Lanka Today-Continuity and Change in the Treatment of Diabetes. Front. Pharmacol. 2018; 9: 1022. PubMed Abstract \| Publisher Full Text\n\nMonami M, Silverii A, Mannucci E: Alternative treatment or alternative to treatment? A systematic review of randomized trials on homeopathic preparations for diabetes and obesity. Acta Diabetol. 2019; 56(2): 241–243. Publisher Full Text\n\nBellavite P, Marzotto M, Olioso D, et al.: High-dilution effects revisited. 1. Physicochemical aspects. Homeopathy: The Journal of the Faculty of Homeopathy. 2014; 103(1): 4–21. PubMed Abstract \| Publisher Full Text\n\nEzzo J, Donner T, Nickols D, et al.: Is Massage Useful in the Management of Diabetes? A Systematic Review. Diabetes Spectr. 2001; 14(4): 218–224. Publisher Full Text\n\nMyers SP, Vigar V: The State of the Evidence for Whole-System, Multi-Modality Naturopathic Medicine: A Systematic Scoping Review. J. Altern. Complement. Med. (New York, NY). 2019; 25(2): 141–168. PubMed Abstract \| Publisher Full Text\n\nGrossman LD, Roscoe R, Shack AR: Complementary and Alternative Medicine for Diabetes. Can. J. Diabetes. 2018; 42: S154–S161. Publisher Full Text\n\nAljasir B, Bryson M, Al-Shehri B: Yoga Practice for the Management of Type II Diabetes Mellitus in Adults: A systematic review. Evid. Based Complement. Alternat. Med. 2010; 7(4): 399–408. PubMed Abstract \| Publisher Full Text\n\nTransforming our world: the 2030 Agenda for Sustainable Development: United Nations. http\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): 467–473. PubMed Abstract \| Publisher Full Text\n\nParsekar SS, Gudi N, Walke SS, et al.: Complementary and alternative system of medicine for type 2 diabetes mellitus and its complications: A scoping review. figshare. 2022. Journal Contribution. Publisher Full Text\n\nCrawford P, Thai C, Obholz J, et al.: Assessment of the effeCt of lIfestyle iNtervention plus water-soluble ciNnAMon extract On loweriNg blood glucose in pre-diabetics, a randomized, double-blind, multicenter, placebo controlled trial: study protocol for a randomized controlled trial. Trials. 2016; 17: 9. PubMed Abstract \| Publisher Full Text\n\nThind H, Fava JL, Guthrie KM, et al.: Yoga as a Complementary Therapy for Adults with Type 2 Diabetes: Design and Rationale of the Healthy, Active, and in Control (HA1C) Study. Int. J. Yoga Therap. 2018; 28(1): 123–132. PubMed Abstract \| Publisher Full Text\n\nKarandish M, Mozaffari-Khosravi H, Mohammadi SM, et al.: Evaluation of the effect of curcumin and zinc co-supplementation on glycemic measurements, lipid profiles, and inflammatory and antioxidant biomarkers in overweight or obese prediabetic patients: a study protocol for a randomized double-blind placebo-controlled phase 2 clinical trial. Trials. 2020; 21(1): 991. PubMed Abstract \| Publisher Full Text\n\nWHO global report on traditional and complementary medicine 2019. Geneva: World Health Organization; 2019.\n\nInternational Day of Yoga 21 June: United Nations. Reference Source\n\nNazarian-Samani Z, Sewell RDE, Lorigooini Z, et al.: Medicinal Plants with Multiple Effects on Diabetes Mellitus and Its Complications: a Systematic Review. Curr. Diab. Rep. 2018; 18(10): 72. PubMed Abstract \| Publisher Full Text\n\nPittler MH, Abbot NC, Harkness EF, et al.: Location bias in controlled clinical trials of complementary/alternative therapies. J. Clin. Epidemiol. 2000; 53(5): 485–489. PubMed Abstract \| Publisher Full Text\n\nDiNardo MM, Gibson JM, Siminerio L, et al.: Complementary and alternative medicine in diabetes care. Curr. Diab. Rep. 2012; 12(6): 749–761. Publisher Full Text\n\nHomeopathy. Australia: NHMRC- National Health and Medical Research Council, Australian Government. Reference Source" }	[ { "id": "168045", "date": "20 Apr 2023", "name": "Shrilatha Kamath", "expertise": [ "Reviewer Expertise Endocrine", "therapeutics", "study designs" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 above average level in terms of scoring. The study is intended with a view of highlighting the necessity of other branches of medicine in integration with allopathic medicine. The review article topic is rightly selected as it is the need of the hour. Many more studies in this regard can definitely contribute to the development of a holistic approach in many such chronic, metabolic, endocrinal, hypersensitivity disorders, degenerative and autoimmune illnesses.\nPositives:\nThe sample size is adequate.\n\nThe study design is suitable for the current topic.\n\nThe disease Diabetes Mellitus is best controlled by the holistic approach integrating different therapies as required by the patient and the reason for the same is rightly analysed.\n\nThe causes of small sample size in many studies in CAM is elaborated and reasoned rightly.\n\nStudy has highlighted the absence of works in siddha system and less by the yoga and naturopathy branches which prompts the necessity of improvising the research in the respective system of medicines.\nSuggestions:\nStudy could have highlighted more details about the perfect research designs among the study samples as a guideline to the upcoming researchers.\n\nIt could have also given more details of few works which are up to the mark during the survey process\n\nIs the work 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": "190280", "date": "25 Aug 2023", "name": "Jun Jie Benjamin Seng", "expertise": [ "Reviewer Expertise Complementary and alternative medicine" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a fairly extensive review of use of CAM in T2DM patients.\n\nAbstract\nPlease include relevant statistics in the results section.\n\nResults can be better elaborated (the conclusion is unusually long with regards to proportion to the results)\n\nIntroduction\nUse mesh terms\n\nThe use of CAM in other populations (e.g. CKD) should be briefly discussed before drawing relevance and applications to T2DM. To cite: Teo et al. (2022)1 ; Chu et al. (2022)2 ; Nahas (2008)3\nMethods\nThe search is 3 years outdated and will benefit from an update.\n\nThe spectrum of CAM studied appears to be limited to some degree.\n\nAuthors need to explain why certain modalities are excluded.\n\nOf note, an important wing of CAM is excluded i.e. Traditional Chinese medicine.\n\nWhile i appreciate the outcomes evaluated by authors, I wonder what are some of the clinical implications of outcomes assessed e.g. serum insulin (which are parameters not routinely assessed for T2DM patients)>\n\nAn important database is missing from this study e.g. Embase.\n\nRisk of bias assessment is important to some degree and it may be helpful to do GRADE assessment for the study.\n\nIt is inadequate to just state in the limitations.\n\nNoted the authors included prediabetes patients in the results.\n\nClear segregation need to be made for the study population as it may render too much heterogeneity in the interpretation of study results.\nResults: Generally well summarized.\nDiscussion\nWhat are important research gaps identified which are unique to T2DM patients as well as could the authors propose a list of outcomes which are critical and important to patients.\n\nWhat are other complications of T2DM not covered in literature that warrants further evaluation?\n\nWhat are the main implication for this scoping review and how will this help future researchers?\n\nIs the work 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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/11-526
https://f1000research.com/articles/12-738/v1	23 Jun 23	{ "type": "Case Report", "title": "Case Report: Multiple atherosclerotic plaques at its extreme in synchrony", "authors": [ "Saket Toshniwal", "Isha Sahai", "Benumadhab Ghosh", "Anuj Chaturvedi", "Gajendra Agrawal", "Sourya Acharya", "Sunil Kumar", "Satish Khadse", "Kashish Khurana", "Saket Toshniwal", "Isha Sahai", "Benumadhab Ghosh", "Anuj Chaturvedi", "Sourya Acharya", "Sunil Kumar", "Satish Khadse", "Kashish Khurana" ], "abstract": "Peripheral vascular disease in association with renal artery stenosis is an important association which predicts the severity of the disease. An increase in the number of vessels affected by peripheral vascular disease increases the chances of renal artery stenosis. In our case, the patient had primarily presented with anginal chest pain with complaints of claudication which on further investigation was diagnosed to be a triple vessel coronary artery disease along with bilateral subclavian and bilateral renal stenosis. On detailed history taking, risk factors like hypertension and chronic smoking was found to be present in our case which predisposed to peripheral artery disease secondary to atherosclerosis which was diagnosed on further investigations. Although the association of renal artery stenosis is not very rare in cases of severe peripheral vascular diseases, the presence of a triple vessel coronary artery disease in synchrony is what makes it unique. Take away lesson from this case report is importance of early diagnosis of dyslipidemia causing atherosclerosis and its complications. Multiple atherosclerotic lesions in synchrony i.e, bilateral renal artery stenosis with bilateral subclavian artery stenosis with coronary artery triple vessel atherosclerotic disease like in our case and its severity should create awareness among health care individuals and early treatment measures including lifestyle modifications should be considered to avoid such drastic events.", "keywords": [ "atherosclerosis", "subclavian artery stenosis", "renal artery stenosis", "coronary angiography", "dyslipidemia", "triple vessel disease", "peripheral vascular disease" ], "content": "Introduction\n\nAtherosclerosis is a progressive systemic inflammatory disease causing stenotic lesions in the walls of arteries due to the formation of fibrofatty plaque which further predisposes to myocardial infarctions, cerebrovascular events and even disabling peripheral vascular diseases.1 There is an increased prevalence of renal artery stenosis in patients with peripheral vascular disease in 60 years and above age group due to atherosclerosis and also due to the presence of various cardiovascular risk factors.2 Peripheral artery disease (PAD), secondary to atherosclerosis is currently the leading cause of morbidity and mortality in the Western world and its risk factors include age, smoking, hyperlipidemia, and hypertension.3 The outcome of Peripheral vascular disease (PVD) patients is substantially determined by the extent of atherosclerotic comorbidities.4,5 Renal artery involvement in peripheral artery disease depicts the increased severity of the disease and hence while investigating for peripheral artery disease, renal arteries should be looked for stenotic lesions.2 In a study performed by Aboyans V. et al,2 681 patients who had got their Digital Subtraction Angiography (DSA) done for peripheral artery disease, 14% were found to be prevalent to renal artery stenosis. The association of coronary artery disease in synchrony due to atherosclerosis is rare and not many cases have been reported with multiple atherosclerotic lesions at multiple sites in synchrony like in this case.\n\nIn this case, we present a 60-year-old man who presented with anginal-type chest pain and was diagnosed with a triple vessel coronary artery disease who on further evaluation for his claudication of upper limbs was found to have severe peripheral artery disease with bilateral renal artery stenosis.\n\n\nCase presentation\n\nA 60-year-old Asian male labourer by occupation, a known hypertensive for 20 years and a chronic smoker for 45 years presented to hospital with anginal-type chest pain radiating to both arms and back with sweating and palpitations associated with breathlessness at rest (NYHA III) with orthopnea and paroxysmal nocturnal dyspnea. The patient also complained of pain in both arms on minimal exertion and while lifting weights which was suggestive of claudication with blackish discolouration of both arms distally.\n\nOn general examination, cold pulseless upper extremities with blackish discolouration of distal upper limbs were noted. Axillary, brachial and radial pulses were non-palpable on examination. Other peripheral pulses of temporal, facial, carotid, femoral, popliteal, posterior tibial and dorsalis pedis arteries were palpable and the pulse rate was found to be 80/minute.\n\nOn systemic cardiovascular examination, both heart sounds were heard with apex beat felt at 5th intercostal space just lateral to midclavicular line with no murmurs, bilateral air entry was heard with basal crepts bilaterally on respiratory examination. The abdomen was soft and non-tender with no organomegaly, no visible veins, normal peristaltic sounds, no arterial bruit or any abnormal enlargement or a pulsatile mass. The patient was conscious and oriented to time, place and person with no neurological deficits.\n\nAn electrocardiograph was done and was suggestive of ST segment depressions in precordial leads with T wave inversions suggesting non ST segment elevation myocardial infarction (NSTEMI).\n\nOn routine blood investigations, raised homocysteine levels (75 mcmol/L with normal range of <15 mcmol/L), C - reactive protein (CRP) (6 mg/dL with normal range of <0.3 mg/dL) with raised cardiac biomarkers (CKMB: 50 IU/L and TropI: 24 ng/ml) raised total cholesterol (400 mg/dl with normal range of <170 mg/dl) with Low density lipoprotein (LDL) (140 mg/dl with normal range of <100 ng/dl) with raised triglycerides levels (754 mg/dl with normal range of <200 mg/dl) were observed. cANCA and pANCA with dsDNA and ANA antibodies were also evaluated to rule out vasculitis and were found to be negative. The ankle-brachial index was >1.4 (normal range of 0.9-1.4) which was suggestive of peripheral vascular disease. A CT Aortogram was also done and was not suggestive of aortoarteritis. A diagnosis of peripheral vascular disease with dyslipidemia and raised cardiac biomarkers were made on routine blood investigations and coronary angiography was advised.\n\nCoronary angiography was done and a triple vessel coronary artery disease was diagnosed as shown in Figure 1; in which, panel A shows stenosis in proximal Left circumflex artery (LCX) with a black arrow, panel B shows a stenotic lesion in proximal Left anterior descending artery (LAD) with a black arrow and panel C shows 100% stenosed Right coronary artery (RCA) with a black arrow. A subclavian puncture was taken to look for a subclavian artery considering the claudication symptoms of the patient and peripheral vascular disease was diagnosed with bilateral subclavian artery stenosis as shown in Figure 2, in which panel A shows right subclavian artery stenosis and panel B shows left subclavian artery stenosis marked with yellow arrows. Considering the severity of claudication pain in our case, bilateral renal and femoral arteries were also checked via aortic flush and bilateral renal artery ostial stenosis was seen. Figure 3 panel A shows right renal ostial stenosis and panel B shows left renal ostial stenosis marked with red arrows.\n\nA final diagnosis of atherosclerotic triple vessel coronary artery disease with severe peripheral artery disease with bilateral renal artery stenosis was made. Multiple severe atherosclerotic stenotic lesions in synchrony at multiple sites is what makes it a unique presentation.\n\nThe patient was advised coronary artery bypass grafting for his CAD-TVD and was referred to CVTS for further management with dual antiplatelets and statins (Tablet Ecosprin 75 mg one tablet once daily, Tab Clopitab 75 mg one tablet once daily and Tab Rosuvastatin 40 mg one tablet once daily at night to be continued), ACE inhibitors (Tab Ramipril 2.5 mg one tablet twice daily to continue), Beta blockers (Tab Metoprolol 25 mg one tablet twice daily to continue) and other supportive treatment.\n\n\nDiscussion\n\nAtherosclerosis and coronary artery disease are responsible for a significant number of global fatalities, primarily attributed to heart attacks and congestive heart failure.6 Atherosclerosis evolves due to repetitive damage to the inner lining of blood vessels and the buildup of lipids over time.6 Renal artery stenosis (RAS) frequently occurs as a complication of atherosclerosis and is often connected to chronic heart failure.7 RAS in individuals suffering from CHF may encompass certain clinical features like high blood pressure, deterioration of CHF symptoms, sudden pulmonary oedema, and declining kidney function.7 Renovascular disease often lacks noticeable symptoms, but it can manifest as hypertension, gradual impairment of kidney function, and a significant increase in serum creatinine levels. Other potential clinical presentations may include atheroembolic disease, proteinuria, sudden pulmonary oedema, and chronic heart failure (CHF).8 Peripheral arterial disease, which arises from insufficient blood flow, typically affects the entire cardiovascular system rather than solely the lower extremity blood vessels. Patients displaying symptoms of peripheral arterial disease should undergo an evaluation to assess their risk of atherosclerosis.9 In its milder form, peripheral arterial disease may only cause intermittent claudication, characterized by pain in the lower extremities during physical exertion that subsides at rest. However, when ischemia becomes chronic, critical, or acute, it significantly heightens the risk of gangrene, tissue death, amputation, and premature death.9\n\nIn individuals with peripheral vascular disease, renal artery stenosis is frequently observed and is associated with a high mortality rate. Nevertheless, it remains uncertain whether incidentally discovered renal artery stenosis is a risk factor for mortality.10\n\nDifferent methods, both invasive and non-invasive, can be employed to study atherosclerotic plaques. These methods include intracoronary optical coherence tomography, intravascular ultrasonography, ultrasonography, CCTA, and magnetic resonance imaging. In our study, we discovered that patients with peripheral vascular atherosclerosis (PVA) affecting two to three regions of atherosclerosis, along with coronary stenosis exceeding 50 per cent and high to very high scores of cardiovascular risk, had a significant rate of coronary artery disease (CAD) as found by CCTA.\n\nCohen et al. conducted a study,11 comparing of the presence of CAD and carotid disease with the help of ultrasonography and CCTA, respectively. They found a strong correlation between carotid plaque and the rigidity of coronary artery calcifications.11 CCTA exhibits high sensitivity in identifying severe stenosis; however, its specificity is inclined to be less, especially in the case of calcified plaques, leading to an overestimation of stenosis.12 CCTA is suitable in patients with a low-to-intermediary risk of CAD to effectively rule out if CAD is present with absolute certainty. However, for patients with high risk for CAD, CCTA is not necessary since it is unable to provide any additional details beyond the already high pre-test probability of CAD. In such cases, subsequent coronary angiography, which remains the gold standard for identifying coronary stenosis, may be required. In recent years, there has been a revived interest in developing new technologies, leading to the expansion of the clinical use of coronary computed tomography angiography (CCTA).13 CCTA, when combined with fractional flow reserve measurement (FFR), offers valuable details for patients having intermediate stenosis.13 This FFR is a measurement that compares the total amount of blood flow in a tapered vessel to the maximum flow threshold in an ordinary vessel.\n\nWhile non-invasive imaging techniques like brightness-mode USG and magnetic resonance imaging of the heart are utilized to assess early indications of atherosclerosis, they do not offer a comprehensive assessment of the arteries.14 Traditional coronary angiography and imaging for myocardial perfusion also have limitations when it comes to depicting atherosclerosis, particularly in their early phases or if the condition is well-established but still did not yet compromise the stability of the arterial lumen through positive remodelling. We discovered that patients exhibiting plaque vulnerability had significantly elevated levels of LDL-C, total cholesterol, uric acid, and triglycerides in their serum. This finding emphasizes implementing regimen lowering for lipids to mitigate the development of atherosclerotic plaque of the carotid artery. Prophylactic and progressive management is essential to control and manage these conditions.\n\n\nConclusion\n\nPeripheral vascular disease and renal artery stenosis had been found to be associated with one another in more cases than reported. Peripheral vascular disease individually as well as in conjunction with renal artery stenosis present a high risk of death due to cardiovascular disease. Therefore, further emphasis must be given to detailed investigation of all cases of peripheral vascular diseases and renal artery disease for any other associated abnormalities to provide prompt treatment so as to reduce mortality and morbidity in such patients. Early diagnosis of dyslipidemia and the literature depicting the severity of such dyslipidemic conditions causing multiple atherosclerotic lesions in synchrony should create awareness among health care individuals and early treatment measures including lifestyle modifications should be considered to avoid such drastic events. There were very few published studies regarding atherosclerotic triple vessel coronary artery disease with severe peripheral artery disease with bilateral renal artery stenosis in the literature research done for this particular case report. Thus, the identification and publication of such unique reports are equally important to add to the knowledge of medical professionals.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nLibby P, Buring JE, Badimon L, et al.: Atherosclerosis. Nat. Rev. Dis. Primers. 2019; 5(1): 56. Publisher Full Text\n\nAboyans V, Desormais I, Magne J, et al.: Renal artery stenosis in patients with peripheral artery disease: prevalence, risk factors and long-term prognosis. Eur. J. Vasc. Endovasc. Surg. 2017 Mar 1; 53(3): 380–385. PubMed Abstract \| Publisher Full Text\n\nHardman RL, Jazaeri O, Yi J, et al.: Overview of classification systems in peripheral artery disease. Seminars in interventional radiology. Thieme Medical Publishers; 2014 Dec; (Vol. 31(04): pp. 378–388). Publisher Full Text\n\nSchlager O, Amighi J, Haumer M, et al.: Inflammation and adverse cardiovascular outcome in patients with renal artery stenosis and peripheral artery disease. Atherosclerosis. 2009; 205(1): 314–318. 0021-9150. PubMed Abstract \| Publisher Full Text\n\nMissouris CG, Buckenham T, Cappuccio FP, et al.: Renal artery stenosis: A common and important problem in patients with peripheral vascular disease. Am. J. Med. 1994; 96(1): 10–14. Publisher Full Text\n\nLin SJ: Risk factors, endothelial cell turnover and lipid transport in atherogenesis. Zhonghua Yi Xue Za Zhi (Taipei). 1996 Nov; 58(5): 309–316. PubMed Abstract\n\nde Silva R , Nikitin NP, Bhandari S, et al.: Atherosclerotic renovascular disease in chronic heart failure: should we intervene? Eur. Heart J. 2005 Aug 1; 26(16): 1596–1605. PubMed Abstract \| Publisher Full Text\n\nScoble JE: Renal artery stenosis as a cause of renal impairment: implications for treatment of hypertension and congestive heart failure. J. R. Soc. Med. 1999 Oct; 92(10): 505–510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwedish Council on Health Technology Assessment: Peripheral Arterial Disease – Diagnosis and Treatment: A Systematic Review. Stockholm: Swedish Council on Health Technology Assessment (SBU); 2008 [cited 2023 May 9]. (SBU Systematic Review Summaries). Reference Source\n\nMui KW, Sleeswijk M, van den Hout H , et al.: Incidental renal artery stenosis is an independent predictor of mortality in patients with peripheral vascular disease. J. Am. Soc. Nephrol. 2006 Jul; 17(7): 2069–2074. Publisher Full Text\n\nCohen GI, Aboufakher R, Bess R, et al.: Relationship between carotid disease on ultrasound and coronary disease on CT angiography. JACC Cardiovasc. Imaging. 2013 Nov; 6(11): 1160–1167. PubMed Abstract \| Publisher Full Text\n\nEckert J, Schmidt M, Magedanz A, et al.: Coronary CT angiography in managing atherosclerosis. Int. J. Mol. Sci. 2015 Feb 9; 16(2): 3740–3756. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZarins CK, Taylor CA, Min JK: Computed fractional flow reserve (FFTCT) derived from coronary CT angiography. J. Cardiovasc. Transl. Res. 2013 Oct; 6(5): 708–714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim WY, Stuber M, Börnert P, et al.: Three-dimensional black-blood cardiac magnetic resonance coronary vessel wall imaging detects positive arterial remodeling in patients with nonsignificant coronary artery disease. Circulation. 2002 Jul 16; 106(3): 296–299. PubMed Abstract \| Publisher Full Text" }	[ { "id": "181682", "date": "10 Nov 2023", "name": "Vishakha Jain", "expertise": [ "Reviewer Expertise Non communicable diseases", "Diabetes mellitus", "CAD", "NAFLD", "medical education", "Echocardiography" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 must congratulate the authors on a well written case report with relevant information about the existing literature and good discussion points.\nThe topic under consideration is very important and highlights the need of screening patients with PAD for CAD/coronary stenosis as well, the ABI has been a good tool for the same.\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": "227820", "date": "15 Dec 2023", "name": "Daniel Sykora", "expertise": [ "Reviewer Expertise inflammatory cardiomyopathy", "cardiac sarcoidosis", "peripheral artery 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\nToshniwal et al. present a case of a 60-year-old male with hypertension and nicotine use evaluated for angina and upper extremity claudication. Initial evaluation is concerning for cardiac and upper extremity ischemia, and objective evaluation with laboratory and electrocardiography raises concern for NSTEMI. Upon undergoing angiography, he is found to have multivessel coronary artery disease, bilateral subclavian and renal artery stenoses. Ankle brachial index studies also revealed PAD with poorly compressible arteries in the lower extremities. Given the severity of his CAD, the patient is referred for bypass grafting and started on medical management with dual antiplatelet therapy, statin, ACE inhibitor, and beta blocker.\nThe strengths of this case report include a highly detailed presentation of a patient with multifocal atherosclerosis. The case is interesting and uncommon presentation of multivessel CAD, bilateral upper extremity arterial disease, and bilateral renal artery stenosis. The authors also include high quality angiogram images that add value to the case. This case is an important reminder to all clinicians that atherosclerosis often involves multiple arterial territories.\nThe weaknesses of the case report include significant omitted details regarding the history, physical examination, and objective findings. The timeline of the patient’s management is also unclear. The final outcome of the case (outcome of CABG, decisions regarding intervention for his other stenoses) are omitted. Most importantly, much of the discussion appears unrelated to the case report and focuses on CCTA and FFR and implies that this text is a study, instead of a case report. Due to these concerns, the case presentation and discussion both require crucial substantial revisions, and all the above should be addressed to make this case scientifically sound. The text would also benefit from extensive grammatical and stylistic editing.\nIntroduction:\nThe authors use the terms “peripheral artery disease” and “peripheral vascular disease” intermittently in the text. While these terms are often used interchangeably, the authors should use one term only for clarity. The summary statement of the case at the end of the introduction fails to mention the bilateral subclavian artery stenosis, which is an important finding in the setting of upper extremity claudication.\nCase presentation:\nThe past medical history of the patient is described but baseline medications are not mentioned. It would be valuable to know if the patient was taking antihypertensive medications at baseline and the degree of control of hypertension, as treatment refractory hypertension is a common manifestation of renal artery stenosis, which is later diagnosed in this patient. The acuity of the chest pain is not mentioned. Given the clinical concern later mentioned in the case report for NSTEMI, the acuity and time course of the chest pain should be described. Likewise, the time course of the upper extremity claudication symptoms should be described, as should the presence/absence of lower extremity claudication symptoms (especially given later confirmed lower extremity PAD with poorly compressible arteries). The patient is described as having NYHA Class III heart failure symptoms, but the text does not mention if assessment of his cardiac function was later performed. If investigated, the authors should describe if the patient had heart failure and how this was diagnosed, especially since the discussion of this paper extensively mentions CHF. The authors should mention whether the patient had cerebrovascular symptoms given the possibility of vascular steal syndromes in the setting of later discovered subclavian artery stenosis. The “general” and “systemic cardiovascular examination” sections should be combined into a single physical examination section, especially since the current “general examination” section details pulse examination. The authors describe “cold pulseless upper extremities with blackish discolouration of distal upper limbs” and note absence of “axillary, brachial and radial pulses.” These initial physical exam findings are concerning for acute limb ischemia. The authors should describe how acute limb ischemia was ruled out (e.g. were pulses subsequently detected via Doppler assessment?). The authors should use the term appropriate medical terminology “cyanosis” as opposed to “blackish discolouration” when describing their physical exam findings. The word “electrocardiograph” should instead read “electrocardiogram.” The description of the ECG should be more detailed, at minimum including description of which leads showed ST segment depressions and T wave inversions. If space permits, the ECG should be added as a figure. The authors mention the ECG results are concerning for NSTEMI. As NSTEMI diagnosis necessitates the presence of dynamic cardiac biomarkers, this should be rephrased to “suggesting acute myocardial ischemia.” The paragraph which begins with “On routine blood investigations…” requires significant revision. In the prior paragraph, the authors mention their suspicion for NSTEMI based on ECG results. Therefore, this paragraph should focus on the initial laboratory investigations pertinent to the concern for NSTEMI that would have informed immediate management decisions, namely cardiac biomarkers. The authors mention the CK-MB and troponin I, but do not mention whether these biomarkers were trended over time and whether dynamic changes were observed. This is crucial to know to confirm the diagnosis of NSTEMI. The patient’s creatinine and estimated GFR should be mentioned in this paragraph, given the later diagnosed bilateral renal artery stenosis. CK-MB and troponin I reference ranges should be mentioned. The timeline of the other studies mentioned (homocysteine, CRP, cholesterol, vasculitis labs, ABI, CT aortogram) in relation to the coronary angiogram is unclear. If the authors were concerned for NSTEMI, the timeline of angiography after admission should be made clear. If other studies were pursued later in the care of this patient, they should be mentioned later in the text. If the concern was for NSTEMI, the priority of these studies seems lower than coronary angiogram so it is confusing to present them earlier in the text. If the patient was diagnosed with NSTEMI, the management prior to angiography should be described. Did the patient receive loading doses of aspirin, clopidogrel, or start on a heparin infusion? These details should be mentioned. The ABI should be described for both extremities. The authors are correct in stating that ABI > 1.4 is suggestive of peripheral artery disease, but this finding is usually seen in poorly compressible arteries due to chronic kidney disease or diabetes mellitus, as opposed to atherosclerotic lower extremity PAD. Whether the patient had these comorbidities should be mentioned. The repetition of the figure descriptions for Figure 1,2, and 3, (e.g. description of arrows) in the text is redundant and can be removed. The results of the femoral artery angiography are not mentioned in the text and should be added. The degree of multifocal atherosclerosis seen in this patient at his age is impressive, and it would be helpful to mention whether he had any family history of early coronary artery disease or whether any laboratory testing for inherited atherosclerosis risk factors (e.g. Lipoprotein(a)). The sentence “Multiple severe atherosclerotic stenotic lesions in synchrony at multiple sites is what makes it a unique presentation” should be removed from the case presentation as this section should focus on presentation of objective data regarding this case as opposed to author interpretation, which should be reserved for the discussion. The abbreviations “CAD-TVD” and “CVTS” are not defined elsewhere in the text and should be either defined or removed (preferably, as there are not universally recognized abbreviations). Generic medication names should be used (aspirin and clopidogrel) instead of trade names for ease of readability. The specific type of metoprolol used should be specified (e.g. Metoprolol tartrate). Based on the history provided, the patient had evidence of (at least) severely symptomatic chronic bilateral upper extremity limb ischemia. Was endovascular intervention for the subclavian artery stenoses considered? If not, was surgical revascularization later considered or pursued? Likewise, the rationale behind pursuing medical management only (as opposed to stenting) for the renal artery stenosis should be mentioned. The outcome of the case, including the outcome of CABG and need for any subsequent procedures, should be added. The blood pressure and cholesterol target goals for this patient should be mentioned since the patient was started on statin and antihypertensive therapy.\nDiscussion:\nThe term “heart attacks” in the first paragraph should be replaced with more appropriate medical terminology: “myocardial infarction.” The first paragraph focuses on the relationship between RAS and heart failure, but this patient was never diagnosed with heart failure. This should be either removed, or the patient’s diagnosis of heart failure should be clarified in the case presentation section. This discussion does not fit the content of the case presentation. For example, consider the sentence: “In our study, we discovered that patients with peripheral vascular atherosclerosis (PVA) affecting two to three regions of atherosclerosis, along with coronary stenosis exceeding 50 per cent and high to very high scores of cardiovascular risk, had a significant rate of coronary artery disease (CAD) as found by CCTA.” This sentence appears unrelated to the case report as CCTA is not mentioned in the case presentation. A large portion of the discussion also focuses on CCTA and FFR, which is not relevant to the case and was not used in the diagnosis of this patient, who proceeded directly to coronary angiogram. In the last paragraph of the discussion, the sentence “We discovered that patients exhibiting plaque vulnerability had significantly elevated levels of LDL-C, total cholesterol, uric acid, and triglycerides in their serum” is also unrelated to the case presentation.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "10777", "date": "17 Jun 2024", "name": "Saket Toshniwal", "role": "Author Response", "response": "Respected sir, Thank you for reviewing my article and adding your valuable inputs. The article has been revised according to the instructions and they are as follows Revisions: Introduction The interchangeable terms 'peripheral artery disease' and 'peripheral vascular disease' have been rectified and only one term has been used i.e; 'peripheral artery disease' 'Subclavian artery stenosis' has been added to the introduction section. Case presentation Past history of the patient has been revised and more detailed past history has been added to the text with comment on degree of control of hypertension . Acuity of chest pain has been added along with description of chest pain and time course Time course of the upper extremity claudication symptoms has been added with comment on lower limb claudication symptoms. Heart failure was diagnosed on 2d echocardiogram and this has been added to the text. The patient had no neurological deficits or any cerebrovascular symptoms which has been added to the text. General and systemic examination sections have been combined. Acute limb ischemia was diagnosed via doppler assessment after clinically suspecting it. This has been added to the text. 'Blackish discolouration' has been replaced with 'cyanosed' while describing physical examination. 'Electrocardiograph' has been replaced with 'electrocardiogram' ECG has been described in details as instructed NSTEMI rephrased as \"acute myocardial ischemia' as instructed The paragraph starting with \"On routine blood investigations...\" has been revised and structured as instructed. Cardiac biomarkers has been mentioned before routine investigations which lead to the diagnosis of NSTEMI. Also, serum creatinine and estimated GFR have been mentioned in subsequent paragraphs. Reference range for CKMB and TROP-I have been mentioned The timeline of the study in the article has been revised as instructed and cardiac biomarkers and cardiac evaluation following coronary angiography have been mentioned at first. Management prior to angiography has been added. ABI for both extremities have been mentioned. ABI has been revised and mentioned in the text. The repetition of figure description in the text has been removed. Results of femoral artery angiography has been added. Inherited atherosclerotic factors (Lipoprotein a ) has been ruled out, with non significant family history's been added to the text. The sentence \" multiple severe atherosclerotic stenotic lesions in synchrony at multiple sites is what makes it a unique presentation\" has been removed from case presentation section. Abbrevations such as CAD-TVD and CVTS has been removed. Generic medication names has been used. Specific type of metoprolol detail has been added \"Endovascular intervention was considered and advised to the patient but were not willing for the same\" added to the text. Renal artery endovascular stunting was advised but the patient was not willing for the same. Outcome of the case couldn't be tracked as the patient failed to maintain follow up. This has been added to the text. Target blood pressure and cholesterol levels have been mentioned. Discussion: \"Heart attacks\" have been replaced with \"myocardial infarction\" The patient was diagnosed to have heart failure with reduced ejection fraction which has been added in the case presentation section. The sentence \"In our study, we discovered that patients with peripheral vascular atherosclerosis (PVA) affecting two to three regions of atherosclerosis, along with coronary stenosis exceeding 50 per cent and high to very high scores of cardiovascular risk, had a significant rate of coronary artery disease (CAD) as found by CCTA\" has been removed. CCTA and FFR though not used in the diagnosis of our case has been given importance in the discussion section in view of advancement in diagnosis with newer modalities and to shed light upon the literature related to it for the practitioners. The sentence in the last paragraph has been revised. Kindly review the revisions made. Thank you Yours sincerely, Dr. Saket Toshniwal" }, { "c_id": "10850", "date": "11 Jan 2024", "name": "Saket Toshniwal", "role": "Author Response", "response": "Respected sir, Thank you for reviewing my article and adding your valuable inputs. The article has been revised according to the instructions and they are as follows Revisions: Introduction The interchangeable terms 'peripheral artery disease' and 'peripheral vascular disease' have been rectified and only one term has been used i.e; 'peripheral artery disease' 'Subclavian artery stenosis' has been added to the introduction section. Case presentation Past history of the patient has been revised and more detailed past history has been added to the text with comment on degree of control of hypertension . Acuity of chest pain has been added along with description of chest pain and time course Time course of the upper extremity claudication symptoms has been added with comment on lower limb claudication symptoms. Heart failure was diagnosed on 2d echocardiogram and this has been added to the text. The patient had no neurological deficits or any cerebrovascular symptoms which has been added to the text. General and systemic examination sections have been combined. Acute limb ischemia was diagnosed via doppler assessment after clinically suspecting it. This has been added to the text. 'Blackish discolouration' has been replaced with 'cyanosed' while describing physical examination. 'Electrocardiograph' has been replaced with 'electrocardiogram' ECG has been described in details as instructed NSTEMI rephrased as \"acute myocardial ischemia' as instructed The paragraph starting with \"On routine blood investigations...\" has been revised and structured as instructed. Cardiac biomarkers has been mentioned before routine investigations which lead to the diagnosis of NSTEMI. Also, serum creatinine and estimated GFR have been mentioned in subsequent paragraphs. Reference range for CKMB and TROP-I have been mentioned The timeline of the study in the article has been revised as instructed and cardiac biomarkers and cardiac evaluation following coronary angiography have been mentioned at first. Management prior to angiography has been added. ABI for both extremities have been mentioned. ABI has been revised and mentioned in the text. The repetition of figure description in the text has been removed. Results of femoral artery angiography has been added. Inherited atherosclerotic factors (Lipoprotein a ) has been ruled out, with non significant family history's been added to the text. The sentence \" multiple severe atherosclerotic stenotic lesions in synchrony at multiple sites is what makes it a unique presentation\" has been removed from case presentation section. Abbrevations such as CAD-TVD and CVTS has been removed. Generic medication names has been used. Specific type of metoprolol detail has been added \"Endovascular intervention was considered and advised to the patient but were not willing for the same\" added to the text. Renal artery endovascular stunting was advised but the patient was not willing for the same. Outcome of the case couldn't be tracked as the patient failed to maintain follow up. This has been added to the text. Target blood pressure and cholesterol levels have been mentioned. Discussion: \"Heart attacks\" have been replaced with \"myocardial infarction\" The patient was diagnosed to have heart failure with reduced ejection fraction which has been added in the case presentation section. The sentence \"In our study, we discovered that patients with peripheral vascular atherosclerosis (PVA) affecting two to three regions of atherosclerosis, along with coronary stenosis exceeding 50 per cent and high to very high scores of cardiovascular risk, had a significant rate of coronary artery disease (CAD) as found by CCTA\" has been removed. CCTA and FFR though not used in the diagnosis of our case has been given importance in the discussion section in view of advancement in diagnosis with newer modalities and to shed light upon the literature related to it for the practitioners. The sentence in the last paragraph has been revised. Kindly review the revisions made. Thank you Yours sincerely, Dr. Saket Toshniwal" } ] } ]	1	https://f1000research.com/articles/12-738
https://f1000research.com/articles/12-399/v1	13 Apr 23	{ "type": "Research Article", "title": "Antenatal care utilization on low birth weight children among women with high-risk births", "authors": [ "Diah Puspita Sari", "Mario Ekoriano", "Resti Pujihasvuty", "Sari Kistiana", "Sri Lilestina Nasution", "Irma Ardiana", "Edy Purwoko", "Yuli Puspita Devi", "Muthmainnah Muthmainnah", "Diah Puspita Sari", "Mario Ekoriano", "Resti Pujihasvuty", "Sari Kistiana", "Sri Lilestina Nasution", "Irma Ardiana", "Edy Purwoko", "Yuli Puspita Devi" ], "abstract": "Background: Low birth weight (LBW) is a major public health problem in Indonesia, while LBW is a leading cause of neonatal mortality. Adequate antenatal care (ANC) utilization would help to prevent the incidence of LBW babies. This study aims to examine the association between ANC utilization and LBW children among women with high-risk birth criteria. High-risk birth criteria consisted of 4T which were too young (mother's age <20 years old), too old (mother's age >35 years old), too close (age gap between children <2 years), and too many (number of children >2 children). Methods: This study utilized calendar data from the women’s module from the 2017 Indonesia Demographic and Health Survey (IDHS), with the unit analysis only the last birth of women of childbearing age (15–49), which numbered 16,627 women. From this number, analysis was done by separating the criteria for women with high-risk birth. Multivariate logistic regression analyses were employed to assess the impact of ANC and socio-demographic factors on LBW among women with high-risk birth criteria. Results: This study revealed that only among women with too many children criteria (>2 children), adequate ANC utilization was significantly associated with LBW of children, even after controlling for a range of socio-demographic factors (p < 0.05). In all four women criteria, preterm birth was more likely to have LBW than those infants who were born normally (above and equal to 2500 grams) (p < 0.001). Conclusions: According to WHO, qualified ANC standards have not been fully implemented, including in the case of ANC visits of at least eight times, and it is hoped that ANC with health workers at health facilities can be increased. There is also a need for increased monitoring of pregnant women with a high risk of 4T to keep doing ANC visits to reduce LBW births.", "keywords": [ "antenatal care", "risky maternal", "low birth weight", "pregnant women" ], "content": "Introduction\n\nOne of the focuses of the National Mid-Term Development Plan (RPJMN) in 2020–2024 was reducing the maternal mortality rate (MMR) and infant mortality rate (IMR).1 Neonatal mortality rate (NMR) and IMR are indicators of child mortality, this figure shows an improvement since 1990. NMR decreased from 20 per 1,000 live births in 2002 to 15 per 1,000 live births in 2017 and IMR from 35 per 1,000 live births in 2002 to 24 per 1,000 live births in 2017.2 However, this figure still has not reached the 2024 target, where NMR is expected to decrease to 10 per 1,000 live births and IMR to 16 per 1,000 live births.1\n\nThe main causes of neonatal death in developing countries include low birth weight (LBW) and premature birth. Data showed that LBW and premature births were 19% in 2016.3 Babies with LBW are defined by WHO as babies born less than 2,500 grams regardless of gestational age.4 In Indonesia, the percentage of LBW has decreased slowly, from 11.2% in 2000 to 10.2% in 2012 then to 10.0% in 2015.4\n\nBabies with LBW have a higher risk of stunting, low intelligence (IQ), and death in the first 28 days of life.4,5 In addition, the risk of death at the age of under 1 year is 17 times greater than that of infants with normal birth weight.6 In adulthood, infants with LBW are at risk for obesity, heart disease, and diabetes.4\n\nLBW can be caused by premature birth (<37 weeks), babies with small gestational age (SGA), or a combination of both.4,7 The lower the gestational age, the lower the baby’s birth weight automatically because physiologically and anatomically the fetal organs have not grown and developed perfectly, and the risk of illness and death will increase.8\n\nPremature births and fetuses that fail to thrive in the womb are influenced by four maternal factors, namely maternal malnutrition, maternal health problems during pregnancy, maternal characteristics, and other factors.4 In addition, obstetric factors such as maternal age, both too young and too old, significantly affect LBW.9,10\n\nPregnancy in women with “4T,” namely “too young (gave birth <20 years), too old (gave birth >35 years), too close (short birth gap), and too many (a large number of children)” can have a positive effect on both the mother and the fetus being born.8,11–15 This risk can be prevented or minimized by performing qualified antenatal care (ANC). ANC is important to prevent, detect and treat maternal and fetal health problems.3,8,11,12,16–18\n\nSince 2016, WHO has recommended pregnant women to have a minimum of eight pregnancy check-ups.19 WHO provides guidance for pregnant women to have a healthy pregnancy (positive pregnancy) through five interventions and 19 recommendations as well as several recommendations for specific cases.\n\nSince 2020, it is agreed in Indonesia for pregnant women to make ANC visits at least six times, with at least two contacts with doctors in the first trimester to screen for risk factors/pregnancy complications; and in the third trimester for one-time delivery risk factor screening. Based on IDHS in 2017, National Family Planning stated that the coverage number of ANC visits (>4 times) in Indonesia was 90.6% and as many as 75% carried out pregnancy checks by health workers.2\n\nThe difference between this study and other similar studies is to look at the effect of ANC on women with 4T with the incidence of LBW. Therefore, this study aimed to determine the effect of ANC on women with 4T on the incidence of LBW. The hypothesis that is built is that qualified ANC in women of childbearing age with 4T reduces the risk of LBW events.\n\n\nMethods\n\nThis study used the 2017 Indonesian Demographic and Health Survey (IDHS) calendar data source, the women of childbearing age module. This study is mostly retrospective data, which requires each respondent to report their experience in ANC at the time of pregnancy and birth history. This study analyzed 49,627 women of childbearing age (15–49 years) with a total of 16,627 last births because the LBW number available in the IDHS was the last birth history.\n\nThe criteria for the unit of analysis were “4T”, among others; “too young” (gave birth <20 years) totaled 843; “too old” (gave birth >35 years) totaled 2,530; “too close” (spacing of fewer than two years) totaled 5,300; and “too many” (bearing more than two children) totaled 873.\n\nThe independent variables being analyzed were ANC quality, area of residence, education level, wealth level, work status, ANC examination place, ANC examiner staff, and access to information media. The qualified ANC indicator in the WHO guidelines is positive pregnancy.19 There were only five recommendations that allow for analysis, namely getting iron, getting bacteria in the urine, getting tetanus toxoid (TT) injections during pregnancy, visiting at least eight ANC, and screening of smoking history. The dependent variable was the incidence of LBW in women with 4T.\n\nThe data analysis of this study used the IBM SPSS application version 21. The analysis of this study was carried out descriptively and inferentially. Descriptive analysis through univariate and bivariate analysis was conducted to determine the frequency distribution of the variables studied. Inferential analysis was carried out through multivariate analysis with binary logistic regression models (crude OR and adjusted OR) to determine the effect of the independent variables on the dependent variable.\n\nAccording to the DHS Program, “the procedures and questionnaires for standard DHS surveys are reviewed and approved by The Institutional Review Board (IRB) of ICF International while country-specific DHS protocols are reviewed by the IRB of ICF International and typically by an IRB in the host country”. The IRB of ICF International ensures the protection of human subjects from the survey complies with the U.S. Department of Health and Human Services regulations, while the host country IRB ensures that the survey complies with the laws and norms of the nation. While downloading the data, the names and addresses of the respondents are de-identified. The data have been obtained by registering and requesting with the Demographic and Health Surveys (DHS) website (https://dhsprogram.com).\n\n\nResults\n\nThe results of the univariate analysis presented a description of social, economic, and demographic characteristics as shown in Table 1. Descriptively, women in this study were relatively more middle-educated in each category (69% “too young”, 52% “too close”, 49% “too many”, and 45% “too old”). Based on the area of residence, the majority of women living in rural areas were “too young” (66%), “too many” (53%), and “too close” (51%), while women with “too old” (> 35 years) live more in urban areas (51%) than in rural areas (49%).\n\nBased on the wealth index, most of the women were in a low wealth index category: women who were “too young” (60%), “too many” (44%), and “too close” (47%). Based on employment status, more than half of the women were not working, namely, women who were “too young” (69%) and “too close” (52%). In addition, more than half of the women underwent pregnancy checks at health facilities, namely women with “too old” (79%), while “too young”, “too much” and “too close” were 77% each. The results of the descriptive analysis also showed that four out of five women had relatively more ANC check-ups with health workers in each 4T category. Table 1 also shows that women who perform qualified antenatal care in each 4T category have a percentage of less than 15%.\n\nBased on the birth status of the children, almost all of them were born at term (normal) with a percentage above 90% in each 4T category. Likewise in all 4T categories, more children were born with non-LBW status (above 90%).\n\nTable 2 shows the percentage distribution of ANC quality among women with 4T in each category according to background characteristics. Most women with a 4T have unqualified antenatal care. Just under 20% of women with a 4T perform qualified ANC. Women with “too old” performed qualified ANC (17%) more than women with other 4T.\n\nAmong women with 4T categories, 82% have unqualified ANC. While the higher the education of women, the more women who perform qualified ANC in each 4T category. Most of the women with qualified ANC were found in high education in the “too old” (23%) and “too close” (12%) categories. Meanwhile, in the “too many” and “too young” categories, most of the women with qualified ANC were found in secondary education, 16%, and 13% respectively.\n\nBased on the place of residence, more women who live in urban areas perform qualified ANC for each of the “4T” categories compared to women who live in rural areas. Furthermore, based on wealth status, the higher the wealth index, the more women who perform qualified ANC in each 4T category. Based on employment status, it is seen that women who are not employed are more likely to do qualified ANC at “too young” and “too many” respectively (14%). Meanwhile, among women with “too old” (17%) and “too close” (9%), most of the women with qualified ANC were working. Less than a fifth of “4 Too” women perform qualified ANC at health facilities in each 4T category and all of them are handled by health professionals.\n\nWomen who performed qualified ANC were relatively higher among those exposed to information through the media, among women “too old” (21%), “too many” (15%), and “too close” (10%). Meanwhile, when viewed from birth status, women who gave birth to children at term/normally had relatively more qualified ANC for each 4T category compared to women with premature births of their last child. Based on the LBW category, relatively more women with non-LBW babies perform qualified ANC in each 4T category compared to women with LBW babies.\n\nTable 3 shows the results of the logistic regression model testing between the characteristics and quality of ANC variables on the incidence of babies born with LBW in women with 4T. The effect of several variables on the incidence of LBW in each 4T risk model shows mixed results. The quality of the ANC only affects women with “too many” children on bivariate testing or together with other variables. Preterm birth status has a significant influence on the incidence of LBW in all groups of women with 4T compared to the quality of ANC and other variables. Babies born prematurely in the “too close” group of women have the greatest chance of LBW incidence compared to babies born normally in the other 4T category, as well as exposure to information through the media.\n\n* Significant 0.1.\n\n Significant 0.05.\n\n* Significant 0.01.\n\nIn women with “too young” status, last childbirth status, women’s exposure to media, and wealth index showed a significant effect when tested per variable or simultaneously on the incidence of LBW babies born. Women with the premature birth of their last child had a 10.48 times greater tendency to give birth to LBW babies compared to women who gave birth to a normal last child (AOR: 10.48; 99% CI; 4.74-23.16). In addition, women who were not exposed to the media had a 2.72 times greater tendency to give birth to LBW babies compared to women who were exposed to the media (AOR: 2.72; 90% CI; 0.87-8.48). Based on social characteristics, women with a middle wealth index were 0.19 times less likely to give birth to LBW babies than women with a high wealth index (AOR: 0.19; 95% CI; 0.05-0.71).\n\nIn the second model, “too old”, the status of the last child’s birth, education level, wealth index, and media exposure had a significant effect on the incidence of LBW both on the test per variable and simultaneously. The qualified ANC in the “too old” group of women did not show a significant effect on the incidence of LBW, as well as the area of residence, place of ANC examination, ANC examiner staff, and employment status. As with the previous model, women in the “too old” group with the premature birth of their last baby had a 16.63 times greater chance of giving birth to LBW babies compared to women who gave birth to a normal last child (AOR:16.63; 99% CI; 10.42-26.53). In addition, women with low levels of education have a 2.76 times greater chance of giving birth to LBW babies than women with higher education levels (AOR: 2.76; 99% CI; 1.30-5.88). Women with a low wealth index were more likely (1.60 times) to give birth to LBW babies compared to women with a high wealth index (AOR: 1.61; 90% CI; 0.97-2.65). Not only education level and wealth index, but media exposure in this group also has a significant effect on the incidence of LBW. Interestingly, women who were not exposed to the media were 0.56 times less likely to give birth to LBW babies compared to women who were exposed to the media (AOR: 0.56; 95% CI; 0.87-8.48). In fact, the opportunity is even greater when tested simultaneously with other variables.\n\nIn the third model, women with “too many”, birth status, birth rate, ANC quality, wealth index, and area of residence had a significant influence on both the tests per variable and simultaneously. Women with preterm birth had a 15.03 times greater chance of giving birth to a LBW baby compared to women who gave birth normally (not-preterm birth) (AOR: 15.03; 99% CI; 10.76-21.00). Furthermore, women with unqualified ANC were 1.47 times more likely to give birth to LBW compared to women with qualified ANC (AOR: 1.47; 90% CI; 0.98-2.20). Interestingly, women living in urban areas were 1.37 times more likely to have LBW babies than women living in rural areas (AOR:1.37; 95% CI; 1.06-1.79). Furthermore, women with a low wealth index have a 1.43 times greater chance of giving birth to LBW than women with a high wealth index (AOR: 1.43; 95% CI; 1.05-1.95).\n\nEducation level becomes an important variable in the group of women with “too many” and “too close”. The higher the education level of women, the lower the tendency to give birth to LBW babies. Women with low and middle education levels in the group of women with “too many” were more likely to give birth to LBW babies by 2.85 times (AOR: 2.85; 99% CI; 1.65-4.93) and 1.77 times (AOR: 1.77; 95 % CI; 1.04-3.01) compared with women with higher education levels.\n\nIt was quite different because the quality of the ANC in the fourth model with “too close” did not show a significant effect on the incidence of LBW. In addition to education level, media exposure and preterm birth status were variables that consistently affect the incidence of LBW. Interestingly, preterm birth status has a nearly double chance of developing LBW in this risk group compared to other risk groups. Women with “too close,” where the distance between the last two children was less than two years and gave birth prematurely, had a 21.72 times greater chance of giving birth to LBW babies than normal births in the simultaneous test. Likewise, women who were not exposed to media in the “too close” group had a greater chance of giving birth to LBW babies than other risk groups.\n\n\nDiscussion\n\nIndonesia has tried to reduce infant mortality. One of the strategies is to prevent the incidence of babies with LBW. The results of this study showed that the birth incidence of LBW babies was almost the same in each 4T category, which is around 6 to 7%. This figure is lower than other Asian countries, such as India.18 While comparing it to African countries, the LBW in Indonesia is lower.17\n\nThe results indicate that ANC quality only affects LBW births in the category of too many children. Even so, previous studies also showed a significant relationship between ANC utilization and mothers who were too old (>35 years), whereas mothers who were too old were higher in using ANC.20 However, mothers who were too young had higher knowledge than mothers who were too old.21 A previous study showed that most adolescent births were from mothers with a low education level.22\n\nWomen with too many children and unqualified ANC will have a 1.47 times higher chance of giving birth to LBW babies than women with too many children and qualified ANC. This was in accordance with research conducted in Padang, mothers with less than four ANC visits were more likely to give birth to LBW babies compared to mothers with four ANC visits.23 Similarly, studies conducted in India18 and China8 also stated that a comprehensive antenatal examination was associated with a reduced risk of LBW in infants. Studies conducted in Rwanda,24 Ethiopia,12,16,17,25 and Sri Lanka11 found that lack of ANC visits was associated with low infant weight. In a comprehensive ANC, including a complete number of visits, pregnant women carry out regular checkups, practice healthy living habits and obtain iron intake during pregnancy. Thus, they can detect problems, diseases, or complications during pregnancy early, including reducing the incidence of babies with LBW.12,13,16–18,24,25\n\nThe results of this study showed that the age of childbirth has a significant effect on infants with LBW. Premature birth had the most significant impact on infants with low birth weight in the four 4T categories, namely “too young”, “too old”, “too many”, and “too close”. The World Health Organization (WHO) stated that premature birth is the cause of about one-third of LBW babies. Studies conducted in Yemen15 and Ethiopia26 showed the same. Likewise, in Abu Dhabi, babies born prematurely have an 18 times higher risk of becoming LBW.7 This happens probably because fetal growth and weight gain mainly occur in the late period of pregnancy, so premature babies receive less nutrition which causes low birth weight.\n\nSeveral socioeconomic and demographic characteristics such as education level, wealth index, and area of residence were significantly associated with the incidence of infants with LBW. The education variable has a significant relationship with the incidence of LBW in all four categories, except for women with births too young, which does not have a relationship with LBW incidence. In line with research conducted by Nuryani and Rahmawati, 2017 in Gorontalo Regency, there was a significant relationship between education level and the incidence of LBW (p=0.017).27 This finding is in agreement with several studies conducted in other developing countries such as India,18 Ethiopia,25 and Ghana.28 Generally, women with higher education were more informed about the risks of not receiving health care during pregnancy and paid more attention to nutritional intake during pregnancy,18,28,29 On the other hand, women with low education generally had less access to health facilities, especially economically.25 However, the research conducted by Sharma et al. (2015) in Nepal and by Rahim FK and Muharry A (2018) in Kuningan showed different things, where maternal education was not associated with the incidence of LBW.30,31\n\nRegarding the wealth index in this study, among women with too old and too many, it is seen that low wealth index are more at risk for giving birth to babies with LBW compared to high wealth index. Studies in India18 and Sri Lanka11 showed similar results, where the incidence of LBW decreases with an increasing wealth index.\n\nThe residence variable is seen only in the category of too many children, which has a significant effect on LBW births. Women with too many children who live in urban areas are 1.37 times more likely to give birth to LBW babies than women in rural areas. In line with research conducted by Mohammed S et al. (2019), the probability of giving birth to an LBW baby was significantly higher in urban residents.32 This is different from the results of the study by Kaur et al. (2019) that found that LBW was more common in rural areas than in urban areas (9.8% vs. 2.0%, p=0.03)33 and several studies conducted in Ethiopia.16,17 This may be related to the education level of women who generally have low and middle education in this study.\n\n\nConclusion\n\nBased on bivariate testing or together with other variables, qualified ANC only has a significant effect on the incidence of LBW in women with the “too many” criteria. It is known that the most influential variable on LBW in women with 4T is premature birth. Besides that, it is known that with a low level of education women who give birth too close have the highest chance of giving birth to LBW compared to the other “4 too” criteria. Likewise, women who are “too old” and “too many” with a low wealth index and women who are “too many” who live in urban areas have the highest chances of giving birth to LBW.\n\nThe findings show that the recommendations for qualified ANC according to WHO standards have not been fully implemented. In the case of ANC visits of at least eight times, it is hoped that ANC with health workers at health facilities can be increased. It is also necessary to increase the monitoring of pregnant women with the risk of 4T to continue making ANC visits to reduce the risk of preterm and reduce LBW births. Moreover, increasing education and counseling related to maturing the age of marriage, reproductive health, family planning (spacing), and the dangers of 4T to reduce the risk of LBW events in women with 4T in various information media. It is necessary to review the coverage of ANC in the National Health Insurance (NHI) mechanism, which is only four times, especially for the poor and with low education.", "appendix": "Data availability\n\nThe dataset of the Indonesia Demographic and Health Survey 2017 is available from the DHS Program website (https://dhsprogram.com/data/available-datasets.cfm). The data can be obtained after registering and requesting permission to download the dataset through the website. The authors did not have any special access privileges that others would not have.\n\n\nAcknowledgments\n\nWe gratefully acknowledge the National Population and Family Planning Agency, Central Bureau of Statistics, and Ministry of Health in Indonesia which have provided the data in this study. We also acknowledge the BRIN (National Research and Innovation Agency), Universitas Airlangga, and Universitas Indonesia as the collaborative research team in this study.\n\n\nReferences\n\nKementerian Kesehatan RI: Indikator Program Kesehatan Masyarakat Dalam Rpjmn Dan Renstra Kementerian Kesehatan Tahun 2020-2024. Kemenkes RI:2020.\n\nBKKBN, BPS, Kemenkes, USAID: Survei Demografi dan Kesehatan Indonesia 2017. BKKBN. Jakarta:BKKBN;2018; 1–446.\n\nKementerian Kesehatan RI: Sample Registration System (SRS) 2016. Jakarta:Kementerian Kesehatan RI;2018.\n\nUNICEF & WHO: Low birthweight estimates. World Health Organization;2019.\n\nAhishakiye A, Abimana MC, Beck K, et al.: Developmental outcomes of preterm and low birth weight toddlers and term peers in Rwanda. Ann. Glob. Health. 2019; 85(1): 1–11.\n\nNugroho PMC, Dewiyanti L, Rohmani A: Tingkat keparahan asfiksia neonatorum pada bayi berat lahir rendah (bblr). Kedokt Muhammadiyah. 2015; 2: 43–46.\n\nTaha Z, Hassan AA, Wikkeling-Scott L, et al.: Factors associated with preterm birth and low birth weight in Abu Dhabi, The United Arab Emirates. Int. J. Environ. Res. Public Health. 2020; 17(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXi C, Luo M, Wang T, et al.: Association between maternal lifestyle factors and low birth weight in preterm and term births: A case-control study. Reprod. Health. 2020; 17(1): 1–9.\n\nSetiati AR, Rahayu S: Faktor Yang Mempengaruhi Kejadian BBLR (Berat Badan Lahir Rendah) Di Ruang Perawatan Intensif Neonatus RSUD DR Moewardi Di Surakarta. J. Keperawatan Glob. 2017; 2(1): 1–61.\n\nLatifah L, Anggraeni MD: Hubungan Kehamilan Pada Usia Remaja Dengan Kejadian Prematuritas, Berat Bayi Lahir Rendah Dan Asfiksia. Kesmasindo. 2013; 6(1): 26–34.\n\nAbeywickrama G, Anuranga C: A decomposition analysis of inequalities in Low birth weight in Sri Lanka: findings from the Demographic and Health survey- 2016. Ceylon Med. J. 2020; 65(1–2): 15–22. PubMed Abstract \| Publisher Full Text\n\nMingude AB, Gebretsadik W, Misker D, et al.: Determinants of low birth weight among live birth newborns delivered at public hospitals in Gamo Gofa Zone, South Ethiopia: Unmatched case control study. SAGE Open Med. 2020; 8: 205031212094054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBater J, Lauer JM, Ghosh S, et al.: Predictors of low birth weight and preterm birth in rural Uganda: Findings from a birth cohort study. PLoS One. 2020; 15(7 July): 1–16. Publisher Full Text\n\nHüseyin ÇH, Muazzez H, Yadigar P: A study of low birth weight prevalence and risk factors among newborns in a public-hospital at Kilis, Turkey. Afr. Health Sci. 2020; 20(2): 709–714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIdris I, Sheryan M, Ghazali Q, et al.: Reproductive and behavioural risk factors of low birth weight among newborns in Al Thawra Hospital, Sana’a, Yemen. East Mediterr. Health J. 2020; 26(11): 1415–1419. PubMed Abstract \| Publisher Full Text\n\nBekela MB, Shimbre MS, Gebabo TF, et al.: Determinants of Low Birth Weight among Newborns Delivered at Public Hospitals in Sidama Zone, South Ethiopia: Unmatched Case-Control Study. J. Pregnancy. 2020; 2020: 1–8. Publisher Full Text\n\nMehare T, Sharew Y: Prevalence and Associated Factors of Low Birth Weight among Term Newborns in Dilla Town, Southern Ethiopia. Int. J. Pediatr. 2020; 2020: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaveri A, Paul P, Saha J, et al.: Maternal determinants of low birth weight among Indian children: Evidence from the National Family Health Survey-4, 2015-16. PLoS One. 2020; 15(12 December 2020): 1–15. Publisher Full Text\n\nWHO: WHO recommendations on antenatal care for a positive pregnancy experience. Geneva:WHO Press;2016.\n\nTadesse E: Antenatal care service utilization of pregnant women attending antenatal care in public hospitals during the COVID-19 pandemic period. Int. J. Women’s Health. 2020; 12: 1181–1188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDegu A, Nibret G, Gebrehana H, et al.: Knowledge and attitude towards the current pandemic corona virus disease and associated factors among pregnant women attending antenatal care in debre tabor general hospital northwest ethiopia: An institutional-based cross-sectional study. Int. J. Women’s Health. 2021; 13: 61–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDevi YP, Ekoriano M, Sari DP, et al.: Factors associated with adolescent birth in Indonesia: a national survey. Rural Remote Health. 2022; 22(2): 1–11. Publisher Full Text\n\nFatimah N, Utama BI, Sastri S: Hubungan Antenatal Care dengan Kejadian Bayi Berat Lahir Rendah pada Ibu Aterm di RSUP Dr. M. Djamil Padang. J. Kesehat. Andalas. 2018; 6(3): 615. Publisher Full Text\n\nFayehun O, Asa S: Abnormal birth weight in urban Nigeria: An examination of related factors. PLoS One. 2020; 15(11 November): 1–11. Publisher Full Text\n\nBaye Mulu G, Gebremichael B, Wondwossen Desta K, et al.: Determinants of Low Birth Weight Among Newborns Delivered in Public Hospitals in Addis Ababa, Ethiopia: Case-Control Study. Pediatr. Heal. Med. Ther. 2020; 11: 119–126. Publisher Full Text\n\nSema A, Tesfaye F, Belay Y, et al.: Associated Factors with Low Birth Weight in Dire Dawa City, Eastern Ethiopia: A Cross-Sectional Study. Biomed. Res. Int. 2019; 2019: 1–8. Publisher Full Text\n\nNuryani N, Rahmawati R: Kejadian Berat Badan Lahir Rendah di Desa Tinelo Kabupaten Gorontalo dan Faktor yang Memengaruhinya. J. Gizi dan Pangan. 2017; 12(1): 49–54.\n\nQuansah DY, Boateng D: Maternal dietary diversity and pattern during pregnancy is associated with low infant birth weight in the Cape Coast metropolitan hospital, Ghana: A hospital based cross-sectional study. Heliyon. 2020; 6(5): e03923. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnggraeni D, Utomo B, Suryawan A: Risk Factors Of Low Birth Weight (LBW) Incidence. A Case Control Study. Folia Medica Indones. 2014; 50(4): 270–277.\n\nSharma SR, Giri S, Timalsina U, et al.: Low birth weight at term and its determinants in a tertiary hospital of nepal: A case-control study. PLoS One. 2015; 10(4): 1–10. Publisher Full Text\n\nRahim FK, Muharry A: Hubungan karakteristik ibu dengan kejadian (bblr) di wilayah kabupaten kuningan. J. Ilmu Kesehat. Bhakti Husada. 2018; 09(02): 125–131.\n\nMohammed S, Bonsing I, Yakubu I, et al.: Maternal obstetric and socio-demographic determinants of low birth weight: A retrospective cross-sectional study in Ghana. Reprod. Health. 2019; 16(1): 1–8.\n\nKaur S, Ng CM, Badon SE, et al.: Risk factors for low birth weight among rural and urban Malaysian women. BMC Public Health. 2019; 19(Suppl 4): 1–10. Publisher Full Text" }	[ { "id": "170911", "date": "25 May 2023", "name": "Gouranga Dasvarma", "expertise": [ "Reviewer Expertise Demography", "including infant and child mortality", "maternal mortality", "fertility", "population and development", "population and environment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nThis is a useful study of the determinants of low birth weight babies in Indonesia based on data collected at a recent national survey, namely The Indonesia Demographic Health Survey 2017.\n\nThe authors have approached the problem of low birth weight babies (LBW) by sensibly selecting the groups of women who are at the highest risk of giving birth to babies with less than the recommended weight of 2,500 grams. Such groups of women comprise those with the following characteristics at the birth of their children, namely women (i) who are too young (less than 20 years of age), (ii) too old (more than 35 years of age), (iii) have too many children (3 or more) and too close (birth interval less than 2 years).\n\nThe aim of the study is to examine the effects of antenatal care (ANC) on the prevalence of LBW in each of the high-risk groups of women mentioned above, with the hypothesis that qualified ANC reduces the risk of LBW babies in the high-risk women.\n\nThe justification of the study appears to be that, by assumption the prevalence of LBW is high in the high-risk groups of women and that it can be reduced by good ANC. However, the prevalence of LBW is 7.1% as at the 2017 Indonesia Demographic and Health Survey, and it seems that Indonesia may be on track to achieving a 30% reduction in LBW between 2015 and 2030 as one of the Sustainable Development Goals of the United Nations. Therefore, further justification is needed for the present study.\n\nMoreover, a similar study, based on data from the 2017 Indonesia Demographic and Health Survey exists (see Safitri et al., 20221), which identifies ANC as a determinant of LBW in Indonesia, although the present manuscript focuses on low birthweight among high-risk groups of women. But reference should be made to the Safitri et al. study.\n\nThe manuscript needs a major revision, particularly a revision of Table 2 and rewriting the discussion of Table 2 findings.\n\nThe manuscript also requires a thorough editing for English.\n\nSeveral other, specific comments are made in the body of the text, which is returned for revision - please also find two attachments of the manuscript with my comments linked here (Attachment 1 and Attachment 2).\n\nSpecific comments:\nTitle. A similar study based on data from the 2017 Indonesia Demographic and Health Survey exists (see Safitri et al., 20221), which identifies ANC as a determinant of LBW in Indonesia. Acknowledgement and appropriate references should be made of the Safitri et al. study.\n\nWhat new information does your study provide to the field of knowledge? Is it the analysis of low birthweight among high-risk groups of women?\nAbstract. Background, Line 2. Rewrite as \"and is a leading cause of neonatal mortality\".\n\nAbstract. Methods, Line 3. \"unit of analysis\".\n\nAbstract. Methods. Line 3. pre-term birth is not included in the four criteria (4Ts) mentioned above.\n\nAbstract. Conclusions, Lines 1-2. The sentence reads as if WHO has found that qualified ANC standards have not been fully implemented (in Indonesia). But it is a finding of your analysis, is it not?\n\nMethods. Lines 11-12. How was quality of ANC (qualified ANC) determined? There are no data in the 2017 IDHS about the quality of ANC.\n\nMethods. Line 12. Do you mean \"positive pregnancy experience\"?\n\nMethods. Line 12. Do you mean to say, \"Data were collected at IDHS 2017 only for five WHO recommendations\"?\n\nMethods. Line 14. All four Ts together?\n\nResults. Line 3. The \"too many\" and \"too old\" categories of women (as well as the \"too close\" category\") also have high proportions in the High education category. This is notable.\n\nResults. Lines 3-4. This is not correct. Please rewrite this part as \"\"the majority of women in the \"too young\" and \"too many\" categories are rural dwellers, almost equal proportions of women in the \"too close\" category are urban and rural dwellers, and the majority of women in the \"too old\" category are urban dwellers.\"\n\nResults. Line 6. This is true only for the \"too young\" women. Please re-write correctly.\n\nResults. Lines 10-11. Where is it shown that “four out of five women had relatively more ANC check-ups”?\n\nResults. Lines 11-12. How is this true? The table shows that 85% or more of the women each category had a Health worker as their ANC provider. Do you mean to say that most of the Health workers are not qualified?\n\nResults. Lines 13-14. This result contradicts your hypothesis that women in the 4T categories run the risk of giving birth to babies with pre-maturity and low birth weight.\n\nTable 1. Usually, the dependent variable (in this case women in each criterion group) should be shown on the horizontal axis and the independent variables on the vertical axis. This can be done by formatting the layout of the table as landscape. Also, try to put the entire table on one page (i.e. do not split a table between pages. Reduce the font size if needed).\n\nTable 2. Line 1. \"ANC quality\" or qualified and unqualified ANC? The two terms are different.\n\nResults. First paragraph after Table 1. Table 2, as presented here shows the distribution of each socio-economic characteristic (independent variable) according to qualified ANC and unqualified ANC for each of the 4T categories of women. But in actual fact, you should show the distribution of qualified ANC and unqualified ANC according to each socio-economic characteristic. In other words, show the column percentages instead of row percentages. Therefore, please re-do Table 2 and re-write its description. Please also show the association (chi-square) between each of the socio-economic characteristics and unqualified and qualified ANC for each of the 4T categories.\n\nTable 3. It appears that you have not used any information derived from Table 2 in performing your analysis in Table 3. Please re-calculate Table 2 as suggested and use the relevant information from that (revised Table 2) to select the pertinent variables for logistic regression (Table 3).\n\nResults. Table 3, Lines 2-3. The \"mixed\" results may be due to the effects of confounding factors. For example, take any one category, such as \"Too old\" - While this group excludes women who are \"Too young\", the \"Too old\" women may have children who are too closely spaced or may have too many children. Similarly, the women who have \"Too many\" children may be \"too old\" themselves or may have children that are \"too closely\" spaced, or the women who are in the \"Too close\" category may have too many children or may be too old themselves. Only the \"Too young\" women would not be subjected to confounding factors like too many or too old, but they may still have too closely spaced children. It is for these reasons that you should also analyse the group of women who are Too old AND Too Close AND Too Many. The \"Too young\" group may be analysed separately, because \"too young\" women would have very little chances of having too many children or too closely spaced children.\n\nResults. Table 3, Line 3. \"Quality of ANC\". Which variable(s) indicate the quality of ANC?\n\nResults. Table 3, Line 15 and Line 28. Why do you refer to the groups as \"Model\"? Just call them what they are i.e., \"Too old\" or \"Too many\". A model may have the connotation of a separate logistic regression.\n\nDiscussion. Line 1. LBW. Is LBW a major cause of infant death in Indonesia?\n\nDiscussion. Line 1. Indonesia has already reduced its IMR by much, but it is pursuing further declines in IMR.\n\nDiscussion. Lines 4-5. If the prevalence of LBW is already so low in Indonesia (6-7%), then why study it? You should cite the target of LBW in Indonesia or cite the prevalence of LBW in countries which is lower than that in Indonesia and then justify your study.\n\nDiscussion Line 5. So, the results show that your hypothesis (that ANC quality affects the prevalence of LBW) is not true in three out of four categories of women. How do you explain this?\n\nDiscussion. Line 8. What kind of knowledge?\n\nConclusion. Lines 1-2. Assuming that the headings of your Table 1 are correct, according to the numbers of women in each category, women with too many children number 5,300. Thus, qualified ANC affects 55.5% of the women at risk (the number of women with all the Ts is equal to 9,546).\n\nConclusion. Line 8. Re ANC visit of at least eight times. The recommendation of eight ANC visits from the WHO came out in 2016 and probably implemented in Indonesia after the 2017 IDHS was conducted. Therefore, in most cases, at the time of IDHS 2017 the recommendation was to have at least 4 ANC visits. Table 9.2 of the 2017 IDHS Final report shows 90.6% of the women giving birth in the last five years had 4+ ANC visits.\nRecommendation: The authors should address the comments, provide further justification of this study and submit a revised 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "189795", "date": "17 Aug 2023", "name": "Garedew Tadege Engdaw", "expertise": [ "Reviewer Expertise Public health 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\nThe title is very useful and interesting. The paper needs major revision - below are my recommendations and questions for the development of the paper:\nThe sentences require editing in grammar. Needs checking grammar and should be re-edited.\n\nWHO recommends 4 or more ANC visits including 1st ANC in the first trimester. However, the authors focused on the effect of ANC visit on LBW and sociodemographic factors with women on high-risk birth, regardless the number of ANC visits. Some women visit a health facility for ANC just before the delivery. If the authors could compare the effect of LBW among women with no ANC visit to the women with 4 or more ANC visits, the results might be more plausible (NA).\n\nFor the analysis, you provided the definition of LBW to include the literature. You also need to provide the definition of ANC visits for inclusion criteria. If one or more ANC visits were your inclusion criteria, your conclusion may mislead the readers. You need to explain why you selected literatures mentioning ANC visit only, not the number of ANC visits in the limitation section.\n\nEducation level: It is the levels such as preschool, primary school, lower secondary school, upper secondary and higher education (diploma, certificate and above). Better to write the educational level of women’s in a scientific way, unless you have evidence for such classification from table 1. Make words uniform across the document (like Educational level from table …Low, Middle High, while from the prose it says secondary. It lacks consistency).\n\nYour outcome of interest is to see the effect of ANC and socio demographic factors on LBW, however from Table 1 you presented the birth weight status of women’s as NA (what is the importance of presenting this result, if the status is already unknown?).\n\nOperationalize the word Qualified ANC and Non-Qualified ANC, Non-health worker in ANC provider women of childbearing age.\n\nDiscussion:\nFrom the first paragraph you wrote as “This figure is lower than other Asian countries, such as India.18 While comparing it to African countries, the LBW in Indonesia is lower”. Make a correction for this paragraph as in “Asian country” and “African country” - you had only one literature for this evidence. Works for the whole document.\nYou think the two sentences had difference? This figure is lower than other Asian countries, such as India.18 While comparing it to African countries, the LBW in Indonesia is lower” (the figure is “lower than both in Asian country and Indonesia”…)\n\nWrite the possible reason for the discrepancy (in your study and other studies) including the factors for the discussion.\n\nFrom the last paragraph of the discussion you wrote as “several studies conducted in Ethiopia.16,17 This may be related to the education level of women who generally have low and middle education in this study\". You had only two evidences. Generally speaking, 'several' is used to refer to quantities above two or so but not so much that it's a lot or many. Perhaps the most common interpretation or intended sense of several is around three to five, but this can vary greatly depending on the context. Change it to 'some studies'.\n\nConclusion:\n\nIn the case of ANC visits of at least eight times, it is hoped that ANC with health workers at health facilities can be increased. Better to conclude your findings based on your discussion (the number of ANC visit is not related with your study/objective).\n\nIt is necessary to review the coverage of ANC in the National Health Insurance (NHI) mechanism, which is only four times, especially for the poor and with low education. Not related with your objective.\nHow is reviewing the National Health Insurance (NHI) related with LBW and related with ANC? It may be showing only the ANC attendants, number of ANC, the service provided during each ANC…not the low birth weight risks of the mothers on ANC follow up (remove the above two sentences).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10938", "date": "22 Mar 2024", "name": "Muthmainnah Muthmainnah", "role": "Author Response", "response": "Thank you for the comments, For the qualified ANC, we have explained the definition in the methods, and one of the criteria for qualified ANC is visiting at least eight ANC. There were only five recommendations from WHO that allow for analysis qualified ANC in this study, namely getting iron, getting bacteria in the urine, getting tetanus toxoid (TT) injections during pregnancy, visiting at least eight ANC, and screening of smoking history. Besides LBW, we also concerned about ANC in this article so we still analyzing NA-LBW because we can get more information about ANC. We have made several adjustments in the paper regarding your comments which are improving our article, especially in the literature and conclusion. However, the comments are really helpful. We appreciate it and already revised based on the comments." } ] } ]	1	https://f1000research.com/articles/12-399
https://f1000research.com/articles/12-1155/v1	14 Sep 23	{ "type": "Case Report", "title": "Case Report: Non-convulsive seizure following traumatic brain injury — a significant occurrence that needs to be considered due to potential long-term sequelae", "authors": [ "Azra Zafar" ], "abstract": "Introduction/background Non convulsive seizures (NCS) following traumatic brain injury (TBI) may remain undiagnosed due to lack of overt clinical manifestation and can have long-term sequelae due to delay in timely treatment. Occurrence of early NCS is known to have subsequent neurologic sequelae due to excitotoxic neuronal injury. Case report This is a case report of a young girl who sustained a TBI due to a motor vehicle accident (MVA) and was admitted with a fluctuating level of consciousness. Her clinical presentation was attributed to TBI; however as her conscious level did not recover, an electroencephalogram (EEG) was requested, which detected non convulsive status epilepticus (NCSE). Anti-seizure medication (ASM) was started. Her follow-up EEG and magnetic resonance imaging (MRI) were suggestive of the potential adverse effects of prolonged NCSE. Conclusion NCS may remain undiagnosed in TBI due to a paucity of overt clinical manifestations. Every patient with TBI and altered consciousness at presentation should be evaluated by EEG immediately, if possible, in the emergency department to avoid long-term sequelae of NCS in such cases.", "keywords": [ "Nonconvulsive seizure", "diffuse axonal injury", "temporal lobe", "excitotoxicity", "nonconvulsive status epilepticus" ], "content": "Case report\n\nA 13-year-old, medically free student was brought to the King Fahd Hospital of the University (KFHU) after sustaining a motor vehicle accident (MVA) as a car passenger. Due to the side impact, the patient did not sustain any open injuries, and did not eject from the car. No evidence of vomiting, convulsions, nose or ear bleeding was present upon arrival either. However, her consciousness level was fluctuating with intermittent episodes of agitation. On examination, she was drowsy. Her vitals were within normal limits and her Glasgow Coma Scale score was 10/15. Neurological examination did not detect any focal abnormality except a bilateral positive Babinski sign.\n\nRoutine blood works and pan body computed tomography scan were normal. Once the patient was admitted to the intensive care unit (ICU), an EEG was requested to rule out non convulsive seizure (NCS) for unexplained altered sensorium. Her first EEG was performed 65 hrs after admission. It detected encephalopathy and electrographic seizures (ESz) arising from the left anterior temporal region (Figure 1), indicating non-convulsive status epilepticus (NCSE). Levetiracetam (LVT) was initiated, and an MRI scan was subsequently performed which revealed multiple hyperintense foci in bilateral frontal and right temporal and occipital lobe, as well as hemorrhage foci on susceptibility weighted image (SWI). These findings were indicative of hemorrhagic axonal diffuse injury type (DAI) type 2. Despite starting LVT, the patient’s consciousness level did not improve; therefore, EEG was repeated after 24 hrs, indicating that NCSE had not been resolved. At this stage, phenytoin was added, after which the patient’s consciousness level improved. The follow-up EEG showed focal epileptic discharges in the form of sharp waves and spikes in the left centrotemporal head region. Patient was discharged on two anti-seizure medications (ASM).\n\n\nFollow-up diagnostic investigations\n\nDuring her follow-up visits, she was symptoms free. Brain MRI and EEG were repeated nine months after the event to determine if ASM should be discontinued. EEG detected focal epileptic discharges in the left temporal head region, and brain MRI showed significant resolution of changes, except an abnormal focal hyperintense lesion in the left temporal area (see Figure 2). Consequently, LVT was continued (the entire timeline is summarized in Figure 3).\n\n\nOutcome\n\nOne year follow-up, the patient was seizure-free with single ASM and had no symptoms suggestive of cognitive disturbance. She was active in her normal routine life including academics.\n\n\nDiscussion\n\nTBI is a significant cause of preventable deaths in Saudi Arabia, and 95% are attributed to MVA.1 Seizures (including NCS/NCSE) can occur in 20−30% of patients with severe TBI due to cerebral metabolic distress and hippocampal atrophy, which contribute to higher mortality rates.2 However, in clinical practice, NCS following TBI may remain undiagnosed due to the lack of overt clinical manifestation and adversely affect outcomes due to the delay in treatment.3 Occurrence of early NCS can have subsequent neurologic sequelae due to excitotoxic neuronal injury aggravating the injury caused by TBI.4 Therefore, it is mandatory to diagnose NCSE in a timely manner to prevent significant neurological sequelae by performing EEG in TBI victims with altered sensorium. The benefits of this protocol are supported by the findings yielded by a study involving continuous EEG monitoring in 16 patients with severe TBI, allowing NCS to be detected in three cases.5 Similarly, seizures were detected using EEG in 20% of examined patients, more than 50% of whom experienced NCS.6\n\nOur patient had TBI of moderate severity7 and prolonged NCSE which lasted for five days. Brain MRI showed changes related to prolonged NCS, along with DAI. The follow-up EEG detected focal ED in the left temporal region and MRI showed a small localized hyperintense lesion in the left temporal lobe which could be either a sequela of TBI or NCSE. However, the left temporal cortical edema, which could be the result of prolonged NCS, was completely resolved. The relationship between prolonged NCS and structural changes to the brain, particularly the temporal lobe, has been reported previously.2,8 Vespa et al. examined cEEG findings of 140 patients with moderate to severe TBI, and detected acute post-traumatic NCS in 23% of the cohort. Moreover, in a selected group of patients, this finding was significantly associated with long-term hippocampal atrophy.2 In addition, according to one case report, a patient with schizophrenia and NCSE having increased hippocampal volume in an acute setting was later found to have hippocampal atrophy.8 Although NCSE is not uncommon following TBI, its association with anatomical changes leading to hippocampal atrophy in the long term is debatable, given that significant neuronal damage due to diffuse injury itself can be a cause. Thus, further research is required to better understand these phenomena.9 Jorge et al., studied 37 patients with closed head injury and concluded that hippocampal volumes were significantly lower in patients with moderate to severe head injury than in patients with mild TBI.10\n\nIn our patient, follow-up brain MRI showed a significant resolution of findings detected in the initial scan; however, a small abnormal hyperintense signal was persistent in the left anterior temporal region. As volumetric MRI is not available in our healthcare facility, we were unable to assess the volume loss. Still, we posit that—in addition to TBI—prolong NCSE in our patient could be a contributing factor for this finding on follow-up MRI. Although our patient did not have any clinical seizures, follow-up EEG and MRI performed nine months after the initial incident were suggestive of temporal lobe pathology with a heightened risk of temporal lobe-onset seizure. Accordingly, her ASM could not be discontinued. In reporting on this case, our aim is to highlight the importance of close EEG monitoring in patients with TBI in whom consciousness is altered. In this particular case, brain MRI was instrumental for detecting changes not only in DAI but possibly NCS as well.\n\nThis case highlights our limitation of immediate EEG recording in patients with TBI upon arrival. The strength is the identification of uncommon electrographic patterns suggesting NCS during routine EEG recording guiding proper management.\n\n\nConclusion\n\nThis case illustrates the possible association of prolonged NCSE following TBI with temporal lobe structural changes. It further emphasizes the need for immediate EEG monitoring in patients with TBI that present with altered sensorium. All institutes dealing with trauma cases should thus have the resources needed for emergency EEG monitoring to avoid neurological sequelae of NCS, which may otherwise remain undiagnosed.\n\n\nConsent\n\nWe confirm that we have obtained permission from the patients’ father to use images and data included in this article.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nUllah S, Bin Ayaz S, Moukais IS, et al.: Factors affecting functional outcomes of traumatic brain injury rehabilitation at a rehabilitation facility in Saudi Arabia. Neurosciences (Riyadh). 2020; 25: 169–175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVespa PM, McArthur DL, Xu Y, et al.: Nonconvulsive seizures after traumatic brain injury are associated with hippocampal atrophy. Neurology. 2010; 75: 792–798. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinney MO, Craig JJ, Kaplan PW: Non-convulsive status epilepticus: mimics and chameleons. Pract. Neurol. 2018; 18: 291–305. PubMed Abstract \| Publisher Full Text\n\nMaruyama A, Tokumoto S, Yamaguchi H, et al.: Early non-convulsive seizures are associated with the development of acute encephalopathy with biphasic seizures and late reduced diffusion. Brain Dev. 2021; 43: 548–555. PubMed Abstract \| Publisher Full Text\n\nVaewpanich J, Reuter-Rice K: Continuous electroencephalography in pediatric traumatic brain injury: Seizure characteristics and outcomes. Epilepsy Behav. 2016; 62: 225–230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVespa PM, Nuwer MR, Nenov V, et al.: Increased incidence and impact of nonconvulsive and convulsive seizures after traumatic brain injury as detected by continuous electroencephalographic monitoring. J. Neurosurg. 1999; 91: 750–760. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMesfin FB, Gupta N, Hays Shapshak A, et al.: Diffuse Axonal Injury. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 Jan 23. PubMed Abstract\n\nFujisao EK, Cristaldo NR, da Silva Braga AM , et al.: Hippocampal Damage and Atrophy Secondary to Status Epilepticus in a Patient with Schizophrenia. Front. Neurol. 2017; 8: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung BG, Claassen J: Nonconvulsive status epilepticus and brain damage. Further evidence, more questions. Neurology. 2010; 75: 760–761. PubMed Abstract \| Publisher Full Text\n\nJorge RE, Acion L, Starkstein SE, et al.: Hippocampal volume and mood disorders after traumatic brain injury. Biol. Psychiatry. 2007; 62: 332–338. PubMed Abstract \| Publisher Full Text" }	[ { "id": "206929", "date": "16 Oct 2023", "name": "Maimoona Siddiqui", "expertise": [ "Reviewer Expertise Epilepsy", "Stroke" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting case, agreed that NCSE is an under recognized entity.\nCan the author explain the meaning of \"medically free\" mentioned in first line? I think its need rephrasing.\nIt would be better if author elaborate neurological findings like fundus examination, signs of meningism at admission and during the course of illness.\nWhat criteria of NCSE was followed in this case? As EEG showed 8 seconds of focal epileptiform discharges.\n\nIt would be better to upload a more detail EEG epoch as it is the most relevant investigation.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "10407", "date": "24 Jan 2024", "name": "Azra Zafar", "role": "Author Response", "response": "We thank the reviewer for reviewing our paper and giving useful suggestions. We have responded to each comment accordingly and hope it will be satisfying. 1-Can the author explain the meaning of \"medically free\" mentioned in first line? I think its need rephrasing. Response; It will be rephrased as suggested in revised manuscript; A 13-years old student without any prior medical illnesses was brought to the King Fahd Hospital of the University (KFHU) after sustaining a motor vehicle accident (MVA) as a car passenger. 2-It would be better if author elaborate neurological findings like fundus examination, signs of meningism at admission and during the course of illness. Response; will be added; Patient was agitated with fluctuating level of consciousness at time of presentation and did not follow commands. Cranial nerves were intact, fundus examination was attempted but failed as patient could not cooperate due to her clinical state. Tone, power and deep tendon reflexes were normal. There were no signs of meningism. After starting second ASM, patient’s consciousness improved and returned to baseline. Patient became oriented to time, place, and person and had intact language functions. Patient was able to walk independently and had normal motor, cerebellar, and sensory system examination before discharge. What criteria of NCSE was followed in this case? As EEG showed 8 seconds of focal epileptiform discharges. Response; Salzburg Consensus Criteria were applied to diagnose NCSE. Reference will be added. Beniczky S, Hirsch LJ, Kaplan PW, Pressler R, Bauer G, Aurlien H, et al. Unified EEG terminology and criteria for nonconvulsive status epilepticus. Epilepsia. 2013; 54 (Suppl. 6):28–9. These details will be mentioned in revised manuscript; EEG fulfilled the Salzburg Consensus Criteria for NCSE; RTDA (rhythmic theta or delta activity) with spatiotemporal evolution/ and subtle clinical signs. Detailed EEG report has been added. Background activity consisted mainly of diffuse theta activity. Frequent 2-3 Hz left hemispheric ictal delta activity with spatio-temporal evolution was recorded. The activity starting from left hemisphere and propagated after few seconds to involve the right hemisphere. In, a 30 minutes EEG recording, 7 events lasting for 7-20 seconds were recorded .Occasional sharp wave activity was also recorded over the left fronto-central head region with maximal negativity at C3. Some spells of these epileptiform pattern were associated with subtle clinical signs which were left hand automatism and screaming indicating electro-clinical seizures. It would be better to upload a more detail EEG epoch as it is the most relevant investigation. Response; Kindly see the epochs here. Figure 1a Figure 1b 1a- Beginning of electrographic seizure; rhythmic slow waves showing propagation to right cerebral hemisphere. 1b- Continuation and termination of electrographic seizure. We can further add follow up EEG shown below as Figure 3 to document improvement of background rhythm and left temporal epileptic abnormality." } ] }, { "id": "218441", "date": "30 Nov 2023", "name": "Seyed M Mirsattari", "expertise": [ "Reviewer Expertise Electroencephalography", "neuroimaging", "epilepsy", "status epilepticus" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report with an important take home message about non-convulsive status epilepticus as a potential cause of immediate and delayed recovery from a traumatic brain injury. However, the EEG figure is not very convincing. Could a better figure be provided? The need for continuous EEG (cEEG) rather than serial EEGs needs to be emphasized. The authors do not explain the discrepancy between their MRI and EEG changes: EEG showed left temporal focal status epilepticus but MRI findings were bifrontal, \"right\" temporo-occipital and within the corpus callosum. Please clarify if you meant to say, \"left temporal\" lobe as your figure indicates it. You may label your MRI images with R and L to avoid confusion. Important reference: Cartagena AM, et al. Reversible and irreversible cranial MRI findings associated with status epilepticus. Epilepsy Behav 2014 Apr:33:24-301.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "10706", "date": "24 Jan 2024", "name": "Azra Zafar", "role": "Author Response", "response": "We are grateful to the reviewer for reviewing our paper and giving valuable suggestions. We have responded to each comment accordingly and hope it will be satisfying. This is an interesting case report with an important take home message about non-convulsive status epilepticus as a potential cause of immediate and delayed recovery from a traumatic brain injury. However, the EEG figure is not very convincing. Could a better figure be provided? Response; Thanks for your comment, I have added another figure here with more explanation. Description will be added in the manuscript. Background activity consisted mainly of diffuse theta activity. Frequent 2-3 Hz left hemispheric ictal delta activity with spatio-temporal evolution was recorded. The activity starting from the left hemisphere propagated after a few seconds to involve the right hemisphere. In, a 30-minute EEG recording, 7 events lasting for 7-20 seconds were recorded .sharp wave activity was also recorded over the left fronto-central and temporal head region. Some spells of these epileptiform patterns were associated with subtle clinical signs which were left-hand automatism and screaming indicating electro-clinical seizures. Response; Kindly see the epochs here. 1a- Beginning of electrographic seizure; rhythmic slow waves showing propagation to right cerebral hemisphere. 1b- Continuation and termination of electrographic seizure. We can further add follow up EEG shown below as Figure 3 to document improvement of background rhythm and left temporal epileptic abnormality. The need for continuous EEG (cEEG) rather than serial EEGs needs to be emphasized. Response; done as suggested and more references have been added to emphasize the need of cEEG. Whether the electrographic patterns observed in patients with TBI affect functional outcome or not is another challenging question that can be addressed by cEEG. Lee et al. studied 152 patients sustaining non-penetrating moderate to severe TBI with cEEG and observed no correlation ship between rhythmic, periodic, and ictal patterns including Esz and functional outcome at three months. However, only four patients had Esz in this study and therefore further studies including a higher number of patients with Esz are needed to evaluate its effect on outcome. Nevertheless, an independent association was established between cEEG background and functional outcome which defines the prognostic value of cEEG in patients with TBI [Lee H 2019]. Lee H, Mizrahi MA, Hartings JA, Sharma S, Pahren L, Ngwenya LB, Moseley BD, Privitera M, Tortella FC, Foreman B. Continuous Electroencephalography After Moderate to Severe Traumatic Brain Injury. Crit Care Med. 2019 Apr;47(4):574-582. doi: 10.1097/CCM.0000000000003639. PMID: 30624278; PMCID: PMC6639805. In another study describing the acute physiological effect of NCS in moderate to severe TBI by cEEG and cerebral microdialysis for 7 days, ten patients with Esz were compared with ten patients without seizures. The patients with Esz as compared to non-seizure patients were found to have higher mean intracranial pressure (ICP) and a higher mean lactate/ pyruvate ratio suggesting Esz to be the cause of metabolic crisis and raised ICP in addition to TBI itself [Vespa PM 2007]. These findings are important and emphasize the need for cEEG to detect and treat Esz timely to prevent further brain cellular injury which can be permanent [Hirsch LJ 2008]. Vespa PM, Miller C, McArthur D, Eliseo M, Etchepare M, Hirt D, Glenn TC, Martin N, Hovda D. Nonconvulsive electrographic seizures after traumatic brain injury result in a delayed, prolonged increase in intracranial pressure and metabolic crisis. Crit Care Med. 2007 Dec;35(12):2830-6. PMID: 18074483; PMCID: PMC4347945. Hirsch LJ. Nonconvulsive Seizures in Traumatic Brain Injury: What you Don’t See Can Hurt You. Epilepsy Currents. 2008;8(4):97-99. doi:10.1111/j.1535-7511.2008.00254.x In reporting on this case, we aim is to highlight the importance of cEEG monitoring in patients with TBI in whom consciousness is altered and emphasize the implementation of a consensus statement for cEEG by Herman et al. The American Electroencephalographic Society recommends that every patient with acute brain injury having an altered level of awareness should be evaluated by cEEG to detect ESz as it can have an impact on the outcome. [Herman ST 2015]. Herman ST, Abend NS, Bleck TP, et al. Consensus statement on continuous EEG in critically ill adults and children, part I: indications. Journal of clinical neurophysiology : official publication of the American Electroencephalographic Society. 2015. April;32(2):87–95. The authors do not explain the discrepancy between their MRI and EEG changes: EEG showed left temporal focal status epilepticus but MRI findings were bifrontal, \"right\" temporo-occipital and within the corpus callosum. Please clarify if you meant to say, \"left temporal\" lobe as your figure indicates it. You may label your MRI images with R and L to avoid confusion. Response; thanks for pointing out this mistake, it was left temporal lobe as mentioned in the figure. We will correct it in revised manuscript. We have corrected the EEG findings in the text as well. Kindly see the above description for EEG. It was left hemispheric delta activity propagating to the right. We have added R and L in MRI images. Important reference: Cartagena AM, et al. Reversible and irreversible cranial MRI findings associated with status epilepticus. Epilepsy Behav 2014 Apr:33:24-301. Response; thank you for mentioning. This will be added in the discussion in the revised manuscript. Previous studies have reported various MRI changes in SE. A retrospective study investigating MRI findings in patients with SE reported that MRI changes can be either diffuse or focal and can be seen in different extratemporal brain areas including the brain stem, cerebellum, thalamus and basal ganglia. Importantly, the authors recommended that as these changes can evolve after one week, serial MRI should be performed to follow these neuroanatomical changes related to SE and reversibility. [Cartagena AM]. Cartagena AM, Young GB, Lee DH, Mirsattari SM: Reversible and irreversible cranial MRI findings associated with status epilepticus.Epilepsy Behav. 2014; 33: 24-30 PubMed Abstract \| Publisher Full Text It is also worth noting that recent studies find MRI changes useful in diagnosing NCS, especially when EEG is inconclusive or in setups where cEEG is not available [Gelisse P]. Gelisse P, Genton P, Crespel A, Lefevre PH. Will MRI replace the EEG for the diagnosis of nonconvulsive status epilepticus, especially focal? Rev Neurol. 2021; 177:359-69. doi: 10.1016/j.neurol.2020.09.005. Epub 2021 Jan 22. PMID: 33487411. Is the background of the case’s history and progression described in sufficient detail? Yes Thanks for the response Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly Response; further details related to examination, outcome, and follow up EEG will be included in the revised manuscript The patient was agitated with a fluctuating level of consciousness at the time of presentation and did not follow commands. Cranial nerves were intact. Tone, power and deep tendon reflexes were normal. There were no signs of meningism. After starting the second ASM, the patient’s consciousness improved and returned to baseline. Patient became oriented to time, place, and person and had intact language functions. Patient was able to walk independently and had normal motor, cerebellar, and sensory system examination before discharge. At one year follow-up, the patient was perfectly normal and had no clinical seizures or cognitive disturbances. The follow-up EEG detected left temporal epileptic abnormality. MRI images included. Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly Response; We have further elaborated the discussion, hope that you will find it satisfactory now. Is the case presented with sufficient detail to be useful for other practitioners? Partly Response; we have added more details. References 1. Cartagena AM, Young GB, Lee DH, Mirsattari SM: Reversible and irreversible cranial MRI findings associated with status epilepticus.Epilepsy Behav. 2014; 33: 24-30 PubMed Abstract \| Publisher Full Text Response; added Moreover, a retrospective study investigating MRI findings in patients with status epilepticus reported that MRI changes can be diffuse or focal and can be seen in different extratemporal brain areas (brain stem, cerebellum, thalamus and basal ganglia). Importantly, the authors recommended that as these changes can evolve after one week, serial MRI should be performed to follow these neuroanatomical changes related to SE. [Cartagena AM]. Data have identified NCS occurring in acute TBI to be associated with disproportionate hippocampal atrophy in long term which is greater in Hippocampus ipsilateral to the Esz focus as compared to the hippocampus without seizure. [Vespa PM 2010] Vespa PM, McArthur DL, Xu Y, et al.: Nonconvulsive seizures after traumatic brain injury are associated with hippocampal atrophy. Neurology. 2010; 75: 792–798. PubMed Abstract \| Publisher Full Text \| Free Full Text" }, { "c_id": "10743", "date": "13 Apr 2024", "name": "Azra Zafar", "role": "Author Response", "response": "We are grateful to the reviewer for reviewing our paper and giving valuable suggestions. We have responded to each comment accordingly and hope it will be satisfying. This is an interesting case report with an important take home message about non-convulsive status epilepticus as a potential cause of immediate and delayed recovery from a traumatic brain injury. However, the EEG figure is not very convincing. Could a better figure be provided? Response; Thanks for your comment, I have added another figure here with more explanation. Description will be added in the manuscript. Background activity consisted mainly of diffuse theta activity. Frequent 2-3 Hz left hemispheric ictal delta activity with spatio-temporal evolution was recorded. The activity starting from the left hemisphere propagated after a few seconds to involve the right hemisphere. In, a 30-minute EEG recording, 7 events lasting for 7-20 seconds were recorded .sharp wave activity was also recorded over the left fronto-central and temporal head region. Some spells of these epileptiform patterns were associated with subtle clinical signs which were left-hand automatism and screaming indicating electro-clinical seizures. Response; Kindly see the epochs here. 1a- Beginning of electrographic seizure; rhythmic slow waves showing propagation to right cerebral hemisphere. 1b- Continuation and termination of electrographic seizure. We can further add follow up EEG shown below as Figure 3 to document improvement of background rhythm and left temporal epileptic abnormality. The need for continuous EEG (cEEG) rather than serial EEGs needs to be emphasized. Response; done as suggested and more references have been added to emphasize the need of cEEG. Whether the electrographic patterns observed in patients with TBI affect functional outcome or not is another challenging question that can be addressed by cEEG. Lee et al. studied 152 patients sustaining non-penetrating moderate to severe TBI with cEEG and observed no correlation ship between rhythmic, periodic, and ictal patterns including Esz and functional outcome at three months. However, only four patients had Esz in this study and therefore further studies including a higher number of patients with Esz are needed to evaluate its effect on outcome. Nevertheless, an independent association was established between cEEG background and functional outcome which defines the prognostic value of cEEG in patients with TBI [Lee H 2019]. Lee H, Mizrahi MA, Hartings JA, Sharma S, Pahren L, Ngwenya LB, Moseley BD, Privitera M, Tortella FC, Foreman B. Continuous Electroencephalography After Moderate to Severe Traumatic Brain Injury. Crit Care Med. 2019 Apr;47(4):574-582. doi: 10.1097/CCM.0000000000003639. PMID: 30624278; PMCID: PMC6639805. In another study describing the acute physiological effect of NCS in moderate to severe TBI by cEEG and cerebral microdialysis for 7 days, ten patients with Esz were compared with ten patients without seizures. The patients with Esz as compared to non-seizure patients were found to have higher mean intracranial pressure (ICP) and a higher mean lactate/ pyruvate ratio suggesting Esz to be the cause of metabolic crisis and raised ICP in addition to TBI itself [Vespa PM 2007]. These findings are important and emphasize the need for cEEG to detect and treat Esz timely to prevent further brain cellular injury which can be permanent [Hirsch LJ 2008]. Vespa PM, Miller C, McArthur D, Eliseo M, Etchepare M, Hirt D, Glenn TC, Martin N, Hovda D. Nonconvulsive electrographic seizures after traumatic brain injury result in a delayed, prolonged increase in intracranial pressure and metabolic crisis. Crit Care Med. 2007 Dec;35(12):2830-6. PMID: 18074483; PMCID: PMC4347945. Hirsch LJ. Nonconvulsive Seizures in Traumatic Brain Injury: What you Don’t See Can Hurt You. Epilepsy Currents. 2008;8(4):97-99. doi:10.1111/j.1535-7511.2008.00254.x In reporting on this case, we aim is to highlight the importance of cEEG monitoring in patients with TBI in whom consciousness is altered and emphasize the implementation of a consensus statement for cEEG by Herman et al. The American Electroencephalographic Society recommends that every patient with acute brain injury having an altered level of awareness should be evaluated by cEEG to detect ESz as it can have an impact on the outcome. [Herman ST 2015]. Herman ST, Abend NS, Bleck TP, et al. Consensus statement on continuous EEG in critically ill adults and children, part I: indications. Journal of clinical neurophysiology : official publication of the American Electroencephalographic Society. 2015. April;32(2):87–95. The authors do not explain the discrepancy between their MRI and EEG changes: EEG showed left temporal focal status epilepticus but MRI findings were bifrontal, \"right\" temporo-occipital and within the corpus callosum. Please clarify if you meant to say, \"left temporal\" lobe as your figure indicates it. You may label your MRI images with R and L to avoid confusion. Response; thanks for pointing out this mistake, it was left temporal lobe as mentioned in the figure. We will correct it in revised manuscript. We have corrected the EEG findings in the text as well. Kindly see the above description for EEG. It was left hemispheric delta activity propagating to the right. We have added R and L in MRI images. Important reference: Cartagena AM, et al. Reversible and irreversible cranial MRI findings associated with status epilepticus. Epilepsy Behav 2014 Apr:33:24-301. Response; thank you for mentioning. This will be added in the discussion in the revised manuscript. Previous studies have reported various MRI changes in SE. A retrospective study investigating MRI findings in patients with SE reported that MRI changes can be either diffuse or focal and can be seen in different extratemporal brain areas including the brain stem, cerebellum, thalamus and basal ganglia. Importantly, the authors recommended that as these changes can evolve after one week, serial MRI should be performed to follow these neuroanatomical changes related to SE and reversibility. [Cartagena AM]. Cartagena AM, Young GB, Lee DH, Mirsattari SM: Reversible and irreversible cranial MRI findings associated with status epilepticus.Epilepsy Behav. 2014; 33: 24-30 PubMed Abstract \| Publisher Full Text It is also worth noting that recent studies find MRI changes useful in diagnosing NCS, especially when EEG is inconclusive or in setups where cEEG is not available [Gelisse P]. Gelisse P, Genton P, Crespel A, Lefevre PH. Will MRI replace the EEG for the diagnosis of nonconvulsive status epilepticus, especially focal? Rev Neurol. 2021; 177:359-69. doi: 10.1016/j.neurol.2020.09.005. Epub 2021 Jan 22. PMID: 33487411. Is the background of the case’s history and progression described in sufficient detail? Yes Thanks for the response Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly Response; further details related to examination, outcome, and follow up EEG will be included in the revised manuscript The patient was agitated with a fluctuating level of consciousness at the time of presentation and did not follow commands. Cranial nerves were intact. Tone, power and deep tendon reflexes were normal. There were no signs of meningism. After starting the second ASM, the patient’s consciousness improved and returned to baseline. Patient became oriented to time, place, and person and had intact language functions. Patient was able to walk independently and had normal motor, cerebellar, and sensory system examination before discharge. At one year follow-up, the patient was perfectly normal and had no clinical seizures or cognitive disturbances. The follow-up EEG detected left temporal epileptic abnormality. MRI images included. Is sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly Response; We have further elaborated the discussion, hope that you will find it satisfactory now. Is the case presented with sufficient detail to be useful for other practitioners? Partly Response; we have added more details. References 1. Cartagena AM, Young GB, Lee DH, Mirsattari SM: Reversible and irreversible cranial MRI findings associated with status epilepticus.Epilepsy Behav. 2014; 33: 24-30 PubMed Abstract \| Publisher Full Text Response; added Moreover, a retrospective study investigating MRI findings in patients with status epilepticus reported that MRI changes can be diffuse or focal and can be seen in different extratemporal brain areas (brain stem, cerebellum, thalamus and basal ganglia). Importantly, the authors recommended that as these changes can evolve after one week, serial MRI should be performed to follow these neuroanatomical changes related to SE. [Cartagena AM]. Data have identified NCS occurring in acute TBI to be associated with disproportionate hippocampal atrophy in long term which is greater in Hippocampus ipsilateral to the Esz focus as compared to the hippocampus without seizure. [Vespa PM 2010] Vespa PM, McArthur DL, Xu Y, et al.: Nonconvulsive seizures after traumatic brain injury are associated with hippocampal atrophy. Neurology. 2010; 75: 792–798. PubMed Abstract \| Publisher Full Text \| Free Full Text Figures , 1a,1b electrographic seizure and figure 3; follow up EEG (please see the figures linked below): Figure 1a Figure 1b Figure 3" } ] } ]	1	https://f1000research.com/articles/12-1155
https://f1000research.com/articles/11-1134/v1	04 Oct 22	{ "type": "Research Article", "title": "Higher amyloid correlates to greater loneliness during the COVID-19 pandemic", "authors": [ "Abigail Kehrer-Dunlap", "Rebecca Bollinger", "Szu-Wei Chen", "Audrey Keleman", "Regina Thompson", "Anne Fagan", "Beau Ances", "Susan Stark", "Abigail Kehrer-Dunlap", "Rebecca Bollinger", "Szu-Wei Chen", "Audrey Keleman", "Regina Thompson", "Anne Fagan", "Beau Ances" ], "abstract": "Background: Little is known about psychosocial characteristics, including loneliness, anxiety, and depression, present in preclinical Alzheimer disease (AD). The purpose of this cross-sectional study was to examine the relationship between these psychosocial characteristics and amyloid accumulation in cognitively normal older adults with and without preclinical AD during the COVID-19 pandemic.\nMethods: A global Clinical Dementia Rating ®\nScale score of 0 was required for enrollment. Cortical amyloid burden was measured using [11C] Pittsburgh compound B or [18F]-Florbetapir PET tracers. Centiloids were used to synchronize measures. Demographic characteristics and measures of loneliness, anxiety, and depression were collected via self-report. Spearman’s correlation was used to examine relationships between amyloid and psychosocial characteristics.\nResults: The 108 participants had a mean age of 75.0 and an average amyloid burden of 22.2. Higher amyloid accumulation was significantly associated with greater loneliness.\nConclusions: Additional research is needed with a larger, more diverse sample to examine these psychosocial characteristics in preclinical AD.", "keywords": [ "loneliness", "Alzheimer disease", "preclinical Alzheimer disease", "COVID-19" ], "content": "Introduction\n\nLoneliness is a growing epidemic affecting millions of older adults and is associated with poor physical health, depression, anxiety, mortality, and an increased risk of Alzheimer disease (AD; Malhotra et al., 2021; Sundström et al., 2020; Sutin et al., 2018). Psychosocial characteristics such as loneliness, anxiety, and depression are associated with symptomatic AD (El Haj et al., 2016; Pietrzak et al., 2015), but it is unclear whether these emerge during the preclinical phase of AD. Individuals with preclinical AD have Alzheimer pathology, including abnormal cerebrospinal fluid (CSF) amyloid-β 1-42 (Aβ1-42), total tau (t-tau), and/or phosphorylated tau (p-tau181) biomarkers or higher global amyloid burden indices beginning years before the onset of cognitive decline (Dubois et al., 2016). Individuals with preclinical AD are cognitively normal (CN) and show no clinical symptomology consistent with AD, such as memory loss or hallucinations, for years or even decades (Price & Morris, 1999); however, other clinical symptoms, such as loneliness, anxiety, and depression, may already be prevalent.\n\nLimited research with CN older adults has shown that greater loneliness is associated with higher amyloid burden (Donovan et al., 2016) and higher tau pathology (d’Oleire Uquillas et al., 2018). Research is mixed on the associations between anxiety, higher amyloid burden (Pietrzak et al., 2015), and higher tau pathology (d’Oleire Uquillas et al., 2018). The relationship between depression and amyloid-β levels in CN older adults remains unclear; while some studies report that increased amyloid burden measured by positron emission tomography (PET) is linked to depression (Wu et al., 2014), others report that amyloid PET is not (Babulal et al., 2020). Further evidence indicates that emotional and psychological distress may accelerate the onset of clinical symptoms of AD for older adults with preclinical AD (Dubois et al., 2016; Wilson et al., 2007). The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to an increase in the number of older adults reporting loneliness, anxiety, and depression (Reppas-Rindlisbacher et al., 2021); as a result, older adults with preclinical AD may be at an increased risk for exhibiting clinical symptoms of AD. Additional research is needed to understand how at-risk groups, including individuals with preclinical AD, may be impacted by prolonged emotional and psychological distress throughout the COVID-19 pandemic.\n\nTo date, relationships between amyloid burden and loneliness, anxiety, and depression in CN older adults using multiple PET tracers have not been investigated. It is important to understand whether these psychosocial characteristics impact older adults with higher amyloidosis consistent with preclinical AD to identify potential risk factors that may exacerbate the progression of AD. The purpose of this study was to examine psychosocial characteristics (loneliness, anxiety, and depression) in CN older adults during the COVID-19 pandemic using a continuous measure of cortical amyloid burden.\n\n\nMethods\n\nCommunity-dwelling older adults were recruited for this cross-sectional study from an ongoing longitudinal cohort study at the Knight Alzheimer Disease Research Center (Knight ADRC) at Washington University in St. Louis (Bollinger et al., 2021). The inclusion criteria for the cohort study were: (1) ≥65 years old, (2) CN as determined by a Clinical Dementia Rating® (CDR; Morris, 1993) score of 0, and (3) had CSF biomarkers and/or neuroimaging (PET and/or MRI) within two years of study enrollment. Individuals were excluded if they had a medical or psychiatric diagnosis that could interfere with longitudinal follow-up or neuroimaging or adversely affect cognition. Participants included in this secondary analysis had amyloid PET data processed within two years of study enrollment. The Knight ADRC recruitment procedures have been published previously (Morris et al., 2019).\n\nAt the beginning of the COVID-19 pandemic, individuals were approached by phone or e-mail and invited to participate. Written informed consent was obtained, and participants completed surveys between 27 April 2020 and 8 June 2020 regarding their mental health with staff via phone or secure e-mail using a Research Electronic Data Capture (REDCap; Harris et al., 2009) survey link. Upon completion, participants could elect to enter a drawing for a $50 gift card. This study was approved by the Institutional Review Board at Washington University in St. Louis (reference number: 201807135).\n\nThe global CDR score (Morris, 1993) from the participant’s most recent annual clinical visit at the Knight ADRC was used to assess the level of impairment from normal to severe symptomatic AD. The CDR is scored as follows: 0 (CN), 0.5 (very mild dementia), 1 (mild dementia), 2 (moderate dementia), and 3 (severe dementia). A CDR score of 0 was required for enrollment in this study.\n\nPET amyloid imaging was used to obtain AD biomarker data through previously described methods (Su et al., 2013, 2015). Participants were injected with either [11C] Pittsburgh compound B (PiB) or [18F]-Florbetapir (AV45), and dynamic scans were acquired. The post-injection time window for quantification was 30–60 minutes for PiB and 50–70 minutes for AV45. A PET Unified Pipeline was implemented to process the data. In short, a region of interest (ROI) segmentation approach was used with FreeSurfer 5.3 (Martinos Center for Biomedical Imaging, Charlestown, MA, USA). Based on the ROI segmentation, a tissue mask was created, and partial volume correction was used (Su et al., 2013). For each ROI, a standard uptake ratio (SUVR) was acquired with the cerebellar gray matter serving as a reference region. Cortical regions affected by AD were used to obtain a summary value (mean cortical uptake ratio) of the partial-volume corrected SUVR (Mintun et al., 2006). Centiloids were used to synchronize measures from both PiB and AV45 tracers, with global amyloid deposition scaled from 0–100. Centiloid cutoffs for PiB and AV45 mean cortical SUVR were 16.4 and 20.6, respectively (Klunk et al., 2015). A value higher than these cutoffs is considered preclinical AD.\n\nParticipants answered questions related to the COVID-19 pandemic to capture temporally relevant personal and social characteristics. Demographic characteristics and six COVID-19-related questions were collected via self-report. Annual income was recoded to reflect the federal poverty level (Office of the Assistant Secretary for Plannning and Evaluation, 2020) and median household income for adults over 65 in the U.S. (Semega et al., 2020): <$20,000 (low income), $20,000–$50,000 (below U.S. median), and over $50,000 (above U.S. median). COVID-19-related questions included whether the participant had been diagnosed with COVID-19, had practiced social distancing, and four questions to assess financial strain related to the pandemic: “Since the start of the COVID-19 pandemic in the U.S., have you (1) involuntarily lost a job; (2) taken a cut in wage, salary, self-employed income, or similar; or (3) newly applied for public assistance or unemployment?” Participants also rated their financial stress on a scale from 0 (no financial stress) to 10 (severe financial stress).\n\nLoneliness was measured using version 1 of the 20-item UCLA Loneliness Scale (Russell, 1996). Participants reported how often they experienced subjective feelings of isolation, feeling left out, and lacking companionship on a scale from 1–5. Total scores on this measure range from 20–80, with lower scores indicating less loneliness. This instrument has high internal consistency (0.96) and a test-retest correlation of 0.73. In our sample, internal consistency was high, with Cronbach’s α = 0.95.\n\nAnxiety was measured using the Patient-Reported Outcomes Measurement Information System (PROMIS) Emotional Distress–Anxiety short form 4a (Pilkonis et al., 2011) and the Hospital Anxiety and Depression–Anxiety subscale (HADS-A; (Zigmond and Snaith, 1983). The PROMIS Emotional Distress–Anxiety short form 4a measures feelings of being fearful, overwhelmed, and uneasy over the past seven days on a scale from 1 (never) to 5 (always). Total raw scores are converted to uncorrected t-scores and range from 40.3–81.6, with a mean score of 50 and standard deviation of 10. Higher scores indicate greater anxiety. This measure has demonstrated content validity across diverse populations (Pilkonis et al., 2011). The HADS-A measures how often participants have felt tense, frightened, and restless over the past week on a scale from 1 (not at all or only occasionally) to 5 (most of the time or very often; (Zigmond and Snaith, 1983). Total scores range from 0–21, with scores greater than 10 indicating clinically meaningful anxiety. This measure has excellent reliability (intraclass correlation coefficient = 0.92) and good internal consistency (Cronbach’s α = 0.87; (Djukanovic et al., 2017). In our sample, internal consistency measures for the PROMIS and HADS-A were Cronbach’s α = 0.79 and 0.76, respectively.\n\nFeelings of depression were assessed using the Patient Health Questionnaire 9 (PHQ-9; (Kroenke et al., 2001). The PHQ-9 includes all nine Diagnostic and Statistical Manual Fourth Edition (American Psychiatric Association, 2000) diagnostic criteria for major depressive disorder and measures how often a participant has felt bothered by symptoms such as little interest in doing things, trouble concentrating, and feeling bad about oneself in the past two weeks on a scale from 0 (not at all) to 3 (nearly every day). Scores on the PHQ-9 range from 0–27, with higher scores indicating more severe depression (Kroenke et al., 2001). The PHQ-9 has internal reliability (Cronbach’s α = 0.86–0.89), excellent test-retest reliability, and established criterion and construct validity (Kroenke et al., 2001). Internal reliability in our sample was Cronbach’s α = 0.75.\n\nData were analyzed using SPSS 27.0 (IBM Corp., Armonk, NY; IBM Corp., 2017). Descriptive analyses were performed to examine demographics, COVID-19-related questions, and psychosocial characteristics (loneliness, anxiety, and depression). As amyloid accumulation distribution was skewed, nonparametric Spearman correlations were used to examine the association between a continuous measure of amyloid accumulation and psychosocial characteristics of loneliness, anxiety, and depression.\n\n\nResults\n\nOf the 169 participants enrolled in a longitudinal cohort study at Knight ADRC, 150 enrolled in this study, and 108 with amyloid PET data available were included in the analysis. Participants were 75.0 ± 5.5 years old, 53.7% female, and 89.8% non-Hispanic White; 71.3% lived with others, 60.2% were married or partnered, and participants had completed an average of 16.7 years of education. Only one individual in the study reported contracting COVID-19; over 96% of participants reported practicing social distancing to minimize potential exposure to COVID-19. The majority of participants reported annual incomes higher than the U.S. median and had not involuntarily lost a job, sustained a cut in income, newly applied for public assistance, or experienced a high level of financial stress. Mean amyloid accumulation centiloid was 22.2 ± 31.9 (median = 9.2). Table 1 provides a summary of demographic characteristics and COVID-19-related questions.\n\na Amount of financial stress range 0–10 (higher scores indicate greater financial stress).\n\nb UCLA Loneliness Scale range 20–80 (higher scores indicate greater loneliness).\n\nc Patient-Reported Outcomes Measurement Information System Emotional Distress–Anxiety short form 4a range 40.3–81.6 (higher scores indicate greater anxiety).\n\nd Hospital Anxiety and Depression Scale–Anxiety range 0–21 (scores >10 indicate clinically meaningful anxiety).\n\ne Patient Health Questionnaire 9 range 0–27 (higher scores indicate more severe depression).\n\nTable 2 displays results of the correlation analysis. Amyloid accumulation was not associated with anxiety or depression. Higher amyloid accumulation was significantly correlated with greater loneliness (rho = .226, P = .019); however, this association was considered weak. This relationship is depicted in Figure 1.\n\n* Indicates p < 0.05.\n\n*** Indicates p < 0.001.\n\n\nDiscussion\n\nThis study is one of the first to examine loneliness, anxiety, and depression in CN older adults with and without preclinical AD during the early period of the COVID-19 pandemic. We found an association between higher amyloid accumulation and greater loneliness. Although this association was weak, it suggests that older adults with preclinical AD may experience greater loneliness than older adults without preclinical AD. This finding is supported by previous studies showing that loneliness is associated with Alzheimer pathology (d’Oleire Uquillas et al., 2018; Donovan et al., 2016) and may contribute to AD-related cognitive decline (Wilson et al., 2007) among CN older adults. Anxiety and depression were not significantly associated with amyloid burden; it is possible that the short timeframe assessed by these questionnaires (past week, past two weeks) did not capture feelings of anxiety and depression that may have occurred at the beginning of the COVID-19 pandemic, prior to the survey. In contrast, the UCLA Loneliness Scale does not include a specific timeframe and may have captured feelings experienced over the several months prior to survey.\n\nKnowledge of these emotional and psychological symptoms is crucial as the clinical symptomology for preclinical AD, aside from neural pathology, is poorly understood. Furthermore, the physical and psychosocial effects of the COVID-19 pandemic will persist far beyond the conclusion of the pandemic. Healthcare professionals should be aware of these symptoms and anticipate care for older adults. Thus, it is imperative to continue examining psychosocial symptoms and the impact of prolonged loneliness on older adults with preclinical AD.\n\nA strength of this study is that data were collected within a short timeframe during the COVID-19 pandemic. In addition, nearly all of our participants reported practicing social distancing, thus implying that they were operating in similar restricted social behaviors.\n\nOne limitation of this study is the lack of long-term follow-up, which could provide information about participants’ feelings of loneliness, anxiety, and depression throughout the pandemic, as well as progression of preclinical AD. Another limitation is the lack of diversity in income, ethnicity, and race in our sample. While the demographic characteristics of our participants mimic those of the Knight ADRC, our recruitment source, future research is needed with a wider distribution of income and more diverse sample to examine the generalizability of our findings.\n\n\nConclusions\n\nDespite these limitations, this study points to the importance of psychosocial characteristics in the preclinical phase of AD. Healthcare professionals should monitor for characteristics of loneliness in older adults and provide treatments as needed to mitigate the effects. Future research should be conducted with a larger, more diverse sample over time to examine the relationship between these psychosocial symptoms, accelerated cognitive decline, and AD progression in older adults.\n\n\nData availability\n\nThe full dataset supporting the findings of this study could not be made publicly available as some of the data was collected by the Knight Alzheimer Disease Research Center (ADRC) who requires a separate request. This can be made through ADRC’s website: https://knightadrc.wustl.edu/data-request-form/. In their request, applicants will need to provide the following information: purpose, background and preliminary data, methods, inclusion and exclusion criteria, analytic plan, sample size justification and a list of data variables. A list of variables available for request from the Knight ADRC may be found on their website: https://knightadrc.wustl.edu/professionals-clinicians/request-center-resources/guidelines-data-available/.\n\nDigital Commons@Becker: Higher amyloid correlates to greater loneliness during the COVID-19 pandemic. https://doi.org/10.48765/m113-0447 (Kehrer-Dunlap et al., 2022).\n\nThis project contains the dataset of psychosocial measures.\n\nDigital Commons@Becker: Higher amyloid correlates to greater loneliness during the COVID-19 pandemic. https://doi.org/10.48765/m113-0447 (Kehrer-Dunlap et al., 2022).\n\nThis project contains the following extended data:\n\n- Falls&AD_Amyloid_Loneliness_Data_Dictionary-akd.csv (data key)\n\n- COVIDQuestions.pdf (COVID items questionnaire)\n\n- COVIDfinancialimpact.pdf (financial impact questionnaire)\n\n- README.txt\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe gratefully acknowledge the Knight Alzheimer Disease Research Center staff and our participants who made this study possible. We would also like to acknowledge Megen Devine from the Washington University in St. Louis Program in Occupational Therapy for her assistance with copy editing and formatting this manuscript.\n\n\nReferences\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders (4th Ed., Text Revision).2000.\n\nBabulal GM, Roe CM, Stout SH, et al.: Depression Is Associated with Tau and Not Amyloid Positron Emission Tomography in Cognitively Normal Adults. J. Alzheimers Dis. 2020; 74(4): 1045–1055. Publisher Full Text\n\nBollinger RM, Keleman A, Thompson R, et al.: Falls: A Marker of Preclinical Alzheimer Disease: A Cohort Study Protocol. BMJ Open. 2021; 11(9): e050820–e050820. Publisher Full Text\n\nd’Oleire Uquillas F, Jacobs HIL, Biddle KD, et al.: Regional Tau Pathology and Loneliness in Cognitively Normal Older Adults. Transl. Psychiatry. 2018; 8(1): 282. 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Publisher Full Text\n\nOffice of the Assistant Secretary for Plannning and Evaluation: U.S. Federal Poverty Guidelines Used to Determine Financial Eligibility for Certain Federal Programs. 2020.Reference Source\n\nPietrzak RH, Lim YY, Neumeister A, et al.: Amyloid-Β, Anxiety, and Cognitive Decline in Preclinical Alzheimer Disease: A Multicenter, Prospective Cohort Study. JAMA Psychiat. 2015; 72(3): 284–291. Publisher Full Text\n\nPilkonis PA, Choi SW, Reise SP, et al.: Item Banks for Measuring Emotional Distress from the Patient-Reported Outcomes Measurement Information System (PROMIS®): Depression, Anxiety, and Anger. Assessment. 2011; 18(3): 263–283. Publisher Full Text\n\nPrice JL, Morris JC: Tangles and Plaques in Nondemented Aging and “Preclinical” Alzheimer’s Disease. Ann. Neurol. 1999; 45(3): 358–368. Publisher Full Text\n\nReppas-Rindlisbacher C, Finlay JM, Mahar AL, et al.: Worries, Attitudes, and Mental Health of Older Adults During the COVID-19 Pandemic: Canadian and U.S. Perspectives. J. Am. Geriatr. Soc. 2021; 69(5): 1147–1154. Publisher Full Text\n\nRussell DW: UCLA Loneliness Scale (Version 3): Reliability, Validity, and Factor Structure. J. Pers. Assess. 1996; 66(1): 20–40. Publisher Full Text\n\nSemega J, Kollar M, Shrider EA, et al.: Income and Poverty in the United States: 2019. U.S. Department of Commerce;2020.Reference Source\n\nSu Y, Blazey TM, Snyder AZ, et al.: Quantitative Amyloid Imaging Using Image-Derived Arterial Input Function. PLOS One. 2015; 10(4): e0122920. Publisher Full Text\n\nSu Y, D’Angelo GM, Vlassenko AG, et al.: Quantitative Analysis of Pib-PET with Freesurfer ROIs. PLOS One. 2013; 8(11): e73377. Publisher Full Text\n\nSundström A, Adolfsson AN, Nordin M, et al.: Loneliness Increases the Risk of All-Cause Dementia and Alzheimer’s Disease. J. Gerontol. B Psychol. Sci. Soc. Sci. 2020; 75(5): 919–926. Publisher Full Text\n\nSutin AR, Stephan Y, Luchetti M, et al.: Loneliness and Risk of Dementia. J. Gerontol. B Psychol. Sci. Soc. Sci. 2018; 75(7): 1414–1422. Publisher Full Text\n\nWilson RS, Krueger KR, Arnold SE, et al.: Loneliness and Risk of Alzheimer Disease. Arch. Gen. Psychiatry. 2007; 64(2): 234–240. Publisher Full Text\n\nWu KY, Hsiao IT, Chen CS, et al.: Increased Brain Amyloid Deposition in Patients with a Lifetime History of Major Depression: Evidenced on 18f-Florbetapir (AV-45/Amyvid) Positron Emission Tomography. Eur. J. Nucl. Med. Mol. Imaging. 2014; 41(4): 714–722. Publisher Full Text\n\nZigmond AS, Snaith RP: The Hospital Anxiety and Depression Scale. Acta Psychiatr. Scand. 1983; 67(6): 361–370. Publisher Full Text" }	[ { "id": "155218", "date": "05 Dec 2022", "name": "Hui-Fen Mao", "expertise": [ "Reviewer Expertise Occupational Therapy", "Long-tern Care", "Dementia Care", "Assistive technology & Applications" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, which reports the relationship between psychosocial characteristics and amyloid accumulation in cognitively normal older adults with and without preclinical AD during the early period of the COVID-19 pandemic. This topic raises the issue of how the emotional condition of the elderly might accelerate the risk of AD, indicated by a higher amyloid burden.\nThis is a preliminary study with a cross-sectional design without longitudinal follow-up to examine how psychosocial characteristics affect AD occurrences or cognitive deterioration. The statistical analysis used only univariate analysis without considering confounding factors, nor other possible moderating factors of these psychosocial characteristics and amyloid accumulation (i.e., gender, age, living alone or not, premorbid cerebrovascular disease risk). Although a weak correlation between amyloid accumulation and loneliness was found, the results should be interpreted with caution and should be considered to have limited impact on the overall body of research.\nAs the authors mentioned, the lack of diversity in income, ethnicity, and race in the study sample was one limitation: the individuals included, who tended to have higher social and economic status, might not be highly impacted by the COVID-19 pandemic (i.e., in terms of the amount of financial stress) and thus might bias the results.\nRevisions are needed to make findings and implications clearer to readers.\nSpecific Points:\nTopic: The authors may consider adding the characteristic of the participants (cognitively normal older adults) in the title.\nIntroduction:\nDue to the intention and the data collection during the initial phase of the COVID-19 pandemic, the loneliness, anxiety, and depression assessed in this study more accurately resemble the “mood state” while facing social isolation rather than a “trait” (personal characteristic). Therefore, I would suggest using “mood status” instead of “psychosocial characteristics”.\n\n1st Paragraph, second sentence: “Psychosocial characteristics such as loneliness, anxiety, and depression are associated with symptomatic AD (El Haj et al., 2016; Pietrzak et al., 2015), but it is unclear whether these emerge during the preclinical phase of AD.” What does “unclear” mean, here? Please specify, as increasing studies indicate that clinical symptoms, such as loneliness, anxiety, and depression, may already be prevalent in preclinical AD (Lavretsky et al., 2009; Pietrzak et al., 2014). Why were the authors interested in collecting data during the COVID-19 pandemic? Please add some explanations or evidence that the pandemic might aggravate mood problems, specifically for the elderly. The following sentence is unclear: “The purpose of this study was to examine psychosocial characteristics (loneliness, anxiety, and depression) in CN older adults using a continuous measure of cortical amyloid burden during the COVID-19 pandemic.” The authors may rephrase to clarify that the purpose of this study was to examine the relationship between psychosocial characteristics and amyloid accumulation in cognitively normal older adults.\nMethods\nThe authors collected COVID-19-related questions, information about annual income, and financial stress. How were these data used to analyze and interpret the results? In order to better understand the correlation between mood and amyloid accumulation, it would be more effective to use multiple regression analysis to control the other possible confounding factors (i.e., age, economic condition, financial stress, living alone or not).\nResults\nHow many participants were considered to have preclinical AD according to the Centiloid cutoffs for PiB and AV45 mean cortical SUVR? The authors may add the number of participants and their average amyloid accumulation with and without preclinical AD in Table 1 or describe it in the results section. Figure 1. Please annotate the two vertical dotted lines. Are the terms “amyloid accumulation”(the caption of Table 1) and “global amyloid deposition” ( the caption of Figure 1) the same? If so, please make the terminology consistent.\nDiscussions\n\nThe authors may point out that this study’s cross-sectional design lacks consideration of the confounding factors or moderating factors of these psychosocial characteristics and amyloid accumulation; the findings should be explained more conservatively.\n\nTable 2 shows a moderately significant correlation between depression, loneliness, and anxiety. Additional discussions regarding the relationship between them could be included by comparing the results with the relevant literature. For example, loneliness has been considered one significant variable in people with depression (Erzen & Çikrikci, 2018).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "10905", "date": "24 Jan 2024", "name": "Susan Stark", "role": "Author Response", "response": "We thank the reviewer for their time and for providing detailed points of feedback. Item-by-item responses to each comment are provided below. Introduction: Due to the intention and the data collection during the initial phase of the COVID-19 pandemic, the loneliness, anxiety, and depression assessed in this study more accurately resemble the “mood state” while facing social isolation rather than a “trait” (personal characteristic). Therefore, I would suggest using “mood status” instead of “psychosocial characteristics”. RESPONSE: We have re-examined the literature to identify the most appropriate terminology for describing the loneliness, anxiety, and depression experienced by our sample. Upon examining the term “mood states,” we determined that this term can include anxiety and depression but traditionally excludes loneliness (McNair, 1992). To address this feedback, we have removed all mention of “psychosocial characteristics” from the manuscript and instead refer to each term (i.e., loneliness, anxiety, and depression). Reference: McNair, D. M. (1992). Profile of mood states. Educational and Industrial Testing Service 1st Paragraph, second sentence: “Psychosocial characteristics such as loneliness, anxiety, and depression are associated with symptomatic AD (El Haj et al., 2016; Pietrzak et al., 2015), but it is unclear whether these emerge during the preclinical phase of AD.” What does “unclear” mean, here? Please specify, as increasing studies indicate that clinical symptoms, such as loneliness, anxiety, and depression, may already be prevalent in preclinical AD (Lavretsky et al., 2009; Pietrzak et al., 2014). RESPONSE: We have made the following changes to the introduction to provide greater clarity in the context of previous evidence. In addition, we have included changes to the introduction to align with additional requested changes from both reviewers, including a more focused description of the context of loneliness within CN older adults: “The impacts of loneliness among older adults with AD have been associated with AD symptoms, including hallucinations and an increased rate of cognitive decline (El Haj et al., 2016; Wilson et al., 2007). Emerging research suggests that subjective experiences of loneliness may precede clinical symptoms of AD during the preclinical phase of AD (Donovan et al., 2016; d’Oleire Uquillas et al., 2018).” “Individuals with preclinical AD are cognitively normal (CN) and show no hallmark symptoms of AD, such as memory loss or hallucinations, for years or even decades (Price & Morris, 1991). However, these individuals present with Alzheimer pathology, including abnormal amyloid accumulation, that can be reliably characterized using neuroimaging, cerebrospinal fluid, and blood plasma (Klunk et al., 2004; Schindler et al., 2019; Schindler et al., 2018; Wong et al., 2010). Evidence indicates that emotional and psychological distress may accelerate the onset of clinical symptoms of AD for older adults with preclinical AD (Dubois et al., 2016; Wilson et al., 2007). It is important to understand how clinical symptoms, such as loneliness, may manifest during the preclinical phase of AD in order to improve our understanding of disease progression.” “The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to prolonged social isolation and an increase in the number of older adults reporting loneliness (Reppas-Rindlisbacher et al., 2021). We sought to characterize subjective experiences of loneliness among our older adult participants during the early months of the pandemic, particularly among those at risk for progressing from preclinical to symptomatic AD. To date, the relationship between loneliness and amyloid accumulation in CN older adults during the COVID-19 pandemic has not been investigated. As it is important to understand the role of loneliness as a potential risk factor that may exacerbate the progression of AD, the purpose of this study was to examine the relationship between amyloid accumulation and subjective experiences of loneliness among CN older adults during the COVID-19 pandemic.” Why were the authors interested in collecting data during the COVID-19 pandemic? Please add some explanations or evidence that the pandemic might aggravate mood problems, specifically for the elderly. RESPONSE: The following has been added to the introduction to address this point: “The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to prolonged social isolation and an increase in the number of older adults reporting loneliness (Reppas-Rindlisbacher et al., 2021). We sought to characterize subjective experiences of loneliness among our older adult participants during the early months of the pandemic, particularly among those at risk for progressing from preclinical to symptomatic AD. To date, the relationship between loneliness and amyloid accumulation in CN older adults during the COVID-19 pandemic has not been investigated. As it is important to understand the role of loneliness as a potential risk factor that may exacerbate the progression of AD, the purpose of this study was to examine the relationship between amyloid accumulation and subjective experiences of loneliness among CN older adults during the COVID-19 pandemic.” The following sentence is unclear: “The purpose of this study was to examine psychosocial characteristics (loneliness, anxiety, and depression) in CN older adults using a continuous measure of cortical amyloid burden during the COVID-19 pandemic.” The authors may rephrase to clarify that the purpose of this study was to examine the relationship between psychosocial characteristics and amyloid accumulation in cognitively normal older adults. RESPONSE: We have rephrased this statement accordingly in addition to tying it in to the previous suggestion: “As it is important to understand the role of loneliness as a potential risk factor that may exacerbate the progression of AD, the purpose of this study was to examine the relationship between amyloid accumulation and subjective experiences of loneliness among CN older adults during the COVID-19 pandemic.” Methods: The authors collected COVID-19-related questions, information about annual income, and financial stress. How were these data used to analyze and interpret the results? RESPONSE: We initially examined these variables to describe the characteristics of our sample. In response to the below comment (see “Methods,” comment #2), we included the practice of social distancing and level of financial stress as covariates in the multiple linear regression to control for COVID-19-related covariates that may have impacted subjective experiences of loneliness. In order to better understand the correlation between mood and amyloid accumulation, it would be more effective to use multiple regression analysis to control the other possible confounding factors (i.e., age, economic condition, financial stress, living alone or not). RESPONSE: We thank the reviewer for this suggestion and agree that this analysis would be more effective for examining the relationship between loneliness and amyloid accumulation. We have included the results of a multiple linear regression examining the relationship between loneliness and amyloid accumulation in Table 2 in place of the previous Spearman correlation table. In addition, we have detailed the methods and findings from this analysis in the text: “Multiple linear regression was used to estimate the associations between loneliness and amyloid accumulation. We controlled for relevant covariates identified through domain knowledge and clinical interest, including anxiety, depression, age, gender, level of financial stress, living situation (alone versus with others), and practice of social distancing, which were added to the model through forced entry. Because the cohort study included two measures of anxiety (PROMIS and HADS-A), we included only the PROMIS Anxiety measure for anxiety in this model given its higher precision in scoring compared to the HADS-A. Ninety-five percent confidence intervals (CIs) for regression coefficients were calculated for all independent variables.” “Table 2 displays results of the multiple linear regression analysis. The independent variables significantly predicted UCLA Loneliness scores (F(8,99) = 8.69, p < 0.001), and the model explained 41.2% of the variance (R2 = 0.412) in UCLA Loneliness Scale scores. After controlling for anxiety, depression, age, gender, level of financial stress, living situation, and social distancing, we observed a significant positive association between loneliness and amyloid accumulation (β = 0.064, SE = 0.027, 95% CI = [0.011, 0.118], p = 0.018). Figure 1 displays the linear association between loneliness and amyloid accumulation. Significant positive associations were also found between loneliness and depression (β = 1.605, SE = 0.344, 95% CI = [0.923, 2.288], p < 0.001) and level of financial stress (β = 1.157, SE = 0.474, 95% CI = [0.216, 2.097], p = 0.016).” Results: How many participants were considered to have preclinical AD according to the Centiloid cutoffs for PiB and AV45 mean cortical SUVR? The authors may add the number of participants and their average amyloid accumulation with and without preclinical AD in Table 1 or describe it in the results section. RESPONSE: We have added this information to the body of the text: “Mean amyloid accumulation was 22.2 ± 31.9 (median = 9.2), and 33 participants (30.6%) were determined to have preclinical AD based on centiloid cutoffs for PiB and AV45; average amyloid accumulation was 4.9 ± 6.3 for participants without preclinical AD and 61.4 ± 32.1 for participants with preclinical AD.” Figure 1. Please annotate the two vertical dotted lines. RESPONSE: We have annotated these lines using color and text in the figure, as well as added text describing these lines in the legend of Figure 1: “Note: Dashed blue line indicates cutoff for preclinical AD positivity using PiB PET tracer (16.4). Dashed purple line indicates cutoff for preclinical AD positivity using AV45 PET tracer (20.6).” Are the terms “amyloid accumulation”(the caption of Table 1) and “global amyloid deposition” ( the caption of Figure 1) the same? If so, please make the terminology consistent. RESPONSE: These terms refer to the same thing; “global amyloid deposition” has been removed, and “amyloid accumulation” is now used consistently throughout the manuscript. Discussions: The authors may point out that this study’s cross-sectional design lacks consideration of the confounding factors or moderating factors of these psychosocial characteristics and amyloid accumulation; the findings should be explained more conservatively. RESPONSE: We have added the following to address this comment: “Another limitation is the cross-sectional design lacking long-term follow-up, which could provide information about participants’ subjective experiences of loneliness throughout the pandemic, as well as progression of preclinical AD. In addition, the UCLA Loneliness Scale does not include a specific timeframe and may have captured feelings experienced over the several months prior to survey. Additional research is needed to understand how at-risk groups, including individuals with preclinical AD, may be impacted by prolonged loneliness throughout the COVID-19 pandemic. Table 2 shows a moderately significant correlation between depression, loneliness, and anxiety. Additional discussions regarding the relationship between them could be included by comparing the results with the relevant literature. For example, loneliness has been considered one significant variable in people with depression (Erzen & Çikrikci, 2018). RESPONSE: To address the reviewer’s previous comment regarding a multiple linear regression rather than Spearman correlation, Table 2 now reflects the results of the updated analysis. As we detected a significant association between loneliness and depression in addition to amyloid accumulation, we included discussion of relevant literature to offer a possible explanation for this finding: “A significant positive association was also detected between loneliness and depression; this finding is consistent with previous findings and may be attributed to shared symptomology (e.g., helplessness, unhappiness) between loneliness and depression (Mushtaq et al., 2014).”" } ] }, { "id": "207323", "date": "20 Sep 2023", "name": "Paula V Nersesian", "expertise": [ "Reviewer Expertise Loneliness" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 summarizes a secondary analysis of an investigation of psychosocial factors and their association with biomarkers of preclinical Alzheimer disease. The significant finding was an association of higher amyloid and a measure of loneliness.\nThis is a well-written already published, but not yet peer-reviewed article. It lacks basic definitions of the psychosocial factors that it is exploring and in particular, the factor that was shown to have a significant association, loneliness. Although loneliness is the key factor showing a significant association with the amyloid biomarker variable, the authors fail to report the prevalence of loneliness among the study participants. Likewise, the authors fail to discuss the fact that a majority of the study participants (77%) “lives with others” in the discussion section. Is the significant finding therefore being drawn from a minority of study participants? The reader cannot know since the prevalence of loneliness is not reported.\nThe conclusion section should become part of an expanded discussion section. It would be helpful if more concrete information were provided to the reader regarding the signs of loneliness that healthcare professionals should monitor for. Suggesting providing “treatments as needed to mitigate effects” is not as helpful as providing concrete suggestions. You may wish to consult this recent publication for ideas as to what to suggest: https://www.hhs.gov/sites/default/files/surgeon-general-social-connection-advisory.pdf\nWe do know that it is a biased sample lacking in diversity and the authors duly identify that limitation. The authors report that the data are not publicly available, but special arrangements can be made to access it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10906", "date": "24 Jan 2024", "name": "Susan Stark", "role": "Author Response", "response": "We thank the reviewer for their time and for providing detailed points of feedback. Point-by-point responses to reviewer comments are provided below. This article summarizes a secondary analysis of an investigation of psychosocial factors and their association with biomarkers of preclinical Alzheimer disease. The significant finding was an association of higher amyloid and a measure of loneliness. This is a well-written already published, but not yet peer-reviewed article. RESPONSE: We thank the reviewer for their time and for providing us with several suggestions to increase the clarity of this manuscript. It lacks basic definitions of the psychosocial factors that it is exploring and in particular, the factor that was shown to have a significant association, loneliness. RESPONSE: We have added operational definitions of loneliness, anxiety, and depression in each of their respective methods paragraphs: “Loneliness was defined as negative thoughts and feelings of being isolated and disconnected from others (Russell, Peplau, & Ferguson, 1978).” “Anxiety was defined as feeling nervous, worried, or frightened (Marks & Lader, 1973).” “Depression was defined as feelings of sadness, hopelessness, or a lack of interest or pleasure in completing daily activities (Herrman et al., 2022).” Although loneliness is the key factor showing a significant association with the amyloid biomarker variable, the authors fail to report the prevalence of loneliness among the study participants. RESPONSE: Established cutoffs for what is considered “lonely” are not available for the 20-item UCLA Loneliness Scale (Russell, Peplau, & Ferguson, 1978). Therefore, we are not able to report the prevalence of “lonely” versus “not lonely.” Possible scores on this measure range from 20-80, with higher scores indicating greater loneliness. We have added the following language to address this comment: “Total scores on this measure range from 20–80, with lower scores indicating less loneliness. This measure does not have established cutoff scores to indicate levels of loneliness, and it is recommended to examine the full range of scores when using this measure.” In addition, we have reported the prevalence of UCLA Loneliness Scale scores that exceed the average in our sample: “Average scores on the UCLA Loneliness Scale were 31.6 ± 10.8, and 46 (42.6%) of participants had total scores that exceeded the sample average.” Reference: Russell, D., Peplau, L. A., & Ferguson, M. L. (1978). Developing a measure of loneliness. Journal of Personality Assessment, 42(3), 290-294. Likewise, the authors fail to discuss the fact that a majority of the study participants (77%) “lives with others” in the discussion section. Is the significant finding therefore being drawn from a minority of study participants? The reader cannot know since the prevalence of loneliness is not reported. RESPONSE: Based on our updated analysis, we examined the association between loneliness and amyloid accumulation using multiple linear regression while controlling for relevant covariates, including living situation (i.e., living with others versus alone) as well as practice of social distancing (social isolation). As reported above (see comment #3), there are not established cutoff scores indicating presence or absence of loneliness for the UCLA 20-Item Loneliness Scale. However, after controlling for other covariates in our multiple linear regression, a significant association was detected between loneliness and amyloid accumulation, which warrants further investigation in future studies. The conclusion section should become part of an expanded discussion section. It would be helpful if more concrete information were provided to the reader regarding the signs of loneliness that healthcare professionals should monitor for. Suggesting providing “treatments as needed to mitigate effects” is not as helpful as providing concrete suggestions. You may wish to consult this recent publication for ideas as to what to suggest: https://www.hhs.gov/sites/default/files/surgeon-general-social-connection-advisory.pdf RESPONSE: We have added the following in the discussion section to provide more concrete recommendations for addressing concerns with loneliness among older adults: “Assessment of characteristics that impact loneliness, including satisfaction with social relationships and opportunities for social participation, should be integrated in routine clinical evaluations of older adults (National Academy of Sciences, Engineering, and Medicine, 2020). Healthcare professionals working with older adults experiencing loneliness should identify appropriate treatments, such as connecting with community resources to offer social support or removing barriers that hinder opportunities for social connectedness (Gardiner et al., 2018; Perissinotto et al., 2019). They should also adopt prevention approaches to addressing loneliness, including fostering conversations with older adults about maintaining healthy social relationships and including assessments of social activity in annual screenings to monitor social health (Perissinotto et al., 2019).” In addition, we have amended the conclusion section to reflect the above added language: “Despite these limitations, this study found an association between loneliness and amyloid accumulation and points to the importance of examining subjective experiences of loneliness during the preclinical phase of AD. Future research should be conducted with a larger, more diverse sample over time to examine the relationship between loneliness and amyloid accumulation in older adults. Healthcare professionals should monitor for loneliness in older adults and recommend treatments to reduce loneliness and prevent future loneliness.” We do know that it is a biased sample lacking in diversity and the authors duly identify that limitation. The authors report that the data are not publicly available, but special arrangements can be made to access it." } ] } ]	1	https://f1000research.com/articles/11-1134
https://f1000research.com/articles/11-1122/v1	29 Sep 22	{ "type": "Software Tool Article", "title": "Librarian: A quality control tool to analyse sequencing library compositions", "authors": [ "Kartavya Vashishtha", "Caroline Gaud", "Simon Andrews", "Christel Krueger", "Kartavya Vashishtha", "Caroline Gaud", "Simon Andrews" ], "abstract": "Background: Robust analysis of DNA sequencing data needs to include a set of quality control steps to ensure that technical bias is kept to a minimum. A metric easily obtained is the frequency of each of the nucleobases for each position across all sequencing reads. Here, we explore the differences in nucleobase compositions of various library types produced by standard experimental methodologies. Methods: We obtained the compositions of nearly 3000 publicly available datasets and subjected them to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction for a two-dimensional representation of their composition characteristics.\n\nResults: We find that most library types result in a specific composition profile. We use this to give an estimate of how strongly the composition of a test library resembles the profiles of previously published libraries, and how likely the test sample is to be of a particular type. We introduce Librarian, a user-friendly web application and command line tool which enables checking base compositions of test libraries against known library types.\n\nConclusions: Library preparation methods strongly influence the per position nucleobase content. By comparing test libraries to a database of previously published library types we can make predictions regarding the library preparation method. Librarian is a user-friendly tool to access this information for quality assurance purposes as discrepancies can flag potential irregularities very early on.", "keywords": [ "high throughput sequencing", "quality control", "sequencing libraries", "FastQ", "base composition" ], "content": "Introduction\n\nHigh-throughput sequencing is now a routine technology for the analysis of biological phenomena. A multitude of methods have been developed that obtain genome-wide information on the transcriptome, protein-DNA binding, chromatin compaction, chromosomal conformation and DNA modifications to name but a few. While these approaches address different biological questions and employ various sample preparation techniques, the workflow mostly converges at a stage where adapter flanked short DNA sequences, so called libraries, are subjected to Illumina sequencing.1 The resulting raw data should pass a number of quality control (QC) steps before analysis is performed.2–4 These can be roughly split into two categories, pre-mapping QC, for example monitoring of base call quality scores, and post-mapping QC, for example overall enrichment scores in ChIP-seq data. For example, raw sequencing data can be queried for adapter contamination and GC bias3–5 to gauge the quality of the library preparation, or using multi-species alignments to confirm the expected species.5–7 Early detection of technical biases or problems during sample preparation is important for rigorous data analysis and conservation of resources.\n\nFastQ is a file format commonly used for storing unmapped sequencing data. One of the metrics that can be obtained from such files is the summarised base composition across the sequencing reads. For each position in the read the respective content of the bases adenine (A), thymine (T), guanosine (G), and cytosine (C) can be determined. For a theoretic random genomic library the expectation would be four horizontal lines reflecting the overall base composition of the genome. Since the GC content of DNA varies according to species8 sequencing libraries will show different composition profiles depending on which organism was sequenced. Less intuitively, libraries produced by different experimental protocols may show vastly different sequence compositions (Figure 1). A prominent example is Bisulfite-seq used for DNA methylation analysis9: during sample preparation unmethylated genomic Cs are converted to Ts resulting in libraries with a strikingly low C content. Another instructive example is ATAC-seq.10 Here, the fragments to be sequenced are produced by a transposase which shows target sequence preference; ATAC-seq libraries are therefore compositionally biased at the start of the read. Expanding on these observations, we asked if base compositions could be used to distinguish different library types more generally.\n\nBase content across the first 50 positions of the sequencing reads was averaged for 54 ChIA-PET, 436 Bisulfite-seq, 416 ATAC-seq and 449 ChIP-seq libraries from mouse and human. Percentages are plotted for each of the four bases.\n\nThe ‘Per base sequence content’ module of the widely used QC tool FastQC4 provides composition information for individual samples, but makes no comparison. Any judgement of whether a particular composition profile is expected for the analysed sample type would require highly specialised niche knowledge which cannot generally be expected of individual researchers. Using the tool MultiQC,11 researchers can collate composition information from multiple individual FastQC reports and visualise them together. This is useful to compare the base compositions of different samples in an experiment and can flag up outliers, but it does not allow for placing samples in the general base composition landscape.\n\nHere, we describe how sample preparation protocols for sequencing libraries result in characteristic composition signatures, and introduce a new quality control tool to check any sequence library against the expected composition of its preparation method.\n\n\nMethods\n\nTo get an overview of expected library compositions we queried the open Gene Expression Omnibus (GEO) database12 for high throughput sequencing datasets from mouse and human samples for the years 2018, 2019 and 2020.13 Mice and humans are among the most studied species and are similar in overall GC content (42% and 41%, respectively) making them a good choice to look for compositional differences of different library types. Search results were filtered to exclude library type ‘OTHER’ as well as under-represented types (fewer than 25 samples), and over-represented library types (e. g. ribonucleic acid (RNA)-seq) were capped at 500 samples. Figure 2A shows the number of samples per library type for which per position base compositions could be retrieved.\n\nA) Number of samples per library type included in the analysis. B) UMAP representation of library compositions (reference map). C) Tile based probability map for each library type. Colour represents the percentage of a particular library type per tile. D) Heatmap illustrating the specificity of each library type for tiles of the reference map. All samples were assigned to a reference map tile and colour represents the average percentage of each library type for these tiles. E) Librarian tile probability output: Percent of each library found in the reference map tile associated with the test library.\n\nWe then determined how frequently the bases A, T, G and C were found at the first 50 positions in the read (read1 for paired-end data). To visualise sample groupings, the resulting composition data was subjected to Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction14 (using the umap R package with parameters n_neighbors = 15, min_dist = 8) and a two-dimensional representation is shown in Figure 2B (‘reference map’). Interestingly, visually distinct clusters are formed largely along library types, with some library types having very specific base compositions (e.g. Bisulfite-seq, ChIA-PET, ATAC-seq) while others are largely overlapping (e.g. RNA-seq and ssRNA-seq).\n\nTo explore how well represented each library type was in each region of the reference map, we split the map into tiles and calculated the percentage of each library type per tile normalised to the total number of samples. Figure 2C shows that, indeed, some tiles are exclusively occupied by a certain library type while others are less specific. To get an overall measure for how well library types could be distinguished, we first annotated each of the samples included in the analysis with its reference map tile. We then averaged the percentages of the library types represented by the tile across all samples of a particular library type to produce the confusion matrix visualised in Figure 2D. While most tiles are very indicative of a certain library type, we also find tiles which are co-occupied by more than one type, for example ncRNA-seq and miRNA-seq. Base composition similarity of certain library types comes as no surprise as the probed material and involved preparation methods can be largely overlapping.\n\nHaving concluded that different library types result in largely distinct base compositions along the sequencing reads, we propose to include checking library compositions as a pre-mapping quality control step in the analysis of high throughput sequencing data. This will help flag technical irregularities during sample preparation or potential sample swaps early on and avoid bias during downstream analyses. To make this generally accessible, we developed Librarian15 which allows the user to relate the base composition of any newly sequenced library to other samples in the database.\n\nLibrarian will first extract base composition of the first 50 positions of randomly selected 100,000 reads from a supplied FastQ file. It will then project the compositions of the test library onto the manifold created by all libraries in the database as described above, thereby assigning it to a tile on the compositions map.\n\nResults are presented graphically: The location of the test sample is indicated both on the compositions map and on the plots displaying the probability of each library type per tile. Moreover, the percentages for each library type for the tile assigned to the test library are plotted as a heatmap. This lets the user easily gauge how similar the test library is to a collection of published library types.\n\nLibrarian is available as a web app and a command line tool. In the web app, one or more FastQ files are selected and processed locally to produce the library compositions. Client-side processing avoids upload of large FastQ files and potentially sensitive data. The resulting library composition is compared to the database on the server, and the graphical output can be viewed and downloaded in svg format from the web page.\n\nLibrarian can also be run as a command line client application on Linux. Download and install instructions are provided via GitHub (see Software availability). Multiple FastQ files can be processed in the same query and summarised output plots are produced. Just as for the web app, library compositions are compared to the online database to ensure integration of future database expansions with additional library types.\n\n\nUse case\n\nAs a use case we assume that a researcher has submitted three samples for sequencing and has now received FastQ files from the provider (use case input13). They want to check if the data conforms to the expectation of the respective library preparation (i. e. RNA-seq, BS-seq and ATAC-seq). Using the Librarian web app, they choose the FastQ files from a directory on their computer and are presented with a graphical representation of how similar their libraries are to published ones regarding their base composition, and a prediction of how likely these samples are to be of a particular library type (use case output13). Any discrepancy to the expected library type should be considered a red flag and investigated further.\n\nAnother use case would be for a sequencing facility to run Librarian together with other QC packages and provide results to users together with FastQ files as standard.\n\n\nDiscussion\n\nOur analyses demonstrate that the base composition of sequencing libraries is heavily influenced by the method through which the library was prepared. This finding can be used as an early quality assurance step for newly sequenced or publicly available data. A sample not matching its expected composition should raise a red flag and the underlying cause should be investigated before moving on with the analysis. While this could point to a sample swap or problem during library preparation, it is also possible that it is caused by a non-standard preparation method.\n\nOf note, within our database of published sequencing libraries we find a small subset of samples which cluster with a different library type. This is nicely illustrated by a group of RNA-seq samples which fall into a region of the map which is otherwise very specific for ATAC-seq. Closer inspection of these examples reveals that their libraries were produced by tagmentation,16 a process that generates short DNA fragments using the same transposase as ATAC-seq. This clearly demonstrates that sequence bias at the start of the read introduced thereby has more of an impact on base composition than the difference between RNA producing genomic regions and generally open chromatin. The limited number of available tags for library types on public sequencing data repositories means that there is inherent heterogenicity within the groups. The example also illustrates that there is a need to update the library database as new methods are developed and certain commercial library preparation kits change popularity over time. We have therefore built Librarian in a way that can easily incorporate future developments.\n\n\nData availability\n\nZenodo: Librarian manuscript data v1, https://doi.org/10.5281/zenodo.7060217.13\n\nThis project contains the following underlying data:\n\n- Composition data (output from the original GEO database queries, and datasets included in the Librarian database (filtered list))\n\n- Use case input (example FastQ files (subsampled for smaller file size))\n\n- Use case output (Librarian plots generated from the use case input files)\n\nGEO database query parameters: Organism: Mus musculus OR Organism: Homo sapiens AND Platform Technology Type: “high throughput sequencing” AND Publication Date: 2018/010/01 to 2020/12/31.\n\nData are available under the terms of the GNU General Public License v3.0.\n\nSoftware available from: https://www.bioinformatics.babraham.ac.uk/librarian/ [Librarian web app]\n\nSource code available from: https://github.com/DesmondWillowbrook/Librarian [Librarian command line download and install instructions]\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.7003739.15\n\nLicence: GNU General Public License 3.0\n\n\nAuthor contributions\n\nKartavya Vashishtha: Software, Writing – Review & Editing\n\nCaroline Gaud: Software, Writing – Review & Editing\n\nSimon R. Andrews: Conceptualization, Funding Acquisition, Software, Writing – Review & Editing\n\nChristel Krueger: Conceptualization, Formal Analysis, Software, Visualization, Writing – Original Draft Preparation", "appendix": "Acknowledgements\n\nLibrarian was originally started as an open project at the Cambridge Bioinformatics Hackathon (www.cambiohack.uk, 21st-23rd Sep 2020) with initial ideas from many people including Stephen Kanyerezi and Lordstrong Akano. We would like to thank Felix Krueger for useful discussions and critical reading of the manuscript.\n\n\nReferences\n\nSequencing\|Key methods and uses. http\n\nWang L, Wang S, Li W: RSeQC: quality control of RNA-seq experiments. Bioinformatics. 2012; 28: 2184–2185. PubMed Abstract \| Publisher 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: 292–294. PubMed Abstract \| Publisher Full Text\n\nBabraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data. http\n\nHadfield J, Eldridge MD: Multi-genome alignment for quality control and contamination screening of next-generation sequencing data. Front. Genet. 2014; 5: 31.\n\nWingett SW, Andrews S: FastQ Screen: A tool for multi-genome mapping and quality control. 2018. Publisher Full Text Reference Source\n\nWood DE, Salzberg SL: Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014; 15: R46. PubMed Abstract \| Publisher Full Text\n\nLi X-Q, Du D: Variation, Evolution, and Correlation Analysis of C+G Content and Genome or Chromosome Size in Different Kingdoms and Phyla. PLoS One. 2014; 9: e88339. PubMed Abstract \| Publisher Full Text\n\nBernstein AI, Jin P:Chapter 3 - High-Throughput Sequencing-Based Mapping of Cytosine Modifications. Epigenetic Technological Applications. Zheng YG, editor.Academic Press;2015; 39–53. Publisher Full Text\n\nBuenrostro JD, Giresi PG, Zaba LC, et al.: Transposition of native chromatin for multimodal regulatory analysis and personal epigenomics. Nat. Methods. 2013; 10: 1213–1218. 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: 3047–3048. PubMed Abstract \| Publisher Full Text\n\nEdgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30: 207–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVashishtha K, Gaud C, Andrews S, et al.: Librarian manuscript data v1.2022. Publisher Full Text\n\nMcInnes L, Healy J, Melville J: UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. ArXiv180203426 Cs Stat. 2020.\n\nVashishtha K, Gaud C, Andrews S, et al.: Kartavya Vashishtha/Librarian-1.0.4. Zenodo. 2022. Publisher Full Text\n\nAdey A, et al.: Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol. 2010; 11: R119. PubMed Abstract \| Publisher Full Text" }	[ { "id": "151965", "date": "06 Oct 2022", "name": "Andrew Keniry", "expertise": [ "Reviewer Expertise Epigenetics", "development", "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\nVashishtha and colleagues perform an analysis of the base composition from ~3000 publicly available sequencing data sets and show that these segregate by library type in a UMAP analysis of fastq files. The authors suggest that this analysis could be a useful pre-mapping QC step to identify incorrect libraries early in analysis pipelines and provide a tool, Librarian, for performing this test. Such an analysis could certainly be useful and has the potential to be widely adopted. I have a few suggestions that could improve the manuscript:\nThe authors show that Librarian identifies libraries prepared by different techniques, but is it able to identify a failed sample within a specific technique? Can the method be tested on failed samples such as ChIP-seq without enrichment, BS-seq with a low conversion efficiency, or samples with high duplication?\n\nBS-seq seems to segregate into multiple clusters. Is there an easily identifiable reason for this – perhaps enrichment techniques or developmental stage?\n\nI’m not sure of the logistics of this, but Librarian may be more widely used if it was available as an option within the already widely used fastqc.\n\nAn example of the Librarian output would be beneficial.\n\nThe terms ‘reference map’ and ‘compositions map’ seem to be used interchangeably. For simplicity, one term should be used throughout.\n\nFig 1A shows the base composition of ChIA-pet data. As this is a less well known technique, it would be beneficial to have an explanation of the base composition results.\n\nFig 1 is missing a legend explaining which base each colour represents.\n\nI’m not certain what Fig 2E is showing. Could the authors provide more explanation in the legend? There is also no reference to this figure in the text.\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": "10922", "date": "23 May 2024", "name": "Christel Krueger", "role": "Author Response", "response": "Vashishtha and colleagues perform an analysis of the base composition from ~3000 publicly available sequencing data sets and show that these segregate by library type in a UMAP analysis of fastq files. The authors suggest that this analysis could be a useful pre-mapping QC step to identify incorrect libraries early in analysis pipelines and provide a tool, Librarian, for performing this test. Such an analysis could certainly be useful and has the potential to be widely adopted. I have a few suggestions that could improve the manuscript: We would like to thank Andrew Keniry for taking the time to review our manuscript and his thoughtful comments and suggestions. Please find below a point-by-point response to all questions. The authors show that Librarian identifies libraries prepared by different techniques, but is it able to identify a failed sample within a specific technique? Can the method be tested on failed samples such as ChIP-seq without enrichment, BS-seq with a low conversion efficiency, or samples with high duplication? Compositional differences between different library types are relatively large in comparison to variations between samples of one specific technique. For example, in ChIP-seq experiments typically only a minority of reads fall within enriched regions. To illustrate this point, the ENCODE consortium only requires a minimum of 1% reads in peaks for their datasets (Genome Res. 2012 Sep; 22(9): 1813–1831). Therefore, for both well enriched and poorly enriched datasets the majority of reads are background, and the overall base composition is not different enough to separate in base composition analysis. Likewise, technical duplication does not change the base composition, and only if the library consisted of very few original molecules would we expect an impact. Since BS-seq libraries are striking in their low C content, we explored if Librarian could spot low conversion rates: The tested library has very high non-CG methylation (around 20 %), but still the test sample is predicted as BS-seq - most Cs are still converted. In summary, technical noise adds markedly lower variation than library type. This makes Librarian robust against variation in sample preparation quality. Please see Image here: https://f1000research.s3.amazonaws.com/linked/649537.download_1.pdf BS-seq seems to segregate into multiple clusters. Is there an easily identifiable reason for this – perhaps enrichment techniques or developmental stage? The reason behind this is that there are several commonly used library preparation methods which produce data with different base compositions, for example directional vs non-directional libraries or reduced genomic representation. Two easily identifiable groups on the Compositions map are whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). Please see Image here: https://f1000research.s3.amazonaws.com/linked/649538.Download_2.pdf Predating the development of Librarian we explored the option of predicting various bisulfite library types from their base compositions. A proof of concept can be found here: https://github.com/ChristelKrueger/Charades The subgrouping of bisulfite libraries highlights a more general issue with the metadata available on GEO. All the different variations of these libraries carry the flag ‘Bisulfite-Seq’ and further distinction is only found in the method description - if at all. This can easily create problems when re-processing published data as processing pipelines are often specific for a particular sample type. Grouping of disparate sample types is widespread amongst all categories especially as by now a lot of different library preparation methods are on the market. We have now included an explanation of subgroupings and underlying reasons in the discussion. I’m not sure of the logistics of this, but Librarian may be more widely used if it was available as an option within the already widely used fastqc. We discussed this as an option but decided against it. FastQC is deliberately kept lean and without dependencies which Librarian would bring. For more convenient incorporation into standard QC we are now writing out the prediction percentages as a text file. This can be picked up by the widely used tool MultiQC and is then displayed as a heatmap in the MultiQC report. We would like to thank Phil Ewels for incorporating this feature. An example of the Librarian output would be beneficial. Librarian will produce the plots shown in Figure 2B, C, E with circles indicating the map location of test samples. We deliberately did not include output examples in the main body of the manuscript as they are almost identical to the ones shown in Figure 2. Instead, they can be found in the accompanying use case data at https://doi.org/10.5281/zenodo.7060217. The terms ‘reference map’ and ‘compositions map’ seem to be used interchangeably. For simplicity, one term should be used throughout. This has been changed to ‘reference map’ throughout. Fig 1A shows the base composition of ChIA-pet data. As this is a less well known technique, it would be beneficial to have an explanation of the base composition results. Please see 7. Fig 1 is missing a legend explaining which base each colour represents. The text has been changed to briefly introduce the characteristic profiles observed with all four library types shown in the figures, and individual subpanels are now referenced. Details regarding the preparation protocols and how these produce the observed profiles have been added to the figure legend. The figure has been changed to reflect the order of library types in the text and the colour legend has been added. I’m not certain what Fig 2E is showing. Could the authors provide more explanation in the legend? There is also no reference to this figure in the text. An in-text reference has been added in the section about Implementation." } ] }, { "id": "151966", "date": "14 Oct 2022", "name": "Konstantin Okonechnikov", "expertise": [ "Reviewer Expertise Bioinformatics data analysis in pediatric neurooncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes the quality control (QC) tool Librarian that predicts the similarity of sequencing library correctness in comparison to the control cohort based on the analysis of reads. Initially for this purpose, the composition of nucleotide variance in the first 50 bp of read was used as input to create a large reference control cohort from publicly available data. Visualisation of these merged nucleotide profiles via UMAP allows to observe clear groups formation based on the data type of a dataset. Novel sample check is a projection into this reference UMAP. Testing the online tool confirmed its usefulness: from inspection of own data, the majority of cases were distinguished correctly. Such projection of a novel dataset into reference would be a useful QC step for any sequencing experiment. However, the manuscript and the software description could be improved in order to provide more details about the tool as well as explain certain missing blocks.\nIn general, the manuscript clearly describes the technique, however, only one possible limitation of the method is stated (effect of a cut in RNA-seq fragments leading to similarity to ATAC-seq in Discussion). More variance factors could be inspected to avoid misleading conclusions about the analysis results. For example, the tissue materials can be obtained from frozen tissue (FFPE), is there any impact of this preparation procedure? In my inspection, the standard RNA-seq datasets were distinguished quite well, but FFPE RNA-seq demonstrated the closest similarity to MBPD and MeDIP-seq. The scRNA-seq protocols are also included, however, they vary since they could be either full gene body covering or only 5’/3’ segment of a gene. Could this have an impact on read nucleotide variance?\n\nThe reads selection is performed with 100K subsampling - how was this amount selected? What is the effect of the total number of reads? Is it sufficient to provide only a subset of them? In this case, what is the suggested limit?\n\nAlso, 50 bp read segment is used as the reference, but how was this selection made? Currently, the main standard for sequencing is 100-150 bp. Would it be more beneficial to use a larger segment of the read for reference generation? Or do quality issues in longer reads have a negative impact?\n\nHow strong is the species effect? Are there variances observed between mice and human materials in full UMAP, e.g. clusters formation? Does it make sense to create own reference for such a procedure, especially when working on other species, e.g. Drosophila?\n\nFurther additional comments could help to improve the manuscript for easier reading:\nIn Figure 1, the nucleotide type color legend is missing, also segments are not cited in the text directly by suffix (a,b,c,d). Figure 1a demonstrates ChIA-PET, but not clear why it is included since it's not stated in the manuscript text.\n\nFigure 2a: Are the amounts of mice and humans mixed? What is the variance?\n\nFigure 2c: Several enrichment UMAP locations for certain data types are far from each other, e.g. RNA-seq. How to interpret this? Could it be certain subclusters, further splitting the dataset types?\n\nWhen downloading example datasets (sample FASTQ files), the archive cannot be opened. Also, there is no documentation available regarding input format preparation, e.g fastq are not allowed to be gzipped, it’s not clear without testing.\n\nGithub documentation on the establishment/launch lacks some details. Would be useful to extend it especially to state what are the system environment requirements before installation.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "10923", "date": "23 May 2024", "name": "Christel Krueger", "role": "Author Response", "response": "The manuscript describes the quality control (QC) tool Librarian that predicts the similarity of sequencing library correctness in comparison to the control cohort based on the analysis of reads. Initially for this purpose, the composition of nucleotide variance in the first 50 bp of read was used as input to create a large reference control cohort from publicly available data. Visualisation of these merged nucleotide profiles via UMAP allows to observe clear groups formation based on the data type of a dataset. Novel sample check is a projection into this reference UMAP. Testing the online tool confirmed its usefulness: from inspection of own data, the majority of cases were distinguished correctly. Such projection of a novel dataset into reference would be a useful QC step for any sequencing experiment. However, the manuscript and the software description could be improved in order to provide more details about the tool as well as explain certain missing blocks. We would like to thank Konstantin Okonechnikov for reviewing our manuscript and for taking the time to test the tool Librarian. We much appreciate his thoughtful comments and suggestions for improvement. Please find below detailed answers to each of the questions/comments. In general, the manuscript clearly describes the technique, however, only one possible limitation of the method is stated (effect of a cut in RNA-seq fragments leading to similarity to ATAC-seq in Discussion). More variance factors could be inspected to avoid misleading conclusions about the analysis results. For example, the tissue materials can be obtained from frozen tissue (FFPE), is there any impact of this preparation procedure? In my inspection, the standard RNA-seq datasets were distinguished quite well, but FFPE RNA-seq demonstrated the closest similarity to MBPD and MeDIP-seq. The scRNA-seq protocols are also included, however, they vary since they could be either full gene body covering or only 5’/3’ segment of a gene. Could this have an impact on read nucleotide variance? Systematic analysis of all factors influencing the base compositions is hampered by the limited amount of metadata that is provided by GEO, e. g. sample preparation methods or library kits are not included. One idea that we are pursuing for future developments in Librarian is to mine the entire GEO entry (rather than just the metadata) for more detailed sample information. Prompted by your observation we manually found and downloaded five FFPE RNA-seq samples from four different studies (SRR11996379, SRR11996380, SRR21404015, SRR21773166, SRR23245053) and ran these through Librarian with the following results: Please see image 1 here: https://f1000research.s3.amazonaws.com/linked/649544.Image_1.pdf Please see image 2 here: https://f1000research.s3.amazonaws.com/linked/649545.Image_2.pdf We cannot confirm that there seems to be a general issue classifying FFPE samples but we note that for some regions of the reference map RNA-seq shows substantial overlap with other library types which may result in misprediction of the library type. The situation seems to be similar for 3’ RNAseq libraries. We downloaded four 10X libraries from two different studies (SRR18916640, SRR18916641, SRR23254936, SRR23254938). The Librarian results can be found below. Two samples are classified correctly, the other two are from an area in the reference map which is occupied by several library types and therefore have some wobble in the classification. Please see image 3 here: https://f1000research.s3.amazonaws.com/linked/649546.Image_3.pdf Please see image 4 here: https://f1000research.s3.amazonaws.com/linked/649547.Image_4.pdf We have included a section on “My sample doesn't come up as the library type that I expect. How worried do I need to be?” in the FAQs. The reads selection is performed with 100K subsampling - how was this amount selected? What is the effect of the total number of reads? Is it sufficient to provide only a subset of them? In this case, what is the suggested limit? The idea behind subsampling is to find a good balance between faithful representation of the characteristics of a FastQ file and file size / processing speed. Subsampling is employed by several Babraham Bioinformatics QC tools including FastQScreen and some modules of FastQC. For Librarian, the subsample needs to be big enough for the base compositions to have stabilised and not be subject to random variation which is indeed the case for 100 K. Selecting the reads randomly rather than from the top of the file ensures that files in which reads are supplied in a sorted manner can also be dealt with. 100 K reads is the lower limit that Librarian accepts in a file. See below for an example in which 100 K, 200 K, 500 K and 13.5 million reads of an RNA-seq library. The samples are located in exactly the same location on the reference map. Please see image 5 here: https://f1000research.s3.amazonaws.com/linked/649548.Image_5.pdf Please see image 6 here: https://f1000research.s3.amazonaws.com/linked/649549.Image_6.pdf Also, 50 bp read segment is used as the reference, but how was this selection made? Currently, the main standard for sequencing is 100-150 bp. Would it be more beneficial to use a larger segment of the read for reference generation? Or do quality issues in longer reads have a negative impact? Our decision to use the first 50 bp of a read was influenced by two considerations: Firstly, many types show characteristic biases at the beginning of the read while becoming more similar to the general GC content further along the read (compare Figure 1). Thus, using information from longer reads actually reduces the differences between library types. Secondly, while it is true that currently read lengths tend to be longer, there are still many experiments that use 50 bp sequencing read length. These could not be analysed if the reference map had been created using the information from 100 bp. We therefore chose 50 bp to balance accuracy with broad usability. How strong is the species effect? Are there variances observed between mice and human materials in full UMAP, e.g. clusters formation? Does it make sense to create own reference for such a procedure, especially when working on other species, e.g. Drosophila? The magnitude of the species effect will depend on the difference of overall GC content and genome biology. We have included the reference map coloured by species as Supplementary Figure 2. As expected from the similar GC content and similar biology, libraries from mouse and human samples largely show the same base composition. We decided to focus on those two species because of the abundance of available data and prevalence in current studies. Here is an example of RNA-seq libraries from species with substantially different GC contents (Apis mellifera ~35 % GC, Staphylococcus aureus ~ 30 % GC and Streptomyces coelicolor ~70 %). Please see image 7 here: https://f1000research.s3.amazonaws.com/linked/649550.Image_7.pdf Please see image 8 here: https://f1000research.s3.amazonaws.com/linked/649551.Image_8.pdf Please see image 9 here: https://f1000research.s3.amazonaws.com/linked/649552.Image_9.pdf We therefore do not recommend using Librarian for species other than mouse or human although it is very likely that it would perform well on other mammalian species with similar GC content to human and mouse. We have now indicated this in the section on Operation and in the new FAQ section of the documentation. Please also see response to Reviewer 3. Species specific and broader mammalian reference maps are planned for future Librarian releases. Further additional comments could help to improve the manuscript for easier reading: In Figure 1, the nucleotide type color legend is missing, also segments are not cited in the text directly by suffix (a,b,c,d). Figure 1a demonstrates ChIA-PET, but not clear why it is included since it's not stated in the manuscript text. The text has been changed to briefly introduce the characteristic profiles observed with all four library types shown in the figures, and individual subpanels are now referenced. Details regarding the preparation protocols and how these produce the observed profiles have been added to the figure legend. The figure has been changed to reflect the order of library types in the text and the colour legend has been added. Figure 2a: Are the amounts of mice and humans mixed? What is the variance? We now include a file called ‘Supplementary Information’ with the publication which is deposited on Zenodo (along with other data and code). This contains a table showing how many data sets were from mouse and human per library type. The inputs were broadly balanced with the exception of ChIA-PET. Please see image 10 here: https://f1000research.s3.amazonaws.com/linked/649553.Image_10.pdf Figure 2c: Several enrichment UMAP locations for certain data types are far from each other, e.g. RNA-seq. How to interpret this? Could it be certain subclusters, further splitting the dataset types? Apart from technical variation an underlying reason can be grouping of library preparation methods that produce distinct composition profiles. For RNA-seq there are a number of popular commercial kits available which will produce their own biases (e. g. stranded vs non-stranded, random vs poly-A primed). The most striking example of grouping by library preparation method is BS-seq where several commonly used library preparation methods produce different compositions. Two easily identifiable groups on the reference map are whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). Please see image 11 here: https://f1000research.s3.amazonaws.com/linked/649554.Image_11.pdf Predating the development of Librarian we explored the option of predicting various bisulfite library types from their base compositions. A proof of concept can be found here: https://github.com/ChristelKrueger/Charades The subgrouping of bisulfite libraries highlights a more general issue with the metadata available on GEO. All the different variations of these libraries carry the flag ‘Bisulfite-Seq’ and further distinction is only found in the method description - if at all. This can easily create problems when re-processing published data as processing pipelines are often specific for a particular sample type. Grouping of disparate sample types is widespread amongst all categories especially as by now a lot of different library preparation methods are on the market. We have now included an explanation of subgroupings and underlying reasons in the discussion. When downloading example datasets (sample FASTQ files), the archive cannot be opened. Also, there is no documentation available regarding input format preparation, e.g fastq are not allowed to be gzipped, it’s not clear without testing. The example file archive has been fixed and is now working. Librarian accepts both fastq and gzipped files. However, we found that upload of gzipped files is an issue outside of Librarian with some Mac/browser combinations. The workaround is to drag & drop files onto the upload button. We have added the sentence “Librarian accepts fastq and gzipped files. If gzipped files are greyed out in the pop up, try dragging them onto the upload button.” to the web app and have also included this workaround in the FAQs. Github documentation on the establishment/launch lacks some details. Would be useful to extend it especially to state what are the system environment requirements before installation. We have now included more comprehensive documentation including detailed installation instructions and FAQs at https://desmondwillowbrook.github.io/Librarian/. We point to these from the manuscript (section Operation) and the github repository." } ] }, { "id": "151968", "date": "18 Oct 2022", "name": "Karim Gharbi", "expertise": [ "Reviewer Expertise genomics", "next-generation sequencing", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Vashishtha et al describes the implementation of a novel quality control (QC) tool for next-generation sequencing (NGS) datasets, which uses nucleotide composition profiles along sequence reads to infer the likely library preparation method used to generate the data. The authors first demonstrate that nucleotide composition is strongly influenced by library method as recorded in the GEO database for a selection of human and mouse NGS datasets. Having established this result, they implemented a program tool (Librarian) to compare library nucleotide composition profiles to a collection of reference datasets and identify libraries with unexpected profiles, which may be indicative of potential failure during library preparation and/or sample/data mix-ups. The tool, which is available as a web application and command line tool, extracts nucleotide composition from user supplied FASTQ files and returns similarity scores against existing profiles stored in the Librarian database.\n\nThe manuscript is well-written, and the authors provide strong evidence of library method influencing nucleotide composition in the read output files. This will be a familiar observation for those experienced with generating and/or analysing diverse NGS datasets, but the manuscript is a welcome documentation and quantification of these patterns. As a tool, Librarian has the potential to become an important step in the QC of NGS data, alongside other, more generic QC tools, such as FastQC, and help detect quality issues early in data processing. However, I have some concerns about the limitations of the software as currently implemented, which I feel are not sufficiently discussed in the manuscript and could cause significant confusion in the hands of less experienced users. The comments below are intended to help the authors improve the current manuscript and indicate areas for future improvement to increase the usability of the tool.\n\nMajor comments - Please comment on the applicability of Librarian to data generated with other NGS technologies than Illumina. If not tested or not applicable, this should be highlighted in the discussion.\n\n- Please provide a rationale for trimming reads to 50 bases and only considering read 1 to build the database of nucleotide composition profiles, i.e., why is this sufficient to accurately capture the nucleotide composition of each library type. Some methods result in asymmetric library fragments (e.g., 10X Genomics), with different nucleotide compositions in read 1 and read 2, which in itself can be diagnostic of the library type.\n\n- The selection of GEO datasets to build reference profiles seems restrictive and potentially biased. Please can you provide evidence that Librarian is applicable to other species than human/mouse, especially species with divergent GC content.\n- The date range filter (01/01/2018 - 31/12/2020) is also likely to have resulted in more recent library types to be excluded from the analysis and therefore the reference dataset. 10X Genomics library types are highly popular, but surprisingly absent. Other library types may have been missed too.\n\n- Transposon-based library preparation is increasingly popular and applied across a wide range of library types, including single-cell RNA and DNA sequencing, whole-genome sequencing, ATAC-seq, enrichment capture etc. The authors briefly acknowledge this in the discussion, but it appears to be a major limitation of the tool, i.e., transposon-insertion signature at the start of the reads will likely obscure the underlying library type, causing most transposon-based libraries to cluster together. This should be explicitly documented and investigated further, if possible.\n\n- More generally speaking, I would strongly encourage the authors to explicitly identify library types and species \"supported\" by Librarian, indicating that submission of other library types and/or species may result in inconclusive or potentially misleading results (I acknowledge that the software will accept any FASTQ file).\nMinor comments\n\n- Please briefly comment on the observed pattern for ChIA-PET and ChIP-seq libraries, i.e., why are these expected and consistent with the library method. ChIA-PET is not a widely used library method. A short description should be included in the text for context.\n\n- Please add legend to Figure 1 with key matching coloured lines to individual bases.\n\n- I would suggest meta-analysis of public datasets as another important use case for Librarian, e.g., as a clean-up tool prior to meta-analysis or identifying patterns/biases in library type, or subtypes.\n\n- Please clarify whether Librarian can we be set up with a local, custom server in addition to query against an online database via the web app or command line tool.\n- The tabular data in figure 2A shows library types with fewer than 25 samples despite these being classified as under-represented libraries and excluded from the analysis in the text.\n\nOverall, I believe that the premise of Librarian is a very good idea and thank the authors for their efforts in releasing the program as a publicly available tool. I look forward to reading their responses, and future iterations of the software addressing current limitations.\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": "10924", "date": "23 May 2024", "name": "Christel Krueger", "role": "Author Response", "response": "Reviewer Comment: In this manuscript, Vashishtha et al describes the implementation of a novel quality control (QC) tool for next-generation sequencing (NGS) datasets, which uses nucleotide composition profiles along sequence reads to infer the likely library preparation method used to generate the data. The authors first demonstrate that nucleotide composition is strongly influenced by library method as recorded in the GEO database for a selection of human and mouse NGS datasets. Having established this result, they implemented a program tool (Librarian) to compare library nucleotide composition profiles to a collection of reference datasets and identify libraries with unexpected profiles, which may be indicative of potential failure during library preparation and/or sample/data mix-ups. The tool, which is available as a web application and command line tool, extracts nucleotide composition from user supplied FASTQ files and returns similarity scores against existing profiles stored in the Librarian database. The manuscript is well-written, and the authors provide strong evidence of library method influencing nucleotide composition in the read output files. This will be a familiar observation for those experienced with generating and/or analysing diverse NGS datasets, but the manuscript is a welcome documentation and quantification of these patterns. As a tool, Librarian has the potential to become an important step in the QC of NGS data, alongside other, more generic QC tools, such as FastQC, and help detect quality issues early in data processing. However, I have some concerns about the limitations of the software as currently implemented, which I feel are not sufficiently discussed in the manuscript and could cause significant confusion in the hands of less experienced users. The comments below are intended to help the authors improve the current manuscript and indicate areas for future improvement to increase the usability of the tool. Author Response: We would like to thank Karim Gharbi for reviewing our manuscript and taking the time to test the tool Librarian, and we are very pleased by his positive assessment regarding its usefulness. We much appreciate Karim’s thoughtful comments and useful suggestions to improve the usability. Please find below responses to each of his questions/comments. Reviewer Comment: Major comments Please comment on the applicability of Librarian to data generated with other NGS technologies than Illumina. If not tested or not applicable, this should be highlighted in the discussion. Author Response: Our analyses focused on Illumina sequencing as this is by far the most widely used technology and also offers the most diverse library types - with many applications not available on other platforms such as Nanopore or PacBio. We have now made this clear in the discussion and included this as a limitation in the section on Operation. We have also added this to the new FAQ section of the documentation. Reviewer Comment: Please provide a rationale for trimming reads to 50 bases and only considering read 1 to build the database of nucleotide composition profiles, i.e., why is this sufficient to accurately capture the nucleotide composition of each library type. Some methods result in asymmetric library fragments (e.g., 10X Genomics), with different nucleotide compositions in read 1 and read 2, which in itself can be diagnostic of the library type. Author Response: Librarian was designed to check individual FastQ files which is why differences between e. g. read 1 and read 2 are not taken into account despite being informative in some cases. Our decision to use the first 50 bp of a read was influenced by two considerations: Firstly, many types show characteristic biases at the beginning of the read while becoming more similar to the general GC content further along the read (compare Figure 1). Thus, using information from longer reads actually reduces the differences between library types. Secondly, while it is true that currently read lengths tend to be longer, there are still many experiments that use 50 bp sequencing read length. These could not be analysed if the reference map had been created using the information from 100 bp. We therefore chose 50 bp to balance accuracy with broad usability. Reviewer Comment: The selection of GEO datasets to build reference profiles seems restrictive and potentially biased. Please can you provide evidence that Librarian is applicable to other species than human/mouse, especially species with divergent GC content. Author Response: We deliberately restricted the selection of samples to human and mouse species as it is indeed the case that species with different CG content will produce libraries of different compositions. Thus, as a minimum, test samples should have a similar GC content to the samples from the reference map. We decided to focus on human and mouse data for the reference map because of the abundance of available data and prevalence in current studies. Here is an example of RNA-seq libraries from species with substantially different GC contents (Apis mellifera ~35 % GC, Staphylococcus aureus ~ 30 % GC and Streptomyces coelicolor ~70 %). Please see Image 1 here: https://f1000research.s3.amazonaws.com/linked/649555.Image_1.1.pdf Please see Image 2 here: https://f1000research.s3.amazonaws.com/linked/649556.Image_1.2.pdf Please see Image 3 here: https://f1000research.s3.amazonaws.com/linked/649557.Image_1.3.pdf We therefore do not recommend using Librarian for species other than mouse or human although it is very likely that it would perform well on other mammalian species with similar GC content to human and mouse. We have now indicated this in the section on Operation and the new FAQ section of the documentation. Please also see reply to Reviewer 2. Species specific and broader mammalian reference maps are planned for future Librarian releases. Reviewer Comment: The date range filter (01/01/2018 - 31/12/2020) is also likely to have resulted in more recent library types to be excluded from the analysis and therefore the reference dataset. 10X Genomics library types are highly popular, but surprisingly absent. Other library types may have been missed too. Author Response: It is indeed the case that new library types become available all the time. While Librarian is unlikely to ever be able to cater for niche technologies it will need to be updated over time as new library preparation methods become popular. We have therefore made the design decision to have both the web app and the CLI query the reference data on the Babraham server. This is to ensure that users will have immediate access to updated reference maps as they become available. A current limitation is that GEO only offers a set number of metadata tags for library types upon sample submission which results in grouping of distinct preparation methods under one name. (Please also see responses to Reviewer 1 Question 2 and Reviewer 2). Currently, there is no tag for 10X Genomics libraries on GEO and submitters may choose to either label it as ‘RNA-seq’ or potentially ‘OTHER’. We are hoping to be able to query a wider range of metadata in the future and provide a more fine grained classification of library types. Reviewer Comment: Transposon-based library preparation is increasingly popular and applied across a wide range of library types, including single-cell RNA and DNA sequencing, whole-genome sequencing, ATAC-seq, enrichment capture etc. The authors briefly acknowledge this in the discussion, but it appears to be a major limitation of the tool, i.e., transposon-insertion signature at the start of the reads will likely obscure the underlying library type, causing most transposon-based libraries to cluster together. This should be explicitly documented and investigated further, if possible. Author Response: This is indeed a limitation and laid out in the second paragraph of the discussion. We have now also added this scenario explicitly to the FAQ section of the documentation. With the current set of reference data we find that the proportion of transposon-based library preparation for other types than ATAC-seq is very low. As this library preparation method becomes more popular it will feature more strongly and in a more diverse way in future reference maps. Reviewer Comment: More generally speaking, I would strongly encourage the authors to explicitly identify library types and species \"supported\" by Librarian, indicating that submission of other library types and/or species may result in inconclusive or potentially misleading results (I acknowledge that the software will accept any FASTQ file). Author Response: We have now been much more explicit about which samples are suitable for analysis with Librarian: We have added the following statement to the Operations section: “Irrespective of platform, Librarian is only suitable to assess datasets which match the types that the reference map is built on. More specifically, test samples need to have been sequenced with Illumina technology, match any of the included library types and be of mammalian origin (ideally mouse or human).” We also discuss extensively which samples are supported in the new FAQ section of the documentation. Reviewer Comment: Minor comments Please briefly comment on the observed pattern for ChIA-PET and ChIP-seq libraries, i.e., why are these expected and consistent with the library method. ChIA-PET is not a widely used library method. A short description should be included in the text for context. Author Response: A description of the techniques BS-Seq, ATAC-Seq, ChIA-PET and ChIP-Seq and an explanation of the method specific composition biases along the read has been added to the legend to Figure 2. Reviewer Comment: Please add legend to Figure 1 with key matching coloured lines to individual bases. Author Response: This omission has been corrected. Reviewer Comment: I would suggest meta-analysis of public datasets as another important use case for Librarian, e.g., as a clean-up tool prior to meta-analysis or identifying patterns/biases in library type, or subtypes. Author Response: This is a good suggestion and this use case has now been included in the manuscript. Reviewer Comment: Please clarify whether Librarian can we be set up with a local, custom server in addition to query against an online database via the web app or command line tool. Author Response: We provide Librarian as a web tool and with a Linux command line interface. The CLI can be run either in offline mode in which visualisations and predictions are computed locally (this is a new option) or remote mode in which Librarian queries the same web server as the web tool. The latter has the advantage of avoiding R dependencies and ensures that libraries are compared to the latest reference map. The different modes are explained in the section Operation of the manuscript, and in the section Usage in the online tool documentation. Reviewer Comment: The tabular data in figure 2A shows library types with fewer than 25 samples despite these being classified as under-represented libraries and excluded from the analysis in the text. Author Response: This is correct. The search results were filtered to exclude library types with fewer than 25 samples. The table shows the number of samples for which we could retrieve base compositions. We were not able to retrieve sequence compositions for all search hits. Also note that RNA-seq search results were capped at 500 but only 452 FastQ files made it through the analysis. Most samples dropped out at the download stage which is a common thing to happen. Since this was done in high throughput we did not attempt to troubleshoot the process or download manually. Reviewer Comment: Overall, I believe that the premise of Librarian is a very good idea and thank the authors for their efforts in releasing the program as a publicly available tool. I look forward to reading their responses, and future iterations of the software addressing current limitations." } ] } ]	1	https://f1000research.com/articles/11-1122
https://f1000research.com/articles/11-1571/v1	23 Dec 22	{ "type": "Research Article", "title": "The chemotherapeutic activity of areca nut extract increased stromal tumor-infiltrating lymphocytes in 4-nutriquinoline-1-oxide-tumor-induced Sprague-Dawley rats", "authors": [ "Liza Meutia Sari", "Cut Fera Novita", "Poppy Andriany", "Dina Keumala Sari", "Cut Fera Novita", "Poppy Andriany", "Dina Keumala Sari" ], "abstract": "Background: Oral squamous cell carcinoma (OSCC) is one of the most common oral cancers with a high mortality rate. The biodiversity source in Indonesia makes areca nut a potential antioxidant in treating disease. Objective: The study aimed to evaluate the chemotherapeutic effect of areca nut extract in 4-nutriquinoline-1-oxide (NQO)-tumor-induced rats. Methods: Twenty-eight male Sprague-Dawley rats were divided into four groups. Group 1 served as the control group, group 2 was 4NQO-induced rats without treatment, and groups 3 and 4 were given 4NQO-tumor inducer with 500 and 1000 mg/kg BW of areca nut extract, respectively. The rats in groups 2,3, and 4 received 30 ppm of 4NQO tumor inducer in drinking water for 12 weeks. In the end, all rats were euthanized and the tongue was removed. The body, liver, kidney, heart, and lungs weights were measured. Tongue tumor volume and dysplasia lesions were analyzed. The tumor-infiltrating lymphocytes (TILs) in the tumor and stromal area were scored semi-quantitatively associating the infiltrate grade (0-3) and analyzed histologically. Results: There were significant differences in body weight loss between the initial and final phases in groups 1 and 2 (p<0.05). The areca nut at doses of 500 and 1000 mg/kg BW significantly reduced the tumor size compared with groups 1 and 2 (p<0.05). The incidence of OSCC in rats with 500 and 1000 mg/kg BW of areca nut extract after 22 weeks was 0%, but the dysplasia lesions were observed at 28.57% and 85.71%, respectively. The highest mean of stromal TILs was in group 3 and there were significant differences in stromal TILs between groups 2 and 3 (p<0.05). Conclusion: Areca nut extract exerts a chemotherapeutic activity in 4NQO-induced rats by inducing infiltrating lymphocytes in the stromal tumor area on the OSCC lesion of the tongue.", "keywords": [ "Areca nut", "Oral cancer", "Tumor-infiltrating lymphocytes", "4NQO-tumor inducer" ], "content": "Introduction\n\nIndonesia is a rich country in biodiversity including herbal plants. About 80% of the world's herbal plants growing spread across various islands in this country. Areca nut (Areca catechu Linn.) is a nut that contains flavonoids and polyphenolics, especially arecoline, catechin, epicatechin, and quercetin has proved to show antioxidant activity which is beneficial for human health.1 Areca nut is also one of substances that is frequently used by people after tobacco, alcohol, and caffeinated drinks according to WHO.2 Nowadays, the habit of chewing the areca nut as part of the hereditary heritage and a source of encouragement to work is still used by people in several rural and mountain areas in Indonesia, especially in Aceh and North Sumatera, Indonesia. This chewing custom is also often consumed in India in the form of paan masala and gutkha.3 Areca nut chewing is also one of the favorite habits in Taiwan.4 Potency antioxidant activity of areca nut has been well established in the previous in vitro research using several oral cancer cell lines.5 Although some areca nut products such as paan masala (fennel seeds, coriander, sesame, mint leaves, powdered lime, areca nut, cardamom, and pure menthol) and gutkha (chewing tobacco, dried ripe areca nut, paraffin wax, slaked lime (calcium hydroxide), and savory or sweet flavorings) have contribution in pathogenesis of oral squamous cell carcinoma (OSCC), several other studies have been carried out to prove the efficacy and safety of areca nuts depending on the type of phytochemical content possessed by the areca nut species from the certain region.\n\nOral squamous cell carcinoma is a cancer that most often causes a high mortality rate.6 The clinical characteristic of OSCC in a certain population shows that smoking and drinking alcohol are the most frequent factors causing oral malignancies. Other etiology factors that can cause oscc are human papilloma virus (HPV), nutritional deficiency, candidal infection, and genetic predisposition.7 The clinical appearance of oscc is ulcerated lesion with necrotic base in the central area surrounded with elevated border. In the last five years, many new cases of oscc and pharyngeal cancer appeared in all over the world and more than 7,000 cases caused death from these cancers.8 The best current OSCC treatment still depends on surgery combined with chemo/radiotherapy. Although this is still the best option, this therapy has many side effects due to its non-selective activity including mucositis, osteoradionecrosis, xerostomia, dysgeusia, and orofacial pain so research to find alternative therapies derived from herbal ingredients is still being developed.9 This herbal therapy is expected to inhibit the development of malignancy cells but has no destructive effect on healthy cells so that side effects of chemo/radiotherapy in the oral cavity can be minimized. Currently, there are no reports that examine whether the intake of areca nut extract is beneficial and safe for consumption in OSCC patients, considering the effects of OSCC induction by areca nut which are feared for humans when consumed in the long term. However, several studies have been carried out to develop the effects of areca nut on health.10,11 The oral manifestations of OSCC are generally in the form of ulcerated lesions which are infiltrated by lymphocytes and inflammatory cells such as monocytes, macrophages, and plasma cells.\n\nRecently, several new therapies have used reactive anticancer immune responses to cancer to treat breast cancer.12 Tumor-infiltrating lymphocytes (TILs) located in the microenvironmental of the lesion site can predict tumor biology and the prognosis of cancer treatment.13 Several clinical studies have also evaluated the importance of TILs as a determinant and predictive value in mammary cancer prognosis.14,15 A previous study reported that the mean TILs for T lymphocytes were generally lower in OSCC patients who died compared to those who survived.16 Another study has shown high lymphocytic infiltrate, especially infiltration of T and B lymphocytes in ductal carcinoma in situ.17 Currently, a more in-depth research is needed to develop the efficacy of areca nut on the development of oral cancer. To support further in vivo research, an animal model is needed that uses the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) to form OSCC lesions. This 4NQO mouse model can induce OSCC with a similar progression step in OSCC and describes a carcinogenesis process similar to that of chronic tobacco abuse in humans.18 In this study, we investigated the effect of areca nut extract on OSCC on the tongue using a mouse model given 4-NQO tumor inducer in drinking water. This study is expected to provide new information on the efficacy of areca nut in the progression of SCC on the tongue through clinical and histological examination.\n\n\nMethods\n\nThis research was approved by the Ethical Clearance Committee Faculty of Dentistry, Universitas of Syiah Kuala, Banda Aceh, Indonesia, with the number 247/KE/FKG/2021. All research procedures were carried out based on the Organization for Economic Cooperation and Development (OECD), which indicated that all rats would receive human care based on the criteria contained in the section \"Guide for the care and use of laboratory animals (OECD, 2015) and Institutional Animal Care and Use Commitee (IACUC) of this facility. All procedures in this study were reported following the ethical roles, principles, and guidelines of ‘Animals in Research: Reporting in vivo Experiments’ (ARRIVE). All the procedures have passed the ethic commitee and IACUC guidelines.\n\nThe areca nut is from an areca nut plantation in the province of Aceh Besar, Indonesia. Determination of plants in the form of roots, stems, leaves, and nuts, was identified by Dr. Sunaryo at the Herbarium Unit of the Botanical Division of Biological Research Center in Cibinong, West Java, Indonesia, with voucher specimen number 735/IPH.1.02/If.8/IV/2019.\n\nTwo kilograms of ripe areca nut were washed under running water and dried in the sun and open air. The drying process was continued by using an electric oven at 50°C for 30 minutes for 5 days. The dried areca nut samples were crushed into a fine powder and then filtered using a 20-mesh sieve. The 1.7 kg of fine powder was soaked in 96% ethanol at 20-25°C for 7 days. The ethanol extract was then evaporated from the solvent so that it became a dry powder using a rotary evaporator at a temperature of 30-40°C. The dry powder was stored at room temperature. The aqueous extract of areca nut is made by mixing dry areca nut powder with water. The doses used in this study were 500 and 1000 mg/kg BW. This dose was chosen based on a safe or non-toxic dose after oral acute toxicity tests were carried out in our previous acute oral toxicity study. The areca nut extract solution was mixed with distilled water so that it had a liquid consistency and was easy to give to rats by giving it every day which was divided into 2 doses, morning and evening.\n\nA total of 28 male Sprague-Dawley rats that were pathogens free aged 8-10 weeks old, with a body weight of 180-250 g came from the Pharmacology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh. Animals were put in animal cages with stainless steel grid cover (seven rats in a cage) in a stable environment and have free access to drinking water and food which is put in polypropylene bottles. Acclimatization of animals has carried out at a temperature of 25-30°C and with a 12 h light/dark cycle for a week before the experiments. We also feed dietary supplement to increase the stamina of the rats.\n\nThe anticarcinogenic test was carried out based on the guidelines of OECD guidelines for testing chemicals in animals (OECD, 2001). Oral squamous cell carcinoma induction was performed using 4-Nitroquinoline 1-oxide (4NQO Cas No. N8141-5G, Sigma Aldrich). This carcinogenic chemical agent was prepared by mixing 30 ppm 4NQO with drinking water and then given daily to rats in light-shielded water bottles for 12 weeks.19 The bottle was protected from sun exposure to protect it from chemical changes that may affect the 4NQO’s efficacy. The drinking water was changed every week.\n\nThis study was designed to observe the chemotherapeutic effect of areca nut extract on the healing of OSCC on the tongue induced using 4NQO in rats. A total of 28 male Sprague-Dawley rats were randomized into 4 groups. Groups 1 and 2 were control groups. Group 1 was untreated and group 2 was oral cancer induced by 4NQO administration without treatment. Groups 3 and 4 were induced by 30 ppm 4NQO administration and treated with areca nut extract at 500 and 1000 mg/kg body weight (BW), respectively. The areca nut extract treatment was started 1 week after cessation of the 4NQO administration. In this study, the observation of the health condition of the rats was done every day in the morning to identify possible deaths from toxicity from the 4NQO administration during the induction and treatment periods. The body weights were routinely measured every week and at the end of the experimental period. At the end of this experimental study, all rats were anesthetized through the intramuscular injection of 50 mg/kg BW ketamine 100 mg/mL and 5 mg/kg BW of xylazine 100 mg/mL. Prior to the injection stage, the rat’s cages were covered by black cover for 20-30 minutes to reduce stress in rats. The rats were sacrificed followed by excision of the whole tongue to assess the volume of tongue tumor analysis. The experimental protocol is shown in Table 1. The clinical examination also includes the calculation of the weight and condition of the heart, lung, liver, and kidney. The tongue of six rats in each group was longitudinally cut into halves for the histopathological examination. The tongue tissue was then fixed in 10% buffered formalin, embedded in paraffin blocks, and stained with hematoxylin and eosin (H & E).\n\nBasal diet and tap water\n\n4NQO 30 ppm in drinking water\n\n500 mg of areca nut extract in diet\n\n1000 mg of areca nut extract in diet\n\nTongue tumor volumes were analyzed at the end of the experimental study and measured based on the formula length x width x height x π/6.20 The tongue sections were analyzed histologically and classified as dysplasia, hyperplasia, and OSCC in every rat.21\n\nThis study aims to evaluate TILs using a methodology for the TIL scoring system developed by Denkert et al. and the International TILs Working Group based on the H & E slides.22,23 Observations were made on intratumoral and stromal TILs. Intratumoral TILs are lymphocytes in direct cell-to-cell contact with the cancer cells with no intervening stroma.24 Stromal TILs are located clustered or scattered in the stroma among the cancer cells and do not directly interact with the cells.25 The evaluations carried out in both TILs with the assessment of score 0: no infiltrating lymphocytes; score 1: mild increase of infiltrating lymphocytes in the tumor nest or stroma; score 2: increase infiltrating lymphocytes interwoven with tumor issue; score 3: prominent infiltrating lymphocytes separate or incorporated in tumor tissue.26\n\nThe buccal mucosa tissue was fixed in 10% formal saline for 24 hours, the tissue was dried or dehydrated using absolute ethyl alcohol, then embedded in paraffin at a temperature of 50°C. The wax tissue blocks were then sectioned by sliding microtome, stained with H & E, and analyzed under the binocular microscope (Olympus CX23 with an attached digital camera). Slides were subsequently reviewed by the pathologist.\n\nThe data were analyzed using the SPSS version 20. The quantitative results were expressed as the mean ± SD. Body weight was analyzed by using a dependent t-test. Data representing heart and lungs weights, tumor volume, intratumoral, and stromal TILs, were compared using the non-parametric Kruskal-Wallis test with post hoc comparisons made using the Mann-Whitney test to determine the significance of the differences between groups. The liver and kidney weights were compared by using a one-way ANOVA test. The results were statistically significant at a p-value <0.05. The data of incidence of tongue dysplasia were displayed in term of percentage.\n\n\nResults\n\nDuring the research, from the first week of the adaptation period until the end of the treatment, no rats died. The mean weight changes of rats were seen to increase and decrease significantly in the control and Ca-induced control group (Group 1 and 2) when compared before and after treatment for 22 weeks. There were no significant weight changes in other groups (Table 2). Although groups 3 and 4 were Ca-induced with areca nut extract treatment, there was no weight loss observed after the experimental study.\n\nThe study showed that there were no significant differences in heart, lungs, liver, and kidney weights among the groups (Table 3). The heart and lung weights in the 4NQO-induced rat groups appeared to be lighter than in the normal group although this difference was not significant. Liver and kidney weights in groups 2, 3, and 4 were heavier than the normal group.\n\nThe tumor tissue began to appear on the tongue and can be observed macroscopically in the 22 weeks of the experimental period. As shown in Table 4, the treated group with 500 and 1000 mg/kg BW of areca nut extract showed a smaller lesion compared with group 1 (untreated group) and group 2 (untreated and induced by 4-NQO group).\n\na significantly different for Group 1 vs. Group 2, 3, 4.\n\nb significantly different for Group 2 vs. Group 3, 4.\n\nDuring the induction of tumors on the rat's tongue, dysplasia and OSCC lesions occurred on the dorsolateral and posterior surfaces of the tongue. The incidence of tongue dysplasia after treatment with areca nut extract is shown in Table 5. The incidence of OSCC in rats after being treated with areca nut extract at 500 and 1000 mg/kg BW was 0%, but the dysplasia lesions can be observed at 28.57% and 85.71%, respectively. The incidence of hyperplasia lesions in the 500 mg/kg BW treatment group was observed at 71.43%. The specimen that has been diagnosed as having OSCC on a certain area of the tongue, the dysplasia and hyperplasia areas could also still be detected in a few parts of the same lesion.\n\nThe results of the univariate analysis showed that the mean score of intratumoral lymphocyte infiltration in 4NQO-induced rats that were given areca nut extract orally at doses of 1000 mg/kg BW every day showed the lowest average intratumoral TILs among the groups. Meanwhile, the normal and 500 mg/kg BW areca nut extract had the highest mean TILs of 0.83 or with a mild increase of infiltrating lymphocytes in the tumor nest. The study did not find significant differences in the mean of intratumoral TILs in all groups (p>0.05) as shown in Table 6. In a subsequent test performed on stromal TILs, it was observed that the highest mean of stromal TILs was in group 3 (4NQO induced rats with 500 mg/kg BW of areca nut treatment) (Table 6). This study showed a significant difference in stromal TILs between group 2 (4NQO induced rats without treatment) and group 3 (p<0.05).\n\na significantly different for Group 2 vs. Group 3.\n\nEvaluation results of the final histological feature were performed using light microscopy by qualified pathologists. The histological appearance of intratumoral TILs of each group can be seen in Figure 1. Accumulation of inflammatory cells especially polymorphonuclear cells or intratumoral TILs 1 was seen in Figure 1B. The TILs 0 in rats treated with areca nut extract 1000 mg/kg BW is observed in Figure 1D which showed the stratified squamous cell epithelium without inflammatory and tumor cells. The basal layer was intact in the specimen. The histological feature of stromal TILs can be seen in Figure 2. The stromal TILs showed an accumulation of lymphocytes that spread in the stroma area and were not directly in contact with the cancer cells.\n\n(A) The low level of TILs showed the mild increase of infiltrating lymphocytes in the normal tongue; (B) The TILs score 2 in the Ca-induced control group; (C) The score 1 of TILs in tongue treated with 500 mg/kg BW areca nut extract (100x); (D) The score 0 of TILs in tongue treated with 1000 mg/kg BW areca nut extract.\n\n(A) The TIL score 1 in the normal tongue; (B) The TIL score 2 in the Ca-induced control group; (C) The TIL score 1 in tongue treated with 500 mg/kg BW areca nut extract; (D) The score 1 of TILs in tongue treated with 1000 mg/kg BW areca nut extract (100x).\n\n\nDiscussion\n\nThe in vivo assay using an animal model is an important part of investigating the development of new therapy for the disease, especially those from new herbal medicine sources. Currently, the development of herbal plants as oral cancer treatment is still being explored. Up to now, the use of areca nut extract for OSCC treatment has not been reported. This study tried to reveal the role of areca nut extract in aqueous as a chemotherapeutic in OSCC induced by 4NQO-tumor inducer in Sprague-Dawley rats. The experimental research was designed by using randomization in a subject grouping. No rats died in this study. The rats were relatively stable in uniformity in age, sex, body weight, and acclimatization process.\n\nThe current study used 30 ppm 4NQO for 12 weeks followed by chemotherapy treatment for up to 22 weeks. This result is in line with a previous study that reported that administration of 30 ppm 4NQO in drinking water for 4-9 weeks and 36-46 weeks caused OSCC in 25% and 75%, respectively.27 Another study reported that with 20 ppm 4NQO for 10 weeks, the OSCC will experience reproducible power as much as 83% at 26 weeks of administration.28 The choice of 4NQO in drinking water is a trigger for dysplastic and neoplastic lesions because it can trigger tumors that morphologically and histopathologically have similarities to human tumors. The length of time the appearance of cancerous lesions on the tongue varies greatly depending on the period of time of administration and dose of the inducer. In addition to the appearance of oral lesions, we found several potential systemic toxicities in experimental animals including discoloration of the lungs, liver, and kidneys, and darkening of the skin accompanied by weight loss. Based on a previous study, some of the changes that occurred due to the administration of 4NQO were focal hepatic lobular necrosis, renal tubular degeneration, decreased cellularity in the splenic, reduction of body weight and white blood cell count, and increased of liver serum.29\n\nWe found there was significant weight loss occurred in 4NQO-induced rats without treatment. The decrease in body weight is generally caused by a loss of appetite, eating disorder, and an increase in metabolic rate because of the OSCC lesion in the oral cavity.30 Interestingly, this study also revealed that there were increases in body weight after treatment with the areca nut, although they were not significant. This is probably due to the areca nut extract’s ability to increase body weight. Its mechanism of action is that the main component of areca alkaloids is an inhibitor of the gamma-aminobutyric acid (GABA) receptor.31 The inhibitory effects of GABA receptor may increase appetite with eventual fat accumulation in general and central area of the body. Another mechanism that might play role in gain weight is that the areca nitrosamines derived from alkaloids may induce insulin resistance which can cause hyperglycemic conditions. Another study has reported that areca nut chewing has closely associated with metabolic syndrome, general obesity, and type-2 diabetes mellitus.32\n\nThe 4NQO is a synthetic carcinogen derivative of quinoline, sensitive to light, easily affected by temperature changes, and soluble in water. The mechanism of action of the 4NQO-tumor inducer is that it can induce the activation of COX-2 which will release nitric oxide (NO), especially inducible nitric oxide synthase (iNOS) which induces intracellular oxidative stress.33 Its action begins with an enzymatic reduction of the nitro group and then produces its carcinogenic potential by producing additional DNA to form hydroxydeoxyguanosine (8OHdG) that can cause oxidative damage.34 The damage caused by 4NQO is similar to the lesions caused by other carcinogenic substances such as alcohol and tobacco. The morphological and histopathological changes also have similarities with OSCC in humans.\n\nOur results showed that treatment of areca nut extract affected the reduction of tongue tumor size and incidence of tumor dysplasia. This study is a chemotherapeutic study which means that the areca nut may affect the progression phase of carcinogenesis. Areca nut has a high level of polyphenols and flavonoids such as catechin, tannin, and quercetin that act as antioxidants. We did not find much research regarding the anticancer activity of areca nut extract, but many studies have explored the effect of catechin, one of the most abundant phytochemicals contained in the areca nut on carcinogenesis in vitro and in vivo. Xu et al. reported that catechin might inhibit the tobacco-specific nitrosamines induced tumorigenesis through inhibition of the 8OhdG. Another research by Yoshimura et al. reported that catechin especially epigallocatechin gallate can inhibit cancer cell proliferation by inducing apoptosis and inhibiting cell cycle progression.35 Catechin can induce p53-mediated cell death through stabilization and activity of p53, inhibit the release of TNF-α which is a proinflammatory cytokine present in cancer, inhibit proliferation by causing cell cycle arrest in the G0/G1 phase, modulate apoptosis-related signaling, diminish mitochondrial membrane potential, neutralize ROS, and inhibit angiogenesis.36 Arecoline is also the primary phytochemical compound in areca nuts. Arecoline is recognized to cause oral submucosal fibrosis because it contains toxic constituents. This phytochemical content might be an obstacle to healing from oral cancer. Areca nut does not contain carcinogenic ingredients, but the toxic effect comes from nitrosamine products which are produced by the nitrosation process by dry arecoline which is chewed and digested by stomach acid for a long time and uncontrollable. Our research uses the areca nut extract for a certain period, so the possibility of areca nut extract causing cell damage such as fibrosis or pathogenesis of OSCC is quite small. In the crude extract that contained many phytochemical properties, there might be a mutually enhancing effect or vice versa from different chemical interactions in the crude extract. The size of extract volume does not determine the efficacy of phytochemical compounds that produce the extract's antioxidant effect. Fractionation of the extract plant might be one of the solutions to get the original antioxidant content of the extract. This study is in line with the research of Al-koshab et al. that reported that Ficus deltoidea extract could significantly reduce the tongue tumor volume of OSCC at a high dose of 500 mg/kg BW and decreased the expression of key tumor marker cyclin D1 and increased β-catenin and e-cadherin antibodies.30 Another study conducted by Roy M et al. uses black tea extract to prevent carcinogenesis by down-regulating protein tyrosine kinases, reducing ROS level in blood, and resolving DNA damage.37\n\nThe immune system is the main actor in the fight against the pathogenesis of many types of cancer, including OSCC. These complex defense mechanisms involve many varieties of lymphocytes. For OSCC, TIL might be a potential prognostic factor for determining the possibility of success of the treatment. Tumor-infiltrating lymphocytes are a heterogeneous collection of lymphocytes dominated by T cells. These cells generally appear in the stromal area and contain cytotoxicity activity against cancer cells. Caruntu et al. reported that TIL CD8+ lymphocytes and CD56+ in the intratumoral compartment can be predictive indicators of OSCC and they are associated with improved patient outcomes.38 This study could not find a significant difference in intratumoral TILs among groups, but it showed an increase in the level of stromal TILs at 500 mg of areca nut extract treatment. Previous study showed that stromal TILs was found to be better and more reproducible parameters than intratumoral TILs.39 The intratumoral TILs generally present in small numbers and mostly are detected in fewer cases of OSCC, more heterogeneous, and difficult to be observed on H & E-stained slides. Counting stromal TILs is more advantageous because stromal TILs have a clearer density and pattern of lymphocytes in carcinoma. Lymphocytes that are counted are only those in the spaces between carcinoma nests. In the microenvironment of a tumor, TILs may reflect tumor biology and predict the outcome of the treatment. Another study demonstrated that infiltrates of CD4+, CD8+, and FoxP3+ lymphocytes were associated with patient survival and can be potential biomarkers for OSCC.40\n\nAlthough this study has shown that the areca nut extract has a chemotherapeutic effect on 4NQO-induced rats in reducing tongue tumor volume and increasing the number of stromal TILs, there are several limitations to this study. First, this was a chemotherapeutic study that only investigated the efficacy of areca nut extract after tumor formation, while its function as chemopreventive study has not been explored especially in determining the antioxidant effect to prevent carcinogenesis in the initiation phase. Second, we did not explore the type of lymphocyte cells and tumor markers involved in the cytotoxicity mechanism of areca nut extract so we could not determine the exact mechanism of the areca nut extract in this chemotherapeutic activity. A more in-depth future experimental assay is needed to develop the results of this study.\n\n\nConclusion\n\nIn conclusion, we have demonstrated that areca nut extract exerts a chemotherapeutic effect 4 NQO-induced rat tongue when fed during the progression phase of carcinogenesis by increasing the number of infiltrating lymphocytes in the stromal tumor area. Additional works are needed to investigate the chemopreventive effect and the exact molecular pathway involved in the anticancer activity of areca nut extract.", "appendix": "Data availability\n\nDryad: Underlying data for ‘The Chemotherapeutic Activity of Areca Nut Extract Increased Stromal Tumor-Infiltrating Lymphocytes In 4-Nutriquinoline-1-Oxide-Tumor-Induced Sprague-Dawley Rats. https://doi.org/10.5061/dryad.mkkwh713d\n\nThe project contains the following underlying data: body, heart, lungs, liver, and kidney weights, tongue tumor volume, incidence of tongue dysplasia, intratumoral and stromal TILS, and Author checklist-E10 ARRIVE.\n\n\nReferences\n\nSari EF, Prayogo GP, Loo YT, et al.: Distinct phenolic, alkaloid and antioxidant profile in betel quids from four regions of Indonesia. Sci. Rep. 2020; 10(16254): 1–12.\n\nWen CP, Tsai SP, Cheng TY, et al.: Uncovering the relation between betel quid chewing and cigarette smoking in Taiwan. Tob. Control. 2005; 14(Suppl I): i16–i22. 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Publisher Full Text" }	[ { "id": "159547", "date": "02 Feb 2023", "name": "Maizatul Hasyima Omar", "expertise": [ "Reviewer Expertise Phytochemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the manuscript was logically organized with a good flow among the introduction, methods, results, discussion, and conclusion. However, it will be good if the authors could write in more detail in terms of the procedure for animal study, i.e. the exact duration (week/day). The authors also failed to explain the reason for a one week gap in all groups (Table 1). For Table 2, the Initial and Final should refer to the week or day which is more appropriate. For Table 6 the value for the mean is referring to what unit? i.e. 0.83 for Group 1.\nIt was reported that areca nut extract may caused oral cancer (Ji et al., 20141; Wadia, 20222), how should the authors respond to these with their findings in vivo?\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9420", "date": "20 Mar 2023", "name": "Liza Sari", "role": "Author Response", "response": "Dear Reviewer, Thank you very much for the detailed correction. I would like to answer the correction that I have made in this article. The procedure took 36 weeks starting with 1 week for the acclimatization period, 12 weeks for 4NQO-tumor induction, and 1 week for the observation period after the 4NQO-tumor induction. We continued the experiment with 22 weeks for treatment with the areca nut extract period. We have added this statement in the “Discussion” section, 1st paragraph. The observation period which is one week after the 4NQO-tumor induction aims to observe whether there was death or not in rats after being induced by 4NQO-tumor induction. This observation is done to anticipate if there was a dying rat that might happen in the early stage or right before the treatment experiment began. This period is also intended to prepare the rat’s condition before treatment, such as observing the activity, toxicity, consciousness, skin color, and body weight condition. After a week of observation, we continued with by 22 weeks treatment period with areca nut extract. We have added this statement in the “Discussion” section. Initial body weight is the weight calculated on the last day of the 1 week observation period after the 4NQO-tumor induction period was complete. Meanwhile, the final body weight is the weight calculated after 22 weeks of the treatment with areca nut. We have added this statement in the “Body weight analysis” section. This study adopted and modified the TIL scoring system used in previous studies. Firstly, the tumor area was divided into the center of the tumor and the invasive margin according to the criterion in the study of Galon et al. The invasive margin was defined as the junctional area between the tumor-invading edge area and the host stroma. The TIL evaluation was conducted operating in the feature of TIL intensity in the center and the invasive margin of the tumor was incorporated. TILs in each tumor on the tissue were manually using a 20x objective lens. The mean count of the scores for each subject was recorded and used in the analysis. TIL assessment was done by two pathologists who were blinded to the clinical appearance of the tumor. We have added this statement in the “Morphological evaluation of tumor-infiltrating lymphocytes” section. References: 1. Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, et al. Towards the introduction of the 'Immunoscore' in the classification of malignant tumors. J Pathol 2014;232:199–209. 2. Zhang D, He W, Wu C, Tan Y, He Y, et al. Scoring System for Tumor-Infiltrating Lymphocytes and Its Prognostic Value for Gastric Cancer. Front. Immunol. 2019;10(71):1-11. Several differences may cause different results between Ji WT's article and this article: Article written by Ji WT et al. using lyophilization or freeze-drying in extracting areca nut. This method is one of the drying methods to produce a stable powder product. In our study, we did not use this method. We use the maceration technique which has the advantage of being able to retain the phytochemical compounds present in the areca nut. This technique used 95% ethanol and dried again by using a rotary evaporator until we had a solid powder of areca nut. The extract has also been tested for antioxidant activity DPPH with a value of 15.95 µg/mL. This result indicates that areca nut extract has good antioxidant activity. The extract also contains total flavonoids 238,5 mg CE/g, and total phenolic 80.3 mg TAE/g. We also found that there was a high level of catechin and quercetin in the extract (HPLC assay). We concluded that different types of extraction methods and areca nut origin types might give different results in areca nut’s cellular or antioxidant activities in various research. We have also done the experimental regarding the cytotoxicity and apoptosis activity of areca nut in HSC-2, HSC-3, and Hacat cell lines. The results showed good results regarding the capability of areca nut in inducing apoptosis of oral cancer cell lines. It has been published in https://f1000research.com/articles/7-723 and https://scholar.ui.ac.id/en/publications/potential-antioxidant-and-cytotoxic-activities-of-areca-nut-areca. We agree with Wadia R’s article about areca nut and oral cancer. Please let us give our opinion. There are a few reasons why areca nut can cause oral cancer, but in my article, areca nut has anticancer activity. Areca nut does not contain carcinogenic substances, but this carcinogenic effect is caused by nitrosamines product in the long term and is uncontrollable. Nitrosamines are produced by nitrosation of the alkaloids in dried stored nuts, when in the mouth, and especially in the acid conditions of the stomach, in the presence of nitric oxide generated by bacteria. The combination with nitric oxide, produced by bacteria, causes the production of methylnitrosaminoproprionitrile which is proven to cause carcinogenesis in animal studies. This endogen nitrosation is higher significantly in a patient with bad oral hygiene. If areca nut is combined with tobacco, then chewed by people with bad oral hygiene, there will be a very high accumulation of nitrosamines products. This process usually occurs continuously for the long term because the seeds have addictive properties. Some studies also reported increased reactive oxygen species such as hydroxyl oxide in the oral cavity caused by a combination of polyphenol autooxidation from areca nut with high alkaline pH of slaked lime (calcium hydroxide paste). If the areca nut is chewed with piper betel and slaked lime, these two materials will cause mucous membrane erosion so that a carcinogenic substance could easily penetrate cells through the mucous membrane. Some part of communities in India and Pakistan use industrial packaging areca nuts called gutka and pan masala. Gutka contains areca nut, piper betel, tobacco, and slaked lime, while pan masala was prepared without tobacco. Approximately, 40% of gutka and pan masala packaging are contaminated with aflatoxin which has carcinogenic properties from Aspergillus flavus, Aspergillus niger, and Rhizopus spp. fungi. The occurrence of the OSCC's risk depends greatly on the composition of the compound which determines the quality of the seeds, the method of using seeds which are associated with oral hygiene, duration of use, the presence or absence of toxins caused by fungi contamination in the seeds, and the presence or absence of other carcinogenic substances such as tobacco and slaked lime. The occurrence of OSCC could also be influenced by several factors such as intrinsic factors (tumor suppressor gene abnormality or mutation and oncogene) and extrinsic factors (tobacco smoking, vitamin A and iron deficiency, candidiasis, viral infection, and immunosuppression)." } ] } ]	1	https://f1000research.com/articles/11-1571
https://f1000research.com/articles/12-1299/v1	11 Oct 23	{ "type": "Research Article", "title": "Evaluating community-wide temporal sampling in passive acoustic monitoring: A comprehensive study of avian vocal patterns in subtropical montane forests", "authors": [ "Shih-Hung Wu", "Jerome Chie-Jen Ko", "Ruey-Shing Lin", "Chia-Hao Chang-Yang", "Hsueh-Wen Chang", "Shih-Hung Wu", "Jerome Chie-Jen Ko", "Ruey-Shing Lin" ], "abstract": "Background: Passive acoustic monitoring (PAM) has become a popular tool for bird monitoring, with vocal activity rate (VAR) being a key metric to gauge bird populations. However, the effective temporal sampling design at the community level for representative VAR data remains underexplored.\nMethods: In this study, we used vocalizations extracted from recordings of 12 bird species, taken at 14 PAM stations situated in subtropical montane forests over a four-month period, to assess the impact of temporal sampling on VAR across three distinct scales: seasonal, diel, and hourly. For seasonal sampling analysis, we employed hierarchical clustering analysis (HCA) and the coefficient of variation (CV). Generalized additive models (GAMs) were utilized for diel sampling analysis, and we determined the average difference in VAR values per minute for the hourly sampling analysis.\nResults: We identified significant day and species-specific VAR fluctuations. The survey season was divided into five segments; the earliest two showed high variability and are best avoided for surveys. Data from days with heavy rain and strong winds showed reduced VAR values and should be excluded from analysis. Continuous recordings spanning at least seven days, extending to 14 days is optimal for minimizing sampling variance. Morning chorus recordings effectively capture the majority of bird vocalizations, and hourly sampling with frequent, shorter intervals aligns closely with continuous recording outcomes.\nConclusions: While our findings are context-specific, they highlight the significance of strategic sampling in avian monitoring, optimizing resource utilization and enhancing the breadth of monitoring efforts.", "keywords": [ "passive acoustic monitoring", "vocal activity rate", "temporal sampling", "Aves" ], "content": "1. Introduction\n\nBiodiversity is paramount for the sustainable progression of human society and environmental preservation. It contributes either directly or indirectly to all 17 of the Sustainable Development Goals (SDGs) (Blicharska et al., 2019). Monitoring biodiversity is a crucial endeavor that facilitates the comprehension of the current state, alterations, and trends of biodiversity and evaluates the efficacy of interventions aimed at mitigating biodiversity loss (Pereira & Davidcooper, 2006). Birds serve as an ideal indicator taxon for monitoring terrestrial biodiversity due to their detectability, identifiability, diversity, widespread distribution, and migratory characteristics (Fraixedas et al., 2020). Bird monitoring can shed light on the effects of habitat loss, deforestation, climate change, invasive species, light pollution, and illegal hunting (Xu et al., 2019; Northrup et al., 2019; Dueñas et al., 2021; La Sorte et al., 2022; Crespo et al., 2020; Negret et al., 2021), while also highlight the beneficial outcomes of conservation efforts (Cazalis et al., 2020).\n\nBeyond human observations, various technologies including radar, thermal imaging, and passive acoustics have been employed for manual or automatic bird monitoring (Lahoz-Monfort & Magrath, 2021). In recent years, PAM has seen an upsurge in its use for bird monitoring and research (Hoefer et al., 2023). Thanks to decreasing costs, autonomous recording units (ARUs) can now be extensively deployed across diverse environments, recording considerable volumes of soundscape data. These data provide rich biological information and allow continuous, automated monitoring, thereby significantly increasing both the temporal and spatial coverage of these efforts (Ross et al., 2023; Pérez-Granados et al., 2021; Sugai et al., 2019; Shonfield & Bayne, 2017). However, the labor-intensive and time-consuming process of identifying species sounds within soundscape data presents a significant bottleneck for PAM utilization. The rise of automated identification tools, such as BirdNET and SILIC, are steadily alleviating this issue (Kahl et al., 2021; Wu et al., 2022). Despite these advancements, handling large volumes of audio files remains a considerable challenge due to the high energy requirements for extended ARU field operations, the need for extensive storage space, and lengthy analysis time (Zwerts et al., 2021).\n\nSampling has been employed as an effective and commonly used method to decrease the amount of recording data. Sampling design can be categorized into four temporal scales: annual, seasonal, diel, and hourly. An annual sampling design implies recording during one or several time periods within a year. Many bird species exhibit enhanced vocalization during the breeding season; hence, the majority of studies prefer to conduct PAM surveys within this timeframe of a year (Campos-Cerqueira & Aide, 2016; Bateman et al., 2021; Duchac et al., 2020). Alternatively, certain studies opt for acoustic surveys during the non-breeding season, a period characterized by relatively stable detectability and community composition (Metcalf et al., 2021). Seasonal sampling design is often employed in light of limited ARU availability, necessitating a rotational system among diverse survey locations. Typically, after being operated for a predetermined number of days at each location, the ARU is relocated to a subsequent site. This rotation ensures a comprehensive collection of crucial soundscape data from each location throughout the survey season (Jahn et al., 2022; Machado et al., 2017).\n\nThe diel sampling design is typically framed based on the behavioral patterns of the target species. For instance, as Passeriformes frequently vocalize during the dawn and dusk choruses, the recordings are concentrated around these periods (Alvarez-Berríos et al., 2016; Deichmann et al., 2017; Rumelt et al., 2021). For nocturnal birds, recordings are conducted during the night (Jahn et al., 2022; Wood et al., 2020). Hourly sampling design can be categorized into coverage (the proportion of recorded time within an hour) and dispersion (the number of recording segments within an hour). Examples of such strategies might include recording a one-minute segment (Diepstraten & Willie, 2021) or a fifteen-minute segment (Pérez-Granados et al., 2021) within an hour, or perhaps recording one minute every ten minutes (Ducrettet et al., 2020; Melo et al., 2021) or every fifteen minutes (Yoo et al., 2020), and even recording fifteen minutes every half hour (Favaro et al., 2021).\n\nThe vocal activity rate (VAR) of birds, defined as the quantity of vocalizations per unit of time, can be derived from PAM data. VAR is a pivotal metric in acoustic surveys, enabling the estimation of bird abundance or density, which is crucial for monitoring avian population (Pérez-Granados et al., 2019a; Pérez-Granados & Traba, 2021). However, avian vocal activity is modulated by an intricate blend of both exogenous and endogenous factors. This results in diverse vocal patterns that vary by species, gender, age, temporal factors, environmental conditions, site-specific characteristics, habitat types, and social contexts (Catchpole & Slater 2003; Marques et al. 2013; Bruni et al., 2014; Digby et al., 2014; Symes et al., 2022). The VAR is substantially influenced by its temporal sampling design. For studies targeting individual species or a limited group, the temporal sampling can be customized to their specific behaviors (Pérez-Granados et al., 2019b). Yet, when the scope encompasses an entire avian community, an optimally structured survey should capture the most prevalent species and a significant proportion of the less common ones (Franklin et al., 2021). Past research has underscored the profound impact of the recording schedule on assessments of avian community richness and composition. For example, prolonging recording durations generally augments species detection, especially for less common species (Wood et al., 2021; Symes et al., 2022). Concentrating recordings during specific time, like dawn, often capture more species but may miss those from distinct functional groups (Shaw et al., 2022). Nevertheless, the effects of temporal sampling designs on the VAR of individual species within a community remain under-investigated.\n\nThis study aims to evaluate the impact of various temporal sampling methodologies on the identification of VAR patterns in a biotic community, with a focus on avian communities in subtropical montane forests. Our objectives include: (1) to characterize the VAR patterns of 12 representative bird guilds inhabiting these forests; (2) to investigate the influence of three different time-scale sampling designs – seasonal, diel, and hourly – on the perceived VAR patterns; and (3) to provide strategic recommendations for optimal temporal sampling strategies to maximize the utility of limited research resources. By aligning the best practices of sampling strategies with available resources, we believe our findings will promote efficient and effective passive acoustic monitoring, thereby contributing to the conservation of avian communities and terrestrial biodiversity.\n\n\n2. Methods\n\nThis study was conducted in the southern sector of Yushan National Park (YSNP) situated in central Taiwan, encompassing an expanse greater than 100,000 hectares (Underlying data: Figure S1 (Wu et al., 2023)). YSNP is named after Yushan or Jade Mountain, renowned for its highest peak in Northeast Asia with an elevation of 3,952 meters. This national park is pivotal in sustaining high-altitude ecosystems, transitioning from subtropical zones at its base to alpine zones at higher altitudes. The mean annual precipitation recorded is around 3,600 mm. Altitudinal variation influences the average annual temperatures: approximately 20°C at 1,000 meters, around 10°C at 2,500 meters, and roughly 5°C beyond 3,500 meters (Minister of the Interior, 2022).\n\nFor the data collection, 14 PAM stations were deployed within the study area. These stations spanned a distance of approximately 10 kilometers along the Southern Cross-Island Highway, from Meishan (23°15′51″N, 120°49′33″E) to Yakou (23°15′51″N, 120°57′28″E) (Underlying data: Figure S1 (Wu et al., 2023)). The altitude of the initial station, SCIH07, was 1,500 meters, and subsequent stations were set up at 100-meter elevation increments, terminating at an elevation of 2,800 meters with station SCIH20. The distance between any two adjacent stations ranged from 750 and 1,850 meters. This elevation gradient encapsulated four distinctive vegetation types: submontane evergreen broad-leaved forest, montane evergreen broad-leaved forest, montane mixed coniferous-broadleaved forest, and upper montane coniferous forest (Minister of the Interior, 2022) (Underlying data: Table S1 (Wu et al., 2023)).\n\nEach PAM station was equipped with a Song Meter Mini recorder (Wildlife Acoustic Inc.) designed to capture soundscape data. These devices, anchored to trees at an average height of 1.5 meters, operated continuously throughout the day between March and June 2021. This period corresponds with the breeding season of the region’s montane forest avian species. The recording configurations were set to mono mode, capturing audio in a 16-bit WAV format with a sampling rate of 44.1 kHz. To facilitate subsequent analytical procedures, the recordings were segmented and stored in three-minute durations.\n\nTo gain a comprehensive understanding of individual species’ status within a community, we utilized ecological guilds as our primary criterion for selecting target species. Ding (1993) cataloged 59 avian species recognized as montane forest breeders and categorized them into 12 distinct ecological guilds within YSNP: raptorial carnivores (RC), ground graminivores (GG), ground omnivores (GO), ground insectivores (GI), bush insectivores (BI), tree fruitivores (TF), tree omnivores (TO), tree insectivores (TI), bole gleaners (BG), bole peckers (BP), tree hoverers (TH), and air flycatchers (AF).\n\nTo identify our research’s target species, a singular species was selected as a representative from each guild. When a particular guild included more than two species, we used trait data from Tsai et al. (2020) regarding Taiwan’s breeding birds to inform our selection. The species that manifested an altitudinal distribution most congruent with our study’s objectives was then chosen.\n\nAs a result, we designated 12 bird species as our primary focus, including Collared Owlet (Taenioptynx brodiei), Large-billed Crow (Corvus macrorhynchos), Taiwan Bush Warbler (Locustella alishanensis), Grey-chinned Minivet (Pericrocotus solaris), Taiwan Vivid Niltava (Niltava vivida), Eurasian Nuthatch (Sitta europaea), Taiwan Rosefinch (Carpodacus formosanus), Taiwan Yuhina (Yuhina brunneiceps), Taiwan Shortwing (Brachypteryx goodfellowi), Ashy Wood-Pigeon (Columba pulchricollis), Green-backed Tit (Parus monticolus), and Gray-headed Woodpecker (Picus canus). Each of these species was emblematic of the 12 aforementioned guilds (Underlying data: Table S1 (Wu et al., 2023)).\n\nWe selected SILIC, an automated wildlife sound identification tool recently developed based on the YOLOv5 object detection model and spectrogram images (Wu et al., 2022), for detecting bird vocalizations in our study. This choice was primarily motivated by two key attributes of SILIC. Firstly, it can recognize the vocalizations of 141 bird species native to Taiwan, encompassing all of our 12 target species. Secondly, SILIC offers a unique capability to detect each vocalization’s exact start and end time within an audio recording, rather than merely identifying the presence or absence of a certain vocalization within a broad time frame. This feature enables precise computation of the VAR, a crucial metric for our research.\n\nSILIC categorizes sounds into ‘sound classes’ rather than by species. Of the 12 target species we studied, each had 1 to 5 sound classes in SILIC, including ‘song’, ‘call’, ‘drumming’, and ‘unknown’ (a classification with an undetermined function). As song is common during many bird species’ breeding, it was our primary choice. If multiple song classes were provided by SILIC, we consulted experts to select the prevalent one. For species without a song or the song was understated and hard to discern, we chose a frequently observed sound class. Accordingly, ‘song’ represented 9 of the 12 species, ‘call’ 2, and ‘unknown’ 1. The spectrograms representing the selected sound classes are provided in the Underlying data: Figure S2 (Wu et al., 2023).\n\nWe utilized SILIC to extract vocalizations of our target species from the soundscape recordings. Each three-minute recording was segmented into three-second spectrogram clips and analyzed using a one-second sliding window. Due to the overlapping nature of the sliding window, one vocalization might be detected multiple times. For detections of the same species within a single recording, if the intersection area of two overlapping bounding boxes (each bounding box representing a specific vocalization in the spectrogram) divided by the area of the smaller bounding box exceeded 0.25 (Underlying data: Figure S3 (Wu et al., 2023)) or if the intersection area divided by the union area exceeded 0.1 (Underlying data: Figure S4 (Wu et al., 2023)), the two bounding boxes (vocalizations) were combined.\n\nA random sample of 100 detected vocalizations for each species, each tagged with a confidence score (ranging from 0 to 1, indicating the level of certainty that the vocalization belongs to a particular species), was manually reviewed. This set of manually reviewed detections constituted our test dataset for evaluating the performance of SILIC on our soundscape recordings. We created a confusion matrix consisting of four parameters: true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN). Subsequently, we calculated precision as TP/(TP+FP) and recall as TP/(TP+FN). Additionally, we computed the area under the receiver operating characteristic curve (AUC) and the average precision (AP), the latter being equivalent to the area under the Precision-Recall curve. Detailed calculations of these performance metrics can be found in the Underlying data: Appendix S2 (Wu et al., 2023). Finally, we identified the confidence scores corresponding to the maximum F1-score values and designated these as the threshold scores for each species. Detected vocalizations exceeding these confidence score thresholds were classified as positive detections and were incorporated in subsequent analyses.\n\n2.5.1 Environment factors\n\nAs previously stated, bird vocal activity is influenced by multiple factors. In addition to species and seasonality, it is also affected by environmental factors such as climatic conditions, topographic features, and vegetation type. We collected data on each station’s altitude (in meters) and vegetation types (as described in the Soundscape data collection section). Daily climate data, including corrected precipitation (Rainfall; mm), wind speed at two meters (WindSpeed; m/s), temperature at two meters (Temp; °C), relative humidity at two meters (RH; %), and surface pressure (SP; kPa), were downloaded from the NASA/POWER CERES/MERRA2 Native Resolution Daily Data website (https://power.larc.nasa.gov/data-access-viewer/).\n\nWe employed a forward stepwise selection approach to develop four GAMs to evaluate the relationship between the response variable, daily vocal activity rate (VAR_d, representing the quantity of vocalizations per day), and nine exploratory variables. The initial model contained only categorical variables ‘Species’ and ‘Vegetation’, as well as their interaction effect. In the second model, a smooth term for ‘Altitude’ for each ‘Species’ was incorporated. The third model included five additional smooth terms for meteorological variables, namely ‘Rainfall’, ‘WindSpeed’, ‘Temp’, ‘RH’, and ‘SP’. Finally, a smooth term for the day of year (DOY) for each ‘Species’ was included in the final model.\n\nAs VAR_d is discrete and highly positively skewed count data, we chose a negative binomial distribution function and a log link function. The collinearity of explanatory variables was assessed using variance inflation factors (VIF), with variables demonstrating values greater than five being discarded, following Zuur et al. (2007). A Thin Plate Regression Splines (TPRS) smoother was employed for each smooth term as it can capture a wide variety of functional forms, making them suitable for modeling complex nonlinear relationships without having to specify the functional form in advance. The performance of the models was assessed by calculating the deviance explained and Akaike’s Information Criterion (AIC). The analyses were conducted using the “mgcv” package (version 1.9-0) in R statistical software (version 4.3.1).\n\n2.5.2 Seasonal sampling\n\nIn a single survey season, should there be an insufficient number of ARUs for concurrent recordings across all sampling sites, necessitating rotation, discrepancies in temporal setup could introduce biases. Such inconsistencies might undermine the reliability of subsequent analyses. For example, shifts in breeding statuses over time might influence vocalization frequencies (Slagsvold, 1977). Thus, during experimental design, it is imperative to target a timeframe wherein temporal variations in avian vocalizations are minimal. Essentially, intervals should be chosen where daily vocal patterns remain sufficiently stable to complete rotation recording within that period. Given the time constraints associated with ARU rotation, reducing deployment durations at individual sites might allow more rotation cycles. Yet, exceedingly short recording spans might risk data bias (Pérez-Granados et al., 2019b). In the segment discussing seasonal sampling, we address two facets: (1) Time window: pinpointing intervals throughout the PAM survey season when bird acoustic activity is relatively uniform; and (2) Survey duration: ascertaining the required days for a PAM survey to garner representative data.\n\nFor the time window, we employed HCA to group dates with similar vocal activity patterns. We first calculated daily VAR (VAR_d) separately for each station and species, and normalized the data using Z-score normalization to achieve a mean value of zero and a standard deviation of one. Subsequently, we conducted HCA to identify dates with Euclidean distance and Ward’s linkage. The root-mean-square error (RMSE, the square root of the sum of squared distances between any two samples within a cluster divided by the number of samples) and the within-cluster maximum distance (WCMD, the greatest distance between any two samples within a cluster) were computed as indicators of data homogeneity within clusters. The clustering analysis and metric calculations were performed using Python (version 3.10.9), with the SciPy (version 1.10.0) and Scikit-learn (version 1.2.1) packages. Visualization was accomplished using the Matplotlib package (version 3.7.0).\n\nFor the survey duration, we utilized the CV of daily VAR (VAR_d) values across various consecutive survey durations as an evaluation metric to assess the influence of different consecutive survey durations on the reliability of VAR_d values. Initially, we defined the consecutive survey durations between one to 14 days for test based on the PAM deployment durations used in previous works (Machado et al., 2017; Wood et al., 2020; Metcalf et al., 2021; Rumelt et al., 2021; Jahn et al., 2022). For each duration, starting from day one, we extracted the corresponding data, calculating the VAR_d values and their mean. Upon completion of calculations, we moved forward day by day and repeated the extraction and calculation process for the same duration until all days were covered. Finally, from the obtained average VAR_d values, we calculated their standard deviation and mean. The ratio of these two values yielded the CV. We applied this calculation to each homogeneous cluster obtained from the Time window analysis, allowing a comparative assessment of CV differences across clusters.\n\n2.5.3 Diel sampling\n\nTo conserve both the storage capacity of audio files and the time required for analysis, recordings are typically sampled during peak vocalization periods of target species within a day. We divided the day into 24-hour segments. Using a GAM, we evaluated the association between an hourly VAR response variable (VAR_h, denoting the quantity of vocalizations per hour) and three explanatory variables: species (categorical), DOY (smooth term), and hour (smooth term). Due to the same rationales applied in the environmental factor analysis, we employed a negative binomial distribution with a log link function and a TPRS smoother. The collinearity of the explanatory variables was evaluated using the VIF; variables with VIF values greater than five were excluded. To represent the 24 hours in a day, a value of 24 basis dimensions (k) was utilized. All analyses were performed using the “mgcv” package (version 1.9-0) in the R statistical software (version 4.3.1).\n\n2.5.4 Hourly sampling\n\nWe examined the impact of diverse sampling strategies within an hourly timeframe on the VAR value per minute (VAR_m), concentrating on two main dimensions: coverage (representing the ratio of time recorded in an hour) and dispersion (indicating the number of recording intervals within an hour, with an X:Y format signifying cycles of X minutes of recording followed by Y minutes of inactivity). Based on seven coverage patterns, we formulated 21 unique sampling combinations for each hour, as elaborated in Table 1. For every species, date, and hour, we derived the mean VAR_m values from both continuous recordings and various sampling strategies, subsequently determining the difference between these values. In the end, we computed the average difference in VAR_m values across different species and sampling designs. A reduced mean difference indicates closer alignment between the VAR_m values from continuous recording and a particular sampling design.\n\nThis table lists 21 distinct sampling combinations derived from seven temporal coverage designs. ‘Temporal coverage’ indicates the fraction of an hour recorded. In the ‘Sampling combinations’ column, the format X:Y designates cycles of X minutes of recording (ON) succeeded by Y minutes of pause (OFF).\n\n\n3. Results\n\nBetween March 1 and June 30, 2021, spanning 122 working days, a total of 789,986 three-minute audio files were collected across 14 sampling stations, approximately totaling 39 thousand hours. During this period, two stations, SCIH11 and SCIH18, failed to record audio files from mid-May to mid-June due to memory card issues. These two stations were subsequently excluded from further analysis. Of the remaining 12 stations, due to deployment scheduling, equipment operation, and battery management issues, at least one station had days where recorded data did not reach 23.5 hours for a total of 10 days. Data from these specific days (i.e., DOY 60–62, 141, 168, and 177–181) were also omitted from further analysis. Hence, data from 12 stations over 112 days continued for subsequent analysis.\n\nWe employed SILIC (Version exp29) for automated sound detection. Upon manual inspection by experienced bird surveyors of the 1,200 randomly sampled entries, 424 were confirmed as true detections, while 776 were false detections. AUC scores for each species, derived from the test set, ranged from 0.87 to 1.0, and AP scores ranged from 0.85 to 1.0. This demonstrates the excellent detection performance of SILIC within the scope of this study, making it apt for further analysis.\n\nWe selected the confidence score at which the precision score for each species was not less than 0.95 as the threshold to minimize the occurrence of false positives. Sound detection results with a confidence score greater than or equal to the threshold were screened for subsequent analysis. In total, 8,202,731 vocalizations from 12 species were detected, with the Taiwan Yuhina having the highest count at 2,863,838, and the Gray-headed Woodpecker the lowest at 23,312. Detailed data on vocalizations detected by SILIC can be found in the Underlying data: Table S2 (Wu et al. (2023)). For comprehensive information on the test datasets, threshold values, and various performance metrics for each species, please refer to the Underlying data: Table S3 (Wu et al. (2023)). Precision and recall curves are provided in the Underlying data: Figure S5 (Wu et al. (2023)).\n\nCollinearity tests revealed that the variance inflation factor (VIF) for all exploratory variables was below 5, hence all variables were retained for forward stepwise GAM modeling and fitting. When only using species and vegetation types as predictors, the deviance explained is 50.1%. Incorporating altitude as a smoothed term, differentiated by species, increased the deviance explained to 62.9%. Subsequently, by adding five climatic variables as smoothed terms, the deviance explained rose to 68%. Finally, introducing DOY while distinguishing among species further increased the deviance explained to 73.2%. This demonstrates that each variable contributes to the prediction of daily vocal activity rate (VAR_d), as shown in Table 2.\n\nThis table summarizes the outcomes of a forward stepwise variable selection procedure, detailing the Akaike Information Criterion (AIC), adjusted R2, and proportion of deviance explained. The GAM predicts the daily vocal activity rate (vocalizations per day) based on interactions between Species and Vegetation types. The model includes smoothed effects of Altitude (meters, varying by Species), corrected precipitation (Rainfall, mm), wind speed at 2 meters (WindSpeed, m/s), temperature at 2 meters (Temp, °C), relative humidity at 2 meters (RH, %), surface pressure (SP, kPa), and Day of the Year (DOY, varying by species). Weather data were sourced from the NASA/POWER CERES/MERRA2 Native Resolution Daily Data repository, accessible at https://power.larc.nasa.gov/data-access-viewer/. Vegetation data were provided by the Minister of the Interior (2022).\n\nThe model with the lowest AIC, encompassing all nine exploratory variables, was chosen. Most fixed effects and all smoothed terms (including altitude, DOY, and climatic variables) were found to have a significant impact on the predictive capacity of the model, as detailed in the Underlying data: Table S4 (Wu et al., 2023).\n\nWhile all 12 species exhibited significant correlations between DOY and the VAR_d, the patterns of these relationships were not consistent across species. Some species displayed a strong positive correlation in the early stages of the survey period, which shifted to a pronounced negative correlation in later stages. Conversely, other species demonstrated the opposite pattern (refer to the Underlying data: Figure S6 (Wu et al., 2023)). Similar inconsistencies between species were observed in relation to altitude (see the Underlying data: Figure S7 (Wu et al., 2023)).\n\nWhen rainfall was less than 40 mm, there was no apparent influence on VAR_d. However, beyond this threshold, VAR_d showed a rapid decrease, with effects diminishing after approximately 60 mm. Wind speeds of up to 3.0 m/s had no discernible effect on VAR_d, but rates declined sharply beyond this speed. For temperatures up to 20°C, VAR_d gradually increased as temperatures rose, with no apparent effects beyond this threshold. Relative humidity had a slight negative effect on VAR_d once it exceeded 80%. As for atmospheric pressure, the impact on VAR_d shifted from negative to positive as pressure moved from low to high. For more detailed information, please refer to the Underlying data: Figure S8 (Wu et al., 2023).\n\nUpon examining the dendrogram derived from HCA (Figure 1), it becomes evident that at a Euclidean distance of 35, the entire survey period can be partitioned into five distinct clusters. Notably, Clusters 3, 4, and 5 exhibit substantially lower root-mean-square error (RMSE) and within-cluster maximum distance (WCMD) values compared to Clusters 1 and 2, indicating more homogeneous distributions of VAR_d within these clusters (Table 3). An observation of the DOY distribution within the five clusters reveals a largely sequential pattern over time, with only two exceptions (DOY 83 and 119) both falling within Cluster 5. Clusters 1 and 2 encompass 12 and 18 days respectively, approximately aligning with the first and latter halves of March. Conversely, Clusters 3, 4, and 5, each containing no fewer than 25 days, roughly correspond to the months of April, May, and June, respectively (Figure 2).\n\nDendrogram derived from hierarchical clustering of dates based on daily vocal activity. At a Euclidean distance threshold of 35 (indicated by the grey dashed line), five clusters are discerned (numbered circles).\n\nThis table details the five clusters identified based on daily vocal activity rates. The root-mean-square error (RMSE), within-cluster maximum distance (WCMD), number of days, and specific DOYs are provided for each cluster.\n\nThe outer ring indicates vocal activity clusters distinguished by colors: 1 (red), 2 (orange), 3 (blue), 4 (green), and 5 (yellow). Day of Year (DOY) is shown counterclockwise from the right on the ring's periphery. Within this ring, dashed lines represent rainfall (in mm) from the SCIH13 PAM station, centrally located in the survey area. Two outlier dates in Cluster 5 correspond to rainfall events. The inner ring denotes the months: March to June.\n\nIn Figure 3, the CV of mean VAR_d values spanning one to fourteen consecutive recording days is presented. Due to the fewer days encompassed by Clusters 1 and 2, when calculating the CV values for a higher number of days, there might be a tendency to obtain relatively lower CV values owing to the smaller sample sizes. Thus, this aspect is not elaborated upon further. In contrast, Clusters 3, 4, and 5 consist of a greater number of days. Among the three, Cluster 4 exhibits the lowest CV values, where the decline becomes less pronounced after recording for more than seven days. Conversely, the decrease in CV values for Clusters 3 and 5 persists until recordings reach 14 days, with their final CV values still slightly exceeding that of Cluster 4 when the recording duration is set at seven days.\n\nThe graph displays the Coefficient of Variation (CV) against consecutive survey days (X-axis) for distinct clusters identified through hierarchical clustering (represented by colored lines). The number of days in each cluster is indicated in parentheses next to the cluster number in the legend. The Y-axis marks the CV values. Interpreting CV values for clusters 1 and 2 should be cautious, particularly for extended survey durations. Their limited days might result in artificially low CV values, potentially underestimating variation.\n\nCollinearity analysis revealed that the variance inflation factor (VIF) for all explanatory variables was less than 5. Thus, all variables were retained for GAM modeling. The fit of the GAM indicated a deviance explained of up to 80.8% (adjusted R2 = 0.70). All species exhibited a significant influence on VAR_h with respect to the hour (p < 0.001). Dawn and dusk are defined as approximately an hour before and after sunrise and sunset, respectively. Given the study’s duration of four months, sunrise times oscillated between approximately 5 am and 6 am, while sunset times ranged from around 6 pm to 7 pm. Consequently, dawn is represented from 4 am to 7 am, and dusk from 5 pm to 8 pm.\n\nObservations from Figure 4 regarding the hourly impact on VAR_h reveal that, except for the Collared Owlet and the Taiwan Bush Warbler, the remaining 10 species exhibited a significant positive influence on VAR_h during dawn. This influence gradually declined during the day and swiftly transitioned from a positive to a negative impact at dusk. Throughout the night, a consistent, highly negative influence was observed. The Collared Owlet displayed rapid fluctuations in its influence on VAR_h during dawn and dusk, transitioning from a mild positive effect during the day to a negative one, and maintaining a mildly negative influence at night. The Taiwan Bush Warbler transitioned from a strong negative impact on VAR_h during the late night to a positive one, peaking just before dawn and then declining. During the day, it transitioned from a mild positive to a negative influence, and finally, it exhibited intense fluctuations during dusk, soaring from a negative to a pronounced positive influence before plummeting to a strong negative impact.\n\nEach panel displays the GAM-predicted relationship between the hour of the day and the hourly vocal activity rate (VAR_h) for a specific species: (a) Collared Owlet, (b) Large-billed Crow, (c) Taiwan Bush Warbler, (d) Grey-chinned Minivet, (e) Taiwan Vivid Niltava, (f) Eurasian Nuthatch, (g) Taiwan Rosefinch, (h) Taiwan Yuhina, (i) Taiwan Shortwing, (j) Ashy Wood-Pigeon, (k) Green-backed Tit, and (l) Gray-headed Woodpecker. The y-axis represents the smooth effect of hour on VAR_h for each species. The solid blue line represents the predicted deviation with a 95% confidence interval (blue dashed lines). A reference line is shown at Y=0 (red dashed line). A deep gray shade indicates nighttime, while dawn (approx. 4 AM to 7 AM) and dusk (approx. 5 PM to 8 PM) are highlighted in light gray, reflecting variations due to sunrise and sunset times over the study's four-month span. Accompanying each plot are the estimated degrees of freedom (edf) and significance codes (* p < 0.001, p < 0.01, * p < 0.05).\n\nAnalysis of 21 distinct combinations of coverage and dispersion revealed that higher proportions of recording time, coupled with shorter and more dispersed recording segments, result in VAR_m values from sampling more closely aligning with those from continuous recordings. This trend was consistent across all target species, as illustrated in Figure 5.\n\nThe graph showcases the mean differential VAR_m (vocalizations per minute) between continuous recordings and various sampling methods for each species. Each subplot presents six species differentiated by unique colors. The x-axis lists sampling designs, ordered by decreasing coverage (proportion of the hour recorded) and dispersion (denoted as X:Y, indicating X minutes of recording followed by Y minutes of pause). A reduced mean differential suggests a closer match between the VAR_m from the sampling method and the continuous recording.\n\n\n4. Discussion\n\nIn this study, while we only selected 12 bird species representing different guilds and chose a single sound type for each species, both the chosen species and vocalization types constitute only a small fraction of the entire avian community and soundscape. Nevertheless, the extensive data extracted offer insights into the substantial variability in avian vocal activity rates across three distinct temporal scales, underscoring the significance of temporal sampling design for studies primarily employing PAM.\n\nWe demonstrate that avian vocal behaviors in subtropical montane forests are influenced by a complex interplay of internal and external factors. We show that the VAR pattern is strongly affected by individual species’ interaction with vegetation, altitude and DOY. Climate also plays a significant role, impacting VAR across all species. These findings emphasize the significant challenges posed by utilizing PAM to infer the population status and trends of a specific species. These challenges become even more pronounced when the monitoring effort is directed at multi-species bird assemblages, especially when constrained by equipment and time. Consequently, choosing the most appropriate recording sampling design is crucial to ensure data representativeness and comparability.\n\nThroughout a bounded time period, like the breeding season examined here, researchers often consider the entire duration as a closed population, executing repeated data collections and comparative analyses within this window (Baillie, 1991). However, our analysis of seasonal time sampling revealed pronounced species-specific temporal variations in vocal data across a seasonal gradient. We partitioned the survey season into five clusters, each reflecting relatively consistent vocal patterns. The vocal behaviors within the first two clusters displayed notable intra-cluster variability, potentially attributable to temporal nuances in breeding phases and vocalization rates among different species (Slagsvold, 1977). Given the observed variability in vocal behaviors early in the season, we recommend extra care when starting acoustic surveys at the beginning of the breeding season.\n\nFor intervals characterized by minimal daily vocalization frequency shifts, we propose that a continuous 14-day recording effectively diminishes sampling variance. During phases with even more consistent patterns, such as the fourth cluster identified in this study (circa May), a seven-day recording suffices. Moreover, we advise discarding data from days with rainfall exceeding 40 mm and average wind speeds surpassing 3.0 m/s. These environmental conditions have been empirically demonstrated to considerably dampen vocal activity rates, a phenomenon corroborated in other avian studies (Vokurková et al., 2018; Robbins, 1981).\n\nRegarding diel sampling, even though our primary emphasis was on diurnal birds peaking in morning vocalizations, certain species, like the Taiwan Bush Warbler in this study, and approximately 30% of North American birds vocalize nocturnally (La, 2012). We therefore recommend sampling approximately one hour before and after sunrise to coincide with the morning chorus. If resources permit, recording sessions could commence from midnight to encompass vocal peaks of nocturnal species (Schaaf et al., 2023; Pérez-Granados & Schuchmann, 2020; Odom & Mennill, 2010).\n\nIn terms of hourly sampling, our findings align with studies on marine mammals, indicating that longer recording durations combined with shorter, more spaced-out intervals, yield vocal activity rates similar to continuous recordings (Thomisch et al., 2015).\n\n\n5. Conclusions\n\nThis study underscores the significance of optimizing temporal and sampling design in PAM from ecological and conservation perspectives. Through such optimization, we not only ensure efficient use of limited resources but also broaden the scope of the monitoring project in terms of temporal, spatial, and taxonomic. Such refinements enhance our understanding of avian community structures and their responses to environmental changes. However, it is essential to emphasize that our study was specifically conducted on 12 breeding bird species within subtropical montane forests. Consequently, applying these findings to other ecosystems or to a broader range of avian taxa demands careful consideration. Moreover, when the focus of monitoring narrows down to a single species, it becomes crucial to devise a sampling strategy that aligns with the distinctive behaviors of that species. We advocate for future studies to build upon our foundational research, venturing into diverse ecological landscapes and including a broader spectrum of bird species.", "appendix": "Data availability\n\nZenodo: Underlying data for ‘Evaluating community-wide temporal sampling in passive acoustic monitoring: A comprehensive study of avian vocal patterns in subtropical montane forests’, https://www.doi.org/10.5281/zenodo.8304104 (Wu et al., 2023).\n\nThis project contains the following underlying data:\n\n• Supplementary Material.pdf. (This file encompasses Figures S1 to S8, Tables S1 to S4, and Appendix S2 which details the calculations for performance metrics.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nAlvarez-Berríos N, Campos-Cerqueira M, Hernández-Serna A, et al.: Impacts of Small-Scale Gold Mining on Birds and Anurans Near the Tambopata Natural Reserve, Peru, Assessed Using Passive Acoustic Monitoring. Trop. Conserv. Sci. 2016; 9(2): 832–851. Publisher Full Text\n\nBaillie SR: Monitoring terrestrial breeding bird populations.Goldsmith B, editor. Monitoring for Conservation and Ecology. Netherlands: Springer; 1991; pp. 112–132. Publisher Full Text\n\nBateman HL, Riddle SB, Cubley ES: Using Bioacoustics to Examine Vocal Phenology of Neotropical Migratory Birds on a Wild and Scenic River in Arizona. Birds. 2021; 2(3): 261–274. 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Publisher Full Text" }	[ { "id": "224082", "date": "19 Dec 2023", "name": "Jerry S Cole", "expertise": [ "Reviewer Expertise Bioacoustics", "ecology", "ornithology", "automated sound classification" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The authors have produced a timely and well written article that helps to further advance our understanding of how VAR differs across species and in response to environmental conditions. I particularly enjoyed the section that used clustering to divide the full sampling season into distinct segments. It would be nice to see a bit more development of the discussion section, for instance suggestions for what additional avenues researchers might explore other than just replicating this study with other species in other locations. Thanks for the interesting read!\nData Availability: Not sure if I missed this but I don't see a location where the raw data - for instance the output summary of number of bird detections per minute for each species on a given sampling day and hour. I assume that at a minimum the raw output from the classifier would be needed to reproduce the data analyses reported here. I see the supplementary material with plots and overall number of detections, but that would not be enough to reproduce the statistical analyses.\n_________ Introduction\nI enjoyed this introduction. Does a nice job to lead in to the aims of the study.\nParagraph (P) 3 - Might consider saying \"more frequent vocalization\" rather than \"enhanced vocalization\" to be more specific about what exactly is meant - if by \"enhanced\" you mean \"more frequent and louder\". ____________ Results\nSection 3.1, P2 - Does \"1,200 randomly sampled entries\" mean detections by SILIC, or just random sections of the recordings? I think this is probably clarified later in the sentence, but something to think about.\nFig. 1 and 2 - Really enjoyed this method of defining distinct sampling periods through a cluster analysis. __________ Discussion\nGeneral - Should researchers analyze data from only a single survey season cluster period? If so, which period?And what does this mean for multi-species analysis?\nP2 - Not sure what \"interplay of internal ...\" means. Does that mean the biology of the bird is partly responsible for differing VAR?\n__________ Conclusions\nSeems like a good way to wrap it up. I do wonder about how this might apply to a full community of birds, but I suppose that is beyond the scope of this work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10950", "date": "01 Mar 2024", "name": "Shih-Hung Wu", "role": "Author Response", "response": "Dear Dr. Jerry S. Cole, We greatly appreciate your positive remarks and thoughtful suggestions, which will be instrumental in refining our paper. Here are our responses aligned with your comments: Summary: The authors have produced a timely and well written article that helps to further advance our understanding of how VAR differs across species and in response to environmental conditions. I particularly enjoyed the section that used clustering to divide the full sampling season into distinct segments. It would be nice to see a bit more development of the discussion section, for instance suggestions for what additional avenues researchers might explore other than just replicating this study with other species in other locations. Thanks for the interesting read! We have added a visionary suggestion at the end of the 'Conclusions' section, where we express our hope for researchers worldwide to collaboratively develop a universal standard for temporal sampling. This initiative aims to enhance the transferability of PAM data and to bolster the capability for data integration and analysis on a global scale. (Section 5, Para. 3) Data Availability: Not sure if I missed this but I don't see a location where the raw data - for instance the output summary of number of bird detections per minute for each species on a given sampling day and hour. I assume that at a minimum the raw output from the classifier would be needed to reproduce the data analyses reported here. I see the supplementary material with plots and overall number of detections, but that would not be enough to reproduce the statistical analyses. Thank you for the reminder. We have now updated the compressed file 'VAR_m_all_columns.zip' in the \"Underlying Data\" to include the data on the number of calls per minute for each species at each PAM station, along with the associated environmental variable data. (Section 3.1, Para. 3, Line 5) _________ Introduction I enjoyed this introduction. Does a nice job to lead in to the aims of the study. Paragraph (P) 3 - Might consider saying \"more frequent vocalization\" rather than \"enhanced vocalization\" to be more specific about what exactly is meant - if by \"enhanced\" you mean \"more frequent and louder\". We have revised our manuscript in accordance with your suggestions. (Section 1, Para. 3, Line 3) ____________ Results Section 3.1, P2 - Does \"1,200 randomly sampled entries\" mean detections by SILIC, or just random sections of the recordings? I think this is probably clarified later in the sentence, but something to think about. As detailed in Section 2.4, Para. 4, for the evaluation of the SILIC model, we randomly selected 100 detections per species from the results identified by SILIC. Each sample includes a confidence score assigned by SILIC. This approach allows us to assess the performance of the SILIC model and to determine an appropriate confidence score threshold for each bird species relevant to our study. Subsequently, we filtered the total detection results to include only those with a confidence score equal to or exceeding the set threshold score. This process provided us with the necessary data on bird vocal activity for our research. We have revised the text to make the meaning more complete and clear. (Section 3.1, Para. 2, Line 2) Fig. 1 and 2 - Really enjoyed this method of defining distinct sampling periods through a cluster analysis. Thank you for acknowledging the validity of our analytical approach. __________ Discussion General - Should researchers analyze data from only a single survey season cluster period? If so, which period?And what does this mean for multi-species analysis? In our study, Clusters 3 and 4 were identified as the optimal periods for acoustic surveys, as these intervals exhibited the lowest variation in vocalization activity across species. Moreover, these consecutive time frames totaled nearly eight weeks, offering greater flexibility in managing the rotation of recording devices. We have added an explanatory text in Section 4.2, Para. 1, Line 8. P2 - Not sure what \"interplay of internal ...\" means. Does that mean the biology of the bird is partly responsible for differing VAR? We wish to clarify that vocal activity is influenced by a multitude of factors including species, temporal aspects, and external environmental conditions. We have accordingly revised the wording of this paragraph in Section 4.1, Para. 1, Line 1. __________ Conclusions Seems like a good way to wrap it up. I do wonder about how this might apply to a full community of birds, but I suppose that is beyond the scope of this work. We intend to continue exploring the impact of temporal sampling on other species and environments in future work. Additionally, we look forward to results shared by researchers from other regions, which would contribute to the development of more broadly applicable universal standards." } ] }, { "id": "224421", "date": "02 Jan 2024", "name": "Laurel B Symes", "expertise": [ "Reviewer Expertise Bioacoustics", "biodiversity", "speciation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe goal of this paper is to evaluate how the measured vocal activity rate of birds differs depending on the temporal sampling strategy. Evaluating how detection results differ with sampling strategy is useful, particularly across different species and habitats globally, and can inform decisions about whether it is possible to use a generalized sampling strategies across a range of scenarios, or whether sampling strategies need to vary with season, latitude, habitat type, geography and other factors.\nOverall, the paper seems like a solid evaluation of sampling strategies. Currently, the introduction is strongly focused on the temporal aspects of sampling. In the analysis and figures, the authors devote substantial attention to impacts of environmental co-variates, which may well make sense, but could use some set-up and introduction (or could be downplayed or eliminated). In addition, the authors could provide some additional synthesis/interpretation of their findings in the context of designing PAM-based studies. While the authors provide some specific guidance in the discussion, people seeking actionable information for study design may appreciate even more clear direction (such as headings for sections that discuss recommendations for hourly sampling, diel sampling, etc., and/or a summary in the conclusion that focuses on the key recommendations).\nMajor feedback: >From the intro “Sampling design can be categorized into four temporal scales: annual, seasonal, diel, and hourly. An annual sampling design implies recording during one or several time periods within a year.” Do you have a reference for the way that these four types of sampling are defined? I would have anticipated that annual sampling compared one year against another (rather than comparing seasons within a year) and that seasonal sampling would have compared one season to another vs “after being operated for a predetermined number of days at each location, the ARU is relocated to a subsequent site” (which to me would generally imply sampling periods shorter than a season).\n>Hourly sampling design can be categorized into coverage (the proportion of recorded time within an hour) and dispersion (the number of recording segments within an hour).\nDispersion can affect sampling designs other than hourly as well (for example, if only certain days are sampled). You might consider suggest treating the different temporal scales as one set and then separately discussing dispersion (how thoroughly each of those temporal scales are sampled), whether or not you assess how dispersion of sampling affects outcomes at scales above hourly.\nSome more specifics below: Abstract: The goal stated in the abstract background (the last sentence) is unclear. Could the authors rephrase the same to indicate clearly what they are attempting to do here? For example, why does the effective temporal sampling design matter? You need to introduce what vocal activity rate means prior to discussing the ideal design for VAR data.\nIntroduction: Having read your introduction – which reads very clearly, I think you might want to rephrase your abstract in the context of trying to identify the right sampling design or approach & why this is important for different ecological research projects/ecosystems/taxonomic groups.\nI think your objectives should be switched – a) trying to identify how sampling designs impact vocal activity rate patterns and b) provide recommendations.\nBased on the key questions asked – your introduction should introduce sampling design followed by a few sentences on vocal activity rate/analysis of acoustic data – and then you could ask your two key questions in your study area. [sampling design -> why this is important for vocal activity rates/patterns -> ask your questions – how does sampling design impact vocal activity rate calculations?] Methods: Is VAR_d a species-specific measure or is it the total number of vocalizations per day for that site irrespective of the species? Please rephrase/clarify. I am unclear on Table 1 – were all these different sampling strategies deployed/implemented in the field or were these post-hoc analysis based on sub-sampling data? What does Figure 1 mean ecologically? HCA statistically models Euclidean distance, but what does it mean that day 94 is closely associated with day 110 for example?\n>gender, In this context, “sex” may be preferrable to “gender”.\n>Concentrating recordings during specific time, like dawn, often capture more species Concentrating recordings during specific time, like dawn, often captures more species\n>For species without a song or the song was For species without a song or if the song was\n>Secondly, SILIC offers a unique capability to detect each vocalization’s exact start and end time within an audio recording, rather than merely identifying the presence or absence of a certain vocalization within a broad time frame. How precisely is this approach able to pinpoint start time? Is this different from other software options that assign call identity within a 3 or 5 second window?\n>In the discussion, you might address how choosing only one vocalization type (generally song) might influence results, vs something like alarm calls, which might have different temporal distribution\n>What is ‘corrected’ precipitation? (vs measured precipitation)\n>Accordingly, ‘song’ represented 9 of the 12 species, ‘call’ 2, and ‘unknown’ 1. In supplemental materials, indicate which spectrograms corresponded to which vocalization type\n>spanning 122 working days I would remove “working” unless it has specific meaning. (often ‘working’ is used to mean Mon-Fri vs weekend days)\n> Due to the fewer days encompassed by Clusters 1 and 2, when calculating the CV values for a higher number of days, there might be a tendency to obtain relatively lower CV values owing to the smaller sample sizes. I found this sentence confusing. I think that it means that clusters 1 and 2 are smaller and should not be compared to clusters that contain higher numbers of days?\n>Figure 2 is visually appealing but may be more complicated to interpret than a horizonal four-month graph. If the figure is retained, the authors might consider having time flow clockwise rather than counterclockwise.\n\nIs the work 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": "10953", "date": "01 Mar 2024", "name": "Shih-Hung Wu", "role": "Author Response", "response": "Dear Dr. Laurel B Symes, Thank you for your insightful review of our manuscript. We value your comments on evaluating vocal activity rates and sampling strategies across various species and habitats. In response to your suggestions, we have revised the manuscript accordingly and provide detailed responses to each of your comments below: The goal of this paper is to evaluate how the measured vocal activity rate of birds differs depending on the temporal sampling strategy. Evaluating how detection results differ with sampling strategy is useful, particularly across different species and habitats globally, and can inform decisions about whether it is possible to use a generalized sampling strategies across a range of scenarios, or whether sampling strategies need to vary with season, latitude, habitat type, geography and other factors. Overall, the paper seems like a solid evaluation of sampling strategies. Currently, the introduction is strongly focused on the temporal aspects of sampling. In the analysis and figures, the authors devote substantial attention to impacts of environmental co-variates, which may well make sense, but could use some set-up and introduction (or could be downplayed or eliminated). In addition, the authors could provide some additional synthesis/interpretation of their findings in the context of designing PAM-based studies. While the authors provide some specific guidance in the discussion, people seeking actionable information for study design may appreciate even more clear direction (such as headings for sections that discuss recommendations for hourly sampling, diel sampling, etc., and/or a summary in the conclusion that focuses on the key recommendations). We have included a checklist of considerations in the Conclusions section to offer specific guidance for researchers conducting PAM studies in similar domains. (Section 5, Para. 1, Line 4) Major feedback: >From the intro “Sampling design can be categorized into four temporal scales: annual, seasonal, diel, and hourly. An annual sampling design implies recording during one or several time periods within a year.” Do you have a reference for the way that these four types of sampling are defined? I would have anticipated that annual sampling compared one year against another (rather than comparing seasons within a year) and that seasonal sampling would have compared one season to another vs “after being operated for a predetermined number of days at each location, the ARU is relocated to a subsequent site” (which to me would generally imply sampling periods shorter than a season). Indeed, we recognize that our use and definitions of 'annual sampling' and 'seasonal sampling' in the study may have been confusing for readers. We have decided to revise these terms to 'intra-annual sampling' and 'short-term periodic sampling' to avoid such ambiguity. (Abstract - Methods; Section 1, Para. 3, Line 2; Section 1, Para. 4, Line 1; Section 2.5.2, Para. 1, Line 8; Section 3.3; Section 4.2, Para. 1, Line 3) >Hourly sampling design can be categorized into coverage (the proportion of recorded time within an hour) and dispersion (the number of recording segments within an hour). Dispersion can affect sampling designs other than hourly as well (for example, if only certain days are sampled). You might consider suggest treating the different temporal scales as one set and then separately discussing dispersion (how thoroughly each of those temporal scales are sampled), whether or not you assess how dispersion of sampling affects outcomes at scales above hourly. The scope of our investigation was confined to examining the effects of dispersion at the minute level, without delving into higher temporal units such as hours or days. This specific focus was dictated by the prevailing limitation that most ARUs are currently only programmable at the minute level. Additionally, there is a noted scarcity in prior research that has formulated dispersion sampling strategies for intervals extending beyond minutes. Investigations in the future, exploring higher temporal scales, might yield further insights. These could potentially enhance the understanding of sampling methodologies that are capable of reducing the duration of recordings, while simultaneously maintaining the integrity and quality of the data collected. We have added a section in the Discussion regarding the exploration of dispersion sampling strategies across different temporal scales. (Section 4.3, Para. 2, Line 3) Some more specifics below: Abstract: The goal stated in the abstract background (the last sentence) is unclear. Could the authors rephrase the same to indicate clearly what they are attempting to do here? For example, why does the effective temporal sampling design matter? You need to introduce what vocal activity rate means prior to discussing the ideal design for VAR data. We have supplemented the abstract with a definition of VAR and its significance in the context of our study. (Abstract - Background) Introduction: Having read your introduction – which reads very clearly, I think you might want to rephrase your abstract in the context of trying to identify the right sampling design or approach & why this is important for different ecological research projects/ecosystems/taxonomic groups. We have rewritten the background section of the abstract to clearly articulate the primary objectives of our study. (Abstract - Background) I think your objectives should be switched – a) trying to identify how sampling designs impact vocal activity rate patterns and b) provide recommendations. We have made revisions in accordance with your suggestions. (Section 1, Para. 8, Line 2) Based on the key questions asked – your introduction should introduce sampling design followed by a few sentences on vocal activity rate/analysis of acoustic data – and then you could ask your two key questions in your study area. [sampling design -> why this is important for vocal activity rates/patterns -> ask your questions – how does sampling design impact vocal activity rate calculations?] We have made revisions in accordance with your suggestions. (Section 1, Para. 4, Line 4; Para. 5, Line 4; Para. 6, Line 5) Methods: Is VAR_d a species-specific measure or is it the total number of vocalizations per day for that site irrespective of the species? Please rephrase/clarify. VAR_d was calculated as the total daily vocalization count for each station and bird species separately. We have added a section to make the narrative more comprehensive and complete. (Section 2.5.1, Para. 2, Line 3) I am unclear on Table 1 – were all these different sampling strategies deployed/implemented in the field or were these post-hoc analysis based on sub-sampling data? Our study is based on continuous field recording data, and it simulates the outcomes and impacts of data acquisition under different temporal sampling scenarios. We have added explanatory text in the relevant sections to clarify this approach. (Section 2.5.4, Para. 1, Line 1; Explanation of Table 1.) What does Figure 1 mean ecologically? HCA statistically models Euclidean distance, but what does it mean that day 94 is closely associated with day 110 for example? Within each cluster, the VAR_d patterns of different bird species at each PAM station are similar among days within the same cluster but differ from those in other clusters. We have added explanatory text in the relevant section. (Section 3.3, Para. 1, Line 2) >gender, In this context, “sex” may be preferrable to “gender”. Revised. (Section 1, Para. 7, Line 5) >Concentrating recordings during specific time, like dawn, often capture more species Concentrating recordings during specific time, like dawn, often captures more species Revised. (Section 1, Para. 7, Line 13) >For species without a song or the song was For species without a song or if the song was Revised. (Section 2.4, Para. 2, Line 4) >Secondly, SILIC offers a unique capability to detect each vocalization’s exact start and end time within an audio recording, rather than merely identifying the presence or absence of a certain vocalization within a broad time frame. How precisely is this approach able to pinpoint start time? Is this different from other software options that assign call identity within a 3 or 5 second window? SILIC can detect each vocalization at the millisecond level. We have made revisions in Section 2.4, Para. 1, Line 4. >In the discussion, you might address how choosing only one vocalization type (generally song) might influence results, vs something like alarm calls, which might have different temporal distribution We have revised the discussion segment focusing on the potential impacts of species and vocalization type on our research findings. (Section 4, Para. 1, Line 1) >What is ‘corrected’ precipitation? (vs measured precipitation) The corrected precipitation from NASA/POWER CERES/MERRA2 Native Resolution Daily Data is estimated through algorithms (climate models) that interpret satellite observations. These observations are then often adjusted or 'corrected' using various atmospheric data and models to provide more accurate estimates. >Accordingly, ‘song’ represented 9 of the 12 species, ‘call’ 2, and ‘unknown’ 1. In supplemental materials, indicate which spectrograms corresponded to which vocalization type We have added information on the type of vocalization to each corresponding spectrogram. (Supplementary Material_v2.pdf) >spanning 122 working days I would remove “working” unless it has specific meaning. (often ‘working’ is used to mean Mon-Fri vs weekend days) Revised. (Section 3.1, Para. 1, Line 1) > Due to the fewer days encompassed by Clusters 1 and 2, when calculating the CV values for a higher number of days, there might be a tendency to obtain relatively lower CV values owing to the smaller sample sizes. I found this sentence confusing. I think that it means that clusters 1 and 2 are smaller and should not be compared to clusters that contain higher numbers of days? We have revised our discussion to more accurately address the differences in the CV values among clusters with varying sample sizes. (Section 3.3, Para. 2, Line 1) >Figure 2 is visually appealing but may be more complicated to interpret than a horizonal four-month graph. If the figure is retained, the authors might consider having time flow clockwise rather than counterclockwise. We have adjusted the figure to rotate clockwise, and we have also included Figure S9 in the Supplementary Material, which presents the data with a horizontal time axis. (Figure 2; Figure S9)" } ] }, { "id": "224078", "date": "04 Mar 2024", "name": "Bárbara Freitas", "expertise": [ "Reviewer Expertise Bioacoustics", "animal communication", "passive acoustic monitoring", "speciation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript ‘Evaluating community-wide temporal sampling in passive acoustic monitoring: A comprehensive study of avian vocal patterns in subtropical montane forests’, the authors aimed to investigate the impact of different temporal sampling designs on the detected vocal activity rate of 12 bird species. The authors showed that there is substantial variability in these species’ vocal activity rates across three distinct temporal scales (seasonal, diel, and hourly) and then provided specific recommendations for each one of them and for different environmental conditions. Although this study is an interesting case study that provides valuable insights into optimizing temporal sampling designs for Passive Acoustic Monitoring, it needs a more thorough discussion. The recommendations provided appear overly broad and exaggerated, considering the specific scope of this study centred on 12 bird species within subtropical montane forests. The generalizations made may not adequately align with the specific context of this research. This limitation is only briefly mentioned in the conclusion. In this regard, the manuscript could substantially improve by elaborating further on the limitations and potential biases of the study design. Addressing the study's constraints, such as the limited species selection and specific geographical location, would provide a more nuanced understanding of the scope of the conclusions. Furthermore, the manuscript would significantly benefit from discussing the potential applicability of the findings in different ecological contexts. Thus, authors should consider expanding upon how these findings might translate to other ecosystems or a more extensive range of avian taxa, or even other classes. Finally, some recommendations regarding sampling durations and environmental conditions should be further elucidated. Clarifying the reasoning behind specific recommendations would enhance the applicability and understanding of these suggestions.\nGeneral comments\nThroughout the manuscript, there is a notable overuse of acronyms, which impedes readability. This hinders comprehension and creates difficulty for readers. Reducing unnecessary acronyms and ensuring consistent explanations for those used is crucial to enhance the overall readability and understanding of the manuscript. By minimizing the reliance on acronyms and providing clear explanations, the text can become more accessible and easier for readers to navigate In the abstract, certain acronyms are introduced but not subsequently utilized, contributing to confusion. Please also check if all acronyms are explained within the text.\nAbstract There is some important information missing: the place where the data was collected, in which season, and the number of days of the earliest two segments that showed high variability.\nIntroduction The authors state that birds serve as an ‘ideal indicator taxon for monitoring terrestrial biodiversity due to their detectability, identifiability, diversity, widespread distribution, and migratory characteristics’. However, many of these characteristics are also present in other taxa. Emphasis should be given to the detectability and identifiability parts, especially through sound and the availability of automatic detectors for this class. The statement regarding the use of sampling lacks references to support it. Authors should explain better and elaborate more on the exogenous and endogenous factors that modulate avian vocal activity. As it is now, it is not clear to the reader.\nMethods The authors do not indicate if this study was or was not preregistered. This must be provided, according to the journal guidelines: \"Authors must include a statement to indicate if they did or did not preregister the research with or without a data analysis plan at an independent registry\" Target species section – it is not clear which criteria the authors followed to define a species as representative of each guild: which parameters were they taking into account? Regarding the selection criteria when two or more species were available for the same guilt, the authors explain that the species ‘that manifested an altitudinal distribution most congruent with’ the study objectives was chosen, but this is vague. The authors should be more specific and explain the criteria with examples. If, for example, high detectability was a factor used to select species, this should be mentioned. Lastly, Table S1 was indicated in the last part of this section but this table does not mention the 12 guilds. The reader would benefit from having more information about the SILIC software. For example, the five classes ‘song’, ‘call’, ‘drumming’, and ‘unknown’ are labelled by SILIC or they are just grouped and then labelled by the authors? What is the aim or need of combining bounding boxes of the same species within a single recording? It should be indicated the mean of the confidence score of the random sample of 100 detected vocalizations. the definition of AUC should be added as well as an explanation on why this and AP were used. The reference for YOLOv5 should be added.\nResults The number of files and equivalent time for the used for subsequent analyses (from 12 stations over 112 days) should be indicated.\nDiscussion This section is oversimplified and the recommendations lack robust baseline support. The authors made use of words such as behaviors, internal and external factors without really specifying what they refer to. The authors suggest sampling around one hour before and after sunrise to capture the morning chorus, even proposing starting recordings at midnight. However, it's crucial to note that these recommendations might lack empirical validation for this particular field site, so the manuscript should highlight the need for further testing or verification. Regarding diel sampling, results should be put into context with many more different studies on birds and justification for these findings should be discussed. For example, see (Darras et al. 2019)1\nMinor comments\nFig 2- rainfall instead of Corrected Precipitation - names should be the same throughout the text Fig S1 – This figure could be improved by adding a small map of the geographic position of Taiwan in the globe. Please add in the legend what orange means and that the map also depicts the relief. Fig S2 – please include axis in each spectrogram. The legend is not necessary to have in all of them (frequency or time), but the axis is fundamental for visualization of the spectrogram. Fig S5, S6, S7, S8 – Readability and understanding of the figures could be improved if the name of the species would be written as title of each graph. Also, precision and recall should be explained. Table 1 – Please provide a column with the temporal coverage in minutes, to allow for easier understanding. Table S3 – Please specify what positive and negative mean. If relative to detections this should be specific in the column name.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1299
https://f1000research.com/articles/12-1300/v1	11 Oct 23	{ "type": "Systematic Review", "title": "Migrant entrepreneurship in Europe: a systematic literature review", "authors": [ "Dimitris Polychronopoulos", "Anh Nguyen-Duc", "Anh Nguyen-Duc" ], "abstract": "Background: This systematic literature review (SLR) analyzes migrant entrepreneurship in Europe through three research questions (RQs) to understand 1) migrant entrepreneur characteristics in the European context, 2) challenges encountered by migrant entrepreneurs in European host countries, and 3) policies supporting migrant entrepreneurship in Europe. This review addresses gaps in current knowledge in academia as well as issues that policymakers and practitioners face when addressing migrant entrepreneurship support.\nMethods: This SLR employed a search protocol to retrieve published sources from 1970 to 2021, via Scopus (27 March 2022) and Web of Science (7 April 2022). Inclusion criteria targeted migrant entrepreneurship support studies while exclusion criteria eliminated domestic migration and non-European contexts. The authors worked iteratively, aligning the data with the RQs to reduce bias, and adapted Bourdieu's forms of capital to create an analytical framework for the sources included in the SLR, with a table for each RQ to synthesize relevant data for analysis.\nResults: The review examined 91 peer-reviewed papers, with a focus on migrant entrepreneurship in Europe, covering characteristics, challenges, and support policies. It classified migrant entrepreneur challenges and characteristics into financial, human, and social capital, as well as external factors. Common challenges include the local culture and language, network, funding, and adapting to local business practices. Migrant entrepreneurs' stability relates to time in the host country and local language proficiency and reflects past entrepreneurial experience and education. Supportive mechanisms involve local networks, financing, and mentoring.\nConclusions: The SLR's limitations encompass possible oversight of pertinent studies, along with potential bias in data extraction, analysis, and subjectivity due to thematic analysis. Nonetheless, the findings suggest the following research agenda for migrant entrepreneurship support: evaluating and enhancing human and social capital, sharing information, designing support programs, addressing in-group/out-group bias in support programs, and exploring bottom-up migrant entrepreneurship support approaches.", "keywords": [ "Migrants", "immigrants", "entrepreneurship", "integration", "mixed embeddedness", "entrepreneurship support" ], "content": "1. Introduction\n\nMigrant entrepreneurship in Europe has gained increasing attention in recent years as a means for individuals to pursue economic opportunities and contribute to the development of their host communities (Aki Harima and Freudenberg, 2020; Kachkar and Djafri, 2021; Lyon, Sepulveda, and Syrett, 2007). As migrants comprise an increasing proportion of the population in Europe, they are seen to be more likely to be entrepreneurial than the native population (Irastorza and Peña, 2007), especially in certain countries, such as Belgium, Denmark, France, Norway, Sweden, and the United Kingdom (Kushnirovich, Heilbrunn, and Davidovich, 2018). Migrant entrepreneurship can be defined as the process of starting and running a business venture by an immigrant in a host country (Communities, 2003), can have a positive impact on the economy of the host country (Meister and Mauer, 2019) and be a source of innovation, economic growth, and social cohesion, as well as a means of integration and personal development for the entrepreneur (Eraydin, Tasan-Kok, and Vranken, 2010). In addition, migrant entrepreneurship can build international connectivity and reinforce economic activity for the host country (Kourtit, Nijkamp, and van Leeuwen, 2013); Backman, Lopez, and Rowe (2021) see under-utilization of immigrants’ skills as waste of resources. Beyond the above considerations of migrant entrepreneurship, Europe is a unique area to study and merits a special focus for four main reasons: the first is the right to free movement of EU citizens (Collett, 2013) and within the European Economic Area (Peers, 2000); the second is the application of the Dublin agreement in processing asylum applications (Angenendt, Engler, and Schneider, 2013; Murray and Longo, 2018). A third consideration is Europe’s position in 2015 concerning its ability to accommodate a significant influx of new arrivals from outside the European Union/European Economic Area, along with the resulting effects (Scipioni, 2017). Finally, scholars in Europe have recognized the need for a broader societal framework to understand migrant entrepreneurship, in order to account for the unique opportunity structures in Europe, by considering the role of economic and political factors on migrant entrepreneurship (Kloosterman and Rath, 2018).\n\nGiven that the literature on migrant entrepreneurship in is fragmented, with studies focusing on various aspects of migrant entrepreneurship such as ethnic enclaves, mixed embeddedness, motivations, challenges, and outcomes (Ilhan-Nas, Sahin, and Cilingir, 2011), therefore a systematic review of the existing research will help synthesize the current knowledge on migrant entrepreneurship in Europe and identify gaps for future research. Such a review will not only provide a comprehensive overview of the field, but also serve as a useful resource for policymakers, practitioners, and researchers interested in understanding and supporting migrant entrepreneurship in Europe.\n\nThis study aims at synthesizing current knowledge about characteristics, challenges and supporting factors of migrant entrepreneurship in the European context, to develop a conceptual framework and identify research gaps that call for future inquiry. From the above objectives, we will address three research questions (RQs) in this study:\n\n1. RQ1: What are the characteristics of migrant entrepreneurs investigated in primary studies, in the European context?\n\n2. RQ2: What do we know about challenges that migrants face as entrepreneurs in European host countries?\n\n3. RQ3: What do we know about reported policies as support mechanisms for migrant entrepreneurship in the European context?\n\nTo the best of our knowledge, this work offers a most up-to-date and comprehensive view to discern the characteristics and challenges of migrant entrepreneurship in the European context and how policies support migrant entrepreneurship, given the conditions of the characteristics and challenges of the migrant entrepreneurs. Existing systematic literature reviews (SLRs) have called for further work to (1) identify governmental and support policies for migrant entrepreneurs (Malerba and Ferreira, 2020), and their impact on immigrant entrepreneurship (Dheer, 2018), (2) synthesize the potential impacts of regional contextual factors (Dheer, 2018) and country contexts (Dabić et al., 2020), and (3) focus on specific geographical areas. Our current study is the latest and most exhaustive secondary study that contributes to all these points.\n\nThe paper is structured as follows: Section 2 presents the background, including definitions of relevant terms. Section 3 presents our research approach. Section 4 contains our findings. Section 5 shares our discussions and recommendations, and Section 6 concludes the paper.\n\n\n2. Background\n\nTo provide further context surrounding the phenomena studied, it is important to define both the terms, ‘migrant’, and ‘entrepreneurship’. We start by clarifying that this study is not concerned with internal migration within the borders of a single country. The definition of ‘migrant’ in much of the academic literature, means that somebody is: 1) foreign-born and 2) a non-citizen (Gimeno-Feliu et al., 2019). Since the foreign-born can often acquire citizenship in their host country, foreign-born is a more suitable definition for this research. However, the non-citizen aspect is also important, since in addition to becoming citizen of a new country, an individual can be born abroad due to parents’ stay abroad; citizenship in country ‘A’ may also extend to the grandchildren of people who left country ‘A’ for country ‘B’, and in some cases, even to further generations back.\n\nSome of the literature refers to migrant entrepreneurship (Berntsen et al., 2021; Hagos, Izak, and Scott, 2019; Sinkovics and Reuber, 2021; Solano, 2021; Szczygiel, Nunes, and Ramos, 2020), while some refers to immigrant entrepreneurship. (Abbasian and Yazdanfar, 2015a; Bolzani and Boari, 2018; Glinka and Hensel, 2020; Murphy, Bogue, and O'Flaherty, 2020; Širec and Tominc, 2017; Yazdanfar and Abbasian, 2013). For this paper, both ‘migrant’ and ‘immigrant’ literature are suitable, if the condition of foreign-born is met. Throughout this article, the authors will refer to migrants and immigrants, and sometimes switch from one term to the other. This is because of the 91 sources that are included in this systematic literature review, the sources themselves may sometimes be based on the term migrant, while at other times based on the term immigrant. For this study's sake, both terms are relevant, and that is why we include both terms throughout the study. Dabić et al., (2020) reported that ‘migrants’ are people who move to a different country from their usual residence, for a period of at least 12 months and that ‘immigrants’ are born abroad. Dheer (2018) emphasizes that the definition of immigrant denotes a move that is permanent and not transitory. However, for this study, we are interested in the phenomenon of migrant entrepreneurship regardless of whether the intention is to stay permanently in the host country.\n\nIn a review of the literature on self-employment and entrepreneurship, Szaban and Skrzek-Lubasińska (2018) position self-employment within the entrepreneurship paradigm. Amongst authors included in our review, definitions of entrepreneur include creating or establishing a business (Glinka and Hensel, 2020; Širec and Tominc, 2017; Solano, 2021), owning a business (Abbasian and Yazdanfar, 2015; Yazdanfar, Abbasian, and Brouder, 2015), managing one’s one business (Szczygiel et al., 2020), and having an aim to succeed at business (Hagos et al., 2019). Given that migrants who are just getting started in their host countries and seeking support from government entrepreneurship initiatives may not have established a business yet, the broader definition by Hagos et al., (2019) seems the most comprehensive for this study.\n\nSince this research aims to study phenomena of migrant entrepreneurship in a broad sense of business activity and migration across national borders, we recognize that migrants may fall into categories that include the following: moving to the country of citizenship inherited from a parent or grandparent, fleeing a warzone as a refugee, joining a partner in a different country with whom they are in a romantic relationship, repositioning to the host country as an employee, trailing spouse, son or daughter, and staying on as an entrepreneur, seeking better economic conditions, seeking political asylum, adoption as a child by new parents in the host country, and staying in the host country after having studied abroad. Dheer (2018) defines immigrant entrepreneurs as those who “identify, create and exploit economic opportunities to start new ventures in their destination nations” (p. 558).\n\nSome of the literature engages in a narrow focus of migrant entrepreneurship; examples include articles that exclusively study tech startups as well as a single case study of a cheese factory. However, there are many categories of entrepreneurial ventures, and they can overlap. This literature review is interested in all forms of business classifications to encompass entrepreneurship; examples include: business to business, business to government, business to consumer, software tech, hardware tech, deep tech, and consulting services, with offerings as either a service or a physical product. Another context is the locations in the studies we will be reviewing. The academic literature includes primarily studies with advanced economies (Dheer, 2018), although Duan, Kotey, and Sandhu (2021) note a trend since 2012 for research to include emerging economies. Our study aims to gather knowledge from the overall European context, regardless of how advanced the economy is.\n\nExisting literature suggests that immigrants to a new country are lacking in several areas, such as credentials, cultural understanding, and language skills, which create challenges that may push them to seek to earn a living by becoming entrepreneurs (Ensign and Robinson, 2011). Li (2000) also notes that immigrants are often driven into entrepreneurship due to obstacles finding employment in their host countries. In addition, Cobas (1986) and Evans (1989) suggest that immigrant groups arriving in some countries have a greater tendency to become self-employed, while some groups of immigrants tend to make more successful entrepreneurs than others (Zhou, 2010). Given that successful entrepreneurship activity drives economic growth (Carree and Thurik, 2010; Schumpeter, 1949), that immigrant entrepreneurs can help the economies of their host countries (Barth and Zalkat, 2021), that the rate of entrepreneurs is often higher for immigrants than amongst native populations (Dheer and Lenartowicz, 2020), and that foreign-born entrepreneurs bring unique resources yet face specific challenges that the native population does not (Bijedic and Piper, 2019), it follows that policymakers have an interest in helping improve the likelihood of entrepreneurial success for immigrants.\n\nTo understand the need for a new systematic literature review on migrant entrepreneurship, we first conducted an ad hoc review of six systematic literature reviews on this topic. We examined the background information to help develop our research questions. The six literature reviews are by: Aliaga-Isla and Rialp (2013), Dabić et al., (2020), Dheer (2018), Duan et al., (2021), Malerba and Ferreira (2020), and Sithas and Surangi (2021). Table 1 displays the number of studies reviewed, the years covered, the main goals establishing prior to conducting the reviews, key findings, and the authors’ recommendations. Aliaga-Isla and Rialp (2013) extracted primary studies’ objectives, theoretical frameworks, and methodologies, finding that most studies took place in the United States, followed by Europe and Oceania, and that studies from Germany and the Netherlands were dominant for Europe and that Europe had a greater prevalence of empirical studies. Dabić et al., (2020) plot the studies they reviewed into four quadrants by geographical scope, major research themes, methodological approaches, and theoretical approaches, with Europe leaning toward more qualitative studies and the use of embeddedness theory. Dheer (2018) outline the factors influencing the outcomes of immigrant entrepreneurship at the micro, meso, and macro levels, in addition to the process and its outcomes. In addition to the details covered in Table 1, Duan et al., (2021) extracted elements including article type, citation rate, methodology, and extra context (such as gender or generation). The study documented a trend in 2012 for emerging economies to conduct research in immigrant entrepreneurship, as well as a trend starting in 2007 for publications in business and entrepreneurship to cover the phenomenon. They find that push-pull theory dominates the field, with its application in 28 of the 62 articles. Malerba and Ferreira (2020) make a novel contribution with a larger search scope (adding Scopus) and the new focus on the identification and exploitation of opportunities by immigrant entrepreneurs, which Dheer (2018) identified as a key issue for future research. Sithas and Surangi (2021) find that most studies concentrate on immigrant communities in the host countries rather than on ethnic differences inside the native country. They reported a dominance of literature of immigrants from African and Asian countries to Western and European countries and that the dominant theories in the literature are community of practice theory, cultural theory, effectuation theory, and ethnic enclave theory, and demonstrate the interactions of these theories between individual members and the greater community, noting that the ethnic enclave extends support to the individual, while also accounting for factors that enhance the individual’s personality traits. There appears to be some overlap with ethnic enclave theory and Community of Practice Theory and Cultural Theory, since all three postulate that the ethnic group has an impact on the individual member’s personality traits, with effectuation having its place to explain the back-and- forth between the individual and the community (Sithas and Surangi, 2021).\n\n\n\n1) immigrant studies,\n\n2) ethnic entrepreneurial motivations,\n\n3) ethnic startup process,\n\n4) motives of ethnic business success,\n\n5) failure factors,\n\n6) unique challenges, and\n\n7) favorite strong ties\n\nThe six literature reviews we examined provided insight into the topics, theoretical perspectives, empirical contexts, and methodologies employed in current research on migrant entrepreneurship, as well as identified gaps for future research. While there is a growing body of literature on migrant entrepreneurship in Europe, these reviews have not extensively covered the European context, which is the distinction of our review. In addition, both Dheer (2018) and Malerba and Ferreira (2020) emphasize the importance of the impact of policies on migrant entrepreneurship, yet none of the literature reviews set out to do so. We see that immigration is getting more attention from both policymakers and scholars in Europe (Dheer, 2018). While new ventures that immigrants establish in their host countries can help lead to economic development and a decrease in unemployment, we also realize that some of the immigrants get stuck in low-margin businesses, while others fail at their businesses (Dheer, 2018). Considering the above, our literature review will fill the gaps in existing research by examining the characteristics and challenges of migrant entrepreneurs in Europe, as well as the policies and support mechanisms that impact their experiences in host countries and how ecosystems influence these policies.\n\n\n3. Methods\n\nWe adopted an existing well-known methodological framework to ensure that the systematic literature review would provide a comprehensive and structured synthesis of available research on migrant entrepreneurship in the European context, by following the steps described by Kitchenham (2015). First, we developed the search protocol. Second, we identified the inclusion and exclusion criteria for relevant publications. Third, we performed an in-depth search for studies, followed by critical appraisal, data extraction and synthesis of past findings. The next sub-sections describe in detail the previously mentioned stages. We also adopted the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guideline for reporting systematic reviews (Page et al., 2021). We use the PRISMA template (Page et al., 2021) to report the process and outcomes of each step in the search process Along with these guidelines, Figure 1 displays the PRISMA 2020 flow diagram for the study selection process, from identification of studies, to screening of studies, and inclusion of studies, while the PRISMA checklist and PRISMA abstract checklist are publicly available with this report (Polychronopoulos and Nguyen Duc, 2023). See Table 11 for the links to the documents included in this study.\n\nThe eligibility criteria helps with the search strategy to derive relevant literature from the main RQs, with our search string has three parts: MIGRANT (C1), ENTREPRENEUR (C2), and SUPPORT (C3). Under this search string, the idea is to cover migrant under diaspora, refugee, immigrant, exile, displaced or asylum, and to cover entrepreneurship under various forms and include self-employment, startup, venture, new business, enterprise, new firm, or similar, and to cover support under training, as well as forms of acceleration or incubation. The synonyms to these terms were identified in the context of either information systems or entrepreneurship research by interviewing field experts. We presented the list of synonyms related to migrant entrepreneurship to our colleagues. Two of these experts came from the information systems domain, while one had a background in entrepreneurship. We asked them to contribute to the list of synonyms based on their knowledge. Each conversation occurred individually, informally and lasted for circa ten minutes. Their feedback was documented and resulted in a revised list of synonyms. The two authors conducted several trial searches to adjust the scope of the search string, so that we do not include many irrelevant studies from different research fields. The most important information is the supporting mechanisms that were provided to address challenges that migrant entrepreneurs confront. On the other hand, we want to include as many studies as possible. After several trials, we culminated with the list of search words as shown in Table 2. The search string is formed as the formula: C1 AND C2 AND C3.\n\nOur linguistic capabilities meant that we could include studies published in the following languages: Danish, English, French, Greek, Italian, Norwegian, Portuguese, Spanish, and Swedish. However, nearly all articles in the search were published in English, with a few appearing in Spanish. By the time we had concluded the inclusion/exclusion process, we had one article in Spanish and 90 in English. Several electronical databases were suggested by the second author who has conducted several systematic literature reviews before. The list includes Scopus, ISI Web of Science, IEEE Explore, Current Contents, Kluwer Online, Computer Database, Science Direct, Springer Link, Inspec and ACM Digital Library. Considering the popular databases within the entrepreneurship research, previous experiences of reviewers, flexible formulation of search strings with unlimited clauses and easily exporting paper lists in various formats, we decided to select Scopus and ISI Web of Science. The search ranges from 1970 to March 2022. We screened the sources based on the title, abstract, and keyword metadata to help us select studies relevant to our RQs.\n\nTo prepare for the next stage in the process of the eligibility criteria, we developed the following inclusion and exclusion criteria:\n\nInclusion criteria:\n\n• IC1 - the paper should investigate migrant entrepreneurs as the main research topic\n\n• IC2 - the paper should explore either challenges migrant entrepreneurs face in their host country, policy on the topic, or both\n\n• IC3 - the paper should discuss how to train entrepreneurs, factors for migrant entrepreneurship success, or both\n\nExclusion criteria:\n\n• EX1 - the paper does not investigate migrant phenomenon transnationally\n\n• EX2 - the paper does not investigate migrant entrepreneurship as a primary topic\n\n• EX3 - the paper does not investigate entrepreneurship topics\n\nFor completeness, we also excluded papers that were published in 2022 because it was March of that year at the start of the search, and we could not fully represent 2022 within the review.\n\nConcerning information sources, the only databases used to search for sources were Scopus and Web of Science. The beginning of our search yielded 803 results in Scopus on 27 March 2022 and 312 results in Web of Science on 7 April 2022. After merging the results to eliminate the 223 duplicate papers that appear in both Scopus and Web of Science, we were left with 803 Scopus sources and 89 results from Web of Science, for a total of 892 results. We conducted the selection process as shown in Figure 1.\n\nRemove non peer-reviewed documents: after removing the duplicates, we removed four titles published in 2022. We then removed 78 records for not being in the category of articles, book chapters, or conference papers. This left us with a total of 810 records for further screening.\n\nPrior to applying the inclusion and exclusion criteria, the two reviewers (the first and second authors of this paper) performed pilot runs, i.e., pretest, to improve homogeneity. The aim was to ensure that the reviewers had the same interpretation of the inclusion and exclusion criteria, which meant that there was a good understanding of the type of studies that needed to be included and excluded. The first pilot consisted of 20 papers, that the main author and second author were drawn to because they included a mix of theory, entrepreneurial incubation, policymaking, migrant integration, and refugee topics in their titles. At this stage the authors were not yet focused on excluding non-European settings. The reviewers were able to retrieve 19 articles of the 20 articles and held meetings to discuss the them after reading through them. Fleiss’s kappa for agreement on inclusion in the review was 0.84 for the two reviewers (Fleiss, 1971). The kappa value between 0.81 and 1.00 represents an almost perfect agreement (Fleiss, 1971), suggesting a very good agreement among the authors. In case a decision could not be made by two reviewers, we kept on-going discussion until consensus was reached. Eleven of the studies made it past the original pilot review and eight of them did not. Primary studies use different research designs, data collection and analysis methods, which leads to a threat of validity of synthesis process. To reduce the variety in primary studies, we used an extraction form, which is driven by the research questions. The form had been initiated by studies in the initial pool and validated during the trial search process. Only studies that addressed at least two RQs made it through the first pilot review.\n\nThe next step was to eliminate sources that did not cover the European context. At this step, five sources made it through to be amongst the total of 91 studies included in this SLR. They are the following sources: 1) Solano, 2021, 2) Berntsen, de Lange, and Kalaš, 2021, 3) Harima, Freudenberg, Halberstadt, and Hanoeman 2020, 4) Bruzelius, 2020, and 5) Evansluong and Ramírez-Pasillas, 2019.\n\nSelect by title and abstract: we removed articles that clearly have no connection to the topic of migrant entrepreneurship, as well as those articles which clearly were beyond the scope of the European context. Here we excluded articles from the Kazakh, Russian and Turkish local contexts as well, given that most of the landmass of these three countries falls in Asia rather than in Europe, and given our expectations that the conditions surrounding migrant entrepreneurship in these three countries would differ significantly from those of the rest of Europe, considering not only their geography, but their cultural traditions and political structures. By considering the three research questions as we moved forward, we could identify documents that did not cover aspects of migrant entrepreneurship such as characteristics, policies, support, challenges, and success factors. At this stage the lead author reviewed the titles and abstracts and shared the results of the included and excluded articles with the second author. Figure 1 below displays the study selection process.\n\nGeographical context was not always evident from the titles and journals. Thus, the authors recognize that in the next step they would need to check for geographical context in the abstracts. This process allowed us to eliminate 609 sources, leaving us with 201 documents for further scrutiny. At this stage both the lead author and the second author reviewed the titles and abstracts to decide on which articles to include and which to exclude.\n\nSelect by full text: when the selection could not be determined with abstract and titles, it was accomplished by reading full texts. Both authors assessed the papers and sought to understand migrant entrepreneurship as an emerging line of research in the business literature and to understand the success factors and moderators of success. We looked for articles that would help answer the research questions based on the following list:\n\n1. How to stimulate entrepreneurial activity\n\n2. How to improve likelihood of success (decrease early-stage failure)\n\n3. To understand and identify barriers to growth\n\n4. How to incorporate the needs of entrepreneurs in developing and support systems\n\n5. Policymaking for migrant entrepreneurs\n\n6. Migrants becoming self-sufficient either as entrepreneurs or as employees\n\n7. Creating the entrepreneurial aptitude for migrants\n\n8. Understanding entrepreneurial support organisations\n\nThis stage of the review brought us to 108 articles for further scrutiny, with all publication years known and the oldest being from 1994, while 62 of the articles were published from 2018 to 2021. Sweden was the most frequently appearing European country, with 17 articles, followed by Italy at 11, and the United Kingdom at 10. Some articles also covered multiple countries and included European and non-European contexts. We also discovered a duplicate article within Scopus, with the same title, but published in two different journals. ‘A new career in a new town: Entrepreneurship among Syrian refugees in Germany and the Netherlands’, in both the International Journal of Entrepreneurship (Johnson and Shaw, 2019a) and the Journal of Legal, Ethical, and Regulatory issues (Johnson and Shaw, 2019b).\n\nFurther evaluation and selection: the next step was to read through all the articles whose geographical contexts were still unknown, and to include them only if they covered the European context. After sorting through the articles where the geographical context was not available in the abstract, we were able to eliminate another 10 sources. This brought us to 98 articles for further scrutiny with an updated list providing geographical context, with Sweden, UK, Germany, and Italy appearing most frequently. At this stage we went through further examination on the inclusion criteria and found that five of the articles were not close enough to the topic, which are:\n\n1) ‘Migrant women entrepreneurs and emotional encounters in policy fields’ (Webster, 2020) had a narrow focus on emotion and didn’t seem to define ‘policy fields’\n\n2) ‘Social innovation in Refugee Support: Investigating Prerequisites Towards a Conceptual Framework’, (McNally, Apostolopoulos, and Al-Dajani, 2020) consisted of five chapters with focus on social innovation, but no clear focus on characteristics, challenges, and policies.\n\n3) ‘Entrepreneurial cultural affinity spaces: Design of inclusive local learning ecosystems for social change, innovation, and entrepreneurship’ (Savva, Souleles, and Ferreira, 2020), was classified as a conference paper, but turned out to be only lecture notes\n\n4) ‘Factors driving the share and growth of Chinese entrepreneurship in Italy’ (Apa, Noni, and Ganzaroli, 2020}, had a very narrow focus on Chinese entrepreneurs in the fashion industry in Italy, with little focus on characteristics and policy\n\n5) ‘Ubicación espacial de los negocios étnicos en Almería. ¿Formación de enclaves económicos étnicos?’ (Garrido and Olmos, 2007), does not focus on challenges, characteristics, or policy.\n\nWe also detected one study which was not a peer-reviewed article and one study that was withdrawn from the published journal. Furthermore, for three articles we cannot access their full text from our universities, which are 1) ‘Diaspora Africans and entrepreneurial characteristics: A focus on Nigerians in the UK’, 2) ‘Self-employment work and small enterprises as channels of integration for immigrants: The case of the Province of Trent’, and 3) ‘Recent refugee migrations to Western Europe: asylum seekers and refugees in Italy and Greece’. In addition, our Scopus search included a book chapter from Female Immigrant Entrepreneurs: The Economic and Social Impact of a Global Phenomenon (Halkias et al., 2016b). The main author borrowed the book from the university library system to discover that it contained four chapters which are relevant to this SLR, given that each separate chapter is devoted to a specific country context: Cyprus, Greece, France, and the United Kingdom. By adjusting this from single source to four separate sources, it raised the number of sources by three.\n\nAt the end of this step, we emerged with the final set of 91 peer-reviewed sources, of which 90 are in English and one is in Spanish.\n\nThe main author created a spreadsheet to record specific data items from the articles. These data items include the database from which the article was located (Scopus or Web of Science), the year published, the title, the unit(s) of analysis, the authors, the retrieval method (PDF, print, or loan from library), the host country/countries, the reason(s) for inclusion criteria, the source title (name of book, conference, or journal), the characteristics of the migrant entrepreneurs studied, the home country/countries (if mentioned) and whether the study focused on refugees, the policies and support mechanisms to help migrants succeed as entrepreneurs, the challenges migrants face in their host countries, theoretical framework(s) and/or key concepts, research type (qualitative, quantitative, mixed methods, or theory exploration), the abstract, author keywords, and document type (article, book chapter, or conference proceedings). The main author collected the above details and manually input them into the spreadsheet and shared the spreadsheet with the second author. No automation tools were used in this process. The main author took an inductive approach to explore the input from the 91 articles and it emerged from content of the article collection that a breakdown of financial, human, and social capital would provide a framework for analysis in terms of answering the research questions, building a conceptual framework, and sharing key takeaways with the readers. The main author created a table for each form of capital: financial, human, and social, and filled it in with the details from the articles. The second author went through the tables and discuss with the main author where questions arose. Through a process of several meetings, the two authors worked on the table so it would be compact and clear enough for specific details to emerge that help answer the three research questions:\n\n1. RQ1: What are the characteristics of migrant entrepreneurs investigated in primary studies, in the European context?\n\n2. RQ2: What do we know about challenges that migrants face as entrepreneurs in European host countries?\n\n3. RQ3: What do we know about reported policies as support mechanisms for migrant entrepreneurship in the European context?\n\nThe two authors followed guidelines with a systematic approach to reduce individual bias and influence on the data selection process, so that others who follow the same search protocol and procedures outlined in this SLR’s sections 3.1, 3.2, and 3.3, should achieve the same results.\n\nThe main author coded each source from S1 to S91 and created a table for each of the three research questions. Table 7 refers to RQ1 and includes human and social capital as the characteristics mentioned in the primary studies. Table 8 refers to RQ2 and includes financial, human, and social capital, as well as external factors as challenges for migrant entrepreneurs. Table 9 refers to RQ3 and includes financial, human, and social capital, as well as external factors as supporting initiatives and policies for migrant entrepreneurs in Europe. Here the ethnic group itself is sometimes mentioned as having its own social capital and this is noted in Table 9. Therefore, during the scanning of the full texts, the main author identified relevant paragraphs and texts and labeled them with codes that refer to the three research questions, with one table for each research question to record the relevant information from all of the 91 sources included in the SLR. In addition, the main author created tables to record the distribution by country, categories of journals, list of conferences and journals appearing at least twice and shared these tables with the second author for review.\n\nAs for reporting bias assessment, Elshater and Abusaada (2022) emphasize that bias can hinder researchers from creating a proper roadmap for their literature reviews, and define bias as favoring or disapproving a particular topic or author’s perspective. To address these issues, during the synthesis process, the authors worked closely together iteratively by regularly revisiting the research questions, the Tables 7, 8, and 9 where the data was recorded in answer to the research questions, and discussed and returned to the primary studies. The authors updated the tables several times and agreed to add external factors to Tables 8 and 9 (challenges and support) in order to complement the three forms of capital that emerged from the sources: financial, human, and social capital. In addition, the main author merged some of the references to Bourdieu’s forms of capital, such as cultural and economic capital into this framework, with economic capital appearing as financial capital in the tables and the analysis, while cultural capital appears either as human or social capital, depending on the context.\n\n\n4. Results\n\nFor each of the 91 studies included in this SLR, the main author created a unique identifier starting with the letter S for ‘study’ and a number from 1 to 91. The table displays the studies in chronological order from most recent to oldest, based on the order of appearance in the original Scopus search. The studies from Scopus are labeled with codes S1 to S86, followed by the same logic for sources from Web of Science, which received codes S87 to S91. Table 11 displays the codes S1 to S91, the authors and publication years, the host countries, the research type (qualitative, secondary qualitative, quantitative, mixed methods, or theory exploration), the type of document (article, book, book chapter, or conference paper), the home countries (if mentioned) and other key characteristics of the migrant entrepreneurs studied (whether the study focused on refugees or a specific gender), and theoretical frameworks used if mentioned explicitly.\n\nAs for the distribution of the selected primary studies across geographical context, Table 3 demonstrates that some countries have a greater research interest in the topic of migrant entrepreneurship than others. Sweden is not only the country that appears the most frequently in the European literature on migrant entrepreneurship, but it was also the first country to appear in chronological order within our 91 sources, with a publication dating from 1994 (See Figure 2 for the distribution of publication years). It is also of merit to note that Sweden has a significantly smaller population than the next five countries that top the list in sources about migrant entrepreneurship. This means that Sweden alone represents more than 17 percent of the weight in the results of the SLR, despite representing just more than 2 percent of the population of the European Union. It is also worth noting that 11 of the 91 studies consider more than one country. For example, Solano (2021)’s study is a review of measures that foster migrant entrepreneurship in countries across the European Union and Organization of Economic Cooperation and Development (OECD).\n\nTen additional sources also cover multiple countries. 1) Wade (2020) study the Fresh Start program and migrant education in Belgium, Netherlands and UK, 2) Glinka and Hensel (2020) provide an analytical framework from contexts of Belgium, Netherlands, Poland and USA, 3) Johnson and Shaw (2019) look specifically at Syrian refugees in Germany and the Netherlands, 4) Williams and Krasniqi (2018) explore human and social capital for migrant entrepreneurs from conflict zones and in several countries, mostly in the EU, 5) Eroğlu (2018) studies Turkish entrepreneurs in Western Europe, 6) Thandi and Dini (2010) use an interdependent perspective to examine secondary data of migrant entrepreneurship in Europe, 7) Van Delft, Gorter, and Nijkamp (2000) study comparative policy in various cities across six different European countries, 8) Širec and Tominc (2017) study growth determinants in 13 European countries, with an aim to cover less developed and more developed areas for comparative purposes, and 9) Bruzelius (2020) investigates local policy responses in Gothenburg, Hamburg, and Stockholm, for EU citizens moving to these locations. 10) Qin (2021) synthesizes the literature on cognitive dissonance, multiple embeddedness and hospitality to explain the implications on entrepreneurship and refugee business support in developed European economies.\n\nIn addition to discussing the geographical contexts of the host countries for studying phenomena surrounding migrant entrepreneurship, we also wish to share some details about the nature of the studies in terms of gender, refugee status, and home country. Of the 91 studies, 42 were not specific to gender, refugees, or home country. In terms of home country focus, 22 of the studies were specific to a single home country, two of the of the sources studied two home countries, and 67 of the studies covered three or more home countries; in total 28 of the studies had a specific home country or set of home countries. The most frequent home country studied was Turkey, which appeared in six of the studies, followed by Syria which appeared in four studies and Morocco and Pakistan which appeared in three studies each. Thirteen of the studies were specific to female entrepreneurs while one was a case study of a specific male Syrian refugee entrepreneur. An additional 16 sources investigated refugee entrepreneurs.\n\nIn Figure 2, we can see that the first publication is an outlier. This is the study by Najib (1994) on migrant entrepreneurship in Uppsala, Sweden, which was labeled in Scopus as an article but when the main author borrowed it via the university library system, it turned out to be a peer-reviewed booklet. We note that in the United States, there were studies published during the gap between 1994 and 2000, but none for Europe that met our inclusion criteria. This would demonstrate that while Sweden is at the vanguard of migrant entrepreneurship in Europe, that Europe in general followed behind the uptick in migrant entrepreneurship literature in the United States. Figure 2 shows several gaps in publications about migrant entrepreneurship in Europe until 2006, at which point annual publications on migrant entrepreneurship on the topic are the norm.\n\nOf the 91 sources in this SLR, 81 are published in academic journals. Table 4 displays the categories of the journals where these articles have been published. We recognize that it is possible to classify some of the journals into more than one area and noticed that the greatest interest in the topic is from journals that focus on entrepreneurship. The authors developed their own journal categorization rather than using Scopus and Web of Science journal categories, because our coding recognizes the specific areas within management and social science fields to allow us to gauge publishing communities’ interest in migrant entrepreneurship phenomena with more detail and to avoid an attempt at merging Scopus and WoS classification systems. For the journal classifications, which appear in Table 4, the main author checked specifically with the journal websites to read a description of the aims of each journal and created the categories based on these descriptions and shared the table with the second author for review. While it is possible to classify some of the journals into more than one area, we noticed that the greatest interest in the topic is from journals that focus on entrepreneurship. Some disadvantages of our classification system are that we did not set clear boundaries since the categories overlap and are not mutually exclusive and there is some subjectivity in the authors’ interpretations of journal characteristics. However, we believe the advantages outweigh the disadvantages because our classification allows for more detailed insight into the publishing communities that have an interest in migrant entrepreneurship phenomena, allowing for a deeper understanding of the focus and alignment of journals covering the topic. In addition, our classification method allows for greater specificity to allow for a more nuanced understanding of which academic fields are covering migrant entrepreneurship.\n\nWe see that most of the publications fit within the scope of the broader topic of management, which encompasses more specific areas such as entrepreneurship/business and human resources/organizational behavior. This means that 45 of the 91 sources are published in journals which fall under the topic of management, with 35 of the articles published in journals specific to entrepreneurship/business, five under human resources/organizational behavior, and five in journals that cover management issues on a broader level. The next two most common journal topics were immigration/ethnic studies and government/policy and immigration/ethnic studies with 13 articles and 12 articles respectively. The categories in Table 4 are not mutually exclusive, as there is much overlap between the categories. However, we aimed to be as specific as possible when larger volumes of articles are concerned, to demonstrate the range of scope from narrow to broad, based on the description of each journal. Table 4 covers only journal articles, which comprise 81 of the 91 sources in the SLR. Nine of the remaining sources consist of articles from conference proceedings and book chapters (Bashir, 2018; Hartmann and Schilling, 2018; Mason, 2008; Ong and Freeman, 2017; Szczygiel et al., 2020), with four of those book chapters published in Halkias et al. (2016b), and the final source is a peer-reviewed study from Uppsala University (Najib, 1994).\n\nIn Table 5, we see a list of conferences and journals that have published at least two of the 91 sources included in this study. Note that the most frequently appearing source, with five articles, is the International Migration, which is published on behalf of the International Organization for Migration. This is a social science journal with a worldwide geographical scope that covers the gamut of policy regarding international migration concerns (“International Migration Journal Overview,” 2023).\n\nTo synthesize findings and categorize studies based on their scope, an analysis of the different research streams was performed. The first step was to identify the relevant information from each study, using the authors’ original terms. The key information was then organized in a spreadsheet to enable comparison across studies and translation of findings into higher-order interpretations. The following information were extracted: information of the authors and meta-data of the paper, research objective, research method, research type (qualitative/quantitative/mixed), theories and frameworks and how they were applied, the sample size, the instruments used (e.g., surveys, interviews, observations) main findings, characteristics of migrant entrepreneurships, their challenges, supporting policies (and their effect). Regular working sessions were conducted by the first two co-authors during the data extraction and synthesis step.\n\nFor extracting answers for RQs, we adopted a tailored thematic analysis with open coding, where the researchers decide which data to extract from the studies by following the research questions with the process to assemble the findings from the set of studies to draw conclusions (Popay et al., 2006). Of the goals for narrative synthesis outlined by Popay et al., (2006) that we have identified as key for this SLR, are: 1) developing a preliminary synthesis from the 91 studies’ findings and 2) exploring relationships within the data. The first author went through all the papers, extracted the relevant text, and labelled them (open coding). The labels were then renamed and merged across articles, resulting in a united set of first-order code. After that, the codes were grouped in a higher-order code scheme, which are then mapped into answers for RQs. This process is outlined in Figure 3.\n\nOne common threat to systematic literature reviews is not to discover all relevant studies. The reason not to cover all seed studies may be that the search ranges in multiple disciplines, such as Information Systems, entrepreneurship, and business research and innovation management. To reduce this threat, we adopted a quite generic search protocol to retrieve as many documents as possible. This search protocol is outlined in this SLR’s sections 3.1, 3.2, and 3.3 and is aimed to reduce individual bias, so others who follow the same protocol would achieve the same results. A relevant study may be misclassified into a removal group during the selection process and vice versa. To reduce the bias in selection of papers, we defined the review protocol with clear inclusion and exclusion criteria for each selection phases. Although the protocol was not reviewed by an independent reviewer, which would have added rigor to the review process, one of the co-authors has extensive experience in conducting secondary studies, which could reduce procedural risks. Prior to applying the inclusion and exclusion criteria, the two reviewers (the first and second authors) conducted a pilot review, as outlined in section 3.3 of this SLR.\n\nIn the pursuit of a comprehensive SLR, it is imperative to acknowledge and address potential sources of bias inherent within the studies themselves that comprise the SLR. The integrity of our synthesis is inherently contingent upon the methodological rigor and quality of the individual studies under review. Therefore, we recognize that the robustness of our findings cannot surpass the caliber of the selected primary studies. In our evaluation of the studies included in this SLR, a primary concern is the potential for bias to influence the outcomes and conclusions presented, due to the inclusion of studies with deficiencies in study design, participant selection, data collection and/or analytical procedures. Given that 81 of the 91 sources are from peer-reviewed journals and that the other sources also went through peer-review prior to publication, we believe our synthesis represents the overall quality of the studies selected.\n\n\n5. Discussion\n\nResults of the search and limitations\n\nThis section presents our answers to the three RQs in Section 5.1 (RQ1), Section 5.2 (RQ2), Section 5.3 (RQ3), as well as the limitations of this SLR.\n\n5.1.1. Characteristics of migrant entrepreneurship in Europe\n\nWhen discussing the characteristics of migrant entrepreneurship in Europe, it makes sense to revisit the definitions from the beginning of this SLR. We view migrant entrepreneurs as foreign born, following the definition by Dheer (2018), who writes they can “identify, create and exploit economic opportunities to start new ventures in their destination nations” (p. 558). We also mentioned that some of the literature uses the term immigrant entrepreneur while other literature uses the term migrant entrepreneur and that both terms are relevant to this SLR. A large number of studies included in our SLR did not explicitly share a definition of entrepreneur or accompanying adjectives such as ‘migrant’ or ‘immigrant’. Several sources in our SLR did not use the terms migrant entrepreneur or immigrant entrepreneur. They instead either used the term diaspora entrepreneur, ethnic entrepreneur, refugee entrepreneur, or referred to entrepreneurs by their home country. It was often the case that sources with a broader coverage of migrant entrepreneurship or immigrant entrepreneurship did not provide an explicit definition of these terms, despite using them as a focus in their studies. The sources that were most explicit with their definitions are listed on Table 6. Some of the definitions clash with ours, because we do not include second generation immigrants, who are born in the host country, as migrant entrepreneurs. Moreover, “country of origin,” as used by Širec and Tominc (2017), could be subject to interpretation. We also note that most of the 91 sources do not include explicit definitions on migrant or immigrant entrepreneurship. Table 6 displays an overview of the definitions. It is possible to categorize the characteristics revealed in the studies, into those that are unique to migrant entrepreneurs and those that apply to all entrepreneurs. Frequently studied characteristics that apply to all entrepreneurs include formal education, prior vocational experience, sector of business activity, gender, age, nationality, reasons for becoming an entrepreneur, marital status, and parental status. 10 of the studies that focus on migrant background investigate differences between first- and second-generation migrants and sometimes even into the third generation. This is not within the scope of this literature review, which only is concerned with first-generation migrants.\n\nSince previous entrepreneurship literature follows the line of thought from Bourdieu (1984) by looking at the impacts of financial, human, and social capital in establishing and running a new business (Baklanov et al., 2014; Eroğlu, 2018; Grubbström and Joosse, 2021) we also use these categories to classify the characteristics gathered from the studies.\n\nEmphasis in this study is on the characteristics specific to migrant entrepreneurs rather than entrepreneurs in general. Table 7 shows the key factors studied. It is worth noting that the social capital factors are those that are the most specific to migrant entrepreneurs. The implication is that it is important for migrants to learn the local language and establish social capital by learning the local culture and by building up a local network, to establish and operate a business in the host country. It is also evident that it is more difficult for migrants to move to the host country and immediately start a business unless they already have business experience or relevant experience from their home countries (Barth and Zalkat, 2021). The more time a migrant spends in the host country, the easier it appears to establish and run a business in the host country.\n\n5.1.2. Human capital\n\nAs mentioned in the background of this article, migrant entrepreneurs are people who have moved to a different country and have started businesses there, and they have an array of backgrounds and offer a unique set of characteristics, experiences, and skills to run their businesses.\n\nTable 7 shows human capital factors that differentiate migrant entrepreneurs from native entrepreneurs. One of these factors is whether migrant entrepreneurs obtain their work experience in the home country or the host country. Mixed-country work experience can provide migrant entrepreneurs with unique perspectives on how to conduct business in cultural and economic environments and offer advantages by helping bridge gaps between the home country and the host country (Bolzani and Boari, 2018; Van Delft et al., 2000). In this respect migrant entrepreneurs have some advantages over native entrepreneurs, provided that they are embedded with the cultural norms and social networks of both the home and host country (Bolzani and Boari, 2018; Meister and Mauer, 2019). As with work experience in the host country, qualifications from the host country are also an important consideration. When a migrant entrepreneur has obtained educational qualifications and certifiable skillsets in the host country, such credentials not only help establish individuals as experts in their fields, but when the credentials are from the host country, it demonstrates a higher level of local embeddedness which means a larger local network and greater understanding of the local bureaucracy and regulations, which in turn can ease the processes of starting and running a business in the host country. Along these lines, the length of time in the host country and ability in the host country language are all key characteristics which contribute to this local embeddedness and higher level of local understanding, and which also appear on Table 7. Examples include a deeper understanding of the local market (Szczygiel et al., 2020) and a greater likelihood of established relationships with key players in the community. On the other hand, those who are newer to the country may have a fresh perspective and may be more open to trying new things. Whereas local language competence eases communications with customers and clients and may be more successful in building relationships within the community (Nijhoff, 2021). It also means that migrant entrepreneurs will be able to compete on a service level rather than merely on price (Valenzuela-García et al., 2014).\n\n5.1.3. Social capital\n\nSome migrants arrive in their host country with more social capital than others. As mentioned in the background section of this article, there are many reasons why people move to another country. Of the eight articles that explore this area further, the motivations for migration include one investigation of“\"lifestyle migrants”\" who have chosen to immigrate to a new country not primarily for economic reasons, but rather for personal or lifestyle reasons (Munkejord, 2017a). These entrepreneurs may be drawn to the country for its culture, climate, or other non-economic factors, and may be more interested in starting businesses that reflect their personal interests and passions. Seventeen studies focus on refugee entrepreneurship, where the motivation to move to another country is to flee dangerous conditions in the host country. This is also known as forced migration. The forced migration studies by Qin (2021) and Harima and Freudenberg (2020) are listed on Table 7 for their contributions. Harima et al., (2020) add that while entrepreneurship can be a path for the vocational integration of refugees, they need special support beyond what native entrepreneurs would require. Along with motivations for migration, it follows that scholars are interested in knowing whether people were joining family in the host country, entering the host country with others, or traveling alone.\n\nAn important part of building social capital is having legal status in the host country, with citizenship status being the highest level of inclusion. Legal status also frequently appears in the literature. When migrant entrepreneurs lack legal status, it means they are engaging in the underground economy with implications of missed tax revenue for the host country and missing social welfare benefits and protections for the migrant entrepreneurs. When considering legal status, it is also the case that some countries do not allow asylum seekers or refugees to work or engage in entrepreneurship until certain conditions are met (Lintner and Elsen, 2020). This can have severe implications on the integration process, especially if the migrants are kept apart from the host society in isolated processing centers or shelters for extended periods of time. Another study focuses on the European Union (EU) movement of people which makes it easy for EU citizens to gain legal status in other EU member states (Bruzelius, 2020).\n\nA final social capital factor to note is the location in the host country. Some of the studies explore comparative relationships between different locations within the same host country. In some host country communities, a town or city may have a strong ethnic enclave in place (Andersson, 2021). Results show that ethnic enclaves frequently play a role on migrant entrepreneurship activities. For example, there may be an inclination to imitate other co-ethnics who appear to be successful entrepreneurs in a specific industry (Andersson, 2021). On one hand, co-ethnics may be able to help introduce and explain the host culture, society, and regulations to new arrivals and opportunities exist for migrant entrepreneurs to leverage the co-ethnic network to access the market of their co-ethnics (Van Delft et al., 2000). On the other hand, ethnic enclaves can also have an effect of slowing the integration process by hindering the speed at which new arrivals learn the local language and have contact with native society (Andersson, 2021; Valenzuela-García et al., 2014).\n\nSeveral challenges discussed in the literature are not specific to migrant entrepreneurs; these include issues with private life, gender discrimination, competition, marketing issues, and problems with advisors and lack of them, as well as a range of insufficiencies, including professional experience, skills and training, access to resources such as personnel, real estate, and financing. Financing, however, can be more central of an issue to migrant entrepreneurs than to native entrepreneurs, due to higher unemployment rates, a lack of access to bank loans, greater financial uncertainty, and difficulty in receiving funding from mainstream banks. As pertaining specifically to migrant entrepreneurs, Table 8 highlights the main points from the literature. The most frequently mentioned challenges specific to migrant entrepreneurs include discrimination and a lack of the following: cultural and social understanding, local network, proficiency in the local language, understanding local laws and regulations, and legal status in the host country.\n\n5.2.1. Social capital\n\nTable 8 highlights the social capital factors that are challenges migrants face as entrepreneurs in European host countries. Here, lack of cultural understanding frequently appears as a challenge. This challenge can arise in several contexts, whether from the lack of understanding refugee resettlement policies in the host country (Qin, 2021), the creation of in-group/out-groups (Rashid and Cepeda-García, 2021), or a general disadvantage in establishing an enterprise in the host country (Bolzani and Boari, 2018). In addition, Meister and Mauer (2019) and Qin (2021) postulate that refugee entrepreneurs face a greater lack of social capital than other migrant entrepreneurs, which would require a specific support model for them, which is the case for the Fresh Start Programme, which also finds that there is not a one-size-fits-all approach for migrant entrepreneurship in various settings within Europe (Wade, 2020). Discrimination by the host community is another frequently mentioned challenge that migrant entrepreneurs face; this results in missed opportunities (Solano, 2021). Initiatives from civil society to increase contact between native and migrant populations can help build cross-cultural familiarity and reduce discrimination (Embiricos, 2020). Meister and Mauer (2019) focus on the refugee context and point out that discrimination creates a lack of trust that is a part of an overall negative societal perception of refugees, with the greater implications being limited socio-economic involvement and lack of engagement in the legal-institutional environment of the host country. Lack of network with natives in the host country is also frequently mentioned. Van Delft et al., (2000) note that this can lead to migrant entrepreneurs’ exclusion from the mainstream economic activity of the host country.\n\n5.2.2. Human capital\n\nChallenges in the area of human capital are also frequently mentioned in the literature. Lack of proficiency in the language of the host country is the most frequently mentioned issue. This has implications such as communicating with customers, suppliers, submitting tenders, ability to negotiate, and ability to comply with local laws, regulations, and tax regimes. Such issues are also frequently mentioned in the literature as a challenge for migrant entrepreneurs. In addition, lack of credentials can prevent entrepreneurs from being able to operate in areas of their expertise if their qualifications are not recognized in the host country. Lack of resources can fall into the human capital arena, either on an individual or ethnic group level. In other cases, the issue can fall under financial capital, as mentioned in the next paragraph.\n\n5.2.3. Financial capital\n\nLack of resources in the financial capital area include lack of money, difficulty to afford housing, and lack of access to financing. Migrants face higher unemployment rates than the native population (Evansluong and Ramírez-Pasillas, 2019; Szczygiel et al., 2020) which can lead to a lack of financial capital. Barth and Zalkat (2020) point out a lack of financial expertise is also an issue for many migrant entrepreneurs. Financial training, while falling under the human capital area in the previous section, is an important consideration for policy makers and practitioners when looking at funding a new migrant entrepreneurship venture. Meister and Mauer (2019) emphasize that forced migrants are likely to face a lack of financial capital due to the nature of suddenly fleeing from their home country. In a study of migrant entrepreneurship support programs across six European cities, Van Delft et al., (2000) find that despite the financial challenges that migrant entrepreneurs face, programs that emphasize education and training appear to be the most successful, which reinforces the view that financial support should come after ensuring that human capital reaches a sufficient level for migrants to establish and run a business.\n\n5.2.4. External factors\n\nExternal factors are an issue when a migrant has refugee status, because of specific constraints such as rules that do not allow refugees to become entrepreneurs. If integration programs direct migrants to find a job and do not consider the individual desires and motivations of the migrants, who may be interested in entrepreneurship, then this can be a challenge that hinders entrepreneurship (de Lange et al., 2021; Harima and Freudenberg, 2020; Wade, 2020).\n\nFinancial capital initiatives include bank loans, donations, subsidies, and tax relief, with general access to finance, funding, or financial support most often mentioned in the literature. Human capital initiatives include programs that will help aspiring migrant entrepreneurs pass the necessary training for required certifications to operate in their chosen industry, entrepreneurial and language training, co-development between small migrant enterprises and larger companies, and specific support for internationalization. Social capital initiatives build cultural understanding, network, and provide helpful information about entrepreneurial support that is available locally and nationally. Table 9 shows that entrepreneurial training, language training and mentoring are the main activities as human capital support; the table also includes a number of external factors which are “opportunity structures” divided into market, regulatory, and state categories (Berntsen et al., 2021).\n\n5.3.1. Human capital\n\nTable 9 displays various human capital factors to consider for migrant entrepreneurship support initiatives and policies. Education and training, followed by mentorship and advice are the most commonly cited areas of support, followed by language training. The literature shows evidence that to establish and run a business in the host country, migrant entrepreneurs require adequate education and a relevant skillset to navigate the complexities of the local bureaucracy, tax rules, legal issues, and accountancy, in addition to the marketing know-how, resource management and leadership skills required to run a business. Research by Nijhoff (2021) in the Netherlands demonstrates that the issues of language and local bureaucratic complexities should be the first foundations to cover for migrant entrepreneurship support programs. Backman et al., (2021) also place language learning as a priority, in addition to cultural training to learn about the host country society. Beyond these foundations, relevant education and training can help to bring aspiring migrant entrepreneurs to a level of self-sufficiency to help them manage their businesses. Mentorship and business advice are also helpful to fill in knowledge gaps and to answer questions for specific areas where migrant entrepreneurs may need extra help. Although we see evidence that language learning is a cornerstone component in the journey of a migrant entrepreneur, it is less mentioned as a support area in the literature that is covered in the present SLR, and instead appears most often as a characteristic that is required to run a business in a new country. Wade (2020) emphasizes the need for language support and intercultural training to be a part of entrepreneurial support programs that are specific for migrant entrepreneurs, such as the case with the Fresh Start Programme.\n\n5.3.2. Social capital\n\nIn Table 9, there are also social capital factors to consider for migrant entrepreneurship support initiatives and policy, with lack of local networks being a frequently mentioned factor. To set up and operate a new venture in the host country, migrant entrepreneurs will benefit from a local network comprised of both weak and strong ties (Bouk, Vedder, and Poel, 2013). One way that migrant entrepreneurs can build up both weak and strong ties is by joining a local accelerator (Noor, 2021), with Harima et al., (2020) and Meister and Mauer (2019) providing extra emphasis on business incubators for refugee entrepreneurs that explicitly incorporate the need for understanding and tapping into social capital in the host country. Bouk et al., (2013) examine the differences between strong and weak ties for migrant entrepreneurs and find that strong ties provide the key resources during the startup phase for those who have a network of entrepreneurs already in the host country. When migrant entrepreneurs’ business network in the host country is missing, they find that migrant entrepreneurs should build up weak ties to help them in the initial phases of their business journey. Research by Colombelli et al., (2021) shows that successful entrepreneurs as role models who are the same gender as the individual just starting out are also important. Therefore, entrepreneurial support programs should take this into consideration. Entrepreneurial support programs should also consider whether the migrant entrepreneur is married to a local of the host country, since there is an existing set of weak and strong ties from the local network of the spouse (Munkejord, 2017b). Furthermore, research by Mason (2008) demonstrates the need for entrepreneurship support initiatives to be bottom-up, that is to say, originating from the migrants themselves, because the research shows that top-down initiatives do not help migrant entrepreneurs create a viable business within a reasonable time frame, while Lyon et al., (2007) shows that entrepreneurship support should consider the individual social context of migrant entrepreneurs as well, since this leads to different qualities of entrepreneurship within a local host economy, regardless of ethnicity. Additionally, research by Van Delft et al., (2000) leads to the concern that migrant entrepreneurship support’s emphasis is on exploiting economic potential rather than focusing on the problems that migrants face when entering labor markets in their host countries. In this respect, it is important to consider other factors of social capital that appear in this SLR, such as training about the local culture and society (Johnson and Shaw, 2019; Qin, 2021) as well as providing emotional support (Hagos et al., 2019; A. Harima, Freudenberg, and Halberstadt, 2020).\n\n5.3.3. Financial capital\n\nFinancial capital is another part of migrant entrepreneurship support initiatives which frequently appears in the literature. Research by Kontos (2003) shows that a specific support policy in Germany, known as a ‘bridging allowance scheme’ to finance the stabilization phase of a new business, appears to be of greatest benefit to female migrant entrepreneurs, who have been socialized toward self-employment.\n\n5.3.4. External factors\n\nBerntsen et al., (2021) suggest that we can break external factors into three “opportunity structures”: 1) market, 2) regulation, and 3) state. Of these three opportunity structures, those taking place at the state level appear most frequently in the literature, in particular concerning immigration and differentiated assimilation policies. Initiatives such as entrepreneurship visas for qualifying individuals (Noor, 2021) and the European Union’s free movement policies (Bruzelius, 2020) are examples of immigration policies that help qualifying migrant entrepreneurs to establish and run a business in the host country. As for market opportunity structures, a couple of articles provide examples of how new digital platforms have helped migrant entrepreneurs to access customers and formulate pricing models (Wang and Chen, 2021; Webster and Zhang, 2020). Examples of regulation opportunity structures include childcare services for women entrepreneurs (Bashir, 2018; Ozasir-Kacar and Essers, 2021) and educating banks to help migrant entrepreneurs with financing (Piperopoulos, 2010).\n\nSLRs offer valuable insights into research domains, but they are not without limitations. One significant challenge inherent is the potential for overlooking relevant studies. This is a salient issue with this SLR because it bridges two disciplines, entrepreneurship and international migration, which in themselves span diverse disciplines. International migration spans disciplines such as demography, economics, geography, history and sociology (Inglis, Khadria, and Li (2019), and entrepreneurship, spans disciplines in economics, management, psychology, and sociology (Davidsson, 2009). To address this, a comprehensive search protocol was implemented to retrieve a wide range of documents, outlined in sections 3.1, 3.2, and 3.3 of this SLR, aiming to minimize individual bias. However, the risk remains that a pertinent study might be erroneously categorized during the selection process, affecting the review's comprehensiveness. Efforts to mitigate this bias include clear inclusion and exclusion criteria defined in the review protocol for each selection phase. While an independent review of the protocol would have bolstered the review's rigor, the involvement of an experienced co-author with a background in secondary studies helped counterbalance procedural risks. The piloting of the review process by the first and second authors further contributed to refining the selection methodology.\n\nThe integrity of the synthesis conducted within this SLR is contingent upon the methodological rigor and quality of the individual studies included. The authors acknowledge that the robustness of the findings from this SLR cannot exceed the caliber of the selected primary studies or analytical procedures. Despite the inclusion of primarily peer-reviewed journals (81 out of 91) and ten remaining peer-reviewed studies, it is important to recognize that the SLR’s conclusions are tethered to the overall quality of the selected studies. In terms of synthesis, efforts were made to categorize and analyze findings systematically. While the process of extracting information and categorizing studies using original terms and predefined criteria is meticulous, it may still be influenced by researchers' interpretation. The adoption of tailored thematic analysis with open coding adds a degree of subjectivity to data extraction, potentially impacting the objectivity of results. Although systematic working sessions between co-authors were conducted to enhance consistency during data extraction and synthesis, individual biases might subtly influence the process. Collaborative iterations during the synthesis process helped maintain focus on the research questions and ensure a balanced approach where two people working together during the iterations helps reduce the bias of a single person. However, despite these efforts, the inherent subjectivity in identifying relevant paragraphs and texts could introduce unintended bias into the analysis. While systematic literature reviews offer valuable insights, they are susceptible to limitations such as potential oversight of relevant studies, bias in data extraction and analysis, and the dependence on the quality of selected primary studies. The authors aimed to mitigate these limitations through systematic procedures, collaboration, and transparent reporting to enhance the validity of the SLR's findings. However, it is of merit to acknowledge the the inherent challenges of the methodology.\n\n\n6. Implications for Practice, Policy, and Future Research\n\nIn terms of support for migrant entrepreneurs, our review attempts to cut through the complexity of the literature to demonstrate that migrants need to build the foundations for human and social capital in the host country prior to focusing on financial capital and entrepreneurship. This has implications for host country governments and practitioners managing migrant entrepreneurship support programs, since without first having a solid level of human and social capital in the host country, migrant entrepreneurs may be doomed to either failure or low margin businesses (and possibly illegal or non-registered businesses) with long working hours and sub-par conditions (Bashir, 2018; Solano, 2021; Van Delft et al., 2000), rather than contributing strongly to the economies of their host countries. This would indicate that offering training opportunities related to learning the local culture and language, educational advancement, networking, and volunteering should be considered higher priority than providing financial support, except in cases where migrants first demonstrate enough human and social capital to serve as the foundations for a future as entrepreneurs in their host country.\n\nAs noted in the categorization of the data, the results of this study also reveal three key thematic clusters as emerging from the 91 sources included in this review. These are namely: financial capital, human capital, and social capital. The financial capital cluster includes categories such as access to financing, employment status when starting a business, personal wealth, and family wealth. The human capital cluster includes categories such as: previous experience, professional background, education, and skillsets. The social capital cluster includes categories such as: host country experience, local credentials, language competency, local network, length of time in host country and legal residency or citizenship status in the host country. These three clusters serve to understand both the characteristics of migrant entrepreneurs as well as the challenges they face when financial, human, and social capital are missing.\n\nFigure 4 displays some of the key reflections from the findings relating to our research questions.\n\nOn the left of the figure, supporting policies encompass key areas that migrant entrepreneurs require to establish, maintain, and grow a business in the host countries. These support areas cover the range of financial, human, and social capital. Evidence shows that cultural and language training are the first foundations necessary prior to building up the remaining support areas. The support then helps migrant entrepreneurs to face the challenges that the literature emphasizes, starting with cultural and language understanding and then tackling other areas such as acquiring local credentials, understanding local regulations, and building up a local network. Consideration of the migrant entrepreneurs’ previous professional and entrepreneurial experience and educational experience are key when considering what kind of entrepreneurial support to offer. Two key issues that also need to be considered are discrimination and legal status, which along with a migrant entrepreneur’s home country origin, have an impact on the path forward as migrant entrepreneurs. To this extent it is important to individualize the entrepreneurial support offered rather than lumping migrant entrepreneurs into one single group. This may help avoid an in-group/out-group scenario and may help overcome issues discrimination. The idea is that entrepreneurial support will then assist migrant entrepreneurs to establish and run a business that will help them become economically independent and contribute to the economies and societies of their host countries.\n\nThis study thus provides some thoughts for further investigation of key issues in the field which can help academics, policymakers, and practitioners, such as: 1) how to measure the levels of human and social capital that are optimal, prior to providing financial support? 2) how to incorporate human and social capital into entrepreneurial support programs? 3) how to balance human and social capital with the financial needs (employment/entrepreneurship) of migrants, within the integration processes of new arrivals into host countries? 4) how to help migrants become aware of the options open to them upon arrival into their host countries that will allow them to enhance human and social capital and allow them to reach their potential in their host countries to integrate culturally, economically, and socially? 5) how to properly assess migrants for their extent of human and social capital to help them fill in the gaps and/or support them in their efforts to become entrepreneurs? And 6) how to introduce a bottom-up approach to take into consideration the needs and wishes of the migrants themselves to support them on their entrepreneurial journeys?\n\nMoving forward, this review can also help scholars to formulate new research questions that contribute to the development of the fields of migration and entrepreneurship support, which will become ever more relevant as people continue to move to Europe or change countries within Europe. It is of merit to note that three of the sources also raise the question about whether the very construction of ‘migrant entrepreneurship’ creates an otherness which perpetuates stereotypes via the comparison to local/native entrepreneurs. There is a danger of lumping immigrants together in one group despite their uniqueness and individual differences (Högberg et al., 2016; Mason, 2008), which can create an in-group/out-group scenario (Rashid and Cepeda-García, 2021). An additional three sources (see Table 8) also touched on this by pointing out that uniform integration policies are not effective and that individual needs, including home country origins, need to be taken into consideration (Bijedic and Piper, 2019; Hartmann and Schilling, 2018; Ram et al., 2013). Moving forward, future research could investigate these issues and examine how to address the needs of migrant entrepreneurs while recognizing the uniqueness of each individual.\n\nOur findings allowed us to propose a research agenda for practitioners and scholars to consider, along the lines of 1) facilitating human and social capital, 2) information sharing, 3) assessing human and social capital, 4) entrepreneurial support programs, 5) in-group / out-group bias in entrepreneurial support, and 6) a bottom-up approach to migrant entrepreneurship support. Table 10 provides an overview of potential research questions for these six research directions. Along the lines of point 1, facilitating human and social capital, the issue is to contribute to entrepreneurship outcomes which lead to employment and economic growth, rather than illegal entrepreneurship activities or low-margin businesses where migrant entrepreneurs compete only on price and work extremely long hours with low pay. As for point 2, information sharing, the key is helping migrants understand options available to them, that will help them learn the local language, customs, rules, regulations, educational and training opportunities, both inside and outside of entrepreneurship support. Regarding point 3, the concern is with how to assess the human and social capital of migrants to provide them with opportunities to help them build human and social capital in the host country. An example of a tool to help assess social capital is the Position Generator by Chen and Tan (2009), to gain an overview of a migrant’s network quality in both the home and host countries. Human capital can be assessed not only by diplomas and certifications, but also by testing for skillsets required for certain forms of employment and entrepreneurship. Concerning point 4, entrepreneurial support programs, there can be issues where attendees lack human and social capital, as well as motivation, to succeed at entrepreneurship. This is why it is important to assess for these factors and consider restricting access to entrepreneurial support programs unless certain conditions are met. With respect to point 5, several of the studies in our SLR raise concern that migrants are lumped into one category instead of being treated as individuals. It is worth investigating the extent that migrant entrepreneurship initiatives perpetuate stereotypes and create an in-group/out-group phenomenon, and to look at cases where migrants and natives can benefit from participating together in the same cohort of an entrepreneurship support program. As for point 6, since there is not a one-size-fits-all approach to entrepreneurial support, it would merit studies that include approaches such as co-creation (Torfing, Sørensen, & Røiseland, 2016; Van Praag, 2021), that adopt the input of the migrants themselves into how to design an entrepreneurial support program to best suit their needs.\n\n\n\n1. How to measure the levels of human and social capital that are needed prior to offering financial support to aspiring migrant entrepreneurs?\n\n2. How to effectively establish human and social capital in migrants who lack it and aspire to become entrepreneurs?\n\n3. How to incorporate human and social capital into entrepreneurship support programs?\n\n\n\n1. How to help migrants become aware of their options after they arrive in the host country?\n\n2. How can we operationalize the kinds of information that migrants to settle into their host country and start a business?\n\n\n\n1. How to assess human and social capital of migrants arriving at host countries?\n\n2. How to consider each migrant’s individual needs, goals, ambitions, and potential?\n\n3. How to consider the role of the home countries on entrepreneurial potential?\n\n\n\n1. Which aspects of entrepreneurial support programs are specific to all entrepreneurs, and which are unique to migrants?\n\n2. To what extent does home country matter in the content of an entrepreneurial support program?\n\n3. Should admission to entrepreneurial support programs be restricted to those with high enough levels of human and social capital?\n\n\n\n1. How do migrant entrepreneurship support initiatives contribute to the in group/out group bias?\n\n2. How do migrant entrepreneurship support initiatives perpetuate stereotypes?\n\n3. How do migrant and natives in the same cohort of entrepreneurship support programs benefit from joint participation?\n\n\n\n1. How to implement a bottom-up approach to migrant entrepreneurship support programs?\n\n2. How much can the migrants themselves be involved, or co-create entrepreneurship support programs?\n\n3. When is it beneficial for migrants to attend the same entrepreneurial support programs as natives to the host country and when should they attend dedicated programs for migrants?", "appendix": "Data availability statement\n\nThe data for this article consists of bibliographic references, which are included in the References section.\n\nUniversity of South-Eastern Norway: PRISMA and PRISMA for abstracts checklists for ‘Migrant entrepreneurship in Europe: a systematic literature review’. https://doi.org/10.23642/usn.23925102. 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Oxford University Press; 2010." }	[ { "id": "216746", "date": "13 Nov 2023", "name": "Monder Ram", "expertise": [ "Reviewer Expertise Migrant entrepreneurship" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aim to contribute to extant academic and policy studies by examining the characteristics and challenges of migrant entrepreneurs in Europe. The authors adopt a well-established framework to ensure that the systematic literature review is based on a comprehensive and structured synthesis of available research on migrant entrepreneurship in Europe. The manuscript is well-structured and comprehensive. The authors identify three research questions and have addressed them systematically. The article is clearly written and easy to follow. The authors also use relevant and contemporary sources to support their arguments. There are some areas where the article could be improved. Firstly, the authors could provide more information on limitations of their study. It warrants a separate section, rather than a brief mention in the conclusion. More information on the implications of the findings would be helpful too. Overall, the authors make a useful valuable contribution to the field of migrant entrepreneurship in Europe. They identify gaps in the field and offer helpful guidance for future 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? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10912", "date": "22 Jan 2024", "name": "Dimitris Polychronopoulos", "role": "Author Response", "response": "Positive Feedback: Response: Thank you for recognizing the structured and comprehensive nature of our systematic literature review. We appreciate your positive comments on the clarity, systematic approach, and use of relevant sources, as well as your positive assessment of our contribution to the field of migrant entrepreneurship in Europe. Action: None required. Limitations of the research: Response: Thank you for suggesting that the discussions surrounding the limitations of the study merit a dedicated section. Action: We have converted Section 5.4 into Section 6 to emphasize that the limitations merits a dedicated section. In addition, we have expanded the discussion of the limitations with the following three additions to the text: “Indeed, during the open review process, we received feedback that the reviewer was aware of an article that met our search criteria but still did not appear in the SLR.” “An additional limitation of this study is that it does not account for the impact factor of the journals included, which is a measure that the systematic review by Aliaga-Isla and Rialp (2013) achieved. Nor does this study engage in citation analysis to focus on the most impactful articles, which is a measure taken in the systematic review by Sithas and Surangi (2021).” “In addition, Table 11, which lists adopted theories of the 91 studies included in this SLR, does not list every implicit theory that the studies incorporated into their research, nor does it provide an in-depth analysis of how the theories listed are applied. Such an in-depth analysis could be worthy of future research because it would provide an overview of how scholars are applying theory and how theory application relates to research methodologies and the unit of analysis for the studies reviewed.” Implications of the findings: Response: Thank you for pointing out the need for a more thorough exploration of the implications of our findings. Action: Considering that Section 7 of the original version included implications for policy makers and implications for future research, we have expanded Section 7 to include a section for owners of migrant entrepreneurship support programs. It reads as follows: 7.2. Implications for practitioners For those running entrepreneurship support programs, it is important to include clear goals for the programs and to include metrics to evaluate their effectiveness according to the desired goals of the program. A takeaway from this SLR is that improved business performance would be a logical desired outcome for entrepreneurship program participants. So setting up ways to track improved business performance in terms of number of employees and profits are examples of metrics that can accomplish that. Other considerations are the catalysts that help generate improved business performance. Indications are that transnational entrepreneurship, defined as business taking place across national borders (Drori, Honig, and Wright, 2009) would be worth instilling into entrepreneurship support program participants long-term plans because transnational enterprises are more profitable than other forms of migrant entrepreneurship (Solano, 2016). The IntEnt non-profit incubator investigated in the Riddle, Hrivnak, Nielsen (2010) had the uniqueness of being dedicated to transnational diaspora entrepreneurship. Wang and Liu (2015) found that transnational firms have higher payroll per employee than other firms, regardless of whether the other firms are migrant or non-migrant. Entrepreneurship support programs can benefit from the findings by Meister and Mauer (2019) by providing access to network and resources to fill the gaps in structural constraints that migrant entrepreneurs experience in their host country, with the long-term aim of helping participants achieve transnational entrepreneurship." } ] }, { "id": "216744", "date": "13 Nov 2023", "name": "Robert Kloosterman", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is another systematic literature review on migrant entrepreneurship adding to the already existing six – four of which were published in the past three years. This could be seen as sign of the maturing of the field of research on migrant entrepreneurship and, given the multifaceted nature of the topic, there is, in principle, ample room for such meta studies. This study aims at focusing on three questions in a selected part of the literature: 1) the characteristics of migrant entrepreneurs; 2) the challenges they face; and 3) the reported policies. This study limits itself to studies regarding Europe.\nThe study is well-written (a few typos) and the methodology is spelled out in detail – almost too much in detail in my view. I could have done without the many references about the discussions between “the main author” and “the second author”. I did miss at least one article that would fall within the selection criteria of the study. The study provides a useful overview of the selected articles in an accessible way. Notably the information on the geographical distribution of the studies and the growth of these over time (graph on p.13) is helpful.\nI am, however, not entirely convinced of the overall added value of this meta study. First, the study is limited to Europe. There are, obviously, differences along many dimensions in the institutional context between European countries. Still, it would have made sense to group these into say three of four groups on the basis of welfare state arrangements and migration policies as these two features have a significant impact of migrant entrepreneurs. This could then form the basis of a much more in-depth analysis of the studies they look at and can, for instance, be linked to the outlier position of Sweden.\nSecond, the formulation of the three “research questions” is rather awkward. The characteristics of the migrant entrepreneurs are not so much characteristics but issues that the studies focus on. Many of these can indeed be traced back to Bourdieu, but also to Granovetter, Waldinger, Portes and few other pioneers. The “challenges” of the second question are, unsurprisingly, closely linked to these issues (two sides of the same coin), whereas the reported policies also, of course, are mainly targeted to these. It would, then, makes sense to regroup the trawl and position the issues as the foundation for policy making. This would also make room for much more in-depth analysis of policy making instead of just reiterating insights, like on p.35, that have already been stated in many other publications (“To this extent it is important to individualize the entrepreneurial support offered rather than lumping migrant entrepreneurs into one single group” and “how to measure the levels of human and social capital that are optimal, prior to providing financial support”- what would optimal look like: optimal in the short term, long term; in which real-life context?).\nThird, one way of really contributing to the discussion, would be to look at the changes over time. Do we see shifts in the issues and in the type of policies? Do we see geographical patterns regarding these as well. From a meta study, you would expect more than just repeating what other studies have already stated. Link the findings to a broader context. Where, in short, is the meta view in this contribution?\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "10913", "date": "22 Jan 2024", "name": "Dimitris Polychronopoulos", "role": "Author Response", "response": "Acknowledging Positive Feedback: Response: Thank you for recognizing detailed presentation of our methodology. We appreciate your positive remarks about the accessibility of the overview and the valuable insights provided, especially regarding the geographical distribution and growth of studies over time. Thank you for recognising the multifaceted nature of the topic and that there is ample room for such meta studies. With our narrow focus on support and Europe, we hope to contribute to a specific niche within this field. Action: None required. Recognition of the multifaceted nature of migrant entrepreneurship: Response: Thank you for recognizing the multifaceted nature of migrant entrepreneurship and that although the research field of migrant entrepreneurship is maturing, there is ample room for meta studies such as this one. Action: To clarify how this systematic literature review is different from the other six literature reviews on migrant entrepreneurship, and to emphasize the search criteria of the study, we have added the word support to the title and in multiple parts of the text, where we felt that the stress on this specific context was missing. In addition, we added PRISMA to the title to differentiate this study from other literature reviews on migrant entrepreneurship. Given the uniqueness of the PRISMA protocol, its inclusion in the title distinguishes our study on the level of protocol and can thus help future researchers who are looking for a systematic literature review that follows the PRISMA protocol. Some typos throughout the article: Response: Thank you for pointing out that there are some typos in the article. Action: In the new version, we removed the error where the reference to Bourdieu appeared on Table 6, and we also noticed typos such as: entrepreneurship instead of entrepreneur in sections 4.2 and 5.1.1. We also removed the word ‘first’ from the end of the first paragraph in section 4.2., and corrected electronical to electronic in the last paragraph of section 3.1 We also corrected the typos in Table 9, and in sections 3.4, 4.1, and 6. Excessive references about the discussions between “the main author” and “the second author”. Response: Thank you for bringing this issue to our attention. Action: We have addressed this issue by removing references to the first and second author, with replacements such as ‘we’, ‘the authors’, and the passive voice. Institutional Contexts within Europe concerning Welfare State Arrangements and Migration Policies: Response: Thank you for your suggestion to categorize European countries based on welfare state arrangements and migration policies to help provide a more in-depth analysis to cover the impact that such arrangements and policies have on migrant entrepreneurship, which could possibly explain the outlier of Sweden in terms of its early appearance in the field as well as its prolific representation in the literature stream. Action: In consideration of the unique migration policies and welfare state arrangements of Sweden, we have looked through the sources in our SLR that focus on the Swedish context and found several unique features that are worthy of mention. Therefore, have added to the text in section 4 where we shared the results about Sweden’s early entrance and volume of studies. In addition, we provide further detail about the investigations from the other top countries in the SLR: United Kingdom, Germany, the Netherlands, Italy, and Spain. The new version now contains the following text: With Sweden as an outlier both in terms of its early arrival into the migrant entrepreneurship support literature, as well as with its volume of studies over time, it is worth looking into possible reasons for its prolific position. First of all, Sweden has high immigration rates compared to other European countries (Evansluong and Ramírez Pasillas, 2019). Backman, Lopez, and Rowe (2020) state that Sweden had the largest share of asylum seekers between 2002 and 2013, which is one aspect that could help explain the large volume of literature covering the Swedish context. Secondly, Sweden has specific integration policies that emphasize new arrival’s first job (Webster and Zhang, 2020), along with a rigid labor market and generous support system (Backman, Lopez, and Rowe, 2020), with government organizations providing dedicated advice about entrepreneurship to immigrants (Andersson, 2021; Högberg, Schölin, Ram, and Jones, 2016; Yazdanfar, Abbasian, and Brouder, 2014). A third factor is the change in immigration rules in 2015, where Sweden stopped recognizing credentials from the Middle East, thus resulting in higher rates of self-employment amongst impacted new arrivals (Barth and Zalkat (2021). When considering Table 3 for geographical patterns, taking the top six countries, which each have at least five sources, in this SLR of 91 sources, which are dedicated to a single country: Sweden, United Kingdom, Germany, the Netherlands, Italy, and Spain, it can be useful to compare these countries to gain further insights. A common theoretical thread across all six countries is that mixed embeddedness (Kloosterman, van der Leun, and Rath, 1999) appears frequently to help provide an analytical framework for the studies. On the other hand, there are some areas of research which do not seem to have a common thread across countries. For example, exclusive coverage of refugee entrepreneurship is absent from the Spanish literature, whereas exclusive coverage of female entrepreneurship is absent from the German literature. In-depth discussion on the topic of ethnic enclaves is also absent from the German sources, although ethnic communities and ethnic markets do provide context in the article by Bijedić and Piper (2019) and co-ethnic networks in the article by Meister and Mauer (2019) as well as ethnic communities, networks, and resources by Kontos (2003). It is also noteworthy that only Italy and the United Kingdom have articles published about tech startups. The United Kingdom seems to have the highest rate of migrant entrepreneurship, with their rate being three times higher than native-born British (Osaghae and Cooney, 2020). Investigations into entrepreneurship in the agricultural sector appeared in the studies from Germany, Italy, and Sweden, while research on internationalization or transnational entrepreneurship appeared only in the articles from Italy and the Netherlands. Both Germany and Sweden have government organizations exclusively focusing on migrant entrepreneurship, and both countries have literature expressing concern of the ‘othering’ of migrants, creating an in-group/out-group and expectations of inferiority of migrant entrepreneurship compared to non-migrant entrepreneurship (Högberg, Schölin, and Ram, 2016; Mason, 2008; Rashid and Cespeda-Garcia, 2021). Formulation of the Research Questions: Response: Thank you for sharing your concerns about the awkward formulation of our research questions, where characteristics are simply issues that are one side of the coin and the challenges are the other, with the policies targeted toward these. We also appreciate that you pointed out that we neglected to include pioneers besides Bourdieu, who also studied these issues, such as Granovetter, Portes, and Waldinger. Action I: We have included key pioneers in the field with the addition of the following text in Section 5.1.1: Previous entrepreneurship literature follows the line of thought from Bourdieu (1984) by looking at the impacts of forms of capital on the establishment and maintenance of a business. Inspired by the cultural, economic, social, and symbolic capital of Bourdieu (1984), the studies covered in this SLR show a shift into three distinct forms of capital that frequently appear often in assessment of starting and running a business: financial, human, and social capital (Baklanov et al., 2014; Eroğlu, 2018; Grubbström and Joosse, 2021). The concept of social capital is also inspired by Granovetter (1973) whose seminal work, The Strength of Weak Ties, researches how bridges across social networks relate to social mobility and social cohesion. Basit (2017) also cites Granovetter (1985) and Uzzi (1997, 1999) to share how social embeddedness is a framework by which to study economic activity, from which researchers investigate how social relations shape entrepreneurial activities and outcomes. Several sources in the SLR stress the importance of support mechanisms that build up both weak and strong ties, including Bouk, Vedder and Poel (2013), Harima et al. (2020), and Noor (2021). Waldinger (1995) also played an important role in earlier literature on social capital in migrant entrepreneurship, with his study on the construction industry in New York City, and is cited sources in the SLR by Bagwell (2008) and Barth and Zalkat (2020). This SLR uses the categories of financial, human, and social capital to classify the characteristics gathered from the studies. The literature stream considers primarily the positive impacts of the social capital from one’s ethnic group, as we did not encounter many pitfalls mentioned by Portes and Landolt (2000). However, the study by Andersson (2021), which is included in this SLR, does mention that entrepreneurship by new arrivals can depend on the extent of entrepreneurial engagement by co-ethnics already in the host-country. Andersson (2012) and another study included in the SLR, by García, Molina, and Lubbers (2014) also mention that ethnic enclaves can slow down the integration process and new arrivals’ ability to learn the host country’s culture and language. Action II: We added to the paragraph that introduces the research questions to help establish the rest of the study as a basis for analysis of policymaking. The new text reads as follows: “This study aims at synthesizing current knowledge on migrant entrepreneurship support in Europe, by understanding the key issues that migrants in Europe face in their entrepreneurship endeavors and how these issues relate to key policy-making decisions. The study seeks to develop a conceptual framework and identify research gaps that call for future inquiry. From the above objectives, we will address three research questions (RQs) in this study, which we divide into characteristics, challenges, and support mechanisms. 1. RQ1: What are the characteristics of migrant entrepreneurs investigated in primary studies, in the European context? 2. RQ2: What do we know about challenges that migrants face as entrepreneurs in European host countries? 3. RQ3: What do we know about reported policies as support mechanisms for migrant entrepreneurship in the European context? To the best of our knowledge, this work offers a most up-to-date and comprehensive view to discern migrant entrepreneurship support in the European context and positions the issues uncovered as the foundation for policymaking.” Changes over time: Response: We appreciate your call to explore shifts over time as issues and types of policies evolve, and to identify geographical patterns as well, to link the findings to a broader context. Action: We have enhanced the study's meta perspective by incorporating the most prominent changes over time to ensure a deeper contribution and a connection to the broader context. The following text now appears in Section 4.1: As for a temporal analysis of the literature, it is worth mentioning sharing our insights on how the literature has evolved over the years. First of all, the earliest source focusing on support for female migrant entrepreneurs was in 2008 by Mason. It was not until 2016 that female migrant entrepreneurship support sources appeared again. From that point, it appears regularly in the literature. See Table 11 for a full overview of the sources focusing on female migrant entrepreneurship. In addition, the first refugee entrepreneurship support study appeared as a quantitative investigation in 2017 by Širec and Tominc. There has been a steady increase since then, with 14 of the most recent 56 sources in this SLR dedicated to refugees. Table 11 shares the specific authors and publication years. Most of the migrant entrepreneurship support studies on females and/or refugees in Europe have been qualitative, with only two quantitative refugee studies, the one mentioned by Širec and Tominc and the study by Andersson (2021), and one quantitative female study by Halkias et al (2016). In addition, there has been an increase in quantitative methods over time. The first quantitative study appeared in 2000 by van Delft, Gorter, and Nijkamp, followed by three studies in 2009 and more steady appearances since 2011." } ] }, { "id": "216752", "date": "23 Nov 2023", "name": "Surangi H.A.K.N.S.", "expertise": [ "Reviewer Expertise Entrepreneurship" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is good. The abstract is well-written, and the article is well-structured. This is an excellent article.\nThe area suggested in the topic is interesting and promising, however, the research issue is not clearly identified at the outset. Lack of explanation for the research issue and lack of support for the knowledge gap (trying to establish) are the main issues in the introduction. Therefore, the introduction section should be strengthened with elaborations of the knowledge gap focused on this study, and it should be supported by the existing literature. The introduction section should elaborate on why this review is required in this area of research. Further, the introduction section lacks the justification for the research, i.e., how the findings of this study would be beneficial to any stakeholders.\nMethodology: Presenting the method of the study (the complete process as well as descriptions of each step) under the ‘research method’ section is crucial in systematic literature reviews (SLRs) in order to strengthen the validity and reliability of the data (literature) collected. The Methods section of this paper provides a clear and detailed description that is sufficient to serve this purpose.\nContribution: Thus, this paper clearly presents the outcome of the review in line with avenues for future research agendas which can be justifiable on the practical, theoretical, and methodological ground, and a detailed description that is sufficient to serve this purpose.\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": "10914", "date": "22 Jan 2024", "name": "Dimitris Polychronopoulos", "role": "Author Response", "response": "Dear Sarangi H.A.K.N.S. Thank you for taking the time to review our systematic literature review. Below you will find the actions we took to address the issues you raised to us: Positive feedback: Response: Thank you for the positive feedback on the abstract, writing composition, and structure of the article. Action: None required. Elaborations of the knowledge gap and why this review is required in this area of research and justification for the research: Response: Thank you for pointing out the need for us to more clearly identify the research issue and to provide a more robust explanation of the knowledge gap, and for highlighting the lack of justification for the research and for emphasizing the importance of explaining how the study's findings would be beneficial to stakeholders. Action: We have strengthened the introduction section by providing a clear justification for the research, highlighting its potential benefits to stakeholders. We have revised the introduction section to clearly identify the research issue and provide detailed support for the knowledge gap by linking these two concerns to the supporting literature, by adding the following paragraph to the bottom of section 1.1: An increase in people moving to Europe coincides with the demographic trends that require immigration to fulfill labor needs and support the pensions of those who will soon be retiring and those who have already retired (Marois, Bélanger, and Lutz, 2020). Some new arrivals express an interest in becoming entrepreneurs. For example 7% of Ukrainian refugees who settled in Norway report that they aim to become entrepreneurs (Hernes, Deineko, Myhre, Liodden, and Staver, 2022) and the European Commission has incorporated refugee entrepreneurship into its 2020 Entrepreneurship Action Plan and the European Union Qualification Directive 2011/95 (article 26 and 34) states that EU members must allow access to self-employment and consider the specific needs of refugees within integration programs (Lange, Berntsen, Hanoeman, and Haidar, 2021). This SLR sees a knowledge gap surrounding the literature to support migrants of all kinds in their entrepreneurship journey, whether refugee or others. Considering that a literature review on entrepreneurship support by Ratinho, Amezuca, Honig, and Zeng (2020) shows a lack of defined evaluation of the outcomes of entrepreneurial support, this SLR aims to contribute by adding conceptual clarity to the body of literature on migrant entrepreneurship support in Europe. Therefore, with the objectives in the next section, we seek to map out the research issues on this topic. The need for this SLR is based on the context of the demographic trends of Europe, the increase in immigration to Europe, the desire of a portion of the immigrants to Europe to become entrepreneurs, and a lack of understanding surrounding the effectiveness of migrant entrepreneurship support in European contexts (Chilova, Farny, and Salmivaara, 2018; De Noni and Ganzaroli (2013); Lillevik and Sønsterudbråten, 2018), as well as a lack of trust by some migrants in government-run migrant entrepreneurship support programs (Ram, Theodorakopoulos, and Jones, 2008; Mwaura, 2019), and the call by Dheer (2018) for scholars to study policies for their impacts on migrant entrepreneurship." } ] } ]	1	https://f1000research.com/articles/12-1300
https://f1000research.com/articles/12-398/v1	13 Apr 23	{ "type": "Research Article", "title": "Drug effectiveness for COVID-19 inpatients inferred from Japanese medical claim data using propensity score matching", "authors": [ "Shingo Mitsushima", "Hiromasa Horiguchi", "Kiyosu Taniguchi", "Hiromasa Horiguchi", "Kiyosu Taniguchi" ], "abstract": "Background: Earlier studies and clinical trials have shown that the drugs such as antiviral drugs, antibody cocktails, and steroids and anti-inflammatory drugs are expected to prevent severe coronavirus 2019 (COVID-19) outcomes and death.\nMethods: We used observational data for Japan to assess the effectiveness of these drugs for COVID-19. We applied propensity scoring, which can treat the choice of administered drug as a random assignment to inpatients, to the Medical Information Analysis Databank operated by National Hospital Organization in Japan. The outcome was defined as mortality. Subjects were all inpatients, inpatients with oxygen administration, and inpatients using respiratory ventilators, classified by three age classes: all ages, 65 years old or older, and younger than 65 years old. Information about demographical characteristics, underlying disease, administered drug, the proportion of Alpha, Beta and Omicron variant strains, and vaccine coverage were used as explanatory variable in logistic regression.\nResults: Estimated results indicated that only an antibody cocktail (sotrovimab, casirivimab and imdevimab) raised the probability to save life consistently. By contrast, other drugs might reduce the probability of saving life. The results indicated that an antiviral drug (remdesivir), a steroid (dexamethasone), and an anti-inflammatory drug (baricitinib and tocilizumab) might not contribute to saving life even at the pseudo-situation of random assignment. However, this logistic regression at the first step might have only insufficient explanatory power.\nConclusions: We found a high likelihood that antibody cocktails were consistently effective to raise the probability of saving life, though a lesser likelihood in other drugs for older patients with mild to severe severity and all age patients with moderate severity.", "keywords": [ "COVID-19", "mutated strain", "underlying diseases", "antibody cocktail", "antiviral drug", "mortality", "steroid and anti-inflammatory drug" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 caused pandemic throughout the world from December 2019. In Japan, the first case of COVID-19 was detected in January 2020, and then COVID-19 spread throughout Japan. As characteristics of COVID-19 were revealed and treatments for COVID-19 developed, the mortality rate due to COVID-19 in Japan has declined.1\n\nEarlier studies and clinical trials have shown that drugs used against COVID-19, such as antiviral drugs (remdesivir),2 antibody cocktails (casirivimab/imdevimab and sotrovimab),3,4 steroids (dexamethasone),5 and anti-inflammatory drugs (baricitinib and tocilizumab),6–8 statins,9,10 RNA-dependent RNA polymerase inhibitor (molnupiravir),11,12 protease inhibitor (nirmatrelvir/ritonavir),11,13 are expected to prevent severe COVID-19 outcomes and death. For this study, we considered the real-world effectiveness of drugs against COVID-19.\n\nThe National Hospital Organization (NHO) in Japan, an organization of regional core hospitals accounting for about 3.4% of all beds in Japan,14 provides a database of medical claims from 60 representative NHO hospitals: the Medical Information Analysis Databank (MIA). NHO is one of the largest organizations in Japan and each prefecture has one or more NHO hospitals. MIA collects the data on medical insurance claims for outpatients and inpatients. It includes inpatients’ demographical characteristics, underlying diseases, medical interventions including oxygen administration, the use of respiratory ventilators, administration of drugs, and outcomes such as discharge or death.15 We used MIA data of inpatients to verify drug effectiveness. MIA does not have the data on vaccination history or causative strain.\n\nIn the real world, whether a drug is administered or not depends on the patient condition. In other words, administration of drugs is probably not a random assignment. Generally speaking, patients who have a higher likelihood of developing an illness have a higher probability of being administered drugs, which implies, because of selection bias in the observational data, a negative association between drug administration and outcomes. Therefore, non-random choice of drug administration should be taken into account to estimate the drug effectiveness. Propensity score matching might resolve this problem statistically but not experimentally.16 This procedure predicts the likelihood of receiving drugs initially. Then outcomes are compared between a drug-received group and a drug not-administered group, the members of which have an almost identical likelihood of having been administered some drugs. We then examined drug effectiveness using this method.\n\nPropensity score matching is widely used in social sciences for the evaluation of programs: participants choose to join a program spontaneously, such as a job training program or an unemployment payment program. However, in the natural sciences, a researcher can perform experiments and thereafter delete selection bias in the choice of subjects. Nevertheless, such experiments are expensive and require a longer period. Moreover, the number of subjects should be constrained; outcomes tend to be evaluated under less severe condition.17,18\n\nIn medicine, random assignment experiments after launching are not so popular. Therefore, situations change, mutated strains emerge, vaccine coverage and/or developing treatment might affect drug effectiveness, making it impossible to evaluate. In particular, mortality tends not to be used as an outcome for evaluation, even though it should be the endpoint of greatest concern. Therefore, experiments in medicine for updated situations and mortality might be difficult. In other words, propensity score matching methods might be useful even for medicine, particularly for orthopaedic surgery and cardiovascular research.17,18\n\n\nMethod\n\nThis study used MIA for confirmed inpatients including their age, sex, underlying diseases, hospitalization date, administered drug, outcome and whether they received oxygen therapy and/or ventilation.\n\nWe used data for vaccine administration published by the Cabinet Secretariat. Moreover, prevalence in Alpha, Delta and Omicron variant strains were referred from a monitoring meeting in Tokyo because MIA included no information about the patient’s vaccine status and sublineage in SARS-CoV-2.\n\nThe study period was January 2020 through March 2022, using data recorded as of May 2022. This study area was the entirety of Japan.\n\nWe defined demographical conditions as age and sex, underlying diseases as cancer (C00–C90 in ICD10), asthma (J45), chronic obstructive pulmonary disease (COPD) (J44), hypertension (HT) (I10), heart failure (HF) (I50), and diabetes mellitus (DM) (E10). We examined the effects of an antiviral drug (remdesivir),2 an antibody cocktail (sotrovimab and casirivimab/imdevimab),3,4 a steroid (dexamethasone),5 and anti-inflammatory drugs (baricitinib and tocilizumab),6–8 which were proven effective against COVID-19 by previous studies. Because the number of cases with antibody cocktails was insufficiently large, we did not divide them by the name of drug, as sotrovimab, casirivimab, and imdevimab. We considered the overall effects of antibody cocktails. These drugs were the five most common drugs against COVID-19. We assumed that vaccination coverage was the rate of the second dose of vaccine received two weeks prior by age class, as younger than 65 years old, and 65 years old or older. Mutated strains, Alpha, Delta and Omicron variant strains, were measured by percentage at one week before admission. The Alpha, Delta and Omicron variant strains were defined as the proportion of Alpha, Delta and Omicron variant strains. Omicron included BA.2 or a later sublineage. Alternatively, we used a dummy variable for the period of the 4–6th wave instead of the proportion of the mutated strains as an explanatory variable, to check robustness. By this specification, the Alpha variant strain emerged and then dominated in the 4th wave, defined as from 1 March through 20 June 2021. Similarly, the Delta variant strain emerged and then dominated in the 5th wave, defined as 21 June through 21 November 2021. Omicron BA.1 strain emerged and then dominated from the 6th wave, defined as 22 November 2021 to the end of the study period. Death during hospitalization was defined as the outcome.\n\nSubjects were all inpatients confirmed as having SARS-CoV-2. However, aside from pure medical criteria, the criteria of hospitalization for asymptomatic patients or patients with mild symptoms who did not require oxygen therapy were probably affected strongly by medical resource scarcity or social situations such as support for their staying at home and recuperation at home. For that reason, we also limited our study to subjects who were expected to be inpatients with oxygen therapy or respiratory ventilation.\n\nWe conducted estimates separately by the type of drug: remdesivir, antibody cocktail (sotrovimab, casirivimab and imdevimab), dexamethasone, baricitinib, and tocilizumab.\n\nThe first step to assess whether the patient was administered a type of drug or not was performed through logistic regression on their age, sex, underlying diseases, pharmaceutical therapy, vaccine coverage, and prevalence in the mutated strains as explanatory variables. The second step was comparison of outcomes among administered patients and unadministered patients with almost identical likelihood as the prediction done in the first step.\n\nAll statistical analyses were conducted using Stata software (SE 17.0; Stata Corp.). We adopted 5% as the significance level.\n\nIndividual informed consent was not required to conduct this study because the dataset was provided as anonymized data by National Hospital Organization.15 This study was approved by the Ethics Committee of Mie Hospital (Approval No. 2020-89) on 23 October 2020. Specific permission to use MIA data was obtained from the NHO (Registration No. 1201003) on 1 December 2020.\n\n\nResults\n\nA supplementary table (see Extended data19), presents a summary of the estimation results obtained for the first step logistic regression for drug administration. Table 1 presents the estimation results obtained for all inpatients, inpatients with oxygen therapy, and inpatients who used a respiratory ventilator in three age classes. Results show that 24 significance estimators of 90 estimators in all, but 20 estimators were positive, which indicates that drug administration reduced the probability of saving life.\n\nFour estimators indicating that a drug probably contributes to saving life were the following: tocilizumab among younger inpatients with ventilators; baricitinib among younger inpatients with oxygen therapy; and antibody cocktails among all older inpatients and among all inpatients with oxygen therapy. Of three drugs, only the antibody cocktail did not have a positive difference, which suggests an increase in the probability of saving life. No other treatment effect estimator of such drugs was available. Some treatment effects in tocilizumab and baricitinib were positive, especially for all older inpatients or older inpatients with moderate severity. Therefore, the results obtained for these two drugs were mixed.\n\n\nDiscussion\n\nResults showed that antibody cocktails might contribute to saving life. The estimated results for tocilizumab and baricitinib were mixed, but these drugs were presumed to have some probability of saving life among younger but severe patients. However, we were unable to find any evidence that remdesivir and dexamethasone contribute to saving life at all.\n\nThese counterintuitive findings, which are inconsistent with results of earlier studies of the effectiveness of remdesivir and dexamethasone, might result from worse matching at the first step.2,5 Also, endogeneity might not be controlled well in decisions to use drugs. Drugs to be administered against COVID-19 depend on the severity of illness of the patients. For instance, an earlier study showed dexamethasone as effective for moderate or severe COVID-19, but ineffective for mild COVID-19.5 Actually, more than antibody cocktails,3,4 remdesivir2 and dexamethasone5 tend to be administered more for severer patients. To resolve this difficulty, more information indicating severity is needed. Moreover, we did not use test results such as blood pressure, body mass index, and oxygen saturation. We must include such information in the first step. Inclusion of such data remains a challenge for future research.\n\nThe results for antibody cocktails included many “not available” entries, although drug effectiveness was confirmed in well matched cases. That finding might be attributable to the small sample of antibody cocktail use for patients. In Japan, remdesivir, baricitinib, casirivimab/imdevimab, sotrovimab, and tocilizumab were approved for treatment of COVID-19, respectively, in May 2020, April 2021, July 2021, September 2021, and January 2022.20 More recently approved drugs tend to be associated with smaller samples. Data accumulation might resolve that shortcoming.\n\nFirst, we estimated the effectiveness of drugs separately. However, the choice of drug was not actually independent. Therefore, intercorrelation among drugs should be incorporated into the estimation model.\n\nSecond, because MIA is a database of medical claims, data of the prior few weeks might change during a few months. For this study, we collected and analyzed data from January 2020 through the end of March 2022, recorded as of May 2022. Data and therefore estimation results might change over time if the study period were extended. In addition to this, we examined using Japanese vaccine coverage instead of the vaccination history of the patients. We also used data on prevalence of mutated strains in Tokyo as causative strains. If the vaccine history and causative strains of the patients themselves were available, the results might be different.\n\nThird, although we compared mortality among an administrated group and an unadministered group based on the estimated propensity score at the first step, we also control demographical condition and/or underlying diseases, in addition to epidemiological situations for mortality. That remains as a challenge for our future research.\n\nFourth, the drugs considered for this study were not always administered to the most severely ill patients. For example, antibody cocktails were administered to patients with less–severe illness. However, to simplify the analysis used for this study, we did not incorporate the combination of drugs or change from one considered drug to another considered drug. That choice might bias the results to some degree. Such a process or pattern of drug administration might be important to evaluate drug effectiveness.\n\n\nConclusion\n\nThe obtained results showed that antibody cocktails for all older patients and all inpatients with moderate severity, tocilizumab for severely ill younger inpatients, and baricitinib for younger inpatients with moderate illness severity might contribute to saving life. In the latter two drugs, tocilizumab and baricitinib, some treatment effects suggested a reduction of saving life for all or older inpatients. We infer that the effects of these drugs were mixed. In fact, remdesivir and dexamethasone might not contribute to saving life. However, more information, including test results, is needed for better matching and achievement of definitive conclusions.", "appendix": "Data availability\n\nThe data in this study was provided from the National Hospital Organization (NHO), but the availability of these data was restricted because of privacy policy of NHO. This data was used under license for this study and were therefore not available to the public. Authors were permitted to use this data by the Ethical Committee of Mie Hospital and NHO Committee.\n\nFigshare: Drug effectiveness for COVID-19 inpatients inferred from Japanese medical claim data using propensity score matching_supplement data.csv https://doi.org/10.6084/m9.figshare.22102016.v1. 19\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nMiyashita K, Hozumi H, Furuhashi K, et al.: Changes in the characteristics and outcomes of COVID-19 patients from the early pandemic to the delta variant epidemic: a nationwide population-based study. Emerg. Microbes. Infect. 2023; 12(1): 2155250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMozaffari E, Chandak A, Zhang Z, et al.: Remdesivir Treatment in Hospitalized Patients With Coronavirus Disease 2019 (COVID-19): A Comparative Analysis of In-hospital All-cause Mortality in a Large Multicenter Observational Cohort. Clin. Infect. Dis. 2022; 75(1): e450–e458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeinreich DM, Sivapalasingam S, Norton T, et al.: REGEN-COV Antibody Combination and Outcomes in Outpatients with Covid-19. N. Engl. J. Med. 2021; 385(23): e81. Epub 20210929. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta A, Gonzalez-Rojas Y, Juarez E, et al.: Early Treatment for Covid-19 with SARS-CoV-2 Neutralizing Antibody Sotrovimab. N. Engl. J. Med. 2021; 385(21): 1941–1950. PubMed Abstract \| Publisher Full Text\n\nHorby P, Lim WS, Emberson JR, et al.: Dexamethasone in Hospitalized Patients with Covid-19. N. Engl. J. Med. 2021; 384(8): 693–704. PubMed Abstract \| Publisher Full Text\n\nMarconi VC, Ramanan AV, de Bono S , et al.: Efficacy and safety of baricitinib for the treatment of hospitalised adults with COVID-19 (COV-BARRIER): a randomised, double-blind, parallel-group, placebo-controlled phase 3 trial. Lancet Respir. Med. 2021; 9(12): 1407–1418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalama C, Han J, Yau L, et al.: Tocilizumab in Patients Hospitalized with Covid-19 Pneumonia. N. Engl. J. Med. 2021; 384(1): 20–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGordon AC, Mouncey PR, Al-Beidh F, et al.: Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-19. N. Engl. J. Med. 2021; 384(16): 1491–1502. PubMed Abstract \| Publisher Full Text\n\nUmakanthan S, Senthil S, John S, et al.: The Effect of Statins on Clinical Outcome Among Hospitalized Patients With COVID-19: A Multi-Centric Cohort Study. Front. Pharmacol. 2022; 13: 742273. Epub 20220705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBergqvist R, Ahlqvist VH, Lundberg M, et al.: HMG-CoA reductase inhibitors and COVID-19 mortality in Stockholm, Sweden: A registry-based cohort study. PLoS Med. 2021; 18(10): e1003820. Epub 20211014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWong CKH, Au ICH, Lau KTK, et al.: Real-world effectiveness of molnupiravir and nirmatrelvir plus ritonavir against mortality, hospitalisation, and in-hospital outcomes among community-dwelling, ambulatory patients with confirmed SARS-CoV-2 infection during the omicron wave in Hong Kong: an observational study. Lancet. 2022; 400(10359): 1213–1222. Publisher Full Text\n\nJayk Bernal A, Gomes da Silva MM, Musungaie DB, et al.: Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized Patients. N. Engl. J. Med. 2022; 386(6): 509–520. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHammond J, Leister-Tebbe H, Gardner A, et al.: Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19. N. Engl. J. Med. 2022; 386(15): 1397–1408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n令和2 (2020) 年医療施設 (静態・動態) 調査 [Survey of Medical Institutions. 2020]. Tokyo: Ministry of Health, Labour, and Welfare; 2020 [cited 2022 Nov 10]. Reference Source\n\nKanazawa N, Tani T, Imai S, et al.: Existing Data Sources for Clinical Epidemiology: Database of the National Hospital Organization in Japan. Clin. Epidemiol. 2022; 14: 689–698. Publisher Full Text\n\nHeckman JJ, Ichimura H, Todd PE: Matching As An Econometric Evaluation Estimator: Evidence from Evaluating a Job Training Programme. Rev. Econ. Stud. 1997; 64(4): 605–654. Publisher Full Text\n\nKane LT, Fang T, Galetta MS, et al.: Propensity Score Matching: A Statistical Method. Clin. Spine Surg. 2020; 33(3): 120–122. Publisher Full Text\n\nDeb S, Austin PC, Tu JV, et al.: A Review of Propensity-Score Methods and Their Use in Cardiovascular Research. Can. J. Cardiol. 2016; 32(2): 259–265. PubMed Abstract \| Publisher Full Text\n\nMitsushima S, Horiguchi H, Taniguchi K: Drug effectiveness for COVID-19 inpatients inferred from Japanese medical claim data using propensity score matching_supplement data.csv. Dataset. figshare. Publisher Full Text\n\nMinistry of Health, Labour, and Welfare: Clinical Management of Patients with COVID-19 ver. 8.1. Tokyo: Ministry of Health, Labour, and Welfare (in Japanese); [cited 2022 Nov 8]. Reference Source" }	[ { "id": "214688", "date": "10 Nov 2023", "name": "Ramesh Kandimalla", "expertise": [ "Reviewer Expertise COVID-19", "Diabetes", "Alzheimer's", "Mitochondria", "Carbon dots", "Nanospheres" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe methods section is detailed, but it could benefit from additional clarity in explaining the rationale behind using propensity scoring and the choice of variables in logistic regression. This could help readers better understand the study design.\n\nIt would be helpful to provide more information about the reliability and representativeness of the observational data from Japan. Mentioning any limitations or potential biases in the data source would enhance the transparency of the study.\n\nThe abstract implies causal relationships between drug administration and outcomes, but the observational nature of the study might only establish correlations. It's essential to acknowledge the limitations in inferring causation from observational data.\n\nThe abstract mentions that the logistic regression might have \"insufficient explanatory power.\" It would be beneficial to elaborate on this limitation and discuss potential sources of bias or confounding that could affect the study's validity.\n\nWhen discussing the drugs, providing a brief rationale for why certain drugs might be effective or ineffective could enhance the comprehensibility of the results.\n\nThe mention of the Omicron variant is timely, but given the evolving nature of the pandemic, it's important to note the potential for changes in outcomes as new variants emerge. This could affect the generalizability of the findings.\n\nThe abstract could include a brief section discussing the limitations of the study, such as potential confounders not accounted for in the analysis or any challenges in accurately measuring certain variables.\n\nThe use of phrases like \"in Japan\" and \"against COVID-19\" is repeated frequently. Adding variety to the language could enhance the readability of the introduction.\n\nThe timeline of events related to COVID-19 in Japan could be presented more clearly. For example, mentioning the initial surge in cases, the development of treatments, and the subsequent decline in mortality rate in a more chronological order could improve the flow.\n\nThe introduction references earlier studies and clinical trials but doesn't provide specific citations for the mentioned data or findings. Including citations within the text could enhance the credibility of the information presented.\n\nWhile the introduction mentions the decline in the mortality rate due to COVID-19 in Japan, it might be beneficial to include specific statistics or percentages to quantify this change and emphasize the significance.\n\nThe term \"real-world effectiveness\" is used, but a brief explanation or definition could be included to clarify its meaning for readers who may not be familiar with the term.\n\nThe limitations of the MIA data, such as the absence of vaccination history or causative strain information, are mentioned but could be discussed more explicitly. Acknowledging these limitations upfront adds transparency to the study.\n\nThe transition from providing background information to discussing the methods is somewhat abrupt. Adding a sentence or two to smoothly transition into the methods section could improve the overall flow of the introduction.\n\nWhile the introduction mentions propensity score matching, a brief explanation of how it works and its purpose in addressing selection bias could enhance reader understanding.\n\nThe introduction could touch upon the external validity of the study, acknowledging that findings may not be universally applicable due to variations in healthcare systems, populations, or treatment protocols.\n\nThe introduction could explicitly state the research question or hypothesis being addressed by the study to guide readers in understanding the study's purpose from the outset.\n\nThe text repeats certain points, such as the mention of \"propensity score\" and \"saving life,\" multiple times. Reducing redundancy could improve the overall clarity and conciseness of the text.\n\nSome sentences are quite complex, which might make it challenging for readers to follow the arguments. Simplifying the language without compromising precision could enhance comprehension.\n\nThe text occasionally uses language that implies causation, such as \"might contribute to saving life.\" It's important to be cautious about making causal claims based on observational data. Acknowledging the limitations in establishing causation would strengthen the study's validity.\n\nThe text mentions \"not available\" entries in the results for antibody cocktails, attributing it to a small sample size. Providing a more explicit explanation of why certain data is missing and its potential impact on the results would enhance transparency.\n\nThe limitations section is placed towards the end of the text. Moving it closer to the beginning or integrating limitations as they become relevant in the discussion could improve the flow and help readers contextualize the findings.\n\nThe use of terms like \"severely ill,\" \"moderate severity,\" and \"less–severe illness\" could benefit from clearer operational definitions or references to specific criteria used for classification.\n\nThe discussion section covers a range of topics, including limitations and future research, without clear organization. Structuring the discussion into subsections could make it more reader-friendly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10871", "date": "22 Jan 2024", "name": "Shingo Mitsushima", "role": "Author Response", "response": "1. The methods section is detailed, but it could benefit from additional clarity in explaining the rationale behind using propensity scoring and the choice of variables in logistic regression. This could help readers better understand the study design. Response: We added the following sentence to Introduction. “Moreover, after approval, experiments with random assignment are expected to be difficult to conduct in the real world because of ethical considerations: some patients might be unable to receive a potentially effective drug. Therefore, pseudo-experimental method should be applied to evaluate drug effectiveness in the real world.” 2. It would be helpful to provide more information about the reliability and representativeness of the observational data from Japan. Mentioning any limitations or potential biases in the data source would enhance the transparency of the study. Response: We added the following sentence to Introduction. “In our earlier study, although we confirmed the representativeness of fatality cases using MIA data, the data on the numbers of patients with oxygen therapy and respiratory ventilation in Japan have not been published, yet. We have not verified the representativeness of patients with oxygen therapy and respiratory ventilation.” 3. The abstract implies causal relationships between drug administration and outcomes, but the observational nature of the study might only establish correlations. It's essential to acknowledge the limitations in inferring causation from observational data. Response: We revised sentences in Abstract as below. OLD: “raised the probability to save life” “contribute to saving life” NEW: “was associated with raising the probability of survival” “were related to reduced probability of survival” In addition, we added the following sentence to Discussion. “this study was an observational study that was able to show only association between considered drugs and death, not necessarily a causal association.” 4. The abstract mentions that the logistic regression might have \"insufficient explanatory power.\" It would be beneficial to elaborate on this limitation and discuss potential sources of bias or confounding that could affect the study's validity. Response: We added the following sentence to Abstract. “However, propensity score matching method might engender biased results because of a lack of data such as detailed information related to intervention and potential confounders. Therefore, the effectiveness of some drugs might not be evaluated properly in this study.” 5. When discussing the drugs, providing a brief rationale for why certain drugs might be effective or ineffective could enhance the comprehensibility of the results. Response: We added the following sentences to Discussion. “An earlier study indicated that casirivimab/imdevimab, an antibody cocktail, lowered the viral load. Typically, these antibody cocktails were given to patients with mild severity, which means that they were administered before a marked increase in viral load. This pattern of administration might be one reason why antibody cocktails were effective for survival.” 6. The mention of the Omicron variant is timely, but given the evolving nature of the pandemic, it's important to note the potential for changes in outcomes as new variants emerge. This could affect the generalizability of the findings. Response: We added the following sentences to Discussion. “If the study period was extended, the estimation results might change over time because the accumulated data engender more accurate results. Emerging new mutated strains, immunization, and development of new therapies might affect the results.” 7. The abstract could include a brief section discussing the limitations of the study, such as potential confounders not accounted for in the analysis or any challenges in accurately measuring certain variables. Response: We added the following sentences to Abstract. “However, propensity score matching method might engender biased results because of a lack of data such as detailed information related to intervention and potential confounders. Therefore, the effectiveness of some drugs might not be evaluated properly in this study.” 8. The use of phrases like \"in Japan\" and \"against COVID-19\" is repeated frequently. Adding variety to the language could enhance the readability of the introduction. Response: We replaced some words “Japan” and “COVID-19” with alternative words. 9. The timeline of events related to COVID-19 in Japan could be presented more clearly. For example, mentioning the initial surge in cases, the development of treatments, and the subsequent decline in mortality rate in a more chronological order could improve the flow. Response: We added Figure 1 and Figure 2 to show the timeline of COVID-19 events. We also added the following sentences to Result. “Table 1 shows the number of COVID-19 patients from National data and the numbers of inpatients, inpatients with oxygen therapy, and inpatients with respiratory ventilation in MIA data. These data show similar trends. Most notably, several surges occurred from January 2020 through March 2022, as shown in Figure 1. Figure 2 presents the COVID-19 fatality rate in National data and the rate of deaths among COVID-19 inpatients in MIA data. These data were less than 10%. They followed a similar trend.” 10. The introduction references earlier studies and clinical trials but doesn't provide specific citations for the mentioned data or findings. Including citations within the text could enhance the credibility of the information presented. Response: We added the following sentence to Introduction. “In earlier studies, all these drugs were associated with improved survival or reduced the risk of disease progression, but antibody cocktails were effective only for mildly ill patients; dexamethasone was only effective for moderately to severely ill patients.” 11. While the introduction mentions the decline in the mortality rate due to COVID-19 in Japan, it might be beneficial to include specific statistics or percentages to quantify this change and emphasize the significance. Response: We drew Figure 2, which shows fatality rates in National data and rate of deaths among COVID-19 inpatients with specific percentage. 12. The term \"real-world effectiveness\" is used, but a brief explanation or definition could be included to clarify its meaning for readers who may not be familiar with the term. Response: We added the following sentences to Introduction. “The objective of this study was evaluating the “real-world effectiveness of drugs against COVID-19 in Japan.” “Real-world effectiveness of drugs” means the effectiveness of drugs considering several factors including population characteristics, examinations, vaccinations, and healthcare system such as hospitalization and treatment protocols.” 13. The limitations of the MIA data, such as the absence of vaccination history or causative strain information, are mentioned but could be discussed more explicitly. Acknowledging these limitations upfront adds transparency to the study. Response: We added the following sentences about the limitation of vaccination history and causative strain to Discussion. “Second, we examined the data, particularly using Japanese vaccine coverage instead of the vaccination history of the patients. We also used data of the prevalence of mutated strains in Tokyo as causative strains. If the vaccine history and causative strains of the patients themselves were available, then the results might be different.” We also added the sentences about the limitation of MIA data to Discussion. “In our earlier study, although we confirmed the representativeness of fatality cases using MIA data, the data on the numbers of patients with oxygen therapy and respiratory ventilation in Japan have not been published, yet. We have not verified the representativeness of patients with oxygen therapy and respiratory ventilation. Moreover, because MIA is a database of medical claims, data of the prior few weeks might change during a few months. For this study, we collected and analyzed data from January 2020 through the end of March 2022, recorded as of May 2022. If the study period was extended, the estimation results might change over time because the accumulated data engender more accurate results. Emerging new mutated strains, immunization, and development of new therapies might affect the results.” 14. The transition from providing background information to discussing the methods is somewhat abrupt. Adding a sentence or two to smoothly transition into the methods section could improve the overall flow of the introduction. Response: We added the following sentence to Introduction. “An average treatment effect model including propensity matching was applied to create these situations.” 15. While the introduction mentions propensity score matching, a brief explanation of how it works and its purpose in addressing selection bias could enhance reader understanding. Response: We added the following sentence to Abstract. “We applied the average treatment effect model, particularly propensity scoring, which can treat the choice of administered drug as if administration were randomly assigned to inpatients.” 16. The introduction could touch upon the external validity of the study, acknowledging that findings may not be universally applicable due to variations in healthcare systems, populations, or treatment protocols. Response: We added the following sentences to Introduction. ““Real-world effectiveness of drugs” means the effectiveness of drugs considering several factors including population characteristics, examinations, vaccinations, and healthcare system such as hospitalization and treatment protocols. Therefore, the results of this study might not be applicable to other countries.” 17. The introduction could explicitly state the research question or hypothesis being addressed by the study to guide readers in understanding the study's purpose from the outset. Response: We added the following sentence to Introduction. “The objective of this study was evaluating the “real-world effectiveness of drugs against COVID-19 in Japan.”” 18. The text repeats certain points, such as the mention of \"propensity score\" and \"saving life,\" multiple times. Reducing redundancy could improve the overall clarity and conciseness of the text. Response: We did the English proofreading. 19. Some sentences are quite complex, which might make it challenging for readers to follow the arguments. Simplifying the language without compromising precision could enhance comprehension. Response: We did the English proofreading. 20. The text occasionally uses language that implies causation, such as \"might contribute to saving life.\" It's important to be cautious about making causal claims based on observational data. Acknowledging the limitations in establishing causation would strengthen the study's validity. Response: We added the following sentence to Discussion. “Third, this study was an observational study that was able to show only association between considered drugs and death, not necessarily a causal association.” 21. The text mentions \"not available\" entries in the results for antibody cocktails, attributing it to a small sample size. Providing a more explicit explanation of why certain data is missing and its potential impact on the results would enhance transparency. Response: The approval of antibody cocktails was relatively late, and the number of prescriptions was simply low, so there is no potential impact. We added the following sentence in Discussion. “The antibody cocktail results included many “not available” entries, although drug effectiveness was confirmed in well-matched cases. The approval of antibody cocktails was late. The number of prescriptions was simply low, which might affect many cases for which “not available” was reported.” 22. The limitations section is placed towards the end of the text. Moving it closer to the beginning or integrating limitations as they become relevant in the discussion could improve the flow and help readers contextualize the findings. Response: We integrated limitations to Discussion and changed each limitation to understand easily for the reader. 23. The use of terms like \"severely ill,\" \"moderate severity,\" and \"less–severe illness\" could benefit from clearer operational definitions or references to specific criteria used for classification. Response: We added the following sentence to Method. “For this study, mild symptoms were defined as a patient who did not require medical intervention such as oxygen therapy or respiratory ventilation, moderate symptoms were defined as a patient who required oxygen administration, and severe symptoms were defined as a patient who required respiratory ventilation.” 24. The discussion section covers a range of topics, including limitations and future research, without clear organization. Structuring the discussion into subsections could make it more reader-friendly. Response: We have organized discussion structure by issue." } ] } ]	1	https://f1000research.com/articles/12-398
https://f1000research.com/articles/12-900/v1	28 Jul 23	{ "type": "Review", "title": "Association study of melatonin receptor 1 A with litter size in sheep: A review", "authors": [ "Mutasem Abuzahra", "Dwi Wijayanti", "Mustofa Helmi Effendi", "Imam Mustofa", "Mirni Lamid", "Mutasem Abuzahra", "Dwi Wijayanti", "Imam Mustofa", "Mirni Lamid" ], "abstract": "Sheep are a valuable livestock species worldwide, providing meat, milk, and various dairy products. This article aims to review the latest literature on the melatonin receptor 1A (MTNR1A) gene as a potential candidate gene associated with reproductive traits, particularly the litter size trait in sheep, by searching various databases for available literature. Studies have shown that different parts of the MTNR1A gene play various roles in sheep. By identifying marker genes associated with reproductive traits in MTNR1A polymorphisms linked to the litter size trait, breeders can achieve a faster selection response in sheep breeding by recognizing the genomic region where these genes are located and understanding their physiological functions. Therefore, highlighting the literature on these functions and their association with reproductive traits may contribute to improving the genetic makeup during sheep breeding.", "keywords": [ "sheep", "MTNR1A", "litter size", "polymorphism" ], "content": "Introduction\n\nAs a result of the increasing demand for livestock production, there is a growing need for animals that are able to reproduce larger litter size.1,2 The ovulation rate, hormones, and growth factors all play a role in determining the litter size, which is one of the most important characteristics of fertility in animals.3,4\n\nIt has been noted that in sheep breeds where estrous activity peaks during short days, variations in day length may function as a main factor determining seasonal variations in estrous activity.5,6 In the pineal gland, the indole hormone melatonin (MT) is produced,7 which is linked to changes in light signals of the external natural environment.8–12\n\nThe binding melatonin receptor, also known as MTNR, has a variety of biological purposes, some of the most important of which include the regulation of animal sexual behavior, reproduction, and circadian rhythm13 a factor that has a positive influence on reproduction even when the night is longer.8 In most livestock species, mutations in the MTNR1A gene are linked to ovarian function and litter size.11,14,15 Despite the fact that several scholars have investigated the aspects of growth that influence the reproductive characteristics of goats.16,17\n\nMelatonin has an effect on reproduction, which is mediated through receptors that are found in the hypophyseal pars tuberalis.18 Melatonin has an effect on the pars tuberalis, which in turn has an effect on the seasonal reproductive activity of females in a number of different mammalian species.19 This includes the nuclei that are responsible for reproduction.20 Preceding studies have revealed that the polymorphisms in the MTNR1A gene sequence have an effect on the ability of Rasa Aragonesa rams to reproduce both when they are young and when they are adults.21 In particular, the T/T or G/G genotype was shown to be connected with earlier ram-lamb mating activity, and adult rams that carried the T/T or G/G genotype exhibited the highest reproductive behavior in the spring.20\n\nDue to the fact that sheep have a clearly established circadian rhythm,9 it is vitally necessary to investigate the mechanisms behind seasonal reproduction in this species. Melatonin production can be stimulated by exposing the eye to less light for longer periods of time. The pituitary gland’s production of follicle-stimulating hormone and luteinizing hormone is influenced by the amount of melatonin that is present in the bloodstream.9 The purpose of this article is to review the most recent literature on this topic may help to shed light on the mechanisms behind seasonal reproduction in sheep.\n\n\nMTNR1A location\n\nThe gene known as MTNR1A can be found in sheep on chromosome 26. It involves two exons that are split up by an intron that is nearly 22 kilobases (kb) in length and stretches from the position 17,354,820 to the position 17,377,973 in the Oar rambouillet v1.0 genome assembly. This region can be found between the two numbers 17,354,820 and 17,377,973. The length of exon I of the MTNR1A gene is approximately 260 base pairs, whereas the length of exon II is around 970 base pairs.6,22 Ewes’ seasonal reproductive activity is linked to the presence of many polymorphic sites at the MTNR1A locus, which can be found to be variable.5,14 Despite the fact that the ramifications of the MTNR1A gene on reproductive seasonality have only been investigated to a limited extent up until this point, those studies have shown that these effects are tied to three specific mutations discovered in exon II at locations 606, 612, and 706,6,22,23 only sheep of the Sarda and Aragonese breeds have had the remaining portions of this gene examined.11,24–27\n\n\nMTNR1A role by season\n\nThe duration of nocturnal melatonin secretion is affected by photoperiod variations between short and long days, which controls reproductive activity through the hypothalamic-pituitary-gonadal axis in seasonal sheep breeds located in areas with a temperate climate.27 These sheep breeds are bred in climates that are considered to be moderate.28 In the study of Martínez-Royo27 observed that reproductive functions were present in Merino d’Arles only in the month of April, as evidenced by varying progesterone concentrations. It was also shown that the SNP known as rs430181568, which was formerly known as 612/MnlI, is connected with the reproductive patterns that are associated with the different seasons. When analyzing the patterns of ram-ewe mating in the Rasa Aragonesa breed, ewes with the A/A genotype at the rs406779174 gene showed higher fertility between January and August than those with G/A or G/G genotypes. Ewes mating to rams or other ewes had the same results.27 This was the case regardless of whether the ewes were mated to rams or other ewes.\n\nBoth of these (606 and 612) loci have been linked in Indian breeds to ewes having lambs during times of the year when the ewes of these breeds would not normally be in oestrus.19 These two SNPs were found to be associated with the resumption of reproductive activities in the fall, following a period of seasonal anoestrus, in the animals studied, Slovenia is home to a variety of sheep breeds used for both dairy and meat production.15\n\nMelatonin production requires the participation of two distinct receptors; however, simply the melatonin receptor subtype 1A (MTNR1A) is believed to be a potential candidate gene that mediates the photoperiodic reproductive seasonality in sheep.19 Certain genotypes have been linked to reproductive activity that occurs outside of the normal breeding season.27 Despite the fact that sheep milk is a highly significant produce for Greece and the raw material intended for a great deal of products with a protected designation of origin.8 Polymorphisms found within the MTNR1A gene of the Sarda sheep breed, which has a period of anoestrus in the late winter/early spring, led to improvements in the return of reproductive function.6 However, in the Aragonesa breed, the polymorphism at position 612 was the sole one linked to a larger percentage of oestrous cyclic ewes between January and August.27\n\nThe change in annual photoperiod, which governs the production of melatonin and is the primary environmental element that determines reproductive timing.15 Melatonin secretion is lower throughout the long photoperiodic phases of the year, whereas it is higher during the shorter photoperiodic periods. As a result, various biological rhythms exist, such as seasonal reproduction, which is regulated by photoperiodic patterns.15\n\nIn small ruminants, the considerably higher nighttime MT concentrations that generally occur throughout the fall have a beneficial influence on reproductive activity,15 Allelic variations at locations g.15099491C > T and g.15099485 A > G in the MTNR1A gene exon II have been connected with the seasonal reproductive features in a variety of sheep breeds. These sites are located in the MTNR1A gene,8,29 only the g.15099491C > T polymorphism was linked to a greater proportion of oestrous cyclic ewes in the Aragonesa breed between January and August.27 These findings suggest that there is a connection the MTNR1A gene and reproduction season. Breed, management (including the presence or absence of males), and environmental conditions can all affect the timing of a gene polymorphism and the subsequent reproductive season. Thus, the length of time researchers observed subjects before drawing conclusions about the relationship between polymorphisms of this gene and the resumption of reproductive behavior is likely to be variable between studies.30 The findings of all of this research suggested that there was a relationship between the MTNR1A gene and the modulation of seasonal impacts on the reproduction of ewes in the breeds that were investigated.12\n\n\nMTNR1A in female reproduction\n\nAfter ovulation, progesterone is crucial for both the maintenance of pregnancy and the maintenance of a normal estrous cycle. As a further point, the granulosa cells in bigger ovarian follicles secrete the most progesterone.23 Because melatonin causes an increase in the diameter of the corpus luteum, it also causes an increase in the concentration of plasma progesterone.31 Melatonin might well be able to exercise direct control over the ovary due to the existence of melatonin receptors in ovarian follicles.32 Melatonin has been shown to stimulate progesterone secretion in granulosa cells, and it appears that MTNR1A plays a role in this process. However, blocking this receptor significantly reduces progesterone synthesis, melatonin’s effect on progesterone production may be partly due to MTNR1A, Consequently, melatonin’s ability to affect reproductive behavior in sheep stems from its ability to inhibit apoptotic genes while simultaneously stimulating antioxidant genes and progesterone synthesis via the MT1 receptor.33\n\nDue to the fact that melatonin is a hormone that is secreted by the body, its influence on the development of sheep oocytes in vitro is an excellent subject for research into the underlying molecular process. This was demonstrated by an experiment conducted in vitro, which also discovered that the addition of MT to the maturation process in vitro boosted the percentage of cultivated sheep oocytes that developed into blastocysts. The most valuable application of sheep MT is in scientific research.34\n\n\nMTNR1A gene and the reproductive performance of rams\n\nIt is unclear whether the specific gene variations responsible for the seasonal pattern in male sexual behavior have any impact on ram reproduction. Melatonin, produced in the testes, interacts with melatonin receptors in various testicular locations.21 The reproductive behavior of female ewes is influenced by breed, season, and the presence of male rams. However, the application of uniting males with ewes in breeding populations is challenging due to the variable reproductive responses of ewes30 Melatonin receptors MT1 and MT2 in the testes, accessory glands, and sperm suggest a direct effect of melatonin on the ram’s reproductive tract.21\n\nRecent studies indicate that melatonin can be produced in the male reproductive tissues, particularly the testes, explaining the high levels found in ram seminal plasma, especially during the daytime.35 Melatonin concentrations in seminal plasma and follicular fluid can exceed those in blood serum, suggesting a direct influence on sperm function.36 Melatonin affects sperm capacitation and in vitro fertilization outcomes in rams. The effects depend on the dosage, with low doses promoting capacitation and high doses inhibiting it.36 Melatonin acts by reducing cyclic adenosine monophosphate (cAMP) levels, suppressing protein kinase A (PKA) activity, a pathway involved in sperm capacitation.37 Different subpopulations of ejaculated ram spermatozoa exhibit variations in melatonin receptor distribution, suggesting differences in their physiological states. Melatonin inhibits apoptosis and modifies sperm capacitation through the MT2 receptor.35 Melatonin produced in the testes protects developing spermatozoa from oxidative damage, additionally, seasonal changes in ram seminal plasma composition and the impact of melatonin treatment have been observed.38\n\nMelatonin implantation in mature rams stimulates testicular growth, increases testosterone levels, improves semen quality, and enhances overall reproductive performance.39 Melatonin directly affects sperm motility and other characteristics during non-breeding seasons.39 Administering melatonin during the non-breeding season can improve testicular function beyond signaling the onset of the breeding season. Effective doses for enhancing testicular size in mature rams range from 36 to 54 mg.40 Furthermore, melatonin protects testicular tissue and improves semen quality in heat-stressed male goats by reducing oxidative stress.41\n\nIn conclusion, this study highlights the importance of the MTNR1A gene and melatonin in ram reproduction, elucidating their influence on sexual behavior, sperm function, and testicular health. Further research is needed to understand the underlying mechanisms and practical implications for improving ram breeding outcomes\n\n\nMTNR1A function\n\nIn animals, two high-affinity melatonin receptors known as MT1 and MT2 have been discovered; nevertheless, only MT1 is important in the regulation of reproductive behaviors. Melatonin exerts its effects by binding to certain receptors that are situated in a number of organs, the nuclei of the central nervous system are responsible for regulating reproductive activity. Melatonin’s actions are exerted.15\n\nAlthough the Pars Tuberalis (PT) contains a significant number of receptors, this formation is only slightly implicated in the regulation of reproduction.21 However, the PT unquestionably plays important roles in the photoperiodic regulation of prolactin secretion.15 However, only a tiny number of these receptors are found in the premamillary hypothalamus, which is the area of the brain responsible for reproduction when melatonin is present15 is only slightly implicated in the reg.15 The MT1 receptor gene (MTNR1A) has been found to exist in a variety of organisms. It belongs to the class of proteins called G protein-coupled receptors. There are a variety of polymorphisms, in exon II of the MTNR1A gene, that are known to influence the reproductive capabilities of different species according to the changing seasons.35\n\nMTNR1A and MTNR1B are the two subtypes that can be distinguished when referring to melatonin receptors. The suprachiasmatic nucleus and pituitary nodules of the hypothalamus are the primary locations where MTNR1A is found in animals.7 This location is significant since it is associated with the control of animal reproduction.7 The activity of MTNR1B is not particularly high, however MTNR1B-mediated MT may have an effect on insulin secretion.42 Therefore, MT mostly joins with MTNR1A, performing a particular function in the biological world.24\n\nMelatonin activates hypothalamic receptors. The suprachiasmatic nucleus houses the circadian clock, and thepremamillary hypothalamus controls the hormone’s reproductive actions.12 The premamillary hypothalamus has less melatonin MT1 receptors than the pars tuberalis.12 Sperm from non-seasonal animals have melatonin receptors. Their roles in spermatozoa may not be tied to seasonal fluctuations.43 Protection of human sperm from oxidative damage and apoptosis has also been linked to the melatonin receptor MT1,44 MT2, on the other hand, has been linked to the regulation of the capacity of male sperm to reproduce in vitro.35 The plasma of rams from three different sheep breeds was shown to contain melatonin when the rams were exposed to an equatorial photoperiod that changes between wet and dry seasons,45 while the source of this variation persists unidentified.28\n\n\nGenetic association in the MTNR1A gene\n\nMultiple variants of the MTNR1A gene have been discovered in sheep breeds worldwide, some of which have little impact on reproductive ability, while others can improve ovulation rates and litter sizes, or play an important role in male reproduction. Kianpoor et al.46 demonstrated a statistically significant relationship between the morphological characteristics and the genotypes of the MTNR1A gene. However, according to the results of Fathy et al.,47 the MTNR1A gene polymorphisms were not associated with most characteristics related to sperm quality and testicular size. Instead, the Ossimi and Barki breeds had the highest frequency of allele C, while the Rahmani breed had more allele T, with both Rahmani and Ossimi ewes with the MTNR1A SNP showing the shortest age at first lambing. Similarly, Mura et al.48 showed that rs430181568 and rs407388227 were linked to the fecundity of adult Sarda ewes. Moreover, the MTNR1A gene affects ram fertility in both juvenile and adult stages, as seen in the results of Vibha et al.49 and Mura et al.24 However, some studies, such as that of Z. Davari Varanlou et al.,50 did not find any association between this gene and reproductive or growth characteristics.\n\nThe study by Hatami M et al.51 revealed a significant association between MTNR1A/MnlI genotypes and BW1 (P<0.05), with Mm genotypes having a higher BW1 than MM genotypes as per the Least Squares analysis. Further research suggests that both MTNR1A SNPs, g.15118683C > T and g.15118951G > A, may influence the timing of oestrus and pregnancy in certain sheep breeds, with Small Tail Han sheep carrying a homozygous mutation (TT) at the g.15118756C > T locus exhibiting higher litter size.6 However, no correlation between genotype and litter size was.1,2 The ovulation rate, hormones, and growth factors all play a role in determining the litter size, which is one of the most important characteristics of fertility in animals.3,4 In the Istrian Pramenka breed, except for ewes with the other two genotypes, the A/A genotype was positively associated with higher fecundity (P<0.05) and a shorter DRPEL (P<0.05).52\n\nMura et al.24 reported the use of RsaI and MnlI restriction enzyme digestion to identify T606C and A612G polymorphisms in the amplicons. Ewes with the G/G, G/A, C/C, and C/T genotypes exhibited increased fecundity (P<0.05) and shorter intervals between ram introduction and parturition (P<0.05) compared to those with the A/A and T/T genotypes. The study revealed that mutations in the MTNR1A gene affected the timing of the Sarda sheep breed’s return to reproduction in the spring. Furthermore, variations in the MTNR1A gene sequence impacted ram fertility during both juvenile and adult stages of life. T/T and G/G ram-lambs born in the fall had superior reproductive abilities, while T/T and G/G adult rams exhibited the most aggressive mating behavior.21 The SNP rs403212791, which alters the amino acid content from Arginine to Cysteine (R336C), may result in reproductive seasonality trait changes. The rs403212791 T allele in the MTNR1A gene was positively associated with the oestrus cycling months OCM values, which indicate behavioral indicators of oestrus in the Rasa Aragonesa breed, and negatively correlated with TDA values, which reflect seasonality attributes for ovarian function based on blood progesterone levels.22 Three single nucleotide polymorphisms (G735A, G753A, and C845A) were detected in both Chinese Merino and Prolific Suffolk sheep, and an association study revealed a significant association (P<0.05) between these three SNPs and litter size.53\n\nThus, it is crucial to further investigate the true involvement of MTNR1A through additional research with larger sample sizes and a wider variety of sheep breeds from around the world, as well as the mechanism of its SNPs in reproductive traits. MTNR1A gene has a significant effect on the reproductive traits of various sheep breeds. Different alleles and genotypes of this gene have been linked to variations in fecundity, timing of sexual maturation, litter size, and body weight. However, there is still a need for further research to confirm these findings and investigate the underlying mechanisms of the gene’s effects.\n\nIn terms of practical implications, the identified SNPs in the MTNR1A gene may be useful for selecting and breeding sheep with desirable reproductive traits. For example, ewes with certain genotypes may be preferred for earlier age at first lambing or higher fertility, while rams with specific alleles may have superior reproductive abilities or mating behavior. Additionally, the use of genetic markers and genotyping techniques can aid in the identification and preservation of rare or valuable sheep breeds.\n\nOverall, the information presented underscores the importance of genetic factors in sheep reproduction and highlights the potential for genetic improvement of this trait in various breeds. Further studies are needed to fully understand the mechanisms involved and to optimize breeding strategies for desired outcomes.\n\n\nConclusions\n\nMTNR1A SNPs have been extensively studied in previous research, some resulting in an amino acid change. Although three of these connections have a significant relationship with litter size, the majority of previous studies indicate a link between MTNR1A and male reproduction, despite three of these relationships having a strong association with fecundity. Only a few studies have found a correlation between the gene and reproductive attributes, and were not significant. Therefore, it is essential to consider MTNR1A’s impact on both female and male reproduction, as this factor further affects sheep’s litter size. However, this implies that mutations within MTNR1A could be detected in exon 1, a region that was not investigated in earlier works.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWang F, Chu M, Pan L, et al.: Polymorphism detection of gdf9 gene and its association with litter size in luzhong mutto sheep (Ovis aries). Animals. 2021; 11(2): 1–11. Publisher Full Text\n\nAjafar MH, Kadhim AH, Al-Thuwaini TM, et al.: Association of bone morphogenetic protein 15 and growth differentiation factor 9 with litter size in livestock: a review study. Acta Sci. - Anim. Sci. 2023; 45: e57927. Publisher Full Text\n\nTesema Z, Deribe B, Kefale A, et al.: Survival analysis and reproductive performance of Dorper x Tumele sheep. Heliyon. 2020; 6(4): e03840. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "220362", "date": "20 Nov 2023", "name": "Ugbo Emmanuel Nnabuike", "expertise": [ "Reviewer Expertise Medical Microbiology/Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article addressed the purpose of the topic in the area of the function of MTNR 1A in the reproduction performance of animals. I hereby approve for publication.\n\nI also suggest that the below comments should be addressed. Topic should be corrected to be (Association of melatonin receptor 1 A with litter size in sheep: A review).\nThe sub-heading (MTNR 1 A gene and Reproductive Performance of Rams) Paragraph 2 (Recent studies, should be corrected to Studies). Since the Reference (35; is 2016 year) so it is not recent.\nI suggest you add all the conclusion together, not concluding in some of the sub-heading. Pull all the conclusion together to be in the conclusion part.\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": "10934", "date": "22 Mar 2024", "name": "mutasem abuzahra", "role": "Author Response", "response": "Topic should be corrected to be (Association of melatonin receptor 1 A with litter size in sheep: A review). Author: thank you for suggesting, I have corrected according to your suggestion. The sub-heading (MTNR 1 A gene and Reproductive Performance of Rams) Paragraph 2 (Recent studies, should be corrected to Studies). Since the Reference (35; is 2016 year) so it is not recent. Author: corrected upon your request I suggest you add all the conclusion together, not concluding in some of the sub-heading. Pull all the conclusion together to be in the conclusion part Author: corrected upon your request, i gathered all together in the conclusion part" } ] }, { "id": "220370", "date": "21 Nov 2023", "name": "Wenjing You", "expertise": [ "Reviewer Expertise Animal nutrition", "metabolic 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\nAbuzahra et al. aimed to review the latest literature on the melatonin receptor 1A (MTNR1A) gene as a potential candidate gene associated with reproductive traits, particularly the litter size trait in sheep, by searching various databases for available literature. While the subject addressed is important and interesting, further details need to be clarified.\nThe focus should be on melatonin and its receptor MTNR1A, whose roles, sources, structure, and characteristics are little described. Additional information regarding these aspects is needed.\nThe authors should also consider describing the mechanisms involved in the production and secretion control of melatonin by the pineal gland, as well as provide an in-depth review of the mechanism by which melatonin regulates the release of follicle-stimulating hormone and luteinizing hormone through the hypothalamic-pituitary-gonadal axis, thereby regulating seasonal reproductive activity in mammals.\nThe MTNR1A gene has different effects on reproductive traits in different sheep breeds and sexes, therefore, the section reviewing the genetic relevance of the MTNR1A gene in sheep breeds should be more logical and organized.\nPlease provide the full name of the abbreviation, such as SNP, BW1, MM.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "10935", "date": "22 Mar 2024", "name": "mutasem abuzahra", "role": "Author Response", "response": "The focus should be on melatonin and its receptor MTNR1A, whose roles, sources, structure, and characteristics are little described. Additional information regarding these aspects is needed. Author: thank you for your suggestions, I have added to the manuscript a subtitle namely (Mechanisms Involved in the Production and Secretion Control of Melatonin) upon your request. The authors should also consider describing the mechanisms involved in the production and secretion control of melatonin by the pineal gland, as well as provide an in-depth review of the mechanism by which melatonin regulates the release of follicle-stimulating hormone and luteinizing hormone through the hypothalamic-pituitary-gonadal axis, thereby regulating seasonal reproductive activity in mammals. Author: thank you for your suggestions, also this part, I have added to the manuscript under a subtitle namely (Mechanisms Involved in the Production and Secretion Control of Melatonin) upon your request The MTNR1A gene has different effects on reproductive traits in different sheep breeds and sexes, therefore, the section reviewing the genetic relevance of the MTNR1A gene in sheep breeds should be more logical and organized. Author: Thank you for your guidance. I have incorporated your suggestions into the manuscript, and I have reorganized the section on genetic relevance of the MTNR1A gene to enhance its logical flow, as per your recommendations. Please provide the full name of the abbreviation, such as SNP, BW1, MM Author: corrected upon your request" } ] } ]	1	https://f1000research.com/articles/12-900
https://f1000research.com/articles/12-791/v1	06 Jul 23	{ "type": "Study Protocol", "title": "Comparative evaluation of fracture resistance of re-attached teeth using self-adhesive bioactive flowable composite after preconditioning the fractured coronal fragments with different remineralizing agents", "authors": [ "Pratik Rathod", "Nikhil Mankar", "Nikhil Mankar" ], "abstract": "One of the common forms of dental injury is anterior crown fractures, which mainly affects the teenagers and young adults. Fractures of the coronal portion of the permanent incisors characterize 18–22% of total traumatic injuries to dental hard tissues; of which 96% of them comprise the maxillary incisors. Uncomplicated fracture of crown is one of the most common types of dental traumatic injury. Dental trauma has an emotional impact on the patient’s overall health and can serious harm to the dentition. The treatment as well as prognosis of the fracture of coronal portion is a major challenge for a dentist because it has to accomplish various parameters like need to obtain an aesthetical result that approaches itself to its natural form and measurement, opaqueness and translucency of the original tooth structure in obtaining a effective restoration. It is suggested that reattachment of the fractured fragment is the best procedure for restoring an uncomplicated fracture of a crown, if a fragment is present. Reattachment of the fractured fragment offers major advantages over the conventional composite restoration. In reattachment, enhanced aesthetics is obtained because enamel’s true shape, colour, intensity, and surface texture are preserved. It re-establishes the major function and produces the favourable emotive and social response from the side of the patient. The reattached fragments are susceptible to further fracture when the restored teeth undergo further trauma. The resistance of the fractured teeth that has been reattached is the subject of the majority of concerns. Preconditioning the fractured fragments with remineralizing agents may aid in hydration. Thus, study will be conducted to evaluate the resistance of the fracture of a tooth that is reattached and pre-treated with remineralizing agents such as sodium fluoride and casein phosphopeptide-amorphous calcium phosphate and further reattached using one of the self-adhesive bioactive composite.", "keywords": [ "Traumatic dental injury", "Casein phosphopeptide–amorphous calcium phosphate", "Sodium fluoride", "Uncomplicated crown fracture", "Bioactive", "Reattachment technique", "Fracture resistance." ], "content": "Introduction\n\nFracture of the crown of anterior teeth is one of the most common types of dental trauma that primarily affects adolescents and young adults. The location of upper incisors and their pattern of eruption brings a major risk for the dental trauma.1 Dental trauma is more prevalent in case of contact sports, road traffic accidents, outdoor events, and in case of falls. Orofacial trauma comprises about 5% of all injuries to the body, while traumatic dental injuries (TDI) has been reported 15.2% prevalent worldwide.2 With a prevalence of 17% to 48%, fractures of uncomplicated crown are the utmost or frequent types of dental trauma. 18–22% of all traumatic injuries to the hard tissues of dental origin result in fractures of the coronal portion of permanent upper incisors, and 96% of them involves the upper incisors (80% of them are central incisors and 16% of them are lateral incisors). Injuries caused by dental trauma not only affects the dentition, but also have a major impact on psychological status of the patient. According to the International Association of Dental Traumatology reattachment of the fractured fragment is the best method to restore uncomplicated fractures of the crown of permanent teeth, if the fractured fragment is available.3 Many of these problems have been resolved because of the advancement in field of bonded aesthetic dentistry and its ongoing evolution. A better prognosis can result by using intermediate composite material and the lack of further added preparations, which can improve the adhesion of fractured fragments. Although composite restorations of direct and indirect type and prosthesis are other treatment options, but fragment reattachment has been proven to be a preferable alternative for restoring aesthetics, improved functions and natural anatomy of the tooth. In Addition to this, it is cost-effective and time-saving.4 Because the general anatomical shape of the tooth, colour, and texture of the tooth surface are preserved, reattaching the fragment can produce pleasing aesthetics that last for a long time. This procedure is rather simple and fairly conservative. It restores tooth function and encourages the patient to feel better immediately on an emotional and social level.5 If another traumatic incident occurs or when the restored teeth are used in a way that is not physiological, the reattached fragments are vulnerable to breaking again. The resistance of the fractured teeth that has been reattached is the subject of the majority of concerns. The significant factor is whether the fragment of fractured tooth and the residual tooth can form a stable, predictable union. Activa bioactive has rubberized resin component having more fracture resistance and shock absorption and being a smart material adapts to PH cycle. It has all the desired properties such as improved hydrophilicity, sealing ability, bond strength. The conventional flowable composite clinically used in dentistry for reattachment varies in terms of bond strength and adhesion therefore newer materials with improved properties are desired and henceforth in this study the material Activa bioactive will be tested. The time interval between the traumatic injury and reattachment as well as the hydration of the broken fragment, which preserves the vitality and original shine of that of the natural tooth, are the major factors in the achievement of re attachment of fractured fragments. Dentin moisture is necessary to fortify the binding between composite resin and dentin. According to findings from the literature, hydrated fractured tooth fragments have a stronger bond than dehydrated pieces.6 For preconditioning of the fractured fragments use of remineralizing agents not only may aid in hydration but also for up taking maximum of the mineral ions. But, the study about the result of remineralizing agents affecting the bond strength of re attached tooth is limited. Henceforth, a study will be done to assess the fracture resistance of the teeth that is reattached using self- adhesive bioactive composite and were pre-treated with remineralizing agents such as sodium fluoride, casein phosphopeptide–amorphous calcium phosphate (CPP-ACP), prior to re-attachment.\n\n\nObjectives\n\n\n\n1) To evaluate fracture resistance of reattached fractured tooth preconditioning with CPP-ACP luted with self- adhesive bioactive material using universal testing machine.\n\n2) To evaluate fracture resistance of reattached fractured tooth preconditioning with NaF as preconditioning agent luted self-adhesive bioactive material using universal testing machine.\n\n3) To compare fracture resistance of reattached fractured tooth with CPP-ACP and NaF as preconditioning agents luted with self-adhesive bioactive material using universal testing machine.\n\n\nMethods\n\nIn-vitro study.\n\n\n\n1. Casein Phosphopeptide – Amorphous Calcium Phosphate (CPP-ACP), (GC tooth Mousse, GC India)\n\n2. 2% Sodium Fluoride (2% NAF) (SEPTODONT Sodium Fluoride Gel -Flucol Gel)\n\n3. Self-adhesive Bioactive flowable composite material – (ACTIVA bioactive-CEMENT, Pulpdent)\n\n4. Etchant -37% phosphoric acid (Prime etching liquid, India)\n\n5. Adhesive agent (Adper Single Bond 2 Adhesive – 3M ESPE)\n\n\n\n1. Sound upper central incisors removed due to periodontal reasons.\n\n2. Teeth devoid of restorations.\n\n3. Non carious teeth.\n\n\n\n1. Teeth with previous root canal treatment.\n\n2. Extensively carious tooth.\n\n3. Abrasion, attrition, fluorosis, or other enamel defects.\n\n4. Teeth with developmental anomalies.\n\n5. Teeth with external and internal resorption.\n\n\n\n➢ Total of 50 samples will be considered in this study.\n\n➢ Samples will be divided into 2 groups, 25 in each group corresponding to the 1 and 2 sample sets.\n\n➢ Group 1: Casein phosphopeptide-amorphous calcium phosphate as preconditioning agent.\n\n➢ Group 2: Sodium fluoride as preconditioning agent.\n\nFor the study, a total of 50 freshly extracted, sound permanent human maxillary central incisor teeth will be used.\n\nSectioning of the teeth\n\nTo simulate an uncomplicated crown fracture, extracted teeth will be sectioned at incisor third of crown using low speed double sided diamond disk. The direction of diamond disk will be in perpendicular direction to the long axis of the tooth.\n\nPreconditioning of teeth\n\nSectioned fragments will be immersed in agents that are re mineralizable such as sodium fluoride and CPP-ACP for a predetermined contact period i.e., 30 min. Then, the fragments from the coronal portion will be attached to the residual structures of tooth with self-adhesive flowable bioactive composite material.\n\nRe-attachment of fragments\n\nThe fragments of the fractured teeth that are preconditioned and the tooth structure that is residual will be rinsed thoroughly with distilled water. After thorough rinsing, acid etching will be done. Application of etchant i.e., 37% phosphoric acid (Prime etching liquid, india) for 15 sec will be carried out, followed by rinsing of the tooth with water for 15 s. After that, both surfaces will be dried with air at maximum of 5 seconds in order to keep the surface moist. The process of applying adhesive bonding agent to the surfaces will be carried out after acid etching. The first coat of adhesive agent (Adper Single Bond 2 Adhesive – 3M ESPE) will be applied to the surfaces that are sectioned for 10 s, and then, second layer of adhesive agent will be applied. Then coats will be cured by light for maximum of 20 s after being air-thinned to remove any surplus. The fractured fragments will be restored together, reattached, and curing is done for 20 seconds on the labial surface and palatal surface respectively using self-adhesive bioactive composite material (ACTIVA BioACTIVE-CEMENT, Pulpdent). The specimens will be placed in artificial saliva after reattachment and left there until their fracture resistance is assessed.\n\nEvaluation of fracture resistance of teeth\n\nThe tooth samples will be numbered for easy identification and embedded in blocks of cold-cure acrylic resin (2 cm × 2 cm) (DPI RR cold cure, India) up to the cingulum for evaluation of fracture resistance. The universal testing machine will be used to evaluate the fracture resistance.\n\nThe force exerted by the applicator tip will be placed exactly perpendicular to the line of fracture in relation to the surface of crown labially having the speed of crosshead at 1 mm/min.\n\nAnd having cell load of 500 newton. Force that is needed to fracture the tooth will be assessed in unit that is Newton.\n\nFormula Using Mean difference on fracture resistance (FR) in N (newton)\n\nZβ = 0.84 at 5% type I error.\n\nPrimary Variable fracture resistance (FR)\n\n((Fracture resistance (FR) in reattached teeth preconditioned with CPP-ACP group) Mean ± SD. = 215.5 ± 81.16 As per Reference article)\n\n((Fracture resistance (FR) in reattached teeth preconditioned with NaF group) Mean ± SD. = 141.29 ± 54.25 As per Reference article)\n\nAs per reference articles.\n\nTotal samples required = 25 per group\n\nFormula Reference: 6\n\nFor the ease of calculation and statistics, the sample size confirmed to 50 consisting of 25 subjects in each group.\n\nAll the results will be calculated using SPSS version 27 software. All the demographic data variable assessment will be done for quantitative assessment in mean std dev minimum & maximum & in frequency & percentage for qualitative assessment. Data for outcomes variables will be tested for normality using kalmogorov-smirnov. The comparative analysis of the fracture resistance will be evaluated on the measurement of Newton. Unpaired t test will be used to find the significant difference between the mean of the 2 groups. P-value ≤ 0.05 will be considered as significant at 5% level of significance and 95% confidence of interval.\n\nIt is expected that preconditioning of fragment with sodium fluoride may give better results as compared to Casein Phosphopeptide–Amorphous Calcium Phosphate on fracture resistance of reattached fragment with self-adhesive bioactive flowable composite.\n\nThe focus of this study is to assess the fracture resistance of the reattached teeth preconditioned with various remineralizing agents. Preconditioning of fragment may further contribute to the increased fracture resistance of reattached fractured tooth.\n\nEthical approval was received from Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha (Maharashtra), India.\n\nIEC reference number - DMIHER (DU)/IEC/2023/582.\n\nWritten informed consent will be taken from patients who undergo extraction regarding the use of extracted teeth for the study purpose.\n\nNot started yet.\n\n\nDiscussion\n\nThe task is to handle the tooth with utmost care that further lessen the damage of the teeth with coronal fractures of upper incisors. Several restorative techniques of direct and indirect type are in use for the treatment of fractured teeth, although such techniques often compromise a lot of the natural healthy tooth structure.7 If there is only a slight violation in the area of the biological width and a complete fragment of the fractured tooth is available, the re attachment of the fractured fragment method can be considered. Bruschi-Alonso et al. stated that the first choice of treatment for fractured tooth having uncomplicated crown fracture should be the re-attachment of fractured fragment.8 Reattachment offers many benefits over other procedures as it is a minimal invasive in nature, uncomplicated, and cost-effective process. It aids in maintaining enduring aesthetics and is highly accepted by the patient.\n\nHydrating fragments of fracture play a very important role in improvement of the resistance of fracture at re-attachment interface.4,9 Time interval during or after trauma, media that is used for storage, and dry time before the re-attachment of the fractured fragments are crucial aspects and they impact the resistance of fracture and adhesive strength of re-attached fragments. Most commonly affected teeth by dental traumatic injury are maxillary central incisors. Hence maxillary central incisors are selected for the study. In regard to the method used, the fragments of tooth will be attained by segmenting with a diamond disk in spite of fracturing. Badami et al. and Reis et al. stated that the tooth surface that is sectioned is totally different from that of a tooth surface that is fractured.10 The path of the fractured line in case of a tooth that is sectioned is indicated by the position of the disk, it inclines to run in a direction parallel to the direction of prisms of enamel in a region of fragmentation. In a while, because trauma has a nature of not proceeding linearly or with good adjustment, this direction may not correctly depict the accurate circumstances of the trauma. Nevertheless, the standardisation of the fragmented fracture that is necessary to lessen the bias that is of confounding nature was made possible by simulating the fracture by use of a cutting disk. According to Garcia et al. and de Souza et al., fractured fragment is reattached by a technique of no preparation and a bonding system having intermediate resin composite comprising good mechanical properties that have the capacity to restore part of the resistance of the tooth that is fracture.11 Preconditioning of fractured fragments with remineralizing agents may aid in hydration. Thus, study will be conducted to assess the resistance of fracture of teeth that is reattached and are pre-treated with various remineralizing agents such as sodium fluoride and casein phosphopeptide-amorphous calcium phosphate and reattached using self-adhesive bioactive composite.", "appendix": "Data availability\n\nNot applicable as this is a protocol.\n\n\nAcknowledgements\n\nI would like to thank my institute, my Guide, HOD and my colleagues.\n\n\nReferences\n\nShirani F, Malekipour MR, Tahririan D, et al.: Effect of storage environment on the bond strength of reattachment of crown fragments to fractured teeth. J. Conserv. Dent. 2011 Jul 1; 14(3): 269–272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetersson EE, Andersson L, Sörensen S: Traumatic oral vs non-oral injuries. Swed. Dent. J. 1997; 21(1–2): 55–68. PubMed Abstract\n\nDiangelis AJ, Andreasen JO, Ebeleseder KA, et al.: International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 1. Fractures and luxations of permanent teeth. Dent. Traumatol. Off. Publ. Int. Assoc. Dent. Traumatol. 2012 Feb; 28(1): 2–12. PubMed Abstract \| Publisher Full Text\n\nMadhubala A, Tewari N, Mathur VP, et al.: Comparative evaluation of fracture resistance using two rehydration protocols for fragment reattachment in uncomplicated crown fractures. Dent. Traumatol. Off. Publ. Int. Assoc. Dent. Traumatol. 2019 Jun; 35(3): 199–203. Publisher Full Text\n\nAndreasen FM, Norén JG, Andreasen JO, et al.: Long-term survival of fragment bonding in the treatment of fractured crowns: a multicenter clinical study. Quintessence Int. Berl. Ger. 1985. 1995 Oct; 26(10): 669–681.\n\nSurekha GL, Vinay C, Baliga S, et al.: Effect of preconditioning the fractured coronal fragments with remineralizing agents on fracture resistance of re-attached teeth. J. Indian Soc. Pedod. Prev. Dent. 2021; 39(4): 384–387. PubMed Abstract \| Publisher Full Text\n\nReis A, Loguercio AD, Kraul A, et al.: Reattachment of fractured teeth: a review of literature regarding techniques and materials. Oper. Dent. 2004; 29(2): 226–233. PubMed Abstract\n\nBruschi-Alonso RC, Alonso RCB, Correr GM, et al.: Reattachment of anterior fractured teeth: effect of materials and techniques on impact strength. Dent. Traumatol. Off. Publ. Int. Assoc. Dent. Traumatol. 2010 Aug; 26(4): 315–322. PubMed Abstract \| Publisher Full Text\n\nde Sousa APBR , França K, de Lucas Rezende LVM , et al.: In vitro tooth reattachment techniques: A systematic review. Dent. Traumatol. Off. Publ. Int. Assoc. Dent. Traumatol. 2018 Oct; 34(5): 297–310. PubMed Abstract \| Publisher Full Text\n\nLoguercio AD, Mengarda J, Amaral R, et al.: Effect of fractured or sectioned fragments on the fracture strength of different reattachment techniques. Oper. Dent. 2004; 29(3): 295–300. PubMed Abstract\n\nBrasil Maia G, Pereira RV, Poubel DL d N, et al.: Reattachment of fractured teeth using a multimode adhesive: Effect of different rewetting solutions and immersion time. Dent. Traumatol. Off. Publ. Int. Assoc. Dent. Traumatol. 2020 Feb; 36(1): 51–57. PubMed Abstract \| Publisher Full Text" }	[ { "id": "211913", "date": "27 Nov 2023", "name": "Anand Marya", "expertise": [ "Reviewer Expertise Orthodontics", "remineralization", "adhesives" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me to review this paper. I do have the following comments:\nHow was the sample size calculation done? What is the power of the sample?\n\nIs there any control group with no remineralizing agent?\n\nPlease cite references for the methodology? eg. duration of etching etc.\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": "10911", "date": "22 Mar 2024", "name": "Pratik Rathod", "role": "Author Response", "response": "Thank you, respected sir, for reviewing this paper. I have made all the suggested corrections as follows. 1. Sample size calculation is done, taking fracture resistance as a variable, keeping the power of sample 80 and the Difference between two groups 74.29. 2. sir, there is no control group with remineralizing agents. 3. I have added references for the methodology." } ] } ]	1	https://f1000research.com/articles/12-791
https://f1000research.com/articles/13-78/v1	19 Jan 24	{ "type": "Research Article", "title": "Developing a measure of participant experience of trials: qualitative study and cognitive testing", "authors": [ "Nicola Small", "Claire Planner", "Katie Gillies", "Caroline Sanders", "Katrina Turner", "Bridget Young", "Ailsa Donnelly", "Lindsey Brown", "Anjie Holt", "Judith Hogg", "Heather Bagley", "Peter Bower", "Nicola Small", "Claire Planner", "Katie Gillies", "Caroline Sanders", "Katrina Turner", "Bridget Young", "Ailsa Donnelly", "Lindsey Brown", "Anjie Holt", "Judith Hogg", "Heather Bagley" ], "abstract": "Background To encourage participation in trials, people need to have a positive experience. However, researchers do not routinely measure participant experience. Our aim is to facilitate routine measurement by developing a measure that captures the participant perspective, in a way that is meaningful, acceptable and useful to trial teams and participants.\n\nMethods We conducted a focus group and 25 interviews with trial professionals and trial participants to explore how participant experiences of trials should be measured, and to identify domains and items to include in the measure. Interviewees were also asked to comment on a list of candidate domains and items informed by a previous review of the literature on participant experience measures. Interviews were analysed thematically. Candidate domains and items were incorporated into a draft measure. Cognitive testing was undertaken in two rounds to ensure the items were comprehensible and grounded in participant experience.\n\nResults Interviewees and patient and public contributors reported that standardising the measurement of participant experience of trials had the potential to improve trial experience but described issues around the timing of measurement. Cognitive testing highlighted issues with comprehension, recall and response and numerous items were removed or refined. We developed a standard and a short version of the measure for feasibility testing.\n\nConclusions We developed a measure covering important domains of participant experience of trials, which could assist trial teams and participants to improve trial design and enhance delivery of a meaningful participant experience.", "keywords": [ "trial", "participation", "patient experience", "patient satisfaction", "patient-centred trials", "cognitive testing" ], "content": "Introduction\n\nIncreasing participation in trials is a global goal but recruitment and retention remain key challenges,1,2 and there is interest in developing better methods to conduct trials,3,4 including new processes,5 incentives6 and methods to enhance diversity.7 One method of improving trials (which may benefit engagement) is a focus on participant experience.8\n\nIn health contexts, measurement of patient experience is increasingly used in quality improvement,9 and a similar approach could be used in trials.10 Most trials already have extensive platforms to measure participant experience of the intervention (e.g. quality of life and quality of care) but few routinely measure experience of trial participation. If the measurement of participant experience of the trial was routine, it would allow measurement of variation in experience over sites and time, and between groups within a trial. Feedback from participant experience data could support interventions to enhance future participant experience and potentially increase engagement with research.\n\nAn example in a health context is the routine assessment of patient experience of primary care.11,12 Done routinely at scale, experience can be assessed among individual practices, providing policy-makers with extensive data on experience over time and providing practices with data on strengths and weaknesses in their services which could be modified. There could be value in the trials community adopting a similar approach.\n\nOur scoping review of self-reported measures of participant experience concluded that there is no existing standardised measure.10 The review found a number of limitations in the available studies, including the lack of a formal definition of participant experience, and the use of measures without detailed data about their development.\n\nSince 2015, the UK National Institute of Health and Care Research (NIHR) Clinical Research Network13 has embedded a measure (the ‘Participant Research Experience Survey’ - PRES) to assess participant experience of studies (including trials) within each local network. The survey uses a standard set of questions, but the survey is delivered within local networks and across multiple studies currently recruiting within that network. This provides a useful snapshot of experience within that local network but does not provide a systematic, comprehensive assessment of individual trials, and is not designed to provide the detail necessary to compare individual trials. Building in routine measurement and feedback of participant experience into all trials would arguably complement this approach.\n\nOur aim was to develop a standardised measure of participant experience in trials through the application of qualitative research, cognitive testing and patient and public involvement and engagement (PPIE).\n\n\nMethods\n\nThe study had two sequential phases, in line with measure development guidance,14 and the study flow is detailed in Figure 1. Phase 1 involved qualitative interviews to determine broad domains of participant experience and specific item content. Phase 2 involved cognitive testing to explore comprehension of the items. A PPIE advisory group (PAG) supported both phases. The 6 members of the PAG provided input into all stages of the project, as outlined in a strategy. The GRIPP-SF checklist15 details implementation of the strategy (Table 1). Three PAG members had previously taken part in a trial and four had experience of PPIE work. AD and CP were joint PAG co-ordinators. To guide development, we modified an existing definition of patient experience16 to make it applicable to trials, and defined participant experience as: (1) the sum of all interactions in the trial (2) shaped by an organisation’s culture (3) which influences participant perceptions (4) across the continuum of research.\n\nIn summary, we identified candidate domains of patient experience and candidate items from our previous work,10 and explored those through a focus group, semi-structured interviews, and PAG meetings. This generated (a) insights about measuring participant experience in trials (b) a preliminary list of candidate domains of participant experience, and candidate items mapped to those domains. These underwent two rounds of cognitive testing.\n\nFirstly, a focus group with clinical trials unit staff captured their perspectives on measuring participant experience of trials and their views on domains and items. Secondly, semi-structured interviews with trial professionals and participants explored the same issues. For pragmatic reasons, we limited the sample size to a maximum of 25 interviewees. Separate topic guides for professionals and participants were used incorporating questions and prompts covering trial experience and what the questionnaire might look like with core questions that can be asked of anyone participating in a trial, and to facilitate discussion, we presented candidate domains and items from a predefined list.10 Topic guides were piloted with members of the PAG who advised that the questions were appropriate. We have uploaded versions of the measure to the OSF repository.26 The unformatted versions show the item content, which can be used with existing trial materials and matched to local formatting and style requirements. The formatted measure is a suggested format for presentation.\n\nThe study used purposive sampling. We recruited professionals who had worked on any type of trial within the previous 12 months, including clinical trials unit directors, principal investigators, trial funders and Clinical Research Network staff. We also recruited current or past (within the last 5 years) adult participants. We sought diversity in trial participation as well as type, age, gender, ethnicity and education. Recruitment sources were a local consent-to-contact database,17 the Greater Manchester Clinical Research Network and research team contacts using an email from the research team and an advert. Before conducting the research, the researcher and the Director of the Trials Unit and relevant CRN lead identified professional participants who could participate in the focus group by circulating the study information. Trial professional recruitment sources were aware of the researcher’s interest to improve participant experience in trials. There was no existing relationship with previous trial participants; people responded to the advert directly to the research team with no prior knowledge of the researcher’s interest.\n\nOne focus group (n=6) was conducted with professionals by author CP in April 2018. Individual interviews with professionals were conducted in-person at the Trial Unit in the UK or via the telephone (n=10) at the University of Manchester by CP between April and July 2018. Trial participant interviews were also conducted in-person (n=10) or by telephone (n=4) by the lead author, NS, between May and July 2018. Each participant took part in one interview or focus group for approximately one-hour; only the participant/s and researcher were present. Both CP and NS are experienced, female, health services researchers, with PhDs, employed as Research Associates at the time of the study. In-person interviews took place in a convenient and private location, with travel expenses reimbursed and a £10 shopping voucher given as thanks. Of note, no participants refused to participate at interview.\n\nTable 2 shows the focus group and interview participant characteristics for the study. Of the 11 trial professional participants interviewed, 36% were staff at the CRN, 27% were clinical trials unit directors, 18% were principal investigators, and 18% were trial funders. Of the 6 participants in the focus group, the majority were Trial Managers (n = 2) followed by a Clinical Trials Director, a Statistician, a Quality Assurance Trialist, and a Clinical Research Fellow. Of the 14 trial participants interviewed, 64% were male, with age range, 30-78 years, with 50% reporting a postgraduate qualification and 64% White English ethnicity.\n\nInterviews were recorded, field notes were made, and transcribed verbatim, then uploaded to NVivo (version 11) for coding and analysis by NS and CP. Research team members read early transcripts, suggesting codes, and offering avenues to explore with participants. PAG members each read one professional and one participant transcript to add their insights. Of note, transcripts were not returned to participants for comment/feedback and/or correction. Transcripts from trial participant interviews were analysed thematically by NS.18 Themes were generated, coded and categorized according to the developing framework. A document listing the codes from each transcript with excerpts of data relevant to each theme was developed to manage the developing framework. Data from trial professionals were coded separately from participant data by CP, and then compared. This and our previous work10 informed the drafting of the measure to be tested by cognitive testing (consisting of 9 candidate domains and 52 items).\n\nCognitive testing is a method used to assess the performance of a measure by collecting information about respondents’ thought processes, as they answer questions, using ‘think aloud’ techniques to inform adaptation of the measure.19\n\nWe invited trial participants from phase 1 and new participants using the same recruitment methods (via email and advert) and purposive sampling. There is no consensus around sample size for cognitive testing, as this depends on the type of cognitive process examined and the number of ‘rounds’ necessary to adjust the measure.20 Here, a round was considered complete when clear problems were identified with items warranting adjustments to the items and measure.\n\nThe study was conducted within the Centre for Primary Care and Health Services Research at The University of Manchester, UK. Cognitive interviews took place in August 2018 in a private room for approximately one hour with only the participant and researcher, NS, present. Interviews were audio-recorded, with note taking, but not transcribed. As before, participants received travel expenses and a shopping voucher.\n\nNS received training on cognitive testing ahead of the study. The topic guide included a full explanation of the ‘think aloud’ task and open-ended questions and probes (spontaneous and pre-prepared) to assess different cognitive processes: comprehension; recall; judgement; and response.21 We opted for an ‘immediate retrospective think aloud’ approach whereby we asked participants to read each item while thinking aloud and to respond to each item. After doing a review of the topic guide, the research team and PAG judged that the questions and topic areas were appropriate.\n\nCognitive testing took place over two rounds (n= 13 and n = 6) where the results of round 1 informed round 2, with each respondent participating once. No participants refused to participate at interview. Of the 19 participants, 63% were male, with age range, 40–86 years, with 58% reporting a postgraduate qualification and 74% previous trial experience (see Table 3).\n\nFor data analysis, we followed the three-stage process in a modified framework analysis,20 involving: chronicling, condensing and using the data to improve the questionnaire; NS prepared the analysis. PAG members each read cognitive testing guidance prepared by NS to add insights to the analysis at the meeting to refine the items. The research team and PAG made decisions on changes, including changes to items, the items contained within each domain, the domain name (to reflect the revised content), the response scale and measure layout. We followed the published reporting framework for cognitive testing.22\n\nThe study received Proportionate University Research Ethics Committee approval (Ref: 2018-2739-5920). Participants who expressed an interest in participating in the study via the advert were emailed a full Participant Information Sheet outlining study involvement and had the opportunity to consider the information and ask questions ahead of consenting to be interviewed. Those who agreed to participate gave written informed consent ahead of the interview, including how data collected would remain confidential; the use of the audio recording; any data collected may be archived and used as anonymous data as part of subsequent research (also known as secondary data analysis); and the use of anonymous quotes in academic books, reports or journals.\n\n\nResults\n\nWe report themes and illustrative quotes from interviews and focus group participants, and insights from the PAG (each quote is tagged to indicate method of data collection and the participant’s role).\n\n(a) Advantages and disadvantages of measuring participant experience\n\nAll participants mentioned that they thought the standardised measurement of participant experience had potential to improve trial experience.\n\nIf you’re looking at the results of that patient experience questionnaire informing future behaviour and future trials, it would be invaluable, wouldn’t it? We get involved in the design, the application … (focus group, clinical trials unit staff).\n\nWhether this benefit was realised as the trial was running or in future trials was discussed. Some interviewees described the potential to use the experience data, if it highlighted the benefits of participation, to market future trials to potential participants and to promote trials generally.\n\nIt would be good if they do think that being in the trial has helped in anyway, possibly other than response, so added value, it would be great to know that so that we can gather a body of evidence to try to put that information out there for patients more generally. (PR8, interview, clinical research network staff).\n\nThe main concern raised from trial professional interviewees centred around burden to participants, particularly in situations that were stressful.\n\nThey’ve been through emotions of the medical treatment and the emotional trauma of having an early baby … You’ve got people like me who go up to them and say, ‘can we put the baby in a trial?’ … ‘Did they get it right?’ (PR4, interview, chief investigator of a running trial).\n\nThe majority of trial interviewees focused on feasibility, in terms of when would be the easiest to do it for the participant and the usefulness of the experience data captured.\n\nIf it’s a long trial, then some of these [items] would be perhaps partway through, but you’d have to vary the questions…. Close to the end of the trial, so you haven’t forgotten what happened (TP1, interview, previous trial participant, podiatry trial).\n\nThe interplay between the intervention, perception of benefit, and impact of receiving trial results was also a concern expressed by interviewees.\n\nIf they weren’t on that drug and saw the results of the trial that said, this drug worked and they didn’t get it, they may have had a good experience as far as being in research but seeing the fact that actually the drug that they didn’t get worked might influence how they put over their experience (PR8, interview, clinical research network staff).\n\n(b) Domains to be included in the measure\n\nCandidate domains were identified by the research team for inclusion, relating specifically to the different phases in the trial (which the PAG described as ‘the participant journey’).\n\n(b1) Early stage information provision and trial processes\n\nProfessionals were concerned that recall would make assessing information and processes occurring early on in the trial problematic if the measure was administered at the end of the trial. The majority of interviewees saw early stage processes as important to assess.\n\nWhat was the consent process there like? Did they receive enough information? Was the information understandable? So all the sort of things that would support that pre-participation decision-making … Were they allowed enough time to consider participation? (PR7, interview, funder of trials).\n\nTrial participants spoke of having too much information with complicated text, and wanted specific information to manage their expectations of participation.\n\nInformation given was easy to understand but perhaps a little over explained… I would help a trial regardless - but the cost needs to be thought about as it is someone’s time, especially if they have to come out of work to attend appointments. (TP14, interview, current trial participant, inflammatory bowel disease trial).\n\n(b2) Perception of conduct of trial processes\n\nHow a trial was conducted and the practical issues (e.g., waiting times and travel) was seen as important by all interviewees:\n\n… the procedures themselves, it’s saying what you’re doing, it’s all the aspects of the trial itself, how much burden was added onto the parents as a result of it, was it an emotional burden or do they have to do something burden, both of them have obviously got different meaning. (PR4, interview, chief investigator of trials).\n\n(b3) Sharing trial results with participants and perception of trial processes\n\nThere was discussion around whether measures should be administered before or after the sharing of trial results, and frustration described by participants when results were not shared by the trial.\n\n… the guy [trial staff] was really good at explaining everything, really, really good. Because putting all these things on your head is quite a big thing and extremely reassuring, which meant a lot. The only thing he did is he promised he would send me the results of my brain scan, because I was interested and he never did. I was disappointed and I never got the results of the study, which I was really interested to get. (TP12, interview, previous trial participant, mental health trial).\n\n(b4) Engagement with trial team\n\nThis domain relates to the communication between the team and the participant across the trial journey and the extent to which participants ‘feel a part’ of the trial and have confidence and trust in staff. Having a relationship with the trial team was viewed as vital to participants.\n\nAnd during the study, I think this is more about retention and what kind of negative or positive experiences would help people to remain in trials. So again, I think experiences around how they were engaged with the team would be quite useful (PR3, interview, clinical trials unit director).\n\n(b5) Perceived benefits of participation for participants\n\nThis theme specifically relates to the perceived benefits of participation other than satisfaction to increase understanding of why participation in trials is important.\n\nIt would be good if they do think that being in the trial has helped in anyway … It would be great to know that so that we can gather a body of evidence to try to put that information out there (PR8, interview, clinical research network staff).\n\n(b6) Perceived satisfaction with participation\n\nInterviewees discussed the utility of a question on overall satisfaction. Some interviewees and PAG members felt that it would be preferable to ask whether a participant would take part in the same trial again compared to asking about taking part in research more generally.\n\nI would agree with asking about taking part in another research study. About the experience of taking part… you know, had a good experience of taking part, would you participate in a future study (PR9, interview, clinical research network staff.\n\nSome trial professional interviewees referenced the assessment of patient experience of clinical care, which has become a mainstay of quality improvement in the NHS.\n\nWe do ask patients in the NHS about their satisfaction with the care received. We do that anyway so why is it any different? (PR8, interview, clinical research network staff).\n\n(c) Logistics of administering an experience measure\n\nResearch participants and PAG members raised a number of logistical considerations around delivering experience measures. These are summarised as a checklist (Table 4) with a brief summary presented here. The majority of interviewees felt that measuring experience in participants who are no longer in the trial (i.e. lost to follow-up or withdrawn) would provide important data on patient experience. The consensus was that the questionnaire needed to be easy to read and short (between 1-4, A4 sides, and take between 3-10 minutes to complete). A variety of modes were suggested (self-complete questionnaire: paper, electronic via a website or app). Different time-points were suggested by interviewees and can be broadly categorised as ‘during the trial’ or ‘end of trial’. There was no consensus as to which option was best, but flexibility was seen as key.\n\nThe cognitive testing highlighted issues around comprehension, recall and response to the candidate items. In summary, from 2 rounds of cognitive testing, we removed 30 items from the measure. We refined 46 items using respondents’ preferred terms to provide clarity and inserted examples grounded in participants’ experiences in 9 of these items, and 2 items were changed from statements to questions. The response options were enhanced to ensure the measure captured a range of trial experiences: binary responses were inserted in 9 items; graded responses were retained in 14 items; a ‘not applicable’ option was added to 6 response scales, a ‘no opinion’ to 4 scales; and free text was added to 4 items. Figure 1 summarises the development process of the measure, showing the domain and item refinement process throughout phase 1 and phase 2. 3 of 9 candidate domains were amalgamated and 3 were re-labelled.\n\nBy the end of the cognitive testing phase, consensus was reached among the research team that the final 6 core domains, and 25 items (including one final ‘full text’ response) captured a ‘meaningful’ trial participant experience. The short measure was subsequently developed based on those items viewed as preferable by respondents, with 1-9 items tapping each of the 6 domains, totalling 17 items. We have uploaded versions of the measure to the OSF repository.\n\n\nDiscussion\n\nThe study aimed to develop a useful measure of participant experience of trials. We describe a detailed development study including the views of both trial professionals and trial participants, with detailed cognitive testing to assess understanding and acceptability.\n\nThere are limitations to the study. First, we did not include participants in either the phase 1 or 2 sample who had not completed a trial they had been recruited to, either because they had withdrawn or were lost to follow-up. Our sample might be biased towards trial participants who may have had a more positive experience, although there were instances in the data where trial participants reported otherwise.\n\nMost of our participants were white (88%) indicating the need for further work to explore under-represented groups. This supports wider initiatives underway to support trialists to design and conduct inclusive trials.7 We did not formerly assess the readability of the items instead relying on a proxy indicator of educational level for health literacy, and we were also less successful in recruiting a diverse group of participants. The PAG provided an important opportunity to integrate patient insights into development, but all were known to the team through existing networks and had interests in the research topic.\n\nThe data were collected over 4 years ago, with publication delayed by the pandemic and other issues. Although we expect that many of the issues remain pertinent, changes in the delivery of trials, such as moves to more remote methods,23 would not have been captured.\n\nThe measurement of trial participant experience may be important for improving delivery of trials, exploring variation in experience in different sites and over time, and between groups within a trial. However, this will require feedback of the data and resulting change. Effective feedback processes (such as, guidance on how to interpret the results of the measure) needs to be co-produced with stakeholders to ensure it is acceptable and useful.24 It will also be important to assess whether potential disadvantages of participation are realised.\n\nOur measure has been piloted with three ongoing trials in the UK, with results reported in a forthcoming paper. Future work will also need to examine the factors that influence participant experience, and how much of the variation in experience is due to context (for example, condition experienced), trial type, participant characteristics, or aspects of the trial (for example, the intervention and trial procedures). It will also be important to explore the extent to which experience is relevant in all trials. For example, some trials have little active participation either because of the trial design (such as, cluster trials without individual consent) or because of the duration and extent of follow-up. We do not foresee any challenges translating the measure to a digital platform given there are substantial precedents for capturing patient experience and study experience data digitally.25 We report a feasibility study of the use of the measure elsewhere.\n\n\nConclusions\n\nBoth trial professionals and trial participants consider the standardised assessment of participant experience of trials important. In this paper, we have outlined the core domains that should be assessed to measure participant experience in trials and provided measures for further assessment.", "appendix": "Data availability\n\nThe agreements with trial teams did not provide for data sharing beyond the study team. If third parties wish to access the data, the corresponding author (PB) can contact the trial teams on behalf of those third parties, to negotiate an additional agreement for data sharing beyond the original scope of the study.\n\nOSF: PACT. https://doi.org/10.17605/OSF.IO/BCXH2. 26\n\nThis project contains the following extended data:\n\n- Patient experience of trials - short measure formatted.docx\n\n- Patient experience of trials - short measure unformatted.docx\n\n- Patient experience of trials - standard measure unformatted.docx\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\nAcknowledgements\n\nThe authors would like to thank everyone who took part in this study. We would like to specially thank the UK NIHR Clinical Research Network staff, as well as those at the Local Greater Manchester Clinical Research Network, and Eastern Clinical Research Network for help with recruitment. We would also like to thank the Research for the Future team for help with recruitment, and those who were involved in supporting the recruitment of professionals to the qualitative study.\n\n\nReferences\n\nTreweek S, Mitchell E, PitKethly M, et al.: Strategies to improve recruitment to randomised controlled trials. Cochrane Database Syst. Rev. 2010; Issue 1. Art. No.:MR000013. Publisher Full Text\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials. 2014; 15: 399. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 2: how to decide if a further Study Within A Trial (SWAT) is needed. Trials. 2020; 21(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 1: what is a Study Within A Trial (SWAT)? Trials. 2018; 19(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkea ZC, Treweek S, Gillies K: ‘It’s trying to manage the work’: a qualitative evaluation of recruitment processes within a UK multicentre trial. BMJ Open. 2017; 7(8): e016475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParkinson B, Meacock R, Sutton M, et al.: Designing and using incentives to support recruitment and retention in clinical trials: a scoping review and a checklist for design. Trials. 2019; 20(1): 624. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreweek S, Banister K, Bower P, et al.: Developing the INCLUDE Ethnicity Framework—a tool to help trialists design trials that better reflect the communities they serve. Trials. 2021; 22(1): 337. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrocker JC, Ricci-Cabello I, Parker A, et al.: Impact of patient and public involvement on enrolment and retention in clinical trials: systematic review and meta-analysis. BMJ. 2018; 363: k4738. Publisher Full Text\n\nNIHR Dissemination Centre: Improving care by using patient feedback. NIHR Themed Review. 2019. Reference Source\n\nPlanner C, Bower P, Donnelly A, et al.: Trials need participants but not their feedback? A scoping review of published papers on the measurement of participant experience of taking part in clinical trials. Trials. 2019; 20(1): 381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampbell J, Smith P, Nissen S, et al.: The GP Patient Survey for use in primary care in the National Health Service in the UK - development and psychometric characteristics. BMC Fam. Pract. 2009; 10(57). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoland M, Elliott M, Lyratzopoulos G, et al.: Reliability of patient responses in pay for performance schemes: analysis of national General Practitioner Patient Survey data in England. BMJ. 2009; 339(sep29_3): b3851. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDarbyshire J: The UK Clinical Research Network-building a world-class infrastructure for clinical research. Rheumatology. 2008; 47: 745. PubMed Abstract \| Publisher Full Text\n\nStreiner D, Norman G: Health Measurement Scales - a practical guide to their development and use. 1st ed.Oxford: Oxford Medical Publications; 1989.\n\nStaniszewska S, Brett J, Simera I, et al.: GRIPP2 reporting checklists: tools to improve reporting of patient and public involvement in research. BMJ. 2017; 358: j3453. Publisher Full Text\n\nWolf J, Niederhauser V, Marshburn D, et al.: Defining Patient Experience. Patient Exp. J. 2014; 1(1): 7–19. Publisher Full Text\n\nGrady K, Gibson M, Bower P: Can a ‘consent to contact’ community help research teams overcome barriers to recruitment? The development and impact of the ‘Research for the Future’ community. BMC Med. Res. Methodol. 2019; 19(1): 195. Publisher Full Text\n\nGlaser B, Strauss A: The discovery of grounded theory: strategies for qualitative research. Chicago: Aldine; 1967; 17. : 364. Publisher Full Text\n\nBeatty PC, Willis GB: Research Synthesis: The Practice of Cognitive Interviewing. Public Opin. Q. 2007; 71(2): 287–311. Publisher Full Text\n\nWillis GB: Cognitive interviewing: A tool for improving questionnaire design. Thousand Oaks, CA: Sage; 2005. Publisher Full Text\n\nTourangeau R: Cognitive sciences and survey methods. Cognitive Aspects of Survey Methodology: Building a Bridge Between Disciplines. Jabine T, Straf M, Tanur J, et al., editors. Washington: National Academy Press; 1984; 73–100.\n\nBoeije H, Willis G: The Cognitive Interviewing Reporting Framework (CIRF): Towards the Harmonization of Cognitive Testing Reports. Methodology. 2013; 9(3): 87–95. Publisher Full Text\n\nMasoli JAH, Down K, Nestor G, et al.: A report from the NIHR UK working group on remote trial delivery for the COVID-19 pandemic and beyond. Trials. 22: 911. Reference Source\n\nFoy R, Skrypak M, Alderson S, et al.: Revitalising audit and feedback to improve patient care. BMJ. 2020; 368: m213. Publisher Full Text\n\nSanders C, Nahar P, Small N, et al.: Health Services and Delivery Research. Digital methods to enhance the usefulness of patient experience data in services for long-term conditions: the DEPEND mixed-methods study. Southampton (UK): NIHR Journals Library;\n\nBower P: PACT. OSF. November 22, 2023. Publisher Full Text" }	[ { "id": "287335", "date": "10 Jun 2024", "name": "Timothy Pickles", "expertise": [ "Reviewer Expertise Psychometrics", "Statistics", "Clinical Trials" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMethods Study design What does standardized mean? Not defined. What are trial professionals? Not defined in this section. No mention of COSMIN guidelines for evidence of content validity. The following should be referenced as a minimum and detail should be provided about which aspects of content validity are covered by this research. User manual: https://www.cosmin.nl/wp-content/uploads/COSMIN-methodology-for-content-validity-user-manual-v1.pdf Reference: (Terwee et al.,2018)(Ref-1) Please provide COREQ reporting checklist https://cdn.elsevier.com/promis_misc/ISSM_COREQ_Checklist.pdf and add as supplement, providing all the details specified by the checklist in the manuscript Phase 1: measurement development Content of topic guides not mentioned. Assuming from results that this was much wider than just a discussion of the domains and items within domains. Provide as supplement and summarise in manuscript. Great that you have uploaded versions to osf but there should be 5 posted here to reflect all stages and versions shown in Figure 1. There are only 3. One has 16 items so I’m not sure which this is, though maybe it is the short measure listed in Figure 1 as having 17 items. I think another is the standard measure, which has 24 items but Figure 1 says 25 items. A third seems to be the short version again. Data collection Why are the demographics of the focus group and the 11 professionals not important? Should be in Table 2 and described, or a justification made for not reporting. Results Phase 2: cognitive testing Having all versions on osf would allow the reader to assess the progression from start to finish. Please upload all. I would not suggest calling the final full text question an item. There needs to be a differentiation between what would be analysed quantitatively and qualitatively. I would expect to see some results here around comprehensiveness, comprehensibility and relevance (as defined by COSMIN). I would expect that there was discussion of wording, response option and recall period that would be interesting to see in these results. There is no mention of how round 1 informed round 2 Discussion This is very short. There is no mention of the items in the final two versions and whether they could work in all trial situations. There is no discussion of the important logistical issues brought up in Table 4. There is no mention of how data from measure collected from trials will be analysed There is no mention if the measures will just be implemented as they are or if anything else collected alongside the measures or if more interviews will be undertaken Figure 1 Cognitive testing spans phase 1 and phase 2 Split into 2 rounds of cognitive interviews to give numbers by rounds. Figure suggests no decisions were made between two rounds. Show PAG differently if providing input in every phase at all stages. Show where in the process this input was If feasibility testing not part of this research then remove Table 1 Suggest GRIPP-SF as supplement Table 2 Acronyms in footnote not complete Table 3 Acronyms in footnote not complete\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "287337", "date": "19 Jul 2024", "name": "Elizabeth Pellicano", "expertise": [ "Reviewer Expertise Expertise in patient and public involvement", "community perceptions of research", "autism research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study sought to develop a measure to capture participants’ experiences of taking part in research trials. I very much appreciated the premise of this study. This could be a really useful tool for researchers. The degree and nature of patient and public involvement (PPI), and how PPI was reported (using GRIPP-2), was also commendable.\nI have only a few comments for the authors to consider.\nFirst, the Patient Advisory Group (PAG) seemed to be involved quite extensively, “into all stages of the project” (p. 3), suggesting that they made a significant contribution to the study. Could the authors please confirm that the PAG members are also authors on this paper? I couldn’t tell from the author list or the Credit author statement. If they aren’t, it would be helpful to know why they aren’t.\nThird, the authors reported the focus group and interview participant characteristics for both Phases in Tables 2 and 3. I am concerned that including such personal information (or ‘special category data’ in GDPR-speak, e.g., ethnicity) at the individual level could make participants identifiable. In order to maintain participants’ confidentiality, I suggest collapsing this information and presenting it for each phase at the group level in the text. The authors have done this already on page 5, but more information could be provided (e.g., by reporting % of male and female participants, and % of all reported ethnicities, not just white).\nOther comments:\np. 3. The authors note that “most trials already have extensive platforms to measure participant experience of the intervention (e.g., quality of life and quality of care)” but no references are provided to substantiate this claim. This is important because this statement certainly does not hold in my own research field. p. 5. Please provide information about the duration of interviews and focus groups (mean/median and range).\n\nIs the work 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\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-78
https://f1000research.com/articles/12-66/v1	16 Jan 23	{ "type": "Research Article", "title": "Identification of the SIRT1 gene's most harmful non-synonymous SNPs and their effects on functional and structural features-an in silico analysis", "authors": [ "Desy Thayyil Menambath", "Usha Adiga", "Tirthal Rai", "Sachidananda Adiga", "Vijith Shetty", "Desy Thayyil Menambath", "Tirthal Rai", "Sachidananda Adiga", "Vijith Shetty" ], "abstract": "Introduction: The sirtuin (Silent mating type information regulation 2 homolog)1(SIRT1) protein plays a vital role in many disorders such as diabetes, cancer, obesity, inflammation, and neurodegenerative and cardiovascular diseases. The objective of this in silico analysis of SIRT1's functional single nucleotide polymorphisms (SNPs) was to gain valuable insight into the harmful effects of non-synonymous SNPs (nsSNPs) on the protein. The objective of the study was to use bioinformatics methods to investigate the genetic variations and modifications that may have an impact on the SIRT1 gene's expression and function. Methods: nsSNPs of SIRT1 protein were collected from the dbSNP site, from its three (3) different protein accession IDs. These were then fed to various bioinformatic tools such as SIFT, Provean, and I- Mutant to find the most deleterious ones. Functional and structural effects were examined using the HOPE server and I-Tasser. Gene interactions were predicted by STRING software. The SIFT, Provean, and I-Mutant tools detected the most deleterious three nsSNPs (rs769519031, rs778184510, and rs199983221). Results: Out of 252 nsSNPs, SIFT analysis showed that 94 were deleterious, Provean listed 76 dangerous, and I-Mutant found 66 nsSNPs resulting in lowered stability of proteins. HOPE modelling of rs199983221 and rs769519031 suggested reduced hydrophobicity due to Ile 4Thr and Ile223Ser resulting in decreased hydrophobic interactions. In contrast, on modelling rs778184510, the mutant protein had a higher hydrophobicity than the wild type. Conclusions: Our study reports that three nsSNPs (D357A, I223S, I4T) are the most damaging mutations of the SIRT1 gene. Mutations may result in altered protein structure and functions. Such altered protein may be the basis for various disorders. Our findings may be a crucial guide in establishing the pathogenesis of various disorders.", "keywords": [ "SIRT1", "nsSNP", "bioinformatics", "protein modelling" ], "content": "Introduction\n\nSirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that regulate transcriptional activity intracellularly. They are present in a wide range of tissues, such as the adipose, kidney, brain, liver, and muscle tissues.1,2 SIRT1 (Silent mating-type information regulation 2 homolog 1) is known to regulate a variety of cellular processes, including lipid and glucose metabolism, stress tolerance, autophagy, circadian rhythms, and mitochondrial biogenesis, according to several studies.3–5 SIRT1 gene expression modulates its downstream pathways in diabetes, cancer, obesity, inflammation, and neurodegenerative and cardiovascular diseases by focusing on numerous cellular proteins, including nuclear factor-κB (NF-κB), endothelial nitric oxide synthase (eNOS), forkhead transcriptional factors (FoxOs), AMP-activated protein kinase (AMPK), protein tyrosine phosphatase (PTP). However, considerable evidence suggests that in a variety of malignant cell types, SIRT1 is upregulated and that SIRT1 antagonists prevented the development of cancer cells.6–8\n\nSingle nucleotide polymorphisms (SNPs) are variations in the DNA sequence that result from the alterations in a single nucleotide (A, T, C, or G). Around 90% of human genetic variation is made up of SNPs. The three-billion-base human genome has SNPs at every 100–300 bases, with varying densities between regions.9 The genome's coding and noncoding sections can both present SNPs. SNPs can have a wide spectrum of effects on how cells behave, from having no effect to causing disease or altering the reaction to a drug. Since they are responsible for about half of the genetic differences associated with human hereditary diseases, non-synonymous SNPs (nsSNPs) that result in an amino acid residue substitution in the protein product are also of high relevance.10 There may be effects on transcription factor binding, splicing, or gene expression from coding synonymous SNPs (sSNPs) and SNPs that aren't in the gene promoter or coding regions.11,12\n\nHuman reactions to viruses, medications, vaccinations, and other agents are significantly influenced by SNPs. SNPs are therefore useful in biomedical research, the creation of pharmaceutical products, the improvement of medical diagnostics, and the application of personalised medicine.13 SNPs are responsible for specific phenotypes and therefore it is very important to identify them. This is a difficult task as it necessitates repeatedly evaluating thousands of SNPs in candidate genes. Selecting a group of SNPs for a study to determine the role of an SNP in a disease is a challenging endeavour; in these situations, a bioinformatics tool may be very helpful to distinguish between neutral and functional SNPs. They might also show the structural underpinnings of the mutations. These bioinformatics applications are used to assess the SNPs’ functional significance.\n\nTo find the SIRT1 protein's most dangerous nsSNPs, we applied bioinformatics techniques. We hypothesised that SIRT1 protein would be harmful because of nsSNPs on the gene. This is the first study of its kind for the SIRT1 gene to include both protein structure prediction and mutation analysis.\n\n\nMethods\n\nMultiple steps were used to complete the study. The figure below shows the equipment used to complete the task (Figure 1).\n\nThe NCBI SNP database was accessed. Information on the entire SIRT1 gene, including its nsSNPs, was obtained. As the query sequence, filtered nsSNPs from the dbSNP database were examined. The NCBI Protein accession IDs NP_ 036370.2, NP_001135970.1, and NP_002294.2 for the SIRT1 gene were used.\n\nSIFT, Provean, and I-Mutant software were used to identify the impact of spotted nsSNPs on the SIRT1 gene.\n\nSIFT (Sorting Intolerant from Tolerant) server\n\nThe SIFT server is a web-based bioinformatics tool that forecasts the detrimental effects of nucleotide substitution and frame shift (insertion/deletion) on protein function based on the degree of amino acid residue maintenance in sequence alignments obtained from highly associated sequences, with the primary assumption that mutations in evolutionarily conserved regions primarily affect its function.14 The distinct input data order for the SIFT server includes protein sequence, chromosome location, and dbSNP reference number. SNPs and Indels were separated from the overall number in order to use this tool, and they were provided with the chromosome positions for frame shift indels and the residue number (rs) ID numbers for missense, nonsense, and stop gain SNPs. Each residue was given a value from 0 to 1 by the SIFT server, with scores below 0.05 indicating detrimental amino acid changes and scores above 0.05 indicating tolerance.15 The website hosts SIFT version 5.2.2.\n\nProvean\n\nA protein's biological activity may be impacted by an amino acid substitution or indel, according to predictions made by the programme Protein Variation Effect Analyzer (PROVEAN). When filtering sequence variants, PROVEAN is useful for locating nonsynonymous or indel variations that are anticipated to be functionally significant.16 The tool takes as input a protein sequence and several amino acid combinations, runs a BLAST search to find related sequences (supporting sequences), and outputs PROVEAN scores. The interpretation was done using the score thresholds. The default threshold is -2.5, meaning that variants with a score of -2.5 or less are deemed harmful, whereas variants with a score of -2.5 or more are considered neutral. http://provean.jcvi.org/index.php can be visited to access Provean.\n\nI-Mutant 2.0\n\nI-Mutant 2.0 was used in the investigation to analyse the stability of the targeted SIRT1 protein. This website server estimates any mutation-related changes to protein stability.17 By adjusting the pH to 7 and the temperature to 25°C, this technique was used to analyse the SIRT1 protein sequences. It gives the opportunity to forecast how the protein's stability will be altered in response to single-site changes in the protein's structure or sequence. The design of the I-Mutant outcome is as follows: Free energy change value; Delta Delta G (DDG)= 0 is neutral, DDG > 0 is an increase in stability, and DDG <0 is a reduction in stability (I-Mutant website)\n\nTo understand the effect of nsSNPs on the SIRT1 protein structure, the study used Polyphen, HOPE and I-Tasser software. The three most deleterious and damaging nsSNPs of the SIRT1 gene from each of its isoforms were chosen and processed to examine their structural and functional effects on SIRT1 protein.\n\nPolyphen\n\nUsing simple physical and comparative considerations, polymorphism phenotyping v2(PolyPhen) is a method that estimates the potential effects of an amino acid substitution on the structure and functionality of a human protein.18\n\nHOPE modelling\n\nThe HOPE server analyses mutations automatically and can show the structural repercussions of a mutation. In addition to predictions from DAS services, sequence annotations from the UniProt database and calculations on the 3D coordinates of the protein using WHAT IF Web services are just a few of the data sources that HOPE uses to compile its information.19\n\nIterative Threading ASSEmbly Refinement (I-Tasser)\n\nI-Tasser is a software package for protein structure and function modelling. The Template modelling score was used to compare the wild and mutant models. The estimated values of root mean square deviation (RMSD) and melting temperature (TM) allowed for the precise determination of similarity score. According to statistics, a TM-score of 0.17 or less indicates that two randomly chosen structures from the Protein DataBank library are comparable, while a score of 0.5 or more indicates that two structures have a similar topology. Studies have demonstrated a clear correlation between a high level of RMSD value and a high amount of change between wild-type and mutant.20,21 The harmful mutations were then introduced into I-Tasser by adhering to their values.22–24 Chimera 1.11 was used to study the molecular characteristics and interactive visualisation of the final protein structure.25\n\nManaging protein interactions is essential for maintaining the system's homeostasis. STRING's task is to display the total score of interaction genes. In this stage, SIRT1 served as the input target gene, and analysis was completed.\n\n\nResults and discussion\n\nThe number of SNPs of the SIRT1 gene obtained from the NCBI database were 15,865. Three isoforms for SIRT1 were found (isoform a, isoform b and isoform c). Isofrom a (NP_036370.2) comprised 597 nsSNPs, isoform b (NP_001135970.1) comprised a total of 330 nsSNPs and isoform c (NP_001300978.1) 331 nsSNPs. The diagrammatical depiction is shown in Figure 1. The nsSNPs listed in all the isoforms are listed and the duplicates were deleted; the total number of nsSNPs included in all the isoforms was 252 (Table 1).\n\nThe following bioinformatics tools have provided the supplied data to further detect the influence of 252 nsSNPs on the structure and function of the SIRT1 gene. Because the resulting values were lower than the Tolerance Index (0.05), the SIFT software revealed 94 nsSNPs to be intolerant (Table 1).\n\nProtein stability changed depending on which amino acid was substituted and 216 nsSNPs demonstrated a decline in stability based on DDG value received from I-Mutant server (Table 1).\n\nPROVEAN identified 77 nsSNPs as having a negative impact since the final score of the variations was lower than the specified value of threshold (-2.5).\n\nStructural and functional effect of nsSNPs\n\nI-Mutant predicted the three (3) nsSNPs which played a role in decreasing SIRT1 stability (from each isoform - rs778184510, rs76519031, rs199983221), and they were selected for finding the impact of substitution of amino acid on structure and function of human protein (using Polyphen) and for the comparison of protein model (using I-Tasser). To generate the SIRT1 protein structure, SIRT1 protein sequences, single amino acid from the wild type, and mutations were uploaded to I-Tasser, which is the most accurate and sophisticated technique for predicting protein structure (Figure 2). Then, using this technique, five models for each SIRT1 mutation and protein were produced.\n\nWhen the native structure is known, TM score and RMSD can be used to compare the structural similarity of two structures.26 The proposed TM score is supposed to solve the RMSD issue, which is prone to local errors. A local error (such as a mismatched tail) will raise the RMSD score even if the overall topology is good, since the RMSD measures the average distance between all residue pairs between two structures. The TM-score is insensitive to the local modelling error, nevertheless, because the short distance is weighted more severely than the long distance. A model with a proper topology is indicated by a TM-score and IT greater than 0.5, while a random similarity is indicated by a TM-score & IT less than 0.17 (Table 2). These cut-offs are independent of the length of the protein.\n\nBy calculating a confidence score, or C-score, I-Tasser evaluates the accuracy of anticipated models. The convergence parameters from simulations of the structure assembly and the significance of threading template alignments are used to make this determination. A model with a high level of confidence also has a higher C-score. The C-score typically ranges from (-5,2).\n\nIsoleucine turned into threonine in position 4. The mutant residue was more compact than the wild-type residue. The mutant residue was also less hydrophobic than the wild-type residue. The mutation caused the hydrophobic contacts in the protein's core to disappear.\n\nAn overview of the protein is also displayed in the ribbon presentation (Figure 3a). Additionally, there are five detailed pictures of the mutation site (Figure 3b).\n\nThe protein is coloured grey, the side chains of both the wild-type and the mutant residue are shown and coloured green and red respectively. (I4T).\n\nIn this case, isoleucine turns into serine at position 223. The mutant residue was more compact than the wild-type residue. The mutant residue was less hydrophobic than the wild-type residue. This could lead to a lack of interactions with the other genes The mutation may cause the proteins' surface-bound hydrophobic interactions with other molecules to disappear.19\n\nAn overview of the protein is also displayed in the ribbon presentation (Figure 4a). There are also five enlargements of the mutation location (Figure 4b).\n\nThe protein is coloured grey, the side chains of both the wild-type and the mutant residue are shown and coloured green and red respectively (I223S).\n\nThe mutant residue was smaller than the wild-type residue in this instance because alanine has replaced aspartic acid at position 357. In contrast to the wild-type residue charge, which was negative, the mutant residue charge was neutral. The mutant residue was more hydrophobic than the wild-type residue. The wild-type residue is expected to be located in its preferred secondary structure turn, according to the Reprof programme. The local conformation would only be slightly unstable since the mutant residue prefers a different secondary structure. The mutation places a more hydrophobic residue here. Hydrogen bonds may break as a result of this, and it may also prevent correct folding.\n\nSTRING revealed the physical interactions between SIRT1 and other genes in the gene's interactions. In its pathways, it interacted with NFKB1, NFKB1A, DDX5, AURKA, BARD1, RPA1, UBEBA, ARNTL,CLOCK,CRY1, PPARGC1A, FOXO1, FOXO3, RELA, MYOD1, SUV39H1, MDM2, EP300, PPARG and TP53 (Figure 5). The query proteins and the initial line of SIRT1's interaction are represented by coloured nodes on the picture. White nodes are the second interactional shell. Protein-protein interactions are represented by edges. The edges of the known interactions are blue and pink. Others illustrate the predicted interplay between proteins. TP53 is more connected to and interdependent with SIRT1 than any other interaction on the list.\n\nNumerous studies have been done in the past to determine the connection between the SIRT1 gene's polymorphism and a number of conditions, such as cancer, inflammation, obesity, diabetes, and cardiovascular and neurological illnesses. The most harmful nsSNPs in the SIRT1 gene that may be crucial in the development of certain disorders have been explored in this work.\n\nThe SIRT1 gene has 252 nsSNPs, according to our findings. The present study's SIFT findings revealed that the SIRT1 protein contains 94 harmful nsSNPs, 66 of which are detrimental as indicated by PROVEAN.\n\nProvean scores were -4.873, -4.47, -4.39 for rs778184510, rs769519031 and rs199983221 respectively which were higher compared to other SNP’s and were chosen from each isoform of SIRT1 protein. These three nsSNPs, which cause high risk of altering normal functioning of SIRT1 gene, were selected for further evaluation based on the I-Mutant value, from each isoform of the SIRT1 protein.\n\nD357A, I223S, and I4T’s respective Polyphen2 scores, which range from 0 to 1, were 0.983, 0.997, and 1.00, respectively; all three were classified as having probable damage by Polyphen2. Protein structure and functional activity depend on protein stability.27 Thus, I-Mutant, which was used to assess the stability of protein, demonstrated the protein stability for D357A, I223S, and I4T as -2.58, -2.25 and -2.2, respectively, as the lowest values. Thus, these three SNPs affect the function and structure of SIRT1 protein.\n\nBy determining the RMSD values and TM scores for each mutant model, we expanded our analysis. While RMSD aids in calculating the average distance between the carbon backbones of wild and mutant models, the TM score is utilised to assess the topological similarity between wild- and mutant-type models.20,21 The mutant model D357A demonstrated a greater RMSD value, which had a greater deviation from the wild type compared to the other two mutant models. To further establish the detrimental impacts of these nsSNPs, the SIRT1 protein structure was determined using I-Tasser, and the protein's FASTA sequence served as the sole input. Using I-Tasser, the prototypes are acquired, and the protein simulation is carried out. Following the introduction of the mutant models to the HOPE server, the server generated the effects of mutations on the contacts and the structural placement.\n\nMutations can affect a protein's stability, structure, and ultimately, function. Mutations are components of the \"raw material\" of evolution. The majority of, if not all, protein mutations are eliminated by negative, purifying selection, which lowers the probability of subsequent adaptations.\n\nBecause of this, under the influence of positive selection, only a small portion of all potential mutations will be resolved to take on a new function. Randomness or \"neutral drift\" might theoretically cause neutral mutations to randomly correct in small populations. At the level of the organism, the consequences of mutations on fitness are complicated and seldom ever correlate to the characteristics of a single gene or protein. Several levels of redundancy, resilience, and backup decrease the impact of numerous mutations. Understanding and predicting the impact of mutations at the organismal level present important problems for evolutionary biology.28,29\n\nThe stability of the proteins is influenced by the quantity of functional protein present. According to previous research, stability and folding effects are responsible for 80% of the negative consequences of pathogenic mutations.30 Protein dysfunctionalization is mostly caused by mutations that reduce the amount of soluble, functional proteins over a specified threshold (or DDG value).30 Experimental studies on a variety of proteins indicated that between 33 and 40% of the time, a detrimental mutation is likely to occur.29 As mutation rates increase, protein fitness therefore substantially decreases. When five mutations are introduced into a protein, its fitness is decreased by 20%.\n\nProtein evolution rates, and maybe even the rates at which entire organisms evolve, seem to be primarily (though surely not solely) influenced by stability,31,32 particularly but not entirely in connection with the acquisition of new functionalities. Stability appears to be the primary (though surely not the only) driver of how rapidly proteins change, despite the fact that a protein's starting stability might mitigate some of the destabilising effects of mutations.\n\nFor a small number of proteins, experimental datasets are frequently made accessible, and they generally focus on changes in mutation thermodynamic stability (DDG values). Recent developments in computing have made it possible for researchers to predict the DDG values of certain protein mutations. Some prediction methods strongly rely on sequence, whereas others mostly rely on three-dimensional structures.33,34\n\nNew protein functions cannot be developed because of the destabilising impact of mutations. Neutral or non-adaptive mutational drifts have been found to be less disruptive and to occur frequently at buried residues as compared to new function or adaptive mutations.35\n\nThe mutant study shows the decreased thermodynamic stability of the proteins, regardless of whether SIFT and Provean examinations of SNPs in the leptin and leptin receptor genes suggest that they are detrimental or tolerated. This might have an impact on how leptin and leptin receptor proteins function. This conclusion supports previous studies linking leptin, leptin gene polymorphisms, and the incidence of depression in obese individuals.\n\nDespite several studies relating SNPs in different genes to a number of disorders, computational analysis of the functional effects of SNPs in SIRT1 is still lacking. To determine whether an amino acid change will have an impact on protein function, the SIFT technique examines sequence homology across related genes and domains across evolution. The physical-chemical properties of the residues of amino acids are also considered. According to estimates, SIFT has error rates of 31% and 20% for false negatives and positives, respectively. When amino acid changes are used as the test set, SIFT is roughly 80% effective in benchmarking trials and is thought to significantly reduce the residual activity of the variant protein.\n\nHowever, utilizing SIFT and Provean, it is now feasible to analyse gene polymorphisms and forecast how a mutation will alter a protein's functionality. Since most disease mutations have an effect on protein stability, I-Mutant assessed the stability of the mutant proteins.\n\nTo find, characterize, validate, and predict the functional consequences of harmful non-synonymous SNPs (nsSNPs) in the interleukin-8 gene, Dakal et al. carried out a comparable.36\n\nIt may also be deduced that all three of the SIRT1 gene's most harmful nsSNPs eventually interfere with and disrupt the normal function of other expressive genes. Based on their interaction patterns and their correlation profiles with numerous diseases and their pathways, SIRT1 is involved in pathways with genes such as NFKB1, NFKB1A, DDX5, AURKA, BARD1, RPA1, UBEBA, ARNTL, CLOCK, CRY1, PPARGC1A, FOXO1, FOXO3, RELA, MYOD1, SUV39H1, MDM2, EP300, PPARG and TP53, which, therefore indicate its importance.37\n\n\nConclusions\n\nThe SIRT1 protein is essential for the development of numerous diseases, including cancer, inflammation, diabetes, obesity, and cardiovascular and neurological conditions. Therefore, to elucidate this domain’s function, studying its structural conformation is crucial. This in silico examination of the functional SNPs in SIRT1 sheds light on the possible damage that the nsSNPs could do to the protein. In this work, it was predicted for the first time how nsSNPs will affect the structure and function of the SIRT1 protein. The three nsSNPs (D357A, I223S, and I4T) in the SIRT1 gene were the most detrimental alterations, according to our research. Our findings will be a crucial reference point for research into prospective diagnostic and therapeutic approaches, because this protein has been linked to several disorders, necessitating large-scale clinical trials and investigations based on experimental mutational validation.\n\n\nAuthor contributions\n\nDesy TM and Usha Adiga designed the research, performed softwares and wrote the manuscript; Tirthal Rai and Sachidananda Adiga, Vijith Shetty revised the manuscript and involved in data analysis. All authors read and approved the fnal manuscript.", "appendix": "Data availability\n\nBiostudies: Identification Of The SIRT1 Gene's Most Harmful Non-Synonymous SNPs And Their Effects On Functional And Structural Features- An Insilico Analysis; Accession number: S-BSST944. https://identifiers.org/biostudies: S-BSST944\n\nThis project contains the following underlying data\n\n- Table 1: SIFT, I mutant analysis, Provean for the nsSNPs of SIRT1 Gene\n\n- Figure 1: Sorting of nsSNPs of SIRT1\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nCohen HY, Miller C, Bitterman KJ, et al.: Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science. 2004 Jul 16; 305(5682): 390–392. PubMed Abstract \| Publisher Full Text\n\nYang T, Fu M, Pestell R, et al.: SIRT1 and endocrine signaling. Trends Endocrinol. Metab. 2006 Jul 1; 17(5): 186–191. 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Signal. 2011 Dec; 9(1): 1–8." }	[ { "id": "162701", "date": "21 Feb 2023", "name": "Laxminarayana Kurady Bairy", "expertise": [ "Reviewer Expertise My area of research includes pharmacogenomics", "wound healing and neurogenerative 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\nThe article is well presented. The aims and objectives are clearly mentioned.\n\nThe introduction part is well written. However, the cited references are before 2016. In fact, the majority are before 2010. There is no update on the recent developments in the field. Hence, the introduction needs to be rewritten with recent references.\n\nThe study design is appropriate and the work is technically sound. The methodology used is clear, described in detail and is enough for anyone to repeat the experiment. The statistical method is appropriate.\n\nThe source data is not available in the manuscript provided. However, it is not customary to give sorce data in the manuscript. Most of the journals do not ask for source data. However, if it is required, editorial can ask for the same.\n\nThere is some ambiguity in the conclusion drawn. It is a bit vague. This needs to rewritten to make it more lucid.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9434", "date": "03 Apr 2023", "name": "Usha Adiga", "role": "Author Response", "response": "Introduction will be modified with recent references. Conclusion will be modified." } ] }, { "id": "224496", "date": "19 Dec 2023", "name": "Bhushan L. Thakur", "expertise": [ "Reviewer Expertise SIRT1 biology", "DNA replication", "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 study provides a comprehensive analysis of single nucleotide polymorphisms (SNPs) in the SIRT1 protein, identifying the three most deleterious non-synonymous SNPs from the NCBI database. The objective of this research is to identify potentially harmful nsSNPs for SIRT1, which may potentially impact disease biology. The study is executed and written well.\nBelow are suggested changes to improve clarity and structure:\nThe introduction should include citations to the most recent literature. The methods section provides a concise outline of the collection and analysis of non-synonymous SNPs from the dbSNP site using bioinformatic tools. However, to enhance transparency and reproducibility, the authors should include additional details about the specific criteria or thresholds used to select deleterious nsSNPs. In Figure 1, the method flowchart seems to indicate that the authors used SIFT, Provean, and I-Mutant sequentially to identify the final 66 nsSNPs. However, based on the method description and Table 1, it appears that they utilized commonality between high-score predictions from the three tools to identify the top three hits. The authors should clarify this in the method to reduce ambiguity and make necessary changes in Figure 1. Based on Table 1 and the author's selection criteria for SNP selection, it is unclear why the authors did not shortlist the following three SNPs: D83G (rs17855430), P116T (rs775426483), and G52E (rs757804740). The authors should consider including the variants, G52D (rs757804740) and G52E (rs757804740), in the current manuscript or explain their exclusion. The conclusion succinctly summarizes the most damaging mutations, if possible authors are encouraged to explicitly state the potential implications of these findings for understanding the pathogenesis of various disorders, any reports that cite identification of these SNP’s, and connecting them back to the broader significance mentioned in the introduction.\nMinor comments - Correct the typo on page 5: \"Isofrom a (NP_036370.2) comprised 597 nsSNPs, \" should be isoform.\n- Verify the discrepancy on page 16: \"PROVEAN identified 77 nsSNPs as having…\" (Figure 1 shows 76).\n- Correct the typo on page 16: \"..rs778184510, rs76519031, rs199983221)…\" should be rs769519031.\n- Add a key for line colors connecting proteins in Figure 5.\n- Verify and provide a reference or criteria for the claim about I-Tasser in the statement: \"...mutations were uploaded to I-Tasser, which is the most accurate and sophisticated technique for predicting protein structure (Figure 2).\" Consider modifying it to \"...one of the most...\" if needed. -Please clarify, what is “E” in Table 1 in DDG column.\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? 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": [ { "c_id": "10882", "date": "19 Jan 2024", "name": "Usha Adiga", "role": "Author Response", "response": "Comment 1: The introduction should include citations to the most recent literature. Answer: The latest references will be included respectively. Comment 2: The methods section provides a concise outline of the collection and analysis of non-synonymous SNPs from the dbSNP site using bioinformatic tools. However, to enhance transparency and reproducibility, the authors should include additional details about the specific criteria or thresholds used to select deleterious nsSNPs. Answer: All three tools provide valuable insights into the functional consequences of genetic variations, their methodologies and focuses differ. SIFT predicts the functional impact of amino acid substitutions based on sequence conservation. It evaluates how well a particular amino acid is conserved across different species. PROVEAN predicts the impact of protein sequence variations using a combination of sequence homology and structural information. It considers the alignment of the query sequence with homologous sequences and the predicted structure changes caused by the variation. I-Mutant focuses on predicting the impact of amino acid substitutions on protein stability. It employs an energy-based approach, evaluating changes in free energy upon mutation to predict whether a mutation destabilizes or stabilizes the protein structure. Comment 3: In Figure 1, the method flowchart seems to indicate that the authors used SIFT, Provean, and I-Mutant sequentially to identify the final 66 nsSNPs. However, based on the method description and Table 1, it appears that they utilized commonality between high-score predictions from the three tools to identify the top three hits. The authors should clarify this in the method to reduce ambiguity and make necessary changes in Figure 1. Answer: In the SIRT1 gene, three isoforms were identified, resulting in a total of 252 identified Single Nucleotide Polymorphisms (SNPs). Among them, SIFT software identified 94 non-synonymous SNPs (nsSNPs) as intolerant (using a cutoff value of <0.05). Subsequently, these 94 intolerant nsSNPs underwent Provean analysis, revealing 67 nsSNPs as harmful (with a cutoff value of <-2.5). The selected 67 nsSNPs then underwent I-Mutant analysis, which indicated 58 nsSNPs with decreased stability (using a cutoff value of 0). From this subset, we selected three nsSNPs from each isoform, respectively, based on decreasing protein stability. The figure will be modified accordingly. Comment 4: Based on Table 1 and the author's selection criteria for SNP selection, it is unclear why the authors did not shortlist the following three SNPs: D83G (rs17855430), P116T (rs775426483), and G52E (rs757804740). The authors should consider including the variants, G52D (rs757804740) and G52E (rs757804740), in the current manuscript or explain their exclusion. Answer: We have chosen one non-synonymous Single Nucleotide Polymorphisms (nsSNPs) from each of the three isoforms, specifically focusing on those identified as more damaging and exhibiting decreased stability. Comment 5: The conclusion succinctly summarizes the most damaging mutations, if possible authors are encouraged to explicitly state the potential implications of these findings for understanding the pathogenesis of various disorders, any reports that cite identification of these SNP’s, and connecting them back to the broader significance mentioned in the introduction. Answer: There is no existing literature on the specified SNPs of SIRT1 gene. Minor comments - Correct the typo on page 5: \"Isofrom a (NP_036370.2) comprised 597 nsSNPs, \" should be isoform. Answer: Corrected - Verify the discrepancy on page 16: \"PROVEAN identified 77 nsSNPs as having…\" (Figure 1 shows 76). Answer: corrected the number of SNPs after rechecking to 67 - Correct the typo on page 16: \"..rs778184510, rs76519031, rs199983221)…\" should be rs769519031. Answer: Corrected - Add a key for line colors connecting proteins in Figure 5. Answer: The explanation will be included in the text. In evidence mode, an edge may be drawn with up to 7 differently colored lines - these lines represent the existence of the seven types of evidence used in predicting the associations. Red lines indicate the presence of fusion evidence, green lines represent neighborhood evidence, blue lines suggest cooccurrence evidence, purple lines correspond to experimental evidence, yellow lines denote text mining evidence, light blue lines signify database evidence, and black lines represent co-expression evidence. - Verify and provide a reference or criteria for the claim about I-Tasser in the statement: \"...mutations were uploaded to I-Tasser, which is the most accurate and sophisticated technique for predicting protein structure (Figure 2).\" Consider modifying it to \"...one of the most...\" if needed. Answer: The statement is corrected accordingly. -Please clarify, what is “E” in Table 1 in DDG column. Answer: E represents error from the analysis report. Thank you for the valuable comments. All the corrections and suggestions will be incorporated in the manuscript accordingly." } ] } ]	1	https://f1000research.com/articles/12-66
https://f1000research.com/articles/13-77/v1	18 Jan 24	{ "type": "Data Note", "title": "A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence", "authors": [ "Vera Ruíz Moleón", "Maryam Fotouhi", "Charles Alende", "Riham Ayoubi", "Logan M. Bedford", "Kathleen Southern", "Timothy I. Richardson", "Carl Laflamme", "NeuroSGC/YCharOS/EDDU collaborative group", "ABIF consortium", "Vera Ruíz Moleón", "Maryam Fotouhi", "Charles Alende", "Riham Ayoubi", "Logan M. Bedford", "Kathleen Southern", "Timothy I. Richardson" ], "abstract": "Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.", "keywords": [ "Uniprot ID P16885", "PLCG2", "PLC-gamma-2", "PLC-γ2", "Phosphatidylinositol-specific phospholipase C gamma 2", "antibody characterization", "antibody validation", "Western Blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nPhosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is expressed primarily in immune cells.1 It belongs to a family of phospholipases that facilitate the cleavage of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).2 DAG activates protein kinase C3 while IP3 diffuses through the cytosol to the endoplasmic reticulum where it stimulates the release of calcium stores.4 By regulating these second messengers, PLC-gamma-2 regulates cellular functions such as migration, adhesion, calcium signaling, and phagocytosis.5,6\n\nIn the absence of activation, PLC-gamma-2 exists in an autoinhibited state in the cytosol.7,8 In order for PLC-gamma-2 to be recruited to the plasma membrane, phosphorylation is required. This phosphorylation induces domain reorganization of the autoinhibitory core and exposes the catalytic domain to its substrate, IP3, at the inner membrane surface.9\n\nThe particular kinases and scaffolding proteins that mediate the activation of PLC-gamma-2 are dependent on the receptor being activated. Unlike its related family members, PLC-gamma-2 is activated downstream of receptor tyrosine kinases rather than G protein coupled receptors.2\n\nPLC-gamma-2 has been implicated in a variety of diseases. Gain-of function point mutations cause autoinflammatory disease.10,11 Patients with rheumatoid arthritis have upregulated PLC-gamma-2 expression in peripheral blood mononuclear cells.12 PLC-gamma-2 signaling can drive leukemogenesis in cases of Ibrutinib refractory chronic lymphocytic leukemia.13,14 Additionally, PLC-gamma-2 is involved in solid cancers through Wnt signaling.15 Finally, whole-exome microarray data identified a rare hypermorphic variant (P522R) associated with decreased risk of Alzheimer’s Disease (AD).16–20 Conversely, a loss-of-function variant (M28L) is associated with increased risk.21 These risk modifying variants suggest that PLC-gamma-2 may be a potential target for the treatment of AD and related dementia.22,23 Studies aimed at elucidating the mechanistic role of PLC-gamma-2 in signaling pathways relevant to neurodegenerative processes involved in these diseases would benefit greatly from the availability of well characterized, high-quality antibodies.\n\nThis research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data24–26 Here, we evaluated the performance of eleven commercially-available antibodies for PLC-gamma-2 in Western Blot, immunoprecipitation, and immunofluorescence using a knockout based approach. This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of PLC-gamma-2 properties and function.\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.27,28 The first step is to identify a cell line(s) that expresses sufficient endogenous levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available THP-1 cells expressed the PLC-gamma-2 transcript at RNA levels above the average range of cancer cells analyzed. Parental and PLCG2 KO THP-1 cells were obtained from Abcam (Table 1).\n\nFor Western Blot analyses, we resolved proteins from WT and PLCG2 KO cell extracts. Both WT and KO cell lines were treated with and without phorbol 12-myristate 13-acetate (PMA) and then probed side-by-side with all antibodies in parallel (Figure 1).\n\nLysates of THP-1 (WT and PLCG2 KO) either treated (+) or not treated (-) with PMA were prepared and 10 μg of protein were processed for Western Blot with the indicated PLC-gamma-2 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. All antibodies were tested at a dilution of 1/500 except antibody 34264** was tested at 1/200. Predicted band size: 148 kDa. Monoclonal antibody, Recombinant antibody.\n\nAs per our standard protocol, we next used the antibodies to immunoprecipitate PLC-gamma-2 from THP-1 WT and KO cell extracts. The performance of each antibody was evaluated by detecting the PLC-gamma-2 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).\n\nTHP-1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated PLC-gamma-2 antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated PLC-gamma-2 antibody. For Western Blot, 55512* was used at 1/500. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. Monoclonal antibody, Recombinant antibody.\n\nFor immunofluorescence antibodies were screened using a mosaic strategy, as per our standardized protocol. First, THP-1 WT and PLCG2 KO cell lines were labelled with different coloured fluorescent dyes, in order to distinguish the two cell lines, and the eleven PLC-gamma-2 antibodies were evaluated. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested. The images presented in Figure 3 are representative of the results of this analysis.\n\nTHP-1 WT and PLCG2 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated PLC-gamma-2 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Antibodies were tested at 1.0 μg/mL. Antibody dilution used: ab109267* at 1/1300, ab133522** at 1/600, MAB3716* at 1/500, NBP2-52536* at 1/1000, 3872 at 1/1000, 34264 at 1/102, 55512 at 1/50, GTX111178 at 1/100, GTX111293 at 1/700, MA5-35670** at 1/700 and MA5-38600* at 1/1000. Bars = 10 μm. Monoclonal antibody, Recombinant antibody.\n\nIn summary, we have screened eleven PLC-gamma-2 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect PLC-gamma-2 under our standardized experimental conditions were identified. In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. However, the authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret the antibody characterization data in this study are openly available.29\n\nThe underlying data can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.30,31\n\n\nMethods\n\nAll PLC-gamma-2 antibodies are listed in Table 2, along with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.32 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).\n\n Monoclonal antibody.\n\n Recombinant antibody.\n\na Antibodies that have been discontinued following this study.\n\nBoth THP-1 and PLCG2 KO cell lines used are listed in Table 1, together with their corresponding RRID to ensure the cell lines are cited properly.33 Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, cat. number 11875119) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent, cat. number 609065), 100 IU penicillin and 100 μg/mL streptomycin (Wisent, cat. number 450201).\n\nOne set of the THP-1 WT and PLCG2 KO cells were treated with 200 ng/mL of PMA (Abcam, cat. number ab147465) for 2 days. 200 ng/mL of PMA was added to fresh medium on both day 1 and day 2.34 The other set of cells, WT and PLCG2 KO remained untreated.\n\nWestern Blots were performed as described in our standard operating procedure.27,28 THP-1 WT and PLCG2 KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.\n\nWestern Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) Or Clarity Western ECL Substrate from Bio-Rad (cat. number 1705061) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).\n\nImmunoprecipitation was performed as described in our standard operating procedure.27,28 Antibody-bead conjugates were prepared by adding 10 μL of antibodies 3872 and 55512 or 2 μg of the remaining antibodies tested to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). The low concentrations of antibodies 3872 and 55512** can account for why 10 μL was added to make the antibody-bead conjugates, rather than 2 μg. Tubes were rocked ~1 hr at 4°C followed by several washes to remove unbound antibodies.\n\nTHP-1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western Blot on a precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific.\n\nImmunofluorescence was performed as described in our standard operating procedure.27,28 THP-1 WT and PLCG2 KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated in 96-well plate with optically clear flat bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37oC, 5% CO2. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10 ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095), 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0.01% Triton X-100) containing the primary PLC-gamma-2 antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.\n\nImages were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water objective lens and scientific CMOS camera (16-bit, 1.97 mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose35 v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns. Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for PLC-gamma-2, https://doi.org/10.5281/zenodo.10108291. 30\n\nZenodo: Dataset for the PLC-gamma-2 antibody screening study, https://doi.org/10.5281/zenodo.10149969. 31\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.\n\nNeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.\n\nABIF consortium: Claire M. Brown and Joel Ryan.\n\nThank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.\n\nAn earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.10108291).\n\n\nReferences\n\nJackson JT, Mulazzani E, Nutt SL, et al.: The role of PLCγ2 in immunological disorders, cancer, and neurodegeneration. J. Biol. 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Publisher Full Text\n\nRuiz Moleon V, Fotouhi M, Ryan J, et al.: Antibody Characterization Report for PLC-gamma-2 (PLGC2).2023.\n\nSouthern K: Dataset for the PLC-gamma-2 antibody screening study. [Data set]. Zenodo. 2023.\n\nBandrowski A, Pairish M, Eckmann P, et al.: The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023; 51(D1): D358–D367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStarr T, Bauler TJ, Malik-Kale P, et al.: The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium. PLoS One. 2018; 13(3): e0193601. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021; 18(1): 100–106. PubMed Abstract \| Publisher Full Text" }	[ { "id": "300907", "date": "05 Aug 2024", "name": "Abhinay Ramaprasad", "expertise": [ "Reviewer Expertise Biochemistry", "Malaria", "Conditional mutagenesis", "phospholipases", "protein function 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\nThis data note compares the performance of 11 commercially available antibodies for PLC-gamma-2 with the aim to act as a guide for readers to choose appropriate antibodies in their studies and to address antibody reproducibility issues. The authors have performed careful, standardised Western blot, immunoprecipitation and immunofluorescence experiments with wildtype and KO cells to draw comparisons. Whilst they abstain from making recommendations for good reasons, the results clearly show some antibodies outperforming others. The experiments are done well with appropriate controls and reported in clear and informative manner. I have only two minor suggestions that might further improve interpretation by readers- 1) Could the authors explain the use of PMA for general readers not familiar with the field? 2) The authors say \"Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested\". However, I was unable to find this data. It would be helpful if this data is provided as a dotplot in Figure 3 along with the images which represent only few select cells and at times, hard to interpret.\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": "330318", "date": "18 Oct 2024", "name": "Deborah Moshinsky", "expertise": [ "Reviewer Expertise Antibody Characterization and Validation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 scientific basis for examining PLC-gamma-2 antibody performance and tested 11 commercially available antibodies against this target in Western Blot (WB), IP, and IF using standardized protocols. WB, IP, and IF data for each of the 11 antibodies were shown, allowing readers to assess Ab performance. The methods were clearly described and links to the underlying data were provided. This publication should be helpful for researchers of PLC-gamma-2 to assess commercially available antibody performance and choose the most promising antibodies for validation or use in their application.\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/13-77
https://f1000research.com/articles/9-522/v1	04 Jun 20	{ "type": "Research Article", "title": "Factors associated with health facility delivery in Kitui County: a cross sectional study", "authors": [ "Mathew Munyamaara Mutiiria", "Gabriel Gatimu Mbugua", "Doris Marwanga", "Gabriel Gatimu Mbugua", "Doris Marwanga" ], "abstract": "Background: High maternal mortality rate is a major public health concern in developing countries. Skilled birth delivery is central to reducing maternal mortality, yet health facility delivery remains low in Kitui County, Kenya. Our study estimated prevalence of unskilled delivery and identified factors associated with health facility delivery in Kitui County. Methods: A cross-sectional study was conducted December 2017-February 2018. 245 women from five administrative wards were interviewed. A structured questionnaire was used to collect data. Variables that had p value ≤0.05 in bivariate analysis were included in multivariable regression model to assess for confounders. Variables with a p value of ≤0.05 in multivariate analysis were considered statistically significant at 95% CI. Results: We interviewed 245 (240 analyzed) women from the five wards; the majority were 16-25 years age group (45.5%; 110/240). Mean age was 27±6.6 years. Prevalence of health facility delivery was 50.4%. Distance from a health facility, number of children in a household, occupation of the respondent’s partner, number of antenatal clinic (ANC) visits and means of transport were significant factors for not delivering in a health facility. On multivariate analysis, women who lived >5km from health facility were less likely to deliver in a health facility (AOR =0.36; 95% CI 0.15- 0.86). Women who attended ≥ 4 ANC visits were 4 times more likely to deliver in a health facility (95% CI 2.01-8.79). Conclusions: More than half of the respondents delivered in a health facility. A long distance from the health facility is a hindrance to accessing ANC services. Inadequate ANC visits was associated with home delivery. Improving accessibility of health care services and health education on family planning would increase delivery at a health facility. We recommend Kitui County introduce five satellite clinics/ambulatory services for expectant women to access ANC and maternal services.", "keywords": [ "Home delivery", "Health facility delivery", "skilled birth delivery", "delivery practices", "Kitui" ], "content": "Introduction\n\nGlobally, the maternal mortality rate has continued to increase leading to failure by countries to achieve the Millennium Development Goals (MDGs) that were set in year 1990 to 20151. This made it impossible to achieve the MDG 52 that aimed to reduce maternal and neonatal deaths (now Sustainable Development Goal (SDG) 3). However, maternal deaths have reduced from 546,000 in 1990 to 358,000 in 2015 representing a 34% reduction. Between 2016 and 2030, as part of the SDGs, the target is to reduce the global maternal mortality ratio to less than 70 per 100 000 live births3. In order to achieve, universal health care has been recognized as one of four pillars of government policy and is therefore receiving support from many quarters.\n\nAccording to Safe Motherhood Initiative (SMI) introduced by World Health Organization (WHO), hospital deliveries were fewer than home deliveries in many regions of sub-Saharan Africa (SSA). The reason for this is that the initiative lacks a clear, concise realistic strategy4. As a result, a high number of still births (3.2 million), 4 million neonatal deaths and more than half a million maternal deaths, continue to occur. Most of these deaths are preventable4, and when skilled health personnel provide delivery services within health facilities to pregnant women, maternal and neonatal health outcomes improve5.\n\nIn Kenya, skilled birth attendance has remained far below the international target of 90% at 44%. Skilled birth attendance during delivery is a benchmark indicator for safe motherhood6. Delivery by a skilled birth attendant reduces chances of maternal complications. A study by Nyongesa et al. found that the gender of a service provider, cost, number of antenatal visits and education level were strongly associated with client’s intention to deliver with a skilled birth attendant at delivery7.\n\nIn Kitui county, Kenya, skilled birth attendance has equally remained very low. Moreover, Mwingi North Subcounty has recorded a low number of deliveries in the health facilities. The district health information system (DHIS2) reported 1472 normal hospital deliveries in 2015 were 1472, 1594 in 2016, and 1194 in 20178.\n\nEven though delivery by skilled birth attendants have remained low, the immunization coverage at a country and county level have remained above the national government rate, showing that immunization coverage is not proportional to the number of skilled birth attendants. According to the Kenya health information system (KHIS), the number of Bacillus Calmette Guillen (BCG) antigen doses administered in Mwingi north Subcounty in 2017 was 3268, while the number of deliveries recorded by skilled birth attendants was 11548. This means that over 65% of babies were delivered at home without the help of a skilled birth attendant, as BCG vaccines are administered within 6 weeks of birth.\n\nImproving maternal delivery care is an essential element of attaining improved maternal health. In order to achieve this agenda, information about the rates and trends in maternal mortality is essential for resource mobilization, monitoring and evaluation of progress towards the SDGs. However, for this to be attained, maternal health programs should be based on quality evidence. Therefore, this cross-sectional study aimed to identify factors affecting health facility deliveries in Mwingi North Subcounty, Kenya.\n\n\nMethods\n\nThe study was carried out in five administrative wards of Mwingi North Subcounty, which has a catchment population of 160,938. Within the Subcounty, there are two Subcounty hospitals, six health centres, 20 dispensaries, two private nursing homes, and one level 4 hospital. Cadres of staff include medical officers (n=2), pharmacists (1), nursing officers (60), clinical officers (14), public health officers (10), nutritionists (2), and medical laboratory technologists (13). Approximately 34,800 women of reproductive age reside in the Subcounty (KDHS, 2014).\n\nThe study population included women of reproductive age (14–49 years) who had delivered within the preceding two years (1st January 2016 to 31st January 2018). Women who were below the age of 18 years who were not be accompanied by a guardian or a parent, those with psychiatric problems and very ill women were excluded from the study.\n\nWomen who were attending the clinics for routine appointments and who met the inclusion criteria were requested to spare about 15 minutes after their appointment to participate in the interview. The respondents were picked on first come basis, whereby the first 5 attendees of maternal child health services were picked every day until the proportionate sample for each health facility was achieved.\n\nSample size was determined using proportion of home deliveries to proportion of hospital deliveries. This data was collected from DHIS; home deliveries (80%) and in hospitals (20%) in the study area. The standard error was set at 5% and Z score value of 1.96 for 95% confidence interval (CI) multistage and proportionate sampling techniques were applied. The sample size required was 245. Purposively, we selected Mwingi North Subcounty among the eight sub counties of Kitui County. Secondly proportionate sampling was done among the five wards of Mwingi north Subcounty according to their catchment population and the number of health facility deliveries reported.\n\nA structured questionnaire was administered by five research assistants between 1st December 2017 and 31st January 2018. The questionnaire was administered face to face at the health facilities as the respondents were coming to seek maternal child health services. The questionnaire had the following: demographic information, social economic and facility related questions (see Extended data9).\n\nStudy variables were classified as either dependent (outcome) variables or independent (exposure) variables. These included age, marital status, level of education, parity, occupation, number of people living in a house and distance from health facilities,\n\nWe carried out descriptive statistics, Chi square test and calculated odds ratios.\n\nThe data was collected using paper questionnaire, which was later entered into Epi Info™ (CDC) and Microsoft Excel 2010. Analysis was based on the specific objectives of identifying factors affecting delivery, respondents’ characteristics and calculation of prevalence of skilled birth attendant. We ran bivariate and multivariate models using binary logistic regression to assess any relationship between independent variables and health facility delivery. Data was analyzed using Statistical Package for Social Sciences (SPSS) v.25.\n\nWe calculated crude and adjusted odds ratios to ascertain effect sizes for any association between the outcome and predictor variables. The significance of these factors was determined using 95% confidence interval (CI). Variables (independent) found to be significant with p value less than 0.05 on bivariate analysis were included in a multivariate logistic regression model to control for any potential confounding variables.\n\nOur study tools and protocol were approved by the Meru University of Science and Technology Ethical Review Committee (approval number: MIRERC/923/2017), while permission to carry out the study was granted by Kitui county government. Respondents provided both written and verbal informed consents following introduction and explanation on the purpose of study being done. Those who could write, appended a signature to the consent form while others left their thumb prints on the form. They were informed about their right to interrupt the interview at any time or even decline to be interviewed without any future prejudice or facing consequence.\n\n\nResults\n\nWe interviewed a total of 245 women; the data from 240 women were analyzed as 5 records were removed from analysis as most variables were not captured. In a further 8 records, respondents did not know their years or date of birth.\n\nThe mean age of the respondents was 27.6 years (± standard deviation (SD) 6.5). The mean age of respondents reporting a previous health facility delivery was 27.4±6.6, while respondents reporting previous home deliveries had a mean age of 27.5±6.5. Most respondents were in the age group of 16–25 years (110; 45.8%), followed by the age groups 26–35 years (94; 39.2%) and >35 years (28; 11.7%). A small proportion of respondents did not know their age (8; 3.3%).\n\nAmong respondents reporting hospital deliveries, Kyuso ward had the majority of deliveries (91; 37.9%) while Tharaka ward had only 32 (13.3%) (chi2=12.8, df=4, p=0.01). Overall, Ngomeni ward had the highest proportion of home deliveries (22; 9.2%) (Table 1).\n\nANC, antenatal care.\n\nIn terms of education level, most of the women (149; 62.7%) were primary school leavers. Out these women, 32.1% (77) delivered at home. Very few (10; 4.2%) of the respondents did not have any form of education. There was no significant association between level of education and delivery in a health facility (chi2=4.64, df=4, p=0.33).\n\nThe majority of respondents were housewives, for both those reporting deliveries in health facilities and at home (51; 21.3% and 53; 22.1%, respectively). All (3; 100%) respondent who had formal jobs delivered in a health facility, while the majority of women who were farmers/livestock keepers (40; 16.7%) delivered at home and 22 (9.2%) delivered in a health facility.\n\nOn bivariate analysis, occupation of the mother (chi2=13.42, df=4, p=0.01), partner occupation (chi2=18.7, df=4, p=0.001), distance from health facility (chi2=19.7, df=2, p=0.0001) and residence (chi2=12.8, df=4, p=0.01) were factors found to be significantly associated with health facility delivery in Mwingi north Subcounty (Table 1).\n\nOn multivariate analysis, women who lived >5 kilometers away from health facilities were 38% less likely to deliver in a health facility (adjusted odds ratio (AOR) = 0.38, 95% CI 0.15-0.86). Partner occupation was also significant for delivery in a health facility (AOR = 0.67, 95% CI 0.26-1.71) (Table 2).\n\nOR, odds ratio.\n\nRegarding the number of antenatal clinic (ANC) visits, 58% (139) of respondents attended less than four visits. Among these, 21.3% delivered in a health facility while the rest delivered in their homes. The majority of those who attended more than four ANC visits (70; 29.2%) delivered in a health facility as opposed to 12.9% (31) who delivered in their homes. When we carried out bivariate analysis, number of ANC visits was significant for giving birth in a health facility (chi2=23.6, df=1, p=0.001).\n\nA quarter of the respondents (65; 21.1%) had a parity of 1+0, among them 64.6% (42) delivered in a health facility while the rest delivered in their homes. There were 31 respondents who had a parity of >5 (12.9%), of these the majority (24; 10%) delivered in their homes.\n\nRegarding family size (number of children) in household most of the respondents 48.8% (117) had 1–2 children. Of these 64.9% (76) delivered at a health facility while the rest delivered in their homes, however respondents who had more than 7 children were few 6.7% (16) of which 81.1% (13) delivered in their homes Chi2=21.6,df=3, p=0.00007 (Table 2).\n\nIn a multivariate model, four or more ANC visits (AOR=2.914, 95% CI 1.105-7.682), parity of the woman (AOR=0.12, 95% CI 0.01-3.44) and the number of children in the homestead (AOR=1.96, 95% CI 0.06-35.02) did not significantly influence place of delivery (Table 2).\n\nMost of the respondents (86; 35.9%) lived approximately 6 to 10 km away from a health facility. Among these 18.8% (45) delivered at home, while only 17.1% (41) delivered in a health facility. Very few respondents lived a distance less than 5km from a health facility (71; 30.1%) (chi2=19.7, df=2, p=0.0001).\n\nDistance from a nearby health institution, means of transport, cost of transport and presence of traditional practices were factors that were statistically significantly determined place of delivery (Table 1).\n\nIn a multivariate model, only distance that was associated factor for delivery in a health facility. Those who lived over 5km from a health facility were less likely to deliver in a health facility (AOR=0.37, 95% CI 0.19-0.72), presence of traditional practice (AOR=0.36, 95% CI 0.15-0.86) and means of transport (AOR=0.001, 95% CI 2.38-9.56). Those who used an ambulance on referral were more likely to deliver in a health facility than those who used other means of transport (Crude odds ratio (COR)=2.21, CI 95% 1.11-4.54) (Table 2).\n\n\nDiscussion\n\nThe percentage of women who delivered through the help of a skilled birth attendant is one indicator in meeting MDG 5 (now SDG3 – ensuring good healthy lives and promote wellbeing for all ages). In almost all countries where health professionals attend more than 80% of deliveries, maternal mortality rate is usually below 200 per 100,000 live births.\n\nIn Kenya, delivery by skilled birth attendants (health professionals) is available at very few births5. This study therefore considered factors affecting delivery in a health facility. The study has shown that that only 50.4% of the respondents had a delivery conducted by a skilled delivery attendant. Our results are slightly different from those from KDHS, whereby statistics for skilled attendants were 44% in Kenya.\n\nOur study demonstrated that there is no significant relationship between age and delivery in a health facility. However, other studies, e.g. a study by Mrisho et al.10 in Tanzania, showed that a higher percentage of younger women deliver at health facilities in contrast to older women who often choose to deliver at home. A study by Bhattacharyaya et al. on factors associated with preference of a health facility delivery indicated that older women preferred health facility delivery as opposed to younger women11. The reason given for this discrepancy according to studies in Zambia and Tanzania was that younger women were inexperienced and also more afraid of birth complications than older women.\n\nThe present study showed that attendance of >4 ANC visits influenced the place of delivery. This study agrees with a study that indicated that women who attended >4 ANC visits were likely to deliver in a health facility than those who did less visits11. In addition, the study by Bhattacharyya et al. indicated that marital status had little influence on the attendance of ANC during the mother’s last pregnancy and consequently on delivery in a health facility; in this study, single and married women mostly delivered either at home or at a government health facility. A study conducted in Myanmar12 showed similar findings. In this study, the majority of divorced women delivered at home whereas widowed women delivered at either a private or government health facility. Women who make more than 4 ANC visits may have bad obstetric history or perhaps have started ANC visits at earliest gestation12.\n\nIn our study, the number of children (parity) had a negative impact on delivery in a hospital. We found that women with higher parity and having more than two children in the family were more likely to deliver in their homes than their counterparts with low parity and one or two children. These study findings are similar to a study in Myanmar that indicated that women who have given birth more than once are less likely to seek maternal care at a health facility because they feel they can manage the birthing process without the assistance of a health care professional13. It was also shown in the present study that the respondent’s partners played a role in determining place of delivery; those women whose partners had formal jobs were more likely to deliver in a health facility as opposed to those whose partners’ job was casual or otherwise. The findings are similar to those of a study by Okang and Kaseje in Eritrea found that partners who had formal employment were more likely to deliver in a health facility than those with non-formal employment14.\n\nLevel of education is recognized as having an influence on the place of delivery. Women with non-formal education are more likely to deliver at home as opposed to a formally educated woman with a higher probability of delivering at a health facility15, as shown by studies carried out in Nepal, Columbia and Kenya16. Additionally a study by Feyissa and Genemo in Ethiopia showed that the level of education has a significant influence on health facility delivery15. Contrary to above, the results of this study in Mwingi North Subcounty show that education has little or no influence on the place of delivery. From the data obtained from our respondents, most of the women, irrespective of their level of education, delivered either at home or at a government health facility except for women with adult education who exclusively delivered at their own homes. This finding was in contrast to the anticipated results that would have shown a distinction in place of delivery depending on a woman’s educational attainment.\n\nSimilarly according to Addai13, in this study farming women were less likely to seek medical care at the time of delivery than women in other occupations. This was also influenced by the occupation of partners. This may be because partners with formal employment may be able to provide insurance cover; limitation of financial resources and poor access to health services are cited as the two reasons why farming woman give birth at home17.\n\nRegarding health facility-related factors, most of the respondents in this study lived more than 10km from a health centre and therefore delivered at home due to the long distance from their homes to the heath facility. The number of ANC clinic visits were proportionately reduced; as distance increased the number of ANC visits decreased. Our study findings concur with Bhattacharyya et al.11 whose findings in a study in India showed that women who gave birth at home would rather have delivered at a health facility but the long distance accompanied by transport challenges proved to be a hindrance. Moreover, a study in Nepal by Devkota et al.18 drew a similar conclusion, where they found that approximately 18% of women who had intended to deliver at a health facility ended up delivering at home. These findings are in agreement with Kenya’s KDHS that showed that most of the health facilities are over 5km radius from the consumers of services19.\n\n\nConclusions\n\nHealth facilities are the preferred sites for delivery regardless of the level of education, marital status and occupation of the respondents. Multiparous women are less likely to give birth at a health facility in preference of home deliveries. Long distance from the health facility is a hindrance to accessing health services and in turn maternal health care from qualified medical practitioners in Mwingi North Subcounty, Kenya.\n\n\nData availability\n\nFigshare: Factors associated with health facility delivery in Kitui County; A cross sectional study, https://doi.org/10.6084/m9.figshare.12295466.v120.\n\nFigshare: Questionnaires, https://doi.org/10.6084/m9.figshare.12374702.v19.\n\nThis project contains the following extended data:\n\n- Questionnaire in the language it was asked with an English translation.\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 thank the study participants and their families. We also thank all the respondents who took time to respond to our questionnaires.\n\n\nReferences\n\nAlkema L, Chou D, Hogan D, et al.: Global, regional, and national levels and trends in maternal mortality between 1990 and 2015, with scenario-based projections to 2030: a systematic analysis by the UN Maternal Mortality Estimation Inter-Agency Group. Lancet. 2016; 387(10017): 462–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaine D, Rosenfield A: The Safe Motherhood Initiative: why has it stalled? Am J Public Health. 1999; 89(4): 480–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanga LA, Mtuy T, Philemon RN, et al.: Inter-pregnancy interval and associated adverse maternal outcomes among women who delivered at Kilimanjaro Christian Medical Centre in Tanzania, 2000-2015. PLoS One. 2020; 15(2): e0228330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtuahene MD, Arde-Acquah S, Atuahene NF, et al.: Inclusion of men in maternal and safe motherhood services in inner-city communities in Ghana: evidence from a descriptive cross-sectional survey. BMC Pregnancy Childbirth. 2017; 17(1): 419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnuameh YA, Okawa S, Asante KP, et al.: Factors Influencing Health Facility Delivery in Predominantly Rural Communities across the Three Ecological Zones in Ghana: A Cross-Sectional Study. PLoS One. 2016; 11(3): e0152235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoo L, Harlow SD: Intimate partner violence affects skilled attendance at most recent delivery among women in Kenya. Matern Child Health J. 2012; 16(5): 1131–7. PubMed Abstract \| Publisher Full Text\n\nNyongesa C, Xu X, Hall JJ, et al.: Factors influencing choice of skilled birth attendance at ANC: evidence from the Kenya demographic health survey. BMC Pregnancy Childbirth. 2018; 18(1): 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDHIS2 deliveries mwingi north.\n\nMutiiria M, Gathingu G, Marwanga D: Questionnaires. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12374702.v1\n\nMrisho M, Schellenberg JA, Mushi AK, et al.: Factors affecting home delivery in rural Tanzania. Trop Med Int Health. 2007; 12(7): 862–72. PubMed Abstract \| Publisher Full Text\n\nBhattacharyya S, Srivastava A, Roy R, et al.: Factors influencing women’s preference for health facility deliveries in Jharkhand state, India: a cross sectional analysis. BMC Pregnancy Childbirth. 2016; 16: 50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaranja S, Gichuki R, Igunza P, et al.: Factors influencing deliveries at health facilities in a rural Maasai Community in Magadi sub-County, Kenya. BMC Pregnancy Childbirth. 2018; 18(1): 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAddai I: Determinants of Use of Maternal Child Health Services in Rural Ghana. J Biosoc Sci. 2000; 32: 1–15. PubMed Abstract \| Publisher Full Text\n\nOkang CA, Kaseje D: The Pull and Push Factors Influencing Choice of Place and Delivery Attendant in the Urban Slums of Nyalenda, Kisumu East District, Kenya. Int J Hum Soc Sci. 2015; 5(8): 8.\n\nYaya S, Bishwajit G, Gunawardena N: Socioeconomic factors associated with choice of delivery place among mothers: a population-based cross-sectional study in Guinea-Bissau. BMJ Glob Health. 2019; 4(2): e001341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDevkota B, Maskey J, Pandey AR, et al.: Determinants of home delivery in Nepal - A disaggregated analysis of marginalised and non-marginalised women from the 2016 Nepal Demographic and Health Survey. PLoS One. 2020; 15(1): e0228440. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTeferra AS, Alemu FM, Woldeyohannes SM: Institutional delivery service utilization and associated factors among mothers who gave birth in the last 12 months in Sekela District, north west of Ethiopia: a community-based cross sectional study. BMC Pregnancy Childbirth. 2012; 12: 74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeyissa TR, Genemo GA: Determinants of institutional delivery among childbearing age women in Western Ethiopia, 2013: unmatched case control study. PLoS One. 2014; 9(5): e97194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurger NE, Kopf D, Spreng CP, et al.: Healthy firms: constraints to growth among private health sector facilities in Ghana and Kenya. PLoS One. 2012; 7(2): e27885. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutiiria M, Marwanga D, Gathingu G: Factors associated with health facility delivery in Kitui County; A cross sectional study. figshare. Dataset. http://www.doi.org/10.6084/m9.figshare.12295466.v1" }	[ { "id": "92076", "date": "01 Sep 2021", "name": "Ferry Efendi", "expertise": [ "Reviewer Expertise 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\nInteresting study regarding health facility delivery and the associated factors in Kitui County. My feedback as follows:\n1. On the methods section please elaborate the inclusion criteria of this study.\n2. Please explain your outcome and exposure variables along with the category of each variable.\n\n3. The explanation of Table 1 doesn't match with the number on the table itself, please kindly check.\n\n4. I am wondering about the section titled: \"Facility and cultural-related factors affecting delivery in a health facility\". Did you collect the data regarding culturally related factors?\n5. On the discussion section, I think the authors should rewrite the discussion in order of the variables in Table 2.\n\n6. Please state the limitations of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7164", "date": "20 Sep 2021", "name": "Mathew Mutiiria", "role": "Author Response", "response": "Dear Reviewer, Thank you for spending your valuable time looking at this article. Your comments are well noted and I will consider them to appear in version two of the article Regards Mathew - Author" } ] }, { "id": "96056", "date": "19 Oct 2021", "name": "Danish Ahmad", "expertise": [ "Reviewer Expertise Global health", "health system strengthening", "maternal and child health", "healthy aging", "disaster risk reduction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 cross sectional survey data collected from 245 women in a region of Kenya, Kitui county, to assess determinants of institutional delivery. The paper is set against the high rates of maternal mortality and countries having missed UN-MDG targets. The focus on institutional delivery in the study area builds on global evidence that finds reduction in maternal mortality occurs initially as rates of skilled birth attendance and institutional deliveries increase.\nThe original research seeks to identify socio-demographic factors associated with deliveries in health facilities compared to home deliveries using bivariate and multivariate analysis. Women were interviewed at a health facility were they had sought care using a structured questionnaire and after obtaining informed consent. The authors report descriptive statistics for an analytical sample of 240 women and initially chi-square results for bivariate analysis. The bivariate analysis identified mother and partners occupation and distance from health facility and residence as statistically significant factors. Multivariate analysis is conducted with select variables (distance and partner occupation) arising as statistically significant. The paper builds on established maternal health research and provides information that is context specific to the study area. While the paper provides information unique to Kitui county, the generalisability of the paper is limited due to a small sample size and methodological constraints.\nThe authors have done well to use initial set of socio demographic factors to assess institutional delivery however, the broader maternal health literature has established a broader set of factors at individual, household, and community level that the paper could have considered. The authors may like to provide details related to the area selected, Kitui, for the study in context of Kenya’s overall MMR. Readers would like to understand why this area was chosen and also understand the administrative hierarchy of the country.\nThe selection of mothers from health clinics for interviews affects the rigour of the paper as selection bias may impact the associations seen. Women who are likely to seek care from a health facility would be different from those who aren’t seeking care. A limitations section is required in the paper where the authors may like to provide details relevant to the paper. While multivariate analysis is used, additional details are required such as the type of multivariable analysis and the construction of the outcome variable.\nIn papers with small sample sizes, the authors can consider recategorizing variables where the sample size is minimal . Table 1 identifies various categories with only 1 or 2 women that fits these categories. It is an accepted practise to construct categories in such cases so that the analysis is performed on a larger subset of women. Similarly, the construction of the baseline variables needs to be reviewed by the authors to assess which baseline category is most meaningful for their interpretation. The authors provide a wide variety of references which make for an insightful reading. As papers from South Asia and the African context are likely to be different, the authors may like to compare studies from similar regions. The study from Myanmar, though informative, is limited in relevance to a culturally different environment such as Kenya. Overall, the results also need to be interpreted with care as the small sample size of women in the paper may be resulting in statistically insignificant findings.\nThe public health potential of the paper can be better articulated so that findings can lead to meaningful changes in health policy and practise in the study area.\nThe language used was clear and easy to understand though in some places typos and grammatical errors cropped up. References had formatting errors especially where websites were used where citation details were missed.\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": "7333", "date": "16 Aug 2022", "name": "Mathew Mutiiria", "role": "Author Response", "response": "Thank you reviewer for spending valuable time looking at this article Find the following responses to the comments I) The generalisability of the paper is limited due to a small sample size and methodological constraints. Response: Though this may be true, the authors used the formula by Cochran (1977) for sample size determination in which two proportions are available. We, therefore, feel that this sample size was sufficient for this study in this area. ii) Readers would like to understand why this area was chosen and also understand the administrative hierarchy of the country. Response: Kenya has 47 administrative units called counties. Kitui county was among the counties with the lowest number of health facility delivery in the country according to the Kenya district health information system. iii) A limitations section is required in the paper where the authors may like to provide details relevant to the paper. Response: We agree that this section is useful, however, according to the guide by the editors for this journal, a limitation section is not among the 18 sections of the article by this journal. However, we have minimized respondents biasness by ensuring that the persons who were interviewed were within at most 2 years from the date of last delivery and that the questions were well articulated. iv) Similarly, the construction of the baseline variables needs to be reviewed by the authors to assess which baseline category is most meaningful for their interpretation. Response: Our most useful baseline variables were social demographic, social-economic and health facility and social-cultural in that order. v)The study from Myanmar, though informative, is limited in relevance to a culturally different environment such as Kenya. Response: Cultural differences in the two counties are indeed different, but our study referenced Myanmar based on the number of ANC visits which is not entirely a cultural factor. vi)The public health potential of the paper can be better articulated so that findings can lead to meaningful changes in health policy and practice in the study area. Response: To make meaningful changes, we have made some of the recommendations such as The county director of health services Kitui should introduce Health education in health facilities on the importance of attending the full prescribed ANC visits. The County Government of Kitui is encouraged to provide satellite clinics or ambulatory services to ensure that expectant women have access to antenatal care services. Conclusion We hope that these responses will provide a better understanding of the study We will share a version2 of the manuscript." } ] } ]	1	https://f1000research.com/articles/9-522
https://f1000research.com/articles/12-262/v1	10 Mar 23	{ "type": "Research Article", "title": "Effect of polyamine precursors and antioxidants on growth and metabolism of salt-stressed barley", "authors": [ "Eman Eldakkak", "Mohamed El-Shourbagy", "Mohamed El-Shourbagy" ], "abstract": "Background: Salinity is a serious problem that reduces crop productivity by affecting seed germination and seedling growth. It affects plant growth by disrupting plant osmosis, causing ionic toxicity, and metabolic and physiological changes. The aim of the present study was to examine the effect of polyamine precursors, besides the effect of some antioxidants such as glutathione and ascorbate on growth, metabolism, and productivity of two barley cultivars, different in salt tolerance, when subjected to salt stress. Methods: Salt-tolerant G124 and salt-sensitive G119 barley cultivars had their seeds planted in plastic pots with clay and sand soil that measured 10 cm in diameter and 4 cm in height (2:1). The pots were divided into five categories: the grains were presoaked for 24 hours in distilled water, an amino acid solution (Arginine, Methionine, and Ornithine) (1 mM each), glutathione, or ascorbic acid (0.1 mM for each), seedlings were cultivated in 0.1 mM sodium chloride alone, seedlings were cultivated using a combination of salt and glutathione, seedlings were grown using a combination of salt and ascorbic acid, seedlings were cultivated using a combination of salt and amino acid mixture. Results: Salinity has resulted in a reduction in all growth parameters in both barley cultivars. In the seedling, pre-flowering, and yield stages, the exposure to salt decreased photosynthetic pigments, total soluble carbohydrates, weight, and the number of grains, whereas it showed an increase in the activities of catalase and peroxidase, proline content, malondialdehyde, and membrane leakage. Conclusion: The addition of glutathione, ascorbic acid, or amino acid mixture alleviated the harmful effect of sodium chloride and improved barley's defense mechanism against salt stress. The results showed that the alleviating effect was eventually reflected in plant growth, indicating that glutathione might be suggested as an effective treatment to reduce the impacts of salinity on barley.", "keywords": [ "Salinity Stress", "Antioxidants", "Polyamines" ], "content": "Introduction\n\nSalinity is one of the most prevalent abiotic pressures in arid or semi-arid settings (Koca et al., 2007). High salinity has a negative impact on plants at the whole-plant level, resulting in plant mortality or a drop in productivity. Many plants develop mechanisms either to exclude salt from their cells or to tolerate its presence. Because of the negative effect of salt stress on crop productivity, crop productivity loss might exceed 60% of crop yield globally. Salinity stress disrupts physiological processes in plant cells, inhibits plant growth, and causes significant damage to photosynthesis (Yan et al., 2018), resulting in declines in pla nt production quantity and quality (Taize & Zeiger, 2006). Furthermore, salinity has impacted plant growth and development by increasing salt content, particularly ionic chloride cl and sodium Na+, leading to changes in water status, mineral uptake, stomatal behavior, carbon allocation, and ion imbalance, resulting in water and osmotic potential disturbance (Hasanuzzaman et al., 2013). Salt stress reduces stomatal conductance, transpiration rate, and intercellular CO2 concentrations by decreasing photosynthesis (Khoshbakht et al., 2018; Xu et al., 2018). Plants respond to salt stress by producing biochemical compounds and activating molecular systems at the cellular and plant levels (Li et al., 2019). Most cultivated plant species are highly sensitive and either die or display reduced productivity after exposure to salt stress for long periods. Yancey et al. (1982) reported that salt stress induced physiological and biochemical changes in the whole plant so that various compounds are synthesized such as abscisic acid, organic acids, proline, and polyamines (PAs). Polyamines are phytohormones that can be utilized to protect plants from abiotic stresses and to induce flowering (Liu et al., 2015). Because PAs levels change significantly in response to environmental stress, it has been reported that they are part of a plant's stress defense mechanism (Turano & Kramer, 1993). Polyamines are involved in various plant development processes, including cell division, embryogenesis, floral and reproductive organ development, fruit ripening, root growth, leaf senescence, and response to biotic and abiotic stressors such as salt stress (Sarwat et al., 2013). Zhao and Qin (2004) suggested that polyamines improve all morphological and physiological characteristics and prevent chlorophyll degradation while also increasing the accumulation of all organic compounds under salt stress. In recent years, attention has been focused on the possible roles of the conjugated forms of polyamines in plants exposed to unfavorable environmental conditions (Bouchereau et al., 1999), but even so, only a few reports on the response of bound polyamines to salinity stress factors are available.\n\nIt has become widely known that reactive oxygen species (ROS) are to responsible for a diverse variety of stress-induced damage to macromolecules and, ultimately, cellular structure (Sairam et al., 2002). As a result, the role of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT), as well as metabolites such as ascorbic acid, glutathione, -tocopherol, flavonoids, and carotenoids (CAR), in the quenching of ROS becomes critical (Soliman et al., 2019). Non-enzymatic antioxidants (glutathione and ascorbate) accumulated in root tissues of plants treated to salt stress, according to Vaidyanathan et al. (2003). Glutathione (GSH) is the most abundant non-protein thiol found in animal, plant, and bacterial cells. It contributes to the structural integrity of cells and has the potential to be utilized in the redox control of cell division (Hossain et al., 2017). Glutathione's physiological importance in plants comes from its role in sulfur metabolism and defense, where it is a vital source of reduced sulfur (Smirnoff & Wheeler, 2000). Many important plant functions are regulated by it, including photosynthesis, DNA biosynthesis and repair, protein biosynthesis, amino acid transport, and enzyme control and activation. GHS has recently been discovered to play an important role in protecting chickpea plants from drought stress by regulating the antioxidant defense system and osmolyte synthesis (El-Beltagi et al., 2020).\n\nAscorbic acid (AA), is one of the most important and most abundant growth promoters presents in plants (Khan et al., 2011). A small amount of AA produced endogenously is involved in the promotion of the development and growth of plant cells. Ascorbic acid is involved in the phytohormonal mediated signaling pathways and is bound towards a various number of environmental stress conditions along with development and growth (Barth et al., 2006).\n\nIn view of the lack of knowledge regarding the role of polyamines in salt tolerance of grain crops, it is attempted in the present study examines the effect of polyamine precursors; arginine, methionine, and ornithine, besides the effect of some antioxidants as glutathione and ascorbate on growth, metabolism, and productivity of two barley cultivars, different in salt tolerance, when subjected to salt stress.\n\n\nMethods\n\nGrains of eight barley cultivars (Giza117, Giza119, Giza 121, Giza 123, Giza 124, Giza 125, Giza 126 and Giza 2000) obtained from the Egyptian Agricultural Research Center were tested for germination under different concentrations of NaCl (50 mM, 100 mM, 150 mM, and 200 mM). Giza 124 was chosen as the most tolerant cultivar and Giza 119 as the most sensitive one with 100 mM NaCl to be used in the present study.\n\nGrains of two cultivars; salt-tolerant G124 and salt-sensitive G119 of barley (Hordeum vulgare L.) were germinated in plastic pots (10 cm diameter × 4 cm height each), filled with clay sandy soil (2:1). The pots were divided into five groups:\n\n1- Grains were presoaked for 24 hrs in distilled water or amino acid mixture (Arginine, methionine and ornithine) 1mM for each or 0.1 mM for glutathione or1 mM for ascorbic acid.\n\n2- One-week old seedlings were grown with 0.1mM sodium chloride alone.\n\n3- One-week old seedlings were grown under the combined effect of 0.1 mM glutathione plus salt.\n\n4- One-week old seedlings were grown under the combined effect of 1mM ascorbic acid plus salt.\n\n5- One-week old seedlings were grown under the combined effect of 1mM amino acid mixture plus salt.\n\nGrowth criteria\n\nAfter 21 d of exposure to each treatment, plants were harvested and separated into shoots and roots then shoot height, root depth, and leaf area were determined and weighed directly for the fresh weight (FW). Plant parts were dried in an air-driven oven at 80°C until constant weight for 48 hrs to determine the dry weight (DW).\n\nChlorophyll determination\n\nA sample of fresh leaves (0.1 g) was homogenized in 5 ml 85% cold acetone at the seedling and pre-flowering stages, then centrifuged for 15 minutes at 3000 rpm and the pigment extracts were stored overnight in a refrigerator. The acetone extract was diluted to the appropriate volume, and the color intensities were measured at 663, 644, and 452.5 nm with a spectrophotometer to determine chlorophyll a, chlorophyll b, and carotenoids (Metzner et al., 1965).\n\nMeasurement of photosynthetic efficiency\n\nIn the seedling and preflowering stages, photosynthetic efficiency (Fv/Fm) of dark-adapted leaves was measured with a portable pulse amplitude modulation (PAM) fluorometer (PerkinElmer, UK). Mature leaves, morphologically similar, were dark-adapted for 30 min then placed in the leaf clip, to maintain constant angles of incidence between the fibre-optic arm of the fluorometer and the leaf surface. The measurement of photosynthetic efficiency, on the adaxial leaf surface, was carried out as described by (Gonçalves & Santos Júnior, 2005). The leaf was initially exposed to the weak modulated measuring beam (< 0.1 umol/m2 per s), to estimate the initial fluorescence (F0), when the PS II reaction centers are open (oxidized). Thereafter, the leaf was then exposed to 800 ms saturation pulse of high-intensity (>10000 umol/m2 per s white light), to produce a transient closure (reduction) of the PS II reaction centers, at which point the maximum fluorescence (Fm), the variable fluorescence (Fv = Fm – F0), the maximum photochemical efficiency of PSII (Fv/Fm) and time of achieving maximum fluorescence yield (Tm) were obtained. Three replicates of each treatment were measured.\n\nDetermination of total soluble carbohydrates\n\nThe phenol sulfuric acid method has been used to estimate total soluble carbohydrates in the seedling, preflowering, and yield stages according to (Dubois et al., 1956).\n\nBorate buffer extraction of dried samples: One g of dry shoot or root was incubated with 5 ml borate buffer (pH8) for 24 h and then centrifuged for 15 min at 3000 rpm. The supernatant was completed to a known volume, then 0.1 ml of sample solution was transferred into a test tube and 1ml of phenol and 5 ml of sulfuric acid were added, then put in a water bath at 25 °C for 20 min. The color was read at 490 nm.\n\nDetermination of proline\n\nThe techniques according to Bates et al. (1973) and Zúñiga et al. (1989) were applied for the determination of proline in seedling and yield stages. One-half g of dry tissue was homogenized in 5 ml of 3% (w/v) sulfosalicylic acid and then filtered. To 2 ml of filtrate, 2 ml acid ninhydrin was added, followed by 2 ml glacial acetic acid then boiled for 60 min, and the reaction was terminated in an ice bath. The reaction mixture was extracted with toluene and mixed vigorously in a separating funnel for 20 sec. The chromophore containing toluene was aspirated from the aqueous phase and the absorbance was read at 520 nm, using toluene as a blank.\n\nAssaying of antioxidant enzymes\n\nFresh leaves (0.5 g) of each barley cultivar were ground with 8 ml of 50 mM cold phosphate buffer (pH 7.8) and centrifuged at 4000 rpm for 20 min. The supernatant was used for the determination of the activities of antioxidant enzymes in the seedling stage.\n\nCatalase (EC 1.11.1.6) was assayed (Kato & Shimizu, 1987) by measuring the initial rate of disappearance of H2O2. A sample of 3 ml of reaction mixture containing 0.1 M sodium phosphate buffer of pH 7.2, 11.8 mM H2O2, and 0.1 ml enzyme extract. The decrease in H2O2 was followed by a decline in the absorbance at 240 nm and the activity was calculated using the extinction coefficient (40 mM-1 cm-1 at 240 nm) for H2O2. The activity was expressed in units of μM of substrate converted per minute per one gram of fresh weight.\n\nThe activity of peroxidase (EC 1.11.1.7) was assessed using (Kato and Shimizu, 1987). The final assay volume was 3 ml, and the assay medium contained 0.1 M sodium phosphate buffer, pH 5.8, 7.2 mM guaiacol, 11.8 mM H2O2, and 0.1 ml enzyme extract. H2O2 was used to start the reaction, and a change in absorbance was detected at 470 nm.\n\nDetermination of lipid peroxidation\n\nThe lipid peroxidation was measured in the seedling stage by the amount of malonyl dialdehyde (MAD), as a product of peroxidation of unsaturated fatty acid (Linolenic acid, (18:3)). MAD concentration was estimated by the method of (Heath & Packer, 1968). A sample of 0.5 g fresh leaves was extracted in 10 ml 5% (w/v) trichloroacetic acid. The homogenate was then centrifuged at 4000 rpm for 10 min. The supernatant (2 ml) was mixed with 2ml of 0.67% (w/v) thiobarbituric acid, incubated at 100°C in a water bath for 20 min then cooled immediately. Absorbance was read at 532 nm and 600 nm. MAD concentration (μM/g FW) was calculated using the extinction coefficient 155Mm-1 cm-1.\n\nMembrane leakage\n\nFresh leaves were taken and quickly cut into sections measuring about 0.5 cm in length for the assessment of electrolyte leakage in the seedling and pre-flowering stages. After being submerged for one hour in 20 ml of distilled water, one-half g of these leaf fragments was used to test the electrical conductivity in the leaking solution.\n\nMineral analysis of plant material\n\nWe used half a gram of oven-dried plant samples. The sample was digested using a semi-a micro Kjeldahl apparatus and 2 mL of concentrated perchloric acid and 4 mL of concentrated sulfuric acid. Digestion continued until a clear solution without charging was obtained, after which it was filtered using Whatman filter paper NO. 44 and completed up to a constant volume. Phosphorus was determined using the Molybdenum blue method and measuring optical density at 700 nm, nitrogen using the Indo-phenol blue method according to (Tetlow & Wilson, 1964) and measuring at 625 nm, and (K+) and (Na+) using the flame photometer (Allen et al., 1974). Protein nitrogen was calculated by the following equation:\n\nDetermination of biogenic amines by thin-layer chromatography (TLC)\n\nBiogenic amines were extracted and determined in all tested samples according to Maijala and Eerola (1993).\n\nThe yield stage\n\nEach barley cultivar was cultivated until the end of the growing season (starting from December 1st 2010 until the end of April 2011). The yield criteria were measured, including the weight of grains per plant, the weight of 100 grains, and the number of grains per plant.\n\nAll experimental determinations were replicated. The obtained data represented the mean values. Data obtained were analyzed statistically using excel to determine the degree of significance between treatments for each cultivar. The one-way ANOVA method was applied for all data and the least significant difference (LSD) at 0.05 and 0.01 was used to compare means (Steel & Torrie, 1980).\n\n\nResults\n\n\n\n1- Growth criteria\n\nGrains of two barley cultivars, Giza 124, and Giza119 were germinated as described in the materials and methods section. Growth parameters (measured after 21 days), including fresh weight, dry weight, shoot height, root depth, and leaf area of barley seedlings were recorded as shown in Tables 1, 2, 3, and 4. The data showed that salinity stress caused a reduction in all studied growth parameters compared to the control. Also, it was observed that salinity resulted in a reduction in fresh and dry weights of both shoot and root of each cultivar compared to the control (Table 1 and Figure 1). Increases in the fresh and dry weights of each cultivar's shoot and root following treatment with glutathione, ascorbic acid, or an amino acid combination offset the effects of salt. It is interesting to learn that, compared to other treatments, glutathione significantly increased the root dry weight of salt-affected Giza 124 (Table 2 and Figure 2). It was observed that shoot height and root depth in each barley cultivar decreased under salinity compared to control (Table 3 and Figure 3). However, shoot height increased in Giza 124 compared to Giza119 under the effect of glutathione, ascorbic acid or amino acid mixture. Moreover, root depth in Giza 124 reached its highest level under the effect of amino acid mixture compared to other treatments.\n\nResults revealed that in Giza 124, the leaf area expanded significantly under the influence of glutathione but to a lesser extent under the influence of ascorbic acid as compared to control and other treatments (Table 4 and Figure 4), while glutathione showed a slight effect on Giza 119. The succulence ratio was decreased in the shoot and root of both cultivars with salinity but it increased in the root of Giza 119 under salinity and no variation was observed between other treatments (Table 5 and Figure 5).\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n* Significant at P≤0.05.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n* Significant at P≤0.05.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that the effects of salinity, glutathione, ascorbic acid or amino acid mixture with or without salt were highly significant (P≤0.01) on each studied growth parameter compared to the control of each cultivar.\n\n2- Photosynthesis\n\na) Chlorophyll contents: Regarding Giza 119, a remarkable rise of all determined pigments can be observed with the addition of either ascorbic acid or amino acid mixture to salt-stressed seedlings. However, the effect was more pronounced in chl b (Table 6 and Figure 6). Again, Giza 119 showed higher values of chl a, chl b, and carotenoids with the addition of ascorbic acid or amino acid mixture to salt-stressed seedlings and the difference were greater between the two cultivars. However, no remarkable effect of glutathione was observed with salt on most pigments.\n\nb) Photosynthetic efficiency: The addition of either glutathione, ascorbic acid or amino acid mixture with salt showed greater photosynthetic efficiency for each cultivar but the effect was greater with Giza 119 compared to Giza 124, which can be correlated with the pigment content (Table 7 and Figure 7).\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that the effects of salinity, glutathione, ascorbic acid, or amino acid mixture with or without salt were highly significant (P≤0.01) on chlorophyll contents and photosynthetic efficiency of each cultivar.\n\n3- Metabolites\n\na) Total soluble carbohydrates: In each cultivar, there was a decrease in total soluble carbohydrates in shoot and root under salinity compared to the control (Table 8 and Figure 8). With the addition of glutathione, ascorbic acid, or amino acid mixture to salt total soluble carbohydrates increased.\n\nb) Proline content: In both cultivars Salinity has resulted in an increase in proline content compared with the control and the effect was more pronounced in Giza 124 than Giza 119 (Table 9 and Figure 9). However, proline content decreased with glutathione, ascorbic acid, or amino acid mixture compared to control. Proline content decreased also with the addition of each of these compounds to salt compared with salt alone.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that the effects of salinity, glutathione, ascorbic acid, or amino acid mixture with or without salt were highly significant (P≤0.01) the on total soluble carbohydrates and proline content of each cultivar.\n\n4- Antioxidant enzymes: Results indicated that in each cultivar the activity of peroxidase and catalase increased under the effect of salinity compared to the control. The effect of salinity on peroxidase and catalase decreased with the addition of glutathione, ascorbic acid, or amino acid mixture (Table 10 and Figure 10).\n\n5- Malondialdehyde content: In each cultivar, salinity showed an increase in malondialdehyde (MAD) content compared to the control. However, lipid peroxidation has decreased with glutathione, ascorbic acid, or amino acid mixture compared to the control. Giza 119 showed more MDA content compared to Giza 124 under salinity (Table 11 and Figure 11).\n\n6- Membrane leakage: The results of both cultivars were more or less similar. However, salinity stress increased the membrane leakage of Giza 124 and Giza 119, while decreased by the addition of glutathione, ascorbic acid or amino acid mixture (Table 11 and Figure 11).\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that in each cultivar the effects of salinity, glutathione, ascorbic acid or amino acid mixture with or without salt were highly significant (P≤0.01) on the activity of each enzyme, MDA content and the membrane leakage of each cultivar.\n\n7- Mineral analysis\n\na) Phosphorus content: Salinity showed a slight increase in phosphorus content in the shoot and root of both cultivars (Table 12 and Figure 12). However, it increased in Giza 124 shoot under the effect of glutathione, ascorbic acid without salt or amino acid mixture plus salt. On the other hand, it decreased in Giza 124 root under the effect of glutathione, ascorbic acid, or amino acid mixture with or without salt compared to the control, while increased in Giza 119 root under the effect of ascorbic acid alone.\n\nb) Potassium content: It can be noticed that potassium showed a decrease in the shoot and root of each cultivar, especially with salinity alone (Table 13 and Figure 13). However, Giza 124 shoot has experienced a rise in potassium content in most treatments and showed lower root potassium with salinity alone and ascorbic acid with or without salt or amino acids with salt compared to Giza 119.\n\nc) Sodium content: Results showed that sodium content increased under salinity in the shoot and root of both cultivars compared to the control (Table 14 and Figure 14). However it increased with glutathione, ascorbic acid, or amino acids with the salt in the root of Giza 119. Potassium/sodium ratio decreased with salt, while it has been increased with other treatments compared to the control (Table 16 and Figure 16).\n\nd) Nitrogen content: Results showed that nitrogen content increased in shoots of both Giza 124 and 119 under the effect of salinity compared to the control (Table 15 and Figure 15). However, it increased in the shoot and root of each cultivar under the effect of glutathione compared to other treatments.\n\ne) Protein nitrogen: Shoot protein nitrogen showed a decrease with salt in both cultivars and appreciably with glutathione, ascorbic acid, or amino acid plus salt in Giza 124 compared to other treatments (Table 17 and Figure 17). In both cultivars, root-protein nitrogen showed a remarkable increase with glutathione alone in Giza124 and Giza 119.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that the effects of salinity, glutathione, ascorbic acid, or amino acid mixture with or without salt were highly significant (P≤0.01) in phosphorus, potassium, sodium, K+/Na+ ratio, nitrogen, and protein nitrogen.\n\n8- Polyamines (PAs) content: Determination of polyamine content under salt stress indicated an increase in spermidine and spermine in Giza 119 compared to Giza 124 while putrescine increased in both cultivars with salt compared to control (Table 18). Addition of glutathione with salt caused an increase in putrescine and spermidine of Giza 124 and ascorbic acid with salt resulted in higher putrescine content in each cultivar with an increase in spermidine of Giza 119 only. Also, addition of amino acid mixture with salt showed an increase in putrescine and spermidine in each cultivar. The amount of spermine was so small to be detected with other treatments.\n\n\n\n1- Photosynthetic pigments: In Giza 124 Chl a and Chl b were most dominant in all treatments. A remarkable rise has been observed in Chl b with glutathione, ascorbic acid, or amino acid mixture alone in G124 (Table 19 and Figure 18). Regarding carotenoids, Giza 124 has higher levels with glutathione plus salt or ascorbic acid plus salt compared to Giza 119.\n\n2- Photosynthetic efficiency: Photosynthetic efficiency decreased with salt in each cultivar. In general, Giza 124 showed slightly higher photosynthetic efficiency compared to the Giza119 cultivar, particularly with glutathione or amino acid mixture with or without salt (Table 20 and Figure 19).\n\n3- Total Soluble Carbohydrates: At the pre-flowering stage, salinity showed a decrease in total soluble carbohydrates in each cultivar compared to the control (Table 21 and Figure 20). By the addition of either glutathione, ascorbic acid or amino acid mixture an increase was detected in total soluble carbohydrates of each cultivar.\n\n4- Membrane Leakage: Results showed that membrane leakage was increased under the effect of salinity in each cultivar compared to the control (Table 22 and Figure 21). Glutathione plus salt showed a decrease in membrane leakage in G124 while Giza119 showed a decrease with amino acid plus salt compared to other treatments.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nStatistical analysis showed that the effects of salinity, glutathione, ascorbic acid or amino acid mixture with or without salt were highly significant (P≤0.01) on photosynthetic pigments, photosynthetic efficiency, total soluble carbohydrates, and membrane leakage.\n\n\n\n1- Yield criteria: In both cultivars weight of grains per plant and weight of 100 grains has decreased under the effect of salinity compared to the control (Table 23 and Figure 22). Grains per plant were increased in Giza 124 with either ascorbic acid or amino acid mixture and in Giza 119 with glutathione alone or plus salt and amino acid plus salt in Giza 119.\n\n2- Total soluble carbohydrates: It can be noticed (Table 24 and Figure 23) that salinity brought about a drop in total soluble carbohydrates in each cultivar but increased in both with glutathione alone. The addition of glutathione, ascorbic acid or amino acid mixture to salt has resulted in a rise of T.S.C in Giza 119-seeds compared to that of Giza 124.\n\n3- Proline content: In both cultivars, a specific increase in grain proline has been seen when sodium chloride, glutathione, ascorbic acid, or an amino acid mixture are combined with salt in comparison to control (Table 25, and Figure 24). According to statistical analysis, each cultivar's proline content, total soluble carbohydrates, and yield criteria were all significantly affected by salinity, glutathione, ascorbic acid, or amino acid mixtures with or without salt (P≤0.01).\n\n4- Mineral analysis\n\na) Phosphorous content: In each cultivar, phosphorus content was increased under salinity compared to the control (Table 26 and Figure 25). Phosphorus content increased in Giza 124 with amino acid mixture alone compared to 119, while increased in Giza 119 with ascorbic acid plus salt.\n\nb) Potassium content: In both cultivars, potassium content showed a low level under salinity compared to control (Table 27 and Figure 26). On the other hand, potassium content increased in Giza 119 with glutathione, ascorbic acid or amino acid mixture with or without salt.\n\nc) Sodium content: An apparent rise has been detected in Na content of Giza 124 and Giza 119. Other treatments showed a decrease in sodium content in each cultivar (Table 28 and Figure 27). K+/Na+ ratio decreased under salinity in each cultivar, while it increased with ascorbic acid alone in G124 and with glutathione, ascorbic acid or amino acid mixture without salt in G119 (Table 30 and Figure 29).\n\nd) Nitrogen content: In both cultivars, nitrogen content showed a remarkable reduction with salinity (Table 29 and Figure 28). However, the highest content was observed with the amino acid mixture in Giza 124 and ascorbic acid in Giza 119, while the lowest one was observed in both cultivars with glutathione plus salt.\n\ne) Protein nitrogen: In comparison to other treatments, protein nitrogen decreased with salt in both cultivars and noticeably with glutathione in two cultivars, either with or without salt (Table 31 and Figure 30). With ascorbic acid or an amino acid and salt, protein nitrogen increased in both cultivars.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\n Highly significant at P≤0.01.\n\nAccording to statistical analysis, salinity, glutathione, ascorbic acid, or an amino acid mixture with or without salt had a highly significant impact (P≤0.01) on the levels of phosphorus, potassium, sodium, the K/Na ratio, nitrogen, and protein nitrogen.\n\n\nDiscussion\n\nIn the present study, salt stress significantly decreased all growth parameters, including fresh weight, dry weight, shoot height, root depth, succulence, and leaf area in both salt-tolerant and salt-sensitive barley cultivars. The reduction in leaf area under salt stress means that photosynthesis is decreased (Abdel-Farid et al., 2020; Munns & Tester, 2008). Such decrease may be partly due to decreased cell water potential associated with stomatal closure thereby, decreased CO2 assimilation, besides NaCl toxicity and decreased availability of water. These results were in agreement with (Razzaque et al., 2009).\n\nThe current findings supported those of (Asch et al., 2000) who studied various rice cultivars and found that the salt-tolerant cultivar (Giza 124) showed less dry matter degradation than the sensitive one. As anti-oxidant compounds, glutathione (GSH) and ascorbic acid (ASH) were found to be effective in raising all growth parameters, which may be related to their inhibitory effect on the uptake of Cl- and Na+ ions and their stimulatory effect on the uptake of the necessary elements, such as N, Mg, and Fe (Abd El-Hameid & Sadak, 2020; Munir et al., 2021). (Rawia et al., 2011) reported that exogenous application of glutathione or ascorbic acid mitigated partially or completely the adverse effects of salt stress on the growth of canola seedlings and Marigold plants, respectively. In this study, the addition of a mixture containing a number of amino acids; arginine, ornithine and methionine has induced an increase in the shoot and root dry weights of salt stressed seedlings of each cultivar. In this regard (El-Shourbagy, 1964), showed a similar effect of amino acid mixture with the in vitro culture of tomato roots. However (Abo Sedera et al., 2010; El-Zohiri & Asfour, 2009), showed that the effect of polyamine precursors on the protein assimilation and cell formation of some potato cultivars can be attributed to the assimilation of gibberellins biosynthesis.\n\nSalt stress induced a significant reduction in chlorophyll a and b levels in the two cultivars of barley which agreed with the results of (Taïbi et al., 2016) for two genotypes, high-yielding ‘Tema’ and low-yielding ‘Djadida’, commonly cultivated in Algeria. This decrease may be due to increased chlorophyllase activity as reported by (Nazarbeygi et al., 2011) and also may be caused by suppression of specific enzymes responsible for the synthesis of green pigments (Kaur et al., 2014; Mishra et al., 2006). In contrast, carotenoids were significantly increased in the salt stressed barley cultivars and this may be due to their role in the protection of the photosystems by reacting with the lipid peroxidation products (Borghesi et al., 2011).\n\nIn Giza 124, glutathione and ascorbic acid with or without salt significantly increased chlorophyll a levels under salt stress compared to the control. Amini and Ehsanpour (2005) found similar results with canola seeds. In comparison to the control, G119 showed an increase in carotenoids when treated with glutathione, ascorbic acid, or an amino acid combination with or without salt. Glutathione's impacts on pigment levels may be due to their effects on either enhancing photosynthetic activities and chlorophyll biosynthesis or retarding chlorophyll degradation caused by oxidative stress. The addition of amino acids (polyamine precursors) has, on the other hand, resulted in an increase in chl b in both cultivars. Similar results have been reached with garlic plants (El-Shabasi et al., 2005), and sweet pepper (Al-Said & Kamal, 2008). This increase may be due to the role of amino acid mixture in detoxification of harmful accumulated of ROS in the thylakoid membrane during photosynthesis.\n\nThe current study showed a highly significant decrease in photosynthetic efficiency (Fv/Fm) under salt stress, which can be attributed to photochemical quenching in PSII. Salt stress increased reactive oxygen species formation, which damaged PSII and organelles such as chloroplasts, mitochondria, and plasma membranes. As a result, PSII's efficiency decreases. Several authors have reported comparable outcomes with several crop species under salt stress (Alnusairi et al., 2021). When glutathione, ascorbic acid, or an amino acid combination (polyamine precursors) were combined with salt, photosynthetic efficiency increased compared to the control. This could be related to their role in mitigating the harmful effects of salt via ROS scavenging.\n\nThere was a highly significant decrease in the total soluble carbohydrates in shoot and root of each cultivar under salinity stress compared with the control at the three studied growth stages. However, the salt-tolerant cultivar (Giza 124) has generally greater total soluble sugars compared to the salt-sensitive one (Giza 119) and this agreed with the results obtained by Almodares et al. (2008) and Ashraf and Tufail (1995) in sunflower. In each barley cultivar, the decrease in the total soluble carbohydrates under salt stress may be due to that the building up in the chloroplast extent a direct toxic effect on photosynthetic processes (Munns & Tester, 2008) or to a reduction in CO2 assimilation rate and in the stomatal conductance. However, treatment with GSH or ASH has stimulated the accumulation of total soluble carbohydrates which are required as compatible solutes for osmoregulation in plants under water and salt-stresses (Hare et al., 1998). Accumulation of these compatible solutes reduces the osmotic potential in the cytoplasm and contributes to maintaining water homeostasis among several cellular compartments (Sairam & Tyagi, 2004), thus tuning the rate of photosynthesis to match the demand arising from grown inhibition.\n\nThe effects of glutathione or ascorbic acid on the accumulation of total soluble sugars can probably be attributed to their protective effects on the photosynthetic systems. However, GSH plays a protective role in salinity tolerance through the maintenance of the redox status (Chaparzadeh et al., 2004). Proline, an osmoprotectant, has been shown to accumulate more in plants in response to salinity (Ueda et al., 2007). Increased cellular proline levels are strongly correlated with the ability to withstand environmental challenges, such as salinity, and they can also act as an organic nitrogen reserve (Sairam & Tyagi, 2004). Proline levels rose in all barley cultivars when exposed to salinity (Somayeh et al., 2012), but it accumulated more in the salt-tolerant variety (Giza 124), which is related to its capacity to withstand salinity stress. The higher level of proline content in the two cultivars under salinity may be due to the expression of a gene encoding key enzymes of proline synthesis and low activity of the oxidizing enzymes which is controlled by either osmotic or salinity stress (Ma et al., 2015; Somayeh et al., 2012). Proline can act as an enzyme protector or a free radical scavenger. Results revealed that adding glutathione, ascorbic acid, or an amino acid combination reduced the level of proline. Such inverse correlation between these antioxidant compounds and proline in barley cultivars can be due to a deficit of common precursors which agreed with the results of Hoque et al. (2007) and Okuma et al. (2002) on Geum urbanum.\n\nHowever, the tolerant cultivar was found to have larger levels of the ROS-scavenging enzymes catalase and peroxidase than the sensitive one, indicating that the antioxidant system is crucial for plant tolerance against environmental stresses. In this respect (Nawaz & Ashraf, 2007; Sairam et al., 2000), found that variations in the antioxidant systems of wheat and maize genotypes lead to differences in how the plants react to diverse stresses. According to the current findings, salt stress boosted the CAT and POD activities in both barley varieties. The salt-tolerant (Giza 124) showed a greater increase than the salt-sensitive one, showing that the former was a more effective scavenger for the H2O2 radical and provided superior protection against H2O2. Similar results have been observed with Beta maritime (halophyte) compared to the non-halophyte Beta vulgaris (Bor et al., 2003), and wheat (Sairam et al., 2002) differing in salt tolerance. However, the addition of glutathione, ascorbic acid, or an amino acid mixture to salt-treated barley cultivars significantly reduced the activities of catalase and peroxidase, indicating a compensatory or substituting effect for the already present antioxidant enzymes by directly scavenging H2O2 and other ROS.\n\nMalonyldialdehyde (MDA), a criterion for evaluating salt injury, causes lipid peroxidation in a variety of plants (Ma et al., 2015; Somayeh et al., 2012). In response to salinity, both barley cultivars showed a highly significant increase in lipid peroxidation (at the seedling stage) and membrane leakage (at the seedling and Pre-flowering stages), with the salt-sensitive cultivar showing these effects more obvious than the salt-tolerant cultivar. The increase in MDA level when exposed to salt may be caused by oxidative damage to the chloroplasts and mitochondria or by antioxidants' inability to completely neutralize and scavenge all of the active oxygen species produced when exposed to salt stress. The present results agreed with those of (Demiral & Türkan, 2005) for two rice cultivars, (Chaparzadeh et al., 2004) for Calendula officinalis. Increasing lipid peroxidation during salt stress has been reported also (Xing et al., 2019) with Portulaca oleracea L.\n\nIn the two barley cultivars, MDA, as a product of the lipid peroxidation, has decreased with the addition of glutathione, ascorbic acid or amino acid mixture due to their compensating effects on the activities of antioxidant enzymes, acting as scavengers of cytotoxic H2O2 and reacting non-enzymatically with other ROS (Sairam et al., 2002). The amino acid mixture has reduced MDA content and membrane leakage which agreed with the results of Zhang and Kirkham (1996) for sorghum and sunflower seedlings.\n\nSeveral authors have reported increased levels of PAs when plants are exposed to diverse kinds of environmental stress (Bouchereau et al., 1999; Nayyar & Chander, 2004). The present results indicated that under salt stress, PUT increased in the tolerant cultivar (Giza 124) whereas Spd and Spm increased in the sensitive cultivar. In contrast to these findings, (Krishnamurthy & Bhagwat, 1989) found that during salt stress, rice cultivars that were salt-resistant acquired more Spd and Spm than sensitive ones. Additionally, they noted that sensitive rice accumulated more Put under salt than tolerant rice (Erdei et al., 1996) indicate that Spd and Spm accumulation in sorghum is one of the adaptive responses to salt stress. (Chattopadhayay et al., 2002; Willadino et al., 1996) proposed that accumulation of Spd and Spm may contribute to stress tolerance, while PUT accumulation may have no positive effect under salinity stress. Besides, it has been reported that, mainly in cereals, Put accumulation seemed to be related especially to the activation of arginine decarboxylase under stress conditions (Bouchereau et al., 1999).\n\nSalt stress has increased phosphorus concentration in the shoot and root of each barley cultivar which agreed with the results of (Taban, 2000; Yahya, 1998). According to (Roberts et al., 1984) the increase of P level in maize is the result of enhanced rates of uptake by the roots and of translocation to the shoots and not a concentration effect due to growth depression or may be due to ion imbalance between the uptake of sodium and ions including phosphorus (Lutts et al., 1999). However, under the effect of glutathione, ascorbic acid, or amino acid mixture with or without salt, phosphorus content decreased in each cultivar. This decrease may be due to the ameliorating effect or controlling the uptake of phosphorus which is linked with pH.\n\nResults showed that Giza 124 showed a greater accumulation of sodium under salinity compared to Giza 119, and this agreed with the results of (Flowers, 2004; He & Cramer, 1992). They found that maize shoots of Giza 2 (salt-tolerant) have greater levels of Na+ compared to those of Tri-hybrid 321(salt sensitive) under NaCl treatments. Furthermore, the greatest accumulation of sodium by plants at high salt concentrations may be attributed to the fact that selective salt absorption may be replaced by passive absorption which causes abnormal accumulation of salts in plant organs (Bouchereau et al., 1999). They suggested that under salinity sodium influx across the plasmalemma to the vacuole might play a major role in permitting turgor maintenance.\n\nIt may be suggested that the cultivar Giza 124 had the ability to sequester Na+ into the vacuole more efficiently than Giza 119. Several reports indicated that the absence of ion compartmentation may contribute to the toxic effects of ions in the shoot of sensitive plants (Flowers & Yeo, 1997). However, K+ concentration and consequently K+/Na+ ratio were decreased under salt-stress in the leaves of each cultivar. These decreases could be due to the effect of Na+ on K+ transport into the xylem or an inhibition of uptake processes (Gu et al., 2016; Yan et al., 2013). Again, decreasing in potassium content can be due to an antagonistic effect between sodium and potassium which has been confirmed by Azevedo Neto and Tabosa (2000). These antagonistic relations between Na+ and K+ may be taken as an indication of the role played by hormones in modifying K+/Na+ selectively under salt stress (Alpaslan & Gunes, 2001). Studies with the two barley cultivars showed that salinity has a significant reducing effect on the K+ concentration in both shoot and root, which can be referred to the competition and resultant selective uptake between K+ and Na+ which causes an increase in uptake of Na+ at the cost of K+ (Kuiper, 1984) or due to a decrease in sink size with the higher concentrations of NaCl, which strongly inhibited shoot and root growth (Ashrafuzzaman et al., 2000). Gebauer et al. (2003) reported that K+ accumulation was reduced in the stem and increased in leaves and roots with increasing concentration of NaCl. On the other hand (Flowers & Yeo, 1997). Epstein (1998) claimed that enhanced K+ uptake can be an adaptive mechanism that allows the cell to evade K+ starvation in the presence of high NaCl concentrations, which does not agree with the present study.\n\nResults showed an increase in shoot-nitrogen content in each barley cultivar. Flanagan and Jefferies (1988) showed that when plants are subjected to high salinity, higher nitrogen content was associated with osmotic solute or an accumulation of nitrate ions or increased protein degradation. Application of glutathione, ascorbic acid or amino acid mixture with or without salt has led to a reduction in nitrogen content in the shoot of Giza 124 compared to the control and the treatment of the salt alone, while in Giza 119, glutathione without salt and ascorbic acid with salt caused a higher nitrogen content compared to control and salt alone. This difference can be due to impairment of nitrogen metabolism in the sensitive cultivars while the tolerant one was able to use nitrogen for enzymatic defense system against salt. In contrast, root-nitrogen content decreased with salinity in each cultivar.\n\nConcerning the yield, the present results showed that, in both cultivars, NaCl stress resulted in a reduction in grain weight in both cultivars which can be confirmed with the results of Sairam et al. (2000) and Sohrabi et al. (2008) for chickpea and (Taffouo et al., 2009) for some cowpea (Vigna unguiculata). Grain weight reduction can be related to the disturbance in the translocation due to toxic ions or a reduction in the photosynthesis, imbalance in mineral uptake, protein synthesis or carbohydrate metabolism (Al-Garni, 2006). However (Zeng et al., 2002, p. 2), reported that the few differences in grain weight of rice (Oryza sativa L.) genotypes under salinity stress can be related to plant genus and genotype.\n\n\nConclusion\n\nSalinity stress resulted in the reduction of all growth parameters, pigment contents, photosynthetic efficiency, total soluble carbohydrates and grain weight in the seedlings, pre-flowering, and grain yield stages. On the other hand, salinity enhanced the activities of the antioxidant enzymes, catalase, and peroxidase, and increased the proline content of the three growth stages. The addition of glutathione, ascorbic acid, or amino acid mixture led to the enhancement of the defense mechanism of barley against salinity stress which was reflected in all previous parameters. Also, the present study indicated that Giza 124 as a salt-tolerant cultivar was more resistant to the damage caused by salt stress compared to Giza 119 as a salt-sensitive one. This salt-sensitive cultivar showed more MDA as a product of lipid peroxidation and membrane leakage with salt stress. The results indicated that the alleviation effect was finally reflected in the growth criteria of the plants suggesting that glutathione may be recommended as the best treatment to alleviate the effect of salinity on barley.", "appendix": "Data availability\n\nFigshare: Spreadsheets, https://doi.org/10.6084/m9.figshare.22082354.v1 (El Dakkak, 2023).\n\nThis project contains the following underlying data:\n\n- Catalse (seedling).csv\n\n- Chlorophyll (preflowering G119).csv\n\n- Chlorophyll (preflowering).csv\n\n- Chlorophyll-Seedling Stage G119.csv\n\n- Chlorophyll-Seedling Stage-G124.csv\n\n- Growth Criteria Seedling Stage-G124.csv\n\n- Growth Criteria Seedling Stage-G119.csv\n\n- Lipid Peroxidation.csv\n\n- Membrane Leakage.csv\n\n- Nitrogen (yield).csv\n\n- Nitrogen (seedling).csv\n\n- Peroxidase (seedling).csv\n\n- Phosphorous (seedling).csv\n\n- Phosphorous (yield).csv\n\n- Polyamines.csv\n\n- Potassium.csv\n\n- Proline Content (seedling stage).csv\n\n- Proline (yield).csv\n\n- Protein Nitrogen.csv\n\n- Sodium-Potassium.csv\n\n- Succulence – Root.csv\n\n- Succulence – Shoot.csv\n\n- Total Soluble Carbohydrates (Preflowering).csv\n\n- Total Soluble Carbohydrates (seedling).csv\n\n- Total Soluble Carbohydrates (yield).csv\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\nAcknowledgments\n\nI would like to express my deep gratitude to my late advisor Prof. Dr. Mohamed Nabih El-Shourbagy, Professor of Plant Physiology, Botany Department, Faculty of Science, Tanta University for his kind supervision, great help, valuable suggestions and advice during the progress of this work. I am greatly indebted to Prof. Dr. Fatma A. El-Shintinawy, Professor of Plant Physiology, Botany Department, Faculty of Science, Tanta University for her kind assistance during the initial stage of this thesis. I would also want to thank Late Prof. Dr. Wedad Abd El-Aziz Kasim, Professor of Plant Physiology, Botany Department, Faculty of Science, Tanta University for her help and encouragement as well as for the critical reading of manuscripts.\n\n\nReferences\n\nAbd El-Hameid AR, Sadak MS: Impact of glutathione on enhancing sunflower growth and biochemical aspects and yield to alleviate salinity stress. Biocatal. Agric. Biotechnol. 2020; 29: 101744. Publisher Full Text\n\nAbdel-Farid IB, Marghany MR, Rowezek MM, et al.: Effect of salinity stress on growth and metabolomic profiling of Cucumis sativus and Solanum lycopersicum. 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Plant. 2013; 35(10): 2867–2878. Publisher Full Text\n\nYan Z, Ming D, Cui J, et al.: Exogenous GSH protects tomatoes against salt stress by modulating photosystem II efficiency, absorbed light allocation and H2O2-scavenging system in chloroplasts. J. Integr. Agric. 2018; 17(10): 2257–2272. Publisher Full Text\n\nYancey PH, Clark ME, Hand SC, et al.: Living with water stress: Evolution of osmolyte systems. Science. 1982; 217(4566): 1214–1222. PubMed Abstract \| Publisher Full Text\n\nZeng L, Shannon MC, Grieve CM: Evaluation of salt tolerance in rice genotypes by multiple agronomic parameters. Euphytica. 2002; 127(2): 235–245. Publisher Full Text\n\nZhang J, Kirkham MB: Lipid peroxidation in sorghum and sunflower seedlings as affected by ascorbic acid, benzoic acid, and propyl gallate. J. Plant Physiol. 1996; 149(5): 489–493. Publisher Full Text\n\nZhao FG, Qin P: Protective effect of exogenous polyamines on root tonoplast function against salt stress in barley seedlings. Plant Growth Regul. 2004; 42(2): 97–103. Publisher Full Text\n\nZúñiga GE, Argandoña VH, Corcuera LJ: Distribution of glycine-betaine and proline in water stressed and unstressed barley leaves. Phytochemistry. 1989; 28(2): 419–420. Publisher Full Text" }	[ { "id": "186050", "date": "30 Aug 2023", "name": "Nasim Ahmad Yasin", "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\nAuthors of the manuscript “Effect of polyamine precursors and antioxidants on growth and metabolism of salt-stressed barley” observed that salinity stress resulted in the reduction of all growth parameters, pigment contents, photosynthetic efficiency, total soluble carbohydrates and grain weight in the seedlings, pre-flowering, and grain yield stages. On the other hand, salinity enhanced the activities of the antioxidant enzymes, catalase, and peroxidase, and increased the proline content of the three growth stages. The addition of glutathione, ascorbic acid, or amino acid mixture led to the enhancement of the defense mechanism of barley against salinity stress which was reflected in all previous parameters.\nThis study is interesting. However, authors need to discuss the following issues:\nPlease add some data of your findings in the abstract section. Additionally, write benefits of polyamines precursors in treated plants.\n\nWhat is the novelty of this study?\n\nWhat was the hypothesis of this study?\n\nPlease rewrite following statements:\n“The pots were divided into five categories: the grains were presoaked for 24 hours in distilled water, an amino acid solution (Arginine, Methionine, and Ornithine) (1 mM each), glutathione, or ascorbic acid (0.1 mM for each), seedlings were cultivated in 0.1 mM sodium chloride alone, seedlings were cultivated using a combination of salt and glutathione, seedlings were grown using a combination of salt and ascorbic acid, seedlings were cultivated using a combination of salt and amino acid mixture”.\n“The addition of glutathione, ascorbic acid, or amino acid mixture alleviated the harmful effect of sodium chloride and improved barley's defense mechanism against salt stress”.\n“It has become widely known that reactive oxygen species (ROS) are to responsible for a diverse variety of stress-induced damage to macromolecules and, ultimately, cellular structure (Sairam et al., 2002)”.\n“In view of the lack of knowledge regarding the role of polyamines in salt tolerance of grain crops, it is attempted in the present study examines the effect of polyamine precursors; arginine, methionine, and ornithine, besides the effect of some antioxidants as glutathione and ascorbate on growth, metabolism, and productivity of two barley cultivars, different in salt tolerance, when subjected to salt stress”.\n“Giza 124 was chosen as the most tolerant cultivar and Giza 119 as the most sensitive one with 100 mM NaCl to be used in the present study”.\n\nWrite keywords alphabetically. Don’t use that word as a key word which is part of the title.\n\nWhy the cultivars Giza117, Giza119, Giza 121, Giza 123, Giza 124, Giza 125, Giza 126 and Giza 2000 were used for the current study?\n\nPlease clearly describe each treatment.\n\nWhy seedlings exposed to salt stress after a week?\n\nHow antioxidant solution was applied?\n\nWhat do you mean by clay sandy soil (2:1)?\n\nDescribe the physicochemical characteristics of the soil used.\n\nOn what basis chemicals concentrations were selected to observe the stress alleviation response?\n\nPlease discuss the mechanisms through which salinity or antioxidants application resulted physiological/ biochemical or morphological modulations in treated plants.\n\nDo you feel that this study provides an economical and eco-friendly solution for mitigation of salt stress?\n\nWhat are future perspectives of this study?\n\nReplace old references with latest ones.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10851", "date": "18 Jan 2024", "name": "EMAN ELDAKKAK", "role": "Author Response", "response": "1. Why the cultivars Giza117, Giza119, Giza 121, Giza 123, Giza 124, Giza 125, Giza 126, and Giza 2000 were used for the current study? These cultivars were used at the beginning of the study as a first step to determine their tolerance and sensitivity. The decision to choose only two species was made from the beginning, prompting us to assess various cultivars to inform our final selection. 2. What is the novelty of this study? The novelty of this study lies in its investigation into the impact of polyamine precursors and antioxidants on the growth, metabolism, and productivity of two barley cultivars with varying salt tolerance when exposed to salt stress. Specifically, the study explores the effects of presoaking seeds in different solutions, including amino acid mixtures (Arginine, Methionine, and Ornithine), glutathione, and ascorbic acid, in the context of salt-tolerant (G124) and salt-sensitive (G119) barley cultivars. 3. Why are seedlings exposed to salt stress after a week? Exposing the seedlings to salt stress after a week serves a specific purpose in this experiment. The one-week duration allows the seedlings to establish and develop to a certain extent before subjecting them to salt stress. This initial growth period enables the seedlings to form a stable root system and develop basic physiological structures. 4. What was the hypothesis of this study? Hypothesis 1 states that the salt-tolerant barley cultivar (G124) will exhibit better growth parameters, higher photosynthetic pigments, increased total soluble carbohydrates, greater weight, and a higher number of grains compared to the salt-sensitive cultivar (G119) under salt stress conditions. Hypothesis 2 states that activities of antioxidant enzymes (catalase and peroxidase), proline content, malondialdehyde levels, and membrane leakage will be altered in response to salt stress, and the addition of polyamine precursors and antioxidants will mitigate these changes in both barley cultivars. Hypothesis 3 states that the combination of polyamine precursors and antioxidants will act cooperatively in alleviating the harmful impact of salt stress on barley cultivars, leading to enhanced defense mechanisms and improved plant growth. 5. How antioxidant solution was applied? The antioxidant solution (glutathione or ascorbic acid) has been dissolved in water and applied directly to the soil surrounding the plant roots. This method ensures that the antioxidants are taken up by the plant roots and transported throughout the entire plant. 6. What do you mean by clay sandy soil (2:1)? The term \"clay sandy soil (2:1)\" refers to a soil mixture composed of clay and sand in a specific ratio. In this case, the ratio is 2 parts of clay to 1 part of sand. This ratio indicates the proportion of the two soil components in the mixture. 7. Describe the physicochemical characteristics of the soil used. Clay soil is characterized by its fine particles and tends to hold water and nutrients well. However, it can have poor drainage and may compact easily. It has a higher cation exchange capacity (CEC), which means it can hold more nutrients for plant uptake. On the other hand, sandy soil has larger particles and generally provides good drainage. However, it may not retain water and nutrients as effectively as clay. Sandy soils often have a lower CEC. 8. On what basis chemical concentrations were selected to observe the stress alleviation response? We experimented with several salt concentrations to find the most suitable level. For other treatments, we relied on insights from prior research studies and reports in comparable experiments. Additionally, the identification of a salt-tolerant cultivar (Giza 124) highlights the environmentally beneficial practice of cultivating naturally resistant crops. 9. Do you feel that this study provides an economical and eco-friendly solution for the mitigation of salt stress? The study will have a significant economic impact, particularly if the recommended treatments work effectively in real agricultural settings. These treatments might be an affordable choice that benefits farmers financially by raising crop yields and improving crop quality. 10. What are the future perspectives of this study? Future perspectives could include long-term evaluations of the sustainability and practical applicability of the suggested treatments and the salt-tolerant cultivar (Giza 124). Practical insights could be obtained through field trials conducted in various environmental conditions and integrated into precision agriculture procedures (GPS, sensors, etc..). In addition, research in molecular and genetics may reveal underlying systems that aid in the creation of genetically engineered crops." } ] }, { "id": "221298", "date": "30 Nov 2023", "name": "Chandra Shekhar Seth", "expertise": [ "Reviewer Expertise Stress Physiology" ], "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\nReviewer’s comments to Authors-\n\nWhile there is value in compilation the paper needs a revision due to several minor mistakes occurring throughout the manuscript. The written language needs to be improved.\nAbstract:\nThe section describing the method can be crisper while elaborating the result and conclusion\nIntroduction:\nThe objectives of this study are missing in the introduction section.\n\n“Because of the negative effect of salt stress on crop productivity, crop productivity loss might exceed 60% of crop yield globally”. Rephrase the sentence, repetition of crop productivity.\n\n“Furthermore, salinity has impacted plant growth and development by increasing salt content, particularly ionic chloride cl and sodium Na+”. Correct symbol Chloride (Cl).\n\n“Plants respond to salt stress by producing biochemical compounds and activating molecular systems at the cellular and plant levels (Li et al., 2019). Most cultivated plant species are highly sensitive and either die or display reduced productivity after exposure to salt stress for long periods.” Instead of generalized plant impact you should focus on barley salinity stress.\nMaterials and Methods:\n“Grains were presoaked for 24 hrs in distilled water or amino acid mixture (Arginine, methionine and ornithine) 1mM for each or 0.1 mM for glutathione or 1 mM for ascorbic acid.” You mean using the amino acid mixture, glutathione, ascorbic acid alone separately. Rephrase this statement accordingly for clarification.\n\n“One-week old seedlings were grown under the combined effect of 0.1 mM glutathione plus salt”. It means both were applied in the soil or firstly treated with glutathione and grown after in salt applied soil? Please clarify.\n\nFor pigment estimation optical densities are taken of acetone extract on the same day otherwise there is possible changes in the reading. Please confirm the methodology and clarify.\n\nEstimate the initial fluorescence (F0). Initial fluorescence is represented as Fo not as F0. Check and correct.\n\nThe lipid peroxidation was measured in the seedling stage by the amount of malonyl dialdehyde (MAD). Malonyl dialdehyde is abbreviated as MDA not as MAD. Correct at all places.\nReferences:\nMaintain uniformity in referencing. Try to add DOI for each reference.\n\nThe different sections of the manuscript are poorly cited with references and required updating and validation with previous studies. The relevant papers listed below1-5 may be considered to enhance the scientific quality of the manuscript significantly.\nResults and discussion:\nWrite about the statistical analysis, how it is done using what procedure etc. Show the significant differences using alphabets.\n\nConsidering the result obtained the discussion is concise. Enrich it.\nConclusion:\nThe conclusion section of a review should be in accordance to the content.\n\nConclusion section failed to enlighten the spirit of the finding. Revise it precisely.\n\nConclusion section must also include the future perspectives of this study which is lacking in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10852", "date": "18 Jan 2024", "name": "EMAN ELDAKKAK", "role": "Author Response", "response": "1. A comprehensive modification was made to the abstract, adding new information and improving language according to the reviewer's suggestions. 2. The introduction was strengthened by adding the objectives with a more extensive range of references to support the context. 3. I rephrased and revised the sentences as per the reviewer's suggestion. 4. In the methodology section, the initial part was reworded to enhance clarity regarding the treatments and align the pigment approach more cohesively. Furthermore, a detailed overview of the statistical analysis process was provided. 5. The discussion section was enhanced by improvements that provided further details regarding the findings and perspectives from earlier studies. 6. The conclusion section was rephrased, incorporating perspectives for future research directions to provide a more forward-looking conclusion. 7. More citations were incorporated. Additionally, more keywords were added and categorized alphabetically for better search engine performance." } ] } ]	1	https://f1000research.com/articles/12-262
https://f1000research.com/articles/12-531/v1	23 May 23	{ "type": "Study Protocol", "title": "Comparison of growth factors isolated from exosomes present in maternal blood of cleft and non-cleft infants for identification and quantification of growth factors involved in the growth and development of cartilage.", "authors": [ "Nikita Soni", "Priyanka Niranjane", "Pallavi Daigavane", "Biswa Pratim", "Mrudula Shinde", "Priyanka Niranjane", "Pallavi Daigavane", "Biswa Pratim", "Mrudula Shinde" ], "abstract": "Background: Cleft lip and palate (C.L.P.) is a common congenital condition which affects the orofacial region. The aetiology of C.L.P. is multifaceted and involves genetic and environmental factors that impact the shape and position of the nasal cartilage, resulting in an asymmetrical appearance of the nose. Growth factors are crucial in regulating cartilage synthesis and repair. Exosomes have emerged as a promising cell-free drug delivery system that facilitates intercellular communication and maintains tissue homeostasis. This study identifies and quantifies the growth factors present in exosomes derived from lactating maternal blood of cleft and non-cleft infants and compares them to evaluate the differences. The study aims to assess the potential of these exosomes as a therapeutic approach for C.L.P. management. Methods: Blood samples were collected from lactating mothers at Sharad Pawar Dental College and isolating plasma using centrifugation. Exosome and total protein isolation will be conducted in the laboratory, and immunoblotting will evaluate growth factors. Quantitative and comparative protein and growth factor analysis will be conducted separately for maternal blood samples. Results: The growth factors responsible for cartilage growth and development will be present in maternal exosomes, potentially leading to novel therapies for C.L.P. management. The study aims to demonstrate the therapeutic potential of exosomes, as carriers of intra-cellular factors, in transporting growth factors to the required site for managing C.L.P. Conclusions: Overall, by understanding the roles played by growth factors, intercellular communication, and genetic and environmental factors in the aetiology of C.L.P., this research can contribute to creating effective treatment strategies for C.L.P. management. Finding growth factors in exosomes made from the blood of nursing mothers could be a cutting-edge therapeutic strategy for treating C.L.P.", "keywords": [ "cleft lip and palate", "congenital anomalies", "nasal cartilage", "growth factors", "exosomes", "lactating maternal blood", "intercellular communication", "cartilage synthesis." ], "content": "Introduction\n\nCleft lip and palate (C.L.P.) constitute one of the common congenital disabilities associated with inherited genetic conditions affecting the orofacial region of a child. This condition can be present with numerous variations and combinations. The Prevalence of C.L.P. is very high in the Asian and American populations compared to the African population.1 According to a study in 2009, India, with approx., 1.1 billion population yields 24.5 million births per year, with a birth prevalence of clefts around 27,000-33,000 clefts per year. The incidence of C.L.P. is higher in males than in females.1 This congenital disability creates medical, psychological, and social problems affecting individuals and their families. Genetic and environmental variables play a role in the complex multifactorial aetiology of C.L.P.1 The genes responsible for non-syndromic C.L.P. are FGFR2, IRF6, FGF8 and BMP42 and shared environmental factors are maternal smoking, alcohol consumption, consanguineous marriage consumption of certain medications such as Topiramate, Valproic acid during the first trimester, nutritional deficiency like folic acid deficiency, Vit-A deficiency, chemical exposure like cosmetics, pesticides & Psychological factors such as stress and, depression.1\n\nThe anatomy of C.L.P. includes the abnormal shape and position of the nasal septum and nasal tip cartilage, which creates an uneven appearance of the nostril, nasal tip, and nasal dome/contour. The deviated nasal cartilage is moulded and corrected with the help of various P.S.I.O. appliances to mould the nasal cartilage pre-surgically.3 The nasal cartilage is a structure within the nose that provides form and support to the nasal cavity and is made of hyaline cartilage.4 As the cartilage is avascular connective tissue having no direct blood supply, it receives its nutrition by diffusion from the surrounding, and this increases when there is an increase in compressive forces acting on it. The structure and organization of cartilage’s extra-cellular matrix are crucial for the proper functioning of cartilage because cartilage contains particular, differentiated cells whose main job is to maintain the stability of matrix elements. When wounded, its capacity for self-repair is constrained.5\n\nThe growth and development of cartilages are regulated by various growth factors which signal extra-cellular matrix and other components.6\n\nEven after a prosperous cleft palate and lip surgery in a C.L.P. child, there are chances for relapse, possibly due to something lacking on a cellular level. Intercellular communication is an essential function for maintaining homeostasis or direct cell-to-cell interaction.\n\nSince the previous decade, extra-cellular vesicles, particularly exosomes, have drawn more attention for their potential applications.6 Recent research has shown that exosomes can serve as a portal for cell-free drug delivery systems that retain their original characteristics from the parent cell. Extra-cellular vesicles, or exosomes, are found in nearly all cells, tissues, and bodily secretions.7 They support intercellular communication and preserve tissue homeostasis in pathological physiology. Nine thousand seven hundred sixty-nine proteins, 2838 miRNAs, 3408 mRNAs, and 1116 lipids are found in exosomes.\n\nThese exosomes can act as the drug delivery tool as their characteristics are derived from the parent cell. Almost all the cells, blood, tissue, saliva, tear, breast milk, urine, and G.I.T. secretion secrete exosomes. Exosomes can cross the blood-brain barrier and the lipid bilayer guards against immune cells & enzymes. They can regenerate new blood vessels & cellular proliferation.8\n\nExosomes are secreted as extra-cellular vesicles and contain growth factors amongst many other essential cellular components. These exosomes contain signalling growth factors which may have therapeutic potential in the growth and development of nasal cartilage in C.L.P. infants.9\n\nThere is a need to access various factors, illustrated in Table 1 (genes, D.N.A., R.N.A., protein, enzyme, growth factor), responsible for the growth and development of cartilage. It was thought that accessing the quality and quantity of growth factors in the exosomes that a sign has a role in the cartilage’s growth and development.\n\n\n\n• TGF superfamily (β1, β2, β3)9\n\n\n\n• As IL-1 activates matrix-degrading enzymes, suppresses matrix synthesis, and inhibits chondrocyte proliferation, it increases the synthesis of E.C.M. and lowers the catabolic activity of M.M.Ps and IL-1.\n\n• Promotes cartilage synthesis, chondrocytes growth and cartilage repairs as it shows high collagen type II activity\n\n\n\n• BMP-79\n\n\n\n• It promotes the production of E.C.M.\n\n• Decreases IL and MMP activity and expression, lessening cartilage breakdown.\n\n\n\n• FGF-29\n\n• FGF-18\n\n\n\n• Increases chondrocyte proliferation and stimulates ECM.\n\n• Function in post-natal maintenance of cartilage stimulates cartilage development and regeneration of hyaline cartilage.\n\nThe following study is one of a kind. The growth factors present in the mothers are genetically transferred to the child to produce the type of cartilage with specific characteristics. They thought to assess the quality and quantity of growth factors in the mother of a child with C.L.P. and a child with non-cleft. The current conventional treatment regime for C.L.P. management does not address the cellular aspect, especially the cellular signalling for cartilage moulding. This rate-limiting factor towards C.L.P. management may be overcome by providing cartilage growth and development-triggering growth factors.\n\nThe following study is designed with the hypothesis that there may be a difference in the quality and quantity of growth factors in the blood of lactating mothers of cleft and non-cleft infants.\n\nThe study presents a challenging scenario in managing cleft lip and palate (C.L.P.) due to the complex process of nasoalveolar cartilage moulding. The importance of time in the initial 2-3 months of a child’s life for cartilage moulding is emphasized, but C.L.P. infants are deprived of essential nutrients in the mother’s milk. While some developed countries provide fortified milk for C.L.P. infants, developing countries like India rely heavily on animal-derived milk, which has limited nutrients. This lack of essential nutrients can lead to a decline in the crucial 2–3-month period to less than one month, failing mechanical appliances like nasal stunts.\n\nTo identify growth factors present in exosomes of lactating maternal blood of cleft and non-cleft infants.\n\nTo quantify the identified growth factors in the exosomes of lactating maternal blood of cleft and non-cleft infants.\n\nTo compare the identified growth factors present in exosomes of lactating maternal blood of cleft and non-cleft infants.\n\nAnalytical observational study.\n\nThe following study will be conducted in the Department of Orthodontics and Dentofacial Orthopedics at Sharad Pawar Dental College in collaboration with Central Research Laboratory (Center of Translation Sciences), Sawangi, Wardha.\n\nThe lactating mothers visiting the Department of Orthodontics and Dentofacial Orthopedics, Department of Gynecology & Obstetrics and Department of Pediatrics and fulfilling the inclusion criteria will be included in the study.\n\nLactating mother of non-syndromic isolated cleft lip and palate infants less than six months of age.\n\nAge of mothers <35 years.\n\nNo systemic conditions such as diabetes and hypertension.\n\nAge of mother >35 years (due to hormonal changes).\n\nLactating mother with systemic disease.\n\nLactating mother with developmental and congenital disease.\n\nLactating mother of an infant of more than six months of age.\n\nNon-lactating mothers\n\nControl Group: Mothers of healthy infant (non-cleft infant).\n\nStudy Group: Mothers of an infant with cleft lip and palate.\n\nEach lactating mother included in the sample will be explained the need for the study and the entire procedure in their language of understanding, and informed consent will be obtained from them. The blood sample collection will be done in the Department of Oral Pathology/Oral Surgery, Sharad Pawar Dental College, in collaboration with the Department of Gynecology. 2 ml of the blood sample will be collected in EDTA collection tubes. Once sample collection is done, it will be taken to the laboratory in the R&D house (D.M.I.H.E.R.) for evaluation. First, the plasma will be separated through a centrifugation process stored at -80 degree Celsius till further experimentation; the isolation of exosomes from plasma afterwards isolation of total proteins from the exosome will be done following standard protocol and followed by an analysis of growth factors from the isolated total proteins (from both the exosome) through immunoblotting. Once the analysis is done, the quality and quantity of growth factors responsible for cartilage growth and development will be compared for both groups.\n\nImmunoblotting is a technique in which host antibodies identify a target protein via antigen-antibody reaction as it identifies the target protein among the number of unrelated proteins. Proteins are electrophoretically separated and then moved to nitrocellulose membranes. This method makes use of the following three components:\n\n(1) Size division.\n\n(2) Transfer to a firm support.\n\n(3) Using a primary and secondary antibody to identify a target protein visually.\n\nExamples for equipment’s used for each component mentioned above are listed here:\n\n• Electrophoresis apparatus: Bio-Rad Mini-PROTEAN system, Invitrogen XCell SureLock Mini-Cell system, etc.\n\n• Transfer apparatus: Bio-Rad Trans-Blot system, Invitrogen iBlot system, GE Healthcare Amersham Biosciences Hybond Transfer System.\n\n• Blocking buffer: BSA and non-fat dry milk.\n\n• Primary antibodies: Santa Cruz Biotechnology, Cell Signaling Technology, Thermo Fisher Scientific.\n\n• Secondary antibodies: anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP).\n\n• Chemiluminescence substrate: SuperSignal West Pico, SuperSignal West Femto from Thermo Fisher Scientific, etc.\n\n• Imaging system: Bio-Rad ChemiDoc MP Imaging System, Thermo Fisher Scientific iBright CL750 Imaging System, etc.\n\n• After immunoblotting, proteins and growth factors will be analyzed separately for maternal blood of C.L.P. and non-CLP infants, and quantification and comparison for both groups will be made.\n\nOUTCOME: Following are the outcomes with their description for measurements mentioned below:\n\nThe proposed study is intended to evaluate the growth factors like TGF-β and B.M.P., present in exosomes of lactating maternal blood, which can be identified for different quantities and quality.\n\nGiven this, isolated maternal exosomes are expected to contain ample growth factors involved in cartilage growth and development, which can be helpful as a therapy alongside prevalent procedures in C.L.P. management.\n\nBIAS: Bias will be minimized as patients are random and will be selected from the O.P.D. of the Department of Orthodontics and Dentofacial Orthopedics of Sharad Pawar Dental College and Hospital, Wardha\n\nThe sample size was calculated by using Daniel’s formula for sample size:\n\nWhere,\n\nZ∝2 = is the level of significance at 5%, i.e., 95% confidence interval = 1.96\n\nP = Prevalence of cleft lip and palate = 1% = 0.01\n\nd = Desired error of margin = 6% = 0.06\n\nn is the population size\n\nTotal sample size = 30\n\nAll the demographic and outcome data will be presented using descriptive statistics for continuous variables, can be categorized using mean, standard deviation, and median for discrete variables, and frequency and proportion for continuous variables.\n\nThe outcome variable will be tested for normality using KALMOGOROV-SMIRNOV TEST for continuous data. Results will be analyzed on R software version 3.2.\n\nGrowth factors are categorized according to the range that will be distributed for analyzing the data into the normal range and not in the normal range. The chi-square test be used to find the result of an association of growth factors with the blood of mothers of cleft and non-cleft infants.\n\nAn odd ratio will be used to find the risk involved multiple times.\n\nAn Independent t-test will be used to find the results on two groups for outcome variables if data comes under the normal distribution. A non-parametric test will find a significant difference if data does not come under a normal distribution.\n\nThis study aims to evaluate the growth factors present in exosomes of lactating maternal blood for their potential use in cleft lip and palate (C.L.P.) management. The study hypothesizes that growth factors enhance cartilage growth and development, crucial during the first 2-3 months of a child’s life. However, C.L.P. infants are often deprived of essential nutrients in the mother’s milk, leading to a decline in the duration of cartilage moulding. Exosomes in lactating maternal blood can carry essential growth factors, hormones, and estrogen, which can be transported to the required site for therapeutic potential in C.L.P. management.\n\nThe study has yet to start.\n\n\nDiscussion\n\nThe study suggests that exosomes found in lactating maternal blood could be a promising therapeutic option in managing cleft lip and palate (C.L.P.). These exosomes can carry various growth factors, estrogen, or hormones to the site of injury, facilitating the growth and development of cartilage in C.L.P. infants. The study emphasizes the need for innovative approaches to address the challenges in managing C.L.P., particularly in developing countries with limited resources.\n\nNumerous studies have demonstrated the potential of exosomes to carry growth factors. For instance, exosomes produced by stem cells have been studied as a treatment for osteoarthritis (O.A.) due to their potential to protect against chondroprotection, as emphasized by the research by Kwang ho Yoo et al. (2022).10 Additionally, exosomes derived from mesenchymal cells contain various growth factors like TGFβ1, VEGF, H.G.F., cytokines, and proteins, as demonstrated by Kan Yin et al. in 2019.11\n\nStudies have also shown the significance of genes like BMP7 and TGF-BETA2 in promoting the production of cartilage matrix, regulating the endochondral ossification process, and maintaining homeostasis in both healthy and unhealthy states of cartilage, as indicated by Yoo et al. (2022)10 and Mengmeng Duaner et al. (2021),12 respectively. Overall, these studies highlight the immense therapeutic potential of exosomes and genes like BMP7 and TGF-BETA2 in promoting cartilage growth and development. The findings emphasize the need for further research and innovative approaches to managing conditions like C.L.P. and O.A., particularly in developing countries with limited resources.\n\nEthical considerations:\n\nEthical approval received by Datta Meghe Institute of Higher Education and Research, Sawangi, Wardha\n\nIEC ref no.- DMIHER (DU)/IEC/2023/572\n\nWritten informed participant consent will be obtain prior to the study taking place.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nRepository name: Zenodo\n\nTitle of Project: Comparison of growth factors isolated from exosomes present in maternal blood of cleft and non-cleft infants for identification and quantification of growth factors involved in the growth and development of cartilage.\n\nFile Name: STROBE cross-sectional Checklist\n\nDOI: 10.5281/zenodo.7780107\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank the Institute and its colleagues for their support.\n\n\nReferences\n\nOner DA, Tastan H: Cleft lip and palate: Epidemiology and etiology. Otorhinolaryngol-Head Neck Surg. 2020 [cited 2023 Jan 24]; 5(4). Publisher Full Text Reference Source\n\nLeslie EJ, Marazita ML: Genetics of Cleft Lip and Cleft Palate. Am. J. Med. Genet. C Semin. Med. Genet. 2013 Nov; 163(4): 246–258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAttiguppe PR, Karuna YM, Yavagal C, et al.: Presurgical nasoalveolar molding: A boon to facilitate the surgical repair in infants with cleft lip and palate. Contemp. Clin. Dent. 2016; 7(4): 569–573. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaddam P, Bayona-Rodriguez F, Campbell SM, et al.: Properties of the Nasal Cartilage, from Development to Adulthood: A Scoping Review. Cartilage. 2022 Mar 28; 13(1): 19476035221087696. PubMed Abstract \| Publisher Full Text\n\nTuan RS, Chen AF, Klatt BA: Cartilage Regeneration. J. Am. Acad. Orthop. Surg. 2013 May; 21(5): 303–311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang LR, Marston G, Martin A: Anatomy, Cartilage. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2023 Jan 25]. Reference Source\n\nYu W, Serrano M, Miguel SS, et al.: Cleft lip and palate genetics and application in early embryological development. Indian J. Plast. Surg. Off. Publ. Assoc. Plast. Surg. India. 2009 Oct; 42(Suppl): 35. Publisher Full Text\n\nLiu Y, Ma Y, Zhang J, et al.: Exosomes: A Novel Therapeutic Agent for Cartilage and Bone Tissue Regeneration. Dose-Response. 2019 Dec 13; 17(4): 1559325819892702. PubMed Abstract \| Publisher Full Text\n\nExosomes: biogenesis, biologic function and clinical potential\|Cell & Bioscience: 2023 [cited 2023 Jan 25]. Publisher Full Text\n\nYoo KH, Thapa N, Chwae YJ, et al.: Transforming growth factor-β family and stem cell-derived exosome therapeutic treatment in osteoarthritis (Review). Int. J. Mol. Med. 2022 May; 49(5): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFortier LA, Barker JU, Strauss EJ, et al.: The role of growth factors in cartilage repair. Clin. Orthop. 2011 Oct; 469(10): 2706–2715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYin K, Wang S, Zhao RC: Exosomes from mesenchymal stem/stromal cells: a new therapeutic paradigm. Biomark. Res. 2019 Apr 4; 7: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "190178", "date": "01 Aug 2023", "name": "Junichi Iwata", "expertise": [ "Reviewer Expertise craniofacial development", "cell signaling pathways", "animal models", "salivary gland", "tooth development", "microRNA", "cleft lip and palate", "autophagy", "tongue development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript aims to propose a study protocol to analyze exosomes present in blood from mothers with cleft- and non-cleft infants to identify and quantify growth factors involved in the growth and development of infant’s nasal cartilage. Unfortunately, the proposed protocol lacks detailed information for the evaluation of the methodology, data interpretation, potential outcomes, etc. In addition, it has been known that contents of exosomes change in various physiological and phraseological conditions. Currently, there is no preliminary data to support the hypothesis. At least some preliminary studies would be necessary before starting the study in humans (for instance, in any CL/P animal models or in small scale human data). Lastly, it is unclear how the authors identify particular growth factors and signaling networks through this study. Advanced bioinformatic analyses would be required; however, in this proposal it is unclear.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "10260", "date": "18 Jan 2024", "name": "nikita soni", "role": "Author Response", "response": "REVIEWER’S QUESTIONS RESPONSE 1.The proposed protocol lacks detailed information for the evaluation of the methodology, data interpretation, potential outcomes, etc. Methodology - In the methodology section, every step of the procedure is explained, from blood collection to isolating and analyzing growth factors (TGF-BETA, EGF), using the immunoblotting method. Detailed procedure of immunoblotting and the necessary equipment used in it is also elaborated. Data interpretation- Descriptive statistics for continuous variables will be used to offer data interpretations, and the KALMOGOROV-SMIRNOV TEST for continuous data will be used to test the outcome variable. R software version of SPSS software 3.2 will be used to analyze the results. Potential outcomes- identification, quantification, and comparison of growth factors isolated from maternal blood of cleft and non cleft infant mothers. 2.contents of exosomes change in various physiological and phraseological conditions As rightly quoted by the reviewer, due to various physiological conditions like stress, smoking , alcohol, medications etc, the contents of exosomes and accordingly the secretion of various growth factors and their amount may vary in exosomes and correspondingly in maternal blood. The author is also trying to prove that there may be a difference in quantity of growth factors secreted in exosomes in maternal blood of cleft and non cleft infants and this difference in quantity of growth factors in maternal blood can be corelated with the chondrogenesis and occurrence of cleft in infant. 3. There is no preliminary data to support the hypothesis. At least some preliminary studies would be necessary before starting the study in humans There are many articles that discuss the interactions between chemical components found in various exogenous and external stimuli and particular growth factors that change their working pathway and, ultimately, affect the expression of proteins, growth factors, etc.which regulate cell proliferation, differentiation, and migration, leading to a variety of congenital diseases, including cleft lip and palate. For example, The below-mentioned article explains the pathways occurring in pregnancy and their connection with placental nourishment. “Tossetta G, Marzioni D. Signaling Pathways in Pregnancy. Cells. 2022 Apr 20;11(9):1385. doi: 10.3390/cells11091385. PMID: 35563691; PMCID: PMC9101431” There is no evidence available in the literature that directly correlates the alteration in the expression of growth factors and the causation of cleft lip and palate, but indirect evidence depicting the correlation between the amount and expression of growth factors affecting at molecular level and occurrence of congenital abnormalities including cleft lip and palate which forms the basis for the hypothesis investigated in the present study. 4. it is unclear how the authors identify particular growth factors and signaling networks through this study. Advanced bioinformatic analyses would be required The growth factors will be found using the immunoblotting approach. The goal of this research is not to understand signalling networks." } ] }, { "id": "174820", "date": "01 Aug 2023", "name": "Mohsen Sheykhhasan", "expertise": [ "Reviewer Expertise stem cell biology", "extracellular vesicle", "exosome", "cancer", "immunotherapy." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 protocol entitled “Comparison of growth factors isolated from exosomes present in maternal blood of cleft and non-cleft infants for identification and quantification of growth factors involved in the growth and development of cartilage\" appears to be interesting, but there are many flaws and concerns on it.\nStudy can be greatly improved if following suggestions were incorporated:\nTitle: the title is not appropriate. I suggesting making up it.\n\nPaper is replete with grammatical mistakes. Needs rewriting and thorough evaluation.\n\nSome references missing. For example, “Cleft lip and palate (C.L.P.) constitute one of the common congenital disabilities associated with inherited genetic conditions affecting the orofacial region of a child.” And etc.\n\nProtocol section requires more information: To confirm the success of exosome isolation, in addition to the NTA and transmission electron microscopy, it is necessary to confirm using the Western blot analysis and DLS. As a result, it is better to include the results of the NTA, transmission electron microscopy, the Western blot analysis and DLS in the Protocol section of the present paper.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\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": [ { "c_id": "10259", "date": "18 Jan 2024", "name": "nikita soni", "role": "Author Response", "response": "REVIEWER’S QUESTIONS RESPONSE 1. Title: the title is not appropriate. I suggesting making up it. The title of article gives a much clearer summary of my research. It answers questions like: What I’m doing In whom am I doing this study? Reason for choosing and doing this study. 2.Paper is replete with grammatical mistakes. Needs rewriting and thorough evaluation The article has been proofread several times, and software was used to check for grammatical errors. 3.Some references missing. For example, “Cleft lip and palate (C.L.P.) constitute one of the common congenital disabilities associated with inherited genetic conditions affecting the orofacial region of a child.” And etc The words you specified are fully cited under reference (1) and similarly. 4. Protocol section requires more information: To confirm the success of exosome isolation, in addition to the NTA and transmission electron microscopy, it is necessary to confirm using the Western blot analysis and DLS. As a result, it is better to include the results of the NTA, transmission electron microscopy, the Western blot analysis and DLS in the Protocol section of the present paper Even though NTA and TEM yield better results, they will be chosen for subsequent research." } ] } ]	1	https://f1000research.com/articles/12-531
https://f1000research.com/articles/12-214/v1	27 Feb 23	{ "type": "Opinion Article", "title": "Why cost-effectiveness thresholds for global health donors differ from thresholds for Ministries of Health (and why it matters)", "authors": [ "Tom Drake", "Y-Ling Chi", "Alec Morton", "Catherine Pitt", "Y-Ling Chi", "Alec Morton", "Catherine Pitt" ], "abstract": "Healthcare cost-effectiveness analysis is increasingly used to inform priority-setting in low- and middle-income countries and by global health donors. As part of such analyses, cost-effectiveness thresholds are commonly used to determine what is, or is not, cost-effective. Recent years have seen a shift in best practice from a rule-of-thumb 1x or 3x per capita GDP threshold towards using thresholds that, in theory, reflect the opportunity cost of new investments within a given country. In this paper, we observe that international donors face both different resource constraints and opportunity costs compared to national decision makers. Hence, their perspective on cost-effectiveness thresholds must be different. We discuss the potential implications of distinguishing between national and donor thresholds and outline broad options for how to approach setting a donor-perspective threshold. Further work is needed to clarify healthcare cost-effectiveness threshold theory in the context of international aid and to develop practical policy frameworks for implementation.", "keywords": [ "Global health", "health financing", "aid", "priority-setting", "cost-effectiveness", "threshold" ], "content": "Introduction\n\nTo maximise population health for the resources available, and accelerate progress towards universal health coverage, health systems must make use of evidence to identify which interventions and services to prioritise for investment. Indeed, the last two decades have seen increasing use of evidence-informed priority setting to guide resource allocation decisions in many low- and middle-income countries (LMICs). In 2014, the World Health Assembly Resolution WHA67.23 urged countries to consider the use of Health Technology Assessment (HTA) to inform a range of resource allocation decisions from coverage of medicines in formularies to inclusions in benefits packages.1 The use of formal priority setting processes and methods has intensified in the past decade.2 A cost-effectiveness threshold is a decision-rule that can be used alongside a cost-effectiveness analysis (CEA) to determine whether an intervention’s incremental cost-effectiveness ratio (ICER) - that is, the ratio of the additional costs and benefits of an intervention, compared to the next-best alternative – indicates that it would constitute an efficient (i.e., cost-effective) use of scarce resources in a given context. Cost-effectiveness thresholds have gained greater prominence in academic and policy circles in recent years; however, the use of such thresholds is still nascent in many LMICs, in some cases due to the lack of formal processes and institutions to guide coverage decisions.3\n\nThis push for the use of CEA and cost-effectiveness thresholds at the national level stands in contrast to methods applied by donors to inform the allocation of Development Assistance for Health (DAH). In recent years, annual DAH has stabilised at around $40bn ($54bn in 2020 including Covid) and represents about a quarter of health spending in low-income countries.4,5 While some DAH supports research, advocacy, or other activities, the vast majority supports service delivery.4 While many donors have adopted Value for Money (VfM) frameworks6 or other forms of assessments, such as cash benchmarking,7 to our knowledge, few rely substantially on CEA to prioritise their funding allocation to programmes or between countries. The use of explicit decision thresholds by donors is even rarer; with the exception of foundations from the effective altruism movement (i.e., Givewell8 and Open Philanthropy9), we have found none. When such methods are applied, they typically do not consider investments from national decision-makers, which can create issues of fragmentation, lack of alignment with national priorities, displacement of national funds, and duplication of investments.10\n\nThere is much debate over what cost-effectiveness thresholds are meant to represent.11–13 For country thresholds, an emerging consensus is that the threshold should: i) reflect national resource availability, and ii) in application, be equal to the opportunity cost of alternative marginal healthcare spending.12–14 Two papers from Woods et al.,15 and Ochalek et al.,16 provide initial estimates of national thresholds for 182 countries based on this “supply-side” perspective. Some health economists have suggested that donors should align with national cost-effectiveness thresholds.17\n\nIn this piece, we outline the case for distinguishing between donor and national cost-effectiveness thresholds, both in terms of the theoretical basis for the threshold and the potential benefits of clearer separation. We seek to build on and complement existing conversations on the use of cost-effectiveness thresholds in LMICs.14–21\n\nIn short, we argue that donors and national decision-makers should adopt different cost-effectiveness thresholds from one another because they have different decision perspectives, shaped by the following two factors:\n\ni) Different resource constraints. At the country level, the purpose of DAH is to alleviate local resource constraints and increase the fiscal space for health. The broad aim of DAH is to support the provision of health services that otherwise would not be possible. If the (possibly hypothetical) national cost-effectiveness threshold is aligned with national resources, then an (equally hypothetical) donor threshold should be higher, reflecting the additional resources introduced.\n\nii) Different opportunity costs due to the global nature of DAH. Unlike national institutions, which must prioritise their investments within a single country, global health donors may choose between support for health services across many countries. Therefore, a donor’s opportunity cost of investing in intervention A in country X is not only intervention B in country X, but also intervention C in country Y. For example, while investing in Covid vaccines for the over 60’s in Kenya may represent good value compared with alternative investments in Kenya, it may be more cost-effective to support the roll out of bed nets in Malawi.\n\nThe lack of clarity around differences in donor and national cost-effectiveness thresholds is indicative of the lack of clarity in the decision perspectives and the roles that different actors have in funding healthcare in LMICs. Despite global health financing being a multi-billion-dollar sector where rhetoric on evidence-informed priority-setting is commonplace, many donors lack a clear framework for prioritisation. Collaboration between donors and national institutions in countries which receive DAH is often complex, political, and constantly negotiated for both donors and countries. The result is a fragmented system of financial support that impedes national health leaders in their work to develop an efficient and effective health system.22\n\nThe application of separate cost-effectiveness thresholds that reflect the perspective of decision-makers and donors can help to clarify the roles and responsibilities of national vs international funders of health services in LMICs; in other words, it would create a structure for who-should-fund-what. National institutions could design and fund a cohesive core package of the most-cost-effective services up to their national thresholds and “invite” donors to support a top-up package of the next-most-cost-effective services (see Figure 1). The role of DAH would therefore be auxiliary: donors would fund interventions above the national threshold, up to their own threshold (we will discuss what this would look like below). In other words, the national cost-effectiveness threshold would represent a ceiling for a national payers and a floor for donors, below which they would not seek to fund activities in that country.\n\nThis approach could address some of the greatest challenges in global health financing.10 First, it could focus national resources towards funding a core package of the most essential services, which could ensure that funding for the provision of key services is not affected by aid volatility. Second, fragmentation of health financing23,24 (and resulting duplication) could be reduced by a clearer separation of funding responsibilities. Further, the application of separate cost-effectiveness thresholds can avoid displacement of domestic resources by aid. Greater prioritisation of health interventions from a national perspective could be achieved, especially in designing a core package of most essential services – which can maximise the impact of overall health funding, rather than of funding streams operating in silos. The approach would also empower national institutions to set their own priorities, rather than needing to work within the complex and fragmented financing space created by ad hoc donor support. At present, a significant share of health prioritisation is de facto done in donor headquarters and does not necessarily reflect national priorities; which is especially important in countries where the share of DAH in total health expenditure is high. Moreover, in this framework, as domestic finances increase, so might the national health budget and national cost-effectiveness threshold, and health aid is naturally crowded out. Conversely, transition from aid or the ending of specific aid programmes does not disrupt the provision of the most-cost-effective services.\n\nBeyond reforms to within-country resource allocation, clearer frameworks for evidence informed prioritisation could help donors equitably and effectively prioritise investments between countries. A clear donor cost-effectiveness threshold would promote the concentration of funding from global health donors in the programmes and contexts in which the greatest health gains can be made with the resources available. This approach is consistent with the ethical position that all health gains should be valued equally, regardless of where occur and how they are produced.\n\nHow might cost-effectiveness thresholds be set to reflect those two decision perspectives?\n\nFor national thresholds, methodological approaches to setting a threshold have been discussed extensively elsewhere.14,25 In 1993, the World Bank suggested income-group-specific thresholds of US$50-200 per disability-adjusted life-year (DALY) averted.26 These were superseded by the World Health Organisation’s Choosing Interventions that are Cost-Effective (WHO-CHOICE) programme, which suggested that interventions with an ICER below 3x or 1x gross domestic product (GDP) per capita could be considered “cost-effective” or “highly cost-effective”, respectively.21 The use of WHO-CHOICE thresholds is now being discouraged because they appear to be too high and do not adequately reflect the resource limitations of LMICs. Indeed WHO health economists note they were never intended to be used in the way they often were.12,27 Recently, health economists have sought to clarify the theory underpinning thresholds, as we discussed in the introduction, linking it to resource availability and local opportunity costs; with a set of estimates produced for LMICs in two papers.15,16 It is worth noting that only few countries have defined an explicit cost-effectiveness threshold – for example, the UK and Thailand do, while Austria and Kenya do not.28\n\nIn contrast, there is almost no literature discussing what a cost-effectiveness threshold could look like from a donor perspective. Drake (2014) outlines a case for a minimum DALY value to guide donor prioritisation.18 Morton et al., (2017) describe an approach towards subsidising and crowding-in services which are just cost-ineffective from a national perspective, but does not address donor-perspective thresholds.19 GiveWell uses a benchmark that charity programmes should be expected to provide value at least 10 time greater than cash transfers29 and Open Philanthropy require a 1000 fold expected return on investment for their (generally higher risk and upstream) investments.9\n\nIn attempting to set a threshold from a donor perspective, a first question is whether global health donors would all follow a single cost-effectiveness threshold or develop their own, agency-specific threshold. It is tempting to view each donor as having its own decision perspective and institutional mandate and therefore its own threshold. Use of numerous donor-specific thresholds could retain the advantage of improving the efficiency of each donor’s allocation between contexts, but the key challenges to coordination with national institutions and other donors would remain. To realise the benefits of improved donor harmonisation outlined in the section above, a shared donor threshold is necessary. This could be a joint threshold agreed between donors at a country level forum, such as a Sector Wide Approach (SWAp), which would facilitate the benefits of country level harmonisation but would lose the benefits of between-country resource allocation. A generalised global threshold would be required to achieve both within- and between-country benefits.\n\nIn broad terms, how could a global health donor threshold be set?\n\nOption 1: Notional. Many countries and organisations use CEA to guide healthcare prioritisation without formally defining a cost-effectiveness threshold. One option is for donors to use the theoretical possibility of a separate cost-effectiveness threshold to shape policy and the clarify roles with regards to national decision-makers, without quantifying the threshold itself.\n\nOption 2: Supply-side. Supply-side estimation means linking the threshold to the resources available and what they currently achieve in health production, at the margin. That is, a new investment opportunity should be more cost-effective than the next-best alternative that additional funding could support instead. If a cost-effectiveness threshold should reflect the payer’s opportunity costs, then a donor’s threshold should reflect opportunity costs at the global level, and should therefore be the same across all countries in which the donor may consider investing. It may also be possible to use statistical analysis analogous to the techniques used for estimating healthcare opportunity cost at the country level for domestic finances30 to estimate the opportunity cost of marginal health aid globally.\n\nOption 3: Demand-side. In contrast to the resource-linked supply-side approach, a demand-side route to setting a donor-perspective threshold could mean defining an aspirational benchmark that relevant stakeholders agree on. For example, participants in a World Health Assembly could support an aspirational declaration that all countries should be able to provide services that produce health for $X per DALY averted. That is, a minimum DALY value above which services should be considered worthy of investment, regardless of affordability to the national healthcare provider. Such an approach bridges the philosophical position of right-to-health advocates and technical optimisation approaches of health economists by effectively establishing a minimum value on health and therefore a right to services that can produce health for this minimum standard. The drawback of the aspirational target is that it may allow sub-optimal allocation decisions if the demand-side aspiration is radically different from the supply-side reality. However, an important advantage of such a threshold is that it would function not only as an optimisation tool, but an advocacy goal.\n\n\nConclusions\n\nIn this article, we have argued that cost-effectiveness threshold(s) for global health donors should differ from thresholds for national institutions because they have different decision perspectives, budgets, and opportunity costs. We then explored some of the potential benefits of distinguishing explicitly between donor and national thresholds and briefly outlined the options for setting those thresholds. We acknowledge that the approach we propose will entail a major shift in the way donors operate by explicitly moving from maximising the impact/cost-effectiveness of their own investments, towards playing a supporting role to national decision-makers. There are also practical challenges in the application of this framework, including the absence of a set national threshold (or ‘threshold thinking’), lack of country processes and institutions to prioritise interventions and develop a core package of essential services, and the lack of cost-effectiveness evidence. Despite these challenges, developing an improved framework for priority setting in countries where aid constitutes a substantial share of health financing could yield numerous and substantial benefits for the strengthening of health systems in those countries. For this reason, we call for further work to: i) advance methodological theory for national and donor collaboration on resource allocation, and ii) explore the political economy of such reforms.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe authors are grateful to Tony Culyer, Peter Baker and David Bath for comments on earlier drafts of the manuscript.\n\n\nReferences\n\nWorld Health Assembly 67: Sixty-seventh World Health Assembly, Geneva, 19-24 May 2014: resolutions and decisions: annexes. World Health Organization; 2014 [cited 2022 Dec 21]. Report No.: WHA67/2014/REC/1. Reference Source\n\nBump JB: Global health aid allocation in the 21st century. 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PubMed Abstract \| Publisher Full Text \| , Free Full Text Reference Source\n\nSantos AS, Guerra-Junior AA, Godman B, et al.: Cost-effectiveness thresholds: methods for setting and examples from around the world. Expert Rev. Pharmacoecon. Outcomes Res. 2018 Feb 27 [cited 2022 May 30]; 18: 277–288. PubMed Abstract \| Publisher Full Text\n\nThe World Bank: World Development Report 1993: Investing in Health. Oxford University Press; 1993 [cited 2015 Nov 27]; 346. (World Development Report). Publisher Full Text\n\nBertram MY, Lauer JA, Stenberg K, et al.: Methods for the Economic Evaluation of Health Care Interventions for Priority Setting in the Health System: An Update From WHO CHOICE. Int. J. Health Policy Manag. 2021 Nov 1; 10(Special Issue on WHO-CHOICE Update): 673–677. Publisher Full Text\n\nSchwarzer R, Rochau U, Saverno K, et al.: Systematic overview of cost-effectiveness thresholds in ten countries across four continents. J. Comp. Eff. Res. 2015 Sep; 4(5): 485–504. PubMed Abstract \| Publisher Full Text\n\nElie: An update on GiveWell’s funding projections. The GiveWell Blog.2022 [cited 2023 Jan 23]. Reference Source\n\nClaxton K, Martin S, Soares M, et al.: Methods for the Estimation of the NICE Cost Effectiveness Threshold. Health Technol. Assess. 2015 Feb 18; 19(14): 1–504. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "166801", "date": "03 Apr 2023", "name": "Jessica Ochalek", "expertise": [ "Reviewer Expertise Application of economic evaluation principles and methods to inform priority setting in healthcare", "particularly in low- and middle-income countries." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion article aims to provide a neat and tidy solution to the problem of how donors should prioritise their funding for health both within and across countries.\nThe authors start with the assumption that donors should apply a different threshold than that which should be applied by national decision makers allocating government resources for health. The latter, we agree, should reflect the marginal productivity of the healthcare system. Where there is less agreement is around whether donors should use a separate threshold at all and, if they must, what that threshold should be based upon.\nThe authors rightly point out that the opportunity cost of investing in intervention A in country X is not only intervention B in country X, but also intervention C in country Y. One way to consider this is using the net health effect of the intervention within each country and across countries. (See Claxton, K. P., Ochalek, J. M., Revill, P., Rollinger, A. & Walker, D., Informing Decisions in Global Health: Cost Per DALY Thresholds and Health Opportunity Costs, Nov 2016, 4 p. Centre for Health Economics, University of York). Net health effects for each country are a function of the cost to the country (or the cost savings), health impacts and an estimate of health opportunity cost to the country of government expenditure on health. Summing these across countries gives the global net health impact. Once this is known, the donor can select what they regard as the best (based not just the net health effects but also on their distribution across countries).\n\nIt may be helpful to distinguish between general budget support and off-budget support. Where funding is provided in the form of general budget support, it effectively becomes part of the government pool. Therefore, the opportunity cost of such general budget support is most closely the opportunity cost of government expenditure on health. There is no reason for a separate threshold, and donors should consider the marginal productivity of expenditure on health within the country when considering general budget support. Ceteris paribus, an expansion of the budget would result in an increase in the marginal productivity of the healthcare system.\nWhere donors are providing off-budget support, given that one possibility is to give the money for the intervention to countries (on budget), donors need to consider if their off-budget project performs better or, if not, whether there are good reasons to fund it nonetheless.\nWhere donors are providing off-budget support and have an exogenously fixed budget, then a supply side approach might be reasonable. This requires two things: an estimate of a “donor ICER” and an estimate of what is displaced from among existing donor-funded healthcare when the donor funds a new intervention. Where the latter is unknown, a reasonable approach would be to rank by donor ICER. Calculating a “donor ICER” requires first calculating the global net health effects. Then calculating a “donor ICER” from the cost to the donor and the global net health.\nWhere donors are providing off-budget support and have an endogenous budget, they may choose to expand their budget to continue to fund interventions until the donor’s willingness to pay for health is exhausted. Ideally, funding decisions would be taken in order of most to least cost-effective. This better aligns with the objective of health maximisation than the demand-side approach described by the authors in Option 3. Donors taking a demand side approach as described in Option 3 may fund healthcare that would be highly cost-ineffective for the country to fund. The problem with this is that donor funding can negatively impact on the efficiency of systems and sustainability of interventions through “the duplication of services, dilution and distortion of limited human and financial resources, and weak coordination between levels of care” (Barr, A., Garrett, L., Marten, R. et al. Health sector fragmentation: three examples from Sierra Leone. Global Health 15, 8 (2019). https://doi.org/10.1186/s12992-018-0447-5). A better advocacy tool might be to illustrate the extent of possible health gains by funding healthcare closer to the margin of what is generated by the healthcare system as illustrated in Figure 1, where spending the money required to fund aid-supported services would generate more health than spending the same money to fund excluded services.\nWhile the authors are right that focusing national resources towards funding a core package of the most essential services would help to ensure that those services are not subject to aid volatility, a caveat to the proposal for aid supported services to be those which are just cost-ineffective for the country rather than those which are cost-effective for the country, is that many countries are not able to deliver the healthcare interventions they do provide to the full population in need of them. Adding additional services may only further detract from this ability by further diluting human resource capacity etc. Donors might instead consider spending their resources to ensure that the essential services included in the core package are accessible to everyone who requires them.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "10901", "date": "18 Jan 2024", "name": "Tom Drake", "role": "Author Response", "response": "We thank for the reviewers for their commentary in response to our article and appreciate their research in this space. Below we offer point-by-point responses to the feedback using [R] to denote reviewer text and [A] for author text. [R] This opinion article aims to provide a neat and tidy solution to the problem of how donors should prioritise their funding for health both within and across countries. The authors start with the assumption that donors should apply a different threshold than that which should be applied by national decision makers allocating government resources for health. The latter, we agree, should reflect the marginal productivity of the healthcare system. Where there is less agreement is around whether donors should use a separate threshold at all and, if they must, what that threshold should be based upon. [A] We build a case throughout the article (and especially in the first section titled, “Why cost-effectiveness thresholds for global health donors should differ from thresholds for Ministries of Health“) that donors should apply a different threshold from national decision-makers. We give two key reasons for the need for a different approach, namely, the differing resource constraints and the differing opportunity costs, which we explain. To make this clearer we have updated the beginning of this first section to read: “Donors may distribute DAH to pursue various objectives, which may include reducing preventable mortality and morbidity, protecting their own country’s health (e.g., by reducing global infectious burden of disease) or fostering wider political and economic national interests. Where health maximisation is at least one of the purposes of a donor’s DAH, and some form of cost-effectiveness thresholds are therefore potentially relevant, then we argue that the adoption of a separate threshold to national decision-makers becomes useful for the following reasons:” [R] The authors rightly point out that the opportunity cost of investing in intervention A in country X is not only intervention B in country X, but also intervention C in country Y. One way to consider this is using the net health effect of the intervention within each country and across countries. (See Claxton, K. P., Ochalek, J. M., Revill, P., Rollinger, A. & Walker, D., Informing Decisions in Global Health: Cost Per DALY Thresholds and Health Opportunity Costs, Nov 2016, 4 p. Centre for Health Economics, University of York). Net health effects for each country are a function of the cost to the country (or the cost savings), health impacts and an estimate of health opportunity cost to the country of government expenditure on health. Summing these across countries gives the global net health impact. Once this is known, the donor can select what they regard as the best (based not just the net health effects but also on their distribution across countries). [A] Thank you for this comment. We agree that net health (or monetary) benefit is one way to make cross-country comparisons. However, a key limitation to this approach is that the conversion factor between money and health in net health calculations, the health production function, is typically the cost-effectiveness threshold. If the CET should reflect the total resource envelope, then it will also be a product of the donor allocation decision as well as - in this use case - a determinant of it. Secondly, a key element of our proposed approach in the section \"Why does this matter\" is the process of negotiation and partnership that can result from explicit negotiation of who will fund which services and why. A joint threshold, concealed in a net health benefit calculation does not support such an approach. [R] It may be helpful to distinguish between general budget support and off-budget support. Where funding is provided in the form of general budget support, it effectively becomes part of the government pool. Therefore, the opportunity cost of such general budget support is most closely the opportunity cost of government expenditure on health. There is no reason for a separate threshold, and donors should consider the marginal productivity of expenditure on health within the country when considering general budget support. Ceteris paribus, an expansion of the budget would result in an increase in the marginal productivity of the healthcare system. Where donors are providing off-budget support, given that one possibility is to give the money for the intervention to countries (on budget), donors need to consider if their off-budget project performs better or, if not, whether there are good reasons to fund it nonetheless. Where donors are providing off-budget support and have an exogenously fixed budget, then a supply side approach might be reasonable. This requires two things: an estimate of a “donor ICER” and an estimate of what is displaced from among existing donor-funded healthcare when the donor funds a new intervention. Where the latter is unknown, a reasonable approach would be to rank by donor ICER. Calculating a “donor ICER” requires first calculating the global net health effects. Then calculating a “donor ICER” from the cost to the donor and the global net health. [A] Thank you for raising the important practical consideration of how the proposed framework might be affected by the practical approach to financing and delivery. We agree that these are important considerations and in fact are the subject of ongoing follow up work. We disagree that our proposed approach is incompatible with on-budget support. First, tracking and monitoring of on-budget support is technically feasible (if burdensome), enabling donor funding to be earmarked for specific services while being channelled through public systems. Second, thresholds could be used during budget formulation to negotiate and agree levels of on budget support to provide, even if contributions are not then traced through public financial systems. Thirdly, when a donor provides on-budget support to a country, the opportunity cost of that support is the value that could have been gained had that DAH been invested in either a project in that country OR in a project or on-budget support in another country. So the fact that for donors the opportunity cost of on-budget support is not restricted to the recipient country implies a different decision threshold. Public financial management considerations will be the subject of future publications and are beyond the scope of this commentary. We have added a note to the article that “Further work is needed to consider the public financial management implications of on- or off-budget support using this framework.” [R] Where donors are providing off-budget support and have an endogenous budget, they may choose to expand their budget to continue to fund interventions until the donor’s willingness to pay for health is exhausted. Ideally, funding decisions would be taken in order of most to least cost-effective. This better aligns with the objective of health maximisation than the demand-side approach described by the authors in Option 3. Donors taking a demand side approach as described in Option 3 may fund healthcare that would be highly cost-ineffective for the country to fund. The problem with this is that donor funding can negatively impact on the efficiency of systems and sustainability of interventions through “the duplication of services, dilution and distortion of limited human and financial resources, and weak coordination between levels of care” (Barr, A., Garrett, L., Marten, R. et al. Health sector fragmentation: three examples from Sierra Leone. Global Health 15, 8 (2019). https://doi.org/10.1186/s12992-018-0447-5). A better advocacy tool might be to illustrate the extent of possible health gains by funding healthcare closer to the margin of what is generated by the healthcare system as illustrated in Figure 1, where spending the money required to fund aid-supported services would generate more health than spending the same money to fund excluded services. [A] Thank you for this comment. We do not promote Option 3 over and above Options 1 and 2, but rather, attempt to describe the strengths and weaknesses of each. We agree with the potential risks of a demand-derived threshold and acknowledge them in the article, where we write: \"The drawback of the aspirational target is that it may allow sub-optimal allocation decisions if the demand-side aspiration is radically different from the supply-side reality.\" We also allude to the potential for endogenous budgets and the potential for aid budgets to be expanded in line with aspirational targets, that is, indications of what donors’ collective willingness-to-pay should be. At the beginning of the section on options for setting thresholds, we recognise the historic and ongoing issues with using the 1x and 3x GDP thresholds for optimising resource allocation from a country decision-maker perspective. For completeness in describing the options available for setting a donor perspective threshold, it makes sense to include both supply and demand options and the significant differences between the country and donor perspective thresholds, both in terms of the cohesiveness of the budget/payer and how the threshold could inform resource allocation and mobilisation. [R] While the authors are right that focusing national resources towards funding a core package of the most essential services would help to ensure that those services are not subject to aid volatility, a caveat to the proposal for aid supported services to be those which are just cost-ineffective for the country rather than those which are cost-effective for the country, is that many countries are not able to deliver the healthcare interventions they do provide to the full population in need of them. Adding additional services may only further detract from this ability by further diluting human resource capacity etc. Donors might instead consider spending their resources to ensure that the essential services included in the core package are accessible to everyone who requires them. [A] We agree that ensuring effective quality delivery of the core package is a priority and that development partners may have an important role to play in achieving this goal, including by financing (or even directly delivering) these services for a period. However, as part of the development process, countries must be supported to take on the responsibility for financing and delivering the highest priority services and this could easily happen while total budget envelope is still low enough to require financial support to achieve an acceptable essential package of services. In short, we agree that the approach should be a new deal or compact to strengthen country processes for prioritisation, financing and delivery, place countries in the driving seat of the prioritisation process and (implicitly or explicitly) link additional financing for services to those at the margin as defined by countries, not to funder priorities." } ] }, { "id": "183976", "date": "01 Aug 2023", "name": "Chris Sampson", "expertise": [ "Reviewer Expertise Health economics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI see no reason why this opinion piece should not be indexed. There are no factual errors or essential omissions. However, there are several respects in which the article may be improved, and I would encourage the authors to consider the following suggestions for a revised version. My suggestions are - roughly - in order of importance.\n1. The article proceeds on the assumption that health maximisation is the objective of (or at least an important consideration for) global health donors. It isn't clear to me that this is the case. This assumption requires i) clearer articulation as part of the authors' argumentation and ii) further discussion of its validity.\n\n2. Even if health maximisation is the objective of global health donors, it isn't clear to me that the proposal summarised in Figure 1 provides a means of achieving this. It seems far more likely to me that global health donors might prefer to spend on infrastructural investments and not marginal technologies, in part because the former is more conducive to health maximisation. The authors should discuss the scenarios in which a donor's use of a threshold would and would not support health maximisation.\n3. The authors should consider (and describe) the suitability of the bookshelf analogy. In the context of low-income countries - perhaps more so than others - the bookshelf analogy is problematic. In particular, the bookshelf analogy starts to fall apart when we allow for divisibility, which is certainly a relevant consideration in countries in which providers cannot reach all patients.\n4. Some further consideration should be given (and discussion provided) to the claim that a \"donor threshold should be higher\". There are numerous assumptions inherent in this, and the authors have not described them. For instance, it seems to assume that resources are currently being allocated efficiently by national decision-makers. It behoves the authors to outline all of the assumptions in their claim.\n5. Throughout the article, 'thresholds' are characterised as a matter of fact, an observable quantity. This is not correct; thresholds are a policymaking tool with a primarily normative basis. For instance, the title should read that cost-effectiveness thresholds could or should differ, rather than stating that they do differ.\n6. The authors state that few donors rely on CEA. This raises the question of why we should care about cost-effectiveness thresholds at all! The authors should acknowledge the limited applicability and importance of their assertions in this context. Indeed, there needs to be more justification for the idea that donor cost-effectiveness thresholds should be specified at all.\n\n7. The authors refer to the use of decision thresholds by Givewell and Open Philanthropy. It would be helpful to state these thresholds in the text.\n8. The claim is made that \"many donors\" have adopted value for money frameworks, but then only one is cited. Either more citations should be added or the claim should be altered.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "10902", "date": "18 Jan 2024", "name": "Tom Drake", "role": "Author Response", "response": "We thank the reviewer for this helpful review of the article. Below we offer point-by-point responses to the feedback using [R] to denote reviewer text and [A] for author text. [A] We would like to thank the reviewer for his time in providing the feedback, which has helped us to improve and clarify the manuscript. We have taken on many of the comments and adjusted the article accordingly. Point-by-point responses are offered below. [R] I see no reason why this opinion piece should not be indexed. There are no factual errors or essential omissions. However, there are several respects in which the article may be improved, and I would encourage the authors to consider the following suggestions for a revised version. My suggestions are - roughly - in order of importance. 1. The article proceeds on the assumption that health maximisation is the objective of (or at least an important consideration for) global health donors. It isn't clear to me that this is the case. This assumption requires i) clearer articulation as part of the authors' argumentation and ii) further discussion of its validity. [A] Thank you for this helpful comment. To address this point, we have added a sentence that explicitly acknowledges the range of objectives that donors may pursue at the top of the section titled, “Why cost-effectiveness thresholds for global health donors should differ from thresholds for Ministries of Health”: The section now reads: “Donors may distribute DAH to pursue various objectives, which may include reducing preventable mortality and morbidity, protecting their own country’s health (e.g., by reducing global infectious burden of disease) or fostering wider political and economic national interests. Where health maximisation is at least one of the purposes of a donor’s DAH, and some form of cost-effectiveness thresholds are therefore potentially relevant, then we argue that the adoption of a separate threshold to national decision-makers becomes useful for the following reasons:” 2. Even if health maximisation is the objective of global health donors, it isn't clear to me that the proposal summarised in Figure 1 provides a means of achieving this. It seems far more likely to me that global health donors might prefer to spend on infrastructural investments and not marginal technologies, in part because the former is more conducive to health maximisation. The authors should discuss the scenarios in which a donor's use of a threshold would and would not support health maximisation. [A] We agree that investments like infrastructure are outside the scope of this commentary. To the extent that aid is directed towards health services (rather than health systems investments, global public goods and so on), we argue that conceptualising separate country and donor cost-effectiveness thresholds is theoretically clearer and could present opportunities for practical benefits. We have added a clarification in the second paragraph of the introduction to this effect. The sentence reads “While some DAH supports research, advocacy, or other catalytic activities, the vast majority supports service delivery, including both financing for specific services (which we focus on in this article) and health system strengthening such as infrastructure investments”. 3. The authors should consider (and describe) the suitability of the bookshelf analogy. In the context of low-income countries - perhaps more so than others - the bookshelf analogy is problematic. In particular, the bookshelf analogy starts to fall apart when we allow for divisibility, which is certainly a relevant consideration in countries in which providers cannot reach all patients. [A] We think that the bookshelf analogy remains relevant. It depends on how the “books” are specified. Additional sentence added: “Each service – or book in the bookshelf metaphor – could be defined either at the level of the national population or for specific subpopulations for whom the cost-effectiveness differs because of high need, geographical inaccessibility, or other factors.” 4. Some further consideration should be given (and discussion provided) to the claim that a \"donor threshold should be higher\". There are numerous assumptions inherent in this, and the authors have not described them. For instance, it seems to assume that resources are currently being allocated efficiently by national decision-makers. It behoves the authors to outline all of the assumptions in their claim. [A] We have now revised the start of the section, “Why cost-effectiveness thresholds for global health donors should differ from thresholds for Ministries of Health” to articulate more clearly the different objectives donors may pursue and our assumptions about the relevance of health maximisation (albeit not as sole criterion) to decision-making. We certainly do not intend to claim that resources are currently efficiently allocated and our arguments do not depend on such an assumption. Instead, we are arguing that increased use of thresholds could support a transition to more efficient resource allocation. We have also modified our claim in this section to argue only that the differing resource constraints mean that donor and country thresholds should be “different”; we present the wider rationale for why donor thresholds should be higher in the following section, where we are able to outline the considerations more fully. A revised section on why resource constraints differ now reads: “Different resource constraints. National decision-makers face budget constraints which reflect their country’s overall resources; the extent to which their government is able to mobilise those resources; and the prioritisation of health within national budgets. Donors, by contrast, face wholly separate resource constraints. Country donors’ budgets are constrained by their country’s resources and prioritisation of (health) aid to other countries, while private and multilateral donors’ budgets are constrained by their ability to raise revenue from government and private contributors globally. If cost-effectiveness thresholds reflect the scarcity of resources, then the differing resource constraints imply that differing thresholds should be used.” 5. Throughout the article, 'thresholds' are characterised as a matter of fact, an observable quantity. This is not correct; thresholds are a policymaking tool with a primarily normative basis. For instance, the title should read that cost-effectiveness thresholds could or should differ, rather than stating that they do differ. [A] Thank you for raising this important point. We agree and have adjusted the language throughout, including in the title of the article. 6. The authors state that few donors rely on CEA. This raises the question of why we should care about cost-effectiveness thresholds at all! The authors should acknowledge the limited applicability and importance of their assertions in this context. Indeed, there needs to be more justification for the idea that donor cost-effectiveness thresholds should be specified at all. [A] CEA is used quite widely in several countries because the analytic community has done a good job of crafting analytic tools which are rooted in economic theory but which speak directly to the decisions faced by in-country decision makers and are contextually appropriate. We haven’t got there for donor aid yet, we would argue because the approach has been to carry over the in-country CEA paradigm rather than figuring out how to implement those economic principles in the donor environment. On prima facie basis, economic analysis, as the science of resource scarcity should have a greater role in the allocation of donor aid than it currently does. We hope that our commentary will encourage wider use of CEA and appropriate cost-effectiveness thresholds. New text: “Economic analysis has huge potential to help donors maximise the achievement of their objectives in the face of the vast gap between need and the resources available. However, the current CEA paradigm was designed for within-country decision making and has limited read-across to the very different environment faced by donors.” 7. The authors refer to the use of decision thresholds by Givewell and Open Philanthropy. It would be helpful to state these thresholds in the text. [A] We include the following in the text: “GiveWell uses a benchmark that charity programmes should be expected to provide value at least 10 time greater than cash transfers 29 and Open Philanthropy require a 1000-fold expected return on investment for their (generally higher risk and upstream) investments.” These specifics come somewhat later in the text than where these donors were first mentioned, as we felt it fit better with the main flow of the text. 8. The claim is made that \"many donors\" have adopted value for money frameworks, but then only one is cited. Either more citations should be added or the claim should be altered. [A] Thank you for this suggestion. We have now added references to UNICEF and the Global Fund’s value-for-money frameworks, alongside the existing reference to the UK’s framework." } ] } ]	1	https://f1000research.com/articles/12-214
https://f1000research.com/articles/13-74/v1	17 Jan 24	{ "type": "Research Article", "title": "A case study on finding efficient monetary policy to solve Shenzhen's excessive-priced housing", "authors": [ "Jiayi Luo" ], "abstract": "Background The real estate market has developed rapidly since 1980s in Shenzhen, China. This study aims to find and investigate an efficient monetary policy to solve the excessive-priced housing problem in Shenzhen.\n\nMethods By regulating the contractionary monetary policy which are open market operation, the discount rate and legal reserve deposit can decrease the accelerating rate of excessive housing price in Shenzhen. Firstly, the relationship between housing prices and the other three tools of monetary policy has been analyzed. Then, from collecting the secondary data online, models were established by using the ordinary least square to find the correlation between housing price and money supply from 2011 to 2017.\n\nResults This model shows the relationship with the change of the logarithmic form of the amount of M2 and housing index is significantly positive. With the decrease in change of the logarithmic form of the amount of M2, the increase in housing index can be controlled in a relative decelerated rate.\n\nConclusions In the reduced monetary supply, the housing price can be managed to an ideal condition and then it can help the government to achieve the goal of maintaining Shenzhen’s healthy real estate market development.", "keywords": [ "monetary policy", "housing price" ], "content": "Introduction\n\nStarting from the 1990s, China has experienced a booming housing market due to its fast-growing economic engine. As a national economic center in China, Shenzhen ranks third in terms of economic development. This happened when it, as China’s first Special Economic Zones in 1980s, began to experience a series of historical events and reforms such as Deng Xiaoping’s Southern Tour in 1992, Hong Kong’s financial crisis in 1997, and the real estate regulation and control policy in 2005. Overall, according to the National Bureau of Statistics of China, Shenzhen’s housing prices have undergone a relatively high level of acceleration rates and never plummeted significantly.\n\nChina had introduced many policies to control the excessive housing prices in Shenzhen, but the results were not ideal or even below the expectations. This raises the question of how researchers can find better monetary policies to bring down the inflation rate of house prices. To be more specific, what monetary policies can keep the healthy development of real estate industry in Shenzhen in the future?\n\nIn general, excessively priced housing in the market might be harmful to both individuals and society. Therefore, implementing real estate regulation is necessary because it can regulate the distribution of wealth to achieve long-term social stability. However, from the current trend in Shenzhen, it is more difficult to implement the right policies to achieve a slower rise. Essentially, it is the design and flaws of a series of deep-rooted systems that affect the development of real estate. The most important of such systems are the housing system, the fiscal system, and the land system (Zhang, 2019).\n\nIn principle, monetary policies can be implemented to regulate housing prices because of their interdependencies. For instance, housing prices rise more promptly in a country where exchange rate movement is restrained than in countries where it is not (Ohno & Shimizu, 2015). In addition, according to Taylor (2007), a larger increase in the long-term rates would clearly have mitigated the housing boom.\n\nHowever, it is still difficult to apply the tools of monetary policy to the housing price regulation in Shenzhen. The first is that speculation in housing is unending, making it difficult to regulate. The second is that Shenzhen’s economy is growing by the day, and the public has more capacities to afford houses, so they are willing to pay for price premiums. Therefore, to investigate and figure out possible responses to excessive priced housing, this study tries to establish an empirical link between housing price and money supply. Specifically, this method can meet our expectations of reducing the rate of housing price increase.\n\nThe remainder of the paper is organized as follows. The “Literature review” section will analyze the previous literature about how the three methods:open market operation, discount rate, and legal deposit reserve can influence the housing price and thus alleviate them. The “Methods” section will apply a mathematical approach to support our hypothesis. The “Results” and “Discussion” section will discuss the feasibility reasons for the three methods, which will be summarized here in combination with practice and theory. Finally, the “Conclusions” section will conclude the paper.\n\n\nLiterature review\n\nThere have been fierce competitions in China’s housing market. Li et al. (2009) emphasized the scale of domestic real estate development enterprises and market in the process of expanding and becoming mature, and this means that the high profits it brings have resulted in the entrance of many enterprises in the housing market, therefore it stimulates local economic development. Meanwhile, monetary policies have been used to act as one type of the most important tools for regulating housing prices. According to Deleidi and Levrero (2021), monetary policy is the action that Federal fund rate took for the purpose of expanding or contracting money supply and influencing interest rates. There are several subgroups in monetary policy, for instance, rate policy, interest rates, money supply, open market operation or the required reserve. (Dufour & Tessier, 2006; Adjasi et al., 2008). This study uses tools on open market operation, discount rate and legal deposit reverse. Then in the following, the paper will separate them into three individual parts and discuss how they can influence housing prices individually.\n\nIn an open market operation, the central bank swap currency for bonds (Rocheteau, Wright, and Xiao, 2018). In China, this included the typical purchasing and selling of government bonds to expand or tighten base money. In addition, the People’s Bank of China (PBC), the central bank in China, controlled the scale and frequency of the short-termed issuance to depository institutions, which was seen as a method to reduce the supply of base money (Xu and Chen, 2012). The money supply is the main aim of open market operation, the more frequent the open market operations, the lower the money supply. Then, the small amount of money supply will make the housing price with a lower increasing level. Liang and Cao (2007) investigated the relationship between property price and bank leading in China from 1999 to 2016 and found that there was a unidirectional causality running from bank lending to property price. In this case, the housing prices were directly connected with the open market operation, and prior researchers had done numerous studies on the negative correlation between housing prices and open market operation. Xu and Chen (2012) examined the monetary policy variables, including money supply growth, had the impact on the dynamic growth of real estate price in China. They explained that the contraction in the growth of money supply may decline the loan-making ability of commercial banks and altering the credit supply to the real estate sector, then going on to elaborate on the influence on the public’s inflationary expectation and the demand for real estate assets. Finally, they summarized that all these channels result in a positive effect of money supply growth on the change in real estate growth. In further study, Lastrapes (2002) simulated the dynamic response of real house prices to exogenous money supply shocks as predicted by theoretical models. In conclusion, the empirical outcomes consistently demonstrate that the slower money supply growth contributes to the deceleration in the housing price growth subsequently. Therefore, the money supply can act as the key driving force behind the real estate price growth in China.\n\nThe discount rate is the interest rate charged by the central bank for loans of reserve funds to commercial banks and other financial intermediaries. In the analysis of the interest rate approach, the discount rate has shown a negative relationship with housing prices. When the discount rate has increased, then the rate of increase in house price has slowed. Kau and Keenan (1980) found that when the currency authorities raised the interest rate, it caused real estate investment to fall, and then the price of commercial housing to also fall. Abraham and Hendershott (1994) revealed that the rise of housing price was negatively correlated with interest rate by using the model of housing price change considering lag process. Similarly, in the UK, Iwayama, Akiyama and Ishigaki (2003) established a vector autoregressive (VAR) model about the influence of monetary policy on the real estate market. This illustrated that the rise by 50 basis points on interest rate led to a fall in housing prices of about 0.8% in five quarters in the UK. After several years, Meltzer (1995) set up an asset price model which suggested that monetary policy could directly push housing prices up. However, at the same times, he argued that if a central bank fixes as interest rate, which was the common practice in China, and it will then let property prices to rise. Therefore, the results illustrated that the interest rate should reduce when dealing with the high level of housing prices. Despite the economic situations and developments being different compared to China, the linkage between housing prices and discount rate is relatively the same. Tan and Wang (2015) claimed that monetary policy and housing prices interacted with each other through direct and indirect channels. The impact of interest rates on urban housing prices was negative in the long run and gradually weakened (Wang & Guo, 2007). This study established a VAR model to reach the conclusion. According to Chen and Zhou (2011), the increase in interest rates had a restraining effect on the rise in urban housing prices. Meanwhile, interest rates had limitations existing when taking into account regional differences. A study by the Research Group of the Investigation and Statistics Office of Kunming Central Sub-branch (2018) pointed out that the interest rate regulation policy had obvious location differences. The role in regulating the region where the real estate market development was relatively immature and the rigid demand for real estate was relatively weak was more obvious. Nevertheless, combining with the housing market is relatively well established and mature in Shenzhen, the interest rate should still be regulated to an even lower level due to the lack of effectiveness of implementation when the regulation of interest rates is not significant enough.\n\nLegal deposit reserve refers to the reserve that an insurance enterprise must deposit in accordance with the relevant laws and regulations of the state. In China, it has been a frequently used and seen as a highly important tool recently, while in many western countries the tool is viewed differently as they are rarely used. In general, higher reserve ratio leads to reduced money supply (Xu and Chen, 2012). In China, there was a negative causality between legal deposit reserve and housing price as the increase in legal deposit reserve caused the money supply down and then led to the drop in housing price. Collyns and Senhadji (2002) found that credit growth had an important impact on housing prices among Asian countries. According to the analysis of Beijing housing price, Hong-ling (2008) stated that the increase in the statutory reserve requirement ratio has reduced the reserves and loanable funds of commercial banks, reducing the ability of banks to issue loans, and housing consumption loans, thereby reducing the effective demand for real estate and leading to a decrease in real estate prices. By using the error correction model (ECM) model, Pi and Wu (2004) showed that there was a linear causal relationship between the real estate market and the bank credit market in China, as well as a bi-directional association in both the long- and short- term mission effect of money supply. However, there are certain deficiencies in this tool. Firstly, to implement the change, there were many steps that need to undergo for decreasing the housing price (Hong-ling, 2008). Also, Hong-ling (2008) analyzed that the result of adjusting legal deposit reserve in a low rate was not obvious for decreasing the housing price because it ultimately depended on the power of real estate supply and demand. The housing price only decreased after its demand went down. Therefore, individual willingness to purchase houses was important as well. Secondly, the legal deposit reserve sometimes may not be an effective monetary tool. It was believed that when the legal deposit reserve was raised to a certain level, its function in liquidity management and monetary control was weakened (Zhang, Ji and Cui, 2008).\n\n\nMethods\n\nThis study is established on the real estate market in Shenzhen, China. As the prefectural-level city in Guangdong Province and the national economic center, Shenzhen is on the eastern bank of the Pearl River, and is bordered with Hong Kong across the Shenzhen River, making it one of the three largest national financial centers in China. Transforming from a fishing village to one of the most developed cities in China, Shenzhen has made a great effort on its own development and experienced a series of miracle changes, such as the establishment of Special Economic Zones in 1980. As a resident who grew up in Shenzhen, the author would like to make a feasible suggestion for the development of Shenzhen’s housing market. In addition, due to its strong economy and high population density, the demand for houses is also quite high, so the price of houses in my investigation can well demonstrate the effectiveness of the monetary policies approach under the wide range of demands in Shenzhen. Since the economic development in Shenzhen is among the highest in China, the city can provide a better place for research on the housing market, and it serves as examples that other cities can also study and imitate.\n\nTo explore how the money supply could influence the housing price in Shenzhen, this paper uses secondary data from the National Bureau of Statistics of China, rather than survey data from questionnaires due to the lack of feasibility. The variables used in this research are divided into two main categories, the money supply, measured in the amount of M2, and the sales price index of newly built commercial properties in Shenzhen, also called the housing index (set the sale price in the base year to 100). In economics, monetary supply is often proxied by M2, which refers to the sum of cash in circulation, outside the entire banking system, owned by enterprises, and by individuals plus the corporate demand deposits, and then add the quasi money which is the time, resident savings, and other deposits. For this research, it is better to use the amount of M2 as it can typically reflect the variations in aggregate social demand and the future state of inflationary pressures. M2 can exclude the effect of other variables for the study and then can be regarded as a target for money supply regulation. For housing index, it is better to choose the sale price index because it can show a more visual representation of the post-regulation price of the property in question, and it also reduces the need to consider the impact of other conditions on the price of the property. The newer data is more relevant to current developments and can provide better reliability for studying excessive housing prices, whereas the older data is often outdated and lacks accuracy.\n\nThis paper uses the monthly data from 2011 to 2017, 84 months in total. One reason to consider their trends from 2011 to 2017 is the more stable economic development at that time, which could avoid distractions by other factors. The data used in this research were obtained from the National Bureau of Statistics of China.\n\nOverall, the sales price index ranges from 98.9 to 107.1, with a mean of 100.9 and a standard deviation of 1.7. While for the money supply, measured in the amount of M2, its mean is 11.9 with a deviation of 2.9 ten trillion.\n\nFigure 1 plots the correlation between housing price index and M2 from 2011 to 2017. M2 grows steadily over time, and the index has increased at a steady growth rate and at the same time, it has fluctuated in the study period.\n\nFigure 2 considers the change of the logarithmic form of the amount of M2 (ΔlogM2t) and the change of housing price (ΔHIt), illustrating that there is a positive correlation between them. In other words, more money supply is associated with an increase in the housing index.\n\nThis research uses the open-source software RStudio, which is mainly based on Java, C++, and a little bit of JavaScript programming languages, and its current latest version is 2023.06.0+421. The software was used to input the code and insert the data from the National Bureau of Statistics of China, correlation coefficients, error terms, and intercepts of the estimated equations between M2 and housing price can be obtained. The ordinary least square was used to find the best functional match for the data by minimizing the sum of squares of the errors. Then the linear regression in research can estimate the mathematical correlation between the money supply and the housing price in Shenzhen. As the money supply, M2 shows the similar increasing trend with housing price index from 2011 to 2017, the author proposes the following model to explore the correlation between the change rate of monetary supply and the change of housing index in Shenzhen:\n\nIn this model, the change of the housing index in period t is called ΔHIt, and ut is the error term. Then β0 is the intercept of this estimation equation, while β1 indicates the correlation between the change of housing index and change of logM2. If β1is expected to be positive, then the increase in M2 for country can increase the housing price; On the other hand, if it is expected to be negative, then the increase in M2 for country can show the decrease in housing price.\n\nHowever, considering another equation that might discuss the relationship between housing index and monetary supply, this paper also shows another equation:\n\n\nResults\n\nFor the first model, which is represented the sales price index of newly built commercial properties in Shenzhen, and the amount of M2 in China in the given time of 84 months. By establishing the equation about two variables, then investigate the linear regression on them.\n\nThis shows the correlation between the housing index and the M2 is relatively weak to represent the model because the beta1 is 0.008. This illustrates that the increase in every 1% of M2, the housing index in given time increases by 0.008%. Thus, it is the lack of stronger relevance in showing the role of the monetary policy, M2 with housing index, as the decline in monetary supply of M2 has done very little to moderate the fall in housing index. In addition, the p-value from the equation is 0.212, which would show the model is free from chance and is reliable. The p-value is the indicator of test sample confidence and to test whether the model is suitable for implement, as the model can be tested by whether it will reject the original assumed value, and it is normally 0.05. Therefore, the model shows that M2 has no such effect on housing index. As a result, the M2 might lack the ability to control excessive housing prices and the monetary policy may not be useful in regulating the housing price in Shenzhen.\n\nThe second equation represents the change of log-M2 and the change of housing index in given time of 84 months. The result shows that the correlation between them is positive.\n\nThis model reveals that there is a quite strong relationship between ΔlogM2t and ΔHIt. The beta1 is 20.562 and it tells that the increase in change of HIt is 20.562% per change of logM2tpercentage. Where the p-value of beta1 is less than 0.05, 0.0297. Thus, the null hypothesis can be rejected. Therefore, it is obvious that the money supply, M2 can affect the housing index in Shenzhen in a positive correlation as the higher change of log-M2 lets the price level for housing price in Shenzhen accelerated greatly.\n\nFrom the period 2011 to 2017, the implementation of monetary policy can play a certain role in the excessive housing price in Shenzhen. When reducing the amount of M2 in housing market, and it will eventually cause the aggregate demand decreased in China. As a result, this can slow down the growth rate of housing prices in the real estate market. In contrast, the more M2 amount can accelerate the growth of housing price. Overall, to solve the housing problems in Shenzhen, the amount of money supply should be lower so as to slow the housing price acceleration in Shenzhen.\n\n\nDiscussion\n\nThis research focuses on how the tools of the money supply directly affect housing prices through a series of transmission mechanisms, which can show people intuitively the correlation between them. Then, the research gives solutions to the real-life practical situations of high housing prices. However, improving the housing market situation in Shenzhen can be very difficult. According to Wen and He (2015), the low persistency and volatility of money supply shocks lead to the mirror role that money supply played in explaining the housing price fluctuation. The housing market requires the money supply to be under a certain lower level for achieving the goal. However, from the current economic situation, the housing price is still situated at an accelerated level, and it is difficult to control it. By investigating the effectiveness of monetary policy on house prices, Kasai and Gupta (2010) stated that the movements in house prices are sensitive to monetary policy shocks, but with quite modest effect. In addition, to achieve the most efficiency of monetary policy in our society, the other factors which affect the housing price need to be included. From this study, the model used only considered the study time and money supply, M2. There are certain limitations in reality, and it might not be very applicable in solving the problem in the estate market. However, the study might be improved by considering more factors such as land location, culture, neighborhoods, and other factors in the model.\n\n\nConclusion\n\nIn Shenzhen, the housing market has been thriving since the opening-up reform, and the excessively accelerated housing price has become a necessary trend in development. Throughout history, there have been several factors that can lead to a sustained and accelerated climb in house prices. The excessive housing price has become the problem in Shenzhen as the development of the real estate market is unhealthy. In this study, finding the better policy in solving the difficulty of accelerating housing prices is the question that we looked at.\n\nBy implementation of the contractionary monetary policy, including regulating the open market operation, discount rates and legal reserve deposits can decrease the accelerating rate of excessive housing prices in Shenzhen. Based on the situation in Shenzhen, excessive housing prices might be controlled by these monetary policies, which act as the approach to achieve the housing price control objective, as the monetary policy includes the open market operation, discount rate and legal deposit reserve. This study is conducted though the methods of representing the diagrams and establishing the model to analyze the relevance between housing price and monetary policy. As a result, the research shows that the implementation of the reduced money supply would contribute to the decelerating housing price.\n\nThis research uses the regression model to investigate the relationship with the housing index and money supply, M2. The statistical model demonstrates that the M2 can act as the important factor in affecting the housing index due to the effectiveness in controlling the real accelerated housing price in Shenzhen. As a result, the model shows that M2 and housing index have a positive correlation. For this research, the money supply, which is the tool of monetary policy, shows a positive relationship with the housing price in Shenzhen real estate market from 2011 to 2017. However, combining the economic and statistical variables, this research needs to consider the M2 as the form of ΔlogM2t, since we suppose that ΔlogM2t is roughly the growth rate of M2. As a result, the money supply might have a better influence on the housing price as the coefficient is more relevant for it.\n\nSuch a relationship in the housing price and money supply will set the example on the real estate market about how to adjust the excessive housing price. To achieve the sustainable development of the healthy real estate market on Shenzhen, M2 cannot be the only variable to consider for house price changing. In addition, the housing price should not only be regarded as the standard for healthy real estate market development, but also as the happiness index that people live in Shenzhen own. Because for some lower wages dwellers who do not have their own houses in Shenzhen, it is possible to improve their happiness index though monetary policy.", "appendix": "Data availability\n\nThe data in this paper is not generated nor is it owned by the authors. In this paper, we use the publicly available data on the below website: https://data.stats.gov.cn/easyquery.htm?cn=E0104&zb=A0108&reg=440300&sj=202308; https://data.stats.gov.cn/easyquery.htm?cn=A01&zb=A0D01&sj=202308\n\nReaders can log in to the website and search for the keywords “Shenzhen new residential sales price index, and currency and quasi-money (M2) supply (100 million yuan) at the end of the period” to find specific data source.\n\nThe data on this website is under copyright and the terms allow for free access and use of the data.\n\n\nReferences\n\nAdjasi C, Harvey SK, Agyapong DA: Effect of exchange rate volatility on the Ghana stock exchange. African journal of accounting, economics, finance and banking research. 2008; 3(3).\n\nAbraham JM, Hendershott PH: Bubbles in metropolitan housing markets.1994.\n\nChen Z, Zhou L: An empirical analysis of the impact of monetary policy on house prices. Northern Economy. 2011; 17: 85–87.\n\nCollyns C, Senhadji AS: Lending booms, real estate bubbles, and the Asian crisis. Real Estate Bubbles, and the Asian Crisis (January 2002). 2002.\n\nDufour JM, Tessier D: Short-run and long-run causality between monetary policy variables and stock prices (No. 2006-39). Bank of Canada. 2006.\n\nDeleidi M, Levrero ES: Monetary policy and long-term interest rates: Evidence from the US economy. Metroeconomica. 2021; 72(1): 121–147. Publisher Full Text\n\nHong-ling GE: The Influence of the Monetary Policy on the Housing Price in Beijing. Journal of Central University of Finance & Economics. 2008.\n\nIwayama N, Akiyama K, Ishigaki K: Houses as Collateral: Has the Link between Houses Prices and Consumption in the U.K. Changed? Econ. Policy Rev. 2003; 8: 163–177.\n\nKau JB, Keenan D: The theory of housing and interest rates. J. Financ. Quant. Anal. 1980; 15(4): 833–847. Publisher Full Text\n\nLiang Q, Cao H: Property prices and bank lending in China. J. Asian Econ. 2007; 18(1): 63–75. Publisher Full Text\n\nKasai N, Gupta R: Financial liberalization and the effectiveness of monetary policy on house prices in South Africa. Repository.up.ac.za. 2010; VII(4). Reference Source\n\nLastrapes WD: The real price of housing and money supply shocks: time series evidence and theoretical simulations. J. Hous. Econ. 2002; 11(1): 40–74. Publisher Full Text\n\nLi H, Li V, Skitmore M, et al.: Competitiveness factors: a study of the real estate market in China. Constr. Manag. Econ. 2009; 27(6): 567–579. Publisher Full Text\n\nMeltzer AH: Monetary, credit and (other) transmission processes: a monetarist perspective. J. Econ. Perspect. 1995; 9(4): 49–72. Publisher Full Text\n\nNational Bureau of Statistics of China, editor. 2022. Reference Source\n\nOhno S, Shimizu J: Do exchange rate arrangements and capital controls influence international capital flows and housing prices in Asia? J. Asian Econ. 2015; 39: 1–18. Publisher Full Text\n\nPi S, Wu KP: Causality between the Development of Real Estate Market and Economic Growth—An Empirical Analysis of China. Manag. Rev. 2004; 16: 8–12.\n\nRocheteau G, Wright R, Xiao SX: Open market operations. J. Monet. Econ. 2018; 98: 114–128. Publisher Full Text\n\nResearch and Statistics Division, Kunming Central Branch, People’s Bank of China: The Impact of Monetary Policy on Regional Real Estate Price Volatility - Based on DSGE Model. Econ. Perspect. 2018; (5).\n\nTaylor JB: Housing and monetary policy. National Bureau of Economic Research; 2007; (No. w13682).\n\nTan Z, Wang C: House price volatility, identification of monetary policy stance and its response. Econ. Res. 2015; 50(1): 67–83.\n\nWang L, Guo F: An empirical study on the dynamic impact of monetary policy on real estate prices based on VAR models. Studies in Finance and Economics. 2007; 11: 15–19.\n\nWen X-C, He L-Y: Housing demand or money supply? A new Keynesian dynamic stochastic general equilibrium model on China’s housing market fluctuations. Physica A: Statistical Mechanics and Its Applications. 2015; 432: 257–268. Publisher Full Text\n\nXu XE, Chen T: The effect of monetary policy on real estate price growth in China. Pac. Basin Financ. J. 2012; 20(1): 62–77. Publisher Full Text\n\nZhang X, Ji Z, Cui Y: Reserves, reserve tax and monetary control in China: 1984-2007. Econ. Stud. 2008; 7: 65–77.\n\nZhang S: Shenzhen Housing System and House Price Regulation. National Administration Press; 2019." }	[ { "id": "279375", "date": "13 Sep 2024", "name": "Josef Bajzík", "expertise": [ "Reviewer Expertise Monetary policy", "Macroprudential policy", "International Economy" ], "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 discusses the interaction between monetary policy and the regulation of housing prices in Shenzhen. Despite the need for such research, I found several shortcomings, as described below. Regarding the literature review, I think this one is not comprehensive enough.\n\nFor example, I found an article by Ehrenbergerova et al. (2022) [Ref 1] published in the IMF Economic Review and documented tens of studies relevant to the researched topics. Moreover, many articles in the literature review should be published in well-known journals.\nEven though the methodology used in the article is clear, I found it on an evolving level, and I think some more statistically sound methods, such as VAR models, should be used. I am afraid that the proposed equations suffer from omitted variable bias or endogeneity problems. Besides, the standard errors are not reported, and I do not know anything about the robustness of the results and the treatment of the standard errors.\nI found the data mentioned in the paper's appendix rather roughly described again; I would expect a table with all the definitions, sources and summary statistics. The proposed comments on the data are not sufficient from my perspective.\nThe conclusions drawn are summed up nicely but based on the data and methodology described above, so I am hesitant to take them as seriously.\nI suggest redoing all the work again, making the research much more reliable.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-74
https://f1000research.com/articles/13-73/v1	17 Jan 24	{ "type": "Clinical Practice Article", "title": "Perioperative management in haemophilia with extended half-life factors: a case series", "authors": [ "Harshit Khurana", "Shailendra Prasad Verma", "Harshit Khurana" ], "abstract": "Hemophilia B is a hereditary bleeding disorder characterized by deficient or defective coagulation factor IX, leading to a propensity for recurrent bleeding episodes, particularly in the joints. Management of Hemophilia B in surgical settings poses unique challenges due to the risk of excessive bleeding. This paper presents a series of two cases that demonstrate the effective use of perioperative extended half-life (EHL) factor IX products, specifically N9-GP (Refixia), in the surgical management of Hemophilia B. The cases include total knee replacement and total hip replacement. In each case, early initiation of EHL was observed to maintain adequate factor IX levels and control perioperative bleeding effectively, leading to successful surgical outcomes. These findings support the growing body of evidence suggesting the advantages of EHL perioperative treatment in patients with Hemophilia B, particularly in preparation for surgery. The implementation of EHL factor IX products as part of perioperative management plans may contribute to improved surgical outcomes and overall quality of life in Hemophilia B patients.", "keywords": [ "Hemophilia", "EHL", "coagulation factor IX", "perioperative extended half-life (EHL) factor IX", "surgical management" ], "content": "Introduction\n\nHemophilia B is an X-linked recessive bleeding disorder caused by deficient or dysfunctional coagulation factor IX, which is essential for normal blood clotting.1 It is the second most common type of hemophilia, accounting for approximately 20% of all hemophilia cases.2 Patients with hemophilia B experience recurrent and spontaneous bleeding episodes, especially in joints and muscles, which can lead to chronic pain, joint damage, and significant morbidity.3\n\nSurgical procedures in patients with hemophilia B pose unique challenges due to the increased risk of perioperative and postoperative bleeding.4,5 This risk is further amplified in surgeries involving highly vascularized areas or those that involve major joints. Uncontrolled bleeding in these patients can lead to significant complications such as prolonged hospital stay, increased healthcare costs, and in some cases, life-threatening situations.6\n\nOver the years, the management of hemophilia B has evolved significantly with the development of recombinant factor IX products, allowing for safer and more effective prophylaxis.7 Perioperative treatment involves regular intravenous infusions of factor IX concentrates to maintain a minimum level of factor IX activity, thereby preventing spontaneous bleeding episodes.8\n\nThe advent of extended half-life (EHL) factor IX products, such as glycoPEGylated factor IX, has further revolutionized the management of hemophilia B.9 These novel products offer longer-lasting protection against bleeding, reducing the frequency of infusions required and improving patients’ quality of life.10–15\n\nDespite these advancements, performing major surgery on individuals with hemophilia B continues to present challenges. This is because it necessitates repeated bolus dosing and/or continuous infusion to maintain sufficient levels of factor IX (FIX) in the bloodstream, ensuring proper haemostasis until the wound heals. Given the pharmacokinetic (PK) properties of existing FIX products, it is necessary to closely monitor FIX levels during both the intra- and postoperative periods. This monitoring helps adjust replacement therapy to achieve and sustain the required FIX levels.16\n\nIn this paper, we present a series of cases highlighting the successful use of perioperative treatment in hemophilia B patients undergoing various surgical procedures. We aim to shed light on the effectiveness of EHL and prompt a broader discussion on its potential benefits in the surgical management of hemophilia B.\n\n\nCases\n\nThe first case involves a 27-year-old Indian male student pursuing his education. The patient was diagnosed with Hemophilia B at the age of 5 years without any relevant family history of Hemophilia. The patient had been suffering from arthropathy, with significant pain and functional limitation in both knees, elbows, hip, and left ankle. The patient’s Hemophilia B and resultant arthropathy were severe enough to result in walking difficulty, necessitating the use of single support.\n\nDue to the severity of his condition, the patient was advised to undergo a total hip replacement (THR) for his left hip. Recognizing the heightened risk of bleeding associated with Hemophilia B, the patient was treated with Nonacog Beta Pegol (N9GP), 40 IU/Kg once weekly for six months prior to his scheduled surgery. His pre-operative and post-operative X ray images are shown in Figures 1 and 2.\n\nDuring the hospitalization period, a total of 5 doses of EHL therapy were administered. The first dose, given preoperatively, was 80 IU/kg and resulted in an excellent haemostatic response. The second and third doses of 40 IU/kgBW were given on the first and fourth day postoperatively, respectively. The final two doses were administered between the 7th and 12th day, marking the end of the treatment. The patient did not experience any adverse effects, and his overall recovery was good.\n\nThis case represents a 29-year-old Indian male computer engineer diagnosed with Hemophilia B. The patient presented with persistent pain, swelling, and functional limitation in his right knee joint, which has progressively worsened over the last 3 years. Diagnostic investigations revealed severe right knee haemophilic arthropathy, leading to the clinical decision to perform a total knee replacement (TKR).\n\nGiven the patient’s history of Hemophilia B, a perioperative treatment regimen was implemented to mitigate the risk of perioperative bleeding. The patient was administered N9-GP (Refixia), a recombinant human coagulation factor IX glyco-pegylated product. The dosing schedule was strategically designed to optimize haemostatic coverage during the perioperative period. The treatment regimen started with a preoperative dose of 80 IU/kg, followed by two postoperative doses of 40 IU/kg at 1-3 day intervals within the first week of surgery. Weekly doses were subsequently administered until bleeding cessation.\n\nThe desired factor IX levels were meticulously monitored to ensure effective haemostatic coverage throughout the postoperative period (Table 1). Intra-operative images are depicted in Figure 3 and post operative X ray is shown in Figure 4.\n\nThe measured factor IX levels closely followed the desired levels, ranging between 16% and 83.3%, signifying the effectiveness of the N9GP or EHL regimen.\n\nNotably, no additional dosing was required intraoperatively, and haemostasis was successfully achieved. The patient’s recovery was uneventful, and the outcomes were encouraging, with no excessive bleeding or other severe complications observed. This case underscores the potential of EHL treatment in enhancing surgical outcomes in patients with Hemophilia B undergoing major orthopaedic procedures. It stresses the importance of diligent perioperative management, including personalized dosing strategies and close monitoring of clotting factor levels, to ensure optimal haemostatic control.\n\n\nDiscussion\n\nNonacog beta pegol is a recombinant factor IX (rFIX) compound that is synthesized by expressing the human factor IX gene sequence in Chinese hamster ovary (CHO) cells, without the use of any human or additional animal materials. To the FIX activation peptide, a 40-kilodalton polyethylene glycol (PEG) moiety is attached via site-directed glycoPEGylation. During the coagulation process, the activation peptide, along with the PEG, is cleaved, resulting in the production of native-activated factor IX.\n\nIn a single-dose pharmacokinetic trial, nonacog beta pegol demonstrated a five-fold increase in terminal half-life compared to standard factor IX products. This extended half-life enabled the administration of once-weekly regimen in adults and adolescents with hemophilia B, ensuring sustained high levels of factor IX activity. Additionally, this treatment approach was deemed safe and effective for managing and preventing bleeding episodes.16\n\nThe case studies presented in this paper highlight the potential of perioperative treatment with Extended Half-Life (EHL) therapies such as N9-GP (Refixia) and GlycoPEGylated Factor IX in improving surgical outcomes for patients with Hemophilia B. These cases depict various surgical scenarios ranging from total knee replacement to hip replacement. Across these diverse surgical interventions, EHL therapies provided effective haemostatic control, underlining their utility in a broad spectrum of surgical contexts.\n\nA common thread observed across these cases is the successful management of perioperative haemostasis with the use of EHL therapy. This aligns with the findings of several previous studies that have reported the advantages of EHL over standard half-life (SHL) factor IX products due to their longer half-life and lower clearance, allowing for reduced dosing frequency and sustained factor IX activity.12,13\n\nThe ability to maintain factor IX levels within the desired range during the postoperative period, as observed in Case 2, is a crucial factor in preventing postoperative bleeding, which is a significant concern in haemophilic patients undergoing surgery. This is supported by other studies, which have shown that maintaining factor IX levels above a certain threshold significantly reduces the risk of bleeding complications.14\n\nInterestingly, the benefits of EHL therapy extended beyond the immediate perioperative period. For instance, in Case 1, EHL therapy was initiated six months prior to the scheduled total hip replacement surgery, and this approach was effective in ensuring excellent haemostatic response throughout the surgical process.\n\nWhile our case series and existing literature support the use of EHL therapies in the surgical management of Hemophilia B, it is important to recognize that each patient presents a unique clinical scenario. Therefore, the decision on the timing and dosing of perioperative treatment should be individualized based on the patient’s specific condition, surgical procedure, and other relevant factors.\n\nThe successful outcomes observed in these cases underscore the potential of EHL therapy as a perioperative strategy in improving surgical outcomes for Hemophilia B patients. However, larger controlled studies are required to further validate these findings and to explore the optimal timing and dosing strategies for EHL therapy in different surgical scenarios.\n\n\nConclusion\n\nThe two cases presented herein provide compelling evidence supporting the use of extended half-life (EHL) perioperative in the management of surgical patients with Hemophilia B. The administration of EHL products, particularly N9-GP (Refixia), showed promising results in maintaining adequate factor IX levels and controlling perioperative bleeding, thus leading to successful surgical outcomes in all cases.\n\nIn clinical practice, these results underscore the importance of individualized treatment plans and the need for a multidisciplinary approach in managing Hemophilia B patients who require surgery. The appropriate timing, dosing, and monitoring of perioperative treatment are crucial elements that can significantly impact surgical outcomes.\n\nIn conclusion, the implementation of perioperative treatment in surgical management strategies for Hemophilia B patients is likely to improve surgical outcomes and the overall quality of life for these patients. The use of EHL factor IX products, as demonstrated in these case studies, could potentially revolutionize the therapeutic approach towards Hemophilia B.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nWe would like to acknowledge the contribution of the patients who consented to share their experiences for the benefit of others. Their willingness to participate in these case studies has made a significant contribution to our understanding of Hemophilia B management.\n\nThe writing of this article was supported by a medical writer at Medwiz Healthcare Communications Private Ltd.\n\n\nReferences\n\nMannucci PM, Tuddenham EG: The hemophilias—from royal genes to gene therapy. N. Engl. J. Med. 2001; 344(23): 1773–1779. PubMed Abstract \| Publisher Full Text\n\nWhite GC, Rosendaal F, Aledort LM, et al.: Definitions in hemophilia: recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb. Haemost. 2001; 85(03): 560. Publisher Full Text\n\nWalsh CE, Soucie JM, Miller CH: United States Hemophilia Treatment Center Network. Impact of inhibitors on hemophilia A mortality in the United States. Am. J. Hematol. 2015; 90(5): 400–405. PubMed Abstract \| Publisher Full Text\n\nKavakli K, Makris M, Zulfikar B, et al.: Home treatment of haemarthroses using a single dose regimen of recombinant activated factor VII in patients with haemophilia and inhibitors. A multi-centre, randomised, double-blind, cross-over trial. Thromb. Haemost. 2006 Apr; 95(4): 600–605. PubMed Abstract \| Publisher Full Text\n\nPoston JN, Kruse-Jarres R: Perioperative hemostasis for patients with hemophilia. Hematology Am. Soc. Hematol. Educ. Program. 2022 Dec 9; 2022(1): 586–593. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranchini M, Mannucci PM: Inhibitors of propagation of coagulation (factors VIII, IX and XI): a review of current therapeutic practice. Br. J. Clin. Pharmacol. 2011 Oct; 72(4): 553–562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSrivastava A, Brewer AK, Mauser-Bunschoten EP, et al.: Treatment Guidelines Working Group on Behalf of The World Federation Of Hemophilia. Guidelines for the management of hemophilia. Haemophilia. 2013; 19(1): e1–e47. PubMed Abstract \| Publisher Full Text\n\nManco-Johnson MJ, Abshire TC, Shapiro AD, et al.: Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N. Engl. J. Med. 2007; 357(6): 535–544. PubMed Abstract \| Publisher Full Text\n\nMahlangu J, Powell JS, Ragni MV, et al.: Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B. N. Engl. J. Med. 2014; 371(26): 2313–2323.\n\nCollins PW, Young G, Knobe K, et al.: Recombinant long-acting glycoPEGylated factor IX in hemophilia B: a multinational randomized phase 3 trial. Blood. 2014; 124(26): 3880–3886. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNegrier C, Seuser A, Forsyth A, et al.: The benefits of exercise for patients with haemophilia and recommendations for safe and effective physical activity. Haemophilia. 2013 Jul; 19(4): 487–498. PubMed Abstract \| Publisher Full Text\n\nNegrier C, et al.: Enhanced pharmacokinetic properties of a glycoPEGylated recombinant factor IX: a first human dose trial in patients with hemophilia B. Blood. 2011; 118(10): 2695–2701. PubMed Abstract \| Publisher Full Text\n\nSantagostino E, et al.: Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial. Blood. 2016; 127(14): 1761–1769. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollins PW, et al.: Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A. J. Thromb. Haemost. 2009; 7(3): 413–420. PubMed Abstract \| Publisher Full Text\n\nAr MC, Balkan C, Kavaklı K: Extended Half-Life Coagulation Factors: A New Era in the Management of Hemophilia Patients. Turk. J. Haematol. 2019 Aug 2; 36(3): 141–154. PubMed Abstract \| Publisher Full Text\n\nEscobar MA, et al.: Low-factor consumption for major surgery in haemophilia B with long-acting recombinant glycoPEGylated factor IX. Haemophilia. 2017; 23(1): 67–76. PubMed Abstract \| Publisher Full Text" }	[ { "id": "262650", "date": "16 May 2024", "name": "Chai W Phua", "expertise": [ "Reviewer Expertise Hematologic Disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 The article under review presents two case studies demonstrating the use of glycoPEGylated, extended half-life, recombinant FIX (Nonacog Beta Pegol, N9GP) in the perioperative management of Hemophilia B during major orthopedic surgeries—total knee replacement and total hip replacement. It details the clinical context with radiographic images, the administration of N9GP, the monitoring of coagulation factor levels, and the outcomes, which indicate effective bleeding control and successful surgical results. Assessment of Article Quality Relevance and Importance: The topic is relevant, focusing on the significant challenges in managing hemophilia patients undergoing surgery, including real-world experience with N9GP. The surgical management of hemophilia presents a complex challenge due to the unique nature of each surgical case, varied patient responses to factor replacement therapies, and differing laboratory capabilities across treatment centers. This necessitates highly tailored surgical plans that are not easily standardized.\n\nConstructive Comments Strengths: The case studies provide insights into practical applications, adding valuable real-world evidence to the literature on Hemophilia B management. The suggested product monograph dosing regimen of N9GP successfully managed major orthopedic surgical procedures. Areas for Improvement: The rationale for choosing these specific cases for publication should be clarified. Were these instances the first applications of N9GP in the context of major orthopedic surgeries? Detailing Case Histories: The descriptions of the cases would benefit from a more comprehensive presentation of each patient’s medical background. This should include:\nDescribe the severity of Hemophilia (mild, moderate, or severe). Detailed prior treatment modalities (prophylaxis vs. on-demand), including exposure days to factor products if known, informing about inhibitors' potential development. Documentation of target joints, HJHS scores and annualized bleeding rates to provide context on the disease impact. Information on prior pharmacokinetic testing with N9GP, if available. The status of inhibitors to factor IX and whether testing was conducted before the surgical intervention to assess the risk of an immune response to N9GP. Were follow-up testing done?\nSpecifics in Case 1 Presentation: For Case 1, additional specifics can deepen the understanding of perioperative management:\nWere any specific laboratory assessments conducted preoperatively or postoperatively? Details of these assessments can help validate the efficacy of the treatment protocol. Explanation of the six-month preoperative treatment with N9GP. Was this aimed at optimizing joint condition, reducing bleed rates, or achieving a specific pharmacokinetic profile? The duration of the hospital stay, any transfusion requirements, significant hemoglobin drop and details about postoperative prophylaxis or return to on-demand treatment. Discuss any thromboprophylaxis measures taken, considering the hypercoagulable state post-surgery. Any long-term outcomes or follow-up data post-surgery to assess sustained benefits or complications (i.e. inhibitor testing) A more precise definition of the surgical response and a clear justification for the chosen dosing regimen, particularly aiming to achieve peak factor levels of 100% pre-operatively and maintaining at least 50% trough levels post-operatively.\nSpecifics in Case 2 Presentation: As with Case 1, the specifics listed above have pertinence and could further enhance the case presentation. Table 1: The presentation in Table 1 needs improvement for better clarity and understanding. It should specify whether the factor IX levels shown are intended to represent peak or trough levels. The total dosing reported appears to be inconsistent. If the table aims to compare the utilization of standard half-life versus extended half-life factor IX products, presenting the data in terms of total dosing per kilogram could provide a clearer comparison and illustrate the benefits of EHL in reducing the frequency and volume of doses. If the lower doses used achieved the desired levels of FIX, this warrants highlighting in the text. Detail the rationale for the 3-month dosing regimen. Was this specifically pre-planned to maintain factor levels for rehabilitation, or did it transition into a regular prophylaxis schedule post-surgery?\n\nSpecifics in Discussion: To enhance the discussion section of your paper and provide a more compelling analysis of Extended Half-Life versus Standard Half-Life factor products including maintaining stable factor levels throughout surgical procedures, reduced dosing frequency, and sustained protection. These benefits enhance patient outcomes and help mitigate some logistical and laboratory constraints associated with shorter half-life products. Reflect on how the cases presented illustrate the benefits of N9GP.\nConclusion The paper provides insight into the real-world experience of N9GP in major surgical procedures. The case studies contribute to existing knowledge, highlighting the safety and efficacy of EHL products in surgical settings. With some enhancements, the paper could further its impact. The review supports the article be with revisions, as suggested.\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": [] }, { "id": "316871", "date": "17 Oct 2024", "name": "Omotola O. Olasupo", "expertise": [ "Reviewer Expertise Health research methodology", "clinical epidemiology", "hemostasis", "thrombosis", "evidence-based medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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/ Overall Comments: The authors presented two cases demonstrating the effect of perioperative extended half-life (EHL) factor IX, N9-GP (Refixia), in the perioperative management of Hemophilia B. In these cases, early initiation of N9-GP was shown to maintain adequate factor IX levels and control perioperative bleeding. Below are some major points to consider to improve the manuscript.\nTitle\nThis report focuses on hemophilia B. The title should be rephrased to indicate hemophilia B.\n\nHemophilia was spelt differently in the title versus across the manuscript. It is recommended to be consistent with the version of the spelling used across the report.\n\nWhile the sample size needed to label a study case series or case report may not be clearly defined, Patterson et al. suggest that case series should have at least five cases.1,2 This report can be labeled a case report, considering two cases were reported.\n\nIntroduction\nThe use of “various surgical procedures” suggests that a variety of surgeries were being described. Specifying the two surgeries conducted (total hip and knee replacement) will be more accurate.\n\nUse the non-proprietary name of the product nonacog beta pegol (or its shortened form N9-GP) across the manuscript.\n\nMethods\nIf this report is deemed a case series, the use of a standard reporting guideline, such as The Preferred Reporting Of CasE Series in Surgery (PROCESS) guidelines, will be helpful to ensure all necessary elements are reported.3 If this is considered a case report, similar reporting guidelines such as the CARE guidelines (for CAse REports) can be considered.4,5\n\nWas the study registered prospectively? If so, state the research registry number.\n\nState whether the case series is: (1) prospective/retrospective, (2) single/multi-centre, and if (3) cases are consecutive/non-consecutive. Also, provide relevant dates.\n\nMore details are needed on the setting(s) in which the surgeries were conducted (e.g. teaching/district general hospital, community, or private practice).\n\nMore details are needed on study participants e.g. demographics, comorbidities, history of inhibitors, previous exposure to N9-GP, exposure to other hemostatic agents before and within the perioperative period, severity of hemophilia B (mild, moderate, or severe)\n\nProvide details on how hemostatic response (excellent, good, fair, inadequate) was defined.\n\nProvide details on how overall recovery was defined. What is the follow-up time in the postoperative period for the assessment of overall recovery?\n\nConclusions\nThis report (being a case report or a case series) does not provide compelling evidence to support the use of extended half-life (EHL) in the perioperative management of Hemophilia B, as mentioned by the authors. The conclusions need to be reviewed and more rigorous study designs are recommended to make such conclusions.\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? Partly\n\nIs the conclusion balanced and justified on the basis of the findings? No", "responses": [] } ]	1	https://f1000research.com/articles/13-73
https://f1000research.com/articles/12-100/v1	27 Jan 23	{ "type": "Study Protocol", "title": "Protocol of systematic reviews on implementation research on cardiovascular diseases, diabetes mellitus and mental ailments in India", "authors": [ "Hisham Moosan", "Mahendra Thakor", "Arun Kumar Sharma", "S. S. Mohanty", "Ansuman Panigrahi", "Vikas Dhikav", "Suresh Yadav", "Ramesh Kumar Huda", "Mukesh Parmar", "Poonam Singh", "Denny John", "Hisham Moosan", "Arun Kumar Sharma", "S. S. Mohanty", "Ansuman Panigrahi", "Vikas Dhikav", "Suresh Yadav", "Ramesh Kumar Huda", "Mukesh Parmar", "Poonam Singh", "Denny John" ], "abstract": "Introduction: The burden of non-communicable diseases (NCDs) is a major public health concern across the world. Various initiatives have tried to address these with varying degrees of success. Objective: The objective is to assess and collate existing evidence in implementation research done in India on three broad domains of NCDs namely, cardiovascular diseases (CVD), diabetes mellitus (DM), and mental health (MH) in India. Materials and methods: Three systematic review protocols have been drafted to explore and collate extant evidence of implementation research on cardiovascular diseases, diabetes mellitus, and mental health in India, in accordance with the PRISMA-P statement. Academic databases including PubMed, Embase and Science Direct will be searched. Search strategies will be formulated in iterative processes and in accordance with the formats that are specific to the databases that will be searched. In addition, grey literature and non-academic databases will also be explored. Data extracted from the selected studies will be analysed and a narrative summary of the selected articles, using the SWiM (Synthesis without meta-analysis) guidelines will be produced. Intended Outcomes: The outputs of these systematic reviews could help in a better understanding of implementation research gaps and also how to address them. Apart from giving insights into how healthcare initiatives for CVDs, diabetes and mental health could be implemented in a better way, the study could also advocate the need to build and consolidate capacity for implementation research in the country.", "keywords": [ "Implementation Research", "Systematic review", "India", "Cardiovascular Diseases", "Diabetes Mellitus", "Mental Health Ailments", "Implementation Science", "Non-Communicable Diseases" ], "content": "Introduction\n\nThe burden of non-communicable diseases (NCDs) is a major public health issue plaguing India and the rest of the world. Hence, attention to prevention, control, management, and mortality reduction of these diseases is of paramount importance.1,2 Ranging across the whole spectrum of primary, secondary and tertiary prevention measures, various government initiatives have tried to address these with varying degrees of success.3 Considering the fact that many of the strategies may have been proven efficient in various experimental and study settings, it is imperative to explore the real-life technical and allocative effectiveness of these programs, and the possible lacunae in their implementation.\n\nAll recent disease burden estimates point toward the fact that the burden of NCDs is increasing with each passing day. Along with this, it is important to note that the prevention and control of these diseases tend to entail longer and larger social and economic commitments on the part of governments. Thus, with a view to maximizing the utility of the efforts made by the government, it would be desirable to focus on implementation research done in this arena.\n\nNevertheless, it would be important to understand the quantum, diversity, and quality of implementation research done in the field of NCDs in India. Being a considerably larger domain covering a wide range of diseases, it is difficult to look at implementation research on all NCDs; hence it seemed prudent to narrow down the focus of such an inquiry to three among the most prevalent and significant disease domains, from a public health point of view, viz., cardiovascular diseases, diabetes mellitus and mental health ailments. Using a systematic review approach, this study aims to examine how IR was done to advance the prevention and control of the aforesaid three NCDs in India. The main goal of this systematic review is to describe IR that has been done in the domains of these NCDs in India and how evidence from these studies could be better applied to inform policy-level impact actions on NCD interventions in India. In this paper, we are presenting the protocol of IR that was followed in conducting the systematic review of IR in three selected NCDs.\n\nTo synthesize evidence on implementation research related to cardiovascular diseases (CVD), diabetes mellitus (DM), and mental health (MH) conducted in India.\n\nResearch question(s)\n\nWhat is the evidence on implementation research on CVD, DM, and MH in India?\n\n\nProtocol\n\nWith an overarching objective to assess and collate existing evidence in implementation research done in India on three broad domains of NCDs namely, cardiovascular diseases (CVD), diabetes mellitus (DM), and mental health (MH), three systematic reviews of the implementation research conducted in the aforesaid three domains of NCDs will be conducted. In accordance with extant PRISMA standards,4 separate protocols have been drafted for each systematic review, which are also registered on the PROSPERO database. (CRD42021290547 – for Cardiovascular Diseases, CRD42021290574 – for Diabetes Mellitus and CRD42021290583 – for Mental Ailments). All three protocols have been written in accordance with the PRISMA-P statement.5\n\n\n\nThe following study designs will be included:\n\n• Randomised & Non-Randomised Controlled Trials\n\n• Observational analytical study designs, such as case control & cohort studies\n\n• Observational descriptive designs such as cross-sectional studies\n\n• Full or partial economic evaluation studies\n\n• Qualitative study designs including qualitative case studies, narrative studies etc.\n\nStudies reporting implementation research not conducted in India, study designs such as case reports, case series etc, policy briefs, editorials, letters, policy/program documents without details of study design used for implementation research, studies/reports published in any Indian language that is not understood by the investigation team member(s) or will require considerable time and effort to translate, will be excluded.\n\nStudies, fulfilling the aforementioned inclusion and exclusion criteria, conducted during the last 20 years (2001-2021) will be included for all three systematic reviews.\n\nAcademic databases including PubMed, Embase and Science Direct will be searched. Search strategies will be formulated in iterative processes and in accordance with the formats that are specific to the databases that will be searched. In addition, grey literature will be searched through various sources namely websites of programs related to non-communicable diseases and that of various departments of national and state governments, private organisations, private-public partnership organisations and non-government organisations (NGOs) engaged in healthcare. Non-academic databases like Google Scholar and ProQuest will also be explored. A few key government portals will be explored, and relevant government agencies will be contacted to obtain documents pertaining to implementation research, as defined in the methodology of this review. The preliminary search strategies are detailed in the appendix of this article.\n\nThe studies will be compiled into citation managing software. After the removal of duplicates, the titles and abstracts in three domains will be screened and assessed against the inclusion and exclusion criteria by two independent reviewers in each team using Rayyan QCRI.6 Titles and abstracts will then be screened and assessed against the inclusion & exclusion criteria for the review by three independent reviewers using Rayyan QCRI software.6 The eligible studies after the initial screening will be retrieved in full and will be assessed in detail against the inclusion criteria by three independent reviewers. Reasons for the exclusion of full-text studies unable to meet the inclusion criteria will be recorded and reported in the final analysis. Any disagreements between the reviewers will be resolved through discussion or consultation with the designated adjudicator wherever needed. From the articles selected for the final review, data pertaining to implementation research aspects in each of the articles will be extracted and tabulated.\n\nThe articles will also undergo a concomitant risk of bias assessment, using the Standards for Reporting Implementation Studies (StaRI) checklist,7 and a study design-specific checklist (such as STROBE,8 CONSORT9 etc.) depending on the study design of the paper in question. It was also decided a priori to produce a narrative summary of the selected articles, from an implementation research perspective, using the SWiM (Synthesis without meta-analysis) guidelines.10\n\nAll selected studies will be grouped as per their study designs. Common to all the selected studies, data will be extracted with regard to the publication date, authors, location, setting, study population, study period and sample size. In addition to these baseline attributes, data pertaining to various domains for IR shall also be recorded.\n\nInitially, two papers will be purposively identified, and a pilot coding form will be created. This coding form will be put up for discussion within the investigation team and will be finalized for further data extraction. Apart from the above details, the coding form will also include relevant IR outcome measures like effectiveness measures (such as uptake, patient-reported outcome measures, etc.),11 economic evaluation measures (such as cost, cost-effectiveness, cost utility, etc.),12 feasibility measures related to aspects such as technical, operational, economic feasibilities, operational issues (barriers, facilitating factors, etc.), fidelity measures (such as adherence, quality of intervention delivery, participant responsiveness, etc.), and scalability measures in terms of cost scalability, operational scalability, etc.11\n\nAll three systematic reviews shall be separately documented and respective manuscripts shall be sent for publication.\n\nSearch and wetting of the articles completed. Narrative synthesis and manuscript preparation are currently underway.\n\n\nDiscussion\n\nThis study aims to gain a better understanding of the scope and magnitude of extant IR efforts in the realm of NCDs in the country. Such a body of evidence would be integral to understanding the implementation research gaps and could pave the way to explore how they could be addressed. In addition to giving insights into how the healthcare initiatives pertaining to CVDs, diabetes and mental health could be implemented in a better way, the outputs from this study could also be a document that makes a case for the need to build and consolidate capacity for implementation research in the country.\n\nNot applicable.", "appendix": "Data availability\n\nNo data is associated with this article.\n\nFigshare: PRISMA-P Checklist_Protocol of Systematic Reviews on Implementation Research on Cardiovascular diseases, Diabetes Mellitus and Mental Ailments in India.pdf, https://doi.org/10.6084/m9.figshare.21556692.v1. 13\n\nFigshare: Appendix I – Preliminary Search Strategy - Implementation research on Cardiovascular Diseases in India, https://doi.org/10.6084/m9.figshare.21903570.v1. 14\n\nFigshare: Appendix II - Preliminary Search Strategy - Implementation research on Diabetes Mellitus in India, https://doi.org/10.6084/m9.figshare.21903729.v1. 15\n\nFigshare: Appendix III - Preliminary Search Strategy – Implementation research on Mental Health in India, https://doi.org/10.6084/m9.figshare.21903732.v1. 16\n\nFigshare: Appendix IV – Preliminary Data Extraction Sheet, https://doi.org/10.6084/m9.figshare.21903765.v1. 17\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nGeldsetzer P, Manne-Goehler J, Theilmann M, et al.: Diabetes and Hypertension in India: A Nationally Representative Study of 1.3 Million Adults. JAMA Intern. Med. 2018 Mar 1; 178(3): 363–372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSreeniwas Kumar A, Sinha N: Cardiovascular disease in India: A 360 degree overview. Med. J. Armed Forces India. 2020 Jan; 76(1): 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealth Performance: NITI Aayog, National Institution for Transforming India, Government of India[cited 2022 Jun 26].Reference Source\n\nPage MJ, McKenzie JE, Bossuyt PM, et al.: The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021 Mar 29; 372: n71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPRISMA-P GroupMoher D, Shamseer L, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst. Rev. 2015 Dec; 4(1): 1. Publisher Full Text\n\nOuzzani M, Hammady H, Fedorowicz Z, et al.: Rayyan—a web and mobile app for systematic reviews. Syst. Rev. 2016 [cited 2022 Oct 18]; 5(1): 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinnock H, Barwick M, Carpenter CR, et al.: Standards for Reporting Implementation Studies (StaRI) Statement. BMJ. 2017 Mar 6; 356: i6795. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E , Altman DG, Egger M, et al.: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J. Clin. Epidemiol. 2008 Apr; 61(4): 344–349. Publisher Full Text\n\nSchulz KF, Altman DG, Moher D: CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010 Mar 23; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampbell M, McKenzie JE, Sowden A, et al.: Synthesis without meta-analysis (SWiM) in systematic reviews: reporting guideline. BMJ. 2020 Jan 16; 368: l6890. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLewis CC, Mettert KD, Dorsey CN, et al.: An updated protocol for a systematic review of implementation-related measures. Syst. Rev. 2018 Dec; 7(1): 66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHusereau D, Drummond M, Petrou S, et al.: Consolidated Health Economic Evaluation Reporting Standards (CHEERS) statement. BMJ. 2013 Mar 25; 346(mar25 1): f1049–f1049. Publisher Full Text\n\nThakor M, Moosan H:PRISMA-P Checklist_Protocol of Systematic Reviews on Implementation Research on Cardiovascular diseases, Diabetes Mellitus and Mental Ailments in India.pdf. figshare. Preprint. 2022. Publisher Full Text\n\nMoosan H, Thakor M:Appendix I – Preliminary Search Strategy – Implementation research on Cardiovascular Diseases in India. figshare. Preprint. 2023. Publisher Full Text\n\nMoosan H, Thakor M:Appendix II – Preliminary Search Strategy – Implementation research on Diabetes Mellitus in India. figshare. Preprint. 2023. Publisher Full Text\n\nMoosan H, Thakor M:Appendix III – Preliminary Search Strategy – Implementation research on Mental Health in India. figshare. Preprint. 2023. Publisher Full Text\n\nMoosan H, Thakor M:Appendix IV – Preliminary Data Extraction Sheet. figshare. Preprint. 2023. Publisher Full Text" }	[ { "id": "162256", "date": "27 Feb 2023", "name": "Zinia Nujum", "expertise": [ "Reviewer Expertise Infectious Diseases 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\nProtocol of systematic reviews on implementation research on cardiovascular diseases, diabetes mellitus and mental ailments in India\nGeneral comment - This will be a very useful review and will be worth publishing. Since the authors have published the search strategy also and included them as appendices, it will be useful for future references. Hence I recommend that the protocol may be taken up for publication.\nSpecific comments- Here are a few suggestions, which may be considered before publication:\nTitle states “Protocol for systematic reviews on “does the author intend to conduct multiple reviews? In this case, multiple protocols will be required CVD, Diabetes Mellitus and Mental ailments are three very broad areas, the search terms -keywords and search strategies would be different. Also, the databases that are searched could be different, for example for mental ailments - PsycINFO covers psychology and behavioral science. This database may not be useful for other areas. So I would suggest having separate protocols for the three areas.\nIt would be better to mention a Protocol for systematic review- the word FOR sounds better to me than OF.\nSearch strategies will be formulated in iterative processes and in accordance with the formats that are specific to the databases that will be searched- shouldn’t we fix the search strategy a priori.\n“Academic databases including PubMed, Embase, and Science Direct will be searched. “- It would be good to add a few more if possible at least Cochrane Library also can be included since the design of the studies includes trials. Also consider some more databases like CINAHL EBSCO, Global Health Ovid, and HMIC Ovid.\nUnder ethical considerations, it can also be written how the review will be beneficial\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": "217931", "date": "27 Nov 2023", "name": "Bishal Gyawali", "expertise": [ "Reviewer Expertise Non-communicable diseases in Primary Care", "Implementation Science", "Global Health", "Low-and Middle-Income Countries" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI appreciate the opportunity to review the manuscript protocol entitled \"Protocol of systematic reviews on implementation research on cardiovascular diseases (CVDs), diabetes mellitus, and mental ailments in India.\" The relevance of the topic cannot be overstated, considering the underutilization of implementation research in India, where exploration of strategies for integrating evidence-based practices into real-world settings is crucial. I have several suggestions to improve the manuscript.\nIn the introduction, the authors rightly emphasize the significant public health challenge posed by major non-communicable diseases (NCDs), including CVDs, diabetes mellitus, and mental ailments in India, necessitating attention to prevention, control, management, and mortality reduction. However, the references cited to support these statements primarily focus on CVDs and diabetes mellitus, neglecting mental ailments. I recommend including references related to the increasing prevalence of mental health conditions in India.\nThe introduction could be strengthened by reporting the burden and consequences of the escalating NCD burden in India. Additionally, it is suggested that the authors provide a brief explanation of implementation research, its significance, and why it is needed in the Indian context, with a focus on NCDs.\nThe protocol lacks clarity regarding the inclusion of interventions, as the authors mention \"any intervention with an embedded implementation research.\" For instance, interventions could be at the system, organizational, site, or patient level. I recommend the authors define these interventions clearly and provide examples in their review.\nThe rationale for limiting the review to the last 20 years (2001-2021) requires clarification.\nIt is not clear if there are language restrictions in the review.\nThe authors stated that non-academic databases such as Google Scholar and ProQuest will be utilized, but it should be clarified that both Google Scholar and ProQuest are web-based academic search engines that catalog records of academic and grey literature. Please reconsider this statement.\nWhen using acronyms such as PRISMA-P, spell out the full name the first time it is used, and similarly, spell out acronyms the first time they are introduced, avoiding repetition thereafter.\nThe choice of only three databases (PubMed, Embase, and Science Direct) needs justification.\nFinally, it is important to acknowledge and discuss potential limitations in the review. There might be substantial implementation research carried out but never published, potentially introducing significant selection bias to the review.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-100
https://f1000research.com/articles/12-936/v1	07 Aug 23	{ "type": "Software Tool Article", "title": "DrosOMA: the Drosophila Orthologous Matrix browser", "authors": [ "Antonin Thiébaut", "Adrian M. Altenhoff", "Giulia Campli", "Natasha Glover", "Christophe Dessimoz", "Robert M. Waterhouse", "Antonin Thiébaut", "Adrian M. Altenhoff", "Giulia Campli", "Natasha Glover", "Christophe Dessimoz" ], "abstract": "Background: Comparative genomic analyses to delineate gene evolutionary histories inform the understanding of organismal biology by characterising gene and gene family origins, trajectories, and dynamics, as well as enabling the tracing of speciation, duplication, and loss events, and facilitating the transfer of gene functional information across species. Genomic data are available for an increasing number of species from the genus Drosophila, however, a dedicated resource exploiting these data to provide the research community with browsable results from genus-wide orthology delineation has been lacking. Methods: Using the OMA Orthologous Matrix orthology inference approach and browser deployment framework, we catalogued orthologues across a selected set of Drosophila species with high-quality annotated genomes. We developed and deployed a dedicated instance of the OMA browser to facilitate intuitive exploration, visualisation, and downloading of the genus-wide orthology delineation results. Results: DrosOMA - the Drosophila Orthologous Matrix browser, accessible from https://drosoma.dcsr.unil.ch/ - presents the results of orthology delineation for 36 drosophilids from across the genus and four outgroup dipterans. It enables querying and browsing of the orthology data through a feature-rich web interface, with gene-view, orthologous group-view, and genome-view pages, including comprehensive gene name and identifier cross-references together with available functional annotations and protein domain architectures, as well as tools to visualise local and global synteny conservation. Conclusions: The DrosOMA browser demonstrates the deployability of the OMA browser framework for building user-friendly orthology databases with dense sampling of a selected taxonomic group. It provides the Drosophila research community with a tailored resource of browsable results from genus-wide orthology delineation.", "keywords": [ "Drosophila", "orthology", "orthologues", "comparative genomics", "database", "orthologous groups", "gene families", "synteny" ], "content": "Introduction\n\nThe fruit fly, Drosophila melanogaster, is one of the most comprehensively studied model organisms, supported by decades of research, with advanced genetic tools and genomic resources, and a wealth of accumulated knowledge (Adams et al. 2000; Markow 2015). It is therefore a key source of gene functional information that can be tentatively propagated to other species through an evolutionarily-informed framework. Reciprocally, cross-species genomic comparisons help to delineate gene evolutionary histories and thereby further inform D. melanogaster biology by characterising gene and gene family origins, trajectories, and dynamics. This is evident from early cross-phyla perspectives (Rubin et al. 2000; Venter et al. 2001) and over shorter evolutionary timescales such as across the Drosophila genus (Drosophila 12 Genomes Consortium 2007; Hahn et al. 2007; Heger and Ponting 2007). Continued sequencing efforts e.g. (Kim et al. 2021; Suvorov et al. 2022) mean that genome assemblies are now available for some 150 Drosophila species, providing unprecedented resolution for employing comparative approaches to study gene and genome evolution across the genus.\n\nCross-species comparisons to characterise gene evolutionary histories provide a foundation from which to trace speciation, duplication, and loss events leading to the gene repertoires encoded in each species’ genome (Koonin 2005). Arising respectively through speciation and duplication events, orthologues and paralogues together form orthologous groups comprising all genes descended from a single gene in the last common ancestor of the set of species under consideration (Nevers et al. 2020). Numerous methods, broadly categorised as tree-based or graph-based approaches, have been developed to delineate orthologous groups (Altenhoff and Dessimoz 2012), with ongoing efforts to improve quality and scalability of orthology resources (Linard et al. 2021; Nevers et al. 2022). Such resources provide the basis for building evolutionarily-informed hypotheses on gene function, or the so-called transfer of functional annotations. This relies on the baseline assumption of functional equivalency amongst genes that share a common ancestor, which although not without its caveats (Robinson-Rechavi 2020), remains the primary means of large-scale functional annotations.\n\nAs the primary database for researchers using D. melanogaster as a model organism, FlyBase provides access to a wide range of information including genetic, genomic, molecular, and reagent resources (Larkin et al. 2021; Gramates et al. 2022). For cross-species gene repertoire comparisons, FlyBase employs the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT) (Hu et al. 2011), which integrates orthologue predictions for human and eight model organisms obtained from a range of popular orthology delineation tools. For comparisons beyond the core model species, FlyBase displays orthology predictions for other Drosophila species as well as for other selected arthropods sourced from the OrthoDB catalogue of orthologues (Zdobnov et al. 2021). Other publicly available orthology resources containing predictions across multiple drosophilids and hundreds to thousands of other species include eggNOG v5.0 (Huerta-Cepas et al. 2019), OrthoInspector (Nevers et al. 2019), Ensembl Genomes (Yates et al. 2022), and the OMA Orthologous Matrix browser (Altenhoff et al. 2021). Most other online orthology resources emphasise taxonomic breadth over depth of sampling within a given lineage, and therefore usually only D. melanogaster is represented.\n\nTo take advantage of the growing number of available genome assemblies for Drosophila species, and to address the lack of orthology resources supporting genus-spanning multi-species comparative analyses to study fruit fly gene and genome evolution, we developed DrosOMA - the Drosophila Orthologous Matrix browser. DrosOMA uses the OMA (Altenhoff et al. 2021) methodology to delineate orthology and paralogy for 36 drosophilids and four outgroup dipterans with high quality genome assemblies and annotations. The results are browsable in a feature-rich web interface, with gene-, orthologous group-, and genome-centric pages, as well as protein domain architecture and local and global genomic synteny visualisations, extensive gene name and identifier cross-references, and available functional annotations. This demonstrates the deployability of the OMA browser framework for building taxon-targeted orthology databases, here at the genus level, and provides a tailored resource for the Drosophila research community.\n\n\nMethods\n\nDrosophila species with high quality and complete assembled and annotated genomes were selected for inclusion in DrosOMA so as to sample broadly across the genus. Of more than 350 assemblies representing some 140 species at the United States National Center for Biotechnology Information (NCBI), genome annotations were available for 49 species (Sayers et al. 2023). Protein-coding gene annotations for D. melanogaster were sourced from FlyBase (Gramates et al. 2022). All of the source data are available publicly - the accession numbers and version numbers are all given in Table 1. Assessments of completeness performed using Benchmarking Universal Single-Copy Orthologues (BUSCO) (R.M. Waterhouse et al. 2018; Manni et al. 2021) v5.4.0 and sourced from the A3Cat Arthropoda Assembly Assessment Catalogue (Feron and Waterhouse 2022) were used to select only annotated assemblies with Diptera-level BUSCO completeness scores of more than 95%. Filtering to reduce sampling of closely related species resulted in a final set of 36 Drosophila species with high-quality annotated assemblies for orthology delineation, as well as four outgroup mosquito species (Table 1).\n\nAnnotations were sourced from the NCBI, apart from D. melanogaster annotations which were sourced from FlyBase. Only one isoform per gene is used as input for OMA.\n\nAll annotated protein-coding genes from the 40 selected species were used as input for delineating orthologous groups for DrosOMA. Briefly, orthology delineation using the OMA Standalone inference algorithm consists of three main stages (Altenhoff et al. 2019, 2021). Firstly, all-against-all Smith-Waterman sequence alignments are computed using the SWPS3 vectorized implementation of the Smith-Waterman local alignment algorithm and significant matches are retained to define homologous proteins (i.e. sequences with a common ancestry). Before inferring orthology, one representative protein per gene is selected. OMA Standalone uses all isoforms for the first all-against-all alignment stage and selects as the reference protein the isoform with the best matches across all species (this can be considered as the most evolutionarily conserved isoform). Secondly, mutually closest homologues between species pairs are identified based on evolutionary distances to infer orthologous pairs (i.e. homologues related through speciation), while accounting for distance inference uncertainties and for potential differential gene losses. Finally, all identified orthologous pairs are clustered using two different approaches to produce catalogues of OMA Groups and Hierarchical Orthologous Groups (HOGs) (Zahn-Zabal et al. 2020). HOGs are defined as sets of genes that descended from a single ancestral gene at a given taxonomic range. These sets correspond to the idea of subfamilies for a given taxonomic range and can contain more than one gene from a species, i.e. inparalogues. OMA Groups on the other hand are sets of orthologues where each gene is orthologous to one another. The history of such sets should correspond to the species phylogeny and hence they are especially useful as markers to reconstruct the species phylogeny. For this dataset the production pipeline of OMA was employed, but the same clustering can also be performed using OMA standalone. In order to build the browsable DrosOMA instance, the OMA orthologues were converted using the oma2hdf command from the pyoma python package into an HDF5 database. CATH domain annotations (Sillitoe et al. 2021) were computed using the cath-tools v0.16.10 package and with the provided hmm models from CATH release 4.2. Protein cross-references were added by matching the sequences against the full UniProtKB and RefSeq databases, requiring exact matches.\n\nThe OMA browser instance for DrosOMA was set up and is hosted on a virtual machine using docker containers. The virtual machine requires relatively modest resources, i.e. 2 CPUs clocked at 2.25 GHz each, 8 GB RAM and 25 GB storage. The docker images for the OMA Browser were created from the pyomabrowser repository (https://github.com/DessimozLab/pyomabrowser) following the steps described in https://zoo.cs.ucl.ac.uk/doc/pyomabrowser/setup.html. Before building the docker images, the following aspects of the OMA Browser web interface were adjusted in order to make it a Drosophila-specific instance: We removed all the instances of non-drosophila proteins in the search examples by adjusting the Django templates in oma/templates, oma/test/ and oma_rest/. Similarly, we changed the OMA logo by replacing the corresponding file in/oma/static/image. These customisations are mostly cosmetic changes that will make the service more user friendly, and are not strictly needed for website functionality. Finally, paths, deployment type, and rabbitmq/celery credentials were adjusted and hosts were allowed in for_docker/env.\n\nThe species tree was computed using single-copy orthologues identified during the BUSCO completeness assessments of the genomes of the species selected for inclusion in DrosOMA. The protein sequences of BUSCO genes found in at least 38 of the 40 species were aligned using MUSCLE 3.8.1551 (Edgar 2004) with default settings and subsequently trimmed to retain well-aligned regions using TrimAl (Capella-Gutiérrez et al. 2009) with the “-strictplus” option. The 2,891 alignments were merged to build a 40-species concatenated superalignment (1,581,953 columns; 683,285 distinct patterns; 658,691 parsimony-informative; 180,333 singleton sites; 742,929 constant sites) used as input for phylogeny reconstruction using IQ-TREE 2.2.0-beta (Nguyen et al. 2015) with 1,000 bootstrap samples (options: -msub nuclear -B 1000 -bnni). The molecular species phylogeny was time-calibrated by providing calibration dates for the Diptera root, Culicidae, Drosophilini, willistoni-melanogaster ancestor, and navojoa-albomicans ancestor, from the TimeTree database (Kumar et al. 2022) to the functions makeChronosCalib() and chronos(), from the ape R package (Paradis and Schliep 2019), and plotted using the ggtree R package (Yu 2023).\n\nThe DrosOMA Drosophila Orthologous Matrix browser implements for users a feature-rich web interface to explore the results of orthology inference amongst complete genomes. The service is implemented with the django framework, a high-level Python web framework that encourages rapid development and clean, pragmatic design.\n\nThe DrosOMA Drosophila Orthologous Matrix browser operates on standard up-to-date web browsers including Google Chrome, Mozilla Firefox, and Apple Safari. The operational setup of an OMA browser instance such as DrosOMA requires a host that runs docker containers orchestrated with docker compose.\n\n\nResults\n\nApplying OMA orthology delineation to the protein-coding genes from 36 drosophilids and four outgroup mosquito species (see Methods) resulted in the clustering of 93.5% of proteins in OMA Groups and 95.6% in Hierarchical Orthologous Groups (HOGs), with almost 25,000 HOGs at the last common ancestor of all DrosOMA species (Table 2). The OMA Groups are cliques of orthologues based on the orthology graph, meaning that all the components (proteins) of an OMA Group are connected to each other through pairwise orthologous relationships. Although all members of the OMA Groups are orthologous to all other members of the same group, OMA group members are not necessarily 1-to-1 orthologues. The OMA HOGs comprise sets of proteins encoded by genes descended from a common ancestral gene in the last common ancestor of a set of species (i.e. at a specific taxonomic level in the species phylogeny). The “hierarchical” nature of HOGs is due to their being defined with respect to specific clades within the species tree, so HOGs are nested subfamilies with groups delineated for younger radiations being encompassed within larger HOGs defined at older nodes. DrosOMA contains HOGs delineated at the root, three mosquito nodes, and 13 drosophilid nodes including Sophophora, the melanogaster group, and the melanogaster subgroup.\n\nThe fully-resolved time-calibrated species phylogeny (see Methods) defines the relationships amongst the 36 Drosophila species and the outgroup mosquitoes over approximately 260 million years of evolution (Figure 1). Analysis of the root-level HOGs shows counts of proteins per species belonging to universal single-copy HOGs (9.8% of HOGs; 17.1% of proteins), universal but variable-copy-number HOGs (19.6% of proteins), non-universal HOGs with outgroup species orthologues (13.7% of proteins), as well as drosophilid-specific HOGs with orthologues from all (16.8% of proteins), the majority (17.9% of proteins), or the minority (7.7% of proteins) of the 36 Drosophila species. This leaves an average of 527±392 proteins per drosophilid species with no identifiable orthologues, i.e. annotated protein-coding genes that, given the set of species under consideration, appear to be species-specific with no traceable common ancestry.\n\nThe time-calibrated species phylogeny (left) shows the estimated evolutionary relationships amongst the set of 40 species spanning approximately 60 million years since the last common ancestor of the Drosophila genus. The dashed line indicates the Drosophila and Culicidae last common ancestor but for visualisation is not placed according to the timescale. The barchart (right) shows counts of genes per species categorised according to their orthology type based on root-level hierarchical orthologous groups (HOGs). Analysis of the root-level HOGs shows counts of proteins per species belonging to universal single-copy HOGs (Single-copy All), universal but variable-copy-number HOGs (Orthologues All), non-universal HOGs with outgroup species orthologues (Drosophila & Culicidae), mosquito-only orthologues (Culicidae Only), as well as drosophilid-specific HOGs with orthologues from all (Drosophila All), the majority (Drosophila Majority), or the minority (Drosophila Minority) of the 36 Drosophila species. This leaves an average of 527 ±392 proteins per drosophilid species with no identifiable orthologues, i.e. annotated protein-coding genes that, given the set of species under consideration, appear to be species-specific with no traceable common ancestry. Branch lengths are shown in millions of years; all nodes received 100% bootstrap support except * with 95%; D. Drosophila; An. Anopheles; Ae. Aedes; Minority <18 drosophilids; Majority ≥18 drosophilids.\n\nAs DrosOMA uses the same database and interface design and architecture as the OMA browser (Altenhoff et al. 2021), an extensive array of data querying and visualisation options are available to the user. Searches may be performed using gene or protein names, descriptors, or identifiers, or protein sequences, and extensive cross-referencing to public databases allows for searches using identifiers from resources such as UniProt (The UniProt Consortium et al. 2023), RefSeq (O’Leary et al. 2016), EntrezGene (Sayers et al. 2023), Swiss Model (A. Waterhouse et al. 2018), STRING (Szklarczyk et al. 2023), and Bgee (Bastian et al. 2021), in addition to the source FlyBase and NCBI identifiers and annotations (Figure 2A). Search result visualisations are focused on the three main data types, i.e. with views for genomes, groups (Figure 2B), or genes (Figure 2C). Genome-view pages summarise available information per species, e.g. a list of all their genes and of their most closely related species, as well as tools for building pairwise global synteny visualisations. Group-view pages display information about OMA Groups or HOGs, showing filterable lists of member genes with their associated cross-referenced identifiers and cartoon views of protein domain architectures, as well as visualisations of HOG members guided by the species phylogeny. Gene-view pages display information associated with a gene and its protein products, including sequences (protein and cDNA), cross references to other public databases, and available functional annotations in the form of Gene Ontology terms (The Gene Ontology Consortium et al. 2021).\n\n(A) The simple search entry point for DrosOMA allows for text searches with gene names, descriptors, or identifiers, as well as with protein sequences. (B) Visualising information for Hierarchical Orthologous Groups (HOGs) can be guided by the species phylogeny (left) showing counts of orthologues per species, or as a table (right) with protein identifiers and cartoons showing domain architectures. (C) The gene view page displays available information for genes of interest and their mappings to external databases.\n\nOther useful search, visualisation, and download features are described in the DrosOMA “Explore”, “Tools”, “Download”, and “Help” pages, with several examples and explanations for the general use of the OMA browser elaborated in a dedicated primer (Zahn-Zabal et al. 2020). Examples of these extended features include sequence alignment tools (Figure 3A) and local synteny visualisations (Figure 3B). For both OMA Groups and HOGs, the browser can generate multiple sequence alignments of the member proteins that can further be sorted, filtered, edited, and exported by users, for example, to use as inputs for building gene trees for orthologous groups of interest. Synteny, or how orthologues have maintained or shuffled their genomic arrangements throughout evolution, can be visualised at a local level (e.g. from a context of 9 to 19 orthologues) or at global level (along entire chromosomes for pairs of species), both based on comparing the relative genomic positions of orthologues across the species under consideration.\n\n(A) Multiple sequence alignments of proteins from hierarchical orthologous groups (HOGs) or OMA Groups can be generated, visualised, explored, and downloaded using the DrosOMA Browser. (B) Local gene synteny conservation can be visualised to explore how orthologues have maintained or shuffled their local arrangements in the genomes of each considered species.\n\n\nConclusions\n\nThe rapidly growing number of species with sequenced and annotated genomes mean that publicly accessible resources offering results from large-scale comparative analyses such as orthology delineation often prioritise taxonomic breadth over depth when selecting which species to include. This means that despite increasingly comprehensive species sampling within some taxonomic groups, the available genomic data can remain under-exploited as only representative species are included in most taxonomically broad resources. The DrosOMA browser provides a resource aimed at the Drosophila research community that exploits the available high-quality genome annotation data across the genus. The successful deployment of DrosOMA illustrates the feasibility and utility of the OMA browser framework to be applied to other taxonomic groups with rapidly growing numbers of species with genomic data. Future studies taking advantage of increased taxonomic depth of sampling within a given genus, such as previous genus-wide investigations of Anopheles mosquitoes (Neafsey et al. 2015) or Bombus bumblebees (Sun et al. 2021), could therefore benefit from applying the framework to not only obtain orthology data, but to simultaneously build and deploy an interactive browser to further support their research. Yet-to-be annotated genome assemblies are publicly available for almost 100 more drosophilids, and data generation for additional species is ongoing. As more high-quality annotations for high-quality genomes become publicly available, future DrosOMA releases are set to further deepen taxonomic representation within the genus containing the arguably best studied representative of all animals.", "appendix": "Data availability\n\nThe underlying data is available from the DrosOMA Browser (https://drosoma.dcsr.unil.ch/).\n\nAll sequence data used to build the DrosOMA browser database were originally sourced from a public repository, the United States National Center for Biotechnology Information (NCBI). The sources for which have been compiled and are provided on an online repository below:\n\nFigshare: Table S1: Data Sources for DrosOMA, the Drosophila Orthologous Matrix browser. https://doi.org/10.6084/m9.figshare.23622507.v1 (Thiébaut et al., 2023).\n\nThis project contains the following underlying data:\n\n- DrosOMA_Data_TableS1.xlsx (Data Sources for DrosOMA, the Drosophila Orthologous Matrix browser).\n\nData are available under the terms of the Creative Commons Attribution 4 International (CC BY 4.0) license.\n\nThe underlying sequences and annotations from the NCBI may be subject to third-party constraints (some submitters of the original data, or the country of origin of such data, may claim patent, copyright, or other intellectual property rights in all or a portion of the data). Users of the data are solely responsible for establishing the nature of, and complying with, any such intellectual property restrictions, as the authors of this article have done.\n\nThe completeness assessments used to select high-quality public data were sourced from the A3Cat Arthropoda Assembly Assessment Catalogue.\n\n\nAcknowledgments\n\nThe authors thank Nina Thomas for designing the DrosOMA logo.\n\n\nReferences\n\nAdams MD, Celniker SE, Holt RA, et al.: The Genome Sequence of Drosophila melanogaster. Science. 2000; 287: 2185–2195. Publisher Full Text\n\nAltenhoff AM, Dessimoz C: Inferring Orthology and Paralogy.Anisimova M, editor. Evolutionary Genomics. Vol. 855. Methods in Molecular Biology. Totowa, NJ: Humana Press; 2012; pp. 259–279. Publisher Full Text\n\nAltenhoff AM, Levy J, Zarowiecki M, et al.: OMA standalone: orthology inference among public and custom genomes and transcriptomes. Genome Res. 2019; 29: 1152–1163. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSayers EW, Bolton EE, Brister JR, et al.: Database resources of the National Center for Biotechnology Information in 2023. Nucleic Acids Res. 2023; 51: D29–D38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSillitoe I, Bordin N, Dawson N, et al.: CATH: increased structural coverage of functional space. Nucleic Acids Res. 2021; 49: D266–D273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun C, Huang J, Wang Y, et al.: Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits. Wei F, editor. Mol. Biol. Evol. 2021; 38: 486–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuvorov A, Kim BY, Wang J, et al.: Widespread introgression across a phylogeny of 155 Drosophila genomes. Curr. Biol. 2022; 32: 111–123.e5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzklarczyk D, Kirsch R, Koutrouli M, et al.: The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023; 51: D638–D646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Gene Ontology ConsortiumCarbon S, Douglass E, et al.: The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021; 49: D325–D334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe UniProt ConsortiumBateman A, Martin M-J, et al.: UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 2023; 51: D523–D531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThiébaut A, Altenhoff AM, Campli G, et al.: Table S1: Data Sources for DrosOMA, the Drosophila Orthologous Matrix browser. Dataset. figshare. 2023. Publisher Full Text\n\nVenter JC, Adams MD, Myers EW, et al.: The Sequence of the Human Genome. Science. 2001; 291: 1304–1351. 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: W296–W303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaterhouse RM, Seppey M, Simão FA, et al.: BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics. Mol. Biol. Evol. 2018; 35: 543–548. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYates AD, Allen J, Amode RM, et al.: Ensembl Genomes 2022: an expanding genome resource for non-vertebrates. Nucleic Acids Res. 2022; 50: D996–D1003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu G: Data integration, manipulation and visualization of phylogenetic trees. 1st ed.Boca Raton: CRC Press, Taylor & Francis Group; 2023.\n\nZahn-Zabal M, Dessimoz C, Glover NM: Identifying orthologs with OMA: A primer. F1000Res. 2020; 9: 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZdobnov EM, Kuznetsov D, Tegenfeldt F, et al.: OrthoDB in 2020: evolutionary and functional annotations of orthologs. Nucleic Acids Res. 2021; 49: D389–D393. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "219663", "date": "24 Nov 2023", "name": "Berend Snel", "expertise": [ "Reviewer Expertise Comparative genomics. Bioinformatics. Orthology. Evolution of protein complexes." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a novel browsable database for the OMA output specific for the Drosophila genus. The methods, including the more technical computational details and genomic considerations of completeness are very clear. The different features of the dataset that are available and how to browse them are also very clear.\nI have only a few points to discuss.\nFirst I am a bioinformatician myself and not a “drosophila” biologist. I am not sure if the current manuscript sufficiently explains to such an audience what the database and its interface exactly offer and what inferences you can use it for. Maybe collaboration with such an expert can be sought to see if they indeed see the data set as something for them.\nSecond and this is quite big but not essential, we are currently basically redoing all of our normal sequence searches with AF2 structural searches. Especially for the inverse cases of “lineage specific genes” or “lineage specific gene loss” it turns out that a lot more homology and thereby also orthology remains to be discovered. I wonder if this is something to consider for the future (but I guess that is an OMA issue and not a drosOMA issue).\nFinally given also the above considerations, panel B of figure 3 shows synteny and the final gene DROME05445 stands out in its column i.e. a green gene in an orange column. So I checked this situation in drosOMA which was indeed eminently browsable, and could open both that gene and its syntenic (but according to drosOMA non orthologous) gene in uniprot (which the the tools lets me do very easily), and the AF2 predictions are identical leading me to expect that both genes are potentially orthologous. In any case I am always worried about false negatives and the visualization via synteny might highlight potential false negatives.\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": "10886", "date": "16 Jan 2024", "name": "Robert Waterhouse", "role": "Author Response", "response": "The article describes a novel browsable database for the OMA output specific for the Drosophila genus. The methods, including the more technical computational details and genomic considerations of completeness are very clear. The different features of the dataset that are available and how to browse them are also very clear. => We thank the reviewer for the positive comments and for particularly recognising the clarity of the methods and features descriptions. I have only a few points to discuss. First I am a bioinformatician myself and not a “drosophila” biologist. I am not sure if the current manuscript sufficiently explains to such an audience what the database and its interface exactly offer and what inferences you can use it for. Maybe collaboration with such an expert can be sought to see if they indeed see the data set as something for them. => We thank the reviewer for bringing up this point about better explaining to potential users what they might use the resources for. After describing the main features we had only briefly mentioned the use of the data in large-scale genus-wide evolutionary studies, giving the Anopheles mosquitoes and Bombus bumblebees as examples. In the data exploration section we now added a few more specific examples to help explain the DrosOMA browser offerings in more concrete terms. Second and this is quite big but not essential, we are currently basically redoing all of our normal sequence searches with AF2 structural searches. Especially for the inverse cases of “lineage specific genes” or “lineage specific gene loss” it turns out that a lot more homology and thereby also orthology remains to be discovered. I wonder if this is something to consider for the future (but I guess that is an OMA issue and not a drosOMA issue). => We agree that this is a very interesting topic and very relevant for inferences made about gene evolutionary histories that could be missing key information when real homology fails to be detected. Ongoing OMA development continues to address these and other issues, which are faced by all orthology delineation methods. Finally given also the above considerations, panel B of figure 3 shows synteny and the final gene DROME05445 stands out in its column i.e. a green gene in an orange column. So I checked this situation in drosOMA which was indeed eminently browsable, and could open both that gene and its syntenic (but according to drosOMA non orthologous) gene in uniprot (which the the tools lets me do very easily), and the AF2 predictions are identical leading me to expect that both genes are potentially orthologous. In any case I am always worried about false negatives and the visualization via synteny might highlight potential false negatives. => We thank the reviewer for taking the time to carry out this investigation and we are pleased that the browsing experience was easy and useful. We investigated this example and agree with the reviewer that these genes are both syntenic and orthologous. Investigating the rendering of the synteny visualisation revealed that the colour palette we had used did not provide enough colours for proper rendering, we have updated the palette and visualisation is now correct. We updated Figure 3 accordingly with a new screenshot of the local synteny visualisation." } ] }, { "id": "222942", "date": "20 Dec 2023", "name": "Daofeng Li", "expertise": [ "Reviewer Expertise Data visualization", "epigenomics", "Genome Browser" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described a well-developed web portal to allow users query high-quality gene/protein annotations across drosophila genus (and 4 others). The data for ontology annotations used on the website were generated by the published method OMA. The website is fast and user friendly, the manuscript and figures are well written/designed. This could be a valuable resource for the fruit fly research community. Meanwhile, docker container is provided to enable users generate annotations and web interface from their interested species. I only have some minor suggestions: 1) In the query box of the DrosOMA landing page, I hope the authors can implement an example data button to add example query input the search box, especially when user changes the item from the dropdown menu, the suggested example query item should update. 2) The documentation for the DrosOMA website is minimal, if the users can expand the documentation for the website could be great, like how to use the website, how to explain the results, how to download a plot etc. 3) I am wondering if the OMA method could work on other types of gene except protein coding genes, such as rRNA gene, pseudo genes etc. 4) Could the author explain more about how to update the annotation data when trying to add/remove/update any source genomes/annotations.\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": "10887", "date": "16 Jan 2024", "name": "Robert Waterhouse", "role": "Author Response", "response": "The authors described a well-developed web portal to allow users query high-quality gene/protein annotations across drosophila genus (and 4 others). The data for ontology annotations used on the website were generated by the published method OMA. The website is fast and user friendly, the manuscript and figures are well written/designed. This could be a valuable resource for the fruit fly research community. Meanwhile, docker container is provided to enable users generate annotations and web interface from their interested species. => We thank the reviewer for their appreciative summary comments. I only have some minor suggestions: 1) In the query box of the DrosOMA landing page, I hope the authors can implement an example data button to add example query input the search box, especially when user changes the item from the dropdown menu, the suggested example query item should update. => While a few generic examples are provided in the search box and additional examples below, it could indeed be useful to add updatable terms that match the types of query available from the dropdown menu. As this would be specific to each instance of an OMA browser (depending on species, versions etc.) adding this would make more curation work necessary for anyone wishing to build an OMA browser for themselves, so we aim to keep it simple and minimise the customisation burden. 2) The documentation for the DrosOMA website is minimal, if the users can expand the documentation for the website could be great, like how to use the website, how to explain the results, how to download a plot etc. => The menus on the top right of the pages (three horizontal bars on small screens, full drop-down menus on larger screens) provide quick and easy access to the documentation, this includes pages dedicated to how to explore the database, the tools connected to using DrosOMA and OMA in general, a download page showing how to obtain the data, as well as the main help pages - Introduction, Orthology basics; Type of homologs; Access the OMA data; Catalog of tools; FAQ; Q&A on Biostars; the Glossary; the link to the OMA Academy pages; and finally, the About page. At the bottom of the home page there is further help information, e.g. in the box titled “First time here?”, as well as the “OMA tools” and “Download options” information boxes. Several specific pages also provide more targeted help for the user, e.g. the alignment viewer page provides a Help button to access the user manual, the local synteny viewer provides a Help button showing a graphic explaining how the viewer works, etc. In addition, in the manuscript we also point to helpful information for users elaborated in a dedicated primer (Zahn-Zabal et al. 2020). To us this seems already very comprehensive, of course we can and will continue to elaborate these help pages in the context of updates to OMA and DrosOMA, especially to describe new features. 3) I am wondering if the OMA method could work on other types of gene except protein coding genes, such as rRNA gene, pseudo genes etc. => In theory, if equivalent pairwise distances could be computed then the rest of the OMA algorithm could be applied to other types of genes, but the OMA method has not been applied to non-protein-coding genes and is not designed to process them. 4) Could the author explain more about how to update the annotation data when trying to add/remove/update any source genomes/annotations. => For a comprehensive update, all pre-processing steps need to be re-computed, this is especially important because of the number of links to external sources provided by DrosOMA because these databases also update their information. So future releases will include more Drosophila species but the processing and orthology delineation needs to be completely rerun to ensure internal and external consistency and robustness." } ] } ]	1	https://f1000research.com/articles/12-936
https://f1000research.com/articles/11-1581/v1	23 Dec 22	{ "type": "Research Article", "title": "Helicobacter pylori prevalence in Indonesia: Higher infection risk in Eastern region population", "authors": [ "Hasan Maulahela", "Dalla Doohan", "Yudith Annisa Ayu Rezkhita", "Ari Fahrial Syam", "Langgeng Agung Waskito", "Camilia Metadea Aji Savitri", "Marselino Richardo", "Abdul Rahman", "Yoma Sari Namara", "Hamzah Shatri", "Andri Sanityoso", "Gontar Alamsyah Siregar", "Eko Sudarmo", "Tomohisa Uchida", "Ratha-korn Vilaichone", "Yoshio Yamaoka", "Muhammad Miftahussurur", "Hasan Maulahela", "Dalla Doohan", "Yudith Annisa Ayu Rezkhita", "Ari Fahrial Syam", "Langgeng Agung Waskito", "Camilia Metadea Aji Savitri", "Marselino Richardo", "Abdul Rahman", "Yoma Sari Namara", "Hamzah Shatri", "Andri Sanityoso", "Gontar Alamsyah Siregar", "Eko Sudarmo", "Tomohisa Uchida" ], "abstract": "Background: Helicobacter pylori (H. pylori) infection status in Indonesia might be higher than the actual prevalence. A comprehensive epidemiologic study is necessary to explore infection susceptibility by demographic characteristics, especially between Indonesia’s Eastern and Western regions. Methods: We included our recent survey and data from our previous studies with 1,172 endoscopic examinations from 19 cities throughout Indonesia from January 2014 to March 2017. H. pylori infection was diagnosed based on the combination of four different tests. Using a criterion of one positive result between four tests, we found that 190 subjects (190/1,158; 16.4%) were H. pylori positive. A total of 939 subjects were analyzed for risk factors of sociodemographic characteristics to H. pylori infection. Results: Prevalence was slightly higher in the Eastern region compared with the Western region of Indonesia (79/277; 59.4% vs. 54/529; 40.6%). Living in the Eastern region imposes a higher risk of infection (OR 5.33, 95%CI 1.17–24.26). Timor ethnicity had the highest prevalence (15/28; 53.6%), followed by Buginese ethnic (21/74; 28.4%), and Papuan ethnic (18/64; 28.1%) in the Eastern region. As an ethnicity with a high prevalence (37/109; 33.9%), Batak was an outlier among the low prevalence areas in the Western region. In general, age, living in the Eastern region, Batak ethnicity, Protestant, Catholic, and alcohol drinking were independent risk factors associated with H. pylori infection in Indonesia (OR 1.03, 95%CI 1.02–1.05; OR 5.33, 95%CI 1.17–24.26; OR 5.89, 95%CI 1.58–21.94; OR 5.47, 95%CI 2.16–13.86; OR 5.05, 95%CI 1.43–17.80; OR 2.30, 95%CI 1.14–4.61, respectively). Conclusions: Several ethnicities and habits were concluded to increase the risk of infection as the interaction of host, agent, and environment is inseparable in affecting disease susceptibility.", "keywords": [ "H. pylori", "prevalence", "ethnic", "Eastern and Western region", "infectious disease" ], "content": "Introduction\n\nHelicobacter pylori, a stomach-living bacteria, infects more than half of the world’s population.1 While most individuals were asymptomatic, only 10% – 20% had symptoms, including a 1% – 2% chance of developing gastric malignancy.2–4 H. pylori infection is associated with various diseases, including chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer.2,5–7 H. pylori infection was strongly related to hygiene and sanitation,8,9 and transmission was believed to be acquired in childhood.10 Even though no exact prevalence had been known, it was reported to be higher in developing countries.11 Moreover, it is varied between different geographical areas. The variety raised the question of whether particular hosts, agents, and environmental factors increased the risk of infection. Recently, the prevalence of H. pylori infection decreased worldwide due to sanitation improvements in most countries.1 Despite its decreasing prevalence, the prevalence and risk factor analysis updates in countries are still necessary since H. pylori is the only microbiota crowned by the World Health Organization (WHO) to cause cancer. Hence, knowing the prevalence might be able to prevent this.\n\nIndonesia is a developing country in Southeast Asia, with thousands of islands complemented by substantial ethnic diversity. Therefore, it is challenging to determine the accurate prevalence therein. Studies reported that Indonesia had a low prevalence of H. pylori infection.12 Despite the low overall prevalence, there were vast differences in H. pylori infection between regions and ethnicities. For example, the prevalence in Jayapura, Papua Island and Medan, Sumatra Island was significantly higher than in cities on Java Island.12 Several islands in Indonesia were huge and lived by multiple ethnicities with a varied prevalence that guaranteed further analysis. Apart from ethnicities, habits also relate significantly to someone’s susceptibility to infection since the whole interaction of the host, agent and environment is inseparable. Moreover, previous studies also mentioned the significantly high risk of Papuan and Buginese ethnics, Protestant religion, and water drinking sources to H. pylori infection in Indonesia.12 Different areas have different habits; hence it will show a difference in disease susceptibility.\n\nPreceding studies on H. pylori infection’s prevalence in Indonesia might be the tip of the iceberg. There is plenty to be revealed about the association between sociodemographic characteristics and H. pylori infection. Prevalence was scanty as the previous study only used small samples, while Indonesia is a vast country with a 267 million population. Aside from population number, the heterogeneity of ethnics, religions, and habits also affect the susceptibility to H. pylori infection. Hence, this study will extensively analyze H. pylori infection prevalence, especially in Indonesia’s Eastern and Western regions.\n\n\nMethods\n\nThis study continues our previous Indonesia endoscopic study.12,13 In addition, we included our most recent endoscopy samples taken from August 2016 until March 2017. Samples were taken in four cities with a total of 193 samples: 50 subjects from Kolaka and 56 subjects from Palu in Sulawesi Island, 43 subjects from Merauke in Papua Island, and 44 subjects from Ternate in Maluku Island (117 male and 76 female subjects, mean age 44.1 ± 12.1 years). Merauke and Kolaka samples were taken during August – September 2016, while Palu and Ternate samples were taken in March 2017. To provide a thorough analysis, we also included samples from our previous preliminary study of the prevalence of H. pylori infection, taken from six big cities in Indonesia,12 the prevalence in Malay ethnic14 and previous surveys for antibiotic resistance.13 All studies used the same data extraction method as this study. Samples were taken in five cities in Sumatra Island; Aceh (n = 38), Medan (n = 82), Dolok Sanggul (n = 47), Gunung Sitoli (n = 32), Padang (n = 33), Palembang (n = 38), one city in Kalimantan Island; Pontianak (n = 90), three cities in Java Island; Cimacan (n = 22), Jakarta (n = 62), Surabaya (n = 316), one city in Bali Island; Bangli (n = 61), one city in East Nusa Tenggara; Kupang (n = 35), four cities in Sulawesi Island; Manado (n = 57), Makassar (n = 30), Kolaka (n = 50), Palu (n = 56), two cities in Papua Island; Jayapura (n = 22) and Merauke (n = 43), and one city in the Moluccas; Ternate (n = 44) (Figure 1). Finally, we included 1,158 samples collected throughout Indonesia.\n\nBiopsy specimens were obtained from 1,158 patients in Indonesia (created with BioRender.com).\n\nA qualitative questionnaire was given for demographic data analysis (can be found as Underlying data71). Questionnaires were made by us and have been unchanged since the first use of the survey in 2013. Subjects signed consent prior to answering the questionnaire and endoscopic examination. When subjects finished answering all questions in the questionnaire, it was concluded as finished. All recruited subjects underwent an endoscopic examination in the referral hospitals having endoscopic facilities by experienced endoscopists, and a total of four gastric biopsies were collected. Three gastric biopsies were taken from the lesser curvature of the antrum, and one gastric biopsy was taken from the greater curvature of the corpus. One antrum biopsy tissue was used for the rapid urease test, and one was kept in a transport medium and immediately stored at –20°C until used. For histological examination, we used one antrum biopsy tissue and one from the corpus, and both were kept in 10% neutral buffered formalin until used.\n\nEthical approvals were obtained from the Ethics Committee of Dr. Soetomo Teaching Hospital, Surabaya, Indonesia (221/Panke.KKE/IX/2012. date: September 25th, 2012), Dr. Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia (206/112/P1/ETIK/2014. date: April 7th, 2014), Dr. Wahidin Sudirohusodo Hospital, Makassar, Indonesia (208/H4.8.4.5.31/PP36-KOMETIK/2015. date: February 16th, 2015), and Oita University Faculty of Medicine, Yufu, Japan (P-12-10. date: January 18th, 2013). Objectives and procedures of the study were explained and written informed consent was obtained under the guidelines of the Declaration of Helsinki.\n\nH. pylori infection was diagnosed based on the combination of four tests: rapid urease test, histology, immunohistochemistry, and bacterial culture. Patients were considered H. pylori-positive if at least one test showed positive and negative when all tests showed negative results.\n\nAs previously described, one antrum biopsy specimen was used for culture.12–16 Briefly, homogenized antrum specimens were inserted onto H. pylori selective plates (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan; Ref. Number 51035), which were then cultured at 37°C under microaerophilic conditions for up to 10 days (5% CO2, 10% O2, and 85% N2). Sub-culture was performed on Brucella Agar medium (Becton Dickinson, Sparks, MD, USA; Ref. Number 211086) complemented with 7% horse blood but without antibiotics. H. pylori was detected by bacterial colony morphology, Gram staining, and positive reaction of oxidase, catalase, and urease. H. pylori strains were kept at –80°C in Brucella Broth medium supplemented with 10% horse serum (Biosera, France, Code Number BS-110) and 10% glycerol (Becton Dickinson, Sparks, MD, USA, Ref. Number 228220).\n\nAll biopsy specimens for histological examination were fixed in 10% buffered formalin and embedded in paraffin. Hematoxylin eosin and May-Giemsa stain were used to stain serial sections. An experienced pathologist (TU) evaluated gastric biopsies using the updated Sydney system.17 Immunohistochemistry (IHC) was performed as previously described.18,19 Tissue sections were incubated overnight at 4°C with α-H. pylori antibody (DAKO, Denmark; Ref. Number B0471), polyclonal rabbit anti-CagA antibody (b-300) (Santa Cruz Biotechnology Cat# sc-25766, RRID:AB_2184120) or α-EAS Ab diluted for 1:2,000 with diluting solution (DAKO, Denmark) after antigen was retrieved and endogenous peroxidase activity was inactivated. After washing, sections were incubated with biotinylated goat anti-rabbit or anti-rat IgG (Nichirei Biosciences, Inc., Japan; Code. Number 424022), followed by incubation with avidin-conjugated horseradish peroxidase solution (Vectastain Elite ABC kit; Vector Laboratories Inc., Burlingame, CA, USA; Ref. Number PK-6100). H2O2/diaminobenzidine substrate solution was used to detect peroxidase activity. H. pylori were identified by Giemsa staining and anti-H. pylori antibody-positive immunostaining with bacterial loads greater than or equal to grade 1 was considered positive for H. pylori.17\n\nThe IBM SPSS Statistics version 23 (RRID:SCR_016479) (IBM Corp., Armonk, NY, USA) was used for statistical analyses. Separate variables were tested using the chi-square test, while continuous variables were tested using Mann-Whitney U and t-tests. A logistic regression model was used to calculate the odds ratios (OR) for H. pylori infection. A multivariate logistic regression model was used to calculate odds ratios including age, sex, H. pylori infection status, demographic, and environmental data. Both OR and 95% confidence interval (CI) were used to estimate the risk of infection.\n\n\nResults\n\nIn analyzing prevalence, we included 1,158 samples. We found that H. pylori infection prevalence in Palu (Sulawesi), Ternate (Moluccas), Merauke (Papua), and Kolaka (Sulawesi) were 12.5% (7/56), 4.5% (2/44), 20.9% (9/43), and 16.0% (8/50), respectively. When we included all data, the prevalence of H. pylori infection in Indonesia was found to be highest in East Nusa Tenggara (51.4%), followed by Papua Island (30.7%), Sumatra Island (24.4%), Sulawesi Island (18.1%), Bali Island (18.0%), Kalimantan Island (8.9%), Java Island (7.4%), and Moluccas Islands (4.5%). However, positivity rates varied between cities on the same island. In Papua Island, Jayapura had a higher prevalence than Merauke (50.0% vs. 20.9%). Furthermore, Makassar (36.7%) had a twice higher prevalence than other cities on Sulawesi Island, such as Kolaka (16.0%), Palu (12.5%), and Manado (15.8%). Table 171 shows the complete distribution of H. pylori positivity rates in islands and cities in Indonesia.\n\n* Corresponds to our previous study12\n\n Corresponds to our previous study13\n\n* Corresponds to our previous study in Sumatra14\n\nIn this analysis, we divided Indonesia into Eastern and Western regions. The regions were divided based on the Wallace line that separates biogeographical realms between Australia and Asia.20 The Western region consists of Sumatra, Kalimantan, and Java Islands, while the Eastern region consists of Sulawesi, Papua, Moluccas, East Nusa Tenggara, and Bali Islands. Overall, the prevalence was higher in the Eastern region than in the Western region (86/398; 21.6% vs. 104/761; 13.7%). In the Western region, the prevalence of H. pylori infection was only high in two cities, Medan (41/82; 50.0%) and Dolok Sanggul (20/47; 42.6%), both located in North Sumatra province, while in the Eastern region, the prevalence was the highest in Kupang, Timor Island (18/35; 51.4%).\n\nWe examined the prevalence of H. pylori infection in Indonesia’s ethnic groups (Table 2). Among 17 ethnicities, the Batak ethnic from the Western region had the highest prevalence of H. pylori infection in Indonesia (48.6%). While in the Eastern region, Timor ethnicity was the highest (44.2%). Batak in the Western region and Timor in the Eastern region were the two ethnicities with the highest prevalence, 48.6% and 44.2%, respectively. Most Indonesian ethnicities analyzed in this study had a low prevalence of H. pylori infection. There were two ethnicities with 0.0% prevalence (Acehnese and Sundanese), as we reported previously.13–15 The notable phenomenon is that in the middle of low H. pylori infection prevalence on an island, there was ethnicity with high prevalence. For example, Batak, as a predominant ethnicity in North Sumatra, was the only ethnicity in Sumatra with a notably high H. pylori prevalence, compared to other predominant ethnics in other cities in Sumatra, such as Acehnese (0.0%), Malay (5.4%), Minangkabau (2.8%), and Nias (6.1%). Buginese also had the highest H. pylori prevalence in Sulawesi Island (21/81; 25.9%), in contrast to Minahasan and Tolaki ethnic, which showed lower prevalence (15.7% and 4.3%, respectively).\n\n* With no respect to the location of the survey or the place of the samples were taken. The data presented in this analysis includes the samples from our previous studies in Indonesia12–14 with the addition of samples acquired from our most recent surveys in four cities.\n\nWe excluded samples with incomplete questionnaire data. A total of 939 samples were included for further analysis. We analyzed the risk factors in both regions (found as Underlying data71). Both sexes have a similar proportion of H. pylori-positive and negative (14.4% male and 13.9% female subjects). The age of the H. pylori-positive subjects was significantly higher than the negative ones (positive: mean = 50.5 ± 13.1 years, median = 52 years vs. negative: mean = 45.2 ± 13.8 years, median = 45 years; P<0.001). Increased age reflects an increased risk for H. pylori infection (P<0.001).\n\nWe used logistic regression analysis adjusted to sex and age to calculate the association of risk factors and H. pylori infection in Indonesia (Table 3). No significant relationship was found between sex and infection risk (OR 1.09, 95%CI 0.75–1.59). While Islam is the predominant religion in Indonesia, Protestant, Catholic, and Hinduism had a significantly higher risk of infection (Protestant, OR 5.43, 95%CI 3.42–8.62; Catholic, OR 2.68, 95%CI 1.27–5.69; Hinduism, OR 3.47, 95%CI 1.62–7.44), respectively. Analysis of ethnicity by using Javanese as the reference category showed that Chinese, Maluku, Minahasan, Balinese, Buginese, Papuan, Batak, and Timor ethnic had a higher risk of getting infected (Chinese, OR 3.50, 95%CI 1.04–13.90; Maluku, OR 4.49, 95%CI 1.10–19.24; Minahasan, OR 8.42, 95%CI 2.41–29.46; Balinese, OR 9.71, 95%CI 12.95–31.96; Buginese, OR 15.19, 95%CI 4.97–46.48; Papuan, OR 19.25, 95%CI 6.16–60.15; Batak, OR 20.23, 95%CI 6.92–59.11; Timor, OR 34.66, 95%CI 10.34–116.11). No association was found between infection and marital status. Most infected subjects have a monthly income of less than 2.5 million Rupiah (approximately USD 172.2; USD 1 equals IDR 14,520). We used an income range of IDR 2.5 to 5 million as a reference category and found no significant association between monthly income and H. pylori infection status. Almost a quarter of the infected subjects (30/133; 22.6%) were smokers. However, no significant association was found between smoking and H. pylori infection status (OR 1.40, 95%CI 0.89–2.20). A total of 18 subjects (18/133; 13.5%) were alcohol consumers, and a significant association was found between H. pylori infection with alcohol drinking (OR 2.46, 95%CI 1.37–4.39). We also analyzed the source of water consumed by subjects. We divided water sources into three categories; mineral water (commercially packaged water), boiled, and others (water from rainwater, river, and wells, which are consumed directly without boiling beforehand). People who consume boiled water have a higher risk of infection (OR 1.68, 95%CI 1.14–2.47).\n\nHowever, results differ when we analyze the significant results from bivariate analysis, through a multivariate logistic regression model to calculate the odds ratios (OR) that included the subject’s demographic data and H. pylori infection status (found as Underlying data71). We found that in general, age, living in the Eastern region, Batak ethnic, Protestant, Catholic, and alcohol drinking were independent risk factors associated with H. pylori infection in Indonesia (OR 1.03, 95%CI 1.02–1.05; OR 5.33, 95%CI 1.17–24.26; OR 5.89, 95%CI 1.58–21.94; OR 5.47, 95%CI 2.16–13.86; OR 5.05, 95%CI 1.43–17.80; OR 2.30, 95%CI 1.14–4.61, respectively). We performed multivariate analyses in separate regions. We found no significant association between demographic characteristics and infection in the Western region. In the Eastern region, where the prevalence was generally higher than in the Western region (79/356; 22.2%), age, Buginese ethnicity, and Protestant, were independent risk factors associated with infection (OR 1.04, 95%CI 1.02–1.06; OR 10.53, 95%CI 1.28–85.56; OR 4.49, 95%CI 1.21–16.54, respectively).\n\n\nDiscussion\n\nInvestigation of H. pylori prevalence in Indonesia has been done over the years.21–26 However, the prevalence of a disease is inseparable from risk factors, especially in different geographic areas. In this study, we found that living in the Eastern region of Indonesia increases the risk of H. pylori infection. In general, the Western region had low H. pylori prevalence. There were no positive H. pylori subjects of Aceh and Sundanese ethnic; both reside in the Western region, while Timor, Papuan, and Buginese, all primarily located in the Eastern region, were the ethnicities dominating the highest prevalence in Indonesia. Moreover, none of the subjects from those three ethnicities who reside in the Western region had a positive H. pylori infection (found as Underlying data71). Papuan, the predominant ethnicity in Papua Island, is one of the ethnicities with high prevalence, which concord with a previous study in a neighboring country, Papua New Guinea.27 Higher H. pylori infection prevalence in the Eastern region is also congruent with the high prevalence in Australia, especially in the Australian indigenous community28,29 and New Zealand’s Maori and the Pacific.30,31 The diversity between the Eastern and Western regions was noticed years ago and was divided by Wallace’s line.32\n\nIn general, we found Indonesia’s overall prevalence of 14.2% lower than other neighboring countries such as Thailand,33,34 Myanmar,16,35,36 Cambodia,37 and Vietnam.38 It was lower than our previous study (14.2% vs. 22.1%, respectively).12 Nevertheless, the low prevalence was almost similar to Malaysia.39 However, while every other ethnicity in the Western region had a low prevalence, Batak ethnic, the indigenous ethnics in Sumatra Island, exhibit high H. pylori prevalence. Even though Batak had a high prevalence, analysis done separately in the Western region did not show susceptibility to infection in any ethnicity. On the other hand, Buginese who resides in the Eastern region showed an increased risk of infection. Several factors could contribute to this phenomenon, including the host, the agent’s virulence, and environmental factors. Host genetic variants were presumably responsible for inter-ethnic susceptibility to H. pylori infection. Previous studies reported that several single nucleotide polymorphisms might be related to Malay ethnic protection against H. pylori infection.40,41 Malay ethnic descendants in Indonesia mainly reside in Sumatra and Java Island, consisting of Aceh, Minangkabau, and Malay. Sundanese and Javanese were also believed to have a similar ancestral line.14,42 All these ethnicities showed low H. pylori infection prevalence in this study. The similarities in between the ethnicities were due to human migration. Modern humans originated from Africa, probably through a coastal route and finally arrived in South East Asia.43 Modern South Asian humans were related to ancient hunter-gatherers from Iran and South East Asia.44 On the agent side, H. pylori possess various virulence factors responsible for its capability to adapt and successfully colonize the hostile environment of the human stomach.45,46 However, this study did not analyze H. pylori strains infecting the subjects.\n\nSeveral sociodemographic factors are essential in H. pylori infection.47 Of note, we found religion as a significant social factor for H. pylori infection in Indonesia. Our result showed that Protestants and Catholics increased the risk of being infected with H. pylori. This result concurred with our previous study, where Protestantism had the highest risk for H. pylori infection in Indonesia.12 In addition to religion, alcohol drinking is a risk factor for H. pylori infection, especially in the Western region. However, according to Indonesia’s Basic Health Research (Riskesdas) in 2018, the Eastern region generally had higher alcohol consumption, ranging from the lowest of 4.1% in West Sulawesi to the highest of 16.0% in South Sulawesi.48 The relationship between alcohol consumption and certain religions to H. pylori infection found in this study might be related to Islam prohibiting Muslims from consuming alcohol where other religions do not. Moreover, certain ethnicities tended to consume alcohol regularly, as depicted by the Batak ethnicity, who consumed it in daily life and ceremonials.49 Alcoholic beverages may affect the human stomach directly or indirectly by influencing gastric acid secretion and hence, disturbs normal gastric mucosa physiology.50 It contributes to gastric mucosa’s damaging process, resulting in altered mucosal defense.51 Alcohol also interferes with muscle activity surrounding the stomach, resulting in delayed gastric emptying.52 However, the concrete relation between alcohol consumption and H. pylori infection remains unclear as some studies reported it as a protective factor,52,53 while others showed an increased risk of infection.54,55\n\nAccording to Indonesia’s Basic Health Research (Riskesdas), in 2018, 24.3% of Indonesians smoked daily.48 There have been conflicting results on whether smoking could increase the risk of H. pylori infection.56–59 However, we found no association between the two. A pooled analysis also found the same result.56 Studies have found that nicotine increases acid and pepsin secretion, hence making H. pylori harder to thrive in the stomach.58 However, in people who are already infected with H. pylori, smoking was found to increase the risk of developing gastric cancer.56,60,61 Substances in the cigarette contain surmountable carcinogenic compounds that impair gastric mucosa through oxidative stress and allow chronic inflammation. Eventually, a precursor lesion of gastric cancer, such as atrophic gastritis, intestinal metaplasia or dysplasia, will be formed.56,61\n\nIn this study, the age of H. pylori-positive subjects was higher than the negative ones. However, some studies reported that H. pylori infection was more common in older people,62–64 while others mentioned that infections are more common in younger age.65 H. pylori infection was established in childhood through person-to-person or fecal-oral route.66 Hence, the living condition with hygiene and sanitation level is highly correlated with the infection.67 Crowded living conditions, low income, and poor hygiene were risk factors. As the hygiene and sanitation standards are improved in the 21st century, the low prevalence at a younger age might originate from decreased microbial acquisition during childhood.64,68,69 Drinking water sources have also been discussed to be a source of infection. The prevalence was seen to be higher in individuals using unprotected water sources.8,70 Although, in our study, there was no correlation between water source and H. pylori infection. Lower income was an independent infection risk factor in the Eastern region, whereas H. pylori were heavily associated with lower income.57,67 However, lifestyle characteristics might be a better indicator of socioeconomic status. Although the exact transmission mode is still unknown, individual and familial characteristics must be assessed to determine the risk of contracting the infection.\n\nWe have multiple limitations in this study. There were differences in sample numbers between cities; hence, it still could not reflect the actual prevalence. Subjects included in this study were patients referred to endoscopic centers, indicating that our study is only the tip of the iceberg. Moreover, we excluded subjects with incomplete questionnaire data. A future study with adequate samples and complete demographic characteristics is necessary to provide a more accurate prevalence. We did not conclude the detailed analysis on which H. pylori strains infect our subjects. There was also a possibility of false negative/positive testing. However, this is the first comprehensive study of H. pylori prevalence and risk factors in Indonesia with almost a thousand samples. Knowing the risk factors would help disease prevention in the country.\n\n\nConclusions\n\nLiving in the Eastern region of Indonesia imposes a higher risk of H. pylori infection. Moreover, specific ethnicities, religions, and habits were risk factors. Understanding the distribution of H. pylori infection according to geographical location and sociodemographic factors is essential for clinicians to increase the awareness of H. pylori infection and determine the infection status in daily practice.", "appendix": "Data availability\n\nFigshare: Helicobacter pylori Prevalence in Indonesia West and East Region. https://doi.org/10.6084/m9.figshare.21514893. 71\n\nThis project contains the following underlying data:\n\n- Helicobacter pylori Prevalence in Indonesia_Data.xlsx (sociodemographic data of participants)\n\n- Publication License Oct-14-2022.pdf\n\n- PrevIndo_Underlying data.docx (includes risk factor comparison between Western and Eastern regions and multivariate analysis of risk factor associated with H. pylori infection)\n\n- Indonesian Helicobacter Pylori Project_questionnaire.doc\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nHooi JKY, Lai WY, Ng WK, et al.: Global Prevalence of Helicobacter pylori Infection: Systematic Review and Meta-Analysis. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinh TT, Tuan VP, Dung HDQ, et al.: Molecular Epidemiology of Helicobacter pylori Infection in a Minor Ethnic Group of Vietnam: A Multiethnic, Population-Based Study. Int. J. Mol. Sci. 2018 Mar; 19(3). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaur G, Naing NN: Prevalence and ethnic distribution of helicobacter pylori infection among endoscoped patients in north eastern peninsular malaysia. Malays J. Med. Sci. 2003 Jul; 10(2): 66–70. PubMed Abstract\n\nMaran S, Lee YY, Xu SH, et al.: Towards understanding the low prevalence of Helicobacter pylori in Malays: Genetic variants among Helicobacter pylori-negative ethnic Malays in the north-eastern region of Peninsular Malaysia and Han Chinese and South Indians. J. Dig. Dis. 2013; 14(4): 196–202.\n\nSchmidt HMA, Ha DM, Taylor EF, et al.: Variation in human genetic polymorphisms, their association with Helicobacterpylori acquisition and gastric cancer in a multi-ethnic country. J. Gastroenterol. Hepatol. 2011; 26(12): 1725–1732. PubMed Abstract \| Publisher Full Text\n\nMiyazaki K: Javanese-Malay: Between Adaptation and Alienation. Sojourn J. Soc. Issues Southeast Asia. 2000 Dec 1; 15(1): 76–99. Publisher Full Text\n\nStanyon R, Sazzini M, Luiselli D: Timing the first human migration into eastern Asia. J. Biol. 2009; 8(2): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNarasimhan VM, Patterson N, Moorjani P, et al.: The formation of human populations in South and Central Asia. Science (80-). 2019; 365(6457). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamaoka Y: Mechanisms of disease: Helicobacter pylori virulence factors. Nat. Rev. Gastroenterol. Hepatol. 2010; 7(11): 629–641. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamaoka Y: Increasing evidence of the role of Helicobacter pylori SabA in the pathogenesis of gastroduodenal disease. J. Infect. Dev. Ctries. 2008 Jun; 2(3): 174–181.\n\nZhu Y, Zhou X, Wu J, et al.: Risk Factors and Prevalence of Helicobacter pylori Infection in Persistent High Incidence Area of Gastric Carcinoma in Yangzhong City. Gastroenterol. Res. Pract. 2014; 2014: 481365.\n\nBadan Litbang Kesehatan KKR: Riset Kesehatan Dasar Tahun 2018. Badan Penelitian dan Pengembangan Kesehatan. Kementrian Kesehatan;2018; 198.\n\nSiregar GA, Parwati I, Achmad TH, et al.: Association between vegf-634g>c gene polymorphism with gastric premalignant lesions and serum vegf levels in helicobacter pylori gastritis patients. Open Access Maced. J. Med. Sci. 2018; 6(8): 1328–1334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinger MV, Leffmann C, Eysselein VE, et al.: Action of ethanol and some alcoholic beverages on gastric acid secretion and release of gastrin in humans. Gastroenterology. 1987 Dec; 93(6): 1247–1254. PubMed Abstract \| Publisher Full Text\n\nCapurso G, Lahner E: The interaction between smoking, alcohol and the gut microbiome. Best Pract. Res. Clin. Gastroenterol. 2017; 31(5): 579–588. PubMed Abstract \| Publisher Full Text\n\nBrenner H, Berg G, Lappus N, et al.: Alcohol consumption and Helicobacter pylori infection: results from the German National Health and Nutrition Survey. Epidemiology. 1999 May; 10(3): 214–218. Publisher Full Text\n\nKuepper-Nybelen J, Thefeld W, Rothenbacher D, et al.: Patterns of alcohol consumption and Helicobacter pylori infection: results of a population-based study from Germany among 6545 adults. Aliment. Pharmacol. Ther. 2005 Jan; 21(1): 57–64. PubMed Abstract \| Publisher Full Text\n\nLuzza F, Imeneo M, Maletta M, et al.: Smoking, alcohol and coffee consumption, and H pylori infection. Alcohol consumption eliminates rather than prevents infection with H pylori. BMJ. 1998 Mar 28; 316(7136): 1019–1020. Publisher Full Text\n\nSánchez-Cuén JA, Cabrales ABI, Magaña GB, et al.: Helicobacter pylori infection and its association with alcohol consumption: a case-control study. Rev. Gastroenterol. Mex. 2013; 78(3): 144–150. PubMed Abstract \| Publisher Full Text\n\nKumar S, Metz DC, Ellenberg S, et al.: Risk Factors and Incidence of Gastric Cancer After Detection of Helicobacter pylori Infection: A Large Cohort Study. Gastroenterology. 2019/10/22. 2020 Feb; 158(3): 527–536.e7. PubMed Abstract \| Publisher Full Text\n\nRazuka-Ebela D, Polaka I, Parshutin S, et al.: Sociodemographic, lifestyle and medical factors associated with helicobacter pylori infection. J. Gastrointestin. Liver Dis. 2020; 29(3): 319–327. PubMed Abstract \| Publisher Full Text\n\nOgihara A, Kikuchi S, Hasegawa A, et al.: Relationship between Helicobacter pylori infection and smoking and drinking habits. J. Gastroenterol. Hepatol. 2000; 15(3): 271–276. Publisher Full Text\n\nMonno R, De Laurentiis V, Trerotoli P, et al.: Helicobacter pylori infection: association with dietary habits and socioeconomic conditions. Clin. Res. Hepatol. Gastroenterol. 2019; 43(5): 603–607. PubMed Abstract \| Publisher Full Text\n\nVenneman K, Huybrechts I, Gunter MJ, et al.: The epidemiology of Helicobacter pylori infection in Europe and the impact of lifestyle on its natural evolution toward stomach cancer after infection: A systematic review. Helicobacter. 2018; 23(3): 1–16. Publisher Full Text\n\nButt J, Varga MG, Wang T, et al.: Smoking, Helicobacter pylori serology, and gastric cancer risk in prospective studies from China, Japan, and Korea. Cancer Prev. Res. 2019; 12(10): 667–674. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCizginer S, Ordulu Z, Kadayifci A: Approach to Helicobacter pylori infection in geriatric population. World J. Gastrointest. Pharmacol. Ther. 2014 Aug 6; 5(3): 139–147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPilotto A, Franceschi M: Helicobacter pylori infection in older people. World J. Gastroenterol. 2014 Jun 7; 20(21): 6364–6373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreckan RK, Paulssen EJ, Asfeldt AM, et al.: The All-Age Prevalence of Helicobacter pylori Infection and Potential Transmission Routes. A Population-Based Study. Helicobacter. 2016; 21(6): 586–595. PubMed Abstract \| Publisher Full Text\n\nRoberts SE, Morrison-Rees S, Samuel DG, et al.: Review article: The prevalence of Helicobacter pylori and the incidence of gastric cancer across Europe. Aliment. Pharmacol. Ther. 2016; 43(3): 334–345. PubMed Abstract \| Publisher Full Text\n\nHatakeyama M: Helicobacter pylori CagA and gastric cancer: A paradigm for hit-and-run carcinogenesis. Cell Host Microbe. 2014 Mar 12; 15(3): 306–316. PubMed Abstract \| Publisher Full Text\n\nKotilea K, Bontems P, Touati E: Epidemiology, Diagnosis and Risk Factors of Helicobacter pylori Infection. Adv. Exp. Med. Biol. 2019; 1149: 17–33. Publisher Full Text\n\nMiernyk KM, Bulkow LR, Gold BD, et al.: Prevalence of Helicobacter pylori among Alaskans: Factors associated with infection and comparison of urea breath test and anti-Helicobacter pylori IgG antibodies. Helicobacter. 2018; 23(3): 1–8. Publisher Full Text\n\nLorenzo I, Fernández-De-Larrea N, Michel A, et al.: Helicobacter pylori seroprevalence in Spain: Influence of adult and childhood sociodemographic factors. Eur. J. Cancer Prev. 2019; 28(4): 294–303. PubMed Abstract \| Publisher Full Text\n\nAbebaw W, Kibret M, Abera B: Prevalence and risk factors of H. pylori from dyspeptic patients in Northwest Ethiopia: A hospital based cross-sectional study. Asian Pacific. J. Cancer Prev. 2014; 15(11): 4459–4463. Publisher Full Text\n\nMiftahussurur M, Maulahela H, Doohan D, et al.: Helicobacter pylori Prevalence in Indonesia West and East Region. figshare. [Dataset]. 2022. Publisher Full Text" }	[ { "id": "161033", "date": "09 Feb 2023", "name": "Hoda Michel Malaty", "expertise": [ "Reviewer Expertise 1. Helicobacter pylori and associated diseases. 2. IBD" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMaulahela et al reported a study on Helicobacter pylori prevalence in Indonesia. They used endoscopic data from their previous endoscopic study and combined to a prospective recent study they have conducted. The authors assessed H. pylori status by four diagnostic methods, rapid urease test, histology, immunohistochemistry, and bacterial culture. They considered an individual Positive for H. pylori if tests positive of one of the 4 tests.\n\nCritique:\nWhile the authors have good materials for the study and good sample size, there are several weaknesses to this study. The authors need to elaborate more the methodology and present that data in an epidemiological fashion and finally, how to make interpretations of the results. There are many uncomprehensive numbers and statements that can leave many of the readers confused about what the authors are trying to say. For example; in the abstract the authors stated there were 190 subjects tested positive of H. pylori of the total 1,158 examined subjects 190 subjects (16.4%). Then the very following sentence they stated that a total of 939 subjects were analyzed for risk factors. So where is 939 came from? They are neither the positive nor the negative ones. If it a closer number of the negative individuals; whey are the authors examining them for H. pylori risk factors. This is a very simple knowledge of epidemiology.\n\nJava Island population is 400 not 401, another primitive mistake.\n\nIn the results section this sentence “Overall, the prevalence was higher in the Eastern region than in the Western region (86/398; 21.6% vs. 104/761; 13.7%) should be written in an epidemiological fashion as: the prevalence was higher in the Eastern region than in the Western region (21.6% vs.13.7%; OR=??, CI=??-??; P=??).\n\nThe discussion is very primitive and limited, and that authors could not make any interpretation of their data. The authors focused on the region east vs. west but there are certain ethnic group living in the western region have higher prevalence of the infection than in the eastern region.\n\nWhile there the data, the discussion and the presentation can be presented in a much better and sophisticated way, this paper provides valuable information on H. pylori across Indonesia in many ethnic groups whom are living on the many islands of Indonesia.\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": [] }, { "id": "200794", "date": "17 Oct 2023", "name": "Onyekwere Charles Asabamaka", "expertise": [ "Reviewer Expertise Gastroenterology and Hepatology. Internal medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis prospective study of H Pylori prevalence in Indonesia was carried out from 2014 to 2017. It involved consenting dyspeptic subjects presenting in selected hospital accross major cities in the country where after consenting and obtaining demographic information as well as risk factors, they subjects subsequently underwent endoscopy where 4 biopsy specimens were obtained for RUT, culture, histology and molecular studies. Positive result in any of the method was considered a positive result for H Pylori.\nPrevalence was slightly higher in the Eastern region compared with the Western region of Indonesia (79/277; 59.4% vs. 54/529; 40.6%). Living in the Eastern region imposes a higher risk of infection (OR 5.33, 95%CI 1.17–24.26).\nOne major concern is the fact the sampling method for enrollment which determine the criteria for the number recruited from each city is not stated. This may limit the generalization of the reports especially the conclusion that the eastern cities had a higher prevalence of H Pylori\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-1581
https://f1000research.com/articles/12-1398/v1	23 Oct 23	{ "type": "Research Article", "title": "Academic publisher guidelines on AI usage: A ChatGPT supported thematic analysis", "authors": [ "Mike Perkins", "Jasper Roe", "Jasper Roe" ], "abstract": "Background As Artificial Intelligence (AI) technologies such as Generative AI (GenAI) have become more common in academic settings, it is necessary to examine how these tools interact with issues of authorship, academic integrity, and research methodologies. The current landscape lacks cohesive policies and guidelines for regulating AI’s role in academic research and prompting discussions among publishers, authors, and institutions.\n\nMethods This study employs inductive thematic analysis to explore publisher policies regarding AI-assisted authorship and academic work. Our methods involved a two-fold analysis using both AI-assisted and traditional unassisted techniques to examine the available policies from leading academic publishers and other publishing or academic entities. The framework was designed to offer multiple perspectives, harnessing the strengths of AI for pattern recognition while leveraging human expertise for nuanced interpretation. The results of these two analyses are combined to form the final themes.\n\nResults Our findings indicate six overall themes, three of which were independently identified in both the AI-assisted and manual analyses. A broad consensus appears among publishers that human authorship remains paramount and that the use of GenAI tools is permissible but must be disclosed. However, GenAI tools are increasingly acknowledged for their supportive roles, including text generation and data analysis. The study also discusses the inherent limitations and biases of AI-assisted analysis, necessitating rigorous scrutiny by authors, reviewers, and editors.\n\nConclusions There is a growing recognition of AI’s role as a valuable auxiliary tool in academic research, but one that comes with caveats pertaining to integrity, accountability, and interpretive limitations. This study used a novel analysis supported by GenAI tools to identify themes emerging in the policy landscape, underscoring the need for an informed, flexible approach to policy formulation that can adapt to the rapidly evolving landscape of AI technologies.", "keywords": [ "Generative AI", "Academic Integrity", "Inductive Thematic Analysis", "Publisher Policies", "Research Methodologies", "AI-Assisted Authorship", "Ethical Guidelines", "Academic Publishing" ], "content": "Introduction\n\nThe emergence of Generative Artificial Intelligence (GenAI) tools, exemplified by OpenAI’s ChatGPT, has resulted in a significant shift in the academic research landscape. These tools, powered by Large Language Models (LLMs) such as GPT3.5 and GPT-4, are capable of generating human-like text with impressive fidelity, accuracy, and creativity (Cotton et al., 2023; Perkins, 2023a). While they have streamlined research processes and opened new avenues for exploration and discovery, their integration into academia has also raised critical questions regarding academic integrity and originality. The advent of GenAI tools has necessitated a closer examination of the academic integrity of researchers, particularly how they utilise these advanced tools. As GenAI has become more sophisticated and widely adopted in academia, understanding its implications for research integrity and establishing appropriate usage policies and guidelines has become increasingly important. It is also crucial to understand how these tools support the academic research process and how to develop effective strategies and policies to ensure the ethical use of these technologies.\n\nThe advent of digital technology has profoundly impacted academic integrity. Over the past decades, instances of academic misconduct, notably plagiarism, have declined (Curtis, 2022). This positive trend is largely attributable to the role of technological and educational measures in mitigating plagiarism. However, the rise of advanced AI tools, notably Automated Paraphrasing Tools (APTs) and GenAI tools has introduced new complexities. These tools, which are capable of generating and rephrasing content, facilitate subtle forms of plagiarism that challenge traditional detection methodologies (Perkins, 2023a). This has sparked a technological ‘arms-race’ (Roe & Perkins, 2022) in plagiarism detection, with new tools and techniques being developed to keep pace with the evolving capabilities of AI.\n\nThe rapid advancement of AI technologies and their increasing application in various domains has underscored the need for comprehensive policy analysis and ethical considerations. Despite the widespread use of digital and AI technologies, only a small percentage of higher education institutions (HEIs) have developed formal policies surrounding their use since the launch of ChatGPT (Perkins & Roe, 2023; Xiao et al., 2023). Just as HEIs had to quickly develop policies and guidelines on students’ use of GenAI tools, academic publishers were also forced to consider how to manage the use of GenAI tools by authors. The uncertainty still present in the academic landscape in this area highlights the pressing need for additional research on how academic institutions, publishers, and employers must consider how these tools and technologies are being used by academics. The development and implementation of effective policies can guide the ethical use of AI tools in academic studies and research, ensuring that they are used to uphold academic integrity. Furthermore, a clear understanding of these policies can help researchers navigate the complex landscape of AI-assisted research, enabling them to leverage the benefits of these tools while avoiding potential pitfalls.\n\nIn this study, we conduct an in-depth exploration of the policies of 109 academic publishers who are key stakeholders in the production of scholarly work. Using inductive thematic analysis, we dissect these policies to identify key themes across different publishers and discern commonalities and variances, thereby enabling us to better understand the collective stance on the use of GenAI tools in academic research. The insights gained from this policy analysis will not only highlight existing norms but will also provide actionable data that can guide the development of more unified and effective policies for the responsible use of GenAI in academia.\n\nTo assess the potential role of GenAI tools in academic research, this study adopts a dual-method analytical approach, employing both GenAI-assisted, and manual thematic analyses. The aim is to provide empirical evidence to evaluate the effectiveness of GenAI tools, such as ChatGPT, as a research assistant or co-pilot within the academic research landscape. These analyses are designed to determine whether insights generated by GenAI are not only comparable but may also exceed those obtained through human-led analysis, thereby providing evidence-based insights into the capabilities of GenAI tools for areas of academic research that have not previously been explored. Therefore, this study contributes to the ongoing discourse on the use of GenAI tools in academic research, especially as this particular use of GenAI tools in the research process is in direct contrast to the policies of several academic publishing entities, including the Social Sciences Research Network1 (SSRN) and Edward Elgar2.\n\nThe key objectives of this study are as follows:\n\n1. To evaluate the role, capabilities, and effectiveness of GenAI tools such as ChatGPT in academic research by comparing the quality of insights generated through GenAI-assisted and manual thematic analyses.\n\n2. To provide empirical evidence on how GenAI can be responsibly and effectively integrated into the academic research process.\n\n3. To identify emergent themes, commonalities, and variances in academic publishing entities’ stances on the use of GenAI tools in scholarly works.\n\n4. To generate actionable insights that will guide the development of effective and unified policies for the responsible use of GenAI tools in academic research, thereby contributing to the broader discourse on maintaining academic integrity in the era of advanced AI.\n\n\nLiterature review\n\nArtificial Intelligence (AI) relates to the scientific field focused on developing intelligent machines which can reason, communicate, learn, plan, and solve real-world problems for the benefit of society (Debrah et al., 2022). Although AI has been an established field of scientific enquiry for the past sixty years, towards the end of 2019, it began to receive a slew of attention in popular culture. This is attributable to the rapid advancement of computing and processing power, which is currently outstripping Moore’s Law and doubling capacity every six months (Pichai, 2023). The pace of change is so great that major corporations involved in developing AI have warned of the potential of a ‘superintelligence’ being developed within the decade (OpenAI, 2023a). AI has a potentially transformative effect on the global level, leading to changes in world politics, military power, and warfare (Johnson, 2019). At the individual level, it has been posited that the near future will see the merging of human biology and AI technologies, leading to a symbiotic relationship (Jiang et al., 2022).\n\nThe release of Generative AI (GenAI) tools and chatbots, such as OpenAI’s ChatGPT and Google’s Bard, has further stoked conversations around the risks and benefits of AI in multiple contexts. Noteworthy examples include thought leaders such as Google’s CEO Sundar Pichai warning us to ‘brace for impact’ (Pichai, 2023, as cited in Elias, 2023), and the Centre for AI Safety releasing a statement signed by scientists and subject matter experts asking for a halt on AI development and identifying that the risk of human extinction from AI should be seen as a top global priority (CAIS, 2023). Other, less critical, yet important risks include the security and protection of confidential data and the potential of training data containing biases or discriminatory ideologies (King, 2023). As a result, governments and intergovernmental organisations worldwide are taking action on AI. The European Union is currently in the process of passing the AI Act, which represents the first comprehensive set of legislations regulating AI (European Parliament, 2023). This framework prohibits high-risk applications of AI while providing guidelines for language models, such as ChatGPT. It mandates the disclosure of AI-generated content, prohibits the production of illegal content, and requires the publication of summaries for copyrighted data used in training (European Parliament, 2023).\n\nDespite the serious risks outlined above and the push to build and implement regulatory frameworks, AI continues to pick up speed in the private sector. The market value of AI is anticipated to reach $190 billion USD by 2025, and it is having a revolutionary effect on multiple industries, including manufacturing, healthcare, education, transport, and medicine (Jiang et al., 2022). In factory and industrial production, AI enables self-monitoring, autonomous operations, and improved efficiency, with reduced downtime (I. Ahmed et al., 2022). In the field of healthcare, AI plays a major role in advancing nanomedicine for cancer treatment (Tan et al., 2023) and shows the potential for taking over traditionally human-performed tasks, such as the generation of medical discharge reports (Patel & Lam, 2023). AI tools have also played a role in identifying case clusters and predicting infection models in the COVID-19 pandemic (Peng et al., 2022), and AI techniques are also being employed for drug discovery, identification of risk factors affecting patients, and disease diagnosis (Kumar et al., 2023). In architecture, AI contributes to sustainability in green construction practices (Debrah et al., 2022), and the finance sector is beginning to utilise AI in areas such as anti-money laundering and behavioural finance (S. Ahmed et al., 2022), while in relation to farming and food production, AI is being explored for its potential to address animal welfare issues (Bao & Xie, 2022). In the newspaper publishing industry the German tabloid Bild has even reduced its workforce with the goal of replacing some job roles with AI (Henley, 2023).\n\nAcademia is also coming face-to-face with the disruptive potential of AI. AI-generated submissions to academic journals have been described as a ‘coming tsunami’ (Tate et al., 2023) and it has been claimed that publishers need to anticipate the potential of wholly AI-generated submissions (Anderson et al., 2023). To date, much of the focus in academia has been on the use of Large Language Models (LLMs) like ChatGPT, thanks to their human-like text production capabilities (Perkins, 2023a; Perkins & Roe, 2023), although other AI tools such as Elicit have also shown promise in summarising literature and identifying source material (Roe et al., 2023). Such capabilities have fuelled the debate on the positive and negative impacts of GenAI on scholarly work. Some researchers have reportedly co-authored studies with ChatGPT (Pavlik, 2023) and produced papers in which the majority of the text was AI-generated (cf: Biswas, 2023; Cotton et al., 2023). LLMs can be used not only for writing but also for summarising literature, generating titles, locating data sources, and identifying gaps in research (Hutson, 2022). In the classroom context, LLMs may increase the efficiency of assessment creation and provide personalised feedback for learners (Baidoo-Anu & Owusu Ansah, 2023). On the side of publishers, AI tools may be able to assist in identifying suitable reviewers for academic work (Solomon et al., 2023), and concerns have been raised about the lack of editorial policies in using GenAI tools in the peer review process (Garcia, 2023).\n\nThese AI models also carry implications for academic integrity (Perkins, 2023a; Roe et al., 2023). At present, LLMs cannot completely compose a novel academic text or author a research study in the way a human researcher can because although they excel at human-like text production and organisation, they still lack the ability to generate novel ideas or engage in creativity (Hammad, 2023). Furthermore, at this stage, using a GenAI tool like ChatGPT to draft a scientific paper still requires expert supervision, as text generated by AI is claimed to lack the nuances of human-authored text (Salvagno et al., 2023). However, LLMs offer students the opportunity to misrepresent their authorship and engage in surface-level learning when completing written assessments (Cotton et al., 2023; Perkins, 2023a). Likewise, non-academic authors may also pass GenAI texts on their own. As a result, efforts have been made to detect AI-generated text, although present studies show that humans are not adept at identifying AI-generated texts. In one study, reviewers of academic abstracts were only able to identify 68% of the content generated by ChatGPT, with a 14% false-positive rate (Gao et al., 2022). Even advanced AI detection software tools struggle, with a detection rate of only 54.8% in examples of work submitted to the Turnitin platform (Perkins et al., 2023), and inaccuracies in a range of detection tools have been identified and discussed by Weber-Wulff et al. (2023). As a result, such cases have spurred the need for author guidelines on AI use in scholarly publishing (Liebrenz et al., 2023). That said, this is in essence a technological ‘arms race’ (Roe & Perkins, 2022), and likely detection will always be catching-up to GenAI text production. As LLMs continue to develop, it has been envisioned that their capabilities will extend to other tasks, such as generating data visualisations and summaries of quantitative and qualitative data with minimal user input, potentially revolutionising aspects of the research process. This has been demonstrated with the beta release of OpenAIs ‘Advanced Data Analysis’ mode of ChatGPT which has the ability to use built in Python libraries to extend the abilities of the GPT4 model beyond language-based tasks (OpenAI, 2023b).\n\nAt the same time, authors need to be made aware of the potential harm to their work when using LLMs. It is possible that their use could perpetuate prejudice and bias found in their training data (Lund et al., 2023). Researchers should also consider that there is a risk of perpetuating inequality; the efficiency-enhancing effects of LLMs could contribute to the growth of the ‘Matthew Effect’, in which successful researchers receive more attention and citations, while newer scholars struggle to receive attention for their work (Lund et al., 2023). On the other hand, the use of such tools could contribute to a more level-playing field. Some believe that the evolution of the scholar’s role could pivot towards asking the right questions rather than only articulating conclusions (Jabotinsky & Sarel, 2022). In this scenario, if the demanding processes of writing, reviewing literature, and presenting results are taken care of by an LLM or AI tool, then perhaps there is more scope for scholars to focus on other aspects of research quality.\n\nAt the core of the issues of academic integrity and ethics relating to AI and LLM use is authorship and whether non-human actors can claim authorial rights and obligations in the production of text. In Pavlik’s (2023) case, ChatGPT was listed as the second author, and this symbolises some of the areas of debate and confusion. The discussion around AI-generated text and authorship is not new but dates to the production of an article by a program named Racter in 1981 (Lee, 2023). Lee (2023) highlights that from a legal perspective, there are varying stances on non-human authors’ credibility. In China, for example, copyright law does not necessitate that an author is a person, while in the USA and Korea, it does. Examples of this issue playing out in popular media have included the rights of a self-taken photograph by a macaque, a copyright claim which was eventually rejected in the USA (Lee, 2023). While a useful and interesting point of reference, copyright law is distinct from academic ethics, and it seems that at the time of writing the consensus among academic publishers is that tools such as chatbots do not meet authorship criteria, as they cannot be held accountable for the produced text (Lee, 2023). However, the issue has not been resolved, and there have been calls for scholarly organisations such as the APA to draft clear guidelines on the use of AI-generated text in research (Tate et al., 2023).\n\nThe reasons why LLMs are not able to hold authorship roles are best exemplified by the criteria given by the Committee on Publication Ethics (COPE). COPE states that four criteria must be met to attribute authorship: substantial contribution, critical revision, final approval, and agreement on accountability of the work (Pourhoseingholi et al., 2023). LLMs can contribute substantially and engage in the revision of work, but they cannot hold responsibility or accountability for the work. This does not mean that LLMs and AI tools should be banned. Rather, ethical guidelines must be in place to govern their use (Rahimi & Talebi Bezmin Abadi, 2023). To this point, some academic journals are encouraging the use of AI to refine manuscripts and edit papers, provided that the technology’s contribution is appropriately recognised (Crawford et al., 2023). However, these guidelines can only go so far, given that accurately identifying articles produced by tools such as ChatGPT remains a major challenge (Hu, 2023). Therefore, it is crucial that journal publishers bolster plagiarism detection systems and include comprehensive originality checks (Dwivedi et al., 2023).\n\nThis study makes two unique contributions to the existing literature. First, it undertakes a comparative analysis of publisher policies on the usage of Artificial Intelligence, an area that has hitherto not been formally explored. Such an examination is critical for understanding the formal stances taken by academic journals, publishers, and other entities involved in research ethics, thereby offering valuable insights into the evolving norms and guidelines that shape AI’s role of AI in scholarly work. Second, the study itself employs GenAI tools in both data analysis and writing up, thus serving as a practical demonstration of GenAI’s capabilities as a ‘co-pilot’ in academic research. This dual focus not only enriches our understanding of the academic publishing landscape in the context of AI, but also validates the utility of GenAI tools in facilitating rigorous academic enquiry.\n\n\nMethods\n\nThe methodology employed in this study involves a comprehensive web search for the collection and classification of policies from journal and publisher websites. This approach was designed to provide a broad and representative sample of policies currently in place regarding the use of AI tools in academic research.\n\nTo identify our population, existing lists and rankings of academic publishers found online (Nishikawa-Pacher, 2022; Wikipedia, 2023) were combined to form a master list of academic publishers which would be investigated. The master list was edited to remove known or suspected instances of publishers of predatory journals as identified by the most recently available version of Beall’s List (Beallslist.net, n.d.). This list was then supplemented by web searches including terms such as ‘AI/ChatGPT journal/publisher policies’ to identify specific journals, publishers, or publishing related groups/institutions which were found to have some sort of policy related to the use of GenAI tools. This resulted in a master list of 107 entities for review.\n\nManual searches were then conducted on the websites of all entities to explore whether they had a policy in place relating to the use of Generative AI tools. From this manual search, 36 entities were identified as having an AI policy, eight of these were replicas. Replicas were due to either the use of wording from other entities, such as The Committee of Publication Ethics (COPE), or because the publishing houses were imprints of broader publishing groups, and therefore did not have their own policy. This resulted in a final list of 28 unique policies for analysis.\n\nThe primary analytical tool used in this study is inductive thematic analysis, a method widely used in qualitative research for identifying, analysing, and reporting patterns (themes) within data (Braun & Clarke, 2006). Thematic analysis offers a flexible and useful research tool that can potentially provide a rich and detailed yet complex account of data (Braun & Clarke, 2006). In this study, thematic analysis is used to classify policies based on emergent themes. Thematic analysis in policy research is a well-established method that provides a systematic approach for analysing qualitative data (Nowell et al., 2017). It involves the identification of themes or patterns of meaning that arise repeatedly within the data. These themes are then used to draw conclusions about the phenomena being studied. In this study, the phenomena in question are the policies of academic publishing houses regarding the use of AI in academic research.\n\nA thematic analysis involves several steps. First, the data is familiarised through reading and re-reading. Next, initial codes are generated, which are then searched for themes. These themes are then reviewed and defined. Finally, the report is produced, which in this case is an analysis of the policies regarding the use of AI in academic research. In the context of this study, thematic analysis is particularly useful for classifying policies based on emergent themes. This approach allows for the identification of commonalities and differences in the policies of different institutions and publishers, providing a comprehensive overview of the current state of policy regarding the use of AI in academic research. The use of thematic analysis in this study is guided by the principles outlined by Braun and Clarke (2006). This involves a six-phase process of familiarisation with the data, generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing a report. This rigorous approach ensures a thorough and systematic analysis of policies, providing valuable insights into the current state of policy regarding the use of AI in academic research.\n\nIn the analysis, we combined manual coding, analysis, and interpretation with an analysis conducted with assistance from the GenAI tool ChatGPT Plus (using the underlying LLM of GPT-4). In our methodological process, these analyses were conducted separately by the two researchers, and the results were then compared during the final stages of the research process, from reviewing themes to producing the report. Manual analysis without the support of AI software was conducted by Researcher 2 (R2), and Researcher 1 (R1) conducted the analysis with GenAI assistance. The rationale for this approach was first to develop greater insight into the data and determine whether there were significant differences between LLM-supported and traditional data analysis and interpretation processes, and second, to engage in the use of AI in academic research. In this sense, the research demonstrates a way of using AI to support and assist, yet not author, academic work. After combining the results of the two analyses, the final set of themes was selected.\n\n\nResults\n\nIn the following, we begin by presenting the results obtained through traditional, manually generated thematic analysis. This is then contrasted with the initial results obtained through the GenAI tool analysis, before our final set of themes is described. All quotes used in the below results are drawn verbatim from academic publisher policies available online. To preserve the record of the policy wording when they were originally downloaded, the publisher policies and URLs can be found in the associated data for this paper (Perkins, 2023b). The full policy statement for each publisher/journal is shown in column D, with the URL shown in Column H.\n\nThe manually generated thematic analysis with no AI assistive tools was developed using the QDA Miner software by R2. R2 began by reading through the policies in detail, searching for indicators of interest, and identifying features that typified the ‘qualitative richness of the phenomenon’ (Boyatzis, 1998, p1) prior to iteratively and reflexively refining them. Coding took place at the semantic level, looking at surface features of the text rather than trying to identify deeper meanings at the latent level. This choice was made based on the genre of the text: policy guidelines are designed to be explicit, rather than implicit. The coding process was undertaken in the traditional, manual manner. Initial steps involved allocating codes to specific passages. For example, ‘Authors must take full responsibility’ was a relevant recurring code throughout the policies, and this code was later translated into the broader theme of ‘authorship is human’. Although it may seem that publisher policy documents would be formulaic and straightforward, with little room for deeper analysis, three key themes were identified from the data.\n\nManual analysis theme one: Authorship is human\n\nAcross all the studied publisher policies, it became clear that authorship is seen as a uniquely human endeavour. While this is at first glance perhaps unsurprising, the way in which the theme is operationalised is of interest. Almost all of the publishers studied did not disallow the use of ChatGPT and LLMs, or other unspecified GenAI in order to assist with writing. It became clear that, either implicitly or explicitly, most publishers believed that despite their limitations, GenAI tools can make significant contributions to academic work. The example below from Thieme publishing demonstrates this point:\n\nAI tools such as ChatGPT can make scholarly contributions to papers. The use of generative AI tools should be properly documented in the Acknowledgements or Material and Methods sections.\n\nHowever, none of the publishers stated that LLMs or GenAI tools can be listed as authors. This is not because the models are not able to generate meaningful insight, content, analysis, or interpretation. Rather, the major focus is that such tools cannot take responsibility for the written work. The examples below from MIT Press and De Gruyter clearly demonstrate this point.\n\nThe MIT Press does not allow artificial intelligence (AI) tools such as ChatGPT or large language models (LLMs) to be listed as authors of our publications. The emerging consensus of scholarly organizations, including the Committee on Publication Ethics, is that AI tools do not meet the requirements for authorship since they cannot assume ethical and legal responsibility for their work.\n\nPlease note that we do not accept papers that are generated by Artificial Intelligence (AI) or Machine Learning Tools [as authors] primarily because such tools cannot take responsibility for the submitted work and therefore cannot be considered as authors.\n\nManual analysis theme two: Policies are ambiguous\n\nThe second most prevalent theme is ambiguity. Ambiguity in this sense does not cover whether it is unacceptable or acceptable to use a GenAI tool, as it is implied and explicitly stated in several policies that their use is allowed in the preparation of manuscripts for publication. However, whether and how the policies would be enforced is especially unclear, as is the permanence of the current policies. For example, the following extract from Elgar demonstrates that there are ‘verification tools’ that may be used in detecting GenAI-created text in manuscripts:\n\nThe publisher reserves the right to verify the use of ChatGPT (or related platforms) and to reject manuscripts that violate this policy.\n\nWhile the above suggests that tests will be undertaken on writing to detect any GenAI text, there is no further information given about the verification process undertaken and whether it applies to all manuscripts or only those deemed suspicious. Nevertheless, this provides an ambiguous basis for policy enforcement and application. A similar sense of ambiguity is found in the permanence of many policies, as Elsevier, Springer, and Emerald demonstrate.\n\nElsevier will monitor developments around generative AI and AI-assisted technologies and will adjust or refine this policy should it be appropriate (Elsevier)\n\nAs we expect things to develop rapidly in this field in the near future, we will review this policy regularly and adapt it if necessary. (Springer)\n\nWhilst industry standards will likely coalesce in the coming months, Emerald is adopting the following approach, as part of a watching brief on developments in the rapidly evolving AI space. (Emerald)\n\nThis ambiguity recognises that the development of AI and LLMs is happening at an astounding pace, and thus, the current guidelines are subject to change as deemed appropriate. On the other hand, there is little explanation of when the review might be undertaken, and what developments might prompt changes to the policy. While the idea of ‘appropriacy’ and ‘necessity’ is mentioned, this leaves considerable room for interpretation. Emerald publishing suggests that a likely future scenario is the convergence of academic publishing policies and norms, as more about the applications of GenAI in academia are uncovered. Overall, this theme suggests that publishers have attempted to tentatively provide guidelines on the use of AI and LLMs in academic writing and publishing but with a high level of ambiguity and uncertainty.\n\nManual analysis theme three: Transparency is vital\n\nFrom the analysis of publisher policies, the first core theme that can be outlined is that of authorship being a uniquely human endeavour based on the core concepts of agency, responsibility, and ownership of ideas. Second, while this is often conceptualised in concrete, unambiguous terminology, the actual operationalisation of AI and LLMs is allowable, and the politics of detection, enforcement, and use cases are highly open to interpretation and change. In essence, there is a sense from these two themes that both the rigidness of ownership and fluidity of rules can operate together.\n\nThe third theme is a similar all-encompassing requirement: transparency. While there are no prohibitions on using GenAI tools in the writing process in any of the policies examined, there are commitments to principles everywhere, and transparency forms one of the most frequently cited principles in the data. The examples below from COPE, MDPI, and Cambridge University press demonstrate the commitment to transparency as a cornerstone of policies regarding AI tools:\n\nAuthors who use AI tools in the writing of a manuscript, production of images or graphical elements of the paper, or in the collection and analysis of data, must be transparent in disclosing in the Materials and Methods (or similar section) of the paper how the AI tool was used and which tool was used. – COPE\n\nFurthermore, authors are required to be transparent about the use of these tools and disclose details of how the AI tool was used within the “Materials and Methods” section. – MDPI\n\nAny use of AI must not breach Cambridge’s plagiarism policy. Scholarly works must be the author’s own, and not present others’ ideas, data, words or other material without adequate citation and transparent referencing. – Cambridge University Press\n\nIn the extract from COPE, it is clear that the expectation is to list the use of such tools in the Materials and Methods section of the manuscript, which is also found in the requirements of MDPI. This statement suggests that an LLM or GenAI tool is an aid to the research process rather than an author. This is congruent with the perspective that AI tools cannot fulfil the role of an author but does not fully capture the fact that such tools can generate novel text. Furthermore, the extent to which AI tools must be declared is not clear. AI tools can assist in finding resources for literature reviews, summarising existing studies, or providing help with study design or creation. As such, there are gaps in the comprehensiveness of AI policy statements for several publishers, and transparency is considered a core fundamental value. However, it is assumed that authors are aware of the exact limits of transparency in a rapidly evolving AI-driven research landscape which will likely not always be the case.\n\nThis is an important aspect of the theme because it identifies a unique aspect of LLMs and GenAI tools, which seems to be true at present. When posed with question ‘Can you take responsibility for a written work?’, ChatGPT 3.5 (24th July, 2023) provided the following answer:\n\nAs an AI language model, I don’t have personal agency or the ability to take responsibility for any written work. Any text generated by me is a result of algorithms and training data up until my knowledge cutoff date in September 2021. The responsibility for the content and its usage lies with the user, who inputs the text and decides how to utilise it.\n\nTherefore, it seems that the fact that textual output produced by LLMs is not ‘considered’ and does not go through a reflective or fact-checking process is one of the key aspects that leads it to be unable to fulfil authorship criteria. However, this has not been explicitly discussed in many publisher-related policies. Rather, ‘not able to fulfil authorship criteria’ is used as a general sentence, but without a detailed explanation. For those who are unfamiliar with how tools such as LLMs work and why they are unable to produce innovative, novel insights or generate new knowledge, this may be confusing. Although in an ideal world, those considering submitting to a scholarly journal would complete due diligence on such matters, this is not always the case.\n\nIn general, this theme indicates that publisher policies identify the unique humanity of authoring works. This means not just producing texts that are coherent, informative, or sound highly polished and professional but also the agency and capacity to understand the potential ethical, legal, and social ramifications of knowledge production.\n\nNo differences between the different categories of publishers explored in the data were discovered during manual analysis.\n\nIn order to conduct the GenAI assisted thematic analysis, R1 began by accessing OpenAI’s ChatGPT interface, using the paid subscription option ‘ChatGPT Plus’ to access the more advanced underlying LLM, GPT-4. Several modes are available using this subscription, including ‘Advanced Data Analysis (previously named ‘Code Interpreter’). Both the default mode and Code Interpreter/Advanced data analysis mode were used to identify themes at two separate points in time: July 27th and September 11th 2023. This was done to demonstrate the continuously advancing nature of the software, highlighting the importance of authors being specific about their use of GenAI tools in the research process. The nature of the analysis driven by GenAI resulted in the output of completed themes by the software when prompted. This is an important aspect to recognise when using a GenAI tool to assist with thematic analysis: some steps are not immediately accessible or visible to the researcher, or may be skipped over altogether.\n\nThe process began with R1 providing the collected publisher data to the LLM structured using the following prompt:\n\nPerform an inductive thematic analysis on a provided dataset of recently created policies related to generative AI from various publishers. The dataset is structured with the columns ‘policy number’, ‘name of publisher’, ‘publisher category’, and ‘policy’. Compare the differing approaches taken by these publishers based on their categories. The results should be provided in a markdown table with at least two specific examples drawn from the policies to illustrate the themes created. Additionally, provide a text summary of the results suitable for inclusion in an academic journal #here is the table.\n\nTable 1 shows the themes identified in the initial analyses conducted in July, 2023.\n\nMultiple errors were produced during the attempted thematic analysis using the code interpreter mode. This software had only been released for under a week and struggled to handle the Excel data file provided. The themes were also not produced in the same style as the standard model, with simple descriptions absent from the tables. The thematic analysis was also noticeably more limited than that produced by the default GPT4 model, with only three high-level themes being identified.\n\nTable 2 shows the themes identified in the second analysis conducted in September 2023.\n\nThe analysis of publisher policies undertaken in July and September reveals interesting distinctions between the default and advanced data analysis modes available in ChatGPT Plus. In the default mode, the identified themes remained largely consistent, with only one new theme, ‘Complete Rejection of AI Tools’, which emerged in the September analysis. The theme ‘Impact of Evolving AI’ subtly diverged from ‘Policy Evolution,’ indicating a minor refinement rather than a major shift. However, the advanced data analysis tool demonstrated significantly different results, which could point to more rapid advancements or refinements in this beta tool. Rather than increasing the intricacy of the thematic structure, it consolidated themes like “Accountability” and “Authorship” into a singular construct, thus reducing the overall number of themes. Despite operating on the same underlying dataset, these two modes of analysis yielded different thematic outcomes, not due to a changing landscape, but rather due to their respective analytical frameworks. No differences between the different categories of publishers were discovered in any of the analyses.\n\nWhen all themes created by the GenAI tool were compared, similarities emerged that enabled R1 to create their final AI-assisted themes, a process carried out by manually grouping the previous themes that had emerged and asking for ChatGPT (default model) to create a revised name and description for the combined themes. The names and descriptions were then manually edited. The final themes for the AI assisted thematic analysis are shown in Table 3\n\nThe final selected AI-assisted themes, based on the four analyses requested from ChatGPT, are discussed below. Examples of quotes from policies representing the themes were requested in all stages of the data analysis, however, despite multiple prompting attempts over the different modes of analysis, it was not possible to obtain fully accurate examples from ChatGPT, with quotes frequently misattributed to other publishers, condensed, or simply fabricated. These LLM hallucinations demonstrate that, although GenAI tools can offer support in research activities, their results must be carefully checked to ensure accuracy. All quotes from the policies shown below were manually selected and/or verified.\n\nAI assisted analysis theme one: Authorship constraints\n\nThe dominant theme from the analysis emphasises that AI tools lack the capability to qualify for authorship, primarily because of the absence of accountability and legal standing. Examples from Cambridge University Press and Palgrave MacMillan/St Martin’s Press. corroborate this stance: “AI does not meet the Cambridge requirements for authorship, given the need for accountability,” and “ Large Language Models (LLMs), such as ChatGPT, do not currently satisfy our authorship criteria.” Interestingly, the focus here seems less on extolling human qualities of responsibility (as identified in the manual analysis) and more on delineating the limitations inherent to AI in the context of authorship.\n\nAI assisted analysis theme two: Human accountability\n\nThe second theme in the assisted analysis is Human Accountability, specifically the claim that authors are accountable for all the work they produce and submit, including any content produced by AI tools. This was supported in the initial AI results by a quote from MIT Press: “Authors are fully responsible for the content of their manuscripts, including any portions produced by AI tools” and Springer: “Notably an attribution of authorship carries with it accountability for the work, which cannot be effectively applied to LLMs”, and SSRN “Authors are ultimately responsible and accountable for the contents of their work” amongst others.\n\nThis theme is similar to the first theme identified in the manual analysis of ‘authorship is human’ because of the unique human ability to take responsibility for the content of the work. In this sense, although the assisted analysis identified the same element in the policies, this was separated out into an entire theme itself, while R2 included it more holistically under the category of authorship being fundamentally human.\n\nThe third theme identified in the assisted analysis is related to the transparent disclosure of AI tools when submitting a manuscript for publication. Examples include Edward Elgar’s guidelines: “If authors choose to use AI tools to assist in their research, they must disclose this in the manuscript,” and Cambridge University Press’s requirement that “AI use must be declared and clearly explained.” The publishers seem to balance acknowledgement of AI’s utility with its ineligibility for authorship, aligning with the manual theme “Transparency is Vital.”\n\nAcross almost all publishers, it was found that there was a requirement to disclose the use of AI tools, reflecting an evolving consensus that simultaneously seems to acknowledge the use of AI tools and their ability to make genuine contributions, while equally denying that they fit the category of an author. This is similar to the theme identified in the manual analysis of ‘Transparency is Vital’. Again, the analysis indicates that there is a high degree of consistency in the relevance of the themes that have been identified separately, yet also demonstrates that there are nuances in the classification of themes as belonging to a broader category (as with manual analysis) or that they constitute discrete themes in themselves (assisted analysis).\n\nThe fourth theme identified was that the use of AI tools should not compromise research integrity. This was supported by examples from Elsevier Science Ltd: “Authors are accountable for ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved”, and Wiley-Blackwell: “The author is fully responsible for the accuracy of any information provided by the tool and for correctly referencing any supporting work on which that information depends”.\n\nInterestingly, this was not identified as a theme in the manual analysis. Following the discussion and comparison of data coding and analysis, it was identified that R2 felt that the concept of integrity was related more wholly to the theme of transparency and authorship being uniquely human. Following this, if an author was not transparent and did not take responsibility for their work, then this by itself would represent a lack of integrity, and that integrity formed a larger ‘macro theme’ of the entire policy data rather than just the data referencing AI. In this sense, R2’s familiarity with the overall policies may have had an effect, whereas the AI-assisted analysis was based solely on data provided by the extracted AI policy.\n\nThe fifth theme discusses the dynamism inherent in AI policies, acknowledging the speed at which AI technology evolves, and how policies will have to evolve alongside these changes. Examples from Edward Elgar and Emerald indicate keen awareness of this fluid landscape. Edward Elgar remarks, “As this is a fast-moving area, this policy is subject to change” while Emerald notes, “Emerald is adopting the following approach, as part of a watching brief on developments in the rapidly evolving AI space”. This parallels the manual theme of ‘ambiguity’, although the focus is more on the adaptability of policies rather than on their indeterminate nature.\n\nThe sixth and final theme outlines the limits, boundaries, and restrictions imposed on the use of AI tools, sometimes extending outright prohibitions. The sixth theme elucidates the stringent restrictions on the use of AI tools in academic settings. Edward Elgar’s policy is particularly explicit: “The use of AI tools such as ChatGPT (or related platforms) to generate substantive content, such as the analysis of data or the development of written arguments, is not permitted.” This sets a clear boundary on the roles that AI tools can play in academic research, underlining the limitations that publishers believe are necessary to ensure ethical integrity. SSRN also emphasises that “Authors should only use AI technologies to improve the readability and language of their work and not to replace key researcher/author tasks”. In the broader publishing ecosystem, other non-academic publishers appear to go even further than this, as exemplified by Clarkesworld’s firm stance: “We will not consider any submissions written, developed, or assisted by these tools. Attempting to submit these works may result in being banned from submitting works in the future”.\n\nThe themes identified by the AI-assisted analysis as well as from the manual analysis were then used as a basis for further investigation and reflection to support the development of a final set of themes which both authors believed adequately represented the data. Table 4 summarises the development of the final identified themes.\n\n\nDiscussion\n\nThe final six themes were developed and labelled through a collaborative effort involving both AI-assisted and manual coding approaches. This process entailed using ChatGPT to assist in theme identification and subsequent discussions between the two authors to refine and elaborate on these themes, along with cross-checking and contrasting the data collected using the two different methods (manual versus. AI-supported). This resulted in a set of final themes that encapsulate the complexities and nuances associated with the use of AI in academic publishing, with three themes being jointly identified by both the GenAI analysis and the manual analysis, and the three additional themes identified by the AI analysis being retained but expanded and modified slightly. Overall, this methodology seemed productive. While there are risks in terms of using such software to coordinate and undertake analysis, in our experience, human intervention, oversight, and a robust triangulation process with manual coding and analysis helped eliminate the risk of errors.\n\nThe major benefit of the AI-assisted theme development was the broader range of themes that emerged, including some that appeared overly obvious to a human researcher. However, on reflection, the researchers agreed that the AI appeared to capture additional subtle themes present in the data. This said, the data used for this analysis are straightforward, simple, and less complex than, for example, interview data which spans several topics and are longer in duration/text length. Consequently, further studies are needed to examine the differences that might be generated from dual analyses in this manner. We suggest that in the future, in similar studies, it is beneficial to adopt this two-system approach, in which manual analysis is conducted in the traditional sense, and AI-generated analysis is then used to further enrich and triangulate findings.\n\nOne of the major areas of interest in the results was the striking similarity between the themes identified by both methods. The most clearly related of these is the notion of ‘Human-Exclusive Authorship’, which combines the ‘Authorship Constraints’ theme from the AI-assisted analysis and the ‘Authorship is Human’ theme from the manual analysis. Both analyses concur that authorship in academic contexts is a uniquely human endeavour, and this suggests a high degree of certainty in the validity of our themes and the consistency of the data. In summarising the content of this theme, the consensus that appeared to emerge from the policies is that AI tools, including Generative AI technologies such as ChatGPT, can assist in various aspects of research and writing but fall short of assuming the ethical and legal responsibilities that come with authorship. This reflects a broader consensus in the academic community that acknowledges the supportive role of AI tools while underscoring the irreplaceable value of human expertise and ethical judgement.\n\nAnother overlap was clear between the themes of ‘Transparency in AI Utilisation’ and ‘Transparency is Vital’. Both analyses underscored the importance of transparency when using AI tools in academic work. However, the AI-assisted analysis focused more on the need for disclosure as a mandate, whereas manual coding highlighted the principle of transparency. The final theme of ‘Transparency and Disclosure’ encapsulates this nuance, emphasising not only the necessity of disclosure, but also its variable implementation across different publishers. From this, we can see that, while some minor differences occur in the micro-focus of the themes depending on the manual or assisted analysis, the general idea of transparency was picked up by both researchers. The necessity of transparency when using GenAI tools in academic research appears in almost every policy examined. Transparency in this case is not merely a procedural requirement but a fundamental ethical principle that upholds the integrity of academic scholarship and serves multiple purposes. First, it allows for verification of the methods employed, which is crucial for the replicability and validity of the research. Second, it ensures that the contributions of the AI tools are appropriately acknowledged, thereby providing a clear demarcation between human and machine contributions. This is particularly important given that GenAI tools can perform a wide array of functions, ranging from data collection and analysis to generating text for literature reviews. Failure to disclose the extent of AI involvement could inadvertently mislead readers and obfuscate the human intellectual labour involved, thereby undermining the ethical foundations of scholarly work.\n\nThe themes “Policies are Ambiguous” from manual coding and “Evolving Policies” from the AI-assisted analysis converge into the final theme of “Fluid Policy Landscape.” This theme captures the dynamic and likely ever-changing nature of policies concerning the use of AI in academic publishing. Both analyses recognise that these policies are not static; they are subject to change in response to the rapid advancements in AI technology and shifts in ethical understanding and acceptance of these tools as they become more widely known and used in the academic community. This theme encapsulates this complexity, underscoring that, while there is a need for guidelines, these policies are inherently fluid and need to be responsive to the rapidly evolving landscape of AI technologies and their ethical implications.\n\nIn considering the final theme of ‘Constraints and Exclusions’, we find this particularly salient in the context of the ongoing discourse around the legitimacy and ethicality of AI utilisation in academic work that was highlighted by the fifth theme. This theme reflects the often stringent guidelines and explicit prohibitions found in the policies of various academic publishers. This theme highlights the field’s cautious approach to incorporating AI into academic processes, especially in generating a significant proportion of intellectual output, thus closely aligning with other themes that emphasise human responsibility and ethical considerations. This theme illuminates the existence of broader concern, transcending academic circles about the capabilities and limitations of AI in content creation. While the policies are not uniform across all publishers, the overall tenor of the discussion seems to be one of caution, placing the onus on human researchers to navigate the evolving landscape. Therefore, the theme ‘Constraints and Exclusions’ serves as a cautionary note, emphasising that, while AI technologies offer vast potential for enhancing academic work, they are not without their limitations and must be used judiciously.\n\nThese themes offer an in-depth understanding of the complexities surrounding the use of AI in academic publishing. They reflect the collective insights gained from leveraging both AI and human analytical capabilities, thereby providing a robust framework for future discussions and research in this area.\n\nThis research has provided insights into the current systems of thought regarding the use of AI and academic publishing. There is no doubt that this is a complicated, evolving issue, but at the same time, it seems that there is a high degree of consistency among academic publishers about the role that AI tools are currently fulfilling. Equally, it seems that the core principles of academic integrity, authorship, and responsibility have not undergone any form of dramatic change. Rather, it seems that the current view is that such applications are another asset in the toolbox of academic scholars. In addition to the ease of use of these tools, there are also arguments that they may contribute to the democratisation of research and scientific knowledge. AI tools can, for example, benefit non-native speakers of English who conduct research and are under pressure to publish in English-medium journals by providing rapid support and feedback on manuscripts or enable new scholars and students who are engaging in academic publishing for the first time to receive additional guidance and support on writing for publication. Students engaged in postgraduate or doctoral studies may also use these tools to help develop and refine their ideas, presentations, and proposals to enhance their chances of success in the competitive academic field. For authors who are experienced and confident writers or composing text in the first language, there are still opportunities to improve the logic of expression, coherence, style, and quickly change formatting to enable submission to a target journal. Therefore, it is encouraging to see that scholarly publishers have not generally taken the path of prohibition or reactionary banning of AI in the research and writing process.\n\nHowever, alongside these positive implications are challenges that the academic community must navigate. One of these is the phenomenon known as ‘stochastic parroting’, where AI tools may simply repeat training data (Li, 2023) or ‘hallucinations’ where it may inadvertently produce misleading or incorrect information (Bender et al., 2021); a by-product of their training data and algorithms. This raises questions about the reliability and integrity of AI-generated content, mandating extra scrutiny from authors (as demonstrated in the AI analyses insistence on creating false quotes from the policies). The issue compounds when we consider the potential for AI tools to produce voluminous yet generic output, adding an additional layer of complexity to the review and editing process in journal production. Editors and reviewers may find themselves burdened by the task of sifting through and filtering this added volume, questioning the originality and contribution of AI-assisted manuscripts. Moreover, the ease of generating academic content using AI tools can be a double-edged sword. On the one hand, these tools may accelerate the research process by supporting in cognitive offloading (Perkins et al., 2023; Dawson (2022) as cited in Sparrow, 2022); the process of reducing the cognitive demands of a task (Risko & Gilbert, 2016). However, this must be balanced by the risks of authors committing research misconduct by providing false information or disguising authorship. This underscores the need for robust policy frameworks to guide ethical AI use in academic research. For example, policies should clarify the extent to which AI tools need to be disclosed in academic work, as found in our analysis of various publishers’ stances on AI authorship and accountability. This can be conducted within the framework of cognitive offloading if any use of GenAI tools is explained adequately and transparently. Another limitation is the current inability of AI to match human scholarship in terms of nuanced contextual understanding and interpretative abilities. As our thematic analysis revealed, both human and AI-assisted reviews emphasise that, while AI tools can be highly beneficial for specific tasks, they cannot replace human intuition and judgement. This is particularly significant in the realms of hypothesis formation and qualitative interpretation, areas in which human cognition currently has an insurmountable edge.\n\nRegarding the limitations of the study itself, it is crucial to acknowledge that our inductive thematic analysis was confined to a limited set of publisher policies available at the time of the research. The rapidly evolving nature of AI technology and its applications in academia means that the landscape is fluid and policies may have already changed. Similarly, we focused our attention on academic publishing in English, and among flagship and major publishing houses. This also comes at the expense of focusing on non-academic publishing, such as fiction and general readership texts, and publishing guidelines for publications produced by professional and governmental organisations. Further research in this area would be useful to expand the field of study on the use of generative AI in publication at large. This study also relied on a combination of AI-assisted and human analysis, each with its own set of biases and limitations. For example, the use of a single AI tool leaves the door open to software-specific errors or idiosyncratic features; however, this was minimised by using four separate analyses over two time periods. Future research should aim to mediate this by conducting an analysis using a range of GenAI tools and comparing the results. Although the GenAI analysis was capable of processing large volumes of data in a short time span, it may have missed nuanced interpretations that a human researcher could capture. Conversely, human analysis, although in-depth, was limited by the constraints of time and cognitive load, which AI does not face. Furthermore, the study did not empirically test the effectiveness or reliability of AI tools in academic research, which is a potential avenue for further research. Therefore, the findings should be considered a snapshot of the complex and evolving interplay between AI technology and academic publishing.\n\n\nConclusion\n\nIn conclusion, the multifaceted roles and implications of AI tools in academic research warrant a more inclusive and forward-thinking approach than is currently demonstrated. As our study shows, there is broad consensus among publishers that authorship in academic contexts remains a uniquely human endeavour. Human authors are irreplaceable in their capacity to assume ethical and legal responsibilities, and are qualities that current AI technologies cannot replicate. However, this recognition should not overshadow the valuable supportive roles that GenAI tools can fulfil. From text generation and data analysis to linguistic translation and textual editing, these technologies can democratise access to academic publishing and facilitate a more inclusive scholarly ecosystem, mediating inequalities that occur in terms of linguistic ability and experience in publishing academic work.\n\nThe themes identified in our study highlight the complexities and nuances of integrating AI into academic research but encouragingly suggest that publishers are aware of the significant benefits that scholars can reap from using such tools in a transparent and ethical manner. They underscore the necessity for a balanced approach that is both open to AI’s potential and vigilant of its ethical challenges. Such an approach calls for policies that are not merely reactionary, but are formulated based on a nuanced understanding of AI’s capabilities and limitations. A reactionary stance toward AI integration in academic settings risks stifling innovation and could result in guidelines that are more obstructive than facilitative, as seen in the more severe restrictions imposed by some publishers.\n\nMoreover, the fluid nature of AI technologies necessitates that publisher policies are agile and adaptive. Our findings indicate that, while there is a consensus on the non-qualification of AI for authorship and the imperative of accountability and transparency, there is also a shared understanding that these stances will need to evolve in tandem with technological advancements. Dialogues around the use of AI in academic research are intricate and evolving, and while the core values of human authorship and accountability remain constant, there is increasing acknowledgement of the supportive roles that AI can play in academic research. As computational power grows and societal acceptance of AI becomes the norm, it is possible, if not likely, that a shift in policy will take place. Therefore, as we navigate this complex landscape, it becomes crucial for academic institutions and publishers to move beyond mere ‘knee-jerk’ reactions. Instead, the focus should be on developing informed and flexible guidelines that safeguard academic integrity and promote the constructive use of GenAI tools. Such a holistic approach will not only serve to uphold the ethical foundations of academic research but also pave the way for innovative practices that capitalise on the synergies between human intellect and artificial intelligence, as demonstrated in this work.\n\nThis study used Generative AI tools to analyse data, create preliminary themes, produce draft text, and revise wording throughout the production of the manuscript. Multiple modes of ChatGPT over different time periods were used, with all modes using the underlying GPT-4 Large Language Model. The authors reviewed, edited, and take responsibility for all outputs of the tools used in this study.", "appendix": "Data availability statement\n\nFigshare: Generative AI policies for academic publishers https://doi.org/10.6084/m9.figshare.24124860.v1.\n\nThis project contains the following underlying data:\n\n• Gen AI policies Academic Publisher.xlsx. (All data related to AI policies including full policies as downloaded and URLs).\n\nThe data is available under the terms of the CC-BY 4.0 license.\n\n\nReferences\n\nAhmed I, Jeon G, Piccialli F: From Artificial Intelligence to Explainable Artificial Intelligence in Industry 4.0: A Survey on What, How, and Where. IEEE Trans. Industr. Inform. 2022; 18(8): 5031–5042. Publisher Full Text\n\nAhmed S, Alshater MM, Ammari AE, et al.: Artificial intelligence and machine learning in finance: A bibliometric review. Res. Int. Bus. Financ. 2022; 61: 101646. Publisher Full Text\n\nAnderson N, Belavy DL, Perle SM, et al.: AI did not write this manuscript, or did it? Can we trick the AI text detector into generated texts? The potential future of ChatGPT and AI in Sports & Exercise Medicine manuscript generation. BMJ Open Sport Exerc. Med. 2023; 9(1): e001568. 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Health Prof. 2023; 20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Z: The Dark Side of ChatGPT: Legal and Ethical Challenges from Stochastic Parrots and Hallucination (arXiv:2304.14347). arXiv.2023. Reference Source\n\nLiebrenz M, Schleifer R, Buadze A, et al.: Generating scholarly content with ChatGPT: Ethical challenges for medical publishing. Lancet Digit. Health. 2023; 5(3): e105–e106. PubMed Abstract \| Publisher Full Text\n\nLund BD, Wang T, Mannuru NR, et al.: ChatGPT and a new academic reality: Artificial Intelligence-written research papers and the ethics of the large language models in scholarly publishing. J. Assoc. Inf. Sci. Technol. 2023; 74(5): 570–581. Publisher Full Text\n\nNishikawa-Pacher A: Who are the 100 largest scientific publishers by journal count? A webscraping approach. J. Doc. 2022; 78(7): 450–463. Publisher Full Text\n\nNowell LS, Norris JM, White DE, et al.: Thematic analysis: Striving to meet the trustworthiness criteria. Int. J. Qual. Methods. 2017; 16(1). Publisher Full Text\n\nOpenAI: GPT-4 Technical Report (arXiv:2303.08774). arXiv.2023a. Reference Source\n\nOpenAI: Introducing ChatGPT Enterprise.2023b, August 28. Reference Source\n\nPatel SB, Lam K: ChatGPT: The future of discharge summaries? Lancet Digit. Health. 2023; 5(3): e107–e108. Publisher Full Text\n\nPavlik JV: Collaborating With ChatGPT: Considering the Implications of Generative Artificial Intelligence for Journalism and Media Education. Journalism & Mass Communication Educator. 2023; 78(1): 84–93. Publisher Full Text\n\nPeng Y, Liu E, Peng S, et al.: Using artificial intelligence technology to fight COVID-19: A review. Artif. Intell. Rev. 2022; 55(6): 4941–4977. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins M: Academic Integrity considerations of AI Large Language Models in the post-pandemic era: ChatGPT and beyond. J. Univ. Teach. Learn. Pract. 2023a; 20(2). Publisher Full Text\n\nPerkins M: Generative AI policies for academic publishers. figshare. [dataset]. 2023b. Publisher Full Text\n\nPerkins M, Roe J: Decoding Academic Integrity Policies: A Corpus Linguistics Investigation of AI and Other Technological Threats. High Educ. Pol. 2023. Publisher Full Text\n\nPerkins M, Roe J, Postma D, et al.: Game of Tones: Faculty detection of GPT-4 generated content in university assessments (arXiv:2305.18081). arXiv. 10.48550/arXiv.2305.18081.2023.\n\nPichai S: An important next step on our AI journey. Google; 2023, February 6. Reference Source\n\nPourhoseingholi MA, Hatamnejad MR, Solhpour A: Does chatGPT (or any other artificial intelligence language tool) deserve to be included in authorship list? Gastroenterol. Hepatol. Bed Bench. 2023; 16(1): 435–437. Publisher Full Text\n\nRahimi F, Talebi Bezmin Abadi A: ChatGPT and Publication Ethics. Arch. Med. Res. 2023; 54(3): 272–274. Publisher Full Text\n\nRisko EF, Gilbert SJ: Cognitive Offloading. Trends Cogn. Sci. 2016; 20(9): 676–688. Publisher Full Text\n\nRoe J, Perkins M: What are Automated Paraphrasing Tools and how do we address them? A review of a growing threat to academic integrity. Int. J. Educ. Integr. 2022; 18(1): Article 1. Publisher Full Text\n\nRoe J, Renandya W, Jacobs G: A Review of AI-Powered Writing Tools and Their Implications for Academic Integrity in the Language Classroom. J. Eng. Appl. Linguist. 2023; 2(1). Publisher Full Text\n\nSalvagno M, Taccone FS, Gerli AG: Can artificial intelligence help for scientific writing?. Crit. Care. 2023; 27(1): 75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolomon DH, Allen KD, Katz P, et al.: Artificial Intelligence, Authorship, and Medical Publishing. ACR Open Rheumatol. 2023; 5(6): 288–289. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSparrow J: ‘Full-on robot writing’: The artificial intelligence challenge facing universities. The Guardian. 2022, November 18. Reference Source\n\nTan P, Chen X, Zhang H, et al.: Artificial intelligence aids in development of nanomedicines for cancer management. Semin. Cancer Biol. 2023; 89: 61–75. PubMed Abstract \| Publisher Full Text\n\nTate T, Doroudi S, Ritchie D, et al.: Educational Research and AI-Generated Writing: Confronting the Coming Tsunami.2023. Publisher Full Text\n\nWeber-Wulff D, Anohina-Naumeca A, Bjelobaba S, et al.: Testing of Detection Tools for AI-Generated Text.2023. Publisher Full Text\n\nWikipedia: Rankings of academic publishers. Wikipedia. 2023. Reference Source\n\nXiao P, Chen Y, Bao W: Waiting, Banning, and Embracing: An Empirical Analysis of Adapting Policies for Generative AI in Higher Education (SSRN Scholarly Paper 4458269).2023. Publisher Full Text\n\n\nFootnotes\n\n1 ‘In all content submitted to SSRN, authors should only use AI technologies to improve the readability and language of their work and not to replace key researcher/author tasks such as producing insights or theories, analyzing and interpreting data or drawing conclusions and presenting viewpoints.’(Elsevier, 2023).\n\n2 ‘The use of AI tools such as ChatGPT (or related platforms) to generate substantive content, such as the analysis of data or the development of written arguments, is not permitted’(Edward Elgar Publishing, n.d.)." }	[ { "id": "218374", "date": "01 Nov 2023", "name": "Brenda M Stoesz", "expertise": [ "Reviewer Expertise Academic integrity", "research integrity", "policy 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\nWith the emergence of publicly accessible generative artificial intelligence (genAI) tools over the past year, concerns about authorship in research and research articles has emerged. The authors provide a brief background on this technological advancement and question how the policies of publishers have begun to address concerns about genAI tools being listed as an author or otherwise used to support the development of research articles. The authors used two methods to examine 28 policies - human coding and use of genAI - and found similarities and differences in extracted themes using the two methods. Although genAI assisted analysis was useful in some ways, the authors concluded that human input in data analysis and interpretation are important. The authors discuss the ethical and practical implications of their findings as they relate to non-native English speakers and writers, students, and the academic community as a whole.\nHere are some minor points to consider when revising this manuscript:\nSome reorganization of the methods section where thematic analysis is described is warranted as there appears to be some redundancy. Please revise, although this is a minor point.\n\nThe last sentence of results section 1 indicated that there were no differences between different categories of publishers during manual analysis. Please add details about these categories in the methods section - these details may be present in the methods section but perhaps they are not as salient as they should be.\n\nI wonder if the authors considered how prolific use of genAI for conducting and writing up research might impact the interpretations made by systematic review articles and/or meta-analyses.\n\nThere are some punctuation errors and omissions in a few places, and some odd text (i.e., #here) in a few places that need correction.\nOverall, this is an interesting study and readers, especially editors, researchers, and other members of the academic community involved in internal policy making will find this study useful.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10833", "date": "16 Jan 2024", "name": "Mike Perkins", "role": "Author Response", "response": "Thank you very much for your comments and review. Please see below for a point-by-point response. This has been reviewed and edited for clarity. We have added clarification on the categories of publishers considered to the methods section. All publisher categories are shown in the additional data This was not something we had considered. Assuming that GenAI supported research is done with integrity and transparency, there should be no implications for any systematic reviews-the problem would come if there was no oversight and AI hallucinations were present. However, as the methodology we describe is supported by GenAI tools, rather than carried out by them, we do not believe this to be an area of concern. The paper has been reviewed again for punctuation errors. The # marks are direct quotes from the inputs used in the GenAI tools-marks like this can be used to organise complex inputs to GenAI tools, (for example, separating sample text from instructions on what to do with it) as these tools generally do not process line breaks." }, { "c_id": "10939", "date": "14 Jun 2024", "name": "Brenda Stoesz", "role": "Reviewer Response", "response": "Thank you for addressing the comments." } ] }, { "id": "218375", "date": "13 Nov 2023", "name": "Mary Davis", "expertise": [ "Reviewer Expertise Academic integrity", "plagiarism", "inclusion", "Artificial Intelligence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 great deal of current research is focusing on the impacts of AI in academia. The authors make an original contribution by identifying a specific area of importance to researchers: current publisher guidance on use of AI. They focus on an analysis of 28 academic publishers’ guidelines on use of AI in author submissions to establish the main themes prevalent in this guidance. By using an AI-generated analysis to examine guidance on use of AI, as well as human analysis, the authors add an additional level of interest and originality to the paper. They provide a well-documented analysis and discussion leading to the emergence of the final themes in publisher guidance on AI: human-exclusive authorship, author accountability, disclosure and transparency, research integrity, fluid policy landscape and constraints and exclusions.\nI found this to be an interesting study which will be directly useful for researchers and publishers in terms of the emerging themes, but also to everyone interested in guidance on AI and indeed conducting research using AI. It was good to see the linked full data for the analysis with the researchers' notes. I recommend the authors consider some minor revisions for this paper as follows:\n1. Abstract - Background line 4 ‘and prompting discussions’ – this seems ambiguous in the sentence. If it is the lack of policies which prompt discussions, I suggest revising as ‘which has prompted discussions among publishers…’\n2. Introduction paragraph 1 In your introduction to key terms, some more definitions would be useful; in particular, I recommend adding whether you are using the terms ‘GenAI tools’ and ‘AI tools’ interchangeably or for different meanings throughout the paper.\n3. Introduction paragraph 2 line 6 ‘This has sparked a technological ‘arms-race’ – in fact reports of a technological arm’s race have been going a very long time in regard to plagiarism and text-matching tools, so rather than ‘sparked’, it might be better to talk about something like an ‘escalation’ in the ongoing arms race. For background, you may like to look at the work of Betty Leask nearly 20 years ago who talked about these battles in: Plagiarism, cultural diversity and metaphor - implications for staff development Assessment & Evaluation in Higher Education Vol. 31, No. 2, April 2006, pp. 183–199.\n4. Literature review - AI’s role and ethical implications in academic research - Paragraph 2 line 8 ‘Pass GenAI texts on their own’ – I think you mean ‘pass off GenAI texts as their own’?\n5. Literature review - AI’s role and ethical implications in academic research – Paragraph 3 Consider avoiding repetition of ‘perpetuate’ and ‘perpetuating’ lines 2-3.\n6. Results section one – line 1 You mention the manual analysis was carried out using QDA Miner software, so actually the manual analysis was not fully manual. The user guide says it can be used for ‘coding textual and graphical data, annotating, retrieving and reviewing coded data, documents and images’ https://provalisresearch.com/uploads/QDA-Miner-5-User-Guide-V1.2.pdf It would be helpful to the reader for you to clarify further how the software was employed in this study.\n7. Results section one - Manual analysis subsections You don’t provide any tables for your manual analysis calculations in this part, you just incorporate them in table 4 to compare with the AI analysis. It would be helpful to explain or at least signpost to the reader that the data from the manual analysis can be seen in table 4.\n8. Table 3 I am wondering why the line ‘limitations and prohibitions’ is repeated across 2 lines at the bottom of the table?\n9. Limitations and implications – paragraph 2 line 11 I suggest citing Dawson directly rather than involving Sparrow’s article in the Guardian, unless you want to highlight newspaper discussion.\n10. References Bealls list.net, no date – I would suggest Beall’s List, 2021 – the date given on the website for the most recent update.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10834", "date": "16 Jan 2024", "name": "Mike Perkins", "role": "Author Response", "response": "Thank you very much for your review. Please see below for a point-by-point response. Adjusted. AI tools are different to GenAI tools and are used as separate terms in the paper. Further definitions are provided in the literature review, but we choose to not dwell so much on definitions, given the specific objectives of this paper. We have replaced 'sparked' with 'escalated' to more accurately represent the continuous development of the technological arms race in plagiarism detection and added additional citations for this statement. Adjusted. Replaced perpetuating with escalating. This is correct, we have updated it to make it clear that this is an analysis assisted through QDA Miner software at the beginning of the paper, and when we discuss ‘manual’ we are referring to with use of non-GenAI software. We have added signposting language to show where the results are for this section in Table 4. This repeated row is deleted. This news article cites Dawson specifically discussing GenAI tools as a method of cognitive offloading. However, given that other reviewers have also commented on this, we have replaced this citation with Dawson’s original work on cognitive offloading for clarity and to avoid further confusion. Updated in the text and citation." } ] }, { "id": "218378", "date": "15 Nov 2023", "name": "Stephen Gow", "expertise": [ "Reviewer Expertise Academic Integrity", "Assessment", "Policy as they relate to generative AI." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, I found this paper timely, innovative and thought-provoking. It makes a solid contribution to the use of generative AI in research and for publication, and is of use for policy makers, researchers and editors alike. In a suitably meta approach, the papers carries out both of the following:\n\nThe identification and thematic analysis of 28 academic publishers’ policy response to generative AI\n\nThe hybrid human and generative AI analysis of these policies in order to demonstrate and evaluate the use of generative AI for policy/qualitative research.\nThe paper clearly states and meets 4 objectives it set’s out. My main take away is the potential use of generative AI to support research, as the authors say, with “human intervention, oversight and a robust triangulation process…” - I expect some of this message may be lost in the summarisation of this article in media - it is not the simple case of ChatGPT carrying out the research, it is carefully used as a tool but human intuition is needed. This actually reinforces the key theme identified in the thematic analysis of the policies - human exclusive authorship - at the current time. It is apparent at this stage that generative AI requires significant human input in order to avoid errors and fabrications. My second take away is that the there is a fluid policy landscape in which publishers are attempting to allow for the use of generative AI but this is highly complex due to the human exclusive authorship and the need for transparency to clearly demarcate where humans and AI contribute to the research.\nI would certainly recommend researchers, teachers and policy makers to read the paper, however I do have the following 10 areas of critique which I hope the authors can take on board:\nProgressive vs Conservative approach to AI: The authors are clearly in favour of utilising generative AI for the purposes of research, this bias is strong in the paper - with them referring to the “knee jerk reaction” or reactionary nature of policies which do not embrace generative AI as wholeheartedly as they do. They do however acknowledge the fluid policy landscape as a key theme. In this the authors note the publishers' flexibility to adapt to the rapidly changing nature of the technology and its impact on academic publishing. While the paper clearly notes that publishers are not attempting to ban AI and do see the potential benefits, the policies are clearly not as progressive as the authors would like. The authors call for more nuanced policies based on AI’s capabilities and limitations, though this is easier said than done as much guidance at this stage is speculative and quickly outdated. This is where I would use the terms conservative and cautious rather than reactionary, they seem like more fair terms for the publishers. Publishers are considering not only authorship and ethical considerations but legal risks involved in publishing work which they may be culpable for in the future, which provides extra cause for caution.\n\nAI as an entirely new problem for authorship/academic integrity: Judging on the literature referred to in the paper, one may get the impression that questions of authorship and AI are completely new in the past two years. This is not the case as authorship and AI has been discussed, particularly in legal journals, for many years pre-dating ChatGPT’s release. In relation to point 1., this is why it is strange that publishers are suddenly aware of this issue as it has clearly been emerging for many years, for example GPT-3 was reasonably capable a full 2 years prior to Chat-GPT. Furthermore, the references do not fully place the research in the rich body of scholarship of the impact of technology on authorship and academic integrity, the arms race analogy of academic authorship dates back to until at least 2000, even then Betty Leask (2007) discusses why war metaphors may not be helpful with discussions of students plagiarism, I expect similar in this case.\n\nSelf-referential: In relation to point 2, the authors refer to the own work a lot - I know from reading their previous work that they are aware of the literature around technology, authorship and academic integrity. I suspect a rush to publish the work in a fast moving field may be partly behind this, so hope the authors may further contextualise their work within the relevant scholarship, I have provided some suggestions below.\n\nTransparency: The research use of generative AI is clearly set out, however the use of generative AI for drafting is not. This is related to the key theme of transparency in Ai utilisation which they identify. As with any writing now, I am curious about whether something has been written by generative AI and especially so with this paper where is admitted it has been used, the demarcation is not clear - a little more explanation of this may help and help for developing a disclosure mandate as suggested. Even if AI has been used in the drafting, the authors have clearly used it in a responsible manner and it reads well - but, in regards to point 3, if writing has been drafted with AI and references added in after, this raises questions about which references the AI is drawing from, this also relates to the the ethical and copyright concerns of using generative AI at all.\n\nImpact of Generative AI on the research process: The authors clearly state how generative AI has been used, it helped identify both obvious and subtle codes in the data and assisted with theme development. I am curious as to whether it has any advantages over other Computer Assisted Qualitative Data Analysis Software, such as Nvivo? I also question whether the researchers would have come up with radically different conclusions had they not used ChatGPT or used an alternative software. The authors did however compare different versions of ChatGPT, which was useful.\n\nDifferentiation of AI/Machine learning and Generative AI: It seems to be the generative element of AI that leads to the problems with it on a practical and theoretical level - e.g. can it be accountable. With non-generative AI/machine learning models - the function is more specific and issues of authorship less problematic, which raises the question about whether generative AI has actually hindered the adaptation of the world to AI and caused the knee jerk reaction.\n\nCopyright issues of how generative AI is trained: Though the authors discuss ethical and copyright issues, a large part of the debate is the copyright or the data used to train models, I don’t feel this is given enough attention given the authors are using the software.\n\nMinor error: Paper states on p3. That there is an in-depth exploration of 109 academic publishers however - the in-depth analysis is only carried out on 28 policies, as far as I can tell.\n\nCognitive offloading: Why is Dawson’s work referenced with a secondary citation? It’s quite accessible.\n\nFurther research: I’d love to see a similar approach for national and international level university policy - I’d be very interested in working on this!\nSuggested papers and some I refer to in the review which may be of interest:\nBeer, D. (2019) - Should we use AI to make us quicker and more efficient researchers? LSE Impact of Social Sciences Blog https://blogs.lse.ac.uk/impactofsocialsciences/2019/10/30/should-we-use-ai-to-make-us-quicker-and-more-efficient-researchers/\n\nCole, S., & Kiss, E. (2000). What can we do about student cheating. About Campus, 5(2), 5-12. - mentions the arms race in terms of technology and academic integrity.\n\nDawson, P. (2020). Cognitive offloading and assessment. Re-imagining university assessment in a digital world, 37-48.\n\nEaton, S. E. E. (2023). 6 Tenets of Postplagiarism: Writing in the Age of Artificial Intelligence [Infographic]. https://prism.ucalgary.ca/items/86a7af9b-fd25-4d35-b20e-cf225fa2d3f5\n\nGervais, D. J. (2019). The machine as author. Iowa L. Rev., 105, 2053.\n\nLeask, B. (2006). Plagiarism, cultural diversity and metaphor—implications for academic staff development. Assessment & Evaluation in Higher Education, 31(2), 183-199.\n\nRogerson AM, McCarthy G (2017) Using internet based paraphrasing tools: original work, patchwriting or facilitated plagiarism? International Journal for Educational Integrity 13:2\n\nSchafer, B., Komuves, D., Zatarain, J. M. N., & Diver, L. (2015). A fourth law of robotics? Copyright and the law and ethics of machine co-production. Artificial Intelligence and Law, 23, 217-240. https://link.springer.com/article/10.1007/s10506-015-9169-7\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10835", "date": "16 Jan 2024", "name": "Mike Perkins", "role": "Author Response", "response": "Thank you very much for your thought provoking review. Although we do not agree with every one of your points, we have clarified this below in the point-by-point response. A very fair point-we are pushing what is quite a major change to traditional academic practices and have adjusted the language as suggested. Reactionary is left in one case as it is used in the sense of making a policy decision that is not informed by research and a full understanding. We have expanded the volume of references around the ‘arms race’ approach to integrity. We recognise the perspective offered by Betty Leask, but believe this is a relevant analogy-the addition of further references does demonstrate the somewhat common nature of this in the literature. We have broadened the citation scope to include a wider range of scholarship, including some of the specific texts mentioned. Although the ‘machine as author’ argument is an interesting area to explore, we are focusing the work in this case towards the applicability of using these GenAI tools for supporting research efforts. There is an AI usage disclaimer present which sets out how GenAI has been used in this study. Yes, it was used to generate draft text, but for the majority of cases, this was a starting point with rewritings and revisions occurring iteratively to get to the final version. We feel that the current disclosure statement goes beyond the minimum disclosure of the use of the tools to focus specifically on the tasks that were carried out. Going beyond this would, we believe, be excessive. To what extent Ai disclaimers will be necessary in the near future is also an interesting point for discussion-as these tools continue to gain prevalence and acceptance in society, will the need to mention them continue to be a requirement form publishers? As per the discussions on cognitive offloading, we don't mention that we used spell checkers or grammar checkers so where is the line? Again, the ethical and copyright concerns of using AI are a little beyond the scope of this paper. While qualitative data analysis software (e.g. NVivo) is exceptionally helpful for research purposes, we would argue that it can be used in conjunction with GenAI tools. The primary reason for this is that GenAI tools enable a triangulation – they can produce a summary of data analysis and interpretation that can be checked against the researcher-led output derived using tools such as NVivo. Furthermore, although these tools have the potential for user confirmation bias (as ‘stochastic parrots’), there is an advantage to having a GenAI ‘research partner’ or collaborator with which ideas can be experimented. It is possible that using different GenAI tools could have led to different results; this is an area that requires further study in the future but we do not feel invalidates our approach and results. Yes, this is certainly one reason! Others would be the lack of reproducibility of the results, and the issues with hallucination (even challenges with changing the original dataset) Again, this is a much broader issue that is not really within the scope of our paper to address. We refer to the European Commission’s point that copyright implications are still currently unclear (EC, 2023) with GenAI tools such as ChatGPT and the training data used on the model. Thus, while we acknowledge this issue, we do not specifically address it within the scope of this paper. Rather, we have focused the discussion of copyright around authorial rights and obligations, not the ethics of the tools themselves. Simply using the software does not, in our opinion necessitate a discussion around the ethics of the creation of the tool. European Commission (2023) Intellectual Property in ChatGPT. Available at https://intellectual-property-helpdesk.ec.europa.eu/news-events/news/intellectual-property-chatgpt-2023-02-20_en. This is clarified later on, but we have adjusted the text here to make the methodology clearer. We reviewed 107 policies, and carried out an in depth analysis on the 28 which made reference to GenAI tools. This news article cites Dawson specifically discussing GenAI tools as a method of cognitive offloading. However, given that other reviewers have also commented on this, we have replaced this citation with Dawson’s original work on cognitive offloading for clarity and to avoid further confusion. That would definitely be an interesting paper-lets discuss more!" } ] }, { "id": "218377", "date": "20 Nov 2023", "name": "Salim Razı", "expertise": [ "Reviewer Expertise Academic integrity policies", "plagiarism prevention", "text-matching tools" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an inductive thematic analysis by investigating 29 publisher policies regarding AI-assisted authorship and academic work. It is well-written in an engaging and lively style on a hot topic to which they make significant contributions. The authors produced a number of positive and welcome outcomes including the literature review which offers a useful overview of current research and policy and the resulting bibliography which provides a very useful resource for further researchers, policy makers, and publishers. In general, I found the manuscript quite good; however, I would like to call the authors’ attention to a few minor issues that I list below.\nPage 3: The authors use both “academic integrity” and “research integrity” in the first paragraph of the introduction section. It would be nice to inform readers about the interaction of these two related terms. I see that the authors make use of these two terms quite often in the rest of the paper.\nPage 3: Considering the objectives and structure of the study, the authors indicate that they explored “the policies of 109 academic publishers who are key stakeholders in the production of scholarly work”. Did they include any policy that they were able to find in their analysis or did they have any inclusion or exclusion criteria for the inclusion of policies in their corpus? Although the authors provide more explanation under methods section, I wish to see inclusion and exclusion criteria clearly.\nPage 4: “‘brace for impact’ (Pichai, 2023, as cited in Elias, 2023)”. Page numbers need to be provided for direct quotations. In addition, the authors use single quotation marks both to present terminology and to show direct quotations which I found quite difficult to interpret.\nPage 5: “the use of Large Language Models (LLMs).” LLM was already abbreviated on Page 3.\nPage 5: “At the core of the issues of academic integrity and ethics.” To my understanding, academic integrity already covers ethics; so, do we need to mention it separately?\nPage 6: “it is crucial that journal publishers bolster plagiarism detection systems”. I would suggest the authors to avoid using “plagiarism detection systems” since it gives the impression that these systems can detect plagiarism automatically without a careful investigation of matches by an expert.\nPage 7: “In our methodological process, these analyses were conducted separately by the two researchers to [establish intercoder agreement].” I suggest adding the phrase in brackets.\nPage 7: “(Boyatzis, 1998, p1)” should be “(Boyatzis, 1998, p. 1)”.\nPage 8: “Elsevier will monitor…” missing full stop.\nPage 8: “the politics of detection”. I guess this is not “politics” but “policies”.\nPage 8: The authors are referring to the policies of MDPI under “manual analysis theme three”. Previously, the authors indicated that they did not include predatory publishers in the analysis. I am not labelling any publisher as possibly predatory here; however, I would like to call the authors’ attention to “Predatory Reports” available at https://predatoryreports.org/news/f/list-of-all-mdpi-predatory-publications .\nPage 10: “the advanced data analysis tool demonstrated significantly different results.” I would invite the authors to remove “significantly” from this expression as “significant” is a statistical term making a reference to “p” values in quantitative analysis.\nPage 11: “The final themes for the AI…” missing full stop.\nPage 14: “then used to further enrich and triangulate findings.” Since this makes a reference to qualitative studies, “findings” should be replaced with “results.”\nPage 15: “Dawson (2022) as cited in Sparrow, 2022” should be “Dawson, 2022 as cited in Sparrow, 2022”.\n\nIs the work 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": "10836", "date": "16 Jan 2024", "name": "Mike Perkins", "role": "Author Response", "response": "Thank you very much for your detailed review. Although we do not agree with every one of your points, we have clarified where this is the case in the points below. This is definitely useful, and we have added a small section in the introduction referring to how we interpret the term research integrity. We have adjusted this section to clarify-107 publishers was our total population (following the exclusion of known predatory publishers). Any publishers with policies mentioning GenAI were then analysed in more detail. No page numbers are available for this online source. Single quotation marks are used throughout as a stylistic choice consistent with British English. Abbreviations have been checked and adjusted as needed. Policy quotes have been left unchanged. Changed to focus on research integrity. This statement has been removed as it was unclear-we are referring to GenAI detection which has been demonstrated to be inaccurate and have provided additional citations for this. In typical qualitative research, this may be the goal, but here we are specifically focused on the differences between the multiple analyses carried out. We were not therefore seeking to establish inter-coder agreement (although this did occur). Corrected Corrected Corrected! We had discussions regarding the inclusion/exclusion of MDPI as a potential predatory publisher and made the decision not to include this, primarily based on the most updated version of Beall’s list. The use of ‘significant’ is not a term that is restricted solely to statistical usage; however, we have replaced this with ‘major’ to avoid any confusion. Corrected Thank you, but we prefer the original wording-we are referring to findings in the sense of the interpreted results of multiple human and GenAI supported analyses. Any GenAI supported analysis requires human-input and interpretation, therefore we believe that findings is the more appropriate term here given that the raw ‘results’ may not be accurate This reference has been replaced with an alternative primary source from Dawson." } ] } ]	1	https://f1000research.com/articles/12-1398
https://f1000research.com/articles/12-439/v1	25 Apr 23	{ "type": "Method Article", "title": "Development of an in vitro microfluidic model to study the role of microenvironmental cells in oral cancer metastasis", "authors": [ "Alice Scemama", "Artysha Tailor", "Stefania Di Cio", "Matthew Dibble", "Julien Gautrot", "Adrian Biddle", "Alice Scemama", "Artysha Tailor", "Stefania Di Cio", "Matthew Dibble", "Julien Gautrot" ], "abstract": "Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field.", "keywords": [ "microfluidic", "cancer", "metastasis", "vasculature", "HUVEC", "microenvironment", "chip" ], "content": "\n\n\n\nScientific benefits\n\n\n\n• Development of a new in vitro model to study OSCC metastasis via the vascular system\n\n• Co-culture of different cell types in a 3D matrix, making it more biologically relevant than existing models\n\n• Ability to produce an ‘all human’ cellular environment – more disease relevant\n\n• High quality imaging, including the ability to study metastatic events in real time at the cellular level\n\n3Rs benefits\n\n\n\n• Replacement of animal models for metastasis studies, which are moderate to severe procedures (including chemically induced and xenograft mouse tumour models)\n\nPractical benefits\n\n\n\n• Use of cell lines, allowing better reproducibility\n\n• Number of channels and overall design of the device can be amended by the user\n\nCurrent applications\n\n\n\n• Disease modelling of OSCC metastasis\n\n○ Investigating interaction of endothelial and OSCC cells in metastasis\n\n○ Modelling progression of metastasis\n\nPotential applications\n\n\n\n• Scale-up the complexity by adding more cells of the tumour microenvironment (e.g. immune cells, lymphatic cells, etc.) as well as additional ECM types, to understand the role of the tumour microenvironment in controlling tumour metastatic behaviour\n\n• Use patient samples to allow a more personalised approach to cancer treatment development\n\n• Use for testing the efficacy of novel drug compounds and/or combinations\n\n\nIntroduction\n\nHead and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide (Johnson et al., 2020), of which the major sub-type oral squamous cell carcinoma (OSCC) accounts for more than 300,000 cases every year (Choi and Myers, 2008). HPV infections remain a significant driver of OSCC (Jiang and Dong, 2017), as well as tobacco smoking and alcohol misuse. These induce genetic changes in the cells of the oral cavity, eventually leading to the formation of a carcinoma (Rivera, 2015; Scully and Robinson, 2016). OSCC is a type of cancer recognised to have a high impact on the patient’s quality of life, as aesthetic defects, as well as chewing and swallowing disabilities, can be induced by the disease and subsequent treatments administered (Rivera, 2015; Sasahira and Kirita, 2018). No radical change has been observed in the treatment landscape for OSCC over the past few decades, with surgery, radiotherapy and chemotherapy remaining the main options offered to patients (Kerawala et al., 2016). Hence, the overall five-year survival for OSCC has remained at around 50% (Scully and Robinson, 2016; Sasahira and Kirita, 2018).\n\nMore than 50% of patients with OSCC experience metastasis (Choi and Myers, 2008), with the primary site being the lymph nodes of the neck (Pisani et al., 2020). Metastasis is a complex process where cancer cells must detach from the primary tumour and reach the lymphatic system and/or the blood system to travel to the lymph node and/or distant sites, such as the lungs, bones and liver (Pisani et al., 2020). Hence, understanding how these cancer cells reach and utilise the vascular system to create these deadly secondary tumours would allow the development of better targeted treatments which prevent this state of the disease from being reached.\n\nIn vivo models have been extensively used for OSCC studies. For instance, exposing rodents to molecules including 4-Nitroquinolone oxide (4NQO) or Dimethyl-1,2,benzanthracene (DMBA) can mimic the effect of tobacco and lead to the development of tumours similar to human OSCC (Ishida et al., 2017; Sequeira et al., 2020). Genetically engineered mice have also been utilised with, for instance, Vitale-Cross et al. (2004)’s mouse model overexpressing K-ras via keratin 5 and keratin 14 promoters, leading to the development of oral lesions (Vitale-Cross et al., 2004). Xenografts have also been extensively used for metastasis studies, as these have enabled direct study of human cancer cells in immunocompromised rodents. However, the absence of the tumour’s native environment and the lack of immune cells in these models remain significant limitations of xenografts for metastasis studies (Scemama, Lunetto and Biddle, 2022). Furthermore, all these in vivo models have additional limitations, including their poor capture of physiological and pathological processes occurring in a human context, ethical drawbacks, high costs, length of studies and the challenges with imaging.\n\nUnlike in vivo models, in vitro models allow high throughput studies to be performed, at a lower cost and without ethical drawbacks. For instance, by forming an endothelial layer on a membrane, transwells have enabled assessment and quantification of cancer cell ability to cross this vascular barrier (Katt et al., 2016). However, real-time imaging of these events remains challenging with these assays. More complex in vitro models have been developed over the past few years, notably with the emergence of commercialised 3D matrices. For instance, spheroids, recognised as aggregation of cancer cells, present features of tumours and integrate cancer cell-cell interactions in a 3D environment (Katt et al., 2016; Suryaprakash et al., 2020). However, despite the advances of the in vitro field, the study of spatial organisation and interactions between different cell types in a 3D space remains limited.\n\nMicrofluidic devices are a promising tool for metastasis studies. Since the development of the lung-on-a-chip model (Huh et al., 2010), multiple types of microfluidic devices have emerged in the biomedical research field, including microfluidic membranes, microfluidic scaffolds, microfluidic hydrogels, organ-on-a-plate and horizontal compartmentalised microfluidic devices (Zhang et al., 2018). This article focuses on horizontal compartmentalised microfluidic devices, as these appear highly suited for the study of the interactions between OSCC cells and the vasculature during metastasis. Indeed, these devices are composed of adjacent channels in which different cell types and extracellular matrices (ECMs) can be added. The level of complexity of these models can be modulated by the users, therefore enabling assessment of the effect of specific cells, matrix components and secreted proteins on the cancer cells’ phenotype simultaneously or independently (Scemama, Lunetto and Biddle, 2022). Furthermore, the small size of these chips allows high quality imaging to be performed and interactions between cancer cells and microenvironmental cells can be followed in real-time.\n\nMicrofluidic devices remain a relatively new model in the biomedical research field, and require the introduction of materials, such as polydimethylsiloxane (PDMS), with inherently abnormal bioactivity compared to more traditional hydrogels such as collagen or fibrin matrices. Therefore, careful optimisation of the assay is required (Rothbauer, Zirath and Ertl, 2018; Sosa-Hernández et al., 2018). This paper describes how this model has been optimised for the co-culture of human endothelial cells with human OSCC cell lines in a three-channel microfluidic device, to better understand the role of the interactions between these two cell types during OSCC metastasis.\n\n\nMethods\n\nHuman umbilical vein endothelial cells (HUVECs, Lonza, cat. no. C2519A; RRID: CVCL_2959) were obtained from Lonza and cultured in EGM-2 medium (Promocell) at 37°C with 5% CO2. Cells were passaged when a 70–80% confluence was reached. To passage the cells, a wash with phosphate buffered saline (PBS) was first performed and cells were detached using 0.05% Trypsin-EDTA 1X (2.5 ml per T75 flask, Sigma) for 1.5 minutes. EGM-2 was then added to neutralise the trypsin. Cells were used until passage six.\n\nOSCC cells (see Table 1 below for details) were cultured at 37°C with 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM)-F12 (1:1) + GlutaMAX (31331093, Thermofisher Scientific), supplemented with 10% Fetal Bovine serum (FBS, FB-1001, Biosera), 1% Penicillin-Streptomycin (Pen-strep, Sigma-Aldrich) and 1% RM+ (composed of 10 ng/ml Epidermal Growth Factor [EGF-1, Serotec], 10-10 M Cholera Toxin [Sigma-Aldrich], 0.4 μg/ml Hydrocortisone [Sigma-Aldrich], 5 μg/ml Insulin [Sigma-Aldrich], 5 μg/ml Transferrin [Sigma-Aldrich], 2×10-11M 3,3′,5-Triiodo-L-Thyronine Sodium Salt [Sigma-Aldrich] in DMEM-F12 medium). Cells were passaged when a 70% confluence was reached, by washing them with PBS and detaching them using 0.05% Trypsin-EDTA 1X (Sigma). Once detached, growth medium was added to neutralise the trypsin. Cells were used only at low passage from frozen stocks (up to approx. passage eight), and discarded if gross changes in cell and colony morphology in tissue culture were observed.\n\nThe microfluidic chip pattern was designed on AutoCAD® and printed as a film photomask. The device was composed of three channels (700 μm width, ~75 μm height), separated from each other by an array of trapezoidal posts which prevent gel leakage into the side channels whilst allowing cells and signalling molecules to move freely across (100 μm base, 100 μm apart, Figure 1A).\n\nA. 3-channel microfluidic chip device. Medium is added in the side channels whereas the gel is added in the middle channel. Each channel width is 700 μm. The side channels are delineated from the middle channels by an array of posts, which are 100 μm large and 100 μm apart. B. A cut out PDMS block with side channel inlets (black arrows) and central inlets (white arrows), showing the size of the microfluidic device. It will be bonded to a glass cover slip on the bottom.\n\nTo fabricate the device, a silicon wafer bearing the positive pattern of the chip was generated by photolithography. First, the wafer (PI-KEM) was washed, using acetone and isopropan-2-ol, and dried with nitrogen gas. The wafer was then spin-coated with SU-8 2050 photoresist (Kayaku Advanced Materials), by using the following spinning protocol: 500 RPM for 15 seconds with an acceleration of 100 RPM/s and 1700 RPM for 36 seconds with an acceleration of 300 RPM/s. Soft bake was performed by heating the wafer at 65°C for five minutes and 95°C for 15 minutes. The photomask, bearing the pattern of the chip, was placed on the wafer and exposed to UV light (45 Mw/cm2) for one second. The wafer was then heated at 65°C for five minutes and 95°C for 10 minutes, before being immersed in the developing solution, propylene glycol methyl ether acetate (PGMEA, Sigma-Aldrich), for a maximum of five minutes. Finally, a hard bake was conducted by exposing the wafer at 150°C for three minutes.\n\nThe resulting silicon wafer bearing the pattern of the desired chip was placed in a petri dish, covered with PDMS and left to set at 80°C. Once crosslinked, the PDMS block was cut out so that new PDMS could be poured onto the silicon wafer, to make new blocks. Blocks were cleaned using ispropan-2-ol and sealed to a glass coverslip via plasma bonding, to close the microchannels. Devices were placed at 80°C for three days, to allow the hydrophobicity of the PDMS to recover, and autoclaved before use for cell culture assays.\n\nA fibrin gel was prepared using fibrinogen from bovine plasma (10–15 mg/ml in PBS, Sigma) and thrombin from bovine plasma (5 U/ml in PBS, Sigma-Aldrich). The gel was added in the middle channel via one of the two inlets and pipetted down at a slow pace to prevent leakage (Figure 2i, ii). The gel was left to set at 37°C for 30 minutes, before adding medium to the side channels.\n\ni) Microfluidic Device ii) A fibrin gel was added in the middle channel. iii-iv) HUVECs were seeded in the left side channel and left to grow in the fibrin gel of the middle channel. v-vi) OSCC cells were then seeded in the right side channel and left to grow in the middle channel.\n\nAs bubbles may assemble around the posts soon after the addition of medium to the side channels, the devices were left at 37°C overnight to allow these bubbles to dissolve. Medium was then aspirated from all the side channels inlets and 8 μl of a 5 million cells/ml HUVECs suspension was added to one of the inlets of the left side channel (Figure 2iii). To allow cells to adhere to the gel interface, the devices were flipped at 90° for 30 minutes. EGM-2 medium was supplemented with VEGF (50 ng/ml, Peprotech) and added to all the side channels. To enhance sprouting of the HUVECs, to form a developing vascular network within the gel, a flow against the cells was set up by increasing the volume of medium added in the opposite side channel (110 μl of medium in the inlets of the right side channels and 90 μl of the medium in the inlets of the HUVECs side channel). Medium was replaced every 24 hours.\n\nWhen a vasculature was formed (after approx. four days) (Figure 2iv), OSCC cells were added in the opposite side channel (Figure 2v). Medium was aspirated from all the side channel inlets and 8 μl of a 5 million cells/ml OSCC solution was added to one of the inlets of the right side channel. To allow cells to adhere to the gel interface, the devices were flipped at 90° for 30 minutes. EGM-2 medium supplemented with VEGF (50 ng/ml, Peprotech) was added to both medium channels. Note that DMEM-F12 was not used in the co-culture, to allow the maintenance of endothelial cells in the device.\n\nWhen the desired time-point of co-culture was reached (Figure 2vi), cells were washed twice with PBS and fixed by adding 100 μl of 4% paraformaldehyde (PFA) in all the inlets for 15 minutes. Cells were washed twice with PBS and left in PBS at 4°C until stained and imaged.\n\nGFP tagged CA1 OSCC cells were made by Dr Muhammad Rahman (Prof M. Philpott group, Cell Biology and Cutaneous Research, Blizard Institute, QMUL), using the following protocol.\n\nGFP was inserted into a pBabe. Puro vector. The DNA vector was mixed with FuGENE®6 (E2691, Promega) at a 1:2.5 construct to FuGENE®6 ratio, and left at room temperature for 15 minutes with antibiotic free DMEM-F12 medium. The mixture was then applied to Phoenix cells in a dish and incubated overnight at 37°C with 5% CO2. The following day, medium was exchanged with fresh medium supplemented with puromycin (1 μg/ml, ant-pr-1, Invivogen). Once the cells became confluent, medium was replaced with fresh DMEM-F12 medium. Medium was then harvested the following day and filtered (0.45 μm) to remove cells producing the virus.\n\nWhen CA1 OSCC cells reached a 70% confluence in a dish, cells were washed with PBS and then serum and antibiotic free DMEM-F12 medium containing 5 μg/ml prolybrene (TR-1003-G, Sigma-Aldrich) was added into the dish and left for 10 minutes. The retrovirus solution was then added and the plates were centrifuged for 1 hour at 3000 G. Cells were then washed with PBS and left in DMEM-F12 medium at 37°C with 5% CO2. These steps were repeated the following day.\n\nThe next day, drug selection of the cells was performed using a 1 μg/ml puromycin solution to only select the cells expressing GFP. To further ensure that only GFP-tagged cells had been selected, the cells were FACS sorted. To do this, cells were expanded and once confluent, detached from the flask using Tryspin-EDTA. The cells were passed through a 70 μm cell strainer to prevent cell aggregates from blocking the FACS machine. Cells were centrifuged at 380 G for 5 minutes and re-suspended in PBS with DAPI (1/2000 in sterile PBS, 10236276001, Roche). Cells were then transferred to a sterile FACS tube and GFP +ve cells were collected into another sterile FACS tube.\n\nStaining reagents were added into both medium channels. Cells were permeabilised with 0.1% Triton X-100 (Sigma-Aldrich) for 10 minutes and blocked for three hours (3% BSA in PBS), at room temperature. If stained with phalloidin (1/500, Scientific Laboratory Supplies) and DAPI (1/1000 in PBS from 1 mg/ml stocks, 10236276001, Roche) or just DAPI, these stains were mixed with the blocking buffer directly and added in each microfluidic chip inlet (100 μl per inlet) for one hour, at room temperature. Cells were washed twice with PBS and left in PBS at 4°C until imaged. When conjugated antibodies were used (see Table 2, below), the antibody mix was prepared using blocking buffer. After the blocking step, 60 μl of the antibody mix was added in each inlet of the microfluidic chips and left overnight at 4°C. Cells were then stained with DAPI for one hour, washed twice with PBS and left in PBS at 4°C until imaged.\n\nImaging was performed using the IN Cell Analyzer 6000 (GE healthcare). Five to six fields of views, using the 20× magnification, were taken to cover the entire middle channel.\n\nPhotolithography\n\nPlease note: SU8-2050 is light sensitive, steps 1 to 6 need to be conducted in the dark and under a fume hood\n\n1. Clean the silicon wafer (WAFER-SILI-0006W25, PI-KEM) with acetone (100%, 8003, Avantor) and isopropan-2-ol (>99.98%, 59300M, Sigma-Aldrich) and dry it with N2 gas.\n\n2. Add SU8-2050 (Y111072 0500L1GL, Kayaku Advanced Materials) in the centre of the silicon wafer and slightly tilt at different angles to spread the SU8-2050 on ~70% of the silicon wafer.\n\n3. Place the silicon wafer in the spin coater machine (SPIN150i/200i infinite spin coater, Polos) and start the following programme:\n\n- Step 1: 500 revolutions per minute (RPM), 15s, acceleration: 100 RPM/s\n\n- Step 2: 1750 RPM, 36s, acceleration: 300 RPM/s\n\n4. Place the silicon wafer on a heating plate (Isotemp®, fisherbrand) at 65°C for five minutes and 95°C for 15 minutes. Ensure the silicon wafer is kept in the dark as SU8-2050 is light-sensitive.\n\n5. Place the photomask (bearing the desired chip design) on the silicon wafer and put it in the UV machine (UV-KUB 6) for one second (exposure: 100%; ~45 45 mW/cm2).\n\n6. For the post-exposure bake, place the silicon wafer on a heating plate at 65°C for five minutes and 95° for 10 minutes.\n\n7. Under a fume hood, immerse the silicon wafer in the developing solution, PGMEA (484431, Sigma-Aldrich). If the silicon wafer is immersed in it for too long, it will become over-developed and the chip design will disappear. It is preferred to immerse the silicon wafer for 1.5 minutes and then use a squeeze bottle to better target the areas that need to be developed. During this process, regularly wash the silicon wafer with isopropanol.\n\n8. For the hard bake, place the silicon wafer bearing the chip design on a heating plate at 150°C for three minutes.\n\nPDMS pouring and plasma bonding\n\n1. Place the wafer with the desired chip design in a petri dish.\n\n2. Mix polydimethylsiloxane (PDMS; SYLGARDTM 184 Silicone Elastomer Base, Dow Chemical) with its curing agent (SYLGARDTM 184 Silicone Elastomer Curing Agent, 1:10 curing agent to PDMS) and pour it in the petri dish (~1 cm thick). Ensure this is combined well. Once satisfactorily combined, place in a desiccator for 30 minutes for degassing, at room temperature.\n\n3. Pour this in the petri dish (~1 cm thick).\n\n4. Place the petri dish in an oven at 80°C for at least 1.5 hours to allow the PDMS to crosslink.\n\n5. Cut out the solidified blocks of PDMS bearing the chip pattern, using a scalpel, use punch biopsy equipment to create inlets, e.g. 5 mm punch biopsy for side channel inlets and 2 mm punch biopsy for central channel inlets (see Figure 1B).\n\n6. Clean the PDMS blocks by first removing the dust using tape and then immersing them in dH2O and isopropan-2-ol sequentially and drying them with N2 gas in a fume hood.\n\n7. Place the PDMS blocks and 24 × 24 mm (thickness 0.13–0.16 mm) borosilicate glass cover slips in the plasma machine (HPT-200, Henniker Plasma machine) for one minute and seal the channels by placing the PDMS and glass in full contact whilst maintaining a light pressure for 1s.\n\n8. Incubate the devices at 80°C for 72 hours to allow the hydrophobicity to be recovered.\n\n9. Autoclave the devices before using them for cell-based experiments.\n\nAddition of the gel in the microfluidic chips\n\n1. Dilute the fibrinogen type I-S from bovine plasma (F86-30, Sigma) in sterile phosphate buffered saline (PBS) to reach the desired concentration (10–20 mg/ml depending on the fibrinogen batch, each tested to determine the concentration that maintains a consistent degree of angiogenic sprouting) and leave the Eppendorf in the water bath at 37°C for one hour.\n\n2. Filter the fibrinogen solution with a 0.22 μm filter to sterilise it.\n\n3. Prepare the thrombin (T6634, Sigma) by diluting it in sterile PBS to reach the desired concentration (5 U/ml)\n\n4. In a 0.5 ml Eppendorf, add 10 μl of the fibrinogen solution and then add 10 μl of the thrombin solution. Mix it by doing two up-and-downs with the pipette and slowly pipette 10 μl into one of the inlets of the middle channel. Note that less than 10 μl is needed to fill the middle channel, but using higher volumes enables the user to pipette more slowly into the inlet and therefore prevents leakage of the gel to the side channels.\n\n5. Leave the gel to set for 30 minutes at 37°C.\n\n6. Add 100 μl of medium to the top inlets of the side channels.\n\n7. To ensure medium goes through the side channel, cut a 200 μl pipette tip and use it to aspirate the medium via the bottom inlet.\n\n8. Add 100 μl of medium to the bottom inlets of the side channels. At this stage, bubbles may form alongside the gel interface. Leave the microfluidic chips overnight at 37°C to allow the bubbles to disappear before adding the cells into the devices.\n\nAddition of the cells in the microfluidic chips\n\n1. Transfer your cell suspension into a 50 ml falcon and centrifuge it at 1200 RPM for five minutes.\n\n2. Re-suspend in 5 ml of growth medium and count the cells (e.g. using a haemocytometer).\n\n3. Centrifuge the cell solution at 1200 RPM for five minutes and re-suspend in growth medium to reach a final concentration of 5 million cells/ml (note that this may vary depending on the cell type used and desired length of the assay).\n\n4. Remove and discard the growth medium from the inlets of the side channels of the microfluidic chips using an aspirator. All the medium must be aspirated but the channels must not become dry (if dry, the cells will not move towards the gel interface and instead stay in the inlet).\n\n5. Add 8 μl of the cell solution in one of the inlets of a side channel and rotate the device such that the long axis of the channels is parallel with the workbench and the cell solution inlet is superior, in the vertical plane, to the inlets of the opposite side channel. Ensure the device remains in this vertical position for 30 minutes at room temperature to allow the cells to adhere to the gel interface.\n\n6. If the cells have adhered homogeneously to the gel interface, add growth medium in all the inlets of the side channels. In some instances, an interstitial flow can be added, for instance to enhance sprouting of endothelial cells. In that case, a higher volume of growth medium must be added in one of the side channels (for HUVECs: 110 μl in the inlets of the opposing side channel and 90 μl in the inlets of the cells’ side channel).\n\n7. Place the devices at 37°C and 5% CO2.\n\n8. Replace the growth medium every 24 hours.\n\n9. Repeat steps 1–8 to place an additional cell population in the opposite side channel (e.g. cancer cells).\n\nProtocol 1\n\nThe following protocol was used in image analysis to segment two cell types on field of view. One cell type was GFP-tagged (GFP cells). All the cells were stained with phalloidin (red fluorophore). Hence, two groups of cells were segmented: the red-only cells and the red and green cells. First the GFP cells were segmented based on their GFP fluorophore intensity. Then, all the cells were segmented (Cells) based on the phalloidin stain. GFP-negative cells were segmented by subtracting the GFP cells target from the Cells targets.\n\nTarget sets\n\n• Seed\n\n• Object\n\n• Post processing\n\n▪ Erosion (Binary)\n\n▪ Sieve (Binary) - greater than\n\n• Nuclei\n\n• Nuclear segmentation\n\n• Post processing\n\n▪ Clump breaking – second segmentation: seed\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• Measure\n\n▪ Sum of Nuclei\n\n• GFP cells\n\n• Intensity segmentation\n\n• Post processing\n\n▪ Erosion (Binary)\n\n▪ Clump breaking – second segmentation: nuclei\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• Debris\n\n• Intensity segmentation\n\n• Post processing\n\n▪ Sieve (Binary) - less than\n\n• Cells\n\n• Intensity segmentation\n\n• Post processing\n\n▪ Erosion\n\n▪ Clump breaking – second segmentation: nuclei\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• GFP negative cells\n\n• Pre-processing macro – P.Sub_11_10_9\n\n• Intensity segmentation: Minimum (1); Maximum (65535)\n\n• Post processing\n\n▪ Sieve (Binary) - greater than\n\n• Whole GFP negative cells\n\n• Measure\n\n▪ Count Whole GFP negative cells\n\n▪ Sum count Whole GFP negative cells\n\n▪ Area covered by Whole GFP negative cells\n\n▪ Sum area covered by Whole GFP negative cells\n\n• Whole GFP positive cells\n\n• Measure\n\n▪ Count Whole GFP positive cells\n\n▪ Sum count Whole GFP positive cells\n\n▪ Area covered by Whole GFP positive cells\n\n▪ Sum area covered by Whole GFP positive cells\n\nTarget linking\n\n• Whole GFP negative cells\n\n▪ Primary target set: Nuclei\n\n▪ Secondary target set: GFP negative cells\n\n▪ Criteria: overlap\n\n▪ Output: Whole GFP negative cells\n\n▪ Overlap: 80% of primary target within secondary target\n\n▪ Find any matched target\n\n• Whole GFP positive cells\n\n▪ Primary target set: Nuclei\n\n▪ Secondary target set: GFP positive cells\n\n▪ Criteria: overlap\n\n▪ Output: Whole GFP positive cells\n\n▪ Overlap: 80% of primary target within secondary target\n\n▪ Find any matched target\n\n Note: this step allows to subtract all the GFP cells target from the Cells target to segment the red-only cells. The end image result of Cells, GFP cells and GFP negative cells should be set on the 11th, 10th and 9th windows of the programme respectively. GFP negative cells is a subtraction of GFP cells (window 10) from Cells (window 11).\n\nProtocol 2\n\nThe following protocol was used to segment two cell types on a field of view, with two distinct fluorophores. Cell type 1 appears red and cell type 2 green.\n\nTarget sets\n\n• Seed\n\n• Object\n\n• Post processing\n\n▪ Erosion (Binary)\n\n▪ Sieve (Binary) - greater than\n\n• Nuclei\n\n• Nuclear segmentation\n\n• Post processing\n\n▪ Clump breaking – second segmentation: seed\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• Measure\n\n▪ Sum of Nuclei\n\n• RFP cells\n\n• Intensity segmentation\n\n• Post processing\n\n▪ Erosion (Binary)\n\n▪ Clump breaking – second segmentation: nuclei\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• GFP cells\n\n• Intensity segmentation\n\n• Post processing\n\n▪ Erosion (Binary)\n\n▪ Clump breaking – second segmentation: nuclei\n\n▪ Fill holes\n\n▪ Sieve (Binary) - less than\n\n▪ Sieve (Binary) - greater than\n\n• Whole RFP cells\n\n• Measure\n\n▪ Count Whole RFP cells\n\n▪ Sum count Whole RFP cells\n\n▪ Area covered by Whole RFP cells\n\n▪ Sum area covered by Whole RFP cells\n\n• Whole GFP cells\n\n• Measure\n\n▪ Count Whole GFP cells\n\n▪ Sum count Whole GFP cells\n\n▪ Area covered by Whole GFP cells\n\n▪ Sum area covered by Whole GFP cells\n\nTarget linking\n\n• Whole RFP cells\n\n▪ Primary target set: Nuclei\n\n▪ Secondary target set: RFP cells\n\n▪ Criteria: overlap\n\n▪ Output: Whole RFP cells\n\n▪ Overlap: 80% of primary target within secondary target\n\n▪ Find any matched target\n\n• Whole GFP cells\n\n▪ Primary target set: Nuclei\n\n▪ Secondary target set: GFP cells\n\n▪ Criteria: overlap\n\n▪ Output: Whole GFP positive cells\n\n▪ Overlap: 80% of primary target within secondary target\n\n▪ Find any matched target\n\n\nResults\n\nA simple representation of the angiogenesis process was reproduced in the microfluidic chips, by adding HUVECs in the left side channel and leaving them to grow into the fibrin gel in the middle channel. Results showed that endothelial cells formed sprouts in the matrix (Figure 3A). Furthermore, these tubular structures appeared to have a lumen, as when sprouts had grown across the whole middle channel, cells added in the opposite side channels directly migrated into these structures (Figure 3B). Hence, by simply adding HUVECs in our device containing fibrin, we have been able to reproduce a growing vascular network, with features resembling those observed in vivo. This is in good agreement with comparable reports of microvascularised chips, presenting lumenated perfusable structures at this time point (Kim et al., 2013; Dibble et al., 2022).\n\nA. HUVECs in the middle channel of the microfluidic chip at day 10. Red, phalloidin; Blue, DAPI. Posts are highlighted in white (dashed line). Scale bar: 100 μm. B. Migration of cells into the sprouts when added in the opposite side channel. Arrows indicate cells that migrated into the sprouts. Scale bar: 100 μm.\n\nTo study the interactions between the OSCC cells and the vasculature, co-culture of HUVECs and OSCC cells was optimised in the microfluidic chip. To allow the discrimination of these two cell types and accurate analysis of the images, GFP-tagged CA1 OSCC cells were used for this experiment (Figure 4). These OSCC cells were added in the opposite side channel when the HUVECs had grown half way across the middle channel, as assessed daily by light microscopy (typically by day 4). Results showed that this co-culture was successful as OSCC cells invaded towards the endothelial cells in the fibrin gel (Figure 4). This successful co-culture will enable the development of experiments to follow and assess the interactions between OSCC cells and the vascular network to better understand the metastasis of OSCC.\n\nHUVECs and OSCC cells in the microfluidic chip 4 days after adding the OSCC cells. Red, phalloidin; Green (GFP tag), OSCC cells; Blue, DAPI. Posts are highlighted in white (dashed line). Scale bar: 100 μm.\n\nTo better understand the interactions between the cancer cells and the vasculature, the expression of specific markers must be assessed. To assess the expression of markers via imaging, such as the lineage markers vimentin and keratin (marking mesenchymal and epithelial lineages respectively), antibodies are required. Non-conjugated antibodies, with primary and secondary antibodies added separately, have been widely used and optimised for imaging in biomedical research. This method allows flexibility over the fluorophore combinations used when multiple marker expressions are being assessed simultaneously. However, when tested in our microfluidic device, high background noise was detected with non-conjugated antibodies, whereas this was not the case with conjugated antibodies (Figure 5). This background noise may reflect some retention of unbound secondary antibodies in the 3D matrix. Hence, this data shows that it may be preferable to use conjugated antibodies in microfluidic devices, to prevent high background noise and thus better analysis of the images obtained.\n\nStaining of OSCC cells with conjugated and non-conjugated antibodies. Red, Keratin; Green, Vimentin; Blue, DAPI. Posts are highlighted in white (dashed line). Scale bars: 100 μm.\n\nImage analysis of the microfluidic chip experiments can be performed using several methods. For instance, for an automated approach, Developer Toolbox (GE Healthcare, version 1.10) can be employed. This programme can segment cells based on their fluorophore intensity and includes a multitude of tools enabling accurate counting of cells, assessment of the area they cover, their X position in the field of view, their shape, etc. (Figure 6A). This automated tool can successfully segment different cell types within a field of view, as long as the cells can be differentiated with a fluorophore. For instance, in Figure 4, cancer cells are GFP-tagged and all the cells are stained with phalloidin (red). Hence, the red-only cells are endothelial cells and the red and green cells are cancer cells. Other tools can also be used for image analysis. For instance, FIJI version 2.9.0 enables drawing of regions of interest manually on each field of view, such as lines surrounding the front of the cell mass, allowing assessment of the complexity of the shape of these masses invading into the matrix (Figure 6B).\n\nA. Segmentation of each individual cell using Developer Toolbox. B. Drawing of a region of interest in FIJI (line around the cell mass).\n\n\nDiscussion\n\nThe number of publications in PubMed featuring the word organ-on-a-chip has increased by 80-fold since the start of the century, demonstrating the high interest in this new technology for biomedical research. Indeed, microfluidic devices bring the complexity of in vitro models to a new level, by integrating multiple human cell types and enabling study of their interactions in a 3D environment. Furthermore, compared to animal models, microfluidic devices are characterised by their high reproducibility and controllability over the experiments (Ma et al., 2021), while allowing cellular events to be followed in real-time.\n\nHere, a simple model of human OSCC metastasis was developed using horizontal compartmentalised microfluidic devices, with only three elements added to the device: HUVECs, OSCC cells and a fibrin gel. HUVECs were directly purchased from a manufacturer, therefore enhancing the reproducibility of this model; whereas OSCC cells were from patient-derived lines, previously characterised (Biddle et al., 2011, 2016). However, reproducibility of this system was reduced when different fibrinogen batches were used, as the animal origin of this product leads to high batch-to batch variability (Ahadian et al., 2018). Hence, for each new fibrinogen batch used, a range of fibrinogen concentrations should be tested to ensure both HUVECs and OSCC cells exhibit appropriate growth in the device. However, fibrinogen batches do not need to be replaced regularly due to the scale of these devices which allows only small volumes to be used for each experiment. The development of synthetic matrices may also counter this variability, and enables the layering of human matrix components onto the synthetic matrix to generate a fully humanised tumour environment (Ashworth et al., 2020). This would further contribute to the 3Rs through reduced experimental variability and reduced reliance on animal derived components.\n\n\nConclusions\n\nTo conclude, a new in vitro model to study the role of the vascular system in the metastasis of OSCC was developed and optimised, aiming to diminish the use of animal models in this field and eventually replace them. This type of device has been used for other cancer types, including breast cancer, glioblastoma, colorectal cancer, etc. (Lee et al., 2014; Jeon et al., 2015; Sobrino et al., 2016), demonstrating its suitability for metastasis studies regardless of the cancer cell origin. The model developed here has the potential for up-scaling and adding further complexity, such as additional channels in which additional cell types and/or ECM components could be integrated, to further increase its physiological relevance (Lee et al., 2014, 2018).", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAhadian S, et al.: Organ-On-A-Chip Platforms: A Convergence of Advanced Materials, Cells, and Microscale Technologies. Adv. Healthc. Mater. 2018; 7. PubMed Abstract \| Publisher Full Text\n\nAshworth JC, et al.: Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro. Matrix Biol. 2020; 85-86: 15–33. Publisher Full Text\n\nBiddle A, et al.: Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative. Cancer Res. 2011; 71: 5317–5326. 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Publisher Full Text" }	[ { "id": "171696", "date": "15 Jun 2023", "name": "Vivek Thacker", "expertise": [ "Reviewer Expertise Organ-on-chip", "infectious diseases", "live-cell imaging" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Scemama and colleagues describe the design, fabrication, and testing of an organ-on-chip device to visualize the metastasis of patient-derived oral squamous carcinoma cells into vascular networks. The device consists of vascular cells seeded in one channel that form an extended network into the adjacent (central) fibrin-gel containing channel and OSCC cells seeded in the other side channel that interact with the vascular cells in the central channel. The use of such a side-by-side geometry overcomes many of the limitations of layered microfluidic devices. OSCC cells are identified by GFP transduction. Overall, the rationale for the need for such devices is clearly described, and the fabrication processes of the organ-chip device has been described with sufficient detail to enable to reader to replicate these experiments. However, there are shortcomings in relation to the characterization of the devices from a cellular perspective. The availability of patient-derived OSCC cells is a huge asset, but apart from the CA1 line, none of the others described in Table 1 are used in this study. There are several similar designs reported for the culture of vascular networks in co-culture with several other cell types including tumour cells, and the authors should place their device in the context of this work. In addition, the authors do not show evidence for a close co-culture in the central channel between the endothelial and OSCC cells, which is needed if this device is to be used to study metastasis as claimed.\nMajor comments:\nCharacterization of GFP-labelled OSCC cells – the data in Figure 4 suggests that several of the GFP labelled cells have low or no GFP expression whereas others have high GFP expression. Did the authors choose one particular clone for expansion or are the cells a mixed population? Protocols for characterization of these cells are important and should be included so as to enable others to repeat these experiments. Furthermore, the lack of GFP expression in a sub-population of OSCC cells is problematic given that this marker is used to classify the OSCC cells in the chip. Are these cells losing GFP expression over the period of growth in the organ-on-chip? The authors could show data with other epithelial markers like EpCAM or E-cadherin to characterize these cells further.\nCo-culture on-chip – the manuscript claims in Figures 2 (v) and (vi) that a co-culture of a vascular network and the OSCC cells is possible, but this is not supported by the images in Figures 4, 5, and 6. The timepoint at which the chips are imaged in Figures 4, 5, and 6 are not clear – it is likely that these are early timepoints and there has been insufficient time to generate the co-culture. Evidence from later stages needs to be provided to back up this claim.\nVascular network on-chip – the authors claim on page 14 and in relation to Figure 3 that their protocol generates a perfusable vascular network, but there is no functional evidence provided. Please nuance this claim or provide evidence to support it. Furthermore, the authors do not adequately cite other publications in this field, notably from the lab of Prof Roger Kamm who has created several models that have extensive perfusable vascularization, including with the addition of tumor cells. The authors should place their model in the context of this literature.\nTable 1 and OSCC cells – the need for table 1 is unclear since none of the other patient-derived cell lines are used in this study and the protocols that the authors propose requires the use of the GFP-labelling to identify OSCC cells. Inclusion of the patient-derived cells is a strength, and the authors could significantly strengthen the manuscript by comparing the behaviours of these four cell lines in the same device for parameters such as invasion or growth or spread via metastasis.\nImage analysis pipeline – it is unclear how the images generated in Figure 6 are obtained, and how this is useful for the purpose of analysis of metastasis. It is unclear how the cell boundaries in 6A are determined given the low and homogenous GFP expression. This image doesn’t quite correlate with the image in 6B, is this a different field of view? In 6B there are very few GFP+ cells, so is this an area from the vascular side of the device? In any case, it does not appear that a vascular network that interacts with many OSCC cells (as depicted in Figure 2 (vi)) has been generated.\nMinor comments: Page 4, limitations of in vivo models - several limitations are described at the top of page 4, it would be useful to have references to these.\nPage 4, limitations of transwells – these models are described as unsuitable for real-time imaging. The authors should clarify exactly why (transparency of membrane, working distance etc.) to help the reader assess these limitations.\n\nPage 5, UV light – typo, should be mW and not Mw.\nFigure 1 - the schematics are nicely presented, showing an XZ or YZ cross-section would also help the reader appreciate the low working distance from the coverslip afforded by the authors’ design.\nPage 7 – the details of the vector used for GFP expression are incompletely described. Please describe these and provide references for the vector. Typo – ‘polybrene’ and not ‘prolybrene’.\nFigures 3-6 – please avoid the use of primary colours to enable colour-blind readers to appreciate the images.\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "10876", "date": "15 Jan 2024", "name": "Adrian Biddle", "role": "Author Response", "response": "In this manuscript, Scemama and colleagues describe the design, fabrication, and testing of an organ-on-chip device to visualize the metastasis of patient-derived oral squamous carcinoma cells into vascular networks. The device consists of vascular cells seeded in one channel that form an extended network into the adjacent (central) fibrin-gel containing channel and OSCC cells seeded in the other side channel that interact with the vascular cells in the central channel. The use of such a side-by-side geometry overcomes many of the limitations of layered microfluidic devices. OSCC cells are identified by GFP transduction. Overall, the rationale for the need for such devices is clearly described, and the fabrication processes of the organ-chip device has been described with sufficient detail to enable to reader to replicate these experiments. However, there are shortcomings in relation to the characterization of the devices from a cellular perspective. The availability of patient-derived OSCC cells is a huge asset, but apart from the CA1 line, none of the others described in Table 1 are used in this study. There are several similar designs reported for the culture of vascular networks in co-culture with several other cell types including tumour cells, and the authors should place their device in the context of this work. In addition, the authors do not show evidence for a close co-culture in the central channel between the endothelial and OSCC cells, which is needed if this device is to be used to study metastasis as claimed. We thank the reviewer for their enthusiasm for the method presented in our report, and for their thorough review. We will answer the specific comments where they appear below. One thing to note is that this is a methods paper to accompany a research article that is now available as a pre-print (Scemama et al., 2024). Whilst conducting patient validation of the data from the microfluidic device for the main research article, we wanted to publish the method without delay as a resource for the research community. We have now updated this methods paper to link it to the pre-printed research article. Where comments are addressed in the main research article, we will note that below. Major comments: Characterization of GFP-labelled OSCC cells – the data in Figure 4 suggests that several of the GFP labelled cells have low or no GFP expression whereas others have high GFP expression. Did the authors choose one particular clone for expansion or are the cells a mixed population? Protocols for characterization of these cells are important and should be included so as to enable others to repeat these experiments. Furthermore, the lack of GFP expression in a sub-population of OSCC cells is problematic given that this marker is used to classify the OSCC cells in the chip. Are these cells losing GFP expression over the period of growth in the organ-on-chip? The authors could show data with other epithelial markers like EpCAM or E-cadherin to characterize these cells further. The apparent lack of GFP expression in some OSCC cells in Fig. 4 is due to the fact that the level of GFP expression is over enough of a range that the levels in the lower expressing cells cannot be visualised without making the higher expressing cells too bright. Quantitative analysis of GFP expression compared to a negative control (e.g. HUVEC) demonstrates that it is positive in all of the OSCC cells. It is also positive in all OSCC cells when tested by flow cytometry. The protocol for production of GFP+ CA1 cells, and their characterisation by flow cytometry, is detailed in the cited publication (Gemenetzidis et al., 2015). We do not use clonal expansion, as the cancer cell line is a heterogeneous population and therefore single cell expansion results in a non-representative population. EpCAM and E-cadherin expression are investigated in the associated research article (Scemama et al., 2024), and also exhibit a range of expression levels in the OSCC cells. Co-culture on-chip – the manuscript claims in Figures 2 (v) and (vi) that a co-culture of a vascular network and the OSCC cells is possible, but this is not supported by the images in Figures 4, 5, and 6. The timepoint at which the chips are imaged in Figures 4, 5, and 6 are not clear – it is likely that these are early timepoints and there has been insufficient time to generate the co-culture. Evidence from later stages needs to be provided to back up this claim. The timepoints are noted in the figure legends. The evolution of the co-culture after addition of OSCC cells is the specific focus of the associated research article, and is described in detail there (Scemama et al., 2024). The purpose of this methods article is to describe methods for production of the co-culture devices and for quantitative analysis of the resulting cultures. Briefly, the separation of OSCC cells and HUVECs is due to their response to one another. Allowing more complete vascularisation before adding OSCC cells results in OSCC cells flowing through the vasculature (Fig. 3), which may or may not be desirable depending on study aims. Vascular network on-chip – the authors claim on page 14 and in relation to Figure 3 that their protocol generates a perfusable vascular network, but there is no functional evidence provided. Please nuance this claim or provide evidence to support it. Furthermore, the authors do not adequately cite other publications in this field, notably from the lab of Prof Roger Kamm who has created several models that have extensive perfusable vascularization, including with the addition of tumor cells. The authors should place their model in the context of this literature. The functional evidence for the production of a lumenised vasculature is the image in Fig. 3B showing cancer cells flowing directly into the vascular lumen. This happens when we allow the vasculature to progress across the whole width of the middle channel and connect to the far side channel, before adding the cancer cells. Usually, we add the cancer cells before the vasculature reaches this level of development, as shown in the other images presented. We agree that our work builds on important previous work on the development of these microfluidic devices for investigation of cancer interaction with the vasculature. Prof Roger Kamm pioneered these devices, and we have cited his work (Jeon et al., 2015) alongside other key previous studies. This is in the conclusion section in page 17, and in the first results section relating to previous studies demonstrating production of perfusable vascular structures. The particular focus of our own work is on using these devices to study the evolution of tumour cellular heterogeneity and plasticity within a complex tumour microenvironment, and the analysis methods we have developed and presented here are designed to enable the application of microfluidic devices to this area of study. Table 1 and OSCC cells – the need for table 1 is unclear since none of the other patient-derived cell lines are used in this study and the protocols that the authors propose requires the use of the GFP-labelling to identify OSCC cells. Inclusion of the patient-derived cells is a strength, and the authors could significantly strengthen the manuscript by comparing the behaviours of these four cell lines in the same device for parameters such as invasion or growth or spread via metastasis. The inclusion of cell lines not used in this methods paper was a mistake, and we have now removed them. These are the cell lines used in the associated research article (Scemama et al., 2024), and we agree that the inclusion of multiple patient-derived cell lines is an important consideration for research studies using these devices. Image analysis pipeline – it is unclear how the images generated in Figure 6 are obtained, and how this is useful for the purpose of analysis of metastasis. It is unclear how the cell boundaries in 6A are determined given the low and homogenous GFP expression. This image doesn’t quite correlate with the image in 6B, is this a different field of view? In 6B there are very few GFP+ cells, so is this an area from the vascular side of the device? In any case, it does not appear that a vascular network that interacts with many OSCC cells (as depicted in Figure 2 (vi)) has been generated. These images are of control devices with no vasculature. The purpose of this figure was to show the analysis methods used, we therefore used images of OSCC monoculture to make it easier to follow. In 6a, the cells are CA1-GFP. This is to show that with fluorescent cells, Developer can easily be used to segment the cells using the nucleus (DAPI staining). In 6b, the cells are stained with Vimentin/Keratin with Vimentin in green and Keratin in red. The cells used here were normal CA1 (non-GFP CA1) in order to be able to do the double antibody stain. The legend has been updated to make the above details clear. These two images are indeed different but this is because the goal was to show two different methods 1) With fluorescent CA1, it is a lot easier to do an automatic segmentation (as shown in 6a). 2) With non-fluorescent CA1, stained for markers, a manual analysis is preferred (6b). Here we are showing how to calculate the length of the cell front by drawing a line manually. Minor comments: Page 4, limitations of in vivo models - several limitations are described at the top of page 4, it would be useful to have references to these. The reference has been moved to make clear it covers all of these limitations. It is a review that covers these in more detail. Page 4, limitations of transwells – these models are described as unsuitable for real-time imaging. The authors should clarify exactly why (transparency of membrane, working distance etc.) to help the reader assess these limitations. This has been updated with further explanation. Page 5, UV light – typo, should be mW and not Mw. Updated. Figure 1 - the schematics are nicely presented, showing an XZ or YZ cross-section would also help the reader appreciate the low working distance from the coverslip afforded by the authors’ design. This is a good point; we have edited the legend to add this point. Page 7 – the details of the vector used for GFP expression are incompletely described. Please describe these and provide references for the vector. Typo – ‘polybrene’ and not ‘prolybrene’. We have re-considered the inclusion of this methods section, as the GFP line was produced as part of a previous study and not by the authors of the current study. We have instead included a reference to the study where the CA1-GFP were produced (Gemenetzidis et al., 2015). Figures 3-6 – please avoid the use of primary colours to enable colour-blind readers to appreciate the images. Thank you. We will incorporate this point into our future image analysis pipeline. References GEMENETZIDIS, E., GAMMON, L., BIDDLE, A., EMICH, H. & MACKENZIE, I. C. 2015. Invasive oral cancer stem cells display resistance to ionising radiation. Oncotarget, 6, 43964-77. SCEMAMA, A., LUNETTO, S., TAILOR, A., CIO, S. D., AMBLER, L., COETZEE, A., COTTOM, H., KHURRAM, S. A., GAUTROT, J. & BIDDLE, A. 2024. Hybrid cancer stem cells utilise vascular tracks for collective streaming invasion in a metastasis-on-a-chip device. bioRxiv, 2024.01.02.573897." } ] }, { "id": "188653", "date": "03 Aug 2023", "name": "Christopher Jon Hanley", "expertise": [ "Reviewer Expertise Tumour microenvironment research using 3D cultures", "digital pathology and bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article by Alice Scemama et al. outlines an in vitro approach to modelling metastatic spread in human OSCC, using microfluidic devices that enable high-quality real time imaging. The method describes the fabrication of PDMS moulds with three separate microfluidic channels that can be used for the addition of different cells, matrices or media to the culture. Protocols are also described for imaging-based analysis of these cultures using endogenous fluorophores and/or antibody staining followed by digital analysis involving cell segmentation and quantification of fluorescence intensity.\nThe method provides useful insight for device fabrication and use for culturing OSCC cells with endothelial cells. As stated by the authors, this device and culture system replicates others that have been used for studying different cancer types previously, so these insights are not entirely novel. It also remains questionable to what degree the stated research highlights are fully achieved by this article.\nConceptual reservations to approval of this article:\nThe authors propose that this microfluidic system could be used for investigating the process of OSCC metastasis via the vasculature, suggesting that this could be used instead of animal models. To adequately demonstrate this, further evidence of benchmarking compared to alternative in vitro systems and in vivo models is required. For example, additional experiments or data that would be useful include:\nDemonstrating that cell lines with varying metastatic potential in vivo exhibit different phenotypes in this model.\n\nDemonstrating quantitative metrics can be derived from this model system and the imaging-based analyses proposed to assess these phenotypic differences.\n\nComparing the results generated using this system to a Transwell system or other existing systems (e.g. Ibidi chemotaxis slides).\n\nThe authors highlight real time imaging as a major advantage of this system over existing methods for studying OSCC metastasis. However, the data presented exposes some limitations to these approaches and their utility for progressing the investigation of molecular mechanisms underlying these processes. For example,\nThe use of endogenous fluorophores and phalloidin for classifying different cell types is confounded by non-ubiquitous reporter expression leading to potential for mis-classification of cells, which appears to be the case in the micrograph shown in Figure 4.\n\nThe interpretation of intercellular interactions across cellular compartments is reliant on strict lineage specific restriction of marker expression. The example provided in figure 5 demonstrates the use of keratin and vimentin “to better understand the interactions between the cancer cells and the vasculature”. However, the use of vimentin here is likely to be suboptimal, given this gene is expressed in cancer cells undergoing EMT. The authors should elaborate further on how experiments should be designed to enable accurate examination of tumour vasculature interactions and ensure that the interpretations are validated with appropriate controls.\n\nIn addition (and as described above) it is not clear how these imaging-based analyses should be used to quantify steps in the metastatic process. Further evaluation and demonstration of how these approaches can be used to measure phenotypic changes under different experimental conditions should be provided to confirm this system be used in a similar manner to existing assays (e.g. Transwell systems) generating reproducible quantitative data.\n\nTechnical recommendations/comments:\nIn the Angiogenesis assay section:\n“cells added in the opposite side channels directly migrated into these structures” – clarity should be provided regarding what cells were added to opposite side in this example (Figure 3).\nIn the Co-culture of endothelial cells and OSCC cells to study metastasis section:\n“Results showed that this co-culture was successful as OSCC cells invaded towards the endothelial cells in the fibrin gel” - can the authors explain why angiogenic sprouting was not observed in the cancer cell co-culture? Also, for this statement to be valid it should be shown that invasion into the fibrin gel does not occur in monoculture; in the subsequent figures where OSCC cells are shown alone there is evidence of invasion suggesting this process may be independent of the presence of endothelial cells.\nIn the Optimisation of the staining section:\n“To better understand the interactions between the cancer cells and the vasculature, the expression of specific markers must be assessed” – as described above the markers chosen (keratin and vimentin) are not appropriate for this purpose, use of CD31 or an alternative endothelial cell specific marker should be used. Further clarity should also be provided in the legend for figure 5. Is this example showing OSCC cells grown in monoculture or co-culture?\n“This background noise may reflect some retention of unbound secondary antibodies in the 3D matrix.” - can the authors provide rationale for why excess secondary antibody but not primary antibody would be retained within the 3D matrix? Could indirect antibody staining be feasible with further optimisation of the staining protocol? If conjugated antibodies must be used instead of indirect antibody staining the feasibility for analysing different proteins in this system is likely to be significantly reduced, due to limited commercial availability of conjugated antibodies and lack of signal amplification.\nIn the Image analysis section:\nThe description of cell segmentation and analysis methodology is not adequately described for readers unfamiliar with the developer toolbox software. Clear descriptions should be provided for the algorithms used for cell segmentation. Additionally, it is unclear whether the term cell segmentation is used correctly throughout the article. This process refers to demarcation of cellular boundaries but seems to be used to describe both cell segmentation and cell-type/group classification, which are two distinct processes.\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "10877", "date": "15 Jan 2024", "name": "Adrian Biddle", "role": "Author Response", "response": "This article by Alice Scemama et al. outlines an in vitro approach to modelling metastatic spread in human OSCC, using microfluidic devices that enable high-quality real time imaging. The method describes the fabrication of PDMS moulds with three separate microfluidic channels that can be used for the addition of different cells, matrices or media to the culture. Protocols are also described for imaging-based analysis of these cultures using endogenous fluorophores and/or antibody staining followed by digital analysis involving cell segmentation and quantification of fluorescence intensity. The method provides useful insight for device fabrication and use for culturing OSCC cells with endothelial cells. As stated by the authors, this device and culture system replicates others that have been used for studying different cancer types previously, so these insights are not entirely novel. It also remains questionable to what degree the stated research highlights are fully achieved by this article. We thank the reviewer for their encouraging words and thorough review. As mentioned for reviewer 1, this is a methods paper to accompany a research article that is now pre-printed (Scemama et al., 2024). Whilst conducting patient validation of the data from the microfluidic device for the main research article, we wanted to publish the method without delay as a resource for the research community. We have now updated this methods paper to link it to the pre-printed research article. Where comments are addressed in the main research article, we will note that below. The particular focus of our own work is on using microfluidic devices to study the evolution of tumour cellular heterogeneity and plasticity within a complex tumour microenvironment, and the analysis methods we have developed and presented here are designed to enable the application of microfluidic devices to this area of study. Conceptual reservations to approval of this article: The authors propose that this microfluidic system could be used for investigating the process of OSCC metastasis via the vasculature, suggesting that this could be used instead of animal models. To adequately demonstrate this, further evidence of benchmarking compared to alternative in vitro systems and in vivo models is required. For example, additional experiments or data that would be useful include: Demonstrating that cell lines with varying metastatic potential in vivo exhibit different phenotypes in this model. Demonstrating quantitative metrics can be derived from this model system and the imaging-based analyses proposed to assess these phenotypic differences. Comparing the results generated using this system to a Transwell system or other existing systems (e.g. Ibidi chemotaxis slides). An important advantage of this system is the ability to generate quantitative single-cell metrics, due to the superior imaging over existing in vitro and in vivo methods. Methods for the generation of such metrics are presented in this methods paper, and we have presented both the outputs of these methods and patient validation data in the main research article (Scemama et al., 2024). Our focus is on benchmarking against human clinical and pathological data, as in vivo animal data is often an inaccurate representation of the clinical truth. The authors highlight real time imaging as a major advantage of this system over existing methods for studying OSCC metastasis. However, the data presented exposes some limitations to these approaches and their utility for progressing the investigation of molecular mechanisms underlying these processes. For example, The use of endogenous fluorophores and phalloidin for classifying different cell types is confounded by non-ubiquitous reporter expression leading to potential for mis-classification of cells, which appears to be the case in the micrograph shown in Figure 4. Reviewer 1 had the same concern, and we have responded in detail there. Briefly, this concern is due to the inability to display the entire dynamic expression range in an image. The expression is in fact ubiquitous. The interpretation of intercellular interactions across cellular compartments is reliant on strict lineage specific restriction of marker expression. The example provided in figure 5 demonstrates the use of keratin and vimentin “to better understand the interactions between the cancer cells and the vasculature”. However, the use of vimentin here is likely to be suboptimal, given this gene is expressed in cancer cells undergoing EMT. The authors should elaborate further on how experiments should be designed to enable accurate examination of tumour vasculature interactions and ensure that the interpretations are validated with appropriate controls. The reference to keratin and vimentin expression here is in relation to the cancer cells, as we are interested in identifying cancer cells undergoing EMT. The vascular cells do indeed also express vimentin, but this can be separated out by including co-localisation with the GFP lineage reporter in the quantitative analysis. This is elaborated further in the associated research article (Scemama et al., 2024). In addition (and as described above) it is not clear how these imaging-based analyses should be used to quantify steps in the metastatic process. Further evaluation and demonstration of how these approaches can be used to measure phenotypic changes under different experimental conditions should be provided to confirm this system be used in a similar manner to existing assays (e.g. Transwell systems) generating reproducible quantitative data. The assessment of changes under different experimental conditions, using the quantitative methods described in this methods paper, is a key feature of the associated research article (Scemama et al., 2024). Technical recommendations/comments: In the Angiogenesis assay section: “cells added in the opposite side channels directly migrated into these structures” – clarity should be provided regarding what cells were added to opposite side in this example (Figure 3). The cells in the opposite channel were CA1, and we have now clarified this. In the Co-culture of endothelial cells and OSCC cells to study metastasis section: “Results showed that this co-culture was successful as OSCC cells invaded towards the endothelial cells in the fibrin gel” - can the authors explain why angiogenic sprouting was not observed in the cancer cell co-culture? Also, for this statement to be valid it should be shown that invasion into the fibrin gel does not occur in monoculture; in the subsequent figures where OSCC cells are shown alone there is evidence of invasion suggesting this process may be independent of the presence of endothelial cells. Indeed, the invasion of CA1 cells into the device occurs in both the presence and absence of HUVECs. Our point here is that we have established culture conditions that allow the successful co-culture of both cell types within the device such that their interactions can be investigated. Investigation of the effects of this co-culture on both the cancer and endothelial cells is a major focus of the associated research article (Scemama et al., 2024). In the Optimisation of the staining section: “To better understand the interactions between the cancer cells and the vasculature, the expression of specific markers must be assessed” – as described above the markers chosen (keratin and vimentin) are not appropriate for this purpose, use of CD31 or an alternative endothelial cell specific marker should be used. Further clarity should also be provided in the legend for figure 5. Is this example showing OSCC cells grown in monoculture or co-culture? This example of staining is on cells growing in monoculture – this has been added. As we’ve outlined in a previous answer, vimentin and keratin are both used as markers in the cancer cells. CD31 can successfully mark the endothelial cells, as can an RFP marker (Scemama et al., 2024). “This background noise may reflect some retention of unbound secondary antibodies in the 3D matrix.” - can the authors provide rationale for why excess secondary antibody but not primary antibody would be retained within the 3D matrix? Could indirect antibody staining be feasible with further optimisation of the staining protocol? If conjugated antibodies must be used instead of indirect antibody staining the feasibility for analysing different proteins in this system is likely to be significantly reduced, due to limited commercial availability of conjugated antibodies and lack of signal amplification. We tried extensive optimisation of indirect antibody staining before proceeding with direct antibody staining so, whilst we cannot know whether it would be feasible with the correct optimisation, it certainly seems a difficult approach within this system. In contrast, direct antibody staining worked very well. We suggest that, being a 3D system, there is more opportunity for antibody to diffuse away from the intended target than in 2D staining. Certainly, extended time between staining and imaging increases this problem. It may be that the binding of primary antibodies to their target is stronger than the binding of secondary antibodies, with less diffusion away, and of course there is less fluorescence signal overall in the system. In the past, we have relied on indirect staining due to the signal amplification effect and limited commercial availability of conjugated antibodies. However, these advantages of indirect staining no longer hold. Improved imaging systems and techniques have rendered the signal amplification effect of secondary antibodies unnecessary for our applications, and commercial providers now have greatly improved catalogues of conjugated antibodies (including recombinantly produced antibodies) such that the majority of targets of interest have antibodies available conjugated to a range of fluorophores. In the Image analysis section: The description of cell segmentation and analysis methodology is not adequately described for readers unfamiliar with the developer toolbox software. Clear descriptions should be provided for the algorithms used for cell segmentation. Additionally, it is unclear whether the term cell segmentation is used correctly throughout the article. This process refers to demarcation of cellular boundaries but seems to be used to describe both cell segmentation and cell-type/group classification, which are two distinct processes. We have updated this section to improve the description and clearly define the term ‘segmentation’. References: SCEMAMA, A., LUNETTO, S., TAILOR, A., CIO, S. D., AMBLER, L., COETZEE, A., COTTOM, H., KHURRAM, S. A., GAUTROT, J. & BIDDLE, A. 2024. Hybrid cancer stem cells utilise vascular tracks for collective streaming invasion in a metastasis-on-a-chip device. bioRxiv, 2024.01.02.573897." } ] } ]	1	https://f1000research.com/articles/12-439
https://f1000research.com/articles/12-1394/v1	23 Oct 23	{ "type": "Brief Report", "title": "Camptostemon philippinensis, a new record of endangered mangrove species in the Balikpapan Bay, East Kalimantan, Indonesia", "authors": [ "Bina Swasta Sitepu", "Abdul Razaq Chasani", "Mukhlisi Mukhlisi", "Tri Atmoko", "Burhanuddin Adman", "Istiana Prihatini", "Bina Swasta Sitepu", "Mukhlisi Mukhlisi", "Tri Atmoko", "Burhanuddin Adman", "Istiana Prihatini" ], "abstract": "Background\nCamptostemon philippinensis, found in mangrove forests in Indonesia and the Philippines, is listed as endangered on the International Union for Conservation of Nature (IUCN) Red List. It is found primarily in isolated mangrove forests in Kalimantan (Indonesian Borneo) and Sulawesi in Indonesia. Despite significant studies on mangrove biodiversity in this region, the occurrence of C. philippinensis in Balikpapan Bay, East Kalimantan, is not extensively recorded.\n\nMethods The study was conducted by exploring the mangrove forests along Balikpapan Bay. The first survey of about 200 km was conducted to observe mangrove vegetation and found one C. philippinensis tree. The second survey focused on the area around the first discovered C. philippinensis tree to census and record its growth stage and distribution.\n\nResults The study recorded a population of 527 individuals of C. philippinensis in Pantai Lango Village, East Kalimantan, dominated by seedlings. The high number of seedlings indicates good natural regeneration potential, but the low number of trees indicates intense competition for space in a restricted habitat. This species inhabits a small and restricted area in Balikpapan Bay, in the middle area of Balikpapan Bay, and is associated with other mangrove flora, such as Rhizophora apiculata, Rhizophora mucronata, Sonneratia alba, Avicennia alba, Lumnitzera littorea, Osbornia octodonta, Ceriops tagal, and Xylocarpus granatum.\n\nConclusions\nC. philippinensis is vulnerable to habitat damage from anthropogenic activities, which could lead to local extinction. Its natural habitat in Balikpapan Bay also has the potential to be under pressure due to the development of Indonesia’s new capital city (Ibu Kota Nusantara/IKN). It emphasizes the need to understand the ecological role of this protected flora in the natural habitat of protected fauna (the proboscis monkey). Documenting the population of C. philippinensis is crucial for conservation efforts, including propagation and understanding its ecological role.", "keywords": [ "Major mangrove", "conservation", "proboscis monkey", "Penajam Paser Utara", "IKN" ], "content": "Introduction\n\nCamptostemon philippinensis (Vidal) Becc. is an endangered species comprising mangrove forests listed in the endangered category on the IUCN Red List (Duke et al., 2010). This species is one of Indonesia’s two protected mangrove species (MEF Regulation Number 106/2018), so it is a high priority for conservation. C. philippinensis is a rare mangrove species globally distributed in Indonesia and the Philippines, with an estimated number of mature individuals of around 200 (Duke et al., 2010; Primavera et al., 2004). In Indonesia, this species is only found on the islands of Kalimantan and Sulawesi (Duke et al., 2010) and mainly in isolated mangrove forests (Djamaluddin, 2018). That is a lesser-known species, and so far, there are no records of its use by the community. However, the bark and leaves of C. philippinensis have the potential to contain anti-melanogenic and antibacterial chemicals (Gadores et al., 2021).\n\nCamptostemon philippinensis is one of three species in the genus Camptostemon (Malvaceae) (Beccari, 1886). This species was first published as Cumingia philippinensis S. Vidal in 1885, with type specimens originating from the island of Luzon. Beccarii (1886) suggested the move to the Camptostemon genus after observing the flower and fruit organs of the early species and discovering that the features in these two organs in Cumingia were comparable to those in Camptostemon. Van De Brink (1924) reduced the species C. aruensis to a synonym for C. schultzii based on his observations on the organs of flowers and fruits. That makes C. philippinensis and C. schultzii very easy to distinguish based on the morphological character of the leaf shape, where the first species has oblong leaf blades and the last species has elliptical leaf shapes. However, molecular studies have not been carried out specifically to support this opinion.\n\nThe population distribution of C. philippinensis on the island of Borneo is very limited. Previously, records of the distribution of this species in Borneo were only documented on the coast of Berau (Mukhlisi & Sidiyasa, 2014). In Balikpapan Bay, several mangrove floristic studies have been conducted and recorded the richness of mangrove species, ranging from 12 species (Kristiningrum et al., 2019) to 20 species (Warsidi & Endayani, 2017). Although various biodiversity studies have been carried out in the area, the presence of C. philippinensis has never been recorded (Kristiningrum et al., 2019; Sayektiningsih et al., 2012, 2020; Toulec et al., 2022; Willard et al., 2022).\n\nBalikpapan Bay is a mangrove ecosystem in East Kalimantan with an area of around 168 km2 (Toulec et al., 2022), which is an essential habitat for various species of endangered wildlife, especially Borneo’s endemic primate, the proboscis monkey (Nasalis larvatus). However, the threat of mangrove forest conversion is still present (Anwar et al., 2021b; Toulec et al., 2022). The study results show that this area has one of the densest proboscis monkey population distributions, with as many as 60 groups (Toulec et al., 2022). On the other hand, mangrove vegetation has a strong interaction with the presence of proboscis monkeys because the monkeys like young leaves, seeds, and mangrove fruit as food sources (Boonratana, 1993). Atmoko (2022) has reported as many as 41 plant species as food sources for proboscis monkeys from several habitats in East Kalimantan. In addition, proboscis monkeys often choose large mangrove trees on the banks of rivers or seas as sleeping trees to avoid predators. However, until now, information on the ecological role of C. philippinensis in the proboscis monkey’s natural habitats has been limited.\n\nDocumentation of the C. philippinensis population is important in supporting comprehensive conservation efforts, such as propagation efforts, and understanding its ecological role for the animals around it. More importantly, because some of the Balikpapan Bay areas will be developed as a site for the new Indonesian capital city (Ibu Kota Nusantara/IKN), this study can be used as a resource for stakeholders interested in the usage of the area.\n\n\nMethods\n\nA focused or intuitive-controlled survey (Brewer, 2013) was conducted to observe C. philippinensis. This approach emphasizes surveying areas with high potential habitat for the target species, relying on the surveyor’s experience, field intuition, and readiness to steer the survey. We explored Balikpapan Bay to observe the mangrove vegetation in July 2022 and found a tree of C. philippinensis. The second survey in March 2023 focused on C. philippinensis existence. The survey was carried out using a boat along approximately 200 km of the coast of Balikpapan Bay. The study area covers Balikpapan City and Penajam Paser Utara Regency, where some will be part of the IKN Area. In the first survey, only one C. philippinensis tree was found on the shores of Kowangan Island, in the middle of Balikpapan Bay. In the second survey, we focused on the area where C. philippinensis had previously been discovered. We explored within a radius of 50 km from the point where the initial tree was found. We used the AvenzaMaps application on a smartphone to navigate and estimate distances in the field and used a Garmin CSx60 GPS receiver to take coordinates. The soil texture was observed in-situ following a guideline by Stuart-Street et al. (2020).\n\nThe study area was in Balikpapan Bay, covering Balikpapan City and Penajam Paser Utara Regency, where some are part of the IKN Area (0°54′27″S-1°13′59S″ and 116°38′06″E-116°50′18″E). Balikpapan Bay is one of the largest remaining mangrove forests in East Kalimantan Province, with an area of 168 km2 (Toulec et al., 2022). This bay is a semi-closed water body with 56 rivers and tributaries (Kreb et al., 2020). It holds high biodiversity value (Lahjie et al., 2019; Kreb et al., 2020), including fisheries resources that have been utilized by local communities for more than 150 years (Lahjie et al., 2019). A permit to access and collecting C. philippinensis in Teluk Balikpapan was obtained from the Directorate General of Nature Resources and Ecosystem Conservation of the Ministry of Environment and Forestry of Indonesia, Number SK 94/KSDAE/SET.3/KSA.2/5/2023.\n\nA census was carried out by measuring the height and diameter of each C. philippinensis tree discovered in the survey area. We also recorded the coordinates of each individual and growth stage based on diameter at breast height (dbh), namely seedlings (germinated seed to <1.5 m in height), saplings (height >1.5 m; dbh <10 cm), and trees (dbh > 10 cm) (Susilowati et al., 2019) A brief description of the surrounding vegetation was carried out by recording the mangrove species assumed to be associated with C. philippinensis. The herbarium specimens of C. philippinensis were collected and deposited at Herbarium Wanariset (WAN) and Herbarium Bogoriense (BO).\n\nBiogeographical information about the species was gathered from the herbarium specimens published on the GBIF website (https://www.gbif.org/) (GBIF, 2023). A literature review on the species, genus, or mangrove species that indicated the presence of C. philippinensis was also mined to enrich the distribution data and taxonomy information (Noor et al., 2012; Giesen et al., 2006; Primavera et al., 2004). All data and information were used to identify the distribution and describe the novel record of the species in Teluk Balikpapan, East Kalimantan.\n\nEach individual was counted to determine their total number based on growth stage (seedling, sapling, and tree) and is presented in the form of a percentage graph. Saplings and trees were evaluated for their average diameter and height to portray the population profile. This analysis was performed using Microsoft Excel 2016 (RRID:SCR_016137). Furthermore, the distribution of C. phillipinensis was mapped using Quantum GIS 3.30.\n\n\nResults\n\nC. philippinensis was discovered on Pantai Lango Village and Kowangan Island in Penajam Paser Utara District, East Kalimantan (Figure 1) (Mukhlisi, 2023; Sitepu et al., 2023). It was discovered in a narrow area, although the Balikpapan Bay mangrove forest is relatively large, covering around 203 km2 (Toulec et al., 2022). The C. philippinensis habitat is on the west shore, in the middle of Balikpapan Bay. Only one individual was found on a small island (Kowangan Island), whereas most others inhabited the mainland’s western shore in Pantai Lango. The distance between these two locations is around 500 m.\n\nBased on our observations in Pantai Lango, there were around 527 individuals of C. philippinensis that were dominated by seedlings (452 individuals), followed by trees (49 individuals), and saplings (26 individuals) (Figure 2). This demonstrates that even though C. philippinensis has a relatively limited range; its natural regeneration potential is functional. However, the number of saplings found was less than the tree stage, and this may be due to competition for space in a limited habitat. Generally, in mangrove forests, the number of seedlings is more abundant than the saplings, and the sapling stage is more numerous than the trees (Islam et al., 2015; Winata et al., 2017).\n\nThe saplings and trees of C. philippinensis have an average tree diameter of 14.98 ± 7.95 cm and an average height of 9.49 ± 3.59 m (Figure 3). C. philippinensis in Balikpapan Bay has a diameter profile similar to the flora in the area. The tree was observed in the fruiting season in February 2023 (Figure 4); however, no ripe fruits or flowers were observed.\n\nA) Diameter and B) height.\n\nThe license mentioned in the Methods section included permission to collect specimens.\n\nA population of C. philippinensis was found in the middle area of Balikpapan Bay, and the influence of fresh water from river flow was not found significantly around its habitat. In-situ investigation showed that the soil texture around the habitat of C. philippinensis is predominantly sandy, both on the shore of the mainland (Pantai Lango) and on Kowangan Island. This species is frequently observed in environments impacted by moderate to low-intensity sea tides. Balikpapan Bay experiences daily semi-diurnal tides, with a tidal range of almost 2.5 m during spring tides and 1 m during neap tides (Anwar et al., 2021a). At the study site, C. philippinensis in the second layer of mangrove zonation, where the habitat does not flood during low tide. This differs from the vegetation in the first layer, which are observed to remain flooded even during low tide.\n\n\nDiscussion\n\nJ. van Borssum Walkes identified C. philippinensis in Indonesia in 1962 based on herbarium specimens collected by CNJ Welmaar in 1919 from Pulau Laut, South Kalimantan (Bakhuizen van den Brink, 1924; Troll, 1933). In the following period, it was never recorded in various publications or scientific collections, so it was stated that it was no longer present in Kalimantan and grew only limitedly in the Papua region (Macnae, 1968). However, Noor et al. (2012) continued to list this species along with C. schultzii as a type of mangrove plant in Kalimantan until publication by Mukhlisi and Sidiyasa (2014) based on botanical records in Tanjung Batu, Berau. While in Sulawesi, it was reported in Bunaken National Park (Djamaluddin, 2018; Djamaluddin & Djabar, 2022) and Donggala (Wahyuningsih et al., 2012). In the Philippines, however, this species is reported to be widely spread but has yet to be abundant (Primavera et al., 2004).\n\nIn a limited habitat, populations of C. philippinensis tend to form stands in groups. The population is quite dominant at a certain point in a habitat, but not like Rhizophora spp. (Mukhlisi & Sidiyasa, 2014). When compared with populations of other mangrove species in a wider sampling area, the population of C. philippinensis is classified as the lowest (Djamaluddin, 2018; Pototan et al., 2021; Wahyuningsih et al., 2012). Several previous studies have also found a similar phenomenon where the population of C. philippinensis is known to be exclusive and limited to a narrow range of habitats (Djamaluddin, 2018; Djamaluddin & Djabar, 2022; Mukhlisi & Sidiyasa, 2014). Although all mangrove propagules have buoyant abilities, which allow them to spread far through seawater (Van der Stocken et al., 2019), C. philippinensis requires specific ecological compatibility to colonize new habitats.\n\nC. philippinensis often inhabits a small area; its dominant habitat in Balikpapan Bay is restricted to 300 m along the mainland shore. Rhizophora apiculata, R. mucronata, Sonneratia alba, Avicennia alba, Lumnitzera littorea, Osbornia octodanta, Ceriops tagal, and Xylocarpus granatum are among the mangrove flora associated with this species. The mangrove forest formation in this location has C. philippinensis in the second layer, behind the R. apiculata, S. alba, and R. mucronata formations. Meanwhile, one individual of C. philippinensis found on Kowangan Island inhabits the shoreline. On Kowangan Island, mangrove vegetation is situated next to a mountainous terrestrial forest in a narrow configuration that is often sparse. Rhizophora apiculata, A. alba, Pongamia pinnata, Hibiscus tiliaceus, Lumnitzera littorea, and Osbornia octodonta are associated with mangrove species on Kowangan Island.\n\nWe did not specifically observe the endangered species, proboscis monkeys (Nasalis larvatus). However, based on information from the local people (Darman, personal communication, 20 March 2022), he has seen two groups of proboscis monkeys on Kowangan Island and one other around the main island of C. philippinensis habitat. Our observations revealed primate bite scars on the leaves of C. philippinensis. This discovery is assumed to be proboscis monkey bite marks, given that leaves are the primate’s primary food source (folivorous) (Boonratana, 1993). Other mangrove species on Balikpapan Bay reported to have a high preference as a food source for proboscis monkeys are R. apiculata (Tangah & Bernard, 2010), S. alba, and S. caseolaris (Atmoko, 2022).\n\nProboscis monkeys are arboreal primates that rely heavily on the presence of the mangrove tree canopy. They often use large and tall trees as sleeping sites at the water’s edge. The average diameter of C. philippinensis trees in Balikpapan Bay was similar to that in other locations (Primavera et al., 2004; Pototan et al., 2021). In Banaybanay, Philippines, Pototan et al. (2021) found that the average dbh (diameter of breast height) of C. philippinensis was 16.25 cm. It can naturally reach heights of 15 m and diameters of up to 50 cm (Primavera et al., 2004).\n\nThe threat of C. philippinensis populations in Balikpapan Bay is increasing, primarily due to anthropogenic activity. The population is localized in a narrow and extremely limited area; damaging its habitat increases the risk of local extinction (Figure 5). Changes in the status of regions in the mangrove area of Balikpapan Bay and surrounding areas (e.g., the Indonesia’s New Capital City area (IKN)) would lead to alterations to the landscape and human population, which could pose a threat to its habitat. Current threats to C. philippinensis include the conversion of mangrove forests, environmental pollution, illegal logging, and the development of IKN.\n\nConserving C. philippinensis from extinction can be done with several efforts, such as prevention, species and habitat protection and preservation, habitat restoration, and ex-situ conservation. Prevention efforts can be carried out through education and outreach to various parties regarding the existence of C. philippinensis populations in the IKN area so that their habitat can be maintained. Protecting its natural habitat can be done by establishing a local protected area for the local community. Habitat restoration can be carried out by restoring degraded habitats and raising the number of individuals in their populations.\n\nC. philippinensis in Indonesia has been listed as a protected plant under government law, and one of its natural habitats on Bunaken Islands has been designated as a conservation area and assigned as a national park. Unfortunately, no report on habitat restoration and ex-situ conservation efforts has been made until the present. Therefore, it is necessary to save genetic material from existing populations through seed storage and efforts to propagate both generatively and vegetatively, which can be used as a source of genetic material for habitat restoration or ex-situ conservation activities, as well as land rehabilitation. It is also necessary to study the genetic diversity of C. philippinensis in Indonesia, especially to determine conservation strategies, evaluate the success of restoration or reintroduction programs in other populations, and maintain genetic variation that will be important for the survival of this species.\n\n\nConclusions\n\nThis study recorded 527 individuals of C. philippinensis found in Pantai Lango Village. This population has a complete structure, including seedlings, saplings, and trees at all levels. The natural regeneration potency of C. philippinensis is still functioning well, as indicated by the presence of many seedlings. However, intense competition for limited space in the habitat causes saplings to grow into the tree strata. It is under growing threat, primarily as a result of anthropogenic activities.", "appendix": "Data availability\n\nZenodo: All Raw Data of Camptostemon philippinensis in Balikpapan Bay, https://doi.org/10.5281/zenodo.8393832 (Mukhlisi, 2023).\n\nRepositori Ilmiah Nasional (RIN) Dataverse: Data of Camptostemon philippinensis in Teluk Balikpapan, https://hdl.handle.net/20.500.12690/RIN/V8VTQV (Sitepu et al., 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank Darman for his assistance during the expedition in Balikpapan Bay. All authors have equivalent contribution in writing this manuscript and conducting the field works.\n\n\nReferences\n\nAnwar IP, Putri MR, Tarya A, et al.: Variation of water mass exchange on tidal scale in Balikpapan Bay. IOP Conf. 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Flora, N.F. 1933; 28: 348–352.\n\nVan der Stocken T, Carroll D, Menemenlis D, et al.: Global-scale dispersal and connectivity in mangroves. Proc. Natl. Acad. Sci. U. S. A. 2019; 116(3): 915–922. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWahyuningsih EP, Suleman SM, Ramadanil.: Struktur dan komposisi vegetasi mangrove di Desa Lalombi Kecamatan Banawa Selatan Kabupaten Donggala. Jurnal Biocelebes. 2012; 6(2): 84–100. Reference Source\n\nWarsidi, Endayani S: Komposisi vegetasi mangrove di Teluk Balikpapan Provinsi Kalimantan Timur. Agrifor. 2017; 16(1): 115–124.\n\nWillard K, Aipassa MI, Sardjono MA, et al.: Locating the unique biodiversity of Balikpapan Bay as an ecotourism attraction in East Kalimantan, Indonesia. Biodiversitas. 2022; 23(5): 2342–2357. Publisher Full Text\n\nWinata A, Yuliana E, Rusdiyanto E: Diversity and natural regeneration of mangrove vegetation in the tracking area on Kemujan Island Karimunjawa National Park, Indonesia. AES Bioflux. 2017; 9(2): 109–119. Reference Source" }	[ { "id": "218353", "date": "10 Nov 2023", "name": "Inocencio E Buot", "expertise": [ "Reviewer Expertise Botany (ecology and 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\nGeneral comments\nThe paper is very well written actually.\n\nThis is a good report as new occurrence record, but I am suggesting some more information if possible.\n\nThe first paragraph under methods should have a subsection title since all the rest that follows have. Would that be reconnaissance perhaps?\n\nI think Study Area should come first as a subsection under Methods so that the authors can discuss why focus in that mangrove forest when there are so many in Indonesia? This discussion is lacking under Study Area. Were there clues or hints that they could spot the species in the area? Were the first herbarium documentations playing a key role in leading them to the area of investigation?\n\nThe herbarium specimens used for biogeographic studies were not tabulated with details of the exsiccata. I would suggest having that to add veracity of the information. And even all the vouchers examined and mentioned in the results and discussions.\n\nThere was no statement in the method on the use of drone to start of the reconnaissance survey. That could have been resorted to first, before ground truthing and spotting the tree using a boat. But so long as there was a thorough ground truthing anyway, that would be fine.\n\nIn the results, I expect the details of the exsiccata of the herbarium vouchers collected by the authors from the study area and deposited at Herbarium Wanariset (WAN) and Herbarium Bogoriense (BO). If voucher photographs are available, it would be great to see one here in this paper. And of course, photos of the plant are wanting with details of the different organs preferably with measurements, so that readers would not have reservations of its identity.\n\nFig 1 seems to include only field data? Would you not consider including here the distribution record of the species as documented in the herbarium collections in the past as mentioned in the discussion portion of the paper?\n\nIn the discussion, there was no mention of the currently gathered data except for the reference to Fig. 5. Hence, it would appear a review paper since the data collected were not cited well.\nI commend the authors for doing this study and writing the paper. I hope to see this revised and published soon.\n\nIs the work 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? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10696", "date": "13 Apr 2024", "name": "Abdul Razaq Chasani", "role": "Author Response", "response": "1. Thank you for providing a review and comments on this paper. 2. We added more information as suggested. 3. A subsection title (preliminary survey) was added under the methods title. 4. The Study Area subsection moved to the first subsection, followed by the Preliminary Survey subsection. The study area selection has been discussed in the methods and discussion section. The study area is an essential ecosystem located in an area that could be affected by the development of Indonesia's new capital city, so the general mangrove survey is important. A single Camptostemon philippinensis tree was spotted during the preliminary survey, collected as an herbarium, and confirmed as C. philippinensis. A second survey was then conducted to find other C. philippinensis trees in the nearby area. 5. We added the exsiccate and the figure of the herbarium. We also added a map and a table to list the herbarium collection from Indonesia. 6. We did not use a drone for the survey; all of the surveys were conducted on a boat, traveling from upstream to downstream and back. 7. We added the figures of the herbarium deposited in BO and provided the figures of the flower (Fig. 4b) and fruit (Fig. 4c). 8. We have revised Figure 1 and added the distribution record of the species.Capmtostemon philipinensis 9. The present study has described the data collection in paragraphs 2 and 3. 10. We have revised our manuscript and provided more information as reviewer suggestions." } ] } ]	1	https://f1000research.com/articles/12-1394
https://f1000research.com/articles/12-424/v1	20 Apr 23	{ "type": "Review", "title": "Prevalence of stress and its relevance on psychological well-being of the teaching profession: A scoping review", "authors": [ "Shilpa Badrinath Bidi", "Varalakshmi Alapati", "Venisha Jenifer Dmello", "Edwin Weesie", "Mathew Thomas Gil", "Sandeep S Shenoy", "Simmy Kurian", "Ambigai Rajendran", "Shilpa Badrinath Bidi", "Varalakshmi Alapati", "Venisha Jenifer Dmello", "Edwin Weesie", "Mathew Thomas Gil", "Sandeep S Shenoy", "Simmy Kurian" ], "abstract": "Background: Well-being among teachers contributes toward quality work and better student support. Teachers’ well-being persists to be a concern in school settings; there is a lacuna in understanding the concept of well-being among them. This scoping review identifies the stress factors and map their association with the psychological well-being of teachers employed in schools. Additionally, to identify the methodology and the interventions used in reducing teachers' stress and their relevance on their psychological well-being. Methods: First, Pubmed, Web of Science, and Scopus databases were searched for eligible studies with MeSH terms for stress factors, well-being, and teachers from 2010 to 2022. Identified studies were screened thoroughly and excluded or included based on prior established criteria. Data from the included studies were extracted and summarized according to the study protocol.\n\nResults: Among the 60 studies that met our inclusion criteria, the majority were quantitative, with cross-sectional studies. Several studies focused on emotional exhaustion, depersonalization, and diminished personal accomplishment aspect among teachers. Almost half of the included studies focused on organizational and social pressures such as administration workload, classroom management issues, lack of supervisor and team support, students’ behaviour, and pressure from parents. The most used interventions to overcome stress were coping strategies and mindfulness training intervention tools.\n\nConclusions: The findings from the current scoping review will reveal the different stressors which impact psychological well-being. Focus on the most used interventions to overcome stress among schoolteachers. This will also provide recommendations to regulators and management to identify the factors causing stress among teachers and their relevance to their psychological well-being, overcome employee turnover and absenteeism issues. Also, different alternatives available to reduce the stress may benefit the stakeholders and policymakers to confirm a suitable intervention that will benefit the teaching profession.", "keywords": [ "stress factors", "stressors", "well-being", "psychological well-being", "teachers", "educator", "interventions." ], "content": "Introduction\n\nThe process of instruction known as education aims to help people develop their knowledge, attitude, and character for giving them the tools they need to live purposeful lives. Our educational system's most critical connection is the teacher. The effectiveness of the teachers is a major factor in the outcome of any educational program. Well-being as a topic has been investigated since the last two decades (Schellenbaum et al., 2002; Schaufeli & Bakker, 2004; Pritchard et al., 2019) and has become an increasingly popular topic in academic and business settings, with organisations attempting to discover what makes working environments engaging and motivating. The key objective of developing organisational understanding and offering development to the workforce has become a major element. Thereby the responsibility of the future generation, by and large, is bestowed upon the educational institution. There are claims stating that education is a process that directs and manages the behaviour of individuals of all ages irrespective of time and place and really not restricted to schools or educational institutions (Howard, 1993). Even today, there is an existence of strong culture that believes educational systems are accountable for the civilization of society and for the development of individuals (Turkkahraman, 2015). Thus, educational institutions have become vital and indispensable work setup in the contemporary situation. Teachers are considered as a driving force of learning organizations to foster learning than ever before, and thus they become ‘knowledge workers’ to effectively deal with the rapidly changing environment for improving learning outcomes. The welfare and well-being of such knowledge workers require primary focus from the regulators and managements.\n\nStudies conducted in various countries shows high stress levels and mental disorder among teachers (Wu et al., 2006; Yu et al., 2014). Approximately 60% of teachers reported they are stressed (Gomez, 2022) globally. Professional burnout is also caused by continuous teacher stress and inadequate coping mechanisms (Maslach et al., 2001). Research has revealed that over a one third of education professionals are expected to leave their job by 2020, highlighting the importance of teacher mental health and the need to address this crisis (Hazlegreaves, 2020).\n\nWell-being among teachers contributes towards quality work, self-efficacy in the classroom and better student support. In many countries, psychological well-being among teachers has become a growing concern (Seibt et al., 2013; Alhija, 2015; Mahfar et al., 2018; Anderson et al. 2022). Nonetheless, most school-based research has focused on pupils rather than teachers' health and well-being (Moy et al., 2014). Teachers plays an integral role in shaping the future generation in their provided workspace and thus in turn it affects the well-being of the society directly. Hence, our study helps to identify the factors causing stress among teachers and its relevance on their psychological well-being. Also, different measures taken to reduce the stress, which intern may also benefit the stake holders policy makers to adopt the suitable interventions which will benefit the teaching profession.\n\nThe objective of our review is to scope the extent and nature of the articles investigating stress factors on psychological well-being among teachers. We aim to identify gaps in the literature and use the results of our search to inform decisions about directions for future research. A preliminary review on this topic was conducted in April 2022 using JBI evidence synthesis and Open Science Framework. We were unable to find any planned or existing scoping reviews with the same focus as ours.\n\n\nMethods\n\nThe main objective of the study is (a) to explore the stressors influencing the psychological well-being of teachers. (b) to map the measures/interventions which helped in managing the stress. To attain the objectives of the study scoping review was conducted in 2022. The methodological framework adhered to the guidelines proposed by Arksey and O’Malley (2005) and was developed further by Levac and his colleagues (Levac et al., 2010). Besides the Joanna Briggs Institute (JBI) guidelines were followed with the following methodological steps: (a) Identification of research question; (b) Identification of studies which are relevant; (c) Selection of studies; (d) Charting of data; (e) Collation of results, summary and report. The scoping review was not entered into the PROSPERO database in accordance with the study’s design.\n\n(a) To explore the stressors influencing the psychological well-being of teachers.\n\n(b) To map the measures/interventions which helped in managing the stress.\n\nA scoping review is a predominantly adopted technique for appraising and synthesizing literature evidence which gives the broader prospect and the extent of the research activity in terms of the topic proposed, mapping the literature gaps and finally provide future direction to the researchers. Based on the objectives, the review addresses the following research questions:\n\na. What are the descriptive findings of the selected studies?\n\nb. What are the core stress factors influencing psychological well-being of schoolteachers?\n\nc. What are the measures/interventions adopted in managing stress among schoolteachers?\n\nThe preliminary search for the study was conducted in the National library of medicine database to define the search strategy and to identify key terms. Once key terms and MeSH terms were identified, three databases (Scopus, Web of Science and PubMed) were used to collect the relevant articles. These databases were considered as a majority of the content published is indexed in reputed journals. The search strategy was performed using various keywords, phrases and Boolean operators based on the combination of the following: stress OR burnout OR cynicism AND perceived stress OR stress factors OR personal stress OR work-related stress OR job-related stress AND well-being OR wellbeing OR psychological well-being OR psychological health OR mental well-being AND teachers OR instructors OR educators OR academicians OR facilitators OR school teachers OR elementary schools. In addition to the electronic search, a manual search using the snowball technique and reference lists was conducted of studies that qualify. The search terms were restricted to title, abstract and keywords in Scopus and PubMed, in Web of Science title, abstract, author keyword and keyword plus.\n\nStudies were considered and included if they met the following inclusion criteria:\n\n\n\n• Articles published in peer-reviewed journals.\n\n• Review articles bibliography was checked as additional search.\n\n• Articles that adopted Quantitative, Qualitative and a Mixed approach\n\n\n\n• Articles and book chapters that were not peer reviewed.\n\n• Review articles.\n\n• All reports, conceptual papers, lectures and conference proceedings.\n\n\n\n• Articles that examined: stress factors measures/interventions to overcome stress in alliance with psychological well-being\n\n\n\n• Articles that examined stress factors in alliance with occupational well-being.\n\n• Measures/interventions not emphasising on overcoming stress.\n\nThe central focus of the study was to understand the impact of stress on well-being mainly due to the burgeoning studies on teachers’ well-being in the past two decades. Research articles mainly focusing on evaluating stress among SEN, language teachers or any other teachers were not considered as it was not in the scope of the study. However, this suggestion is addressed by proposing to future researchers to conduct a comparative review or study by considering the above-mentioned segments.\n\nAll eligible studies were further screened by two researchers independently with the title and abstract in Rayyan software by excluding duplicates and considered the full text for data extraction. Third researcher was involved to resolve discrepancy through discussion. The reference lists of systematic reviews were examined to recognise possible eligible studies.\n\nThe search resulted initially in total of 3564 hits, duplicates were removed in Microsoft Excel which resulted in up to 2502 articles. All the refined articles were added to Raayan software for title and abstract screening. Two independent researchers screened and in any case of any discrepancies, was solved by discussion with the third researcher (Brereton et al., 2007). With the reasons records of other professional samples (1431), records with outcome other than psychological well-being (862), records with review papers (15), records with book chapters (10) and records with foreign language (18) were excluded which resulted to 166 articles were considered for full paper screening. Further, among 166 articles by full paper screening we excluded articles with reasons such as studies not considered psychological well-being only as outcome variable (17), articles not studying the association between stress and psychological well-being (24), articles studying stress factors associated with variables other than psychological well-being (25), articles not solely focused on the teaching profession (12), records with book chapters (14), records requested to authors but not received (14). Finally, 60 articles were considered for the study.\n\nA piloted and standardised data extraction form was used by two reviewers to independently collect the data and third as a checker (Brereton et al., 2007). Finally 60 selected articles were reviewed separately by two authors in order to analyse and record the data. Each article was summarized in Microsoft Excel carefully, by recording following main elements: ‘Bibliographic information such as, extent i.e., year of publication, title, author, country, objective of the study, methodology, sample size, data analysis, findings, stress factors, measures to overcome stress, future scope and limitations. During the process, two researchers compared their Excel forms and discussed if any disagreements in weekly meetings. A third researcher cross checked the information.\n\n\nResults\n\nA summary of the recognised studies is provided in this section as listed in Appendix A. First, we report descriptive statistics related to publication date, research context, geographical setting and theories adopted by the researchers. Next, we categorize different stress factors among the teaching profession and measures/interventions techniques employed to reduce the impact of stress.\n\nWhat are the descriptive findings of the selected studies?\n\nPublication period\n\nFigure 2 shows the annual distribution of selected articles. As seen, the publication of articles on stress factors aligned with psychological well-being among schoolteachers are very few in 2010, a moderate increase can be noticed in 2013. In 2014, there was no studies recorded. From 2016 to 2020 there was a substantial increase in studies. Maximum records were identified recently in the year 2021 i.e., post covid period.\n\nThe geographic distribution\n\nFigure 3 shows the geographic distribution of the selected articles. It can be seen that stress factors and psychological well-being has been studied most in USA (N = 21). Followed by Australia (N = 6), Finland (N = 6), UK (N = 6), Israel (N = 4), Norway (N = 4), Canada and Spain (N = 3). Regarding Colombo, Iran, Italy, India, Turkey, China two each were found.\n\nResearch methodology\n\nThere is tendency to use quantitative research methods in studying stress factors associated with psychological well-being among schoolteachers. In total, 37 publications employed quantitative research methods, whereas 13 publications employed qualitative, and 10 publications employed mixed methods respectively. Most of the studies adopted survey and randomized control trail (RCT) methods to collect the data based on statistical analysis, used SPSS and structural equational modelling (SEM), correlation/regression analysis, ANOVA/ANCOVA and t-test to test the hypothesis. Whereas qualitative method adopted in-depth interviews, semi structured interviews and focus group interviews. Mixed methods adopted both survey and interviews for the better understanding of the research problem. Table 2 presents the main research methodologies adopted in the selected publications.\n\nTheoretical perspectives\n\nTable 3 presents the theory which were adopted by authors among the selected studies. Job Demand and Resource model theory (Demerouti et al., 2001) is the maximum adopted theory by the researchers followed by self-determination theory, teacher performance motivation theory. PERMA theory coined by Seligman (2018) has captured researcher's attention in recent years. Along with these social cognitive theories, theory of planned behaviour, green glass theory, psychological theory of well-being and many more were used.\n\nWhat are the core stress factors influencing on psychological well-being of schoolteachers?\n\nA number of studies have proved teachers are prone to higher level of stress (Boyle et al., 1995; Burke et al., 1996; Bhuin, 2017) exposure to higher stress levels results in adverse consequences on health and well-being which in turn leads to poor performance, low work engagement, negative impact on the quality of student learning/education, employee turnover rates and absenteeism (Kyriacou, 2011; Hoglund et al. 2015; Von der Embse et al., 2016). Various researchers have identified different stress factors that influence the psychological well-being of the teaching profession. Although extensive research has been carried out on identifying the stress factors very few researchers have conducted a scoping review to explore the key stressors influencing psychological well-being. In order to address the research question, factors causing stress were coded and analysed using a thematic analysis approach. The main themes (Figure 5) on the stress factors were arrived on the basis of Bronfenbrenner’s ecological systems framework (Bronfenbrenner, 1992). It has emerged as an effective framework as it captures a wide range of stress factors ranging from micro and macro systems (Farley & Chamberlain, 2021) The stress factors were categorised into intrapersonal, systematic, relational and organisational stressors.\n\nSystematic stress factors\n\nThe systematic stressors theme comprised of national and state level policies that manage the education system operating. Holistic changes in the policies impose burdens on teachers to professionally equip with the required alterations. The review identified approximate 7% of the factors were contributed from systematic stressors like coping with changes in the policies (Yerdelen et al., 2016), Inefficient working policies (Skaalvik & Skaalvik, 2018), Constant changes in policies (Alvarado & Bretones, 2018; Farley & Chamberlain, 2021), Education reforms (Alloh et al., 2019; Hepburn et al., 2021c), National & State level policies (Schnaider-Levi et al., 2017; Carroll et al., 2020; Gearhart et al., 2022).\n\nOrganisational stress factors\n\nThe organisational stress theme comprised of significant factors in the work place which had an influence on psychological well-being. Within this theme around 48% of the factors contributed to various categories of stress. They were multi-tasking (Gold et al., 2009), workload (Gold et al., 2009; Simbula et al., 2012; Vazi et al., 2013; Vesely et al., 2013; Bermejo-Toro et al., 2015; Desrumaux et al., 2015; Yerdelen et al., 2016; Helms-Lorenz & Maulana, 2016; Skaalvik & Skaalvik, 2017; Alvarado & Bretones, 2018; Alloh et al., 2019; Braun et al., 2019; Soykan et al., 2019; Brady & Wilson, 2020; Carroll et al., 2020; Farley & Chamberlain, 2021; Hepburn et al., 2021a; Ibrahim et al., 2021; Tebben et al., 2021; Billett et al., 2022), lack of professional recognition and appreciation, administration overload, role conflict, poor working condition, time pressure, lack of recourses and technology, appeared as organisational factors which contributed to teachers' stress. Workload was the prominent category which was recorded in most of the studies to be the key factor for emotional exhaustion among teachers (Bower & Carroll, 2017) and burn out (Chang, 2009). Often inseparably connected with workload was due to increasing work demands from leadership team, extensive student performance and short deadlines. Factors such as lack of autonomy, job insecurity, technological challenges also were the reasons for creating stress among teachers (Figure 4).\n\nRelational stress factors\n\nWithin the theme of relational factors, there were many categories which contributed up to 21% of the stress factors. Authors mentioned, parent demands and expectations (Carroll et al., 2020), low level of depersonalization (Luk et al., 2010; Brown & Roloff, 2011; Ross et al., 2011; Klassen et al., 2012; Alloh et al., 2019), Student behaviour & massive class strength, interpersonal relations with colleagues and students, maintaining discipline and teaching preparation (Yerdelen et al., 2016) were the contributing factors under this theme.\n\nIntrapersonal Stress factors\n\nThe theme intrapersonal stress comprised of factors that were not related to work directly. Ibrahim et al. (2021) and Gearhart et al. (2022) found emotional exhaustion and lack of coping abilities with challenges (Alhija, 2015; Mahipalan & Sheena, 2019; Carroll et al., 2020; Hepburn et al., 2021c) as the prime factors for intrapersonal stress. Followed by inequity Work/family conflict (Jerrim & Sims, 2021; Billett et al., 2022; Gearhart et al., 2022), Low self-efficacy level (Vesely et al., 2013; Bermejo-Toro et al., 2015; Prilleltensky et al., 2016), Lack of experience (Ibrahim et al., 2021), Low self-efficacy level (Schnaider-Levi et al., 2017; Braun et al., 2019).\n\nSource: Authors own.\n\nWhat are the measures/interventions adopted in managing stress among schoolteachers?\n\nThe number of therapies, interventions and measures have increased in lowering employee stress in recent years. Although ample research has identified different ways to manage stress among teachers very few studies have carried out scoping reviews to explore intervention or measures in align with psychological well-being. To address the research question, three different ways of measures/interventions were coded and analysed using a thematic analysis approach. There were five themes identified from the selected studies. They are mindfulness-based intervention, improved social support, coping mechanisms, improved managerial support and others.\n\nMindfulness-based intervention\n\nMindfulness can be defined as “a state of being attentive to and aware of what is taking place in the present” (Brown & Ryan 2003, p. 822). Under this theme, Cultivating Awareness and Resilience for Teachers (CARE) (Roeser et al., 2012; Jennings et al., 2013), Stress management and relaxation techniques (SMART) (Roeser et al., 2013; Jennings et al., 2013), Co-ordinated Anxiety Learning and Management program (CALM) (Jennings et al., 2013; Jennings et al., 2019; Hepburn et al., 2021a), Inner Resilience Mindful based training (Frank et al., 2013; Beshai et al., 2015; Dave et al., 2020; Bonde et al., 2022), Emotional Intelligence skill training (Vesely et al., 2013; Schoeps et al., 2019), Mindfulness stress reduction practice such as walking, stretching, body awareness reflection, deep breathing (Jennings et al., 2013; Hepburn et al., 2021a), RCT through training sessions (Siu et al., 2014), training sessions on gratitude, happiness day (an activity which consist of planning and carrying out of positive emotions for example activities such as visit to the parlour for self-care, an outing or musical experiences.), savouring experience (“savoring makes a decisive contribution to experiencing and intensifying positive emotions” Bryant et al. (2011)., letter (Rahm & Heise, 2019), social-emotional skill training (Tarrasch et al., 2020), training & classes to promote well-being (Tebben et al., 2021), Mentalising (Schwarzer et al., 2021), intervention - brief PPIs (BPPIs)- (Shankland & Rosset, 2016), Mindfulness training (Bonde et al., 2022) were identified. Recently yoga based intervention (Harris et al., 2015; Telles et al., 2018; Dyer et al., 2020; Hepburn et al., 2021c) was the most commonly adopted intervention to overcome stress.\n\nImproved social support\n\nUnder this theme, the measures which are adopted through social support such as colleagues, students and family were recorded. They are: building of inter-personal relationships (Ibrahim et al., 2021), Positive interactions with students (Ross et al., 2011), collaboration between teachers and colleagues (Helms-Lorenz & Maulana, 2016; Round et al., 2022), Positive social climate (Aldrup et al., 2017), interpersonal support environment (Soykan et al., 2019), mentor and coach collaborative and reflective relationship (Hollweck, 2019; Carroll et al., 2020), Community bonding (Gearhart et al., 2022), Positive and supportive relations with colleagues and parents and collective culture (Skaalvik & Skaalvik, 2017) were identified under this theme.\n\nCoping mechanisms\n\nCoping mechanisms which were employed to overcome stress has been recorded under this theme. Jennings found that emotional skill instruction helped in managing the prevailing stress (Jennings et al., 2013). School enculturation was the technique used by Helms (Helms-Lorenz & Maulana, 2016). Followed by an improvement in communication skills (Carroll et al., 2020), sessions on stress management techniques (Li et al., 2016), cognitive and behavioural coping (Bermejo-Toro et al., 2015; Prilleltensky et al., 2016; Aulén et al., 2021; Turner et al., 2021; Ciuhan et al., 2022) feeling valued and fringe benefits (Gearhart et al., 2022), workplace change (Carroll et al., 2020) and health promotion (Vazi et al., 2013) were gathered from the selected studies.\n\nImproved managerial support\n\nThis theme comprises of the measures which was adopted by management to reduce stress mong teachers. Many researchers found building of interpersonal relationships among colleagues and management helped in reducing stress (Wu et al., 2006; Luk et al., 2010; Ibrahim et al., 2021; Prilleltensky et al., 2016) further, positive interaction with students (Ross et al., 2011), Positive & supportive relations with colleagues and parents, collective culture (Skaalvik & Skaalvik, 2017) and community bonding (Gearhart et al., 2022) were the thoughts adopted by management in lowering stress among teachers (Figure 6).\n\nSource: Authors own.\n\n\nDiscussion\n\nHoudmont, Cox, and Griffiths (2010) argued, the assessment of interventions related to work is challenging; framing, conceptualising and methodology regarding interventions that supports to overcome the problem is still an open debate. \"The decision about which type of intervention to use should be based on a thorough assessment of the specific situation rather than some general principles” (Briner and Reynolds 1999, p. 658). Interventions cannot be carried out purely based on broad theoretical ideas; they must be tailored to the unique organizational context in which they are implemented. Ecologically sound and measurable interventions are required. Taking this viewpoint into account, the goal of this study was to a set out with the main aim of identifying the stress factors and assessing the significance of interventions/measures to reduce stress for the psychological well-being among schoolteachers. Additionally, the study also aimed to ascertain the overview of methodologies, geographical distributions, theoretical underpinnings and publication period with the said topic. Therefore, to address the research questions, scoping review methodology adhering to JBI guidelines was adopted.\n\nThe results of the review showed that the articles on stress aligned with psychological well-being among schoolteachers were few in the year 2010, gradual increase was recorded between 2013 to 2020 and exponential growth was recorded in the year 2021. The significant growth in the articles is associated with the outbreak of the global pandemic, called for incorporating the additional factors such as technological challenge (Farley & Chamberlain, 2021; Ibrahim et al., 2021), lack of work life balance (Billett et al., 2022; Gearhart et al., 2022), student behaviour management (Tebben et al., 2021; Hepburn et al., 2021a) and online teaching (Billett et al., 2022) along with the prevailing factors that elevated stress among the teaching profession.\n\nThe geographical distribution of the study shows that most studies emerged from US (n = 16) followed by UK, Finland and Australia (n = 5). The geographical distribution of the selected articles in the review demonstrates stress and interventions as a measure to overcome stress has captured global attention. However, the review highlights a lacuna of studies emerging from developing countries such as India, Iran and Turkey.\n\n60 studies ranging from the year 2010 to 2022 were selected that fulfilled inclusion and exclusion criteria set out for the study. Among them, 37 studies adopted quantitative method, 13 studies adopted qualitative method and 10 articles adopted mixed methodology. It is apparent from the review that, quantitative research design was extensively used method by the researchers to examine the phenomena.\n\nAn application of a strong theoretical background enhances the outcome of the study in terms of proposing theoretical and practical implications. Through review it was observed that researchers have adopted various psychological, psychosocial, and behavioural theories. JD R theory (Demerouti et al., 2001) strongly guided the stress and well-being literature. However, these results are not surprising as the existing literature has confirmed the application of JD R models from past two decades. Furthermore, positive emotion, engagement, positive relationships, meaning and accomplishment which is termed as “PERMA” (Seligman 2011) was used as a core theory to measure the psychological well-being. This model has exhibited a great heuristic power in stimulating enormous studies due to integration of various theories in positive psychology.\n\nData from teaching and learning survey reports confirmed the existence of high level of stress in teaching profession (Carroll et al., 2022). High level of stress impacts the job performance, mental and physical health. Another study by (Della Valle et al., 2020) expressed chronic stress among employees results in huge economic burden due to low productivity and engagement. The current study emphasised on identifying the drivers of the stress. The stressors were classified into intrapersonal, systematic, relational and organisational stressors. Among the studies included in the review it could be observed that workload (22 studies), time pressure and gap between job demand and availability of resources (6 studies), administration overload (4 studies), role conflict and ambiguity (4 studies), poor working condition (4 studies), job insecurity (3 studies), lack of administrative support (2 studies), and lack of appreciation (1 study) were the commonly studied organisational stress factors. Further, massive class strength, poor relationship between teacher, student, and colleagues (13 studies), managing supervisors (9 studies), low levels of depersonalization (5 studies) and managing parents demand and expectations (2 studies) were highlighted under relational stress factors. Additionally, intrapersonal factors such as Low self-efficacy level (10 studies), emotional exhaustion (8 studies), Inequity Work/family conflict (7 studies), moderate levels of personal accomplishment (4 studies), Lack of coping abilities with challenges (3 studies), Lack of experience (3 studies), Lack of time (2 studies), Lack of motivation (2 studies) Personal challenges (1 study). Among the mentioned factors “workload”, poor relation between students, teachers and colleagues”, low self-efficacy and emotional exhaustion were frequently studied by the researchers. There were relatively lower studies focusing on policy related factors. Overall researchers empirically validated that excessive stress levels caused unpleasant outcomes such as low productivity, dissatisfaction, job turnover, low job performance, increased anxiety levels, sleep disorder, insomnia and chronic health problems. Moreover, the negative consequences of stress did not restrict only to the facilitators but also affected the students' academic performance. Stress management interventions act as an antidote for overcoming the negative consequence of stressors. Various strategies have been developed by researchers to reduce the impact of stress on the well-being of the desired population. Stress management interventions at the workplace emphasise on designing ameliorative tactics to prevent and restore the well-being of employees through promotive wellness programs. Therefore, interventions identified through this scoping review were aligned with the stress factors which were deployed to overcome stress among the teaching profession.\n\nThe most common intervention adopted to overcome organisational, intrapersonal and relational stressors was mindfulness (30 studies). It mainly comprised of CARE, CALM and SMART programmes. These programmes were conducted based on randomised control trials. Study by (Roeser et al., 2012; Jennings et al., 2013, 2019; Carroll et al., 2020) examined the effectiveness of CARE program package such as emotional skills instructions, mindful awareness, caring and compassion practices. It resulted in proximal outcomes such as emotional stability, decrease in psychological and physical distress. The cohort which underwent this program, have shown massive difference in teaching efficacy and reported lower level of anxiety (Jennings et al., 2019). Studies by (Jennings et al., 2013, 2019; Telles et al., 2018; Rahm & Heise, 2019; Dyer et al., 2020; Hepburn et al., 2021a) implemented CALM program. It included sessions on yoga, relaxation, breathing exercises, gratitude, loving and caring, self-care and shared experience with colleagues. The results of CALM and SMART program showed significant increase or improvement in work related functioning, better adaptability towards work climate, reduction in emotional exhaustion (Roeser et al., 2012; Jennings et al., 2013). Recently investigators have introduced innovative mindfulness techniques such as emotive behaviour theory, inquiry-based stress reduction and mentalising programs. It was observed that these interventions manifested positive improvements in productivity and wellness of controlled group participants.\n\nThrough review it was evident that relational and intrapersonal stressors were second contributors for the persistent stress level among teachers. Coping mechanism (14 studies) such as social support (12 studies) and improved managerial (8 studies) consisting of individual and organisational interventions were designed to overcome the above-mentioned stress factors. The interventions were delivered in the form of seminars, training session on stress awareness, stress management tactics, conflict management, school enculturation technique, cognitive and behavioural coping (Bermejo-Toro et al., 2015; Prilleltensky et al., 2016; Aulén et al., 2021), workplace change (Carroll et al., 2020). That resulted in meeting the job demands, having a work life balance and improved student teacher relationship, meeting the expectations of management, colleagues and parents, increase in self efficacy and improved work engagement.\n\nThe current scoping review have certain limitations. At the first instance, the articles are published in English and only three available databases were considered for the study, which resulted in loss of data reported in other languages or the studies from other databases. Secondly, articles that examined stress factors in alliance with psychological well-being were only considered, hence further researchers can explore the association of stress with occupational well-being. Thirdly, this review was limited to only schoolteachers from primary and secondary level school, kindergarten and higher education teachers can be the future scope to study and analyse the stress factors and the interventions.\n\nThough it can be observed, researchers have adopted diverse stress management interventions to cope with the stress and enhance well-being. In summary it can be concluded all the strategies aimed at attaining balance to work life and promote overall growth and development of teachers professionally and personally. Yoga intervention programs were adopted as a mechanism to overcome stress. For instance, Harris et al. (2015) conducted sessions on gentle yoga for educators which included meditation practices. Another group of researchers implemented interventions through residential programs which mainly comprised of bhakti yoga (mediation on Sanskrit syllable), Surya namaskar (sun salutation), Sukshma vyayama (relaxation exercise), shavasana (guided relaxation), pranayama (voluntarily regulated breathing, aasanas (posture) and meditation (Telles et al., 2018; Dyer et al., 2020) designed a 3-days program comprising of physical exercises and yoga postures, sitting meditation and breathing exercises (Hepburn et al., 2021b) introduced meditation practices (samyama), ethical principles (yama niyama), posture asana (asanas). All these yoga interventions focused on eliminating personal stress. Therefore, future researchers can evaluate the effectiveness of these intervention at an organisational level. There were relatively lower studies focussing on the systematic stress factors. Hence, future research can focus on exploring the impact of policy related changes on the well-being of teachers. Additionally, a larger emphasis can be placed on a qualitative approach to better understand the fundamental problems relating to the workplace, work climate, and work culture. Lastly, it was observed that the definition of psychological well-being has been operationalized according to the context of the study by different authors, therefore, future research can explore in framing a common definition through a meta-analysis considering heterogeneity.\n\n\nConclusion\n\nThis scoping review aimed at identifying the stress factors and the interventions/measures followed to overcome the stress among teaching profession. The stress management techniques have identified numerous health benefits when the teachers become able to handle stressful situations. Programs using different approaches are required to help the teaching professionals to deal with different stress factors inherent in their cognitively challenging and physically demanding work. A successful intervention must address the genuine issues such as the social, emotional, or physical challenges that teachers are experiencing and assist them in coping with their demanding circumstances. The study outcome helps the school management to ensure psychological well-being through interventions. The higher the psychological well-being results in overcoming depression, poor performance, poor performance, poor teacher student relationship, lack of self-confidence and improved quality in the classroom. Additionally, this study helps managers working in consultancies and organizations involved in manpower training for education industry to think and apply intervention to attain better harmony of our real drivers of the education sector.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare. Annexure A final.doc DOI: 10.6084/m9.figshare.22324336\n\nThis project contains the following underlying data:\n\nTabular representation of the included studies for the review\n\nFigshare. Supplementary file - Figures.docx DOI: 10.6084/m9.figshare.22324549\n\nThis project contains the following underlying data:\n\n• Supplementary file – Figures\n\n• Prisma check list\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\nReferences\n\nAldrup K, Klusmann U, Lüdtke O: Does basic need satisfaction mediate the link between stress exposure and well-being? A diary study among beginning teachers. Learn. Instr. 2017; 50: 21–30. Publisher Full Text\n\nAlhija FN-A: Teacher Stress and Coping: The Role of Personal and Job Characteristics. Procedia. Soc. Behav. Sci. 2015; 185: 374–380. 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Publisher Full Text\n\nMahfar M, Xian KH, Ghani FA, et al.: Irrational Beliefs as Mediator in the Relationship Between Activating Event and Stress in Malaysian Fully Residential School Teachers. Asian Soc. Sci. 2018; 14(10): 21. Publisher Full Text\n\nMahipalan M, Sheena S: Workplace spirituality, psychological well-being and mediating role of subjective stress. Int. J. Ethics Syst. 2019; 35(4): 725–739. Publisher Full Text\n\nMoy FM, Hoe VCW, Hairi NN, et al.: Cohort study on clustering of lifestyle risk factors and understanding its association with stress on health and wellbeing among school teachers in Malaysia (CLUSTer) – a study protocol. BMC Public Health. 2014; 14(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrilleltensky I, Neff M, Bessell A, et al.: Teacher Stress: What It Is, Why It’s Important, How It Can be Alleviated. Theory Pract. 2016; 55(2): 104–111. 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Publisher Full Text" }	[ { "id": "205242", "date": "05 Oct 2023", "name": "Ana Patricia Ayala", "expertise": [ "Reviewer Expertise My area of research include knowledge syntheses", "transparency in reporting", "development of reporting guidelines" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity of reviewing this article.\nMy observations focus in two main areas: The clarity of the study regarding grammar, and syntax, and in the methodological adherence, and transparency of the review.\nIntroduction\nI suggest adding citations to support the statements regarding teachers' effectiveness.\n\nDefine well-being. This is a broad term and includes numerous aspects that contribute to it.\n\nPlease clarify how developing organizational understanding is linked to bestowing this responsibility to the educational institution. This also a broad term. What does this imply? High schools? Universities?\nMethods\nCan you provide a link to the protocol for this scoping review? Was a protocol developed? All scoping reviews are protocol driven reviews, and an a priori protocol is necessary to mitigate bias.\n\nCan you please indicate what reporting guideline you've used to report the study? I don't see mention of the authors' using PRISMA-SCr, the reporting guideline for Scoping reviews.\nResearch questions\nIt would be extremely helpful if authors kindly clarified what is meant by item a. \"What are the descriptive findings of the selected studies?\"\n\nWhat does descriptive findings mean?\n\nSearch strategy\nCan authors please provide the full search strategies for all databases searched exactly as they were ran? I'm afraid the way that the search has been described is not reproducible or transparent. From the information that is supplied, the search does not seem comprehensive enough to have effectively captured all the information regarding this topic.\nThere is no information regarding the date the search was ran, or updated, if it was updated at all. What platforms were used. Scopus and Web of Science are not databases, they contain many databases and different collections.\n\nI strongly suggest authors use PRISMA-Search to report the findings of their search, as well as their documentation of the search process.\nThe sources of information are not sufficient to capture the topic at hand. Sources like APA PsycINFO, EBSCO CINAHL, and education focused databases should've been searched as well.\nThe exclusion of grey literature should be addressed in the limitations of the study\n\nInclusion and exclusion criteria\nKindly explain why the authors only looked at that date range? What is the justification to only include articles from these years?\nThis should be included as a limitation to the study, and the steps the authors took to ameliorate it.\nCould authors kindly indicate how the first and second level of screening were conducted? In the description under inclusion decision based on inclusion and exclusion criteria, the authors don't explicitly state how they performed each level of screening, and they must do so for transparency purposes.\n\nI also recommend they change the title to \"Study selection process\" to more accurately reflect the actions taken, and in accordance with language used in the methodologies the authors' cite for conduct.\nCharting the data\nPlease describe how the data extraction form was piloted\n\nIs the topic of the review discussed comprehensively in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] }, { "id": "226814", "date": "24 Apr 2024", "name": "Moh'd Shoqeirat", "expertise": [ "Reviewer Expertise - this paper is critical and worth publishing", "as it touches on an essential societal profession. The scientific procedure followed in this study is well-defined and appropriate for such a 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\nReview for the paper: Prevalence of stress and its relevance on the psychological well-being of the teaching profession: A scoping review\n\nThe review covers the period between 2010 to 2022. Although the title implies all the teaching professions, the study sample includes only school teachers. The study scope is well-defined, identifying the stress factors and the interventions/measures followed to overcome the stress in the teaching profession. The criteria for selecting the sample and the exclusion was well defined. I believe the topic of the study is significant as it takes the school teachers as a sample, and the procedures used are suitable for this kind of study. The statistics and layout of the results clarify the scope of the study. The discussion includes increased research in the field (from three countries), theoretical perspective and stress intervention. The authors clearly clarify the limitations of the study. The conclusion: The paper identifies the benefit of higher psychological well-being of the teachers' profession. Also, they stress that the study results help managers working in consultancies and organisations think and apply the intervention better to harmonise our real drivers of the education sector. It seems that the English language may need revision. Finally, this paper is critical and worth for approving, as it touches on an essential societal profession. The scientific procedure followed in this study is well-defined and appropriate for such a study.\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/12-424
https://f1000research.com/articles/12-776/v1	04 Jul 23	{ "type": "Research Article", "title": "Perception and attitude towards online clinical modules: a cross-sectional study among medical students from two countries", "authors": [ "Heraa Islam", "Mohsin Nazeer Muhammed", "Sindhura Lakshmi", "Aditi Kapoor", "Afraz Jahan", "Akhila Doddamani", "Nagaraja Kamath", "Muhammed Ehsan", "Suma Nair", "Heraa Islam", "Sindhura Lakshmi", "Aditi Kapoor", "Afraz Jahan", "Akhila Doddamani", "Nagaraja Kamath", "Muhammed Ehsan", "Suma Nair" ], "abstract": "The coronavirus disease (COVID-19) pandemic has significantly affected the world, including the education system, in various ways. In this study, we intended to explore the merits and demerits of online clinical learning and its effect on medical education from a student’s perspective. The study also assessed final-year medical students’ perception of and attitude towards, online clinical modules. This observational study was carried out in the Department of Community Medicine, Kasturba Medical College, Manipal (KMC) in collaboration with King’s College London, UK (KCL). In our study, a total of 42 students were enrolled, with 37 students from KMC and 5 students from KCL. In total 81% of students reported that they were not willing to continue with the online mode of learning. The abrupt switch to e-learning without prior preparation has exposed some pitfalls that must be attended to. Contrary to other fields, the medical field places much importance on offline clinical teaching, which has recently been impacted by the shift to online teaching. The survey responses were analysed for the improvisation of online clinical modules as well as to come up with better ideas and outcomes since this mode of learning may have to continue till the spread of the disease is under control.", "keywords": [ "E-Learning", "Perception", "Assessment", "COVID-19" ], "content": "Introduction\n\nThe coronavirus disease (COVID-19) pandemic has significantly affected the world, including the education system, in various ways. The COVID-19 pandemic forced many schools and colleges to remain closed temporarily. In several parts of the world, many students missed at least one semester, with some students missing as much as a year of offline teaching. Teaching in medical school has traditionally been didactic, with clinical postings in the hospital. The WHO (World Health Organization) labeled the coronavirus outbreak a pandemic on March 11, 2020.1,2 To battle this, several novel methods were employed like social distancing, masking, hand hygiene, and vaccination. The education sector has undergone substantial changes so that it can cope with the current situation.3 Universities and colleges have employed a transition from traditional face-to-face teaching to online teaching or a hybrid of face-to-face teaching with online modules.4 As the time spent on online education increases, a study in the United States showed that many educators are transforming their face-to-face teaching into an online format.5 Therefore, to build a robust teaching tool to continue medical teaching during the pandemic, it is necessary to involve the students’ ideas and feedback to improve the online teaching modules. Online modules have restricted students from learning through bedside clinical teaching. However, this was necessary as there was a significant risk of medical students contracting the virus and spreading it within the community.6,7 In this study we explored the merits and demerits of online clinical learning, its effect on medical education from the perspective of a student and assessed the perception and attitude of final year medical students towards online clinical modules.\n\n\nMethods\n\nThis observational study was carried out in the Department of Community Medicine, Kasturba Medical College (KMC) Manipal, and King’s College London (KCL), UK. In our study, a total of 42 students were enrolled from KCL (5 students) and KMC (37 students). The recruitment of participants was through Google forms, data from which were directly populated to the primary student researcher from KMC. Final year MBBS students from KCL and KMC college were RANDOMLY picked up or the survey. The authors from KMC analyzed the combined data.\n\nFinal year MBBS students from KCL (who participated in the Virtual Global Health Elective between the two institutions) and students from KMC Manipal who were in Final year MBBS, as of June 30th, 2020, and underwent clinical teaching on an online platform during their academic year.\n\nStudents attempting the final year exam for the second (or more) time were excluded from the study.\n\nA questionnaire was used to conduct the survey. It was structured on an electronic database using Google forms and it was open for one month. The questionnaire was divided into four sections. The first section contained the disclaimer and the informed consent. This was followed by the participant information sheet in the second section. The third section addressed the general information on the participants like gender, which college they belong to, the year of medical school they are in, and the duration of online teaching exposure they have received. The final section of the questionnaire asks the students various questions about online clinical modules, their perception and attitude toward e-learning, and the applicability of online clinical teaching as a modus operandi of teaching in the future. The questionnaire provided to the participants was in English. The characteristics and replies of respondents were analyzed using descriptive statistics such as frequencies and percentages.\n\nInformed consent was obtained from the participants before the study initiation. The study was approved (526/2021, dated July 14th, 2021) by the Institutional Ethics Committee (IEC, Registration No- ECR/146/Inst/KA/2013/RR-19 clearance) from KMC. Data obtained through this survey will not be transferred to the foreign collaborator. Collaboration with KCL was only in place until the collection of data through a Google form created by the primary student researcher. This data will be directly accessible only to the primary student researcher and will not be shared with the foreign collaborator.\n\n\nResults\n\nIn our study, a total of 42 students were enrolled, 24 were male and 18 were female (Table 1 and Figure 1).20 26 out of 42 students received in-person clinical exposure for more than 6 months (Table 2 and Figure 2). Around 16 students were not able to grasp clinical concepts well through online sessions (Table 3 and Figure 3). In total, 39 students felt that their attention had been affected in online mode in comparison with attending clinics in person (Table 4 and Figure 4). In the absence of peers, the motivation level was altered in 36 of our students (Table 5 and Figure 5). Because of online clinical modules a lot of atmosphere distractions were present as per our study (Table 6 and Figure 6). More than half the students disagreed to the fact that they were trained by online class to take clinical cases independently (Table 7 and Figure 7). 28 of 42 students felt that their interpersonal skills were affected due to the pandemic (Table 8 and Figure 8). Giving online presentation to the class was challenging to 17 out of 42 students (Table 9 and Figure 9). At the same time most of the students were able to get their doubts solved during online lectures (Table 10 and Figure 10). Due to online teaching, students also felt that demonstrating a clinical sign on a patient in future would be challenging (Table 11 and Figure 11). 14 out of 42 students were not satisfied with their performance in final clinical evaluation (Table 12 and Figure 12). More than half the students were familiar with hospital procedures like sending investigations and preparing discharge letters (Table 13 and Figure 13). In addition, 22 students were overwhelmed about the responsibilities that they will have to undertake as a junior doctor (Table 14 and Figure 14). More than half the students in the study agreed on the fact that online mode of learning has given them more time to explore extracurricular interests (Table 15 and Figure 15). The online mode of learning was not interesting for students, and they did not wish to continue with this (Tables 16, 17 and Figures 16, 17).\n\n\nDiscussion\n\nDuring the COVID-19 pandemic, we investigated numerous components of e-learning that students at KMC Manipal and KCL encountered in terms of technical preparedness, teaching, and learning experience, engagement and communication, and assessment methodologies. While online learning may be more appropriate for preclinical students, it may not be the most ideal for their senior counterparts during the pandemic. The students from KCL experienced rotations like short- and long-term conditions, acute care, GP, and women’s health online while students from KMC Manipal had online rotations in medicine, surgery, OBGyn, orthopedics, and pediatrics. Importantly, these rotations are vital in imparting clinical knowledge to medical students. Moreover, it is crucial for final year students to have adequate contact with patients and bedside exposure to develop good communication and clinical skills.\n\nSimilarly, as an alternative to clinical placements, students at Imperial College London were exposed to Tele-teaching through computers in hospital settings. However, it was found that student-patient interaction was still lacking.8\n\nIn our study, a total of 42 students were enrolled, 24 were male and 18 were female. Only four students from online classes felt that they were effectively trained to take clinical cases independently, while 37 students reported that they were not effectively trained to take clinical cases independently. Furthermore, 22 students agreed on learning atmosphere distractions and 19 students reported that their interpersonal skills were affected during the pandemic.\n\nEvery e-learning system establishes its foundation of computers, networks, communications, and technical facilities along with information technology professionals to continuously maintain and upgrade the system, train users, and provide technical support.9 Appropriate technological support and maintenance of the available hardware and software are of great value for optimal utilization of technology by both educators and students alike.10–12\n\nE-learning modalities face several barriers and challenges. One of these is poor motivation and an expectation to be able to meet their personal and professional needs and goals.13 Internal factors such as poor engagement, poor perception and motivation, high levels of anxiety and stress, and poor interactions between learners and facilitators all impede the process of learning and motivation.14,15\n\nThe majority of the students reported disinterest in the online mode of learning. The students experienced fatigue from online classes and difficulty in enforcing self-discipline, along with the absence of usual social interaction which led to a decrease in attendance as well as engagement during the virtual sessions. Stress has been more often associated with e-learning than with traditional learning.16,17\n\nIn our study, only 19% of students wish to continue the online mode of learning while 81% of students reported that they were not willing to continue with the online format. Varied results have been reported in the literature when comparing student satisfaction in face-to-face and online courses, with a higher proportion of students preferring face-to-face classes.18\n\nThere were diverse opinions obtained from our survey regarding the experience of students with the technical aspects of online classes. Some felt that these classes helped them explore their extracurricular interests and, as the need of the hour, the classes were good and manageable. However, most students were not satisfied, found it difficult to focus, and felt that it should never be a substitute for the offline practical learning experience. Some of the technical drawbacks included network and connectivity issues, difficulties with the application, and video/audio quality. These issues made it difficult, especially when the students were looking forward to a topic of interest. Moreover, while some facilitators were more adept at using online platforms than others, oftentimes the time taken to set up the online lecture took away from the overall classroom experience. The same professors who had previously engaged the classroom and involved the students during face-to-face lectures had now resorted to monotonous online lectures. Thereby, most of the students participating in this study stated that they would not like to take more e-learning classes if they were given the option. The consensus was that the long and draining online classes had now given the students renewed vigor and zeal to attend face-to-face classes whenever possible.\n\n\nConclusion\n\nThe sudden shift to e-learning without prior preparedness has revealed some pitfalls that need to be addressed. The central hypothesis is that COVID-19 has impacted the academic performance and clinical skills of medical students. The responses were analyzed for improvisation of online clinical modules as well as to come up with better ideas and outcomes since this mode of learning may have to continue till the spread of the disease is under control. In the near future, with the re-emergence of the pandemic and the advent of modernization and the digital world, e-learning and e-teaching should be incorporated in the medical curriculum along with clinical teaching.\n\nMedical education places an emphasis on offline clinical teaching with the most ideal learning known to happen at the bedside. As a result, given the current pandemic scenario, medical educators must develop new approaches to imparting knowledge to medical students; they can no longer rely on markers and whiteboards, or chalks and blackboards.\n\nSince the online mode of learning may have to continue for the foreseeable future, one of the effective methods that can be adopted to create the most effective multimedia learning experiences are Mayer’s 12 principles of multimedia learning.19 These principles can be used as guidelines to develop productive digital learning experiences.", "appendix": "Data availability\n\nFigshare: Perception and attitude towards online clinical modules: A Cross-sectional study among medical students from 2 countries, https://doi.org/10.6084/m9.figshare.21857076.v2. 20\n\nThis project contains the following underlying data:\n\n• Survey (Responses).xlsx\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\nAcknowledgment\n\nWe wish to extend our heartfelt gratitude to the final year medical students from King’s College who were kind enough to complete the survey. The writing of this article was supported by a medical writer at Medwiz Healthcare Communications Private Ltd.\n\n\nReferences\n\nMorens DM, Folkers GK, Fauci AS: What is a pandemic?2009; pp. 1018–1021.\n\nWHO Director-General's opening remarks at the media briefing on COVID-19.2020 Mar. Accessed on March 11 2020. Reference Source\n\nHixon E, Buckenmeyer J, Barczyk C, et al.: Beyond the early adopters of online instruction: motivating the reluctant majority. Internet High. Educ. 2012; 15: 102–107. Publisher Full Text\n\nEdginton A, Holbrook J: A blended learning approach to teaching basic pharmacokinetics and the significance of face-to-face interaction. Am. J. Pharm. Educ. 2010 Jun 15; 74(5): 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrleans M, editor. Cases on critical and qualitative perspectives in online higher education. IGI Global; 2014.\n\nKhasawneh AI, Humeidan AA, Alsulaiman JW, et al.: Medical Students and COVID- 19: Knowledge, Attitudes, and Precautionary Measures. A Descriptive Study From Jordan. Front. Public Health. 2020 May 29; 8: 253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkers A, Blough C, Iyer MS: COVID-19 Implications on Clinical Clerkships and the Residency Application Process for Medical Students. Cureus. 2020 Apr 23; 12(4): e7800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMian A, Khan S: Medical education during pandemics: a UK perspective. BMC Med. 2020; 18(1): 100. Published 2020 Apr 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIbrahim NK, Al Raddadi R, Al Darmasi M, et al.: Medical students’ acceptance and perceptions of e-learning during the Covid-19 closure time in KingAbdulaziz University, Jeddah. J. Infect. Public Health. 2021; 14(1): 17–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWentink MM, Siemonsma PC, van Bodegom-Vos L , et al.: Teachers’ and students’ perceptions on barriers and facilitators for eHealth education in the curriculum of functional exercise and physical therapy: a focus group study. BMC Med. Educ. 2019; 19: 343. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEimer C, Duschek M, Jung AE, et al.: Video-based, student tutor- versus faculty staff-led ultrasound course for medical students – a prospective randomized study. BMC Med. Educ. 2020; 20: 512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulz P, Sagheb K, Affeldt H, et al.: Acceptance of e-learning devices by dental students. Med. 2013 Aug 14; 2(2): e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRegmi K, Jones L: A systematic review of the factors - enablers and barriers- affecting e-learning in health sciences education. BMC Med. Educ. 2020 Mar 30; 20(1): 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFawaz M, Samaha A: E-learning: Depression, anxiety, and stress symptomatology among Lebanese university students during COVID-19 quarantine. Nurs. Forum. 2021 Jan; 56(1): 52–57. PubMed Abstract \| Publisher Full Text\n\nHammarlund CS, Nilsson MH, Gummesson C: External and internal factors influencing self-directed online learning of physiotherapy undergraduate students in Sweden: a qualitative study. J. Educ. Eval. Health Prof. 2015; 12: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazarevic B, Bentz D: Student perception of stress in online and face-to-face learning: the exploration of stress determinants. Am. J. Dist. Educ. 2020; 35: 2–15. Publisher Full Text\n\nHendrix E: Assessing student stress in clinical laboratory science programs: online versus face-to-face. Clin. Lab. Sci. 2016; 29(2): 99–100. Reference Source\n\nGarratt-Reed D, Roberts LD, Heritage B: Grades, Student Satisfaction and Retention in Online and Face-to-Face Introductory Psychology Units: A Test of Equivalency Theory. Front. Psychol. 2016; 7: 673.\n\nMayer RE: Introduction to multimedia learning. Cambridge Handbook Multimedia Learning. 2005; 15(2): 1–24. Publisher Full Text\n\nRaut M: Perception and attitude towards online clinical modules: A Cross-sectional study among medical students from 2 countries. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "198158", "date": "04 Oct 2023", "name": "Gehanath Baral", "expertise": [ "Reviewer Expertise Obsterics", "Gynecology", "Oncology", "Clinical Research" ], "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\nWrite the interpretation of information in a Table or Figure as a summary finding followed by a Table or Figure for easy understanding of the result. At the end of the paragraph, there must be a reference to the relevant Table or Figure in parentheses.\n\nKeep either Table or Figure for the same information. Information is duplicated.\n\nTable/Figure 1 information on the gender of participants can be displayed in text only. There is unnecessary space occupied by these.\n\nTable/Figure 7 information on the “Trained by online classes to take clinical cases independently” should have been verified to publish the result. It means that research should not highlight misperceptions if any. Verifiable information should be presented.\n\nTable/Figure 9 information on “Giving online presentations during class is challenging” doesn’t clarify the presentations delivered by teachers or students to judge in terms of a CHALLENGE.\n\nTable/Figure 12 information on “Performance in final clinical evaluation” is not clear whether it is on patient evaluation or clinical reports or case scenarios; and how the performance was measured or gauged.\n\nTable/Figure 13. Familiar with hospital procedures like sending investigations and preparing discharge letters needs objective verification. Such learning requires a set/validated teaching procedure – is it used or not?\n\nTable/Figure 14 information on “Working as a junior doctor in the following year” seems to be an assumption or hypothetical. For this research, design should be different like time series information on online and offline experiences.\n\nTable/Figure 17. Wish to continue online mode of learning in the future. Without looking at its impact, the data shown here will not be realistic. Because the level of knowledge and skill acquisition by a medical student is for real-time implementation, theoretical knowledge will not suffice for medical education.\n\nDiscussion on technology is not supported by results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10617", "date": "15 Jan 2024", "name": "Mohsin Naseer Muhammed", "role": "Author Response", "response": "1. Write the interpretation of information in a Table or Figure as a summary finding followed by a Table or Figure for easy understanding of the result. At the end of the paragraph, there must be a reference to the relevant Table or Figure in parentheses. Response: Interpretation of information in a Table or Figure as a summary finding was added. 2. Keep either Table or Figure for the same information. Information is duplicated Response: Bar graphs was retained and Tables were deleted as per the recommendation 3. Table/Figure 1 information on the gender of participants can be displayed in text only. There is unnecessary space occupied by these. Response: Table was deleted 4. Table/Figure 7 information on the “Trained by online classes to take clinical cases independently” should have been verified to publish the result. It means that research should not highlight misperceptions if any. Verifiable information should be presented. Response: The question was verified with the students before analysing the result part. 5. Table/Figure 9 information on “Giving online presentations during class is challenging” doesn’t clarify the presentations delivered by teachers or students to judge in terms of a CHALLENGE. Response: The term challenging was changed to difficult task. 6. Table/Figure 12 information on “Performance in final clinical evaluation” is not clear whether it is on patient evaluation or clinical reports or case scenarios; and how the performance was measured or gauged. Response: The Performance in final clinical evaluation is measured on clinical case scenario evaluation individually and it was added now. 7. Table/Figure 13. Familiar with hospital procedures like sending investigations and preparing discharge letters needs objective verification. Such learning requires a set/validated teaching procedure – is it used or not? Response: Such learning requires a set/validated teaching procedure was used for students to familiarize with hospital procedures like sending investigations and preparing discharge letters 8. Table/Figure 14 information on “Working as a junior doctor in the following year” seems to be an assumption or hypothetical. For this research, design should be different like time series information on online and offline experiences. Response: Though the question was hypothetical, since it was a pandemic and necessity of doctors were highly required at the time of study. Hence it was added to know their mind set. 9. Table/Figure 17. Wish to continue online mode of learning in the future. Without looking at its impact, the data shown here will not be realistic. Because the level of knowledge and skill acquisition by a medical student is for real-time implementation, theoretical knowledge will not suffice for medical education. Response: Majority of the medical students strongly disagreed online mode of learning in the future since hands on skills will not be adequate. Hence it was added in result to highlight its importance among medical students. 10. Discussion on technology is not supported by results. Response: Discussion part is modified accordingly." } ] }, { "id": "198153", "date": "04 Oct 2023", "name": "Carlos Trenado", "expertise": [ "Reviewer Expertise Neuroscience", "Neuroeducation", "Clinical Neuroscience. Computational Modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 observational study focuses on differences between online and offline learning in the medical setting. The authors designed a questionnaire in which students expressed their views. The study is valuable in highlighting some issues with the sudden adoption of an educational style, nevertheless some issues remain.\nComments:\n- The authors expressed that participants were exposed to online clinical training, was online training similar to all students at the same institution? Was it different? Please provide more information about the online training received and any differences between them. Was the online lecture presented with slides? Are there any other features such as sound, etc.?\n- What was the duration of a typical online session?\n- While the authors emphasize several elements as contributing factors for a low of interest in online education, it is not clear which one of them was more influential. I strongly recommend a regression analysis to extract information about what factors were more influential in predicting a particular preference.\n- Please provide data on students that suffered from technical difficulties. I think this factor should be considered in the regression model.\n- I recommend the use of bar graphs instead of tables for each of the items described. Please omit tables.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10588", "date": "15 Jan 2024", "name": "Mohsin Naseer Muhammed", "role": "Author Response", "response": "Comment 1. The authors expressed that participants were exposed to online clinical training, was online training similar to all students at the same institution? Was it different? Please provide more information about the online training received and any differences between them. Was the online lecture presented with slides? Are there any other features such as sound, etc.? Response: The online training given in our study was same for all the students in both the institutions. Information regarding online training modules were added. The online lecture was presented with slides in Zoom and Google platforms which includes clinical contents, videos, images, diagrams and flow charts etc. Comment 2. What was the duration of a typical online session? Response: One hour for one topic Comment 3. While the authors emphasize several elements as contributing factors for a low of interest in online education, it is not clear which one of them was more influential. I strongly recommend a regression analysis to extract information about what factors were more influential in predicting a particular preference. Response: Our study utilized a descriptive quantitative design to obtain the opinions of the respondents. The recommendation by the reviewer for regression analysis will be followed to extract information about the factors involved to predict particular preference in our future studies. Comment 4. Please provide data on students that suffered from technical difficulties. I think this factor should be considered in the regression model. Response: The technical difficulties faced by the students during the study was added. Comment 5. I recommend the use of bar graphs instead of tables for each of the items described. Please omit tables. Response: Tables were deleted as per the recommendation" } ] }, { "id": "193973", "date": "04 Oct 2023", "name": "Ivan Buljan", "expertise": [ "Reviewer Expertise Evidence based medicine education", "Science communication", "Psychology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript.\n\nI commend the authors for exploiting COVID situation to assess the attitudes of students towards online learning. However, in my opinion, the reporting needs to be further expanded, including relevant literature sources, reporting guidelines and more straightforward statistical reporting.\nMy comments are the following:\na) In the background section more references are needed, especially scoping reviews of the literature. Further expansion of background in several paragraphs would also be helpful (especially is those would answer the questions \"What is this about and why it is important?\", What have previous studies discovered, Which questions remained unanswered, What did we decide to do?).\nb) Methods section should be more itemized. The recommendation is to use STROBE checklist for observational studies. Here I would emphasize on the importance of sample size calculation, since it seems that the sample of 42 students can be understood as relatively small.\nc) The results section can be shortened, since the analysis is mostly descriptive, and I would recommend that all results are entered in a single table, rather than series of small tables. Moreover, Likert type scales can be entered as medians and interquartile ranges, rather than frequencies. Also, all percentages in the description of the sample should be removed and presented as simple frequencies, because there are less than 100 participants in the sample.\n\nd) Finally, the discussion should rely on the evidence granted from the Results, which it does. The recommendation is to restructure it, by putting the most important results first, and providing more precise explanations for findings, with literature sources.\nOnce again, thank you for the opportunity to review this manuscript.\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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10618", "date": "15 Jan 2024", "name": "Mohsin Naseer Muhammed", "role": "Author Response", "response": "1. In the background section more references are needed, especially scoping reviews of the literature. Further expansion of background in several paragraphs would also be helpful (especially is those would answer the questions \"What is this about and why it is important?\", What have previous studies discovered, Which questions remained unanswered, What did we decide to do?). Response: Further paragraphs and references are added in background section as per the suggestion. 2. Methods section should be more itemized. The recommendation is to use STROBE checklist for observational studies. Here I would emphasize on the importance of sample size calculation, since it seems that the sample of 42 students can be understood as relatively small. Response: Method section was itemized and modified accordingly. 3. The results section can be shortened, since the analysis is mostly descriptive, and I would recommend that all results are entered in a single table, rather than series of small tables. Moreover, Likert type scales can be entered as medians and interquartile ranges, rather than frequencies. Also, all percentages in the description of the sample should be removed and presented as simple frequencies, because there are less than 100 participants in the sample. Response: The result section was shortened and corrected as per the suggestions. 4. Finally, the discussion should rely on the evidence granted from the Results, which it does. The recommendation is to restructure it, by putting the most important results first, and providing more precise explanations for findings, with literature sources. Response: Discussion section was edited and restructured providing the relevant evidence first." } ] } ]	1	https://f1000research.com/articles/12-776
https://f1000research.com/articles/13-68/v1	15 Jan 24	{ "type": "Study Protocol", "title": "To study the utility of tumor budding as a histopathological marker in comparison to various histopathological parameters and TNM staging in breast carcinoma", "authors": [ "SOUMYA AGRAWAL", "Sunita Vagha", "Samarth Shukla", "Sunita Vagha", "Samarth Shukla" ], "abstract": "Background Breast cancer is the leading cause of death in Indian females. Detection of breast cancer in later stages leads to poorer prognosis and therefore decreases patient survival. Various new modalities such as mammography and USG guided FNACs are developed and many new markers are available to diagnose breast cancer; however, tumour budding is a cost-effective method which can be helpful in early diagnosis. Tumour buds are found to have a positive correlation with various histopathological prognostic markers in breast cancer. The present study will be conducted to evaluate tumour buds as a prognostic marker in breast cancer. This study aims to compare tumour budding with histopathological prognostic markers, TNM staging and IHC phenotypes.\n\nMethods The study will be observational, cross- sectional, and prospective, will include 60 cases and will be conducted at Jawaharlal Nehru Medical College (JNMC) Wardha in the Pathology Department.\n\nResults Data will be collected and combined together over a period of two years and will be analysed statistically for tumour budding as a marker and its correlation with breast prognosis.", "keywords": [ "Breast cancer", "Tumor budding", "histopathological markers", "TNM staging" ], "content": "Introduction\n\nBreast cancer is among the commonest cancers diagnosed in females.1 Breast cancer is the main cause of death and is the most diagnosed cancer, exceeding lung cancer. Breast cancer is ranked first among Indian females,2 and men contributes 0.5 to 1% of breast cancers.3 In 2020 breast cancer was diagnosed in 2.3 million women, and 6,85,000 related deaths were reported globally in the same year. Among all known cancers, breast cancer leads to loss of most DALYs (disability adjusted life years) in women. A total of 7.8 million women who diagnosed with breast cancer in the past 5 years were alive at the end of 2020.1,3 Breast cancer is a risk in both rural and urban India and it accounts for 14% cancers in India. A breast cancer case in Indian women is diagnosed every four minutes. Breast cancer stage 3 and 4 are present in more than 50% Indian women.\n\nBreast cancer patients’ survival five years after diagnosis varies greatly –in high income countries it is higher than 90%, 66% in India, and 40% in South Africa.1,3 Women in their early 30s and 50s are at a higher risk of having breast cancer. Breast cancer incidence is at peak in the 50-64 years age range. One in 28 women in India is likely to develop breast cancer and it is more prevalent in urban populations than in rural populations. Approximately 30% of breast cancer patients have distant metastasis at first diagnosis.4\n\nEarly detection and treatment have led to reduce mortality in the last few decades and have proved to be successful in countries with higher incomes; therefore, they should also be made available to countries with limited resources. In later stages of cancer, chances of patient survival are lower. The presence of lumps or masses that suggest cancerous outgrowths can be diagnosed by women themselves by self-examination. Patients’ line of treatment is determined by various factors including clinical, pathological and genetic factors.5\n\nTumor budding is defined as a small clusters of one to five cells which are present at the invasive edge of the tumor mass. Tumor buds undergo transition in the mesenchymal epithelium which is an early event in the process of metastasis6 and therefore considered as representation of Epithelial Mesenchymal Transition (EMT).7 In tumor buds, mesenchymal markers are upregulated and epithelial markers are downregulated. Tumor budding is a relative prognostic marker in carcinoma breast and is one of the mechanisms of distant metastasis and invasion.4 Presence of tumor buds is suggestive of progression in cancer and is an important risk factor in breast carcinoma.4 Tumor budding in colon cancer shows a positive correlation with lymph nodes and poor prognosis.4 Tumor budding as a risk factor can be diagnosed routinely and is therefore both cost and time- effective. As tumor budding is related with local and regional metastasis, it may also be used in therapeutic decision making.4 High magnification is preferred for diagnosing tumor buds and should not be confused with tumor border. Correlation between tumor budding and positive lymph nodes indicates a poor prognosis in breast carcinoma patients.6 Therefore tumor budding in breast carcinoma is a parameter suggestive of aggressive tumor behavior, growth and distant spread, predicting a poor outcome.8\n\nThe present study is proposed to explore tumor budding as a histopathological marker in breast cancer in correlation with other poor clinicopathological parameters, which include-lymphnode involvement, lymphovascular invasion, tumor size, ER, PR, and Her2 expression.\n\nTumor buds may be peritumoral, which means they are near the tumor margin, or intratumoral, i.e., inside the tumor mass.5 Detecting tumor buds in early metastasis would be of immense clinical value which would lead to or provide better survival of the patient.\n\nTumor budding is associated with poor clinicopathological parameters and usually indicates a poor prognosis in patients diagnosed with breast cancer. Early evaluation of tumor budding will provide better outcome for the patient and is also a cost-effective diagnostic marker, as IHC markers are not affordable to everyone. Distant metastasis is linked with high- gradetumors budding, and therefore its evaluation, scoring and inclusion in Tumor, Node and Metastasis TNM grading will improve the diagnosis of the patient.\n\n\n\n1. To confirm histopathological diagnosis of breast malignancy.\n\n2. To evaluate other histopathological prognostic markers as per CAP guidelines.\n\n3. To determine the stage of breast carcinoma as per TNM classification by the American Joint Committee of Cancer (AJCC).\n\n4. To assess the tumour bud status in carcinoma breast and histopathological examination.\n\n\nMethods\n\nThis study will be conducted at JNMC Wardha in the Pathology Department, in coordination with Department of General Surgery, Acharya Vinoba Bhave Rural Hospital, Sawangi.\n\nIt will be an observational, cross-sectional, and prospective study, and has received clearance from the institutional ethics committee (approval no. DMIMS (DU)/IEC/2022/1058). It will be carried out during a two-year period (June 2022 to June 2024).\n\n\n\n1. Histopathologically- confirmed cases of breast carcinoma.\n\n2. Specimens of all modified radical mastectomy specimens.\n\n3. Females diagnosed with breast carcinoma.\n\n4. Newly diagnosed cases of breast cancer.\n\n\n\n1. Patients diagnosed with lesions other than breast carcinoma-, e.g., myoepithelial tumors, fibroepithelial tumors, mesenchymal tumors, benign tumors and mastectomy specimens.\n\n2. Patients with a history of -lobectomy and biopsy specimen including both Tru - cut biopsies and wedge biopsies.\n\n3. Male patients diagnosed with breast cancer.\n\n4. Patients with a history of neo adjuvant therapy, e.g. Trastuzumab, Pertuzumab.\n\nPrior written informed consent will be taken from the patients participating in the study. Keeping in mind the inclusion and exclusion criteria of the study, new and previously diagnosed cases will be noted and for newly diagnosed cases clinical history and physical examination will be carried out. Modified radical mastectomy specimens of clinically confirmed breast carcinoma patients will be studied for histopathological diagnosis. The specimens will be initially kept for fixation followed by proper gross examination and dissection. Sections from soft tissue and skin margins, nipple areola complex, tumour mass and different levels of axillary lymph nodes will be taken. Sections will then be stained using standard haematoxylin and eosin stains.\n\nSteps for grossing a mastectomy specimen:\n\nIdentification of the carcinoma breast specimen will be done with the patient’s requisition form. Orientation of specimen will be done by the surgeon. If the specimen is not oriented it will be then oriented in accordance to its anatomical position. The specimen will then be incised and kept for fixation. Sectioning of the specimen is done and it is dissected into slices of 1 cm. Tumour examination will be done if the tumour is identified, dimensions are noted, its quadrant is identified and other features like consistency, haemorrhage and presence of necrosis will be identified. Longitudinal and coronal serial sections will be taken from the nipple areola complex. Other quadrants of the breast will be examined for micro calcification, satellite nodules and any other abnormalities. Axillary lymph node dissection will be conducted, the largest lymph node will be identified and its cut surface will be noted.\n\nIdentification of the carcinoma breast specimen with the patients requisition form.\n\nSample size\n\nSample size formula9:\n\nZα2 = level of significance at 5% = 1.96\n\np = incidence = 28.54% = 0.2854\n\nd = desired error of margin = 7%\n\nThe study will include approximately 60 confirmed breast carcinoma modified radical mastectomy specimens received from the Department of Histopathology, JNMC. Sections from the tumour mass of these specimens will then be fixed in formalin (10%) and embedded in paraffin blocks. Grossing instruments required includes- grossing tray, knife, scalpel, measuring tape, plain forceps and toothed forceps. Automated tissue processing assembly will be conducted, followed by staining with haematoxylin and eosin. Antibodies of ER/PR, Her2/neu of Brand-PATH NSIT2 will be used. Glass slides (Blue Star) with dimensions: 7.5 x 2.5 cm for microscopy assessment using binocular research microscope Magnus MLXi pro will be used.\n\nStaining protocol: Haematoxylin and eosin staining\n\nStaining protocols will be conducted by the following methods.10 Sections will be deparaffinised in three sets of xylene, 10 minutes for each set followed by dewaxing - and rehydration by decreasing grades of alcohol. They will then be watered and kept in Harris haematoxylin stain for 10 minutes. Sections will then be washed under running water for 2-3 minutes and then kept in 1% acid alcohol for a few seconds. They will then be washed in alkaline tap water for 5 minutes and then stained for 1 minute in 1% aqueous eosin. Finally, sections will be dehydrated by 90% alcohol and then mounted using Dibutylphthalate Polystyrene Xylene (DPX).\n\nImmunostaining\n\nFormalin- fixed paraffin- embedded tissue sections will be mounted on slides coated with Poly l- lysine followed by deparaffinization and rehydration. A pressure cooker will used for heat-induced epitope retrieval. Sections will then thrice rinsed with phosphate buffer saline (PBS). Then to block peroxidase activity, sections were kept in 3% hydrogen peroxide for 10 minutes for 30 – 40 minutes, ER/PR primary antibody will be placed on top and is kept for reacting at room temperature, followed by washing with PBS thrice. For the next 30 minutes it will be allowed to react at room temperature with the secondary antibody (streptavidin biotin), then sodium phosphate buffer, and for next 10 to 20 minutes they will be kept in DAB (3, 3-diaminobenzidine). After washing with tap water, it will then be kept in haematoxylin at room temperature for 2 minutes. Slides are mounted after clearing and drying.\n\nThe tumour will be staged in accordance with TNM Classification determined by AJCC, eight edition.11\n\nCombined T, N and M Stage-\n\n• Stage 0 - Tis\n\n• Stage I - T1N0\n\n• Stage II - T2N0, T3N0 T0N1, T1N1, or T2N1\n\n• Stage III - Invasion into skin and/or ribs, matted lymph nodes, T3N1, T0N2, T1N2, T2N2, T3N2, any T N3, T4 any N, locally advanced breast cancer.\n\n• Stage IV - M1, advanced breast cancer.\n\nER/PR status assessment -12\n\nProportion score\n\nIntensity score\n\nAllred score (total score) = Proportion score + Intensity score\n\nHer2 scoring13\n\nGrading of tumor budding14\n\n≤ 4/10 HPF – low tumor budding\n\n> 4/10HPF – high tumor budding\n\nThe present study received approval from the institutional ethics committee (IEC) with the reference number DMIMS (DU)/IEC/2022/1058, dated 27 June 2022.\n\nInformed written consent will be taken from the patients participating in this study.\n\nStatistical analysis will be carried out ‘using Chi square’ test to analyse the relationship between tumour budding grading and clinicopathological, histopathological indices and immunohistochemical markers. Multiple linear regression analysis will be used to clarify the relative factors for metastasis. A value of P<0.05 considered to indicate statistical significance.\n\n\nDiscussion\n\nBreast cancer is a considerably heterogenous disease.4 A range of factors – are considered, such as- disease development, treatment and patient survival. Breast cancer is the major cause of death linked to cancer in females. There are many different subtypes of breast cancer, and they present with various different clinicopathological and biological features which leads to different prognosis.7 Early metastasis leads to poor prognosis in patients with breast carcinoma although many advances have been made in treatment. Approximately 30% patients are diagnosed with distant metastasis in their first breast carcinoma diagnosis.4 Interpretation of tumor buds is made difficult in breast carcinoma due to many interstitial reactions as these reactions leads to tumor A necrosis, fibroblast, proliferation, inflammatory cell infiltration-, among others.4\n\nTumor buds are defined as cells which are detached from the main tumor mass. Tumor bud can be a single cell or cluster of upto 5 cells.7 It easy to evaluate in routine diagnosis and is both time and cost effective.5 Tumor budding is one of the parameter that might help in decision making therapeutically.5 Tumor buds are invasive cells that migrate through peritumoral connective tissue, evade the host defense mechanism leading to invasion of lymphatics and blood vessels which leads to local and distant metastasis. Tumor budding is also linked with various different cancers like cancers of head and neck, colorectal, lung, gastric and esophageal.6 Raised tumor bud count is related with shortened survival in breast cancer and various other cancer cells. Tumor buds that have undergone epithelial mesenchymal transition are more invasive and prone to metastasize. Tumor budding is therefore the first step of metastasis. Tumor buds during EMT lose their epithelial characteristics and acquire mesenchymal characteristics which also includes expression of mesenchymal proteins and therefore becomes motile. EMT is a dynamic process in which loss of epithelial characteristics and acquired mesenchymal characteristics are not permanent but temporary.4\n\nBelow are discussed a few studies that were conducted in the last few years which showed tumor budding as a histopathological prognostic marker in early stages of breast cancer, and its association with poor clinicopathological characteristics and worst clinical outcome. These studies showed that early detection of tumor budding would be helpful in treating patients diagnosed with breast cancer.\n\nHuang et al., carried out a study titled “Tumor budding is a novel marker in breast cancer: the clinical application and future prospects” in the year 2022. The study showed that tumor buds which have an invasive behavior biologically provides tumor cells an increased ability to migrate and invade. The tumor buds can help predict patients’ prognosis as they are associated with lymph node involvement and lymphatic invasion.4\n\nMohamed Gabal et al., conducted a -study titled “Tumor budding and MMP-2 expression in Breast Invasive Ductal carcinoma” in 2018. It was a cross-sectional, observational study in which specimens of 61 cases of either mastectomy or conservative breast surgery were taken from females diagnosed with invasive ductal breast carcinoma aged from 22 to 82 years. The study emphasized the prediction of poor outcomes in patients as tumor budding is a parameter which denotes the aggressiveness, growth and distant spread of tumor. It also showed positive correlation of tumor budding with ill-defined border, low mitotic count, expression of MMP- 2 and positive metastasis of lymph nodes.8\n\nMasilamani et al., carried out a study titled “-Evaluation of clinicopathological significance of tumor budding in breast carcinoma” 2018. They conducted a retrospective observational study in which 107 surgically resected specimens of breast carcinoma were collected. Whole tissue sections were taken from specimens and analysed using hematoxylin and eosin staining. The study concluded that tumor buds are associated with poor prognosis in breast cancer and that further standardization, proper framing of scoring parameters and cut off criteria for tumor budding is required prior to its inclusion in the histopathological reports.6\n\nSriwidyani et al., conducted a study titled “-Tumor budding in breast carcinoma: relation to E-cadherin, MMP-9 expression and metastasis risk” in 2016. It was a cross -sectional nested case control type study in which 35 cases of breast cancer with metastasis and 35 cases of breast cancer without metastasis were selected during a period from January 2012 to June 2015, with a mean age of 48.6 years (ranging from 23-74 years). The study concluded that tumor budding had no correlation with E-cadherin expression, while it was related to the level of MMP-9 expression. It showed high grade tumor budding is an independent risk factor of metastasis in breast cancer and shows features of EMT.7\n\nSalhia et al., conducted a study titled “High tumor budding stratifies breast cancer with metastatic properties” in 2015. It was a retrospective study over a period of seven years from January 2005 to December 2011, in which tumor budding was analyzed in patients with known status of lymph nodes. The study included 148 cases resected surgically and 99 core biopsies. A correlation between tumor budding in breast cancer and invasion of lymphatics and positive lymph node status was found in the study.5\n\nAssociation between tumor budding and breast cancer prognosis is suggested by the above- mentioned studies. In the present study we will observe consistent correlation of tumor budding with lymph node involvement, lymphovascular invasion, ER, PR, and tumor size.\n\nWe intend to publish the study in a peer reviewed journal. The data will be prepared in excel sheet without patient identifiers and will be shared as and when requested by concerned journal/publishers/statistians. If required these data files will be uploaded in open data repository like Zenodo.\n\nRecruitment has started, with currently 24 patients enrolled in the study.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: STROBE checklist for protocol ‘To study the utility of tumor budding as a histopathological marker in comparison to various histopathological parameters and TNM staging in breast carcinoma’. DOI: 10.5281/zenodo.8337107.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nI would like to sincerely thank and acknowledge all the patients that participated in my study, all my teachers, technical staff and department of Oncology surgery without whom this study would not be possible.\n\n\nReferences\n\nBreast cancer: Who.int.2022 [cited 7 June 2022].\n\nMalvia S, Bagadi SA, Dubey US, et al.: Epidemiology of breast cancer in Indian women. Asia Pac. J. Clin. Oncol. 2017 Aug; 13(4): 289–295. Publisher Full Text\n\nBreast cancer: Who.int.2022 [cited 7 June 2022].\n\nHuang T, Bao H, Meng YH, et al.: Tumour budding is a novel marker in breast cancer: the clinical application and future prospects. Ann. Med. 2022 Dec 31; 54(1): 1303–1312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalhia B, Trippel M, Pfaltz K, et al.: High tumor budding stratifies breast cancer with metastatic properties. Breast Cancer Res. Treat. 2015 Apr; 150(2): 363–371. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasilamani DS, Kanmani P: Evaluation of clinicopathologic significance of tumor budding in breast carcinoma. Int. J. Clin. Diagn. Pathol. 2019; 2(1): 171–173. Publisher Full Text\n\nSriwidyani NP, Manuaba IB, Alit-Artha IG, et al.: Tumor budding in breast carcinoma: relation to E-Cadherin, MMP-9 expression, and metastasis risk. Bali. Med. J. 2016 Jan 1; 5(3): 150. Publisher Full Text\n\nGabal SM, Bassam AM, Sedqi ME, et al.: Tumour Budding and MMP-2 Expression in Breast Invasive Ductal Carcinoma. J. Clin. Diagn. Res. 2018 May 1; 12(5). Publisher Full Text\n\nChada V: Sample size determinant in health studies. NTI bulletin. 2006; 42/3 and 4: 55–62.\n\nBancroft JD, Gamble M: Theory and practice of histological techniques. Elsevier Health Sciences; 2008.\n\n1 greene f, edge s, schilsky r, gaspar l. ajcc cancer staging manual 8th edition. 8th ed. springer; 2018.\n\nRai PD, Vagha S, Shukla S, et al.: Comparison of various scoring systems by immunohistochemistry for evaluating hormone receptors (Estrogen receptor and progesterone receptor) in carcinoma of breast. Journal of Datta Meghe Institute of Medical Sciences University. 2020 Apr 1; 15(2): 202. Publisher Full Text\n\nCornolti G, Ungari M, Morassi ML, et al.: Amplification and overexpression of HER2/neu gene and HER2/neu protein in salivary duct carcinoma of the parotid gland. Archives of otolaryngology–head & neck surgery. 2007 Oct 1; 133(10): 1031–1036. PubMed Abstract \| Publisher Full Text\n\nSalhia B, Trippel M, Pfaltz K, et al.: High tumor budding stratifies breast cancer with metastatic properties. Breast Cancer Res. Treat. 2015 Apr; 150(2): 363–371. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "239730", "date": "07 Feb 2024", "name": "Vivek Gupta", "expertise": [ "Reviewer Expertise General Pathology", "Surgical Pathology", "Molecular Pathology", "Oncopathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rationale for the study is well-defined and has clarity. It highlights the gap in the literature and the research question. The objectives are in sequence and lead to clarity in assessing the tumor bud in breast carcinoma. Objective 4 needs to be reframed to “Assessing the tumor bud status in carcinoma breast.” The histopathological examination may be removed as the same has already been mentioned in earlier objectives.\nThe study design is apt for study. It mentions inclusion and exclusion. They have graded tumor budding as ≤ 4/10 HPF – low tumor budding and > 4/10 HPF – high tumor budding. However, it can be graded as ≤ 4/10 HPF, 4 – 9/10 HPF, and >10/10 HPF. An optimal cut-off for the number of tumor budding and lymph node metastasis can also be correlated.\nThe protocol provides sufficient details for the evaluation of tumor budding. Microscopic pictures of high and low tumor buds can be more effective.\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": [] } ]	1	https://f1000research.com/articles/13-68
https://f1000research.com/articles/13-62/v1	12 Jan 24	{ "type": "Research Article", "title": "In silico identification of quorum sensing inhibitors against LasR protein in a clinical isolate of multidrug resistant Pseudomonas aeruginosa DMC-27b", "authors": [ "Md. Hasanul Banna Siam", "Ahmad Salman Sirajee", "Md. Belayet Hasan Limon", "M. Anwar Hossain", "Munawar Sultana", "Md. Hasanul Banna Siam", "Ahmad Salman Sirajee", "Md. Belayet Hasan Limon", "M. Anwar Hossain" ], "abstract": "Background: Pseudomonas aeruginosa is an opportunistic pathogen that uses quorum-sensing (QS) and biofilm formation to subvert antibiotic therapy. Antibiotic resistance has led to a demand for alternative methods of treatment, and destabilizing the LasR-OdDHL binding with inhibitors offers a potential solution. Methods: This study aimed to construct a homology model of the LasR protein using the genetic sequence of the P. aeruginosa DMC-27b strain (GenBank: SMRY00000000.2). Molecular docking, molecular mechanics-based binding free energy calculation, and pharmacokinetic analysis were performed on 1900 3D structures collected from synthetic and natural compound databases to identify three potential lead molecules. These compounds were evaluated using ADMET (absorption, distribution, metabolism, excretion, toxicity) analysis, and molecular dynamics protocols were used to refine the results. Results: The three lead compounds showed higher binding capability with the LasR receptor than the native ligand and passed the ADMET evaluation stage. In total, 44 properties remained within the range of 95% of known drugs, indicating their potential efficacy as drugs against P. aeruginosa and other bacteria that use a similar QS system. Conclusions: This study provides insights into potential drug designing and development against clinical isolates of emerging P. aeruginosa strains and other bacteria that use a similar QS system.", "keywords": [ "Quorum sensing", "Virtual screening", "Pseudomonas", "Antibiotic resistance", "Docking" ], "content": "Introduction\n\nPseudomonas aeruginosa, a species of considerable medical interest, is wreaking havoc in humans due to its wide role in infections. This encapsulated, gram-negative, rod-shaped bacterium has emerged as a multidrug-resistant superbug rendering antibiotics ineffective during treatment (Lyczak et al., 2002). The opportunistic bacterium causes a plethora of diseases including cystic fibrosis, urinary tract infection, acute leukemia, endocarditis, meningitis, pneumonia, and septicemia (Bodey et al., 1983). The infections are more common in immunocompromised patients – making P. aeruginosa the second most common organism isolated in nosocomial pneumonia (17% of cases) and the third most common organism isolated in both urinary tract infection (UTI) and surgical site infection (11% of cases) (Richards et al., 1999). As a mechanism of antibiotic resistance, the bacteria can form slime-enclosed biofilms as a protective barrier (Lyczak et al., 2002). The development of biofilm is closely connected to another process termed as Quorum Sensing (QS) (Solano et al., 2014). QS is a cell-to-cell communication process through which bacteria can sense population density and regulate gene expression accordingly. Biofilm development, biofilm dispersion and upregulation of the synthesis of surfactant molecules are deeply intertwined with QS circuit (Lee & Zhang, 2014).\n\nThere are four QS systems in P. aeruginosa, namely Las, RhI, Pqs and Iqs (Scoffone et al., 2019). The Las remains at the top of the circuit in the hierarchy and is comprised of LasI and LasR – the former synthesizing an autoinducer molecule i.e. N-(3-oxododecanoyl)-L-homoserine lactone or OdDHL, and the latter receiving the OdDHL to carry out transcriptional regulation (Lee & Zhang, 2014). The binding of OdDHL to LasR initiates the cascade of signals that regulate further signaling in Rhl, Pqs and Iqs systems. The quorum sensing network in Pseudomonas aeruginosa also employs three other signaling molecules: N-butyryl-L-homoserine lactone (BHL) synthesized by the RhlI AHL (Acyl-Homoserine Lactone) synthase which binds to the RhlR receptor; 2-heptyl-3-hydroxy-4(1H)-quinolone (PSQ) binds to PqsR; and 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) binds to IqsR. The two receptors, LasR and RhIR, are transcriptional regulators that regulate the expression of nearly 300 genes of P. aeruginosa genome (Feltner et al., 2016). The LasR protein has two domains: ligand-binding and DNA-binding. When OdDHL binds to the LasR receptor, it binds to the ligand-binding domain and activates the protein. Consequently, the DNA-binding domain attaches to the target DNA to regulate the transcription. The production of rhamnolipids and the virulence factors such as elastase, exoprotease, pyocyanin are all regulated and influenced by the initial interaction and complex formation of LasR/OdDHL (Ochsner et al., 1995).\n\nThe search for potential quorum sensing inhibitors (QSI) has been of great interest and research showed that QSI could compete against the native ligand for binding with LasR and disrupt the QS-signaling cascade and the subsequent biofilm formation (Annapoorani et al., 2012; Zeng et al., 2008). The LasR inhibitors can be classified into three classes: non-AHL-like antagonists, AHL-like antagonists, and covalent binders (Scoffone et al., 2019). Both natural products and synthetic compounds have been researched for potential candidates. A recent study has found two potential chemical compounds that can work as quorum sensing inhibitors (Nain et al., 2019). In another study, a halogenated furanone compound from a marine alga Delisea pulchra was shown to have good anti-QS property (O’Loughlin et al., 2013). But most of the halogenated furanone is reactive and hence too toxic to be used for treatment in human (Hentzer & Givskov, 2003). There are other natural products having anti-QS activity such as patulin, penicillic acid from Penicillium species (Rasmussen et al., 2005), ajoene, a sulfur-rich molecule from garlic (Jakobsen et al., 2012), ellagic acid derivatives from Terminalia chebula Retz (Sarabhai et al., 2015), and Coumarin. Coumarin is obtained from plant extract and it has strong anti-virulence activity. It is also effective against protease and pyocyanin production and it blocks biofilm formation (Monte et al., 2014).\n\nIn our study, we used Pseudomonas aeruginosa DMC-27b (GenBank: SMRY00000000.2), a clinical strain isolated from a urine sample of an ICU patient at Dhaka Medical College hospital, Bangladesh. The original data collection took place as part of a previous study by our lab (Jahan et al., 2020), and the strain was found to be resistant against a total of 20 antibiotics with having chromosomal inheritance of all four classes of beta-lactamases (Jahan et al., 2020). The significance of using this Multi-Drug Resistant (MDR) isolate from Bangladesh offers an opportunity for a case study and investigate into any possible mutation in the LasR protein that might have an impact on the ligand binding modes. Considering the emergence of new MDR Pseudomonas. aeruginosa strains across the globe, and how south Asian countries like Bangladesh are acting as epicenters of AMR (Antimicrobial resistance) spread, it is critical to survey changes in the LasR receptor and continue to enrich the pool of new inhibitors.\n\n\nMethods\n\nThe cheminformatics analyses were carried out in Schrodinger Maestro v11.8 (Schrodinger, LLC, New York, NY. There is a free academic version of this license that can be used to replicate this work. In addition, readers can use AutoDock Vina as an alternative software that carries out a similar function). This study utilised a desktop PC with Intel (R) Core™ i7 7700/4.0GHz processor, 8GB DDR4/3200 MHz RAM, and 4GB AMD Radeon RX 570 graphics with support for OpenCL 2.0 running Windows 10 Professional operating system. The entire workflow was segregated into three stages (Figure 1). Stage-I included genome retrieval and annotation, gene profiling, homology modeling of protein, and database search for AHL-like compounds. The stage-II included the protein and compounds preparation leading to molecular docking, molecular mechanics based binding free energy calculation. Stage-III began with further analysis comprising of ADMET analysis, induced fit docking and molecular dynamics. Finally, the lead compounds were comparatively analyzed with respect to the protein from the reference strain.\n\nSP, Standard Precision; XP, Extra-Precision; MM-GBSA, Molecular mechanics-Generalized Born Surface Area; ADMET, absorption, distribution, metabolism, excretion, toxicity; RAST, Rapid Annotation using Subsystem Technology.\n\nThe genomic data were retrieved from National Center for Biotechnology Information (NCBI) for the strain Pseudomonas aeruginosa DMC-27b with the GenBank accession number SMRY00000000.2 (Pseudomonas Aeruginosa Strain DMC-27b, Whole Genome Shotgun Sequencing Project, 2019). The raw data was uploaded to the publicly available web server RAST (Rapid Annotation using Subsystem Technology) for automatic annotation. The presence of LasI/LasR and the proteins involved in the QS system were confirmed by RAST and were furthered validated by custom Basic Local Alignment Search Tool (BLAST) searches inside the RAST SEED viewer version 2.0 (Overbeek et al., 2014).\n\nThe amino acid sequence of the LasR protein of P. aeruginosa DMC-27b was submitted to the publicly available SWISS-MODEL web server for homology modelling (Waterhouse et al., 2018). The protein template PDB: 3IX3 was selected owing to its high resolution (i.e. 1.4 Å), standard range R-free value (i.e. 0.207) and an excellent score in Ramachandran outliers (i.e. 0.0%). The generated 3D homology model (created using SWISS-MODEL) was created in homodimer containing two chains: A and B and both contained the co-crystallized ligands. The quality of the resulting protein structure was validated using QMEAN-Z score and Ramachandran plot analysis.\n\nThe original ligand (i.e. N-3-Oxo-Dodecanoyl-L-Homoserine Lactone) for the LasR protein was used to observe the protein-ligand interaction, and to identify the core structure of the ligand. A total of three chemical databases were used to download chemical compounds. PubChem (Kim et al., 2019) and ChEMBL (Davies et al., 2015) were used to find and download chemical structures similar to N-3-Oxo-Dodecanoyl-L-Homoserine Lactone. Next, a complete SancDB database was downloaded (Hatherley et al., 2015) in the form of SANCDB SDF structure PubChem is a massive open repository of experimental data and ChEMBL is a manually curated chemical database of compounds with drug-like properties. SancDB, which stands for South African Natural Compounds Database, is a database containing compounds isolated from the plant and marine life in and around South Africa. PubChem and ChEMBL returned 1080 compounds, and SancDB returned 820 compounds totaling the number of compounds to reach 1900 - see Underlying data (Siam, 2023) for the full list (re-uploading permissible under copyright).\n\nThe protein model (LasR_DMC27b.pdb, generated using SWISS-MODEL by keeping 3IX3 as a reference) was not suitable for molecular docking in its unprepared state (Kalia et al., 2017) because an unprepared protein structure may contain gaps, missing atoms, and other structural irregularities that may affect the accuracy of the docking simulation. The protein structure was prepared by Schrodinger’s Protein Preparation Wizard (part of Schrodinger Maestro). During preparation, proper bond orders were assigned, hydrogens atoms and missing residues were added, water molecules beyond 5.00 Å distance were removed from hetero groups, and hetero states were generated using Epik at pH: 7.0 ± 2.0. The protein was further refined by optimizing the H-bond assignment and running restrained energy minimization of the structure using force field Optimized Potentials for Liquid Simulations (version OPLS_2005) until the heavy atoms converged to the RMSD value of 0.30 Å (Banks et al., 2005).\n\nAll the selected 1900 compounds were downloaded in their three-dimensional structure-data file (SDF) format. A phase database was created using Schrodinger Maestro v11.8 to assemble the potential ligand-like compounds. The Schrodinger LigPrep v2.2 module (part of Schrodinger Maestro) was applied to generate tautomers and ionization states at pH: 7 ± 2.0 and to minimize energy using force field OPLS_2005. The Epik v4.6 (part of Schrodinger Maestro) was used to compute the predicted pKa values for drug-like molecules; and reactive functional groups were eliminated from the result which might give false positive during screening (Shelley et al., 2007). To filter the unsuitable compounds, Schrodinger QikProp module (part of Schrodinger Maestro) was applied to test the Lipinski’s Rule of five (Lipinski et al., 2001) and Jorgensen’s rule of three (Jorgensen, 2004).\n\nThe grid-box was generated using the Schrodinger Glide v8.1 module (part of Schrodinger Maestro) based on the co-crystallized ligand structure in LasR-DMC27b PDB model. For flexible docking, the active site was confined to a larger enclosing box in proportion to the defined smaller box (27×27×27 Å) around the potential binding site of interest using the centroid native ligand. The Van der Waals radius scaling factor was set to 1.0 Å and the partial charge cutoff was kept at 0.25. There was no constraint or rotatable group set during the grid-box generation.\n\nThe Schrodinger Glide v8.1 was used to run Standard Precision (SP) docking for 1900 compounds employing the generated receptor grid (Friesner et al., 2004). Based on the literature review, an initial cutoff value of -7.5 kcal/mol was considered for filtering which returned 270 compounds (Nain et al., 2019). For the second phase, the compounds were docked using XP (Extra Precision) method and this time, the cutoff value was set to -8.75 since the native ligand (OdDHL) binds to the LasR_DMC27b with an XP score of -8.72 kcal/mol. In both cases, the flexible ligand sampling scheme was used. Compounds with more than 500 atoms and 1000 rotatable bonds were skipped and Epik state penalty was added to the docking score. In post docking minimization, the number of poses per compound was set to five. After XP docking, a total of 45 compounds were assessed comprising of different variations of PubChem compounds: K5D, K5G, K5J, OHN, and 443433.\n\nThe Schrodinger Prime v3.3 [Prime, Schrödinger, LLC, New York, NY, 2019], part of Schrodinger Maestro, was used to calculate MM-GBSA (Molecular Mechanics-Generalized Born Surface Area) based End-Point Binding Free Energy Calculation, which is an algorithmically rigorous, computationally intensive and superior to molecular docking. The MMGBSA methodology yields a credible assessment of binding free energy, expressed in Kcal/mol units, and may serve as a suitable approach for post-docking validation purposes. Here, GBSA continuum solvent model, as outlined in the work of (Lyne et al., 2006), was utilized to conduct simulations on a set of 45 compounds. Schrodinger Prime incorporates a surface generalized Born (SGB) model that utilizes a Gaussian surface in place of the Van der Waals surface to enable a more accurate representation of the solvent accessible surface area. Subsequent to the docking procedure, the binding free energy (ΔGbind) for each compound was calculated. Schrodinger Prime uses a surface generalized Born (SGB) model employing a Gaussian surface instead of Van der Waals surface for better representation of a solvent accessible surface area. Based on the docked complex, the binding free energy (ΔGbind) of each compound was calculated. All 45 compounds showed lower binding free energy than the native ligand, thus validating the result of XP docking.\n\nThe 45 selected compounds were tested for drug-likeness and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties using the Schrodinger QikProp v5.8 module (Ioakimidis et al., 2008). The druggability prediction is a crucial step in filtering the compounds that might be unsuitable for use in the human body. Unlike fragment-based approaches, QikProp’s predictions are based on the full 3D molecular structure, providing accurate results on par with the properties of the 95% of known drugs. The QikProp’s result yielded 44 properties for the chemical compounds, of which, ADMET properties and molecular descriptors were taken into consideration particularly octanol/water and water/gas log Ps, log S, log BB, overall CNS activity, Caco-2 and MDCK cell permeabilities, log Khsa for human serum albumin binding, HumanOralAbsorption and log IC50 for HERG K+-channel blockage. Since most halogenated furanone compounds are too reactive for use as drugs for their high electrophilicity, they were carefully removed from the result letting only 3 compounds to pass through the filters based on the QikProp’s results.\n\nThe stability and intermolecular interactions between protein and molecules were investigated using molecular dynamics (MD) simulation (Liu & Kokubo, 2017) techniques with a 100 ns time interval using the complexes obtained from docking studies to further investigate the protein and ligand binding mode. MD simulations were run with the Desmond module from the Schrödinger Release 2020-4 (Academic edition) suite (Molecular dynamics can also be done using GROMACS which is free to use). A TIP3P water model (density: 0.997 g/L) at physiological conditions (298 K, pH 7.4) with an orthorhombic box shape as the boundary was used to represent the complex protein-ligand interaction (Mark & Nilsson, 2001). On all three axes, the minimum distance between the protein surface and the boundary was set to 10 Å. The system was neutralized by adding 0.15 M Na+ and Cl- salt concentrations (Paul et al., 2023). Before simulation, NPT parameters (constant particle number (N), pressure (P), and temperature (T)) are utilized representing an isothermal-isobaric ensemble, which simulates the most typical experimental conditions. The MD simulation was run with the OPLS-2005 force field at constant pressure (1.01325 bar) and temperature (310 K), with recording intervals of 100 ps. These simulation conditions were set to mimic human cell microenvironment. The Simulation Interaction Diagram (SID) of the Desmond module of the Schrödinger program was used to analyze the trajectories acquired from the MD simulation. Based on the simulated trajectories, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and Protein-Ligand interactions were utilized to assess the stability of the ligand-protein complexes.\n\nA comparative docking analysis between our laboratory strain and the NCBI reference strain was done. The LasR_DMC27b.pdb was obtained from Pseudomonas aeruginosa DMC27b and the reference protein 3IX3 was obtained from Pseudomonas aeruginosa PAO1 and downloaded from the Protein Data Bank (PDB). The selected inhibitors were used against the reference protein 3IX3. Molecular docking and MMGBSA were performed in case of both proteins using the selected compounds and the native ligand for comparison.\n\n\nResults and discussion\n\nThe quorum-sensing circuit in P. aeruginosa DMC-27b showed substantial resemblance to P. aeruginosa PAO1 except for RhlL and PhzD proteins. All the required proteins responsible for QS were found to exist in P. aeruginosa DMC-27b indicating capacity for quorum sensing and biofilm formation. The presence of LasI, LasR, RhlR, RhlI and RhlL were manually confirmed by BLASTp search. The activation of the Las system further activates the Rhl system that produces rhamnolipids for which the contributing proteins (RhlI, RhlR, RhlA, RhlB, RhlC, RhlE, RhlL, RhlG) were all present in the P. aeruginosa DMC-27b. A notable difference was found in Rhl system as P. aeruginosa DMC-27b had both Rhl-I and Rhl-L proteins that contribute to N-butyryl-L-homoserine lactone synthesis and N-acyl-L-homoserine lactone synthesis, respectively. This property might provide the bacteria with an evolutionary advantage over the reference strain (PAO1) as the Rhl system can make use of quorum sensing signals by generating both types of homoserine lactones. Similarly, the absence of PhzI in the phenazine system indicates that the PhzR could be co-activated by different acyl-homoserine lactones. The production of exoprotease (encoded by lasA), elastase (encoded by lasB), biofilm dispersal mediator (C-di-GMP binding protein), the regulator of hydrogen cyanide synthase (ANR) were also present that play role in virulence. In the Phz system, PhzR was present, but the PhzI was not identified.\n\nThe quality of the resulting protein homology structure (denoted here as LasR_DMC27b) was checked and the QMEAN-Z score was 0.99 and the all-atom score was 1.92. One mutation was reported in the ligand-binding domain at the position of amino acid no. 171 (Glycine instead of Valine) and it was further confirmed by way of EMBOSS matcher 6.6.0. The LasR protein has two domains: amino-terminal ligand-binding domain and a carboxy-terminal DNA-binding domain. The mutation in our strain is at the position of amino acid no. 171 (Glycine instead of Valine) at the terminal region, far away from its natural active site, hence it might not have considerable effect on the ligand-binding activity as seen from our study. The solvation and torsion scores were within an acceptable range. In Ramachandran Plot analysis, the number of residues in the favored region was 319 (99.1%); the number of residues in the allowed region was 3 (0.9%); and the percentage of outlier was 0.0% (Figure 2).\n\n(A) In Ramachandran Plot, about 99% of residues are in the favored region with 0.0% outliers. The image is generated and viewed using SWISS PDB viewer. (B) the Pairwise Alignment between LasR_DMC27b & 3IX3 has Identity: 171/172 (99.4%), Gaps:1/172 (0.6%), and Score: 892; performed using EMBOSS matcher 6.6.0.\n\nThree compounds were obtained as potential inhibitors denoted as L1, L2 and L3 after sequential identification process in order of- molecular docking, MMGBSA, molecular dynamics, ADMET prediction and were further improved with induced fit docking.\n\nThere was slight difference in the SP and XP docking scores of the selected compounds. All three compounds (L1, L2. L3) showed higher scores in SP, XP docking compared to the native ligand (OdDHL). The molecular docking result is shown in Table 1. The extra precision (XP) docking score for the OdDHL was found to be about -8.7 kcal/mol which is on par with other studies (Nain et al., 2019). All three selected compounds showed better binding capacity against both the clinical strain (i.e. DMC27b) and the reference strain (i.e. PAO1). The potential compounds showed greater free binding energy than the native ligand and had reasonable ligand strain energy. The compound L2 and L3 had variation in ligand strain energy, 5.198 kcal/mol and 8.041 kcal/mol, respectively, despite having a very similar nature of chemical structures.\n\nIUPAC, The International Union of Pure and Applied Chemistry; SP, Standard Precision; XP, Extra-Precision.\n\nIn the binding mode analysis, the selected compounds were well within the protein’s active site. Compounds no. L1, L2 and L3 showed similar patterns of hydrogen bond interaction (Figure 3). The potential quorum-sensing inhibitors (QSI) were bound to the protein’s active site by way of hydrogen bonds to the amino acids Tyr56, Ser129, Asp73 and Trp88. This showed a crucial difference in comparison to the native ligand (OdDHL) which binds to the protein using Tyr56, Ser129, Asp73 and Trp60 (Table 1). Furthermore, LasR can bind an array of compounds containing the lactone head ring. Furthermore, the primary binding site for OdDHL is a combination of hydrophilic and hydrophobic interactions where the lactone head group facilitates hydrogen bonds stabilizing the structure while the acyl tail contributes to hydrophobic interactions. Reports show that LasR has two binding modes: one that mimics the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150° (Paczkowski et al., 2019). The selected three compounds lack the lactone head group but facilitate strong hydrogen bonds with the receptor. All the compounds hydrogen-bonded with Tyr56, Ser129, Asp73 and Trp88. The tyrosine-56 and Serine-129 are the two most defining amino acid residues for LasR binding to its ligand. But, the native ligand binds with the receptor using Tyr56, Ser129, Asp73 and Trp60 instead of Trp88 found in the L1, L2 & L3 inhibitors. This difference in the binding mode of the potential inhibitors could play a key role in inhibiting the quorum sensing signaling.\n\n(A) Compound L1 hydrogen-bonded with Tyr56, Ser129, Asp73, Trp88 residues of the LasR receptor. Similarly, (B) Complex-L2 showed HB interactions with Tyr56, Ser129, Asp73, Trp88 residue, and (C) Complex-L3 shared HBs with Tyr56, Ser129, Asp73, Trp88 residues, and (D) the reference compound shared HBS with Tyr56, Ser129, Asp73, and Trp60 residues.\n\nIn the MMGBSA analysis, the three compounds showed lower binding free energy than the native ligand (Table 2). The free energy for the native ligand (OdDHL) binding to the receptor LasR was -115.08 kcal/mol, and the binding free energy for L1, L2 and L3 ranged from -128.65 kcal/mol to -127.51 kcal/mol which is lower than OdDHL. The obtained coulomb energy affinity was significantly higher than the native ligand, -65.27 kcal/mol to -74.75 kcal/mol versus -33.9 kcal/mol, respectively. The compound L1 had the lowest ΔGBind energy, but compound L2 had comparatively better coulomb energy as well as the solvation energy of the complex. The native ligand had more ligand strain energy (SELig) than the selected compounds.\n\nAll three compounds retained satisfactory results with values in the range of 95% of known drugs. The ADMET properties of the selected compounds were within the acceptable range for optimal drug candidates. None of the compounds violated Lipinski’s rule of five or Jorgensen’s rule of three. The HERG value, central nervous system (CNS) activity, binding to human serum albumin and other properties were within limit. The human oral absorption of the compounds was above 80%. The QPlogHERG values for L1, L2 and L3 were -4.964, -4.59, -3.688 respectively. A value below -5.00 raises concern as to whether the compound might act as hREG inhibitor for K+ ion channel. Since the L1 compound is close to the threshold level for QPlogHERG value, it must be experimentally checked for hREG inhibition activity. The human oral absorption rate for all three compounds was excellent (>80%) and the compounds did not show acute oral toxicity. The ADMET profile of selected three compounds is provided in Table 3. Furthermore, the molecular descriptors for the compounds were analyzed and shown in Table 4.\n\nNo suitable inhibitor was found from ChEMBL and SancDB databases. Despite some compounds having good XP score, all of them were eliminated in the ADMET screening stage. The chemical structures of the L1, L2, L3 and OdDHL are presented in Figure 4. The compounds lack the ester functional group in the ring implying that there is no lactone headgroup which is the characteristic of the native ligand, OdDHL.\n\nThe L2 and L3 chemical compounds have similar chemical structures with one ring while L1 contains two rings. All selected compounds lack the characteristic lactone headgroup of OdDHL.\n\nMolecular dynamics were run using the LasR_DMC27b protein and the three selected ligands as well as the reference ligand, OdDHL. After 100 ns simulation period, the ligand-protein RMSD (root mean square deviation) results indicated that all three complexes were close to the permissible limit of 3.0 Å with respect to the reference ligand complex. However, complex-3 had a value >4.0 Å during the 20 ns and later at 95 ns simulation period (Figure 5(A)) and it was not equilibrated until 50 ns. All the complexes were equilibrated after 30 ns and found to be stable, albeit the reference complex was not found to be equilibrated at the beginning but after 80 ns, it had the lowest RMSD, indicating lesser fluctuation and improved stability. Complex-1 has shown superior stability and is closer to the reference complex than the other experimental complexes. In terms of the LasR-DMC27b protein’s stability, all three selected ligand-protein complexes were quite stable as they showed an RMSD value lower than 3 Å. Compared to all, the complex-2 showed better protein stability (Figure 5(B)). The conformation changes occurring to the protein’s side chain were analyzed using the root mean square fluctuation (RMSF) values. Greater fluctuations are usually found in the protein’s N-terminal and C-terminal domains, and this was also the case for the LasR-DMC27b protein and the docked complexes. Except for a few spikes, the complexes exhibited low fluctuations overall indicating their potential to be used as antagonist compounds (Figure 5(C)).\n\n(A) Ligand-protein complex RMSD (root-mean-square deviation), (B) Protein Cα RMSD and (C) Protein Cα RMSF (root-mean-square fluctuation) of the backbone-atoms of the docked LasR-DMC27b protein.\n\nUsing the default parameters of the Desmond module, the MD trajectories of the LasR protein, three possible lead compounds, and the reference complex were analyzed. In protein-ligand interactions, Complex-1, Complex-2, and Complex-3 hydrogen bonded with residues including ASP_73, THR_75, THR_115 and SER_129, while reference complex further formed hydrogen bonds with TYR_56 & TRP_60. Moreover, complex-3 formed hydrogen bonded with TYR_64, TRP_88 & TYR_93 exclusively. There was a weak ionic bond with ASP_73 in complex 3. There were hydrophobic bonds and water bridges present in various residues in all the complexes (Figure 6).\n\nIn the post-molecular dynamics MMGBSA analysis, out of the three compounds, L2 compound showed lower binding free energy (ΔGBind = -88.97 kcal/mol) than the native ligand (Table 5). The ΔGBind free energy for the native ligand (OdDHL) binding to the receptor LasR was -80.58 kcal/mol, and the binding free energy for L1, L2 and L3 compounds ranged from -69.7 kcal/mol to -88.97 kcal/mol. This is lower compared to MMGBSA analysis prior to molecular dynamics which ranged from -128.65 kcal/mol to -127.51 kcal/mol. The compound L2 also had the lowest hydrogen bond energy of the complex as well as the lowest Coulomb energy of the complex. This indicates that L2 compound was a better drug candidate compared to L1 and L3.\n\nOur study reported no major mutation in the LasR protein in P. aeruginosa DMC27b strain that could make it functionally different from the reference strain. We further performed a comparative analysis between the two strains to identify if docking score changes between two proteins due to single amino acid mutation in the terminal region. Our comparative analysis showed that the selected three compounds (L1, L2, and L3) had consistently better binding capacity to both receptors than the native ligand OdDHL. This finding further strengthens the hypothesis that ligand screening in single isolate could be relevant for use in other strains. A further in-depth study could be performed to observe how inhibitors reported by other studies would act against LasR across different strains of P. aeruginosa. Table 6 shows the comparison of interaction of selected compounds and the native ligand with both receptors, LasR_DMC27b and 3IX3.\n\nXP, Extra-Precision.\n\nOur study has its limitations: first, the research design was based on similarity search to find potential ligands, and this has its inherent limitation that potential inhibitors must be further chemically synthesized and experimented in-vitro for efficacy. Second, we could not perform longer than 100 ns simulation in molecular dynamics due to resource constraints. However, the selected compounds in this study were found to rigidly dock within the binding pocket of the receptor and they had shown higher binding capability to the LasR than the native ligand. The study thus indicates that the docked complexes can be considered as stable compounds for further in-vitro experiments.\n\n\nConclusions\n\nThe clinical isolate Pseudomonas aeruginosa DMC-27b has a genetic profile of a complete QS network with biofilm-forming capacity. Three lead compounds that were proposed in this study will likely be generalizable for other P.aeruginosa strains due to a lack of significant mutation in the LasR protein. But both in-vitro and in-vivo studies are required to check for the potentiality of the suggested QSIs. Based on the findings, this study will continue to provide insights for further research in drug designing and development against clinical isolates of P. aeruginosa as well as other bacteria that use a similar QS system.", "appendix": "Data availability\n\nGenBank: P. aeruginosa DMC-27b genome. Accession number, SMRY00000000.2; https://www.ncbi.nlm.nih.gov/nuccore/SMRY00000000.2.\n\nProtein Data Bank: LasR-OC12 HSL complex used to generate LasR_DMC27b.pdb file at Swiss Model. Accession number, PD3IX3; https://www.wwpdb.org/pdb?id=pdb_00003ix3.\n\nPubChem: Three druggable candidates used in this study. Accession numbers, 119321904, 119321914, and 119321910.\n\nFigshare: Chemical Library_Potential antagonist of LasR receptor_HSB1900. https://doi.org/10.6084/m9.figshare.22579234.v1 (Siam, 2023).\n\nThis project contains the following underlying data:\n\n- Chemical Library_Potential antagonist of LasR receptor (1900 chemical structure files in sdf text-based format).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAnnapoorani A, Umamageswaran V, Parameswari R, et al.: Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa. J. Comput. Aided Mol. Des. 2012; 26(9): 1067–1077. PubMed Abstract \| Publisher Full Text\n\nBanks JL, Beard HS, Cao Y, et al.: Integrated Modeling Program, Applied Chemical Theory (IMPACT). J. Comput. Chem. 2005; 26(16): 1752–1780. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "274961", "date": "19 Jun 2024", "name": "Sudipta Shaw", "expertise": [ "Reviewer Expertise I am a trained microbiologist and protein biochemist." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAntibiotic resistance has become a pressing global health problem and bacterial quorum-sensing plays a significant role in it. Authors of this manuscript have focused on quorum-quenching of a multi-drug resistant clinal isolate of Pseudomonas aeruginosa DMC-27b. They have identified 3 potential inhibitor molecules through a multiple in silico screening process. Although there are several limitations of this study as those have been rightfully pointed out by the authors themselves, however, those 3 compounds do seem genuine candidates for future in-vitro and eventually in-vivo studies. This is a nicely written manuscript with proper scientific evidence and I would recommend that it should be published in this journal. I would just like to point out that some of the figures are hard to read, especially the amino acid labels on Figure 3 are quite small and there are some minor irregularities between the images, such as using arrows and lines for the insets in the same panel. Authors should modify those accordingly.\n\nIs the work 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/13-62
https://f1000research.com/articles/11-347/v1	22 Mar 22	{ "type": "Research Article", "title": "Can Positive Emotions Predict Consumer Satisfaction in Adverse Services?", "authors": [ "Nahima Akthar", "Dr. Smitha Nayak", "Dr. Yogesh Pai P", "Nahima Akthar", "Dr. Yogesh Pai P" ], "abstract": "Background: Managing emotions during hospital visits is important to enhance patient satisfaction. The purpose of this paper is to explore the relationship between patients’ happiness and satisfaction through patients’ mood and perceived service quality at a healthcare setup. Methods: This study was conducted in a tertiary care hospital located in coastal Karnataka during the period from November to December 2021. Primary data was collected through a structured questionnaire from 227 respondents. “Statistical Package For The Social Sciences (SPSS) 27.0” and “SmartPLS 3.0” software was used for data analysis. Results: Hypotheses proposed in this study were examined by comparing the direct effect of patients’ happiness level on perceived service quality and the indirect effect of the level of patients’ happiness on patient satisfaction. The influence of all the exogenous latent variables namely, happiness, mood, perceived service quality, on the endogenous latent variable of patient satisfaction is estimated to be moderate (R2=62.5%) Conclusion: This study empowers hospital managers to recognize how patient satisfaction is dependent on patients’ happiness. In order to enhance patient satisfaction, the quality of care provided by health services, human resources, and infrastructure must be improved. As a result, the entire service encounter can be made enjoyable to the customers by reducing the distress caused by adverse services. Managers can utilize the outcomes of the study to develop marketing strategies to influence patients’ emotions in the healthcare setup by modifying the servicescape elements.", "keywords": [ "Happiness", "mood", "patient satisfaction", "perceived service quality", "negative services" ], "content": "Introduction\n\nThe service sectors are growing faster as compared to other sectors in developing nations (Mukherjee, 2015; Latha, 2016; Service sectors in India, 2021). The services industry not only accounts for the bulk of India’s Gross Domestic Product (GDP), but it also attracts significant foreign investment, contributes significantly to export, and employs a vast majority of the population. In the financial year 2021, the services sector in India contributed 54 percent of the total Gross Value Added. Hence the service sector is an integral component of economic growth in India and necessitates investigation on various economic and behavioral dimensions.\n\nThe service industry comprises routine, positive, and negative services. “Routine or neutral services” raise familiar experiences that they navigate frequently. For example, the housekeeping and beauty services. “Positive services” are those associated with travel and entertainment. Tourism and hospitality services are examples of positive services. “Negative services” are those associated with unpleasant circumstances. Law and healthcare services are examples of negative services (Morgan & Rao, 2006).\n\nThe healthcare industry is one of the fastest-growing service industries in recent years. In the last few decades, extensive attempts have been made to incorporate “information and communication technology” (ICT) into healthcare operations (Fullman et al., 2018; Aayog, 2019). It is projected that India’s healthcare industry is expected to grow up to 372 billion dollars by 2022. The country’s healthcare market had grown rapidly to become one of the largest sectors in terms of income and jobs (Statista Research Department, 2021). This necessitates studies on the healthcare sector.\n\nThe services offered by law and healthcare are considered to be unpleasant (Hellén & Sääksjärvi, 2011). These services are referred to as “adverse services” or “negative services” since the clients are exposed to uncomfortable situations such as overcrowding, long waiting times, anxiety, and risk of infections. In adverse services customers are exposed to these situations which cause an element of uneasiness which further influences their service experience (Morgan & Rao, 2006; Schwartz, 2015; Bahadori, Teymourzadeh, Ravangard, & Raadabadi, 2017). Although healthcare services are hostile, many people are forced to use them at some point in their lives (Hellén & Sääksjärvi, 2011). Negative emotional states such as worry, uncertainty, and unease are frequently created in hospital settings (Morgan & Rao, 2006; Berry & Bendapudi, 2007; Miller, Luce, Kahn, & Conant, 2009). Researchers have examined the association between emotions and service quality evaluations (Mattila & Enz, 2002; Slåtten, 2011; Naami, & Hezarkhani, 2018). Ben-Ze’ev (2000) opined that positive emotions such as joy or happiness express satisfaction, while negative emotions such as rage or guilt express dissatisfaction.\n\nScholars in the field of “positive psychology” have defined happiness as “a summary appraisal of one’s life” (Diener, Scollon, & Lucas, 2009). Happiness is described as a feeling, sentiment, or transitory form of joy (Labroo & Patrick, 2008; Labroo & Mukhopadhyay, 2009). An individual’s happiness level impacts how happy and unhappy people acquire, understand, and evaluate the same situation. The happiness level of the consumer predicts their ability to cope in difficult conditions (Boehm, Ruberton, & Lyubomirsky, 2017). According to past research, consumers’ attitudes vary depending on the services they have received (positive or negative). Happiness, on the other hand, seems to remain consistent over time and in various situations (Hellén & Sääksjärvi, 2011). During service encounters, consumers have a strong tendency to pursue psychological needs such as hedonistic needs, inner joy, and happiness (Boehm et al., 2017). Researchers revealed that consumers with positive emotional states are more likely to evaluate services in a positive way (Ali, Amin, & Cobanoglu, 2016).\n\nService quality (SQ) evaluation is a cognitive process in which clients compare service quality expectations to the actual services obtained (Lee, Lee, & Yoo, 2000; Abedniya, & Zaeim, 2011). Therefore, hospitals must ensure that good quality services are provided to meet their client’s expectations (Suki, Lian, & Suki, 2011; Rechel, Doyle, Grundy, & McKee, 2009). Marketers need to explore the precursors of service quality evaluation. In service marketing literature, precursors of service quality evaluation by the customers have received considerable attention over the recent decade (Kiran & Diljit, 2017; Tan, Benbasat, & Cenfetelli, 2013; Sultan & Wong, 2011; Shamdasani, Mukherjee, & Malhotra, 2008).\n\nService quality is critical for healthcare organizations, and it has a significant impact on patient satisfaction (Dagger, Sweeney, & Johnson, 2007; Zaim, Bayyurt, & Zaim, 2010). The major outcomes of a business are SQ and customer satisfaction (CS), in this case, patient satisfaction. Patients are treated as guests who are seeking positive outcomes as well as quality service experiences (Otani, Waterman, Faulkner, Boslaugh, & Dunagan, 2010; Marzo et al., 2021). Delivering exceptional services leads to a high CS, which leads to customer retention (Otani et al., 2010; Loureiro, Miranda, & Breazeale, 2014; Oluwafemi & Dastane, 2016).\n\nPatient satisfaction (PS) is an individual opinion of the standard of care received (Otani, Kurz, Harris, & Byrne, 2005). PS is a critical analysis of patients’ happiness with the quality of health care they receive both in and out of the doctor’s office. PS is a major determinant of the quality of healthcare outcomes (Marzo et al., 2021). CS is essential to every industry since satisfied customers are loyal and bring in new business. The healthcare industry is no exception to this. Many studies believe that happy patients are more likely to tell their friends about their doctors and return when they need help again (Otani et al., 2010). CS reflects the feelings of healthcare patients about the quality of service they expect in comparison to what they currently experience. It’s also possible to presume that the satisfaction level of the patient is determined by the number of expectations and realities learned from the health services he/she has received (Kotler & Keller, 2016).\n\nIn the field of psychology, the importance of emotions has acquired a lot of attention, but it is lacking in the marketing literature (Hellén, 2010). Positive services are well researched when compared to negative services (Morgan & Rao, 2006; Berry & Bendapudi, 2007; Miller et al., 2009). Thus, the goal of this study is to determine the relationship between patients’ happiness and satisfaction through patients’ mood and perceived service quality at a healthcare setup. This research contributes to the service marketing literature by demonstrating how patient satisfaction is enhanced in an unpleasant service context by mood and perceived service quality. Healthcare consumers are susceptible to deal with the adverse features of the service differently and to evaluate the quality of service encounters through emotions to develop satisfaction. The hospital management can support healthcare consumers to improve satisfaction by changing the servicescape of the hospital.\n\nThis research article begins with a review of literature on the constructs of the study. In the subsequent sections the methodology adopted and the data analysis is presented followed by the major findings, limitations of the study, and directions for future research.\n\n\nLiterature review\n\nAshby and Isen (1999) proposed that positive affect has a consistent impact on a variety of cognitive activities. Many of these effects are explained by the neuropsychological theory, which claims that the pleasant effect is linked to higher levels of dopamine in the brain. They have suggested that “positive affect influences olfaction, the consolidation of long-term or episodic memories, working memory, and creative problem-solving”. For example, the idea claims that higher dopamine release in the “anterior cingulate” promotes “cognitive flexibility” and allows the choice of “cognitive perspectives”, which helps with creative problem-solving. The resulting theory has several advantages over other methods to the study of positive affect now in use. First and foremost, it offers a neuropsychological explanation for a variety of well-known positive affect occurrences. Second, it predicts “positive affect” influences on tasks that have never been studied by the researchers. Third, it lists several tasks in which positive emotion is not expected to have an impact on performance. Fourth, it connects positive emotion research findings to previously unconnected neuropsychological studies. For example, it compares cognitive processing in healthy people with cognitive processing in some neuropsychological patient groups.\n\nSubjective wellbeing (SWB) is a synonym for happiness (Lu, Gilmour, & Kao, 2001; Chang & Nayga, 2010). In the positive psychology literature, happiness is defined as “a summary appraisal of one’s life” (Diener et al., 2009; Veenhoven, 2010). Diener stated that “the term happy in common English usage refers to a transient, positive state of mind brought on by a specific experience, such as a nice social engagement” (Diener et al., 2009). One of the most important aspects of a human being’s life is their mental state of mind (Diener, Lucas, & Oishi, 2002).\n\nVarious factors influence happiness levels, such as individual feelings of joy, positive well-being, and a sense of a good and meaningful life (Lyubomirsky & Ross, 1997; Lyubomirsky & Tucker, 1998; Lyubomirsky, Tucker, & Kasri, 2001). Lyubomirsky et al. (2001) and Lyubomirsky, King, and Diener (2005) have found that happy people make more positive decisions than unhappy people. As a result, happiness plays a crucial role in determining outcomes. Happy customers are more likely to be pleased when they make a specific decision. Happy people have a more consistent reaction to life situations, as they are considerably better at dealing with stressful conditions than unhappy people (Hellén & Sääksjärvi, 2011). Happy people are more likely to be connected by more positive life circumstances, and as a result, they have more positive outcomes in their lives (Kahneman, Krueger, Schkade, Schwarz, & Stone, 2006).\n\nCustomers’ moods are transient states of emotion that prompt them to evaluate services precisely (Pieters & Raaij, 1988). When compared to other emotional states that endure longer, mood has a lower intensity, is more distributed, and is unintentional (Bagozzi et al., 1999). Lane and Terry (2000) have defined mood as “a collection of experiences that are fleeting in character, vary in intensity and length, and frequently involve multiple emotions”. This definition states that mood is a chain of expressive emotions that forms a frame of mind to change incoming events gradually through day-to-day activities, as happy customers are in a better mood, they have a greater opinion of service quality than disgruntled customers (Hellén & Sääksjärvi, 2011). Therefore, happiness is defined as an emotional state of well-being that is stored in the memory as a mood rather than an emotion (Belanche, Casaló, & Guinalu, 2013).\n\n“The customer’s assessment of an entity’s total excellence or superiority can be defined as service quality” (Zeithaml, 1988). Consumers evaluate the service quality of an organization by comparing their perceptions to their expectations (Sivakumar, Li, & Dong, 2014). Service providers must ensure that service recipients have pleasant service interactions, as negative experiences will be shared with others (Petzer, Meyer, Svari, & Svensson, 2012). According to recent research in the service industry, negative emotions experienced by service recipients during the service contact have an impact on their loyalty levels (Kiran & Diljit, 2017; Ali et al., 2016; Park, Lee, Kwon, & Del Pobil, 2015; Son, Jung, & Lee, 2015).\n\nService quality is a critical factor in determining whether or not a service provider is favored, hence it must be carefully measured and improved (Javed & Ilyas, 2018; Marzo et al., 2021). According to researchers, hospitals must now meet their criteria and deliver the greatest health care services to patients as a result of growing expectations for common facilities (Padma, Rajendran, & Lokachari, 2010; Pai & Chary, 2014). PSQ in the healthcare sector has received a lot of attention. It should be mentioned that in both public and private institutions, patients’ perceptions of healthcare services are influenced by the quality of care they receive (Shabbir, Malik, Malik, & Wiele, 2016).\n\nOne of the most widely researched topics in literature is satisfaction (Sawyer et al., 2013; Barnett et al., 2013). According to Oliver (2000), satisfaction is “a post-consumption judgment by the consumer that a service provides a pleasant level of consumption-related fulfillment, including under or over-fulfillment.” Patient satisfaction is one of the most often reported outcome indicators for quality of care in the healthcare sector, and it can be referred to as consumer satisfaction (Marzo et al., 2021). Patient satisfaction is described as “meeting or exceeding the requests and expectations of the patient” (Akbulut, 2016; Yilmaz, 2011). This situation may arise as a result of patients’ inability to assess the medical element of the treatments provided. During the examination, physicians’ compassion, empathy, and other related abilities have a beneficial impact on patient satisfaction (Akbolat, Sezer, Ünal, & Amarat, 2021). Patients who are happy with the service they receive will share their experience among people they know (Juhana, Manik, Febrinella, & Sidharta, 2015).\n\nThe hypotheses proposed in this study are shown in Figure 1. The model proposes that patients’ happiness influences their satisfaction via mood and PSQ.\n\nHappiness causes recurrent pleasant moods because happy persons are more likely to have optimistic ideas (Lyubomirsky et al., 2005; Diener & Biswas-Diener, 2005). According to psychologists, a cheerful individual might experience a bad mood. Moods fluctuate to some extent as a result of positive and negative events. Even when their moods vary, the happy person adapts to events while maintaining a positive attitude (Diener et al., 2009). Happiness is important in healthcare services because it protects consumers from the harmful consequences of those services. Individuals who are happy cope with stress better and can maintain a positive attitude in adverse services (Hellén & Sääksjärvi, 2011). As a result, the association between happiness and mood is likely to be substantial in healthcare services.\n\nHappiness is positively related to mood in adverse services.\n\nPrevious researchers have discovered an association between mood and PSQ (Kocabulut & Albayrak, 2019; Pornpitakpan, Yuan, & Han, 2017; White, 2006). Experts have documented that the impact of mood is greatest when the buyer is uninformed of the goods or services. Individuals are more likely to rely on their feelings in uncertain situations (Dubé, & Morgan, 1996). According to a comprehensive understanding, patients find it difficult to measure service quality in unpleasant services, thus they lean on their moods for assessment (Collier, 1994). Similarly, Darke, Chattopadhyay, and Ashworth (2006) have demonstrated that when data is lacking, mood serves as a clue, and this conclusion is consistent with customers’ tendency to grasp actual opinions. These findings show that when additional evidence is absent, people rely on their mood for assessment. According to previous research, evaluating adverse services is difficult for clients (Morgan & Rao, 2006). As a result, it is hypothesized that mood and perceived service quality in healthcare services have a substantial link.\n\nMood is positively connected to perceived service quality in adverse services.\n\nCustomer’s emotion is a vital factor in evaluating services that they received (Tsaur, Luoh, & Syue, 2015). Patients depend on their moods when evaluating healthcare services. Researchers believe that happiness affects PSQ through mood since happy people are more likely to be in a positive frame of mind, and hence are more likely to rate service quality positively (Hellén & Sääksjärvi, 2011; White, 2006), as they are bound to encounter positive emotional states while in adverse circumstances, a happy customer may be more likely to experience enhanced SQ (Hellén & Sääksjärvi, 2011). As a result, it is hypothesized that happiness and service quality perceptions in healthcare services are mediated by mood.\n\nThe association between happiness and perceived service quality is mediated by mood.\n\nThe impact of healthcare quality on PS has been thoroughly researched by scientists (Marzo et al., 2021; Akbulut, 2016; Badri, Attia, & Ustadi, 2009; Zineldin, 2006; Amin, & Nasharuddin, 2013). Various researchers in the field of marketing have found hypothetical as well as practical links between SQ and other user behaviors such as satisfaction, value, purchase/revisit intentions, and so on (Akbolat et al., 2021; Lee & Carter, 2011; Theodorakis & Alexandris, 2008; Kouthouris, & Alexandris, 2005).\n\nThe disparity between clients’ thoughts and expectations of the services is referred to as hospital service quality (Aagja & Garg, 2010). Patients are the hospital’s most valuable asset in a healthcare setting. Hospital service quality has grown increasingly important as a means of satisfying and sustaining patients (Alhashem, Alquraini, & Chowdhury, 2011; Arasli, Haktan, & Turan, 2008). Scholars have discovered a link between PSQ and PS, demonstrating that if healthcare SQ is higher, patient satisfaction will be higher (Yesilada & Direktör, 2010; Fatima, Malik, & Shabbir, 2018). PS is utilized to define SQ in a healthcare context. SQ and satisfaction are found to have a substantial relationship (Shabbir et al., 2016). Furthermore, it is assumed that greater services are required to satisfy customers (Fatima et al., 2018; Marzo et al., 2021). As a result, a considerable relationship between PSQ and PS in healthcare services is envisaged.\n\nPerceived service quality is positively related to patient satisfaction.\n\n\nMethods\n\nThis research work is highly focused on patient satisfaction in a healthcare setting to empirically validate the hypotheses framed. A cross-sectional research design was applied in the study.\n\nThe top four districts of Karnataka, India (Bangalore Urban, Dakshina Kannada, Udupi, and Mysore) were identified based on the Human Development Index (HDI) details made available by the Government of Karnataka (HDI report, 2015). High HDI was considered a significant inclusion criterion as it strongly captures the three dimensions of health, literacy, and standard of living. Research evidence suggests a strong association between HDI and happiness and a stronger positive relationship between HDI and life satisfaction. There is a significant positive association between HDI and the happiness index of a region as reported by Leigh and Wolfers (2006); UNDP Human Development Report authored by Hall, Helliwell, and Helliwell (2014). Applying the simple random sampling technique, Udupi district was selected for this study. The study was conducted in a tertiary care hospital located in this district from November to December 2021.\n\nThe participants included in the study were outpatients who had more than two visits to the hospital, were aged between 18-65 years, and spoke English or Kannada. The outpatient departments (OPDs) considered for the study were medicine and medical specialties and surgery and surgical specialties. Pediatric and psychiatric OPDs were excluded. Participants were approached at the pharmacy, which is their final point of outpatient service encounter at the hospital.\n\nThe sample size was calculated based on the number of items on the rating scale which is multiplied by 10 (Hair, Sarstedt, Ringle, & Gudergan, 2017) i.e. 1710 = 170. Accounting for a non-response rate of 20%, 170+34=204. So, it was approximated to 210. The total sample size of the study was 227.\n\nEthical approval was obtained from the Institutional Ethics Committee (IEC) of Kasturba Medical College and Kasturba Hospital Manipal, Karnataka, India (IEC: 868/2020). Total confidentiality of the data is maintained by not using participant identifierss. This has been included in the participant information sheet (Clause No.12).\n\nPrimary data was collected through a structured questionnaire. The questionnaire contained a set of scales evaluating the level of happiness of the participants (Lyubomirsky & Lepper, 1999). This was rated on a seven-point Likert scale. The set of scales evaluating participants’ mood (Peterson & Sauber, 1983), service quality perceptions (Brady & Cronin, 2001; Parasuraman, Zeithaml, & Berry, 1988), and patient satisfaction (Greenfield & Attkisson, 1989; Oliver, 1997) were rated on a five-point Likert scale. The questionnaire had a total of 23 questions of which 6 captured demographic details of the participants, and 17 were related to the constructs of the study. After the questionnaire was finalized, it was translated into Kannada by a language expert. Both the English and Kannada versions of the questionnaire were created using Microsoft Word 2013, then printed for participant use. This hardcopy is used to collect data from the participants. Data collection was carried out at the hospital pharmacy since it is the last point of contact in outpatient services. The participants were selected purposively. They were informed about the purpose and procedures of the study through a participant information sheet. Written consent was obtained from the participants and then the researcher distributed the questionnaire to participants. The researcher instructed the participants to tick the appropriate response on the questionnaire.\n\nBefore collecting the data, a pretest procedure was carried out which involved pre-testing of a survey questionnaire to evaluate the complete questionnaire using validity and reliability checks. Validity is the accuracy with which an instrument measures what it is supposed to measure. In this research endeavor, the following validity checks have been implemented.\n\nFace validity: This was checked to see whether at face value the questions/items appeared to be measuring the construct or what is intended to measure.\n\nContent validity: A panel of judges who were experts in healthcare management, marketing, and operations evaluated the draft questionnaire. The survey items were rated based on the clarity, relevance, appropriateness, and redundancy of the items. Out of the 10 experts approached, six of them responded with their comments and suggestions. Suggestions given were incorporated.\n\nConstruct validity: This involved convergent and divergent validity checked after receiving data from the final sample.\n\nThe data was collected from 47 respondents during the pilot study. Data was collected through a structured questionnaire. A copy of the questionnaire can be found in the Extended data (Akthar et al., 2022). The questionnaire was administered personally. The written consent was obtained from the participants before giving the questionnaire. The respondents were approached in September 2021.\n\nReliability is the consistency or repeatability of the measure. The pilot study helped to determine the construct reliability.\n\nInternal consistency: The reliability within a scale was checked to see whether all the items were designed to measure a particular construct. The reliability scores, Cronbach’s α value of the constructs are as follows: Happiness (0.812), Mood (0.643), Perceived service quality (0.756), and patient satisfaction (0.862) and all the values are well above the threshold limit (Hair et al., 2017).\n\nWhile conducting the pilot study it was observed that a few participants perceived difficulty in responding to two questions (Items H3: Some people are generally very happy. They enjoy life regardless of what is going on, getting the most out of everything. To what extent does this characterization describe you? and H4: Some people are generally not very happy. Although they are not depressed, they never seem as happy as they might be. To what extent does this characterization describe you?) pertaining to the construct happiness.\n\nThese items were reworded and the cognitive interviewing technique (involving two methods i.e. think-aloud interviewing and probing) was adopted (Willis, 2004) to check the validity of the statements. In order to check the accuracy of the reworded statements, it was subjected to 10 respondents. The paraphrased statements are as follows- H3: People are mostly happy and enjoy their life no matter what is going on to make the most out of everything. Does this describe you?; H4: People are mostly not very happy and not once appear happy as they may be. Does this describe you?\n\nDescriptive statistics were calculated using IBM SPSS statistics 27 (IBM SPSS Statistics, RRID: SCR_016479; Armonk, NY: IBM Corp). The proposed hypotheses were tested and the mediation analysis was performed using the SmartPLS 3 (SmartPLS, RRID: SCR_022040). The results are represented in the form of tables and figures in the subsequent section. SmartPLS 3.0 software was used to analyze the data of this research endeavor. “Partial Least Squares Regression-Structural Equation Modeling” (PLS-SEM) adopts the SEM technique and has many similarities to regression. In addition, PLS also models the theoretical association between the latent variables and also the relationship between the latent variable and its indicators (Chin, Marcolin, & Newsted, 1996). PLS was also preferred to other covariance-based techniques, like LISREL, as it can be run on smaller sample sizes.\n\n\nResults\n\nDescriptive statistics are estimated and the output is presented in Tables 1 & 2. The full dataset can be found in the Underlying data (Akthar et al., 2022).\n\nThe measurement model was estimated using the data and it is presented below (Figure 2).\n\nThe construct reliability was established by estimating Cronbach’s alpha, factor loadings, and composite reliability (Table 3). Composite reliability is said to be a better accurate measure of internal consistency as the measure of composite reliability doesn’t tend to increase with the addition of every new item. The threshold value of internal consistency reliability is 0.8 (Daskalakis & Mantas, 2008), which is established in this research endeavor. In a reflective model, the outer loadings of all indicators have to be above 0.7 (Henseler et al., 2014), which is evident in this case. Further, the “average variance extracted (AVE)” of each construct must be above 0.5 (Wasko & Faraj, 2005; Wixom & Watson, 2001) indicating at least 50 percent variance of each construct could be explained by the indicator. These criteria have been fulfilled and presented in Table 3.\n\n p<0.01.\n\n p<0.05.\n\n* p<0.1.\n\n“Discriminant validity” is verified by comparing the AVEs with the squared multiple correlations of each latent variable (Chin, 1998). In this analysis, Fornell and Larcker criterion is adopted (Table 4).\n\nAs the AVE of each construct is higher than the squared multiple correlations, it is concluded that the constructs of this research endeavor exhibit discriminant validity. Collinearity among the constructs was tested using the “Variance Inflated Factor (VIF)” guidelines. The predictor variables displayed VIF values below 5 (Table 3). This implied that collinearity is not a constraint in this structural model.\n\nHypotheses proposed in this study were examined by comparing the direct effect of patients’ happiness level on PSQ and the indirect effect of the level of patients’ happiness on PS. The results are displayed in Table 5. H1 proposed that the patient’s happiness level positively influences mood and it is supported (β=0.26, t=3.770, p<0.01). H2 proposed that mood positively influenced the PSQ and is supported (β=0.552, t=10.957, p<0.01). H3 proposed that happiness positively influenced PSQ and this hypothesis is also supported (β=0.212, t=3.958, p≤0.01). H4 proposed a direct positive effect of PSQ on PS (β=0.791, t=29.516, p≤0.01). The path values (β values) and the empirical t values of all the hypotheses are above the cutoff value of 0.2 and 1.96 respectively, which substantiates the proposed hypotheses of this research endeavor. The influence of all the exogenous latent variables namely, happiness, mood, perceived service quality, on the endogenous latent variable of patient satisfaction is estimated to be moderate (R2=62.5%) (Hair et al., 2017).\n\n* p<0.01.\n\n p<0.05.\n\n* p<0.1.\n\nThe effect size, f2 of all the exogenous latent variables was calculated (Table 5). The effect size measures the extent of influence of the variables independent of the scope of the sample analyzed (Cohen, 1988). Cohen (1988) proposes a threshold to gauge the extent of the effect of the constructs. Effect size above 0.35 is reported as a large effect; value in the range of 0.15 to 0.35 is reported as moderate effect and values below 0.15 is reported as a low effect. In our research endeavor, the effect size of mood on PSQ (f2=0.484) and the effect size of PSQ on PS (f2=1.669) are estimated to be large. Model Fitness is assessed with the help of the value of “Standard Root Mean square Residual (SRMR)” as proposed by Henseler et al. (2014). The threshold value of model fitness is 0.8 (Hu & Bentler, 1998). The SRMR value of this model is reported as 0.073 which indicates a good model fit.\n\nA mediation analysis was undertaken to assess the mediating effect of the construct ‘mood’ between the constructs of happiness and PSQ (Figure 3).\n\nThe direct effect of happiness on perceived service quality (0.163) is significant and the indirect effect of happiness on PSQ through mood (0.116) is also significant. The VAF (Variance Accounted for) value of 41.58 percent indicates a partial mediation between happiness and PSQ (Table 6).\n\n* p<0.01.\n\n p<0.05.\n\n* p<0.1.\n\nImportance Performance Matrix Analysis (IPMA) provides researchers with an insight into the relative importance of the performance of the exogenous latent variables in their association with the endogenous latent variables and was first proposed by Martilla and James (1977). This method enables the researchers to examine the importance of an item in addition to its performance. The rationale of this analysis is to identify the total effect of the predecessor constructs (mood, perceived service quality, and happiness) in forestalling the target endogenous construct (patient satisfaction) (Hair, Hult, Ringle, & Sarstedt, 2016, p. 276; Hair, Sarstedt, & Ringle, 2018, p. 105). The total effect establishes the importance of the constructs while the mean value of their scores reflects their performance (ranging from 0, which is the lowest, to 100, which is the highest) (Höck, Ringle, & Sarstedt, 2010, p. 201).\n\nThe results of IPMA are presented in Figure 4 and Table 7. Analyses demonstrate that PSQ is ranked high on performance (81.48) in comparison to the other exogenous constructs. In addition, the total effect of PSQ on PS is 0.791 which is also high. Thus, a unit increment in the performance of PSQ from 81.448 to 82.448 will result in an increase in the performance of PS from 77.662 to 78.453. The total effect and performance of the exogenous construct mood are 0.437 and 77.313 respectively. Thus one unit increment of mood from 77.313 to 78.313 would increase the performance of PS from 77.662 to 78.099. Similarly, the total effect and performance of the exogenous construct happiness are 0.281 and 74.553. Thus, an increment of one unit of happiness from 74.553 to 75.553 would yield an increment in patient satisfaction to 77.943. This study found that the total effect of PSQ has the strongest and most significant effect on patient satisfaction followed by Mood and then Happiness. This is an important implication to healthcare service providers.\n\nThe Importance Performance Map is presented below (Figure 5).\n\n\nConclusions/Discussion\n\nIn this research endeavor, we intended to explore the role of emotions in adverse services. We proposed to explore if mood mediates the relationship between happiness and service quality perception which had a direct bearing on patient satisfaction. Happiness was explored as a significant antecedent to perceived service quality of adverse services, such as hospitals, and it provides a significant foundation to determine patient satisfaction. These findings enable us to conclude that happy people are most likely to experience better service quality and patient satisfaction. We can also conclude that patients’ emotions (mood) at the hospital play an instrumental role in developing service quality perceptions and indirectly strengthening patient satisfaction.\n\nThe results of this research endeavor to uphold the results of previous research from positive psychology and service marketing literature showing that the happiness of people significantly influences their mood which directly or indirectly influences their service quality perception, especially in adverse services such as hospitals, legal services, etc (Morgan & Rao, 2006; Diener et al., 2009; Hellén & Sääksjärvi, 2011; Badri et al., 2009; Yesilada & Direktör, 2010; Fatima et al., 2018). However, this research endeavor extends the literature by displaying that happiness is a significant predictor of mood and mood mediates the association between happiness and perceived service quality in adverse services. This study contributes to the body of knowledge by highlighting the role of patients’ moods in predicting service quality and thereby patient satisfaction.\n\nThese results could be used to deduce the following. Firstly, happy people would experience positive affective states, and consequently, a person who scores high on happiness is more likely to report a positive mood. Secondly, the mood is a reflection of their happiness and hence the results could be biased (Diener, 2009). This study contributes to the body of knowledge by highlighting the role of patients’ moods in predicting service quality and thereby patient satisfaction. This result does not echo the outcomes of past research such as Kumar and Oliver (1997) and Oliver (1993), who proposed that there is no significant relationship between consumers’ affective response and service quality. However, White (2006) has proposed a positive association between mood and service quality, and our findings are in line with this research output.\n\nThis research also presents significant managerial implications to industry, especially to the services of adverse nature. We recommend that marketers of adverse service must design strategies to enhance the mood of their patients or customers. Every element of the servicescape in adverse services must be designed such that it enhances the mood of customers. From service providers (doctors and nurses) and support staff to peripheral service encounters, there should be effective management that contributes to elevating the customer’s mood. Doctors and nurses can be trained to handle customers’ queries about the line of treatment and medication effectively. Health care providers or medical teams, environmental conditions, and hospital completeness are all elements that influence patient satisfaction. To enhance patient satisfaction, the quality of care provided by health services, human resources, and infrastructure must be improved. As a result, the entire service encounter can be made more enjoyable for the customers by reducing the distress caused by adverse services.\n\nThis study is also subject to limitations. First, this study was conducted in a tertiary hospital of a high HDI district. There is a significant positive association between HDI and the happiness index of a region as reported by Leigh and Wolfers (2006). Future research would benefit from conducting a comparative study amongst high HDI and low HDI districts. Second, this study adopted a quantitative approach. Future researchers would consider improving patient satisfaction by exploring the elements of servicescape in adverse services through an experimental approach. The study design adopted in positive psychology research endeavors consists of a two-step process. In the preliminary stage, happy and unhappy subjects are identified and are then subject to the experimental setting. This paves the way for effective comparison in both groups. In this scenario, service quality perceptions could have been effectively captured in the controlled group and the experimental group. Thirdly, this study utilized the scale developed by Peterson and Sauber (1983) to measure the construct mood which might be a cause of concern. This scale fails to capture the extent of influence of elements of servicescape on a patient’s general mood. Thus there could be a possibility of an element of error in capturing the patient’s mood. However, it can also be argued that a patient’s mood captured at the hospital is attributed to elements of servicescape.\n\nIn conclusion, the concept of consumer emotions and its implication on service quality evaluation had gained momentum. Taking a step in this direction, this research endeavor explored the impact of consumers’ happiness on service quality perception at a hospital, which is considered to be an adverse service by nature. This research outcome indicated that consumers’ mood partially mediated the association between happiness and service quality perception. This outcome provides significant evidence that goes against the theoretical underpinnings of positive psychology theories which suggest that happy people significantly experience situations more positively. However, outcomes of this research endeavor contribute to service marketing literature which highlights the role of servicescape to modify the mood of consumers thereby influencing their service quality perception. Hospitals must design their servicescape effectively, to trigger positive emotions (mood) among patients that will have a direct bearing on service quality evaluation and thereby patient satisfaction. This is of paramount importance, especially during the current COVID-19 pandemic. COVID-19 has created significant distress economically and emotionally across the globe. Mental health has become the focal point of discussion and concern. Hence, hospitals must ensure that all their marketing strategies revolve around creating a positive affective state (mood) among their patients which will enable them to perceive adverse services in hospitals in a positive way.\n\n\nData availability\n\nFigshare: Underlying data: Can positive emotions predict consumer satisfaction in adverse services? An empirical investigation. https://doi.org/10.6084/m9.figshare.19360625.v3 (Akthar et al., 2022)\n\nThis project contains the following underlying data:\n\n- Dataset.csv\n\nThis project contains the following extended data:\n\n- Questionnaire.docx\n\n- Informed Consent.docx\n\n- Participant Information Sheet.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": "References\n\nAagja JP, Garg R: Measuring perceived service quality for public hospitals (PubHosQual) in the Indian context. Int. J. Pharm. Healthc. Mark. 2010; 4(1): 60–83. 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I am presently working as a professor", "in management studies at VIT Business School", "VIT", "Vellore." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am very much pleased to add my review report on the title 'Can positive Emotion predict consumer satisfaction in adverse services' submitted by Nahima Akhar, Dr. Sunitha Naayak and Yogesh Pai P. First of all, I congratulate the authors for choosing the topic, which is more beneficial to the society at large. The sequence of the paper is well prepared. The reviews conducted for the research gap are also self demonstrative, the methodology and tools used in this study add more relevance to the present context. The analysis part of the paper sounds good. The interpretations given about the study results project the validity of the paper to all stakeholders involved in it.\n\nThe entire article was prepared without any flaws in the language used and was also informative, to the entire researchers' community. The themes of the paper showed unique by comparing the direct effect of patients' happiness level on perceived service quality and the indirect effect of the level of patients' happiness on patients' satisfaction. Further, this study also compared all the exogenous latent variables like happiness, mood, and perceived service quality with the endogenous latent variables of patient satisfaction.\n\nAt the outset, the study conducted demonstrated how patients satisfaction is dependent on patients' happiness. The implication of the study is also well explained. Once again by congratulating the authors of the study, I place my recommendation for indexing the article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "8147", "date": "25 Apr 2022", "name": "Nahima Akthar", "role": "Author Response", "response": "Dear reviewer, we appreciate the time taken by you to go through our article and submit a detailed review of it. We will go through the paper again and further check for any lapses, and rectify them if any. Thank you." } ] }, { "id": "162772", "date": "09 Jan 2024", "name": "Blessy Prabha Valsaraj", "expertise": [ "Reviewer Expertise Mental Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written. Methodology can be more explained, the design, setting (about the hospital), data collection process etc.\nSuggestions: Can add more recent literature.\nUNDP Human Development Report : Setting: Expand UNDP at the first usage in the article.\nPossible limitations of the results:\n1. Happiness is one type of mood itself, so I think these two variables are greatly overlapped, further proven by the positive correlation in the study.\n2. Mood can be a reflection of the person's personality trait, such as perceiving the world around as positive and thus assuming optimistic perspectives on all the circumstances in life, including adverse. But is known that environment also has an impact in the mood and happiness of the individual. Thus, it could be due to the positive hospital environment (being one of the HDI) it would have triggered the happiness? (A random selection of the hospitals and the settings would have minimized this bias).\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": [ { "c_id": "10875", "date": "12 Jan 2024", "name": "Nahima Akthar", "role": "Author Response", "response": "Dear Reviewer, We appreciate the time taken by you to go through our article and provide a detailed review of the article. We have incorporated all the changes suggested by you. The response to the reviewer's comments is mentioned below: 1. Thank you for your suggestion. The methodology section has been revised as suggested. 2. This article was submitted in February 2022. Therefore, the articles published till 2021 which are related to this study were included. 3. Thank you for the suggestion. This has been incorporated as suggested. 4. Thank you. When we look at the variables happiness and mood, they look similar. However, as per positive psychology literature happiness and mood are two different constructs. Hence, we have proved statistically that there is a significant relationship between happiness and mood. 5. Thanks for the suggestion. Yes. A positive hospital environment does impact patient's happiness and mood. In this research, the hospital is selected through a simple random sampling." } ] } ]	1	https://f1000research.com/articles/11-347
https://f1000research.com/articles/12-299/v1	17 Mar 23	{ "type": "Research Article", "title": "Load-induced blood marker kinetics in patients with medial knee compartment osteoarthritis are associated with accumulated load and patient reported outcome measures", "authors": [ "Annegret Mündermann", "Corina Nüesch", "Simon Herger", "Anna-Maria Liphardt", "Frédérique Chammartin", "Enrico De Pieri", "Christian Egloff", "Corina Nüesch", "Simon Herger", "Anna-Maria Liphardt", "Frédérique Chammartin", "Enrico De Pieri", "Christian Egloff" ], "abstract": "Background: This study aimed to quantify the mechanoresponse of 10 blood marker candidates for joint metabolism to a walking stress test in patients with knee osteoarthritis and to determine the association among marker kinetics and with accumulated load and patient reported outcomes. Methods: 24 patients with knee osteoarthritis completed questionnaires, and a walking stress test with six blood serum samples and gait analysis. Concentrations of cartilage oligomeric matrix protein (COMP), matrix metalloproteinases (MMP)-1, -3, and -9, epitope resulting from cleavage of type II collagen by collagenases (C2C), type II procollagen (CPII), interleukin (IL)-6, proteoglycan (PRG)-4, A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and resistin were determined by enzyme-linked immunosorbent assays, Joint load (moments and compartmental forces) was estimated using musculoskeletal modeling using gait analysis data. Results: COMP and MMP-3 showed an immediate increase after the walking stress followed by a decrease. MMP-9 and resistin showed a delayed decrease below pre-stress levels. ∆COMP correlated with ∆MMP-3 for most time points. ∆MMP-9 correlated with ∆resistin for most time points. The load-induced increase in blood marker levels correlated among blood markers and time points. C2C and resistin correlated positively and C2C/CPII and MMP2 correlated negatively with load during gait. Immediate relative ∆CPII and ∆MMP1 and delayed relative ∆COMP, ∆IL6, ∆C2C, ∆CPII, ∆MMP1 and ∆MMP3 correlated with the load accumulated during the walking stress. Baseline C2C levels correlated with Knee Osteoarthritis Outcome Score (KOOS) subscales and load-induced changes in MMP-3 with KOOS and Short Form 36 quality of life subscores (P<0.05). Conclusions: The distinct and differentiated physiological response to the walking stress depends on accumulated load and appears relevant for patient reported osteoarthritis outcome and quality of life and warrants further investigation in the context of disease progression. ClinicalTrials.gov registration: NCT02622204", "keywords": [ "Knee osteoarthritis", "articular cartilage", "serum biomarkers", "stress test", "in vivo mechanoresponse", "ambulatory load", "PROMs" ], "content": "Introduction\n\nThe response of biomarkers of tissue biology to mechanical stimuli in physiological and pathological conditions has attracted increasing attention in recent years.1 Introducing a walking stress test involving a 30-minute walking exercise, Mündermann et al.2 reported consistent immediate effects of the walking stress on cartilage oligomeric matrix protein (COMP) with a return to pre-stress levels within 30-minutes after the exercise in young healthy adults. This mechanoresponse was clearly attributed to the mechanical stimulus confirmed by a control experiment without a stimulus. Patients with medial compartment knee osteoarthritis (OA) also showed an immediate increase in COMP but continuously decreasing levels below pre-stress levels after the walking stress without differences to an age-matched asymptomatic control group.3 The change in COMP at 3.5 and 5.5 hours,4 in a collagen degradation marker C1,2C and in the synthesis marker CS846 5.5 hours after the stimulus5 predicted changes in articular cartilage thickness supporting the potential relevance of load-induced changes in blood markers in the progression of knee OA.\n\nTo date, most studies only investigated changes in COMP and reported an increase in COMP levels during a 20 to 45 minute walking exercise or up to 5000 steps2–4,6–15 with a return to pre-stress levels within 30 minutes2,3,7,9,11 or 60 minutes after the exercise.10 Although load-induced marker kinetics may be affected by OA and differ in magnitude and timing between markers,3–5 only few studies have reported on the kinetics of other markers to walking, marker kinetics beyond 60 minutes after the exercise or other mechanical stimuli in patients with OA.1 Interestingly, Jayabalan et al.14 reported an increase in COMP but not in tumor-necrosis-factor-α (TNF-α) after a 30-minute walk in patients with unilateral knee OA, and hence not all blood marker candidates may respond to mechanical stimuli in patients with knee OA. Correlations among blood marker kinetics may reveal sets of biomarkers involved in the same physiological pathways.\n\nCandidates for mechanosensitive blood markers of joint pathology include markers of cartilage synthesis, cartilage degradation, joint lubrication or inflammation including the C-propeptide of type II procollagen (CPII),16 the C2C epitope cleavage of type II collagen,17,18 COMP,19 matrix metalloproteinase (MMP)-120 or MMP-3.21 A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-4),22,23 proteoglycan 4 (PRG-4) or lubricin,24 interleukin 6 (IL-6),25,26 and resistin.27–30\n\nBaseline patient reported osteoarthritis outcome and quality of life (QoL) may predict future changes in blood markers of articular cartilage as suggested in a study on volleyball players,31 and selected blood and urinary markers appear to be related to radiographic evidence of joint damage and/or clinical symptoms of OA.32 To date, it is unknown whether the response of blood markers to load is associated with patient reported osteoarthritis outcome and QoL in patients with knee OA. Moreover, as none of the previous studies estimated the load accumulated during the respective exercise stress, to date the association between accumulated load and load-induced marker kinetics is unknown. Our exploratory study aimed (i) to quantify the mechanoresponse of this array of potential blood markers for joint pathology (COMP, MMP-1, MMP-3, MMP-9, CPII, C2C, C2C/CPII, ADAMTS-4, PRG-4, IL-6 and resistin) to a walking stress test in patients with knee OA and to determine the correlation (ii) among the kinetics of these blood markers, (iii) with accumulated knee load during the walking stress, and (iv) with patient reported osteoarthritis outcome and QoL.\n\n\nMethods\n\nData presented in this exploratory cross-sectional laboratory study were collected as part of the baseline assessment of a larger study on the effect of high tibial osteotomy on load-induced changes in cartilage biomarkers.33 Participants were recruited from the Department of Orthopaedics and Traumatology at the University Hospital Basel. Inclusion criteria were radiographically diagnosed and isolated symptomatic medial compartment knee OA and being scheduled for high tibial osteotomy. Exclusion criteria were: use of walking aids; inability to walk for 30 minutes; age < 18 years (before maturation) or age > 70 years (risk of advanced general sarcopenia (degenerative loss of muscle mass in aging)); body mass index (BMI) > 35 kg/m2; active rheumatic disorder; prior neuromuscular impairment (e.g. stroke); conditions other than knee OA that could cause abnormal patterns of locomotion; prior hip, knee, and ankle prosthesis or osteotomy of the lower extremities; prior spine surgery; other major medical problems; and current enrollment in another experimental (interventional) protocol. The study was approved by the regional ethics board (August 19, 2015; Ethikkommission Nordwest- und Zentralschweiz EKNZ 2015-224) and conducted in accordance with the Declaration of Helsinki, and participants provided written informed consent prior to participation.\n\n24 patients (16 male, 8 female) with medial compartment knee OA met the inclusion criteria and participated in this study. Patient demographics, disease characteristics (Kellgren Lawrence (KL)34 grade) and the patient reported outcomes measures (PROMs, patients reported function: Knee Injury and Osteoarthritis Outcome Score (KOOS)35; patient reported quality of life: 36-Item Short Form Health Survey (SF36)36; KOOS and SF36 assessed immediately prior to the walking stress test) are shown in Table 1. We considered all KOOS subscores but used only the total SF36 score and its subscores physical function and pain for further analysis.\n\nAll participants completed a walking stress test at the Functional Biomechanics Laboratory at the Department of Orthopaedics and Traumatology the University Hospital Basel, Switzerland (March 2016 to November 2019).33 Briefly, patients were asked to refrain from physical activity during the 24 hours prior to the experiment and perform minimal walking activity before the study visit. Participants rested in a seated position for 30 minutes before the walking stress. The walking stress comprised walking at self-selected walking speed on a flat treadmill (mercury® 3p, h/p/cosmos sports & medical GmbH, Nussdorf-Traunstein, Germany). Blood samples were collected immediately before (T0), immediately after (T1), and 0.5h, 1.5h, 3.5h, and 5.5h (T2–T5) after the walking stress. During the walking stress, spatiotemporal parameters (walking speed, cadence, step length) were collected using the built-in pressure plate (Zebris FDM-T, Zebris Medical GmbH, Isny, Germany) and the manufacturer’s software. For the 5.5 hours after the walking stress, participants rested in a seated position to preclude blood volume distribution.\n\nBlood samples (7.5 ml each) were obtained from an antecubital vein. A thin catheter was placed and remained in their vein for 6 hours. As described in the protocol,33 to prevent clogging by clotted blood, we flushed the catheter with 10 ml isotonic saline solution (0.9% NaCl) after every blood draw. The first 3 ml of each sample was discarded to avoid dilution by the injected saline solution. Blood samples clotted in the blood tubes (S-Monovette® 7.5ml Z-Gel, Sarstedt AG, Nürnbrecht, Germany) at room temperature for 30 minutes. They were then centrifuged (Sarstedt AG &Co SMC6) for 15 minutes at 2016 g, separated into aliquots, frozen (-20°C), and transferred to a -80°C freezer within 48 hours until assays were performed.\n\nAfter the final blood draw, participants completed gait analysis with a 3-dimensional motion capture system (Vicon Motion Systems Ltd., Oxford, UK) and force plates (Kistler Instrumente AG, Winterthur, Switzerland). We attached reflective skin markers to selected anatomical landmarks according to the Plug-In Gait marker set.37 After collecting data for a standing trial (anatomic upright position), data for three gait trials at self-selected walking speed were collected for each participant (own regular walking shoes; none of the participants used prescribed orthoses or insoles).\n\nSerum biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays (ELISAs, Table 2). Investigators were blinded to the samples, which were analyzed in duplicate and in random order. Differences due to inter-assay variation were eliminated by comparing concentrations within participants and testing all samples of any participant on the same plate.\n\nAccumulated load was estimated using two different approaches:\n\n(1) Approach 1: Maximum, rate of increase and impulse of the vertical ground reaction force during treadmill walking multiplied by number of steps taken during the walking stress. The vertical ground reaction force was measured for each step using the pressure plate built into the treadmill (Zebris FDM-THM-S pressure plate, Zebris Medical GmbH, Isny, Germany). Vertical ground reaction force trajectories normalized to body weight were computed for each step by the manufacturer’s software and exported. The vertical ground reaction force impulse for each step was calculated as the area under the vertical ground reaction force trajectory during loaded stance phase and normalized to body weight.\n\n(2) Approach 2: Maximum and impulse of the knee adduction moment, knee flexion moment, total knee compressive force, medial and lateral compartment compressive force as well as medio-lateral shear force were calculated from musculoskeletal modeling for the affected knee during overground walking multiplied by number of steps taken during the walking stress. As previously described for the same cohort,38 kinematic and ground reaction force (GRF) data were filtered using a second-order low-pass Butterworth filter with a cut-off frequency of 5 Hz and 12 Hz, respectively. Gait events were determined from the GRF measurements using force thresholds (>20 N for foot-strike and <20 N for foot-off). Marker trajectories and GRF data were used as input for an inverse dynamics analysis in the AnyBody Modelling System (AnyBody Technology A/S, Aalborg, Denmark). Personalized models for each subject were created as previously described39 from a detailed generic model of the lower limb40 (based on a cadaveric dataset41) scaled to match the overall anthropometrics to each subject and the marker data collected during the standing reference trial of the same subject.42 We computed joint kinematics from the measured marker trajectories, and calculated the required muscle activations, resulting knee total, flexion and adduction moments, as well as the proximo-distal compressive force, medial and lateral compartment compressive force, and medio-lateral shear force by an inverse dynamics analysis based on a third-order-polynomial muscle recruitment criterion.43 All moments were reported as external net moments, and all forces and moments were normalized to body weight. The impulse of each variable was calculated as the area under the curve during loaded stance phase for each step.\n\nFor the biomarkers PRG-4 and ADAMTS-4, 23 and 63 measurements were below the detection limit, respectively. We imputed these measurements by using the midpoint between 0 and the lower detection limit.44\n\nLoad-induced changes in biomarkers were defined as\n\nTo assess the association between accumulated load and load-induced blood marker kinetics, relative changes in blood markers were computed as\n\nWe only interpreted associations between blood marker levels and their load-induced changes with patient reported osteoarthritis outcome and QoL or loading parameters if significant correlations were observed with at least two KOOS or SF36 subscores or at least two loading parameters to account for the possibility of detecting correlations by chance.\n\nADAMTS-4 was log transformed to address skewness. Because we intended to only interpret results that are in concordance with previous evidence for mechanosensitivity,45 we refrained from applying Bonferroni corrections and set the significance level to 0.05. Statistical analysis was performed in R version 4.0.3 (The R Foundation, Vienna, Austria). Assuming that a true correlation has an absolute value of 0.60 or greater (0.60 being considered an overall moderate correlation), a sample size of 19 is required to determine if the correlation coefficient differs from zero with a power of 80% and an alpha significance level of 5%.\n\n\nResults\n\nDuring the 30-minute walking stress, patients walked at a median speed of 0.94 (IQR, 0.70–1.04) m/s with a cadence of 100.6 (92.1–107.3) steps/minute and step length of 0.56 (0.45–0.61) m, and took 2416 (1410–2945) steps. Descriptive statistics of the loading parameters per step and the estimated accumulated load are shown in Table 3. Detailed loading trajectories for a subset of patients have been published previously.39\n\nA summary of each blood marker at the different time points is given in the extended data 1.65 Compared to pre-walking stress serum concentrations, COMP and MMP-3 levels differed significantly between time points. Levels increased from pre-stress to 0h, returned to pre-stress levels within 0.5h and then continued to decrease further until 5.5h post walking stress (Figure 1). MMP-9 and resistin levels were significantly lower at 1.5h and 5.5h post walking stress than pre-stress levels (Figure 1). Although significant differences in ADAMTS-4 and IL-6 between several time points were observed, there was no clear pattern of a loading response (Figure 1). Few to no significant differences in MMP-1, C2C, CPII, C2C/CPII, and PRG-4 levels between time points were observed. As a sensitivity analysis, we excluded measurement for ADAMTS-4 and PRG-4 that were below the detection limit. Results were coherent with the results from the imputed dataset. Please note that a subset of this data has been previously presented at a conference.46\n\nBoxplots show median, 25% and 75% quartiles and individual data points. p<0.05; p<0.01; p<0.001; **p<0.0001. Grey bars illustrate the walking stress.\n\n∆COMP correlated with ∆MMP-3 from before to immediately after (T1-T0, ρ=0.514, P=0.011), from 0.5h to 1.5h (T3-T2, ρ=0.465, P=0.023), and from 1.5h to 3.5h (T4-T3, ρ=0.465, P=0.023) after the walking stress (Figure 2). ∆MMP-9 correlated with ∆resistin from 0h to 0.5h (T2-T1, ρ=0.517, P=0.011), 0.5 to 1.5h (T3-T2, ρ=0.523, P=0.010), 1.5h to 3.5h (T4-T3, ρ=0.480, P=0.018) and 3.5h to 5.5h (T5-T4, ρ=0.641, P=0.001) after the walking stress (Figure 3).\n\nCross-correlations of change from baseline concentrations (T0) among all markers and time points (T1 to T5) are shown as correlogram in Figure 4. ∆COMP from baseline correlated positively with ∆MMP-3, ∆C2C, ∆CPII, ∆ADAMTS-4, ∆PRG-4 and ∆C2C/CPII ratios from baseline and negatively with ∆IL-6 and ∆resistin from baseline for most time point comparisons. Several other positive and negative correlations among change from baseline among markers and time points were observed (Figure 4).\n\nPositive correlations are displayed in blue and negative correlations in red color. Color intensity and the size of the circle are proportional to the correlation coefficients. The legend color shows the correlation coefficients and the corresponding colors. Only statistically significant correlations are shown (P<0.05).\n\nC2C at T0 correlated negatively with the peak total joint moment (ρ=-0.51, P=0.013) and the knee medio-lateral shear force impulse (ρ=-0.52, P=0.011; Figure 5). C2C/CPII correlated negatively with the total joint moment impulse (ρ=-0.42, P=0.042). MMP1 at T0 correlated negatively with the knee medio-lateral shear force impulse (ρ=-0.47, P=0.023). Resistin at T0 correlated positively with the first peak knee total compressive force (ρ=0.42, P=0.045) and vertical ground reaction force (ρ=0.43, P=0.033). None of the other blood marker concentrations at T0 correlated with any parameter describing load exerted during a single step.\n\nMax1—first maximum of loading parameter trajectory during gait cycle; imp—impulse of loading parameter for one gait cycle (=integral of loading parameter over time); GRF—ground reaction force; PBW—per body weight; KFM—knee flexion moment; KAM—knee adduction moment; TJM—total joint moment; med—medial; lat—lateral. Color intensity and the size of the circle are proportional to the correlation coefficients. The legend color shows the correlation coefficients and the corresponding colors. Only statistically significant correlations are shown (P<0.05).\n\nCOMPT4rT0 correlated positively with accumulated peak total joint moment (ρ=0.52, P=0.014) and accumulated total joint moment impulse (ρ=0.43, P=0.047; Figure 6). IL6 T4rT0 correlated negatively with accumulated peak knee adduction moment (ρ=-0.49, P=0.020) and impulse (ρ=-0.43, P=0.045), with accumulated peak total joint moment (ρ=-0.47, P=0.028) and impulse (ρ=-0.46, P=0.031), and with accumulated medial compartment compressive force impulse (ρ=-0.46, P=0.030). C2CT34rT0 correlated positively with accumulated peak total joint moment (ρ=0.52, P=0.014), peak medial compartment compressive force (ρ=0.52, P=0.012) and impulse (ρ=0.45, P=0.038), peak total joint force (ρ=0.48, P=0.023), and ground reaction force impulse (ρ=0.48; P=0.024). CPIIT1rT0 correlated negatively with accumulated vertical ground reaction force impulse (ρ=-0.67, P=<0.001) and lateral compartment compressive force impulse (ρ=-0.58, P=0.005), and positively with accumulated knee flexion moment impulse (ρ=0.62, P=0.002). CPIIT3rT0 correlated positively with accumulated peak medial joint force (ρ=0.45, P=0.035) and impulse (ρ=0.53, P=0.011), medio-lateral shear force impulse (ρ=0.43, P=0.047) and total compressive force impulse (ρ=0.43, P=0.047). MMP1T1rT0 correlated negatively with accumulated peak knee adduction moment (ρ=-0.44, P=0.039), peak total joint moment (ρ=-0.44, P=0.039), peak medial compartment compressive force (ρ=-0.43, P=0.043) and total compressive force impulse (ρ=-0.44, P=0.039). MMP1T5rT0 correlated negatively with accumulated knee adduction moment impulse (ρ=-0.54, P=0.010) and knee medio-lateral shear force impulse (ρ=-0.45, P=0.036), and positively with accumulated lateral compartment compressive force impulse (ρ=0.45, P=0.036). MMP3T4rT0 correlated positively with accumulated peak total joint moment (ρ=0.49, P=0.021), peak medial compartment compressive (ρ=0.44, P=0.043), medio-lateral shear (ρ=0.54, P=0.009) and total compressive force (ρ=0.51, P=0.015). Correlations of ADAMTS5 were not further considered because of the high number of imputed data points.\n\nMax1—first maximum of loading parameter trajectory during gait cycle; imp—impulse of loading parameter for one gait cycle (=integral of loading parameter over time); GRF—ground reaction force; PBW—per body weight; KFM—knee flexion moment; KAM—knee adduction moment; TJM—total joint moment; med—medial; lat—lateral. Color intensity and the size of the circle are proportional to the correlation coefficients. The legend color shows the correlation coefficients and the corresponding colors. Only statistically significant correlations are shown (P<0.05).\n\nC2C levels before the walking stress (T0) correlated negatively with the KOOS subscales symptoms (ρ=-0.439, P=0.032), pain (ρ=-0.503, P=0.012), activities of daily living (ρ=-0.607, P=0.002), and recreation/sports (ρ=-0.677, P<0.001; extended data 2 (65)). Patients with higher C2C levels had lower osteoarthritis outcome scores (greater pain respective limitations). ∆IL-6 (T1-T0) correlated positively with the total SF36 score (ρ=0.430, P=0.036) and the SF36 category physical function (ρ=0.467, P=0.022; extended data 3 (65)). Patients with a smaller load induced change in IL-6 had lower SF36 scores. ∆MMP-3 (T1-T0) correlated positively with the KOOS subscales QoL (ρ=0.429, P=0.036), and with the SF-36 category pain (ρ=0.444, P=0.030). Patients with a smaller load induced change in MMP-3 had lower QoL scores (greater pain respective limitations).\n\n\nDiscussion\n\nThe purpose of this exploratory study was (i) to quantify the mechanoresponse of this array of potential blood markers for joint pathology (COMP, MMP-1, MMP-3, MMP-9, CPII, C2C, C2C/CPII, ADAMTS-4, PRG-4, IL-6 and resistin) to a walking stress test in patients with knee OA and to determine the correlation (ii) among the kinetics of these blood markers, (iii) with accumulated knee load during the walking stress, and (iv) with patient reported osteoarthritis outcome and QoL. Only COMP and MMP-3 showed a consistent increase immediately after the walking stress while COMP, MMP-3, MMP-9 and resistin all showed consistent delayed decreases below pre-stress levels up to 5.5h post walking stress. COMP and MMP-3 kinetics in response to the mechanostimulus correlated as did load-induced MMP-9 and resistin kinetics. Moreover, we observed correlations of change from baseline levels among several blood markers for several time points, between baseline levels and load per gait cycle, between load-induced changes in selected blood markers and accumulated load during the walking stress, and between baseline levels and load-induced changes in selected blood markers with patient reported osteoarthritis outcome and QoL.\n\nThe results are in line with previous reports of an initial increase in COMP above pre-stress levels during a 30-minute walking exercise2–4,6–15 followed by – on average – a continuous decrease in COMP well below pre-stress levels in the 5.5 hours after the walking stress but with a large variability in patients with OA and in older adults.3,4 Previous work including a control experiment where COMP remained stable has shown that the increase in COMP after the walking stress is clearly attributed to the walking stress.2 The pattern of a prolonged decrease contrasts observations of the mechanoresponse of COMP in a walking stress test in young healthy adults.2 The consistent results in patients with knee OA are particularly relevant as the delayed mechanoresponse of COMP has been shown to predict future changes in knee cartilage morphology in patients with OA4 and in older adults.47 Erhart-Hledik et al.4 reported that in 17 patients with medial compartment knee OA changes in COMP levels from pre-stress levels to 3.5h and 5.5h after the walking stress varied largely among patients (ranging from 40% decrease to 40% increase) and that those with higher COMP levels 3.5h and 5.5h after the walking stress experienced a greater loss in articular cartilage five years later. Moreover, in that study neither pre-stress COMP levels nor changes in COMP levels immediately after the walking stress correlated with cartilage thickness changes over five years.\n\nIn another study on 12 asymptomatic persons aged 45 years and older, changes in COMP levels from pre-stress levels to 5.5h after the walking stress varied largely among persons (ranging from 54% decrease to 22% increase) and that those with higher COMP levels 5.5h after the walking stress experienced a greater loss in articular cartilage seven to nine years later.47 This association was not as strong as that in patients with medial compartment knee OA.4 These results suggest that patients with knee OA and older asymptomatic adults show a large variability in load induced COMP kinetics and that those with prolonged elevated COMP levels will experience greater cartilage degeneration in subsequent years. However, the factors determining the kinetic patters are not understood and it is unknown how load induced COMP kinetics evolve throughout a person’s lifetime. In our study, baseline COMP did not correlate with joint load during one gait cycle but patients with greater accumulated load during the walking stress still had higher relative COMP levels 3.5 hours after the walking stress suggesting the relevance of the characteristics of a mechanostimulus for of the response of COMP to this stimulus. This result is in agreement with a recent study showing that the response of blood markers (i.e. type II collagen degradation and formation-degradation ratio) to exercise depends on characteristics of the exercise (impact—running versus no impact—cycling).48 Overall, COMP kinetics in response to a walking stress appear may be determined not only by the presence of OA or the specific mechanostimulus but also influenced by age as previously suggested,3 and relevant for degenerative processes of articular cartilage.\n\nSimilar kinetics of COMP and MMP-3 and correlations with accumulated load observed here are in line with previous reports in studies on the response of blood biomarkers to extreme ambulatory exercise49 and to immobilization during bed rest.50,51 Changes in COMP were linearly related with changes in MMP-3 throughout an ultramarathon race (4486-km during 64 running days without any rest days) where in 68% of runners, ultramarathon-induced changes in MMP-3 levels explained more than 30% of ultramarathon-induced changes in COMP levels.49 Liphardt et al. showed in two separate studies involving 5-day51 respective 21-day50 bedrest under highly controlled conditions that COMP and MMP-3 decreased by 10 to more than 20% within the first 24 hours of bedrest and both returned to pre-bedrest levels within 24 hours after bedrest. Moreover, COMP and MMP-3 levels in serum respective in synovial fluid were higher in patients with diagnosed knee OA and that patients with more severe knee OA had higher COMP and MMP-3 levels than patients with less severe knee OA.52,53 MMP-3 activates other MMPs such as MMP-1 and MMP-13,54 which may lead to an initial high release of type II collagen fragments55 and COMP cleavage.56 The strong correlation among the load-induced kinetics of COMP and MMP-3 and with the accumulated load in our study and their metabolic relationship suggest that COMP and MMP-3 may not only be involved in the pathophysiology of OA but also that MMP-3 may play a role in regulating COMP according to mechanical demands. The latter is further supported by our finding that the load induced changes in MMP-3 correlated with patient reported osteoarthritis outcome and QoL.\n\nInterestingly, MMP-9 showed similar load-induced kinetics as resistin, and both had a tendency towards a negative correlation with accumulated load. This pattern clearly differed from that of COMP and MMP-3 without an immediate but with a delayed response. We did not observe an association of MMP-9 and resistin levels at baseline. Hence, the similar patterns in load-induced kinetics can be clearly attributed to the walking stress. In contrast, a more than twofold increase in resistin (and association with load-induced changes in MMP-3) in lean adults has been reported for marathon running.57 Clearly, marathon running represents a much greater physiological stress than walking for 30 minutes. Resistin levels are higher in obese persons and in patients with severe knee OA.58 Moreover, resistin may lead to an overexpression of MMPs.28,29 Hence, a decrease in resistin (and MMP-9) levels elicited by daily activities such as walking may be particularly relevant in patients with OA and/or obesity. In our study, we observed a decrease in resistin and MMP-9 levels in response to walking stress. This finding may reflect stress-induced suppression of resistin and downregulation of MMP-9, which could indicate a chondroprotective function of exercise by impeding the proteolytical digestion of matrix components such as gelatin. The potential relevance of this mechanoresponse is further supported by the observation that associations of changes in response to load for COMP, ADAMTS-4, and PRG-4 levels and C2C/CPII ratios with resistin and their correlations with accumulated load only emerged 0.5h after the walking stress.\n\nIn our study, the walking stress did not elicit consistent changes in kinetics for MMP-1, C2C, CPII, C2C/CPII, IL-6, PRG-4, and ADAMTS-4. Previous evidence suggested that COMP, MMP-3, MMP-9, ADAMTS-4, CPII and IL-6 could suitable for assessing in vivo cartilage mechanosensitivity.45 It is possible that these markers may reflect individual disease progression despite of the lack of group results. Joint load assessed during gait analysis can be considered a surrogate of the typical loading of an individual’s joint. The observed negative correlations of catabolic blood markers (MMP1, C2C) with joint load during gait assessed using gait analysis may suggest that those patients who have lower joint level loads tend to have higher joint metabolism or – as indicated by C2C/CPII – a greater misbalance towards cartilage destruction. These results agree with previous evidence on the role of underloading in early knee OA after an ACL injury.59\n\nInterestingly, we did not observe a clear pattern of loading response of IL-6 to the walking stress nor an association with any of the other blood makers except a moderate correlation with the response to load for COMP and MMP-3 at time points after the walking stress. Almost all patients included in this experiment had mild to moderate OA with moderate symptoms. The data on IL-6 are not sufficient to rule out the possibility that inflammation played a role in the current disease process of this population or in their metabolic response to the walking stress. For instance, Atkinson et al.60 have recently shown the presence of local inflammation using knee effusion-synovitis volume and a correlation between the change in the load-distribution and change in knee effusion-synovitis volume in patients undergoing high tibial osteotomy suggesting the phenomenon of mechano-inflammation in patients with knee OA. While such data cannot be retrospectively produced in the current study, future analyses may assess other inflammatory markers to further elucidate the role of inflammation in this population.\n\nWe observed an association of load-induced kinetics among these blood markers and the association of C2C and ∆MMP-3 (both markers of tissue degradation) with patient reported osteoarthritis outcome and QoL. These associations may provide novel insights into disease activity and metabolic processes. The association of C2C with patient reported osteoarthritis outcome and QoL is consistent with previous reports of negative correlations of urine C2C levels with KOOS subscores in women with knee OA.61 Moreover, urinary C2C was one of the markers predictive of worsening pain and radiographic OA over 2 years.62 C2C levels also differ between patient with multijoint and those with single-joint OA63 and correlate with disease severity as determined by magnetic resonance imaging.64 The average C2C levels in patients with knee OA in our study were comparable to those obtained in healthy athletes.65 The negative correlation between C2C levels and patient-reported OA outcomes suggests that patients with lower OA outcome scores have higher catabolic activity, possibly reflecting increased tissue turnover or tissue degradation. Further insight into this association could be obtained in patients undergoing treatment for knee OA, including joint-preserving procedures such as corrective osteotomy.\n\nMMP-3 consistently changes with ambulatory load and immobilization45,49–51 and is relevant for OA.52,53 Our finding of an association between ∆MMP-3 and patient reported outcome measures supports the importance of MMP-3 in the context of cartilage mechanosensitivity. MMP-3 degrades collagen types II, III, IV, IX, and X, proteoglycans, fibronectin, laminin, and elastin21 and is thus critical for tissue turnover. MMP-3 is also involved in metabolic processes in other tissues than articular cartilage. Nevertheless, higher load-induced systemic concentrations may influence healthy or pathological cartilage metabolism. The dose-response of load-induced changes in MMP-3 was stronger than those in COMP in healthy persons.45 Most studies on in vivo mechanosensitivity of articular cartilage in health and disease have focused on COMP.2–4,6–15 Greater ambulatory load lead to a greater load-induced increase in MMP-3 in healthy persons.45 Our result that patients with a higher QoL had a greater load-induced increase in MMP-3 suggests that metabolic processes are related to QoL. Our observations of correlations of blood marker kinetics with the accumulated load during the walking stress emphasizes the need to quantify and consider these parameters in future studies employing loading stress tests. In particular, immediate or delayed positive correlations of some blood markers (C2C, CP2, MMP3, PRG4) with accumulated load and negative correlations of others (IL6, MMP1, MMP9) point towards differential metabolic response to a walking stress. Future longitudinal studies in patients with knee OA are warranted to shed light on the relevance of this finding in the context of future disease progression.\n\nParticipants were instructed to minimize their physical activity during the 24 hours prior to the experiment but their activity more than 30 minutes prior to the start of the walking stress was not monitored, and hence a potential effect of pre-stress physical activity cannot be excluded. Participants walked at their preferred speed for 30 minutes, and the speed and hence the number of steps taken during the walking stress differed among participants. We decided to prescribe the length of the physical stress rather than the number of loading cycles as to date the relevance of one over the other on the metabolic response to a mechanical stimulus is unknown and to facilitate comparison with the literature. However, we addressed this presumed limitation by estimating the load accumulated during the walking stress test and assessing the effect of the accumulated load on blood marker kinetics in response to load. Blood markers reflect contributions of each marker from all joints in the body, and it is thus possible that the serum biomarker kinetics presented here reflect the effects of walking stress on all joints, not just the knee affected by OA. Previous studies4,47 showing a correlation of load-induced blood marker kinetics (e.g., COMP) with subsequent changes in articular cartilage thickness. Moreover, even if elevated blood markers (e.g., enzymes) in response to load may originate from other tissues, their systemic presence may still affect articular cartilage. These considerations suggest that the results reported here are relevant despite these limitations. Here, we report on selected blood markers for joint pathology using ELISAs. Applying other approaches such as untargeted or targeted metabolomics or proteomics to a controlled experiment as presented here may be useful for identifying other blood markers relevant for in vivo mechanobiology of articular cartilage in health and pathology.\n\n\nConclusion\n\nThis exploratory study represents a comprehensive analysis of the in vivo response of several blood markers of joint pathology to a mechanical stimulus and novel insights into associations of load-induced blood marker kinetics and accumulated load. Changes in blood marker levels in response to load correlated among COMP, MMP-1, MMP-3, MMP-9, CPII, C2C, CPII/C3C, IL-6, ADAMTS-4, PRG-4, and resistin. Similar load-induced kinetics of COMP and MMP-3 as well as those of MMP-9 and resistin, respectively, suggest the presence of distinct and differentiated metabolic responses to a walking stress. While the data presented here cannot be directly linked to pathophysiological processes, the associations with accumulated load during the walking stress and with patient reported osteoarthritis outcome and QoL emphasize the relevance of not only assessing concentrations of (single) blood biomarkers but also considering the mechanoresponse of (arrays of) blood markers in the context of OA pathomechanics.\n\nThe study was approved by the regional ethics board (August 19, 2015; Ethikkommission Nordwest- und Zentralschweiz EKNZ 2015-224) and conducted in accordance with the Declaration of Helsinki, and participants provided written informed consent prior to participation.\n\n\nAuthor contributions\n\nAM designed the study. CN recruited the participants and collected the data; EDP conducted the musculoskeletal modeling computation; CN, SH and FC prepared the data for statistical analysis; FC performed the statistical analysis; AM, SH, AML, CN and CE were involved in data interpretation; AM, SH and CN prepared the manuscript; AM, CN, AML, SH, FC, EDP and CE contributed to reviewing and revising the manuscript, and approved the final draft.", "appendix": "Data availability\n\nZenodo: Patient reported outcome measures, load-induced blood marker kinetics, and ambulatory knee load in patients with medial knee compartment osteoarthritis, https://doi.org/10.5281/zenodo.7648802. 66\n\nThis project contains the following underlying data:\n\n• Mastertable_all_pre.xls: Patient demographics, patient reported outcome subscores, raw concentrations for each marker (COMP, MMP-1, MMP-3, MMP-9, CPII, C2C, C2C/CPII, ADAMTS-4, PRG-4, IL-6 and resistin), parameters describing walking stress, and ambulatory knee load; metadata describing all parameters.\n\nZenodo: Patient reported outcome measures, load-induced blood marker kinetics, and ambulatory knee load in patients with medial knee compartment osteoarthritis, https://doi.org/10.5281/zenodo.7648802. 66\n\nThis project contains the following extended data:\n\n• Extended_data.pdf: Median (interquartile range) serum concentrations for all time points; cross-correlations between baseline blood marker levels and patient reported outcome measures; and cross-correlations between load-induced change in blood marker levels and patient reported outcome measures.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nTREND checklist for ‘Load-induced blood marker kinetics in patients with medial knee compartment osteoarthritis are associated with accumulated load and patient reported outcome measures’, https://doi.org/10.5281/zenodo.7648802. 66\n\n\nAcknowledgements\n\nThe authors express their gratitude to Prof. G. Pagenstert for referring patients and providing radiographic data.\n\n\nReferences\n\nRoberts HM, Law RJ, Thom JM: The time course and mechanisms of change in biomarkers of joint metabolism in response to acute exercise and chronic training in physiologic and pathological conditions. Eur. J. Appl. Physiol. 2019; 119: 2401–2420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMundermann A, Dyrby CO, Andriacchi TP, et al.: Serum concentration of cartilage oligomeric matrix protein (COMP) is sensitive to physiological cyclic loading in healthy adults. Osteoarthr. Cartil. 2005; 13: 34–38. PubMed Abstract \| Publisher Full Text\n\nMundermann A, King KB, Smith RL, et al.: Change in serum COMP concentration due to ambulatory load is not related to knee OA status. J. Orthop. Res. 2009; 27: 1408–1413. 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Publisher Full Text" }	[ { "id": "195180", "date": "08 Sep 2023", "name": "Stephen Cornish", "expertise": [ "Reviewer Expertise Biomarkers", "skeletal muscle", "aging", "sarcopenia", "osteoarthritis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study evaluated the correlations between a number of blood based biomarkers and patient reported outcomes as well as accumulated loads during a 30-minute walking protocol in patients with knee OA. The number of biomarkers assessed is a strength in the study design. I only have a few comments for the authors to consider in making revisions to the manuscript:\nI realize this was an exploratory study; however, it would be appropriate to include a hypothesis statement in the introduction.\n\nIn the methodology it is mentioned that the patients were asked to refrain from physical activity for 24 hours prior to testing. I believe this would be nearly impossible, so should be rephrased or eliminated from the methodology.\n\nFigure 1 is very difficult to see in its present form. Could this be enlarged to show where the significant differences are located more clearly in the 11 biomarkers?\n\nIn the methodology a more thorough explanation of how the ELISAs were completed should be included.\n\nMinor comment - sentence 2 of the conclusion should read:\"... CPII/C2C, IL-6...\" not 'C3C' I believe.\nOverall, this is a well written manuscript with some interesting and novel 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10873", "date": "12 Jan 2024", "name": "Annegret Mündermann", "role": "Author Response", "response": "“This research study evaluated the correlations between a number of blood based biomarkers and patient reported outcomes as well as accumulated loads during a 30-minute walking protocol in patients with knee OA. The number of biomarkers assessed is a strength in the study design. I only have a few comments for the authors to consider in making revisions to the manuscript.” We have carefully considered your comments and hope to have addressed these to your satisfaction. “I realize this was an exploratory study; however, it would be appropriate to include a hypothesis statement in the introduction.” We rephrased our study aims to include hypotheses that are in line with the published study protocol (reference 33). Specifically, we tested the hypotheses that (i) some but not all blood markers will respond to the walking stress, and that the kinetics in response to a walking stress correlates (ii) between some but not all markers, (iii) with accumulated knee load during the walking stress, and (iv) with patient reported osteoarthritis outcome and QoL. “In the methodology it is mentioned that the patients were asked to refrain from physical activity for 24 hours prior to testing. I believe this would be nearly impossible, so should be rephrased or eliminated from the methodology.” It is correct that patients were not asked to refrain from physical activity as this would be nearly impossible. We have corrected this statement to the actual instructions given, which were to minimize their physical activity for 24 hours prior to testing. “Figure 1 is very difficult to see in its present form. Could this be enlarged to show where the significant differences are located more clearly in the 11 biomarkers?” We agree that the figure is compact in its printed form. To enlarge the individual graphs, the figure would have to be split into three separate figures which would have the disadvantage of losing the difference in mechanoresponse to the walking stress between the different blood markers. Since the figure can be enlarged as desired in the online and pdf versions, we prefer to leave the figure as it is. “In the methodology a more thorough explanation of how the ELISAs were completed should be included.” All ELISAs were performed according to the manufacturers’ instructions. We have included this information in the Methods section. All manufacturers are listed in Table 2, and the instructions are available on the manufacturers’ websites. Therefore, we do not believe that describing the ELISA for each blood marker in detail is necessary or indicated. However, if the reviewer or the editor would prefer to include this information in the article, we would be happy to do so. “Minor comment - sentence 2 of the conclusion should read:\"... CPII/C2C, IL-6...\" not 'C3C' I believe.” Thank you for noting this oversight. We have corrected this typo. “Overall, this is a well written manuscript with some interesting and novel data.” Thank you. “Is the work clearly and accurately presented and does it cite the current literature? – Yes” Thank you. “Is the study design appropriate and is the work technically sound? – Yes” Thank you. “Are sufficient details of methods and analysis provided to allow replication by others? – No” We assume that this comment referred to the information on the pre-stress test activity, the duration of the stress test and the ELISAs. We have included this information as requested. “If applicable, is the statistical analysis and its interpretation appropriate? – Yes” Thank you. “Are all the source data underlying the results available to ensure full reproducibility? – Yes” Thank you. “Are the conclusions drawn adequately supported by the results? – Yes” Thank you." } ] }, { "id": "206343", "date": "17 Oct 2023", "name": "Amelie M Lutz", "expertise": [ "Reviewer Expertise MSK Radiology", "peripheral nerve imaging" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written manuscript on the an explorative study of load-induced blood marker kinetics in patients with medial knee compartment osteoarthritis are associated with accumulated load and patient-reported outcome measures.\nThe authors measured six blood serum biomarkers of cartilage synthesis, cartilage degradation, joint lubrication, or inflammation after a walking test for an extended period up to 5.5 h after the walking test. Of these six blood biomarker, only COMP and MMP-3 showed a consistent increase immediately after the walking test, and COMP and MMP-3 kinetics in response to the mechanostimulus correlated as did load-induced MMP-9 and resistin kinetics.\nThe main limitation of the study is the low patient number. The authors list additional limitations.\n\nSpecific comments for the authors :\n\nIn the introduction, please change the listing fot he evaluated blood biomarkers according to their category, meaning list which ones belong to the cartilage synthesis, cartilage degradation groupds and so on. This will make it a much more valuable manuscript also to more general physician readers.\n\nMaterials and Methods:\nHow long was the walking activity? I did not see a defined time period of walking in this section, only briefly mentioned in a subsentence in the discussion (30 minutes?). Was the time duration the same for everybody?\nHow were the time intervals for blood draws chosen? Please explain.\n\nHow accurate do the authors think their estimates are of the load accumulated during the walking stress test and assessing the effect of the accumulated load on blood marker kinetics in response to load? Can this really correct for the variability of stress at a self-paced walking exercise?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10874", "date": "12 Jan 2024", "name": "Annegret Mündermann", "role": "Author Response", "response": "“This is a well-written manuscript on an explorative study of load-induced blood marker kinetics in patients with medial knee compartment osteoarthritis are associated with accumulated load and patient-reported outcome measures. The authors measured six blood serum biomarkers of cartilage synthesis, cartilage degradation, joint lubrication, or inflammation after a walking test for an extended period up to 5.5 h after the walking test. Of these six blood biomarkers, only COMP and MMP-3 showed a consistent increase immediately after the walking test, and COMP and MMP-3 kinetics in response to the mechanostimulus correlated as did load-induced MMP-9 and resistin kinetics.” Thank you. “The main limitation of the study is the low patient number. The authors list additional limitations.” Thank you. We have focused our discussion on limitations that are potentially relevant to the interpretation of our data. Specific comments for the authors: “In the introduction, please change the listing of the evaluated blood biomarkers according to their category, meaning list which ones belong to the cartilage synthesis, cartilage degradation groups and so on. This will make it a much more valuable manuscript also to more general physician readers.” Thank you for this suggestion. We have restructured this section accordingly. Materials and Methods: “How long was the walking activity? I did not see a defined time period of walking in this section, only briefly mentioned in a subsentence in the discussion (30 minutes?). Was the time duration the same for everybody?” Thank you for noting this oversight. The walking stress test include a 30-minute walking activity. We have added this information to the Abstract and Methods section for clarity. “How were the time intervals for blood draws chosen? Please explain.” The time intervals were chosen based on the literature described and cited in the Introduction section. We have added this information to the Methods section accordingly. “How accurate do the authors think their estimates are of the load accumulated during the walking stress test and assessing the effect of the accumulated load on blood marker kinetics in response to load? Can this really correct for the variability of stress at a self-paced walking exercise?” In this study, we assessed accumulated load using two different approaches. In approach 1, the maximum, rate of increase and impulse of the vertical ground reaction force during treadmill walking were multiplied by the number of steps taken during the walking stress. In the more advanced approach 2, the maximum and impulse of the knee adduction moment, knee flexion moment, total knee compressive force, medial and lateral compartment compressive force as well as medio-lateral shear force were calculated from musculoskeletal modeling for the affected knee during overground walking multiplied by the number of steps taken during the walking stress. The number of steps taken during the walking stress and hence the number of load cycles was counted and hence are an accurate measure. Ideally, one would measure the load directly at the tissue level, but this is not possible in vivo in humans for obvious reasons. Any musculoskeletal model used to estimate joint level load is based on some assumptions. In our models, we scaled the generic model to patient-specific anthropometrics and adjusted the varus/valgus alignment estimated from the marker position as described in the cited articles. The accuracy of these models can be further improved by increasing the personalization of the models (i.e., person-specific morphology taken from medical imaging such as magnetic resonance imaging vs. scaled based on anthropometrics) and estimating compartment-specific tissue level pressures. The latter was beyond the scope of this study but will be considered in future projects. We have added this potential limitation to the Limitations section. The reviewer is correct that there may be some variability in tissue level stress during a self-paced walking exercise within an individual. In this study, we measured blood marker kinetics after the walking stress and not during the stress, and we were primarily interested in the variability in blood marker kinetics in response to the walking stress and the variability in accumulated load between participants, which we found to be related. Overall, while the accuracy of the estimates depends on the underlying assumptions, but the observed correlations of blood marker kinetics and accumulated load supports our hypothesis. “Is the work clearly and accurately presented and does it cite the current literature? – Yes” Thank you. “Is the study design appropriate and is the work technically sound? – Yes” Thank you. “Are sufficient details of methods and analysis provided to allow replication by others? – Partly” We have added information on the methods and analysis as requested that is important for allowing replication by others. “If applicable, is the statistical analysis and its interpretation appropriate? – I cannot comment. A qualified statistician is required.” All statistical analyses were performed by an experienced biostatistician. “Are all the source data underlying the results available to ensure full reproducibility? – Yes” Thank you. “Are the conclusions drawn adequately supported by the results? – Partly” We hope that with the revisions made, the reviewer agrees that our conclusions are supported by the results." } ] } ]	1	https://f1000research.com/articles/12-299
https://f1000research.com/articles/13-60/v1	12 Jan 24	{ "type": "Research Article", "title": "Standard versus mini amplatz size in patients undergoing pediatric percutaneous nephrolithotomy through 16 years of experience: A retrospective study in single-center experience", "authors": [ "Nur Rasyid", "Syifa Fauziah Fadhly", "Widi Atmoko", "Ponco Birowo", "Nur Rasyid", "Syifa Fauziah Fadhly", "Widi Atmoko" ], "abstract": "Background: Pediatric urolithiasis is associated with considerable morbidity and a very high recurrence rate of approximately 67%. Current guideline recommendations for standard pediatric percutaneous nephrolithotomy (PCNL) remain a problem due to several complications, such as hemorrhage or renal damage, especially in the pediatric population. However, mini-PCNL is regarded as a safer and more effective method for pediatric patients. This study compares PCNL with mini-PCNL, along with their efficacy and safety, in the Cipto Mangunkusumo General Hospital, Indonesia. Methods: This observational retrospective study was performed on pediatric patients aged < 18 years (pediatric patients) who underwent PCNL with a standard 22-30 Fr sheath or mini 15-21 Fr sheath. We reviewed postoperative outcomes, including stone-free rate (SFR), presence of complications, and postoperative drainage method. Data analysis was performed using SPSS version 26.0. Results: Our study included a total of 42 pediatric patients (mean age: standard, 4.61 ± 3.52 years; mini, 8.0 ± 3.57 years; p<0.05). The stone-free rate was significantly higher in the mini-PCNL group (87.9%) than in the standard PCNL group (59.1%, p <0.05). All patients treated with mini-PCNL were tubeless, while some patients in the standard group still needed a postoperative drainage tube (p=0.012). Complications, such as infundibulum laceration, were rare, and their differences between groups insignificant (p=1). Even though it has been demonstrated that standard sheath PCNL is considered effective for children, smaller PCNL sheaths offers more experience. Mini-PCNL provides a similar complication rate, while remaining effective in treating urolithiasis; thus, it is thought to be a non-inferior option for treating pediatric populations, especially younger and smaller children. Conclusions: Mini-PCNL is non-inferior to standard PCNL in terms of efficacy and safety for the treatment of pediatric urolithiasis. Mini-PCNL resulted in higher SFR and totally tubeless follow-up.", "keywords": [ "Mini-PCNL", "standard PCNL", "pediatric urolithiasis", "efficacy", "safety" ], "content": "Introduction\n\nAn increasing amount of evidence has shown an increasing incidence of pediatric urolithiasis, with a reported incidence of approximately 50 cases in 100,000 children.1,2 Although a mere 2%–3% of the pediatric population will develop urinary calculus, pediatric urolithiasis is associated with a considerable morbidity and a high recurrence rate (approximately 67%)3,4; thus, it is important for this population to receive appropriate treatment to keep them stone-free.\n\nGuidelines by the European Association of Urology have recommended pediatric percutaneous nephrolithotomy (PCNL) as the primary procedure for stones larger than 20 mm and stones larger than 10 mm located at the lower renal pole.5 Despite achieving a high stone-free rate (SFR), ranging from 50%–98.5%,5–9 PCNL using a standard sheath of 24-30 Fr for renal access is associated with severe complications, including hemorrhage, blood transfusion, sepsis, damage to renal parenchyma, and a postoperative need for analgesia due to pain.10,11\n\nPCNL in pediatric patients is always a challenge for urologists because of the smaller anatomical structures and fragility of patients compared to adult patients. Various strategies to decrease morbidity post-PCNL have been described, focusing on reducing the tract size to minimize the risk of kidney parenchymal injury, which provides fewer morbidities without changing the therapeutic efficacy.12,13 Recently, mini-PCNL, which uses a smaller sheath size of 14-22 Fr has been regarded as a safer and more effective treatment option for pediatric nephrolithiasis, with studies reporting a promising SFR, as well as reduced bleeding and decreased hospital stay, compared to standard PCNL.6,12,14–16\n\nIn Indonesia, pediatric PCNL began in 2005 using the protocol and instruments of standard PCNL. Even though mini-PCNL has been around since 1997, when it was first described by Helal et al.,17 the equipment was only introduced to our institution, the Cipto Mangunkusumo General Hospital, Indonesia, in 2014. Thus, this study aimed to describe the experience and development of pediatric PCNL in a tertiary referral hospital in Indonesia, comparing the effects of the use of different sizes of instruments (standard PCNL vs. mini PCNL) on the outcomes of SFR, presence of complications, and postoperative use of drainage tubes.\n\n\nMethods\n\nThe study design was approved by the Health Research Ethics Commitee - Faculty of Medicine Universitas Indonesia and Cipto Mangunkusumo Hospital (HREC-FMUI/CMH) on April, 18th 2022 which waived the requirement for obtaining informed consent with the ethical No. KET-371/UN2.F1/ETIK/PPM.00.02/2022.\n\nThis observational retrospective study was conducted at a single tertiary referral hospital in Indonesia. The records from 2010 to 2021 were obtained in one instance prior to conducting the research in 2022. The inclusion criteria for this study were patients aged <18 years (pediatric patients) who underwent PCNL with either the standard 22-30 Fr sheath or mini 15-21 Fr sheath. Our exclusion criteria for this study were incomplete general characteristics of the patients, intraoperative and postoperative data of the patient. We collected general characteristics of the patients such as sex, body mass index (BMI), stone’s side, number of PCNLs sessions, stone number, and stone burden. Collected intraoperative data were the patient’s position during PCNL, calyceal puncture, and length of operation. Collected postoperative data were stone clearance, postoperative drainage tube, and complications. The general characteristics of the patients, intraoperative and postoperative data were all collected from medical records by two of our researchers. The collected data were then reviewed by one of our researchers. We excluded data if there were missing data due to our exclusion criteria. Basic PCNL techniques were applied to both instruments. Total sampling was used for the recruitment for subjects in this study. We also implement the STROBE Statement in our study.\n\nData entry was performed using Microsoft Excel 2022 version 16.69.1, while data analysis and validation were performed using SPSS Statistics for Windows version 26.0 (IBM, USA). The normality of continuous variables was confirmed using the Shapiro–Wilk test. Mean and standard deviation were used to describe normally distributed continuous variables, while median and range values were used to express non-normally distributed variables. Categorical variables are presented as counts and percentages. An independent t-test was used to compare the means between normally distributed continuous variables, while the Mann–Whitney U test was used to compare non-normal variables. The statistical analyses of categorical variables were performed using the chi-squared test or Fisher’s exact test, depending on the number of observations. Statistical significance was set to p < 0.05.\n\n\nResults\n\nA total of 42 patients under the age of 18 years underwent PCNL, 27 of which underwent mini-PCNL, while the other 15 underwent standard PCNL. The general characteristics of both groups are shown in Table 1. Comparing the two common characteristics, there were no significant differences between the two groups in terms of patients’ sex, as most patients were male in both groups, number of PCNL sessions, BMI, stone burden, and stone number. Meanwhile, there was a significant difference between both groups in age (p < 0.05); the mean age in the mini-PCNL group was 4.61 ± 3.52 years, while that in the standard PCNL group was 8.0 ± 3.57 years. Moreover, most patients underwent only one PCNL session in both groups.\n\n* p<0.05.\n\nIntraoperative and postoperative outcomes are shown in Table 2. The outcomes were recorded for all surgeries performed on patients who underwent either mini-PCNL or standard PCNL. From the table, it can be observed that most patients were placed in the prone position in both groups. Access to the calyxes was mostly from the lower calyx in both groups, even though there was no significant difference between groups. The length of operation was significantly longer in the mini-PCNL group than in the standard PCNL group (97.55 vs 71.82 min, p = 0.008). Stone clearance was higher in the mini-PCNL group (SFR, 87.9%) than in the standard PCNL group (SFR, 59.1%); these differences were statistically significant. Generally, patients in the mini-PCNL group were mostly discharged totally tubeless, while they were mostly tubeless in the standard PCNL group, which led to a statistically significant difference (p = 0.012) between both groups in terms of the use of postoperative drainage tubes. In terms of complications, there was no significant difference between groups, and most surgeries did not induce any complications.\n\n* p < 0.05.\n\n\nDiscussion\n\nPediatric urolithiasis has a high recurrence rate, and its prevalence is growing globally in all age groups. The use of PCNL in the treatment of pediatric urolithiasis was initially shown by Woodside et al.18 in 1985, with an SFR of 100% in a single session using standard adult instruments. However, the use of a larger tract size in standard PCNL is associated with disadvantages, such as increased bleeding that necessitates transfusions, renal damage, or organ damage, including potential pneumothorax.16,19 Therefore, the use of mini-PCNL has grown in prominence due to recent efforts to reduce the morbidities associated with using adult instruments, while maintaining high efficacy. The hypothesis for utilizing smaller devices through smaller diameter sheaths is that the narrower the PCNL tract, the less injury is caused to the renal parenchyma; consequently, related morbidity is reduced without affecting therapeutic efficacy.8,20,21\n\nIn this study, we observed that older patients mostly underwent standard PNCL compared to younger patients who mostly underwent mini-PCNL, and the differences between the two means were statistically significant. A growing body of studies has put forward the perspective that larger sheaths cause more dilatation and, inadvertently, parenchymal and vascular injury in smaller kidneys.20–22 Thus, following this theory, it is a logical step to treat younger patients with a smaller sheath size to avoid these damages and the potentially long-term effects of using larger sheaths, which could explain this observation. However, the studies were mostly observational; hence, more scientific support from molecular studies and randomized controlled trials would allow this theory to be accepted universally.\n\nAnother significant observation in this study was the intraoperative position of patients, that is, most patients were placed set in a prone position. Prone position is preferred given the familiarity over the supine position and the larger field of operation.16 Furthermore, the success rate was high with acceptable complication rates.23 However, given the disadvantages of prone position such as possible cervical trauma24 and ventilatory difficulty due to restricted thorax and abdomen mobility,25 there has been great interest towards the application of supine position. Initial reports have shown non-inferiority in the supine position. A study by Zhan et al.23 has described equal safety and effectiveness in both supine and prone positions. Another study by Gamal et al.24 evaluated the efficacy and safety of the supine position using mini-PCNL (sheath size, 19 Fr) and reported a high SFR of 92.5%, with two of 27 patients experiencing intraoperative complications. Furthermore, a study by Nerli et al.25 reported similar results regarding the safety and efficacy of the supine position in pediatric PCNL. As these reports were observational, further subgroup analysis or future comparative studies should be conducted to ascertain their effects on the outcome of surgeries. Thus, this observation highlighted the importance of the intraoperative positions as a factor affecting procedural outcomes.\n\nThus, despite the current study and the aforementioned studies being observational, this study highlighted the importance of the intraoperative position as a factor affecting procedural outcomes one of the factors affecting the outcomes of the procedures. Further subgroup analysis or future comparative studies could be conducted to ascertain their effects on the outcome of surgeries.\n\nWe observed that operational duration was significantly longer in the mini-PCNL group than that in the standard PCNL group (97.55 vs. 71.82, p < 0.05). This result was in accordance with the results of other studies by Unsal et al.,26 Bilen et al.,7 Celik et al.,6 Ozden et al.,16 and Mahmood et al.27 The prolonged duration could be attributed to the limited visual field of smaller endoscopes. The need to extensively shatter the stones into smaller pieces to remove the pieces through the smaller sheath might also prolong operational length.\n\nIn this study, the stone clearance rate was significantly higher in the mini-PCNL group than in the standard PCNL group (87.9% vs. 59.1%, p < 0.05) in a single session. Similar SFR results were reported in several other studies that compared stone clearance using equipment of different sizes (i.e., mini-PCNL vs. standard PCNL); the SFR of mini-PCNL ranged from 76%-91.4% to 50%-98% for standard PCNL.6,16,20,26,27 Nevertheless, in these studies, they obtained a higher SFR in the standard PCNL group, while we reported a higher SFR in the mini-PCNL group. Be that as it may, a study by Bilen et al.7 reported similar findings; they reported that the SFR in the 20-Fr sheath group and 26-Fr sheath group were 80% and 69.5%, respectively; the differences, however, were not statistically significant. To summarize, an increasing amount of evidence agrees that mini-PCNL is not inferior to standard PCNL in SFR.\n\nA plausible reason for the higher SFR in the mini-PCNL group could be the smaller stone burden in the mini-PCNL group than in the standard PCNL group, although the distribution was not significantly different. Indeed, an association between stone burden and SFR has been reported.28,29 A study by Hussain et al.30 reported a trend where single treatment success decreased as stone size increased.\n\nAnother possible reason for the higher SFR in the mini-PCNL group was the lower BMI, which was observed in this study; the mean BMI in the mini-PCNL group was lower than that in the standard PCNL group (17.0 vs. 17.6, p > 0.05). Although no prior studies have compared the outcomes of mini-PCNL vs. standard PCNL in obese vs. non-obese pediatric settings, the Clinical Research Office of the Endourological Society (CROES) study by Fuller et al.,31 where standard PCNL was performed on 4 different groups based on BMI, demonstrated a lower SFR in the morbidly obese group than in the obese group (65.6% vs. 78.9%). Other similar studies also reported similar results, where SFR after standard PCNL seemed to be lower in patients with a higher BMI.32,33 However, the effect of BMI on PCNL outcomes remains debatable, as several studies by Ferreira et al.,34 Alyami et al.,35 and Akbulut et al.36 showed that PCNL outcomes were independent of BMI. Thus, a large population-based study or randomized controlled trial is needed to ascertain the effect of BMI on PCNL outcomes, especially in the more recent mini-PCNL group.\n\nThe standard procedure for drainage after pediatric PCNL includes inserting nephrostomy tubes and ureteric stents. Given the use of standard postoperative tube procedures, alternative procedures, such as tubeless and totally tubeless PCNL, began to gain traction. Instead of utilizing both ureteric stents and nephrostomy tubes, tubeless PCNL only uses ureteric stents, while totally tubeless PCNL forgoes both nephrostomy tubes and ureteric stents. These procedures have been associated with a shorter hospital stay and reduced need for analgesics.37,38 Ultimately, the use of tubed or tubeless PCNL depends on the current complications, bleeding, and residual fragments. In this study, most patients were discharged totally tubeless after mini-PCNL, and the proportion was significantly different from that of standard PCNL. In contrast, with standard PCNL, most patients underwent a tubeless procedure. These results demonstrate the shifting trend of tubeless procedures after PCNL. Furthermore, the use of a totally tubeless procedure would imply that mini-PCNL induced minimal postoperative complications and thus continued to show the safety of mini-PCNL.\n\nSimilar complication rates were observed after mini-PCNL and standard PCNL (15.2% vs. 13.6%), even though the differences were not significant. Major complications, such as pneumothorax and other organ injuries, were not recorded; however, lacerations of kidney parts, such as the infundibulum and inferior calyces, were observed. Signs of infection, such as pus, were documented in one patient who underwent mini-PCNL. While other common complications such as hemorrhage, fever, and the need for transfusion were not detailed in this study, preliminary data showed a similar safety profile between mini-PCNL and standard PCNL, which is in accordance with other studies that reported a similar overall complication rate between mini-PCNL and standard PCNL.6,13,27\n\nThis study has some limitations. As this was an observational study, the results might suffer from selection and information biases and be undermined by confounding factors. This was true for age, which is one of the general characteristics of the populations and should be similar between the two populations. Data were also retrieved from a single institution with a limited sample size. Hence, further comparative research, particularly randomized controlled trials, are needed to corroborate the conclusions of this study.\n\n\nConclusion\n\nThe management of pediatric urolithiasis has evolved over time. Mini-PCNL, which is a miniaturization of conventional PCNL, is the fundamental result of this evolution. In this study, we demonstrated that mini-PCNL is non-inferior, if not superior, to standard PCNL in safety and effectiveness in treating pediatric renal calculi. Mini-PCNL provided a high SFR and tubeless follow-up.", "appendix": "Data availability\n\nOpen Science Framework: RAW DATA of Standard versus mini amplatz size in patients undergoing pediatric percutaneous nephrolithotomy through 16 years of experience: A Retrospective Study in single-center experience. https://doi.org/10.17605/OSF.IO/E9UWC. 38\n\nThis project contains the underlying patient data in xlsx format. Where two values are entered for a single patient, this refers to the same patient undergoing the procedure twice.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nOpen Science Framework: STROBE checklist for ‘The data and STROBE checklist for ‘Standard versus mini amplatz size in patients undergoing pediatric percutaneous nephrolithotomy through 16 years of experience: A Retrospective Study in single-center experience’. https://doi.org/10.17605/OSF.IO/E9UWC.\n\n\nReferences\n\nSharma A, Filler G: Epidemiology of pediatric urolithiasis. Indian. J. Urol. 2010 Oct-Dec; 26(4): 516. Publisher Full Text\n\nSas DJ, Hulsey TC, Shatat IF, et al.: Increasing incidence of kidney stones in children evaluated in the emergency department. J. Pediatr. 2010 Jul; 157(1): 132–137. PubMed Abstract \| Publisher Full Text\n\nTasian GE, Copelovitch L: Evaluation and Medical Management of Kidney Stones in Children. J. 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PubMed Abstract \| Publisher Full Text\n\nAtmoko W, Birowo P, Rasyid N: Factors affecting stone free rate of primary percutaneous nephrolithotomy on staghorn calculi: a single center experience of 15 years. F1000Res. 2016; 5: 2106. Publisher Full Text\n\nFerreira TAC, Dutra MMG, Vicentini FC, et al.: Impact of Obesity on Outcomes of Supine Percutaneous Nephrolithotomy. J. Endourol. 2020 Dec 1; 34(12): 1219–1222. PubMed Abstract \| Publisher Full Text\n\nAlyami FA, Skinner TAA, Norman RW: Impact of body mass index on clinical outcomes associated with percutaneous nephrolithotomy. Can. Urol. Assoc. J. 2013 Apr; 7(3–4): E197–E201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkbulut F, Kucuktopcu O, Kandemir E, et al.: Efficacy and safety of mini percutaneous nephrolithotomy in obese patients. Springerplus. 2016 Dec; 5(1): 1148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRasyid N: RAW DATA of Standard versus mini amplatz size in patients undergoing pediatric percutaneous nephrolithotomy through 16 years of experience: A Retrospective Study in single-center experience. [Dataset]. 2023, January 9. Publisher Full Text" }	[ { "id": "253448", "date": "14 May 2024", "name": "Arun Chawla", "expertise": [ "Reviewer Expertise Endourology", "Reconstructive and Functional Urology", "Renal Transplant" ], "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\nComments\n\nBeing a retrospective single non-randomized study with a small sample size of 42 patients, who are unequally matched in the two groups, reduces the reliability and reproducibility of the findings of this study\n\nThis line in the abstract “Even though it has been demonstrated that standard sheath PCNL is considered effective for children, smaller PCNL sheaths offers more experience” Needs to be reconsidered. What does the author mean by offers more experience? The significant difference in age between the Mini-PCNL and standard PCNL groups raises concerns about the validity of the comparison and subsequent analysis, thereby raising the question of Selection Bias.\n\nWhile the mean stone burden of both groups appears comparable, the criteria for opting Mini-PCNL or standard PCNL is unclear.\n\nThe mean access tract size for both groups has not been specified, despite Mini-PCNL and standard PCNL having wide range from 15-21 Fr and 22-30 Fr, respectively. Though the authors state that prone positioning was preferred over supine, the rationale provided lacks clarity. There are no details on the determining factors that led the surgeon to consider prone over supine. The authors need to address how they justify their finding of significantly higher stone-free rates in Mini-PCNL compared to standard PCNL when all the existing evidence points otherwise, especially considering the absence of any mention of additional modalities such as suction used alongside Mini-PCNL. This raises questions about the technique of the operating surgeon. Also, the imaging modality for declaring the patient stone free is not mentioned. Complications mentioned are not classified. Classification systems such as Clavien-Dindo would make the findings more comparable. Description of the type of complication is missing. One patient is quoted to have had “signs of infection, such as pus”, but there is no mention as to whether this pus was in the surgical site, perinephric or in the kidney and also there is no mention on the management of these complications. The authors state that there was a higher need of nephrostomy tube placement in the standard PCNL group, but there is no clarity on what were the factors that make them consider nephrostomy tube placement.\n\nBased on these comments, I find major limitations in this study and I would not approve this Manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "290928", "date": "23 Jul 2024", "name": "Davide Campobasso", "expertise": [ "Reviewer Expertise Endourology", "mini-invasive surgery", "uro-oncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors reported a retrospective series of 55 PNL in pediatric patients (33 mini PNL and 22 standard PNL). They did not found any difference in terms of complication, but stone free rate and totally tubeless procedures were in favour of the mini PNL group. Instead standard PNL had a shorter operative time. Despite the topic is interesting, I feel that your manuscript could be reconsidered for indexed should you be prepared to incorporate some revisions.\nMinor revisions\n1- What about the number of surgeons involved and their individual variation of expertise level in supine or prone approach for PNL? This could be a bias, if several surgeons with different levels of expertise were involved.\n\n2- What were the reasons to perform some patients in prone or supine PNL? This is an interesting aspect\n3- In supine patients did you perform a combine approach (ECIRS) or only a anterograde approcah (PNL)?\nMajor revisions\n4- What is the defintion of stone burden? This is an important data to know.\n5-Please mention pre-operative and residual stones follow up imaging and protocol (US, CT scan, others)\n6- Was location and number of stones also incorporated in the final analysis as in previous decade many papers have discussed importance of Guys stone score and other stones scores to overcome the compounding variables. (PMID:29631882). This data are important to undestand the complexity of the cases treated.\n7- Discuss more contemporary papers on supine and prone in pediatric PNL (PMID:37962271 PMID:35962906).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/13-60
https://f1000research.com/articles/13-59/v1	12 Jan 24	{ "type": "Research Article", "title": "Thoroughness of philosophy and return to religion in Tanabe Hajime", "authors": [ "Hiroto Doi" ], "abstract": "Japanese philosopher Tanabe Hajime (1885–1962) not only discussed religion in ways that were rooted in the fundamental claims of his writings, such as Shinran in Philosophy as Metanoetics (1946), but he also made proposals on religion, as in Demonstration of Christianity (1948), where he advocated for the need for a “second religious reformation.” For Tanabe, philosophy and religion (along with science) are fused in a specific way, and it is possible to find a vision of religion to be aimed for that is worth considering as a theory of religion among Japanese philosophers. To examine the theory of religion in Tanabe, this study focuses on the work, Demonstration of Christianity, and other works from the perspective of religious studies. In Demonstration of Christianity, the concept of absolute religion toward the second religious reformation is envisioned based on Tanabe’s dialectics of the absolute mediation, and a discussion on his world religion can be found, although it is limited to Christianity and some Buddhist sects. Reflecting his absolute dialectic, Tanabe says that this world religion was established through the absolute mediation of Christianity with “Nembutsu-Zen” (the unification of Pure Land Buddhism and Zen Buddhism). One cannot deny the impression of Christocentrism in these discussions, but they are in fact the result of Tanabe’s thoroughness in philosophy. What can be drawn from these discussions is that Tanabe's theory of religion is strongly connected to Platonism, which he considered the root of his dialectics. For example, by the third article in Existence, Love, and Practice (1947), “Self-Transcendence in Platonism and Faith in Gospel.” Tanabe’s argument reveals the characteristics of conceiving a new religion that combines Eastern and Western ideas, philosophy, and religion through a creative interpretation of Platonism, the source of Western thought where philosophy and religion are inseparably linked.", "keywords": [ "Tanabe Hajime", "absolute religion", "world religion(s)", "Platonism" ], "content": "Introduction\n\nNishida Kitarō (1870–1945), a well-known Japanese philosopher, and Tanabe Hajime (1885–1962),1 who was influenced by Nishida but later criticized his philosophy, were known for their originality and deep thoughts on not only philosophy but also on religion. The fourth section of Nishida’s An Inquiry into the Good (Zen no Kenkyū, 1911) is titled “Religion”, and it is often noted that Zen Buddhism has substantially influenced him.2 His lecture notes also suggest that he was aware of contemporary theories on religious studies in the Western world.3 Although Tanabe did not adhere to any particular faith,4 religion was fundamental to his thinking. His interests shifted from Zen to Pure Land Buddhism, then Christianity, and again Zen Buddhism. Although this attitude is sometimes considered to indicate Tanabe’s lack of a core religion (like Zen in Nishida), it also signals the purity and thoroughness of his philosophical speculations.5 The theme of religion is crucial to both Nishida and Tanabe.\n\nA particular characteristic of Tanabe’s philosophy is the appearance of religion at major points in his treatises, as in the case of Shinran in Philosophy as Metanoetics (Zangedō to shiteno Tetsugaku, 1946).6 In his Demonstration of Christianity (Kirisutokyō no Benshō, 1948),7 he advocates for the “second religious reformation” (THZ 10, 11, 17, pp. 319–320). Tanabe’s proposals on religion are so aggressive to seem unusual for a philosopher. In his vision, philosophy and religion join. As he states, “Religion and philosophy have different starting points and paths, but in the end, they come to a point of unity from opposite directions” (THZ 11, p. 430). In Tanabe’s work, philosophy and religion (and science) exhibit a fusion—not simply an identification, but a mediation in his dialectic—8 which reflects his ideal conception of religion. This view suggests that Tanabe’s thought is worth considering as one of the Japanese philosopher’s theories of religion.\n\nThe aim of this study is to examine Tanabe’s theory of religion, which is unique among Japanese philosophers, by first examining the absolute religion related to the second religious reformation as a keyword. However, the question arises as to how his unique absolute religion pursues a universal theory of religion. Therefore, we examine Tanabe’s world religion and investigate the elements that are underlying it. Through this process, this study helps clarify the uniqueness and significance of Tanabe’s theory of religion from the perspective of religious studies.\n\n\nMethods\n\nTo achieve the aforementioned research objectives, we examine various documents by the Japanese philosopher Tanabe, from the perspective of religious studies. Although Tanabe has numerous writings, our chosen scripts are limited to later works containing relatively further developed theory of religion. This study first examines Demonstration of Christianity, which calls for absolute religion and anticipates a second religious reformation, thus clearly showing Tanabe’s attitude towards the future of religion and its formation. However, as the title suggests, the book may seemingly have a close affinity to Christian theology. For a discussion not limited to Christianity, but open to other religions, Tanabe’s other works need consideration. We then discuss Existence, Love, and Practice (Jitsuzon to Ai to Jissen, 1947). The focus of this study is on how Tanabe develops a theory of religion not limited to Christianity. For this purpose, the third article in the same book, “Self-Transcendence in Platonism and Faith in Gospel,” containing the corresponding argument, is selected for our study.\n\nThe basis of this study is the textual interpretations, but the author also focuses on letters and recollections that clues the understanding of how Tanabe constructed his theory of religion. The literature that is referred to, comprise of papers focusing on Tanabe’s later works. The perspective of this study’s analysis is Tanabe’s view of Platonism. One reason for adopting this, is the author’s previous research on Plato and Neoplatonism. In addition, Platonism crucially bridges philosophy and religion in Tanabe’s work. To examine his theory of religion without limiting it to a specific religion, it is presumably insufficient to compare Tanabe’s views on a specific religion with its doctrines. Focusing on Platonism and analyzing its relation to religion, is a necessary process to find a theory of religion in Tanabe’s works, that is not limited to a specific religion.\n\nBased on the above discussion, we explore how Tanabe’s contemplation of religion is also a practice of forming religion, by referring to Platonism as a model for bridging philosophy and religion in Tanabe.9\n\nAlthough religion has an important role in Tanabe’s thoughts, he states, “Since the principle of nothingness in the dialectic is the principle of philosophy, philosophy must come to it by negating itself, and must be restored to the philosophy of religion as an occasion of negation as religious faith” (THZ 9, p. 335). In other words, Tanabe’s thoughts focus on the philosophy of religion, not religion itself.10 When addressing the religious aspect of Tanabe’s work, previous studies have mainly discussed it as a philosophy of religion from the perspective of a study of Japanese philosophy.11 Few authors have examined it from the perspective of religious studies.\n\nThe word syūkyō (宗教) was first used in Japan in the early Meiji era as a translation of “religion.” In Japan, religion is the product of various discussions and attempts, mainly by intellectuals with religious backgrounds, such as Buddhism and Christianity, in turbulent circumstances.12 However, previous studies have not fully clarified the relationship between these debates and the views expressed by Japanese philosophers. The study of this relationship falls between the analysis of Japanese philosophy, which attempts to understand the philosopher’s thoughts from within, and the study of religion, which addresses intellectuals with religious beliefs.13\n\nThere are several possible reasons why the theory of religion in Japanese philosophers has not been well studied. First, although both Nishida and Tanabe established their scholarly careers at a time when the concept of religion was still being formed in Japan, their use of the word “religion” does not seem very strange from a modern perspective. It is conceivable that this makes it unnecessary for us to be very aware of how their concept of religion was defined. Another possible factor is that Nishida and Tanabe both mainly focused on so-called world religions, such as Buddhism and Christianity, and less on folk religions and practices.14 Of course, one could criticize this point of view by saying that the two Japanese philosophers overlooked areas that do not belong to world religions or “higher” religions with established doctrines and scriptures. One could also argue that because this is a philosopher’s theory of religion, it is not necessary to discuss religions other than well-established world religions. With Tanabe in particular, compared to Nishida, there is little attempt to take a reflective consideration of religion itself. However, this does not mean that there is no need to examine the theory of religion in Tanabe, but rather that there are still areas that have not been explored by previous studies.\n\nConcerning Tanabe, it seems significant today to consider the religion that he contemplated and attempted to form as part of his practice. This is because, while the religious intellectuals of his time were struggling to establish a new religion for a new era, Tanabe was seeking to do so from a different point of view. Tanabe’s discussion of religion is unlikely to be limited to the acceptance of the religious views of the time but is assumed to be unique. This is because Tanabe’s thoughts, expressed in the symbolic form of the vortex (kadō 渦動), denies being static or fixed, and always places himself as a practitioner in dynamism. This study sheds light on aspects of Tanabe’s unique religion that have not yet been examined from the perspective of how religion is bridged by philosophy.\n\nTo examine Tanabe’s concept of “religion,” we acknowledge how strongly religion is considered in his theory. If Tanabe considered religion weakly, it would have been difficult to discuss his theory of religion alongside the arguments made by the religious intellectuals of his time.\n\nOne of Tanabe’s major characteristics as a philosopher is his emphasis on practice (praxis) rather than contemplation (theōria). His practice does not refer to acts such as Zen meditation in Nishida. Still, it reflects his thorough contemplation of religion dialectically as a practitioner of philosophy15 rather than a mere scholar. Therefore, even if it does not involve specific religious acts, such as rituals, Tanabe’s way of life and attitude may only be described as praxis. After retiring from Kyoto Imperial University, Tanabe moved to Kita-karuizawa, known for its cold climate in winter. Here, he devoted himself to philosophical research and writing. His disciples were concerned about his health and suggested that he move to a warmer climate, but he disagreed.16 As an example of his philosophy in practice, Tanabe moved his life to a cold region as a punishment for his former actions. In addition, during Tanabe’s tenure at Kyoto Imperial University, he was criticized by Minoda Muneki and others, who were known as nationalists and attacked many scholars in the humanities. However, Tanabe published his rebuttal in their magazine17 and engaged in a direct dialogue with Minoda. This episode shows Tanabe’s sincerity, which contrasts with Nishida’s advice18 not to deal with Minoda. Tanabe put himself at risk by engaging with these aggressors.19 Hence, although human factors are involved, his response to Minoda and others20 demonstrates the practical side of Tanabe’s speculation.\n\nTanabe’s thoughts are also oriented toward transforming society. During the war, his insistence on “the logic of species” was fraught with danger, as it could be interpreted as a message that the nation should be respected.21 Nevertheless, Tanabe’s pursuit of a thorough practice-oriented philosophy includes religion as an object. Hence, exploring his thoughts as a Japanese philosopher’s theory of religion is of great significance.\n\nTanabe’s vision is explicitly expressed in Demonstration of Christianity. As Itō points out,22 this work shows Tanabe’s intention to form a new religion23 oriented toward social practice by extending Philosophy as Metanoetics, which lacked socially oriented practice. Published in 1948, the book begins with a somewhat distinctive introduction in which Tanabe recollects his entry into high school (the old school system).24 At that time, the First Higher School25 students were confronted with the “inevitable problem” of Christianity, and Tanabe was no exception. This phenomenon was due to the influence of Christian intellectuals such as Uchimura Kanzō (1861–1930), Uemura Masahisa (1858–1925), and Ebina Danjō (1856–1937). Their influence was more significant than those of Buddhist intellectuals such as Kiyozawa Manshi (1863–1903) and Chikazumi Jōkan (1870–1941). Thus, Christianity “responded even better to the humanist demands of the youth” (THZ 10, p. 4). A science student at the time, Tanabe was not convinced by “mythical content that seems to be the product of fantasy, incompatible with scientific attitudes” (THZ 10, p. 6). Even after listening to lectures by Raphael von Koebel (1848–1923) and Hatano Seiichi (1877–1950) in university, he did not become a Christian. However, after his “Metanoetics” (zangedō) lecture in 1944,26 which culminated in Philosophy as Metanoetics, Tanabe “felt as if his eyes had been opened for the first time to the truth of the Christian gospel which I had not been able to see before” (THZ 10, p. 8). He decided to “confront Christianity which had been his longstanding issue” (THZ 10, p. 9) and wrote Demonstration of Christianity.\n\nThis work develops from the question “Jesus or Paul?” by William Wrede (1859–1906). The argument that Christianity, Marxism, and Japanese Buddhism, which are dissimilar, mediate and unite as a dialectic is, from today’s perspective, a reflection of the world at the time. Since this study focuses on the development of religion in Tanabe, we begin with “Appendix: Christianity, Marxism and Japanese Buddhism,” in which the theme appears several times. Although Demonstration of Christianity was published in 1948, this appendix was published the previous year, in 1947, and could be considered a stronger reflection of Tanabe’s awareness of the issues. In this appendix, Tanabe’s reference to the second religious reformation and absolute religion27 stands out as a remarkable theory of religion.\n\nIf Protestantism ends its protest halfway, it will naturally lose its life. It must first die to itself for its resurrection. It must live in nothingness with a thoroughgoing self-negation. This is Christianity, but no longer Christianity. But it is not Buddhism, of course, and it can only be called absolute religion or absolute faith. I believe that it is in this position that the second religious reformation is required to be carried out today. (THZ 10, p. 319)\n\nAlthough the second religious reformation that Tanabe discusses has Protestantism in mind,28 it is also mediated by Buddhism through negative occasions, becoming an absolute religion that “is Christianity but is not Christianity.” As Liao points out, the “absolute” in this absolute religion is derived from Georg W. F. Hegel29. Therefore, Tanabe’s absolute religion is based on Christianity, the religion of history, and Christianity’s position as an absolute religion that is a “religious faith through a concrete historical mediation” (THZ 10, p. 322).\n\n… I am sure that everyone is aware that the concept of absolute religion mentioned above is derived, of course, from Hegel. My use of it is the same as his, and while it involves historical mediation to a great extent, it must also exhibit the character of nothingness more thoroughly than it did in his case. If you think about it for a moment, absolute religion might seem to refer to a religion such as Zen, which absolutely denies any limitation and is universally free and flexible. Such universality, however, remains a so-called abstract universality, not a concrete universality. (THZ 10, p. 320)\n\nBuddhism is not given sufficient significance in Hegel’s theory of religion in the Phänomenologie des Geistes. However, in this quote, Tanabe emphasizes the negative occasion of nothingness that appears in Zen and other forms of Buddhism and tries to give it a role by concretely and universally establishing absolute religion. After pointing out that “it is also clear that, relying on today’s developments in religious studies, Buddhism represents the position of nothingness as opposed to the position of being in traditional Christianity, and is the essential occasion for the mediation of the truly dialectical absolute religion” (THZ 10, p. 323), Tanabe goes on to state:\n\nIt must be said that only by including this can absolute religion truly become absolute. For without the occasion of nothingness, the absoluteness of religion cannot be realized against itself. In this sense, Mahāyāna Buddhism is truly representative of the absoluteness of absolute religion. However, as we have often seen, the absolute must be mediated by the relative, or else it loses its absoluteness. To demonstrate its content as an absolute mediation in the modern age, Buddhism must not simply return to the past, but rather be rooted historically, mediated by the religious needs of the present. (THZ 10, p. 323)\n\nTanabe advocates a thoroughgoing absolute religion from the standpoint of a philosophy of religion based on Hegel’s theory of religion by adding the Buddhist concept of nothingness to the dialectic of that philosophy. Tanabe’s absolute religion, with Christianity in mind and Buddhism added to it, differs from Hegel’s view of the history of religions. However, this reference to absolute religion, characteristic of Tanabe’s theory of religion, does not clarify what kind of a universal religion he aims for. Hence, we address Tanabe’s reference to “world religion(s)”30 in our discussion.\n\nAbsolute religion is used in Tanabe as if it were a synonym for world religion. In his lecture, “The Limits of Culture,” for example, Tanabe states, “The question of death forms the central problem in all religions, especially in Buddhism and Christianity, which are called absolute or world religions” (THZ 8, p. 281).31 World religions in Tanabe are not based on population or regional distribution but are limited to “Buddhism and Christianity, which today are recognized as two opposing world religions” (THZ 11, p. 447). Previous studies also identify Buddhism and Christianity as world religions in Tanabe.32 Furthermore, Christianity is representative of world religions in his works. Paul superseded Judaism, a tribal religion (THZ 10, p. 66), and became the driving force in establishing Christianity as a world religion (THZ 10, pp. 174, 237).33 An essential element in this world religion is that it “is not concerned with the comfort of self alone, but intends the comfort and salvation of all humankind” (THZ 10, p. 147), namely, it “advocates the salvation of humanity” (THZ 8, p. 432). Tanabe also states that “all world religions recognize love at the root of existence, and they generally preach love as the principle of spirituality as the highest being of human” (THZ 13, p. 189).\n\nFrom these assertions, it would seem that Tanabe is simply defining world religions based on Christianity. Passages such as “there can be no religion apart from theology in world religions” (THZ 10, p. 255) seem to confirm this view. However, in his later work, Introduction to Philosophy, Tanabe not only states that theology that “organizes the content of the beliefs of some particular religion into a logical doctrine” is essential for evangelism but also that it is appropriate for “a theology that attempts to logically formulate the absolute universally” to be called a philosophy of religion if it moves beyond the specifics of particular religions (THZ 11, pp. 38–41). Hence, in Tanabe’s view, world religions are defined by the degree to which they reflect his dialectic.34 From the standpoint that “it is essential to clarify the essential differences between Buddhism and Christianity and the point of unity between the two religions from the standpoint of religious philosophy” (THZ 10, p. 451), Tanabe argues the ideal religion as below.35\n\nPure Land Buddhism, which is included in Nāgārjuna and other branches of Mahāyāna Buddhism, shows that Buddhism as a religion cannot lack the occasion of salvation by Other-power (tariki 他力). The fact that it is thoroughly realized as the position of nothingness in Zen on the one hand, and the return to the love of grace by Other-power on the other, can be understood as the reason for the realization of the essence of the religion of nothingness as love and love as nothingness. In this sense, Zen and Nembutsu should be regarded as opposing occasions that establish the religiosity of Buddhism. If a concrete synthesis of the two, which may be called Nembutsu-Zen,36 is realized, it can be understood as the most concrete and universal stage of Buddhism. However, it is a logical necessity that the creative development of such a synthesis requires a religious form corresponding to the historicity of the particular, mediating between the universal of nothingness and the individual of love. To think that this is offered in Christianity should be expected from what has already been said. In the mediation of these three, the unifying content of absolute mediation is embodied, and this alone is the task of religious innovation at present. (THZ 10, p. 34)\n\nIt is not difficult to imagine that the absolute mediation of Zen, Pure Land Buddhism, and Christianity would result in the aforementioned absolute faith or religion, but one cannot deny the impression that the philosopher Tanabe is creating a religion by cherry picking. Tanabe’s attitude toward including religion in the philosophical discussion became more pronounced after the zangedō period. In “The Logic of Place and the Religious Worldview (Bashoteki Ronri to Shūkyō-teki Sekai-kan, 1945),” written shortly before his death, Nishida’s statement that “a philosopher should not fabricate religion based on his system”37 may be considered a reflection of Tanabe’s attitude.38 How can a philosopher form a religion, whether fabricated or not? As noted above, the main theme of Demonstration of Christianity is not the formation of religion by philosophers or the bridge between philosophy and religion. The book is a realization of Tanabe’s long-considered confrontation with Christianity in his unique demonstration. However, this awareness of the problem can be traced back to Tanabe’s earlier writings, which are also discussed at the beginning of Demonstration of Christianity.\n\nI have already mentioned that I have come to feel a kinship with Christianity since I was converted to “philosophy as metanoetics.” Moreover, when I reflected on the dialectic of Platonism that I had adopted in the past, it became clear to me that I could no longer stop there. I then became aware that it was inevitable that I should go on to the position of paradoxical dialectics and come to faith in the gospel. In other words, the dialectic of good must now be turned into the dialectic of love. (THZ 10, pp. 11–12)\n\nHere, “the dialectic of Platonism that I had adopted in the past” is a clear reference to the third article in Existence, Love, and Practice, “Self-Transcendence in Platonism and Faith in Gospel.” In addition, with the “dialectic of good,” he probably has the “idea of good” in Plato’s Republic in mind, extending it to the Christian dialectic of love and Buddhism. As noted earlier, world religions are based on love (THZ 13, p. 189) as a requirement for the salvation of humanity (THZ 8, p. 432). Therefore, Existence, Love, and Practice seems to suggest how philosophy is connected to the formation of world religion and its universality from Tanabe’s standpoint. In the next section, we will examine how his conception of world religion is bridged through philosophy.\n\nThe third article in Existence, Love, and Practice, “Self-Transcendence in Platonism and Faith in Gospel,” is vital for understanding how philosophy is connected to religion in Tanabe. The article opens with a chapter titled “Socrates’ Tragic Death Hymn and Philosophy,” which discusses Socrates’ “practice of death” as depicted in Plato’s Phaedo:\n\nIn philosophy, we attain the wisdom of liberation through the path of nothingness. Its starting point is in accord with the criticism of science, and its point of return is in accord with religion as a standpoint for liberation and salvation…. To die, that Plato demanded of the true philosopher, must be to stand in such a standpoint of nothingness. (THZ 9, pp. 405–406)39\n\nThe unity of the point of return of philosophy and religion is presented by Socrates. Then, daemōn’s voice to Socrates is explained as the intervention of the Other-power, which urges repentance as absolute conversion. Tanabe asserts that “philosophy must be the very practice of absolute nothingness” (THZ 9, p. 430). In Demonstration of Christianity, the occasion of absolute nothingness through the death and resurrection of Jesus is pointed out as the reason why Judaism, which remained within the bounds of tribalism, was transcended and became a world religion called Christianity. Zen is also a religion of nothingness for Tanabe, and Pure Land Buddhism is a religion of Other-power salvation. Thus, “Self-Transcendence in Platonism and Faith in Gospel” refers to Socrates in the same way as the world religions in Demonstration of Christianity. Moreover, since Socrates is also regarded as an ordinary man (bonbu 凡夫) who obtains salvation through repentance (zange懺悔) in Other-power religions (THZ 9, p. 29), his practice of death comes even closer to salvation through Other-power religions as the only way possible, even for ordinary people.\n\n“Self-Transcendence in Platonism and Faith in Gospel,” which begins with the death of Socrates, discusses such themes as the development of Plato’s Idealism, the significance of matter as an occasion of self-negation in Plato’s philosophy, its similarity to Buddhism, and the fact that the existential philosophy of the time, including Karl Jaspers, became an overreach of Plato’s dialectics as well as Neoplatonism. Toward the end of the work in chapter six, “The Mediation of Zen of Learning Death in Nothingness as Love,” Tanabe refers to Suzuki Shōsan in the early Edo period to make practicing death possible even for ordinary people. This argument may seem rather far-fetched, but Tanabe sees in it a connection between philosophy and religion that transcends both East and West. Suzuki Shōsan was a Zen monk of samurai origin and was regarded as an advocate of economic ethics due to his insistence that daily life was a Buddhist practice. Tanabe contrasts Suzuki’s experience of constantly facing death with that of Socrates and interprets it as follows:\n\nI find it very significant that on the method of learning death, we can recognize the process by which philosophers practice death, something that Socrates seems to have accomplished with almost superhuman innate gifts and training. … Therefore, if we learn death by dying in death, as Shōsan dōjin has shown, then true death, no longer an object of conception, becomes the inner occasion that makes us subject. This learning from death is the only way to practice death that is open to us ordinary people. (THZ 9, pp. 473–474)\n\nFurthermore, Tanabe, referring to Suzuki Daisetsu, interprets the emphasis on knowing shame as a characteristic of Zen of learning death as “an emphasis on repentance not normally found in Zen” (THZ 9, p. 474). He praises Shōsan Zen as “deeply committed to the absolute state of religion, without any doubt” (THZ 9, p. 475). However, Zen was a standpoint pursuing absolute nothingness through self-power practice and treating others with love was thin and abstract (THZ 9, pp. 477–478). Here Tanabe states that by applying his dialectic principle to Zen, the Zen of self-power and the Nembutsu of Other-power go together to arrive at the original state of being.\n\nIn this way, the belief in self-power, a characteristic of Zen, becomes against itself through transcendence as immanence and is developed into self-power as Other-power through the mediation of the method learned from death. Thus, combined with the social nature of love mentioned above, Zen’s demand for self-centered liberation, as rooted in the mercy of Buddha and the fundamental vow of Tathāgata, is transformed into gratitude for the other and proceeds to the phase of returning to the other. (THZ 9, pp. 478–479)\n\nIn Tanabe, because repentance by Other-power is the essence of Shinran, Suzuki Shōsan’s Zen practiced by ordinary people who repent may be connected to Nembutsu (THZ 9, p. 476). Hence, with Socrates as a starting point, Zen and Nembutsu are strongly linked, and religious salvation is not limited to self-salvation but is also linked to social practice. This also corresponds to the fact that Plato’s dialectic, which began with Socrates, is oriented toward self-transcendence and necessarily leads to social practice, hence the claim that Platonism is completed by the Christian gospel.\n\nIn seeking a satisfactory and possible way to meet the inevitable demand of the Platonic dialectic to transcend the self through dialectical methods, I have taken up and interpreted, and critiqued ideas such as existential philosophy and Zen of learning death, which have much in common with Plato’s philosophy. I realized, however, that these are still one-dimensional abstractions and that we must demand the opposite positions of social liberation and Other-power return as an occasion, and that they must also be mutually combined through them. Thus, as a concrete religion that mediates and integrates these two or three opposing occasions, I have no choice but to take up faith in the gospel. (THZ 9, p. 481)\n\nThis statement also seems to be an assertion that world religions, including philosophies, are unified under Christianity as the standard. However, as discussed in Demonstration of Christianity, the element of dialectic mediated by the negative occasions allows Christianity to break away from the tribal religion of Judaism to become a world religion. In the same book, Plato is indicated as the founder of that dialectic (THZ 10, p. 28). Plato’s later philosophy establishes the dialectic common to Christianity that practically mediates individual existence toward the social organization (THZ 10, p. 29). Although one cannot deny the impression of Christocentrism, Platonism can be seen as the framework for the world religion that Tanabe calls for in the second religious reformation, in which Christianity and Buddhism are linked. In other words, Platonism, as the root of dialectics, is the mediator between philosophy and religion and the condition for forming what Tanabe calls a world religion.\n\nThis insistence on moving from Platonism as a dialectic philosophy to a world religion that must include a dialectical occasion is also found in Dialectic of the Logic of Species, published in 1947. However, there are few references to religions other than Christianity. In this work, published slightly earlier than Existence, Love, and Practice, Plotinus is frequently mentioned critically for his emphasis on contemplation. In contrast, Plato is mentioned positively for his emphasis on practice. However, Tanabe also noted that the person who best understood and succeeded Plotinus was Meister Eckhart, known as the Christian mystic of medieval Germany, who overcame Plotinus by emphasizing practice (THZ 7, p. 310).40 As in “Self-Transcendence in Platonism and Faith in Gospel” from Existence, Love, and Practice, when Platonism is correctly and thoroughly implemented in Tanabe’s dialectic, we observe a structure that leads to a world religion with Christianity as its main axis.\n\n\nConclusion\n\nIn this discussion, we have seen that in the Japanese philosopher Tanabe’s theory of absolute dialectic, the way of absolute religion is envisioned for the second religious reformation, and that there is a discussion of world religions, though limited to Christianity and some sects of Buddhism. The religion of the new era as envisioned by Tanabe, is a universal one in which philosophy and religion are bridged through dialectics and where Platonism is presented as a model for realizing this. Tanabe’s discussion reveals the following aspects that have not received much attention so far. The Japanese philosopher who confronted Western philosophy with intensive contemplation made a “creative”41 interpretation of Platonism as an idea that inseparably links philosophy and religion and envisions a religion for a new era connecting East and West, philosophy, and religion.\n\nTanabe’s conception of religion probably does not go beyond the scope of an intellectual’s religion. The scope of his discussion of world religions is narrow, and his attention to folk customs and practices is limited. This would indicate that even if Tanabe’s thoroughness in philosophy had led him to religion, he would not have escaped the limitations of a “religion” that is mainly based on doctrinal and speculative aspects. However, this does not mean that Tanabe’s inquiry was not directed toward practices such as folk customs that today’s religious studies would naturally include in the realm of religion, but rather that Tanabe was directed toward practices as a philosopher. Although it may appear to be an empty theory, for Tanabe, it was an existential practice and not an empty argument as shown in Demonstration of Christianity.42\n\nThe confrontation with Minoda also evidences the intensity of Tanabe’s determination to pursue his practice of philosophy. The fact that the thoroughness of his philosophy, on which he stakes his existence, is somewhat aligned with religion, may be considered a practice of retracing the path of Socrates, the founder of the dialectic, as Tanabe calls him. Platonism, which originates in the practice of death by Socrates, is, according to Tanabe, the driving force behind the development of Christianity into a world religion of humanity and a borderline position that may diverge into Neoplatonism as an overreach of Plato, which Tanabe criticizes.43 Thus, Platonism in Tanabe’s philosophy, as a prototype of dialectics, is the basis of his philosophy. It also plays an interesting role as a mediation between his philosophy and world religions that aim at universal salvation.44\n\nHatano Seiichi, whose lectures Tanabe also attended, wrote of Neoplatonism, including Plotinus, that “this school of philosophy sought to satisfy the truth-seeking mind on the one hand and the religious mind on the other. In a word, it is the religion of scholars,” in his The History of Western Philosophy (Seiyō Tetsugakushi-yō, 1948).45 Plotinus was a philosopher whom Tanabe criticized for his bias toward contemplation. Tanabe contended that Christianity had overcome it. Aside from “unification” (henōsis), Tanabe and Plotinus may have something in common in that they have practiced philosophy with a religious mind in seeking salvation and that, in the end, the thoroughness of their philosophical inquiry has led them to religiosity. What does the thoroughness of the philosopher’s inquiry and its practice at a turning point bring to religion? As the above discussion shows, this question is of particular interest to Japanese philosophers. However, to examine this theme in Japan, the trend concerning (self-) cultivation (shūyō 修養) should be considered, which was common among both scholars and religious intellectuals from the middle of the Meiji era. This point could not be dealt with adequately in this study and should be explored further in the future.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nFootnotes\n\n1 Tanabe’s work referred to in this study is based on Tanabe Hajime Zenshū (Collected Works of Tanabe Hajime), Tokyo: Chikuma Shobō, 1976 (3rd. pr.), abbreviated as THZ. When citing or referring to the work, the reference is indicated by the THZ volume number and page number. The quotations in parentheses ( ) are the original text, and brackets [ ] indicate the author’s additions.\n\n2 It has also been pointed out that Nishida’s strong influence from Zen has not always been fully demonstrated (Sakamoto Shin-ichi, “Nishida Tetsugaku no Futatsu no Keishō: Kyōto Gakuha to Chisan Gakuha (Two Inheritances of Nishida Philosophy: Kyoto School and Chisan School),” Hikaku Shisō Kenkyū (Studies in Comparative Philosophy), No. 48, 2022, pp. 115–116).\n\n3 The new edition of the Collected Works of Nishida Kitarō, Vol. 14, includes a record of Nishida’s lectures on religious studies. The section “The Rise of Religious Studies and Its Development” lists the scholars of religious studies since Max Müller. Takeda Atsushi, Klaus Riesenhuber, Kosaka Kunitsugu, Fujita Masakatsu (eds.), Nishida Kitarō Zenshū (Collected Works of Nishida Kitarō), Tokyo: Iwanami Shoten, 2004, Vol. 14, pp. 37–42.\n\n4 In Demonstration of Christianity, for example, Tanabe recounts his relationship with religion, including Christianity, and states that he never held any religious beliefs to maintain his position as a philosopher (THZ 10, pp. 3–9). In his Introduction to Philosophy ( Tetsugaku Nyūmon ), published in 1951 and based on lectures given from 1948 to 1951, Tanabe stated that although he had no specific religion or belief, “I cannot suppress the religious urge to be aware of my relationship with something absolute” (THZ 11, p. 444). This attitude should not be taken as a suspension of judgment concerning value or a neutral attitude toward religion but rather as an indication of the thoroughness of his philosophy.\n\n5 Himi Kiyoshi, Tanabe Tetsugaku Kenkyū: Shūkyō Tetsugaku no Kanten kara (Studies in the Philosophy of Tanabe Hajime: From the Perspective of the Philosophy of Religion), Tokyo: Hokuju Shuppan, 1990, p. 20.\n\n6 The year of publication may mislead readers into thinking that this work was written in response to Japan’s defeat in World War II. However, it is based on a lecture titled “Metanoetics” (zangedō) given in 1944 after the setback of the logic of species (shu no ronri), and it is mentioned in a commentary by Ōshima Yasumasa that the book was still in the process when Japan lost the war (THZ 7, pp. 382–384).\n\n7 Tanabe distinguishes between the conscious “benshō (辯証)” and the objectified “benshō (辨証),” merely seen from the outside, and adopts “benshō (辯証)” for the book’s title (THZ 10, pp. 38–40).\n\n8 It should be noted that Tanabe’s dialectic is his theory, which he calls “absolute dialectic.” Therefore, when Tanabe uses the term “dialectic” to refer to Plato in particular, he includes many of his interpretations that depart from Plato’s text.\n\n9 It is important to consider the division of Tanabe’s thoughts into periods, which differs among scholars. In this study, the main subject under consideration is Tanabe’s writings after his lecture “zangedō” in 1944, which belongs to the period after which Tanabe realized the inadequacy of the logic of species. This later period of Tanabe is strongly characterized as a philosophy of religion. In line with Himi’s interpretation, this study takes the position that Tanabe maintains a consistent problematic awareness of religion, even though the subject matter he addresses changes over time (Himi, op. cit., pp. 19–20).\n\n10 It should be noted that Tanabe’s philosophy of religion has a different connotation compared to the philosophy of religion in general terms, such as Kant’s philosophy of religion, for example. Tanabe also refers to the philosophy of religion in philosophy in general, for example, in “Supplement III: Philosophy of Religion and ethics” in his Introduction to Philosophy. He states that the philosophy of religion is a touchstone for each philosophy to question its true value (THZ 11, pp. 429–432).\n\n11 As works that thematically discuss Tanabe’s philosophy of religion, we can mention Himi, op. cit., and Liao Chin-ping, Shūkyō Tetsugaku no Kyūsai-ron: Kōki Tanabe Tetsugaku no Kenkyū (The Soteriology of Religious philosophy: A study of Tanabe Hajime’s Late Philosophy), Taipei: National Taiwan University Press Center, 2018.\n\n12 For a study of these religious figures and their theories, see Hoshino Seiji, Kindai Nihon no Shūkyō Gainen: Shūkyōsha no Kotoba to Kindai (The Concept of Religion in Modern Japan: Religious Figures and Modernity), Tokyo: Yūshisha, 2012.\n\n13 The claims made by modern Japanese religious figures in Hoshino ibid. diverge from Tanabe’s theory of religion. This difference may be explained by a difference in positions: while some of the religious figures mentioned in the book approach philosophy from the perspective of religion, Tanabe identifies himself as non-religious and approaches religion from the perspective of philosophy.\n\n14 In Demonstration of Christianity, Tanabe states that religion apart from theology cannot be a world religion and excludes indigenous beliefs from his consideration (THZ 10, p. 255). Citing Edward B. Tyler and Cornelis P. Thiele, Nishida also mentions primitive religions, and states that the essential classification of religions is not monotheism/polytheism or natural religion/revealed religion, but “according to the content of vital demand” (Collected Works of Nishida Kitarō, Vol. 14, 2004, pp. 80–86).\n\n15 Nakano Hajimu (ed.), Kindai Nihon Shisō Taikei 23 Tanabe Hajime Shū (Modern Japanese Thought Series 23: Tanabe Hajime Collection), Tokyo: Chikuma Shobō, 1975, p. 449.\n\n16 Tanabe’s consistent refusal to move to a warmer climate when advised by Karaki Junzō and others close to him are documented in letters to Karaki dated July and October 1947 (Tanabe Hajime Karaki Junzō Ōfuku Shokan (Tanabe Hajime and Karaki Junzō Reciprocal Letters), Tokyo: Chikuma Shobō, 2004, pp. 62–63, 72). For background on Tanabe’s stubborn stance, see the discussion by Itō Susumu (Ai to Shi no Tetsugaku: Tanabe Hajime (The Philosophy of Love and Death: Tanabe Hajime), Tokyo: Hokuju Shuppan, 2005, pp. 139-179).\n\n17 Tanabe Hajime, “Minoda-shi oyobi Matsuda-shi no Hihan ni Kotau (Response to Mr. Minoda’s and Mr. Matsuda’s Criticisms),” THZ 8, pp. 13–31.\n\n18 Letters to Mutai Risaku and others (Collected Works of Nishida Kitarō, Vol. 22, 2007, pp. 150, 170).\n\n19 For the discussions between Minoda and Tanabe, see Uemura Kazuhide, “Nihon” heno Toi wo meguru Tōsō: Kyōto Gakuha to Genri Nihon Sha (Conflict over the Question of Japan: Kyōto School and Genri Nihon Sha), Kashiwa Shobō: Tokyō, 2007, pp. 142-144.\n\n20 As to why Tanabe responded to the debate, Ōshima Yasumasa speculates that one of the attackers, Matsuda Fukumatsu, may have once been a student of Tanabe (THZ 8, p. 466).\n\n21 See Himi, op. cit., pp. 111–117.\n\n22 Itō, op. cit., pp. 122–123.\n\n23 Mutō Kazuo explains this as follows: “Tanabe did not advocate a new religion himself, so to speak, but rather sought a deep self-reflection of Christianity and Buddhism from the standpoint of philosophical demonstration and hoped for creative development originating from within them” (THZ 10, p. 333). Depending on one’s view of the distinction between philosophy and religion, it is difficult to say whether Demonstration of Christianity was an attempt to establish a new religion or a proposal for religion from the standpoint of philosophy. However, as the practice of philosophy that Tanabe aimed at was in line with religion (THZ 11, p. 430), we argue that it was a practice aimed at some form of religion from the standpoint of philosophy.\n\n24 When Tanabe was a high school student, discourses on (self-) cultivation (shūyō 修養) were being developed by Christian and Buddhist intellectuals. Some studies note that during this period, there was a movement among scholars of religion, such as Anesaki Masaharu, to search for a new form of religion from the perspective of cultivation (Kurita Hidehiko, “The Notion of Shūyō and Conceptualizing the Future of Religion at the Turn of the Twentieth Century,” Religious Studies in Japan, 2018, Vol. 4, pp. 74–75). However, the word shūyō rarely appears in Tanabe. The few examples are his letter to Karaki in December 1947 (Tanabe and Karaki, op. cit. pp. 76-77).\n\n25 The school was known as the predecessor of the Collage of Arts and Sciences at the University of Tokyo and others, with its dormitories being famous for their self-governing system. This spirit had already faded by the time Tanabe entered the school (THZ 10, p. 3).\n\n26 Liao interprets this as a religious experience that occurred to Tanabe during this period. The author agrees with Tanabe’s description from the perspective of religious studies (Liao, op. cit., p. 44).\n\n27 For a discussion of how absolute religion in Tanabe is constructed through his absolute dialectic, see Liao, op. cit. pp. 167-175. This study focuses on the aspect of absolute religion as a theory of religion rather than its dialectical construction, then then discusses how Platonism bridges between philosophy and religion in Tanabe.\n\n28 Protestantism “dynamically testifies to eternity insofar as it constantly renews itself in creative repetition and innovative circulation” (THZ 10, p. 322), but even in Hegel’s time, such a spirit was lost. Hence, the second religious reformation must restore its originality, as Tanabe suggests.\n\n29 Liao, op. cit., p. 66, 72, 110.\n\n30 A well-known study of world religions is the one by Masuzawa Tomoko (The Invention of World Religions: Or, How European Universalism Was Preserved in the Language of Pluralism, Chicago and Lindon: The University of Chicago Press, 2005). Tanabe’s world religions are quite different from those discussed in the book, especially the issue of the plurality of languages, which was becoming well known at the time but seemingly not reflected in his books.\n\n31 This lecture was given to the national elite, First High School students, the year before Japan’s defeat in the war. It should be noted, therefore, that the “question of death” here was also given in the context of total war, a situation of urgency quite different from those in which philosophy and religion are discussed today.\n\n32 See Himi, op. cit., pp. 113, 116. Regarding the narrow range of religions that Tanabe took up in his discussion of world religions (although Tanabe states in THZ 11, p. 448 that Islam, like Judaism, is not a world religion because it is unique to a particular people), Himi said that it was unavoidable given the state of research in Japan at the time, but that Tanabe’s failure to mention Shintoism, perhaps due to insufficient awareness of the issue of the Emperor System, greatly reduced his persuasiveness (Himi, op. cit., pp. 182-183). The author speculates, in contrast, that Tanabe may not have placed Shintō in the category of religions that he can discuss, either concerning the so-called state Shintō or to Shintō as a custom. This may be attributed to the fact that Shintō was not considered a religion by scholars of the time, as discussed in a 1949 dialogue between Ienaga Saburō and Yanagita Kunio: (Ienaga) “Especially about Shintō, written thought is no good at all.” (Yanagita) “I am painfully aware of this” (Ienaga Saburō, Tanabe Hajime no Shisōshi teki Kenkyū” Sensō to Tetsugakusha (An Intellectual History of Tanabe Hajime: War and Philosopher), Tokyo: Hōsei Daigaku Shuppan Kyoku, 1974, p. 423). In any case, it is possible that Tanabe did not intentionally include Shintō in his discussion.\n\n33 Tanabe’s interpretation of this establishment is also based on his dialectic (THZ 10, p. 90).\n\n34 The death and resurrection of Jesus is a strong manifestation of this element. In Demonstration of Christianity, Tanabe states that “it would be impossible to find an equivalent occasion in Buddhism” (THZ 10, p. 35) and mentions other fundamental differences between Christianity and Buddhism (THZ 10, p. 48). However, despite the differences, the fact that a sect that lacks a dialectic cannot become a world religion and remains Japanese Buddhism (THZ 10, p. 309) is connected to the assertion that Buddhism is a world religion by his dialectic.\n\n35 In Tanabe, Pure Land Buddhism is the closest to Christianity as a religion of Other-power salvation (THZ 10, p. 33), and Zen Buddhism is considered to have the occasion of nothingness corresponding to the death of Jesus.\n\n36 For Nembutsu-Zen in Tanabe’s context and its dialectic construction, see Liao, op.cit., pp. 84-91.\n\n37 Collected Works of Nishida Kitarō, Vol. 10, 2004, p. 295.\n\n38 Nakano, op. cit., p. 457. In response to these points made by Nakano, Himi observes that since Tanabe is not named, “it would be sufficient to accept it honestly as a very general phrase of admonition.” Himi, op. cit., p. 28. However, as Nakano explains, the following words of Tanabe in “I Ask for Professor Nishida’s Instruction” in which he turns to a criticism of Nishida, can be interpreted as a rebuke directed at Tanabe in his later years: “I cannot help but be suspicious of the religiousization of philosophy” (THZ 4, p. 328) and “I think that the religiousization of philosophy cannot avoid the tendency to make philosophy an imperfect imitation of religion, instead of recognizing its authority. By its very nature, philosophy aims at a standpoint free from all standpoints. Religion is also a standpoint, and therefore should be transcended” (THZ 4, p. 313).\n\n39 Tanabe states that “Socrates was a hero and a superhuman, not a philosopher like Plato himself” and that “in philosophy, which is thoroughly concerned with the dialectic of consciousness of thought, he only taught its starting point and the direction it should go, but it cannot be said that he fully developed it” (THZ 9, p. 413), and that the dialectic was perfected by Plato who succeeded Socrates. He then positions the Neoplatonism of Plotinus and others as an “overreach” of Plato by transferring the negative occasion of nothingness to the position of life (THZ 9, pp. 92, 398–399, 432). This indicates that Tanabe regarded Socrates, Plato, and Plotinus as a continuous Platonism. Tanabe was referring to research trends in ancient Greek philosophy and was critical of the theory of the differences between the philosophies of Socrates and Plato (Burnet=Taylor theory) that was being proposed at the time (THZ 9, 411, pp. 414–415).\n\n40 A similar statement is found in Philosophy as Metanoetics (THZ 9, p. 258).\n\n41 The bracketed “creative” is because Tanabe’s argument is formed by including a misreading of Plato. An example is his interpretation of the “practice of death (Plato’s Phaedo)” in “Self-Transcendence in Platonism and Faith in Gospel” (THZ 9, p. 406).\n\n42 “My demonstrations are inevitably criticized as philosophical fantasies by genuine Christians just as Buddhists call it an unserious argument, I must be willing to accept criticism from Christians that it is fanciful. I can only endure in despair but not despair, relying on the words, “he that shall endure unto the end, the same shall be saved” (Mark 13: 13). In that case, Paul’s conversion will be a great help to me. No, not only that. I believe that the truth of the dialectic is already being proven in the execution of this task, as it is an unavoidable philosophical demonstration for me, even if it is criticized by others” (THZ 10, pp. 92–93).\n\n43 On the positive and negative influences from Plato and other Greek thought on Christianity as a world religion, see THZ 10, pp. 78–79.\n\n44 After Demonstration of Christianity, in which Tanabe praised Christianity, especially Protestantism, he took a new turn by approaching Buddhism and Zen in particular. The awareness of the Master as a relative person that was developed in Demonstration of Christianity (THZ 10, pp. 82, 89) led to a shift from Christianity, which was limited to salvation by the Savior and Master, to Bosatsudō, in which the many relativities of ordinary people worked together to save one another. What made this development possible was probably Tanabe’s theory of world religions, which, from Philosophy as Metanoetics to Demonstration of Christianity, understood Zen as a religion of Other-power and connected it to Christianity. However, a discussion of this theme is beyond the scope of this study and should be explored in future research.\n\n45 Hatano Seiichi, Hatano Seiichi Zenshū (Collected Works of Hatano Seiichi), Tokyo: Iwanami Shoten, 1968, p. 150. Circle points were added on “the religion of scholars” in the original text." }	[ { "id": "241410", "date": "26 Feb 2024", "name": "Seiji Hoshino", "expertise": [ "Reviewer Expertise History of religions in modern Japan" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPaper Summary\nThis paper delves into Tanabe Hajime's evolving conception of \"religion,\" particularly during his later years, mainly focusing on Existence, Love, and Practice (1947) and Demonstration of Christianity (1948). While Tanabe has been primarily identified as a philosopher, his philosophical inquiries frequently made references to religious traditions. Scholars have previously documented his shifting interest in historical religions from Shinran (Pure Land Buddhism) through Christianity to Zen Buddhism. The author, however, diverges by focusing on Tanabe's theoretical understanding of \"religion\" itself, examining Tanabe's use of key terms such as \"second religious reformation,\" \"absolute religion,\" and \"world religion.\" Tanabe posited the notion of a universal religion, which he called \"absolute religion,\" envisioning it as a dialectical synthesis of Christianity, incorporating elements of Pure Land and Zen Buddhism. While acknowledging that this may appear somewhat Christocentric, the author argues that it resulted from Tanabe's rigorous philosophical investigation, particularly his pursuit of the idea of \"negation.\" More specifically, the paper contends that Tanabe's conception of \"absolute religion\" was deeply rooted in his understanding of Platonism. According to the author, Platonism is a philosophical tradition where the boundaries between philosophy and religion are not distinctly demarcated, and the author concludes that Tanabe's formulation of \"absolute religion\" emerged from Tanabe's creative interpretation of Platonism.\nImportance of this paper\nJapanese philosophy of religion has often been described and studied in the context of how it has incorporated Eastern thought into the vocabulary and thinking of Western philosophy. Intensive scholarly works exist on the relationship between Kyoto School philosophers and Buddhist thoughts; Tanabe's understanding of Shinran has been examined. However, this paper argues that Tanabe's conception of \"religion\" is drawn from his philosophical pursuit and his creative reading of Platonism. The argument makes an interesting case study of Tanabe's thought.\nSuggestions\n-Context\nThat being said, I am a bit wondering how this paper's argument could connect to a broader context. For example, how does Tanabe's conception of religion fit into the history of \"religion\" discourse in modern Japan? As the author noted that Tanabe's argument would be within \"the scope of an intellectual's religion,\" what role did such an \"intellectual's religion\" play contemporarily?\n\nI understand this is not the main issue the author attempted to address. However, the author could provide his own perspective on the context of Tanabe's \"religion.\" (Perhaps the author could develop his reference to Tanabe's youth or the idea of \"shūyō\" mentioned in the conclusion.)\n-Tanabe's scope\nI want to make one more point. It is about the readers of Tanabe, or whom Tanabe tried to address; this relates to the question of Tanabe's scope. This paper argues that Tanabe's \"absolute religion\" involved a certain \"practice,\" namely, a thorough pursuit of philosophical speculation. At first glance, such a \"practice\" appears to be a personal ethics (for example, Tanabe's residence in Kita-Karuizawa and his attempt at dialogue with Minoda). However, providing that \"social practice\" and \"social improvement\" are repeatedly mentioned in the paper, Tanabe's \"absolute religion\" was, from what I understood, more than a personal ethics. Then, how did Tanabe think his \"absolute religion\" should be embodied in this world (or, more practically, how should it interact with the actual historical religious traditions of his time)? Of course, Tanabe was a philosopher, not a social scientist; thus, his arguments did not need to be directly connected to the real world. However, the readers might want to know Tanabe's vision regarding this.\n- Regarding the section \"Tanabe as a \"practitioner\"\nTanabe's \"practice\" explained in the section is difficult to understand at first read. To grasp the idea in this section, such that Tanabe's \"thorough contemplation\" was his (religious) \"practice,\" readers need the author's explanation of Tanabe's understanding of Platonism provided before the conclusion. Thus, I suggest the main argument of this section could be moved after the \"Platonism as a framework for world religion in Tanabe\" section, though this revision requires some rewriting.\nAlso, as pointed out above, readers may need an explanation of the relation between Tanabe's \"practice\" with \"social practice\" and \"transforming society\" or of the practical goals of \"social practice\" in Tanabe's conception.\nOther minor comments\n- Regarding Notes 12, 13\nThe author can provide more explanation regarding the differences between Tanabe's argument of religion and the arguments of the latter half of the 19th century.\n- Regarding Notes 14 and Tanabe's attitude towards \"folk religions and practices\"\nThe author can elaborate on Tanabe's understanding of evolutionary views of religions. Tanabe's attitude towards \"folk religions and practices\" appears to be influenced by the idea of the evolution of religion. What did Tanabe think about the evolutionary views of religions prevalent in his time? Or was Tanabe's view different from the views of his contemporaries?\n- Regarding the section \"Absolute religion in Tanabe\"\n\"This phenomenon was due to the influence of Christian intellectuals such as Uchimura Kanzō (1861–1930), Uemura Masahisa (1858–1925), and Ebina Danjō (1856–1937). Their influence was more significant than those of Buddhist intellectuals such as Kiyozawa Manshi (1863–1903) and Chikazumi Jōkan (1870–1941).\"\nI have some concerns in the author's description above, especially the evaluation of the influence of Buddhist figures because they were also very influential among young intellectuals at that time. If it is Tanabe's retrospect, it is understandable (apparently, those who were influenced by Kiyozawa or Chikazumi would look back differently). Please add citations or revise the description.\n- Tanabe's mention of the second religious reformation\nThere is a quote on Tanabe's mention of the second religious reformation (THZ 10, p. 319). The author can elaborate on Tanabe's claim about what points in existing Christianity should be reformed (or negated).\n- Tanabe's views of \"religious studies\"\nThe author cited Tanabe's brief phrase: \"relying on today's developments in religious studies\" (THZ 10, p. 323). Considering the theme of this paper, the author can explain Tanabe's views of the achievements of religious studies of his time.\n\nIs the work 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? Yes", "responses": [] }, { "id": "294163", "date": "27 Jun 2024", "name": "Ellie Palmer", "expertise": [ "Reviewer Expertise Japanese philosophy", "Tanabe Hajime and Watsuji Tetsuro", "philosophy of death and dying", "self and identity", "world philosophy", "community and ethics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPaper Summary\nThis paper focuses on the apparently inextricable relationship between philosophy and religion in the writing of Tanabe Hajime, centred upon his later works, in particular “Demonstration of Christianity” in which Tanabe’s notion of “absolute religion” is expounded. The author of this paper takes this concept as synonymous to “world religion”, whilst acknowledging the restriction of Tanabe’s discussion to Christianity and Buddhism. The author’s focus lies in interpreting this combination of Buddhism and Christianity with Platonism as the “bridge” between Tanabe’s theories of philosophy and religion. The author claims this “bridging” finds its basis in Tanabe’s indication that Plato is the founder of his dialectic. It is this dialectic which forms the fundamental mediation between philosophy and religion that defines Tanabe’s theory of the philosophy of religion. The author defines world religion, in an interpretation of Tanabe, based not only on Christianity but by the degree to which they reflect Tanabe’s Platonic dialectic, concluding that it is Platonism, despite the apparent Christocentrism in Tanabe’s work, that can ultimately therefore be considered the framework for Tanabe’s concept of world religion and, further, is itself a world religion.\nContribution to field\nThis paper makes a unique and valuable contribution to the field by choosing to examine not merely Tanabe’s theory of religion in isolation but specifically acknowledging it within the context of Tanabe’s reading of Platonism and the inseparable connection to his wider philosophy. The author also engages in discussion of more specific concepts in Tanabe’s philosophy of religion that are, in fact, key to its understanding yet often overlooked, such as the notion of an “absolute” or “universal” religion and the “second religious reformation” discussed in Demonstration of Christianity. In general, Japanese philosophy, including Tanabe’s theory of religion is frequently examined in this comparative context between “east” and “west”, and other times, as the author points out, exclusively from the perspective of Japanese philosophy. Interestingly, however, this paper situates Tanabe’s theory of religion as grounded in his interpretation of Platonic theory. This arguably makes for a stronger foundation to both portray and discuss Tanabe’s theory, rather than evaluating Tanabe’s theory in reference to a particular religion or tradition, or being tempted by applying this to a comparison of eastern and western religion, which would fail to preserve Tanabe’s fundamental aim of establishing a universal theory of religion. Discussion and development of Tanabe’s theory of religion is a particularly welcome topic today, where the world is increasingly secular, yet many still yearn for faith and the societal and meaning-based role that religion plays in life in the way Tanabe describes through his work on the philosophy of religion.\nRecommendations\nContent\nI’m not entirely convinced whether the section on Tanabe as a “practitioner” adds much to the overall paper. The idea that Tanabe’s philosophy of religion required a certain “practice” is relevant but why this is was not entirely clear within this particular section. This section could be expanded in terms of what this practice entails and how the theories the author discusses should be employed in a wider societal, practical and ethical context, and/or condensed in terms of the background content.\nContext and scope\nThe author mentions throughout this paper the fact that Tanabe’s theory of religion was not only extremely “unique” or “unusual”, but even radical and “aggressive”. It would be beneficial to draw on precisely why this was, through situating Tanabe’s work within some further discussion and analysis of the previous attitudes and views on the link between philosophy and religion before Tanabe’s new theory, to elucidate why it was considered so radically different. This may be limited to the attitudes of Japanese philosophers, or expand to explicate more generally those ideas in the east and west since the author does discuss that Tanabe’s theory is a combination of eastern and western ideas. It seems there is an ideal space to develop this within the section on the ‘Range of Tanabe’s discussion and religion in Japanese philosophers’. Although this may have been beyond the remit of this paper, I would have enjoyed some engagement with some of Tanabe’s earlier theories on the philosophy of religion. The author acknowledges Tanabe as a dynamic practitioner, and in footnote 9 the importance of considering the division of Tanabe’s thoughts into various periods, but some discussion of his earlier attempts to define the philosophy of religion in Outline of Philosophy 哲学通論 (1933) could have made an interesting contribution to the paper.\n\nStructure\nIn this same footnote, footnote 9, the author states that this paper assumes Himi’s position that “Tanabe maintains a consistent problematic awareness of religion, even though the subject matter he addresses changes over time (Himi, op. cit., pp. 19–20)”. Some further discussion of why the author holds this position within the main body of this paper may be beneficial, also because another citation referencing Himi in the main body mentions Tanabe’s philosophy as being “pure” and “thorough” which seems to confuse the author’s position. This critical position is mentioned within the conclusion and very briefly in the “Methods” section rather than having a dedicated place to evaluate this criticism in more depth.\nReferences\nReference appears to be missing from the third paragraph in the section ‘Absolute religion in Tanabe’ (“is Christianity but is not Christianity”) The comment made in the section “Absolute religion in Tanabe”, that the influence of Christian thinkers such as Uchimura Kanzo and Uemura Masahisa etc. was “more significant than those of Buddhist intellectuals such as Kiyozawa Manshi (1863–1903) and Chikazumi Jōkan (1870–1941).” should be more carefully rephrased or better referenced if the author is intending to represent some opinion by Tanabe that this was the case. The statement referenced, that “Christianity “responded even better to the humanist demands of the youth” from THZ 10 pg. 4 does not quite seem sufficient for such a sweeping statement.\nOther comments\nFinally, I would like to briefly draw attention to the general desire for philosophers to depart from what could be considered problematic usage of the “east” vs. “west” distinction, which is made a couple of times in the paper. Even though the author might disagree with these kinds of criticisms of characterising philosophy in this way often inspired by Said and McEvilley et al., this should still be acknowledged as a criticism that may crop up, and this distinction isn’t entirely necessary for or central to the fundamental arguments in this paper so can/should be easily avoided. What the author seems to want to say at various points in the paper can be said whilst avoiding this and simply referring to, for example, Buddhist vs. Christian tradition. Making more specific reference to, for example, the distinctions in Buddhist vs. Christian thought not only avoids this criticism of east/west essentialisation but would aid in the clarity, indicating precisely what the author is trying to draw our attention to about this difference. A further argument could be made specifically in the context of Tanabe that this distinction seems counterproductive to his aims and the idea propelled in this paper that Platonism connects “east and west”.\n\nIs the work 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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-59
https://f1000research.com/articles/12-1356/v1	18 Oct 23	{ "type": "Review", "title": "Effect of extrusion cooking on the chemical and nutritional properties of instant flours: a review", "authors": [ "Remigio Yamid Pismag", "María Paula Polo", "José Luis Hoyos", "Jesús Eduardo Bravo", "Diego Fernando Roa", "María Paula Polo", "José Luis Hoyos", "Jesús Eduardo Bravo", "Diego Fernando Roa" ], "abstract": "Satisfying the nutritional requirements of consumers has made food industries focus on the development of safe, innocuous, easy-to-prepare products with high nutritional quality through efficient processing technologies. Extrusion cooking has emerged as a prominent technology associated with the nutritional and functional attributes of food products. This review aims to establish a theoretical framework concerning the influence of extrusion parameters on the functional and nutritional properties of precooked or instant flours, both as end-products and ingredients. It highlights the pivotal role of process parameters within the extruder, including temperature, screw speed, and raw materials moisture content, among others, and elucidates their correlation with the modifications observed in the structural composition of these materials. Such modifications subsequently induce notable changes in the ultimate characteristics of the food product. Detailed insights into these transformations are provided within the subsequent sections, emphasizing their associations with critical phenomena such as nutrient availability, starch gelatinization, protein denaturation, enhanced in vitro digestibility, reduction in the content of antinutritional factors (ANFs), and the occurrence of Maillard reactions during specific processing stages. Drawing upon insights from available literature, it is concluded that these effects represent key attributes intertwined with the nutritional properties of the end-product during the production of instant flours.", "keywords": [ "precooking", "instant powders", "instant preparation", "instant consumption" ], "content": "Introduction\n\nThe market for precooked flours has witnessed significant growth both in developed and developing countries, providing opportunities for industries engaged in offering these products (Allied, 2020). Developing nations, particularly, seek viable alternatives that facilitate the processing of region-specific raw materials using cost-effective and highly efficient techniques. The aim is to enable the development of instant preparation foods that effectively cater to the nutritional needs of the population (Ali, Singh, & Sharma, 2016; Mouquet, Salvignol, Van Hoan, Monvois, & Trèche, 2003; Onyeoziri, Torres, Hamaker, Taylor, & Kock, 2021; Sebio & Chang, 2000). Currently, consumers are increasingly aware of their food consumption, and the ingredients employed in their formulation. The response of the food industry has been offering secure and nutritionally superior products while employing processing technologies that ensure efficiency and productivity (Akande et al., 2017; Sandrin et al., 2018). In this context, instant flours have gained prominence as indispensable products or ingredients that contribute to the development of foods with versatile nutritional, functional, and textural properties. Instant flours, often referred to as pregelatinized flours, undergo hydro-thermal modification processes to attain starch gelatinization in the raw materials (Sebio & Chang, 2000). Pregelatinized flours primarily are made of starch-rich raw materials, such as cereals, tubers, and selected fruits. However, it is vital to acknowledge that plant-based flours encompass additional constituents besides starch, including proteins, fibers, lipids, and bioactive compounds (Akande et al., 2017; Espinosa et al., 2021). Hence, the presence of these constituents must be considered during their processing, due to their influence on the physical, chemical, nutritional, and functional properties of the processed flours (Transparency, 2019).\n\n\nExtrusion parameters to produce instant flours\n\nExtrusion is a widely used technique in the creation of instant preparation foods such as beverages, porridges, infant foods, and desserts. Additionally, the raw materials employed in this process encompass a wide range, including cereal flours, legumes, fruits, tubers, and customized blends (see Table 1). The common raw materials used for producing precooked flours are rice, wheat, and maize. They have priority due to their wide availability and ease of being processed on an industrial scale. (Transparency, 2019). Extrusion induces starch modification through the process of gelatinization (Heredia et al., 2019). However, it’s common to employ additional processes such as germination and enzymatic hydrolysis for starch modifications (Tovar et al., 2017; Pismag et al., 2023). These supplementary procedures contribute to the creation of distinct functional properties in instant preparation products, with the objective of achieving effortless solubility and optimal water retention (Sandrin et al., 2018; Espinosa et al., 2021). However, ensuring solubility and suspension formation may not be enough, it is essential to consider the target audience when developing instant foods. In the pursuit of nutritional adequacy, the inclusion of legume-derived proteins or defatted raw materials is put forward by certain researchers (Espinosa et al., 2021). In some cases, animal-based proteins, such as dairy proteins, have also been explored to achieve balanced nutritional profiles (Heredia et al., 2019).\n\nTable 1 provides an overview of instant preparation food types obtained through extrusion. It outlines key process conditions that significantly influence product characteristics. Parameters less frequently discussed in the literature encompass extrusion temperature, material moisture content, and screw speed. Additionally, less extensively studied factors, including die opening, feed rate, and the composition ratios of raw material mixtures, warrant further investigation. Notably, both single-screw and twin-screw extruders are employed in processing these raw materials, showcasing the versatility, and expanded horizons of extrusion-based techniques for innovative food development.\n\n\nChemical and nutritional properties\n\nExtrusion cooking is a sequential and staged process that allows for progressive modification of the materials fed into the extruder. The raw materials used in the production of precooked or instant flours has a significant amount of starch (from cereal processing), high protein content (from legume processing) or it is also possible fiber content (mixed with cereal bran) (Ali et al., 2016; Akande et al., 2017; Kaur et al., 2019; Otondi et al., 2020). Moreover, these raw materials exhibit a potential complementary component such as lipids, vitamins, minerals and micronutrients (Ali et al., 2016).\n\nUnderstanding the behavior of main components during extrusion cooking is essential to predict the characteristics of the final products. The extent of modification or structural changes in these components depends on the thermo-mechanical conditions regulated or adjusted through process parameters in the extrusion equipment (Ali et al., 2016; Nahemiah et al., 2018). Consequently, the resulting characteristics of the products can vary significantly, leading to desirable attributes such as starch gelatinization, protein denaturation, increasing the in vitro digestion, enhanced nutrient availability (including sugars, polypeptides, phenolic compounds, and vitamins), increasing the soluble dietary fiber and reducing the level of antinutritional factors (Chulaluck et al., 2008; Otondi et al., 2020). Conversely, some papers describe the occurrence of Maillard reactions promoting a reduction in the protein digestibility as same as decreasing the content of thermolabile compounds like vitamins and phenolic compounds (Alam & Aslam, 2020; Offiah, Kontogiorgos, & Falade, 2019; Singh, Gamlath, & Wakeling, 2007). This section specifically focuses on elucidating the influence of process parameter modifications on the chemical and nutritional properties of instant preparation flours.\n\n\nFree sugars\n\nExtrusion cooking promotes the formation of low molecules weight from the starches. The main phenomena is called gelatinization, which is a particular process in extrusion that occurs at low moisture contents (12–45%), and involves the starch granule swelling, the leaching of amylose fractions and low molecular weight polysaccharides is also promoted. However, as thermo-mechanical conditions become more aggressive, fusion, depolymerization, and dextrinization of starches are induced (Dalbhagat, Mahato, & Mishra, 2019; Yadav, Dalbhagat, & Mishra, 2022). Both amylose and amylopectin are fully exposed to the mechanical effect of the screws, leading to the production of low molecular weight structures, including the generation of sugars (Singh, Gamlath, & Wakeling, 2007).\n\nTable 2 demonstrates that the production of free sugars can occur during the extrusion process. High-temperature conditions, elevated moisture contents, and increased screw speeds contribute to significant levels of free sugar production in flours. Mild or low extrusion conditions, characterized by low temperature profiles, low moisture content, and high screw speeds, do not induce structural changes in starches, as reported by (Martínez, Calviño, Rosell, & Gómez, 2014b; Martínez, Rosell, & Gómez, 2014a). Conversely, reversing these conditions during extrusion facilitates structural modifications in starches, leading to increased availability of free sugars in the extruded products (Cheftel, 1986). This is relevant in legume extrusion, where the structural modification of oligosaccharides, such as raffinose, results in the increased production of reducing and non-reducing sugars (Chauhan & Bains, 1988; Pham & Del Rosario, 1984). Controlling sugar levels during the extrusion process is crucial for ensuring the nutritional and sensory quality of extruded products (Yadav et al., 2022). The production of sugars must be regulated to prevent adverse effects on protein digestibility during extrusion processes, unless fermentation is desired (Singh et al., 2007).\n\n\nProtein solubility\n\nProtein is a representative fraction of cereal or legume flours, which suffer modification depending on the extrusion process conditions. During extrusion the hydrothermal and mechanical conditions cause denaturation and aggregation of proteins, leading to reduced solubility (Silvestre, Espinosa, Heredia, & Pérez, 2020). The treatment of proteins at high temperatures and high moisture contents, unfold their quaternary structures and become part of the melted material. Subsequently, these modified proteins polymerize, cross-link, and reorient themselves, forming longer and fibrous structures. This mechanism is particularly noteworthy in obtaining textured proteins. Most of the extrusion processes reported in Table 3 demonstrate that protein solubility tends to decrease with increasing temperature. This is likely due to the thermal effect inside the extruder, which promotes cross-linking of shorter protein units (Pelembe, Erasmus, & Taylor, 2002).\n\nHowever, some authors report an increase in protein solubility associated with higher barrel temperatures (Silvestre et al., 2020; Zhang et al., 2017). This may be attributed to the breakdown of protein aggregates at high temperatures (Singh, 2021). Comparing the reports of Singh (2021) and Silvestre et al., (2020) where the maximum temperature and moisture content are similar, the screw speed is the determining factor for increased nitrogen solubility. The mechanical effect of this parameter is rarely reported, but it may contribute to the breakdown of protein aggregates, thereby increasing protein solubility. According to Table 3, solubility losses are evident with increasing moisture content (Zhang et al., 2017).\n\nAccording to Zhang et al. (2017), the molecular weight distribution of canola proteins undergoes redistribution after the extrusion process, resulting in a decrease of higher molecular weights and increased concentration in intermediate molecular weights due to the formation of protein conglomerates, similar findings were described by Pelembe et al., (2002). Although a reduction in protein solubility is widely reported, Li and Lee (2000) describe the associations or conglomerates formed under high extrusion conditions (temperature, and moisture content) are weak molecular bindings composed of non-covalent or fragile bonds easily breakable. Protein solubility is an outstanding characteristic in the formulation of instant foods. Nonetheless, this techno-functional attribute can be influenced during the extrusion process (Table 3). Hence, the regulation of processing temperature and moisture content is in the interest of regulating the solubility of these processed flours.\n\n\nBrowning index\n\nBrowning index is the quantification of non-enzymatic browning reaction. This chemical reaction is developed when a reducing sugar interacts with amino groups in protein fractions, resulting in brown pigmentation in products exposed to high-temperature cooking processes such as extrusion (Singh et al., 2007). The quantification of non-enzymatic Maillard browning holds significant importance not only due to its desirable attributes in food manufacturing, resulting in appealing colors and aromas that attract consumer preferences (Ruiz et al., 2008). However, it becomes crucial to control this reaction as it negatively impacts protein digestibility and causes amino acid depletion from a nutritional perspective (Osen, Toelstede, Eisner, & Schweiggert, 2015). The development of brown color during the extrusion process is partly associated with lysine depletion caused by interactions with simple sugars. Lysine is an essential amino acid in protein synthesis (Iwe, Van Zuilichem, Stolp, & Ngoddy, 2004). While the evaluation of non-enzymatic browning relies on color parameters determined through the CIElab protocol, the predictions derived from this index demonstrate remarkable precision in assessing the behavior of Maillard reactions production (Ruiz et al., 2008).\n\nMost of the cereal, legume, or tuber flours encompass readily available free sugars within their matrix, which increase during the extrusion process due to gelatinization and the mechanical impact of the screws within the extruder equipment. Among the sugars documented as susceptible to Maillard reactions, glucose > d-mannose > d-fructose are considered more reactive as fundamental units compared to their dimers. A similar pattern of reactivity is observed for proteins, which exhibit enhanced responsiveness in Maillard reactions when present as basic constituents, namely amino acids (Singh et al., 2007; Yadav et al., 2022). Maillard reactions are intensified when the available protein content in the raw material is higher, this is particularly noticeable in legume flours that are ready for such reactions (Yadav et al., 2022).\n\nTable 4 shows the effect of extrusion conditions on the generation of Maillard reactions. It is apparent that during the extrusion of flours to produce instant foods, intensified Maillard reactions occur with higher process temperatures and screw speeds. This can be attributed to the thermal and mechanical effects, which break down both starches and proteins, resulting in simpler components that promote Maillard reactions. As a result, there is an increase in brown color, quantified by the browning index, correlated with a decrease in luminosity (L) and increasing the red (a) and yellow (b*) colors (Waramboi, Gidley, & Sopade, 2013; Yadav et al., 2022). High screw speed promotes a mechanical shearing force on the melted material as it crosses the barrel in the extruder, developing the damage of polysaccharides (starch), which can contribute production of the Maillard reaction (Iwe et al., 2004; Yadav et al., 2022). On the other hand, it becomes apparent that the browning index decreases as the moisture content increases during the extrusion process. Although moisture content starts the Maillard reaction, a higher level of moisture increases the flow of the melted material, thereby diminishing its residence time and controlling the formation of this reaction. Water acts as a plasticizer in the process reducing the viscosity of the melted material (Camire, 1991).\n\n\nIn vitro starch digestion\n\nEnglyst, Kingman, and Cummings (1992) developed a colorimetric method to describe the three starches fractions in food matrices. Rapidly Digestible Starch (RDS), indicating starch that undergoes digestion within 20 minutes based on hydrolysis rates; Slowly Digestible Starch (SDS), denoting starch that is digested between 20 and 120 minutes; and Resistant Starch (RS), which can be analyzed in three fractions and corresponds to the starch fraction that remains undigested after 120 minutes. This method relies on controlled enzymatic assessment using pancreatin and amyloglucosidase. The evaluation of these starch fractions provides information into the in vivo starch digestion rate (Smrckova et al., 2014; Zhang et al., 2019).\n\nThe evaluation of in vitro starch digestibility is one of the extensively studied process parameters in extruded foods. this method is capable to reveal the changes that occur in the starch fraction of raw and processed materials, as same as their potential nutritional effects. Moreover, the rate at which modified starches can be digested, the proportion of starches available for digestion, and indirectly their relationship with glycemic index (GI) associated to the blood sugar content. This can be critical when RDS levels are elevated as they could negatively affect consumer health (Qi et al., 2021; Smrckova et al., 2014). The available information regarding in vitro starch digestion in extruded instant flours is limited. Based on Table 5, it is possible to assess the impact of modifying the parameter process during extrusion on the starch fractions in the raw materials. In a general, it can be established that the modification of process parameters such as temperature, moisture, and screw speed exhibit an inverse correlation with the quantification of the Rapidly Digestible Starch (RDS) fraction, in comparison to the Slowly Digestible Starch (SDS) and Resistant Starch (RS) fractions. Within the extrusion process, alterations take place in each starch fraction because of gelatinization and shear forces. Consequently, RDS starches tend to convert into dextrin and/or simple sugars, while the SDS and RS fractions exhibit a decline content and may be quantified as RDS due to those modifications.\n\nThe variability in the in vitro starch digestion depends on the severity of the applied extrusion conditions to the raw material. Increasing the extrusion temperature leads to a high RDS content, attributable to modifications in the SDS and RS fractions (Zhang et al., 2023). The extrusion temperature constitutes one of the principal factors responsible for the gelatinization of available starches, causing the loss of their crystalline structure and the fragmentation of amylose and amylopectin chains due to the thermo-mechanical impact of the screw. This facilitates amylase accessibility during digestion (Lai et al., 2022). Conversely, increasing moisture content during the extrusion process reduces the mean residence time of the material inside extruder, resulting in higher quantifications of SDS and RS than RDS content. These results agree with the reduction in the in vitro starch digestion (Zhang et al., 2023). Water acts as a plasticizer capable to modify the gelatinization temperature of starches (Koa et al., 2017). Screw speed represents the factor with higher influence on the mechanical and shear forces during the extrusion process. Heightened screw speed enhances the starch structure modification through mechanical and shear-induced depolymerization, which impact the in vitro starch digestibility (Koa et al., 2017). Table 5 demonstrates that higher screw speeds yield a greater amount of RDS while reducing the proportion of SDS and RS.\n\n\nIn vitro protein digestion\n\nIn vitro protein digestion is an indirect assessment of protein nutritional quality (Akande et al., 2017; Omosebi et al., 2018). Plant proteins generally exhibit lower digestibility compared to the animal-derived proteins due to their amino acid configuration and the presence of antinutritional factors (Atukuri et al., 2019). The digestibility of plant proteins can be enhanced through treatments such as extrusion cooking. The fitted process parameters during extrusion and their intensity effect in the extruder influence protein digestibility. These factors impart different characteristics to the protein fractions found in the raw materials. From a nutritional perspective, the protein digestion extent is related to their level of denaturation, wherein their quaternary structures lose order and transition into lower-order structures, promoting the intestinal proteolysis during digestion (Qi et al., 2021; Ruiz et al., 2008). Based on the studies in Table 6, it can be observed that, in most cases, an increase in temperature correlates with enhanced in vitro protein digestibility. However, some reports showed a reduction in the in vitro protein digestion when the raw materials were exposed to extremely high temperature conditions during the extrusion process (see Table 6).\n\nBetween the most reported reaction involving proteins during the extrusion process encompass Maillard reactions, protein crosslinking, racemization, degradation, and the generation of lysinoalanine (Martínez et al., 2014a; Nahemiah et al., 2017; Omosebi et al., 2018; Ruiz et al., 2022; Silvestre et al., 2021). The quantification of these reactions tends to increase when proteins undergo excessive cooking during extrusion with a reduction in the in vitro protein digestion (Reyes et al., 2003). Furthermore, it can be observed that an increase in moisture content generally leads to a reduction in protein digestibility. This is attributed to the low residence time effect, which hinders structural modifications of the proteins. Conversely, the high screw speed increases protein digestibility due to the mechanical impact exerted by the screws on the raw material. Considering that the extrusion process requires a direct exposure of raw materials to high temperatures and relatively reduced moisture levels, a substantial quantity of complexes resulting from the reaction between reducing sugars and free amino acids is generated, leading to the occurrence of Maillard reactions. Then it becomes an important condition that should be regulated. The Maillard reaction imparts costumer pleasing flavors and colors, but as a uncontrolled reaction imposes limitations on protein availability and digestibility (Alam & Aslam, 2020; Atukuri et al., 2019). In extreme conditions it’s possible the formation of compounds such as acrylamides, which pose health risks and are synthesized when process temperatures overpass the threshold or when exposure time is long, even under low moisture content conditions during the extrusion process (Alam & Aslam, 2020). Furthermore, higher screw speed during extrusion generates high shear forces, thereby boosting protein denaturation within the raw material and enhancing enzymatic hydrolysis during digestion (Atukuri et al., 2019). The excessive thermo-mechanical effect made during the extrusion process can also give rise to protein crosslinking. In addition, the high moisture content during the extrusion process prompts protein swelling and softening, consequently facilitating their denaturation and fractioning due to the thermo-mechanical impact within the extruder (Atukuri et al., 2019).\n\nA few factors affecting in vitro protein digestibility during the extrusion process have been discussed thus far. However, it is notable to acknowledge that in plant-based raw materials, there exist other interfering elements that can also influence protein digestibility (Qi et al., 2021; Ruiz et al., 2008; Silvestre et al., 2020; Pico et al., 2019). The presence of polyphenols in plant matrices can directly interact with digestive enzymes (such as amylase, trypsin, chymotrypsin, lipases), diminishing protein functionality and, consequently, reducing nutrient digestibility (Omosebi et al., 2018). However, the extrusion process can significantly decrease the presence of these interfering compounds in protein digestion and decrease the molecular weight of proteins to facilitate their digestion.\n\n\nTotal phenolic compounds\n\nThe quantification of total phenolic compounds in vegetable matrices include free and bounded fractions and during extrusion process is possible to modify the relationship between the quantity of both fractions. Furthermore, it has been possible to observe a high quantification of total phenolic compounds when the extrusion conditions effectively break down the cell walls of the matrix (Pico, et al., 2020; Pico et al., 2019). The increase of the temperature and screw speed during extrusion promotes the thermo-mechanical movement of the melted material inside the extruder, facilitating the release, extraction, and quantification of bound phenolic compounds, which then become part of the free phenolic fraction. Some phenolic species released include hydroxybenzoic acids, flavonols, catechins, and low molecular weight proanthocyanidins. However, it is important to note that certain studies report an increase in total phenolic compounds, while others report a decrease in this parameter, suggesting that the response may depend on the specific characteristics of the raw material and the employed extrusion conditions (see Table 7).\n\nOn the other hand, it has been observed that increasing the moisture content during the extrusion process tends to reduce the quantification of total phenolic compounds (Akande et al., 2017; Sarawong et al., 2014). Water acts as a plasticizer, preventing the structural breakdown of the cell walls in the matrices and making it difficult to quantify the fractions of bound phenolics (Jan et al., 2017; Sarawong et al., 2014; Wang et al., 2022). However, it has also been reported that an increase in moisture content enhance the preservation of released phenolic fractions, as low moisture contents during the extrusion process can cause structural disruption or alteration of phenolic compounds (Cheng et al., 2020). Nevertheless, it is imperative to optimize the operational parameters of the extrusion process to prevent excessive temperatures and/or shear conditions. Most of the phenolic compounds are sensitive to temperature, which can induce their structural modifications, leading to the loss of their functionality and potential quantification as functional constituents in foods (Wang et al., 2022).\n\n\nAntinutritional Factors (ANFs)\n\nPlant-based raw materials used in food production possess small quantities of a distinctive chemical compound, which are synthesized primarily for the plant’s survival and multiple physiological functions (Elizalde et al., 2009). Among these group of compounds are phytic acid, lectins, protease inhibitors (trypsin and chymotrypsin), saponins, oligosaccharides (raffinose and stachyose), and phenolic compounds (see Table 8). Consuming these components in high concentrations can lead to digestive issues, intoxication, allergies, and, in some cases, damage to intestinal epithelial cells, all these compounds have been grouped as antinutritional factors (ANFs) (Elizalde et al., 2009). ANFs possess high activity and exhibit the capability of conformation complexes with molecules of nutritional importance. Trypsin inhibitors disrupt the enzymatic activity during protein hydrolysis, thereby impeding its absorption (Thirunathan & Manickavasagan, 2019). Lectins, as glycoproteins, readily bind to red blood cell and intestinal epithelial cell walls, causing harm and an adverse effect in the intestinal microbiota (Vidal et al., 2022). Phytic acid hinders mineral absorption at the intestinal level by forming complexes involving phytate-mineral-protein, which exhibit low solubility (Vega et al., 2010). Certain phenolic compounds are regarded as ANFs due to their capacity to bind with proteins, reducing protein solubility and digestibility (Vidal et al., 2022). Additionally, they influence consumption and acceptance due to production of bitter tastes and dark coloration (Akande et al., 2017; Omosebi et al., 2018; Vidal et al., 2022). Oligosaccharides, such as raffinose and stachyose, are high molecular weight sugars commonly found in most legumes (Thirunathan & Manickavasagan, 2019). These sugars are indigestible due to their structure and the presence of α-1-6 galactose linkages that resist intestinal hydrolysis. Legume grains generally contain higher proportions of these sugars, which, according to some studies, display reduced sensitivity to temperature effects (Elizalde et al., 2009). However, their behavior can be influenced by processing conditions. Saponins, classified as glycosides of steroids or triterpenoids, display distinct characteristics such as imparting a bitter taste to quinoa and soybean seeds and their ability to generate foams, which interfere with mineral absorption in the intestine (Elizalde et al., 2009; Gee et al., 1993).\n\nTannins, categorized as phenolic compounds and recognized as ANFs, are present in the structure of various plant-based raw materials. Their consumption interferes with protein and mineral digestion as tannins form complexes with carbonyl groups in dietary proteins or enzymes (Duguma, Forsido, Belachew, & Hensel, 2021; Nikmaram et al., 2017; Qi et al., 2021).\n\nThe ANFs in plant matrices interfere on nutrient absorption. Therefore, thermal treatments like extrusion are employed to mitigate their effects and enhance the overall digestibility of nutrients in pre-cooked cereal and legume flours. Table 8 shows the behavior of oligosaccharides, such as raffinose and stachyose, and the influence of the processing conditions applied during extrusion. It has been observed that increasing the temperature leads to a decrease in raffinose content while increasing stachyose content (Ciudad et al., 2020; Thirunathan & Manickavasagan, 2019). The reduction in raffinose can be attributed to structural modifications, including the cleavage of 1-2 furanosidic bonds, which occur due to exposure to high temperatures. On the other hand, the increase in stachyose at higher temperatures and screw speeds is associated with the thermo-mechanical effect on the food matrix, which facilitates the release of bound oligosaccharides from other macromolecules and the disruption of cell walls, allowing their extraction as soluble components (Cotacallapa et al., 2021). Similarly, Table 8 demonstrates that high temperatures inactivate the content of phytic acid due to its thermosensitivity and the formation of insoluble complexes with other components (Omosebi et al., 2018; Batista et al., 2010). According to Rathod and Annapure (2016), both temperature and moisture content during the extrusion process contribute to the structural modifications of phytates in the raw materials. These changes may involve the cleavage of covalent bonds between amino groups and the hydrolysis of peptide bonds in aspartic acid residues, leading to the inactivation of phytates.\n\nLikewise, the content of saponins exhibits a decrease with increasing temperature and moisture content during the extrusion process (see Table 8). This behavior is attributed to the shear and mechanical energy during extrusion, which alters the original structure of saponins, resulting in smaller chemical fragments and a reduction in the pungent or astringent taste in quinoa-based products (Kowalski et al., 2016). In addition, Table 8 shows that the trypsin inhibitors tend to decrease under intense thermal and mechanical effects during extrusion, leading to molecular degradation (Batista et al., 2010; Ciudad et al., 2020; Konstance et al., 1998). However, the trypsin inhibitors content increases when the extrusion conditions are less aggressive. The only condition that modifies this behavior is an increase in moisture content. While some reports suggest classifying ANFs based on their temperature sensitivity, it has been observed that the most affected by thermal and mechanical effects are protease inhibitors (trypsin and chymotrypsin inhibitors), including phytic acid and lectins (Akande et al., 2017; Balandran et al., 1998; Camire, 1991; Elizalde et al., 2009). Tannins can develop complexes during the extrusion process under strong processing conditions. High temperatures and high screw speeds induce a thermal and shear effect that modifies the structure of these phenolic compounds. However, when the extrusion conditions are milder, achieved by increasing the moisture content, the preservation of tannins in the final product is higher (Ummadi et al., 1995).\n\n\nConclusions\n\nThis review has demonstrated the usefulness of the extrusion cooking process in obtaining instant flours known as pregelatinized flours. The extrusion cooking process conditions such as temperature, moisture content of the material and screw speed, directly affect the behavior of the raw material components during the process. The most used raw materials to obtain pre-cooked flours have in their composition a significant amount of starch, protein, lipids, and others like fiber, micronutrients such as vitamins and minerals, all of them exposed to be modified by the thermo-mechanical conditions. These modifications influence the nutritional and functional characteristics of the final product.\n\nThe final results of the process will be associated with the modification of the operative extruder parameters, finding as results characteristics that become beneficial for the final product such as starch gelatinization, protein denaturation, increased in vitro digestibility, nutrient availability, increased soluble dietary fiber and reduction in the content of anti-nutritional factors (ANF), all of these characteristics are linked to the nutritional and functional properties of the instant flours. However, there is a lack of information that must be investigated regarding the already mentioned process conditions and other new ones, in the same way, to study their effect on the processed raw materials. There are even raw materials whose behavior is unknown, and which may be important in obtaining instant flour by extrusion.\n\nIt should be noted that the moisture content and the temperature of the process influence the solubility of the proteins and the gelatinization of the starch, which is fundamental in the development of instant preparation foods; therefore, it is necessary to modulate the moisture content and the process temperature to keep the solubility of these processed flours under control. On the other hand, the increase in temperature increases the digestibility of protein in vitro and by extrusion cooking it is possible to improve the digestibility of proteins of vegetable origin. In the same way, the phenolic content is modified by increasing in availability of thermostable bonded species due to cell wall broken of raw materials due to thermo-mechanical conditions during extrusion process.\n\nAs it has been established so far, the presence of ANFs in plant matrices represents an important limitation on the levels of nutrient absorption, it is for this reason that heat treatments such as extrusion reduce their effect and enhance the effective digestibility of available nutrients from precooked cereal and legume flours.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAi Y, Cichy KA, Harte JB, et al.: Effects of extrusion cooking on the chemical composition and functional properties of dry common bean powders. Food Chem. 2016; 211: 538–545. 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Publisher Full Text" }	[ { "id": "220802", "date": "15 Nov 2023", "name": "Mercedes Pedrosa", "expertise": [ "Reviewer Expertise Food Technology", "extrusion", "bioactive compounds." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have reviewed the paper entitled effect of extrusion cooking on the chemical and nutritional properties of instant flours: a review.\nIn general, the paper is of interest and it is well written. However, the discussion according to the literature has to be improved.\nI do not agree with the sentence on page 3 that affirms that the parameters of extrusion are less discussed in the literature. There are many papers that compare the effect of temperature, moisture, speed screw, etc. on different compounds. Please revise this fact and add the corresponding references.\nThe main criticism is about the values of the parameters that the authors discuss. The authors include the different values of the parameters chose in the table 1; however, these values do not appear in the other tables nor in the text.\nFor example, in table 2. The first line the authors write that barrel temperature, moisture and screw speed affect the free sugars of wheat flour. However, they only write in the table the moisture values.\nThis problem can be observed in all the tables. Moreover, from table 4 to 8, the values do not appear.\nIn page 8, the authors write that table 5 demonstrates that higher screw speeds yield a greater amount of RDS….. However, in the 5 and in the text they do not write the values of these parameters.\nSomething similar can be observed in the text. For example, the authors write about an increase in the temperature or moisture produce an XXX effect. However, it is impossible to know which the range of this increase is, or they write about milder extrusion conditions, but they do not say which are the values of the different parameter necessaries to establish these mild conditions.\nOn page 5, the authors say that extrusion produces free-sugars. Please add an example of these sugars.\nIn page 9 when they write about ANF, please correct the name oligosaccharides by galacto-oligosaccharides or oligosaccharides of raffinose family...\nTable 6. Many of these papers studied more than an ANF, please include them in the table. The raffinose and stachyose resit the intestinal hydrolysis because mammals do not have the enzyme galactosidase. Please correct the sentence.\nPhytic acid is not very thermosensitibe compounds.\nPage 11, the authors say that extrusion produced an increase of trypsin inhibitors; however there are many papers that establish the opposite trend.\nThe extrusion reduces some ANF and the smallest amount present in the extruded products can exert a positive health effect. Please add some sentence.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\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": "10870", "date": "12 Jan 2024", "name": "Remigio Pismag", "role": "Author Response", "response": "I do not agree with the sentence on page 3 that affirms that the parameters of extrusion are less discussed in the literature. There are many papers that compare the effect of temperature, moisture, speed screw, etc. on different compounds. Please revise this fact and add the corresponding references. R/ Dear reviewer, In this paper, we are discussing about the reduced amount of information about the extrusion process in the develop of instant food products specifically. We carry out a searching focused on these topics: instant powders, instant porridge, instant beverages, and other same products all of them obtained by extrusion. However, we had clarified this issue on page 3 following your recommendation. The main criticism is about the values of the parameters that the authors discuss. The authors include the different values of the parameters chose in the table 1; however, these values do not appear in the other tables nor in the text. R/ We have adjusted the table with the information required. In addition, we had extended our discussion around the table 1. For example, in table 2. The first line the authors write that barrel temperature, moisture and screw speed affect the free sugars of wheat flour. However, they only write in the table the moisture values. R/ We have adjusted the table with the information required. This problem can be observed in all the tables. Moreover, from table 4 to 8, the values do not appear. R/ We have adjusted the tables with the information required. In page 8, the authors write that table 5 demonstrates that higher screw speeds yield a greater amount of RDS….. However, in the 5 and in the text they do not write the values of these parameters. R/ We have adjusted the tables with the information required. Something similar can be observed in the text. For example, the authors write about an increase in the temperature or moisture produce an XXX effect. However, it is impossible to know which the range of this increase is, or they write about milder extrusion conditions, but they do not say which are the values of the different parameter necessaries to establish these mild conditions. R/ We have adjusted all the tables with the information required. We are following your recommendation. On page 5, the authors say that extrusion produces free-sugars. Please add an example of these sugars. R/ We describe the type of sugars generated during extrusion of cereal flours and we added examples of free sugars reported in extrusion of legume flours. In page 9 when they write about ANF, please correct the name oligosaccharides by galacto-oligosaccharides or oligosaccharides of raffinose family... R/ We are following your recommendations, and this issue was fitted. Table 6. Many of these papers studied more than an ANF, please include them in the table. The raffinose and stachyose resit the intestinal hydrolysis because mammals do not have the enzyme galactosidase. Please correct the sentence. R/ We added the ANFs available in the papers and all of them were included. On the other hand, we included the sentence adjusted in our discussion. Phytic acid is not very thermosensitibe compounds. R/ We are following your recommendations, and this issue was fitted. Page 11, the authors say that extrusion produced an increase of trypsin inhibitors; however there are many papers that establish the opposite trend. R/ We have fitted this issue, and we have explained the soft extrusion condition promotes the release and preservation of trypsin inhibitors. The extrusion reduces some ANF and the smallest amount present in the extruded products can exert a positive health effect. Please add some sentence. R/ We are following your recommendations, and we added a sentence." } ] }, { "id": "220796", "date": "08 Feb 2024", "name": "Martha Isabel Sanchez", "expertise": [ "Reviewer Expertise Food Engineering", "Food Science and Technology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 presents in a comprehensive and organized way the theoretical framework concerning the influence of extrusion parameters on the functional and nutritional properties of precooked or instant flours, both as end-products and ingredients. It highlights the pivotal role of process parameters within the extruder, including temperature, screw speed, and raw materials moisture content, among others, and elucidates their correlation with the modifications observed in the structural composition of these materials.\nBelow are some recommendations from the text:\n1. I recommend including a figure of the extrusion process/equipment where parts and some operating conditions are mentioned, considering that throughout the manuscript, reference is made to process/equipment operating conditions.\n2. It is necessary to include an explanation in the footer of each Table indicating the information and the meaning of the symbols (+) and (-).\n3. In the section on Maillard reactions, it is necessary to mention in more detail the type of raw materials involved, what type of colors are desirable or undesirable in the final product, and their effect on consumer preference.\n4. Conclusion \" However, there is a lack of information that must be investigated regarding the already mentioned process conditions and other new ones, in the same way, to study their effect on the processed raw material\" does not take into account information previously mentioned in the text, is broad, and has no basis in the text to be included as a conclusion of the manuscript.\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": [] } ]	1	https://f1000research.com/articles/12-1356
https://f1000research.com/articles/13-57/v1	12 Jan 24	{ "type": "Study Protocol", "title": "Assessment of knowledge, attitude and practices of self-medication among the geriatric population of the Wardha district in central India: A cross-sectional study", "authors": [ "Khushal Ghodkhande", "Dr. Abhay Gaidhane", "Dr. Abhay Gaidhane" ], "abstract": "Background The incidence of self-medication varies greatly for each country. It is a widely used method across advanced and developing nations worldwide. Self-medication is taking pharmaceuticals without seeking physician advice for a diagnosis or treatment. This study will evaluate geriatrics’ knowledge, attitudes, and practices regarding self-medication. The objectives of the current study are to examine the practice of self-medication and assess the knowledge and attitude toward the same in the senior population, as well as the socio-demographic aspects influencing its usage and the reasons for its use. We also aim to test the correlation between demographic issues and self-medication.\n\nMethods A cross-sectional study will be conducted in the rural community area of the Wardha district. A survey will be used to evaluate the entire sample of geriatric age groups to determine the socio-demographic characteristics and associated risk factors for practicing self-medication. A semi-structured, pre-validated questionnaire Self-Medication Scale (SMS) will be used to understand the participants’ practice and attitudes regarding self-medication. The frequency and percentages of descriptive statistics will be determined. The correlation among variables will be discovered using the Chi-Square Test. Tables, graphs, and charts will present the data analysed.\n\nStudy implications The present study will help formulate guidelines on medicine access and reinforce them with strong policy implementation mechanisms. The study will also help provide medical insurance or free consultations for populations in need.", "keywords": [ "geriatric people", "self-medication", "drug", "o-morbidities", "attitude", "knowledge" ], "content": "Introduction\n\nThe World Health Organisation (WHO) states that self-medication involves the use of medicinal products by the consumer to treat self-recognized disorders or symptoms or the intermittent or continued use of a medication prescribed by a physician for chronic or recurring diseases or symptoms.1 To lessen the load on healthcare service facilities, which are sometimes understaffed and difficult to reach in rural and isolated locations, and in order to manage symptoms effectively and quickly, the World Health Organisation encourages self-medication without consulting a doctor.2 But regularly using over-the-counter medicines and self-medication can result in a wide range of serious side effects. The most frequent issue is the chance of adverse drug reactions (ARDs) with the drugs that are being given.3 Self-medication is broadly used among urban and rural people worldwide and growing nations like India because it offers individuals a cheap alternative and allows for dispensing various pharmaceuticals without a prescription over the counter.4 Several elements, including socioeconomic position, way of life, easy access to medications, and the larger accessibility of medications in developing nations, might cause self-medication.5 As far as a person’s health is concerned, it is an essential health problem. Self-medication must be supported by valid medical knowledge to prevent unreasonable drug abuse; it can ultimately result in the waste of medicinal products and elevated pathogen resistance, leading to major health risks such as extended discomfort, adverse drug reaction, and medication dependency.6\n\nThe incidence of self-medication varies greatly for each country. Prevalence rates in the evaluation of 140 study, which included 189,278 people of all ages, ranged from 0.1% to 100%. Various studies examined the high rate of self-medication, such as sixty-five percent in Finland, seventy-eight percent in Yemen, Saudi Arabia (S.A.), also Uzbekistan, 88-92% in Thailand,7 and 60% in central India.8\n\nThe worldwide elderly population is increasing, even in developing countries. By 2025, the number of older adults in the globe will increase to 1.2 billion, up from 550 million in 1996.9 In India, the percentage of older individuals is predicted to climb from 8% in 2015 to 19% by 2050, with elderly individuals contributing to one-third of the overall population by the last decade of the century. Multi-morbidity becomes increasingly prevalent as people age and is more prevalent in older adults. According to reports, multi-morbidity affects older persons generally in a range between 24%to 83%.10 Due to co-morbidities, the aging population is more likely to self-medicate. They are not conscious of the dangers of self-medication.9 Elder people are more likely to experience adverse drug reactions (ADRs) due to physiologic variations in metabolism caused by ageing.11\n\nMost of the research was conducted on people from the general public, professionals, hospital visitors, or medical students. There are few investigations of elderly self-medication conducted in the community, especially from central India. The current study’s findings will help the government determine the scope of elderly self-medication and devise measures for boosting public awareness of the risks associated with self-medication overuse. Hence, this research project aims to assess people’s understanding, attitudes, and use of self-medication in the rural community area of Wardha District in the Indian state of Maharashtra.\n\nThis study evaluates knowledge, attitude and practice of self-medication among the geriatric population in the Wardha district’s rural community region (Table 1).\n\n\n\n• Name\n\n• Gender\n\n• Age\n\n• Highest level of education\n\n• Marital status\n\n• Chronic illness\n\n\n\n• Percentage of geriatric people aware of self-medication\n\n\n\n• Part of self-care\n\n• No need for training\n\n• Recommend self-medication to others\n\n• Easy access\n\n• Availability and belief in safety\n\n\n\n• Duration of last use\n\n• Frequency of self-medication use\n\n• Name of drug purchase\n\n• Indication of drug use\n\n• Source of information\n\n• Any side effects\n\n\n\n1) To assess the socio-demographic features influencing self-medication usage and the rationale for its use.\n\n2) To assess elderly patients’ knowledge, attitudes, and self-medication practices.\n\n3) To evaluate the relationship between demographic factors and self-medication.\n\n\nMethods\n\nThe current study will be descriptive and cross-sectional.\n\nThe research will be conducted in the School of Epidemiology and Public Health field practice area, DMIHER, Wardha.\n\nThe study will be carried out between August 2023 and May 2024.\n\nThe review of the literature started in August 2023 and, currently, the study proposal has been submitted for the IEC approval. As soon as the ethical approval is received, we will start the study.\n\nGeriatric individuals aged 60 and above who live in Wardha district’s rural community areas.\n\nInclusion criteria:\n\n1) The study will comprise all people aged 60 years and above.\n\n2) Those who will be able to give answers to the questions asked by the interviewer.\n\n3) People who will be willing to participate in the study and give consent.\n\n4) Geriatric people who understand at least one Marathi, English, or Hindi language.\n\nExclusion criteria:\n\n1) People below the age of 60 years will not be a part of this study.\n\n2) The study will not include those who are hesitant or unwilling to participate.\n\nThe prior research study, which was utilized as a mother research study, found that the majority had inadequate knowledge (88%) regarding consuming self-medication, the estimated proportion, i.e., P value is 0.88, and the following articles were used to estimate the number of participants for this investigation.12\n\nWith the help of following formula, the sample size was calculated:\n\nEstimated proportion (p): 0.88\n\nEstimated error (d): 0.05\n\nAlpha (α): 0.05\n\nSample size: 163\n\nAs the outcome of 163 sample size elderly peoples will be selected for the data collection.\n\nAs a result, a sample size of 163 older people will be used to collect data and examine self-medication knowledge in senior adults living in rural locations.\n\nThe technique known as convenience sampling will be used to interview people.\n\nThe research will examine variables related with socio-demographic data, knowledge, attitude, and practice.\n\nDemographic characteristics of the sample such as age in year, sex, highest level of education, occupation, chronic illness, and health insurance.\n\nThe study using the Self-medication scale (SMS)13 and includes a semi-structured interview survey, employing inquiries in the local languages (Marathi/Hindi) and English to evaluate knowledge, attitude, and practice of self-medication among the geriatric age group.\n\nThere are three sections in the structured questionnaire.\n\nSection A: Consists of 8 questions related to knowledge of self-medication.\n\nSection B: Consists of a five-point Likert scale scoring the attitude of the sample from 1 to 5 from strongly disagrees to strongly agrees respectively strongly.\n\nSection C: Comprises 13 questions that represent the practise of self-medication.\n\nEvery home in the targeted zone will be visited until a sufficient sample size is reached. The purpose of the interview will be explained to the participants in their local language so they can understand the objectives of study properly and can decide if they are willing to participate in the study. House-to-house visits will be done and information will be obtained using interviews. The planned data recording technique will be used for each participant separately, in which the investigator asks the questions, provides the options as specified in the proforma, and ticks the responses provided by the participants.\n\nRecall bias may influence results. The current study included a six-month memory span, which could have resulted in recall bias. We’ll reduce this prejudice by employing a well-made, straightforward, and basic questionnaire.\n\nThe study will commence after receiving approval from the DMIHER (DU) Institutional Ethical Committee. Before our research, we will obtain written consent from the participants and explain the purpose of the survey to them. During the interview, using a questionnaire, we’ll ensure the interviewee is at ease and enjoys privacy during the conversation.\n\nThe data will be analysed using R Statistical software, a statistics program, after being imported into Microsoft Excel. The frequency and percentages of descriptive statistics will be determined. The correlation among variables will be discovered using the Chi-Square Test. Tables, graphs, and charts will present the data analysed.\n\nStudy status\n\nThe study is not yet started. The proposal of this study has been submitted for ethical approval to the IEC department of DMIHER (DU) and is under process.\n\nExpected outcomes/Results\n\nThe knowledge, attitude, and self-medication practices of elderly individuals will be assessed in this study. Also, examine how socio-demographic factors impact the use of self-medication and the reasons for its use and test the association between demographic characteristics and self-medication.\n\nTo raise awareness among the general public, this study will assist in the conduct of numerous studies, such as public awareness campaigns, education, and group discussions.\n\n\nDiscussion\n\nA cross-sectional research was done in rural Sahaswan, Northern India, with the goals of studying the association between research factors and self-medication, identifying the kinds of medications used for self-medication, and identifying typical illnesses for self-medication, Indicated that men, those above 15 years individuals with a lesser education or illiteracy, and those with limited financial resources were more likely to self-medicate.2\n\nAl Ghofaili and Fatimah (2021) did a cross-sectional study on self-medication habits in Qassim Province, Saudi Arabia. The goals of this study are to investigate OTC medical practises in the general community, report on their quantity, most commonly purchased medications and reasons for purchase, and raise public awareness about self-medication. In the study region, there was a significant prevalence of non-prescribed drugs purchased from community pharmacies, according to the findings of this study.3\n\nCross-sectional descriptive research was conducted in Kermanshah, Iran, by Faranak Jafari, Alireza Khatony, and Elham Rahmani (2015) to look into older people’s use of self-medication and the factors that may be associated with it. The study included 272 participants. According to this survey, 83% of people self-medicate.11\n\nAlmalki et al. (2022) performed a cross-sectional study in western Saudi Arabia to assess knowledge, attitude, and practise of self-medication, determine its prevalence, and identify variables and sources of information impacting such practise. This survey included 647 respondents from three main cities: Jeddah, Makkah, and Taif. According to this study, around 67.7% of individuals use self-medication.7\n\nVarun Kumar et al. (2015) did a cross-sectional study on the ‘prevalence and trend of self-medication as practices in an urban region of Delhi, India’. This study aims to determine the prevalence and pattern of self-medication in metropolitan areas. According to this survey, 92.8% of individuals used self-medication, most of which were allopathic medications.14\n\nA cross-sectional study on self-medication in Saudi Arabia was carried out in 2019 by Yahya Ibrahim Mahzari et al. It intends to assess the prevalence and types of self-medication in the Saudi Arabian Kingdom. According to this survey, the frequency of self-medicating in KSA is dangerously high, with analgesics being the most commonly utilized drug with a prescription.15\n\nThe present study will help formulate guidelines on medicine access and reinforce them with strong mechanisms to implement the policy. This could aid in preventing the widely accepted detrimental effects of self-medication among older adults. Also, it helps to provide medical insurance and free consultations for people in need.\n\nData will be collected using interviews, which may affect the accuracy of the results. The study may be affected by selection bias, measurement bias, or confounding factors, distorting the impact and reducing their validity and generalizability. This study does not consider long-term changes or trends in knowledge, attitude, or self-medication practice because it only provides a one-time measurement of the variables. The results of the study may be affected by seasonal or cyclical variations.\n\nThis study protocol will be published in an indexed journal and will be presented in seminars and conferences.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWorld Health Organization: Guidelines for the regulatory assessment of medicinal products for use in self-medication. World Health Organization; 2000 [cited 2022 Oct 22]. Report No.: WHO/EDM/QSM/00.1. Reference Source\n\nAhmad A, Patel I, Mohanta G, et al.: Evaluation of Self Medication Practices in Rural Area of Town Sahaswan at Northern India. Ann. Med. Health Sci. Res. 2014; 4(Suppl 2): S73–S78. PubMed Abstract \| Publisher Full Text\n\nAlGhofaili F: Patterns of self-medication in Qassim Province, Saudi Arabia: A cross-sectional study. Ann. Med. Surg. 2021 Mar 17; 64: 102207. Publisher Full Text\n\nHussain S, Malik F, Hameed A, et al.: Exploring health seeking behavior, medicine use and self medication in urban and rural Pakistan. South Med. Rev. 2010 Oct 1; 3: 32–34.\n\nWorld Health Organization: The Role of the pharmacist in self-care and self-medication: report of the 4th WHO Consultative Group on the Role of the Pharmacist, The Hague, The Netherlands, 26-28 August 1998. World Health Organization; 1998 [cited 2022 Nov 3]. Report No.: WHO/DAP/98.13. Reference Source\n\nKasulkar AA, Gupta M: Self medication practices among medical students of a private institute. Indian J. Pharm. Sci. 2015; 77(2): 178–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlmalki ME, Almuqati FS, Alotaibi MO, et al.: A Cross-Sectional Study of the Knowledge, Attitude, and Practice of Self-Medication Among the General Population in the Western Region of Saudi Arabia. Cureus. 2022 Oct 5 [cited 2022 Nov 4]; 14(10). Publisher Full Text Reference Source\n\nRathod P, Sharma S, Ukey U, et al.: Prevalence, Pattern, and Reasons for Self-Medication: A Community-Based Cross-Sectional Study From Central India. Cureus. 2023 Jan 18 [cited 2023 Jul 28]; 15(1). Publisher Full Text Reference Source\n\nParmar Z, Malhotra S, Patel V: Prevalence and pattern of self-medication in elderly individuals. Int. J. Basic Clin. Pharmacol. 2015 Jan 1; 1095–1099. Publisher Full Text\n\nKshatri JS, Palo SK, Bhoi T, et al.: Prevalence and Patterns of Multimorbidity Among Rural Elderly: Findings of the AHSETS Study. Front. Public Health. 2020 [cited 2023 Aug 1]; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJafari F, Khatony A, Rahmani E: Prevalence of Self-Medication Among the Elderly in Kermanshah-Iran. Global J. Health Sci. 2015 Mar; 7(2): 360–365. PubMed Abstract \| Publisher Full Text\n\nBabu PS, Balu V, Maheswari BU, et al.: A Comparative Study to Assess the Prevalence, Knowledge of Impact, and Practice of Self-Medication Among Adults in Urban and Rural Communities in Bangalore. Cureus. 2023 May 29 [cited 2023 Jul 28]; 15(5). Reference Source\n\nAlduraibi RK, Altowayan WM: A cross-sectional survey: knowledge, attitudes, and practices of self-medication in medical and pharmacy students. BMC Health Serv. Res. 2022 Mar 17; 22(1): 352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar V, Mangal A, Yadav G, et al.: Prevalence and pattern of self-medication practices in an urban area of Delhi, India. Med. J. Dr. Patil Self-Medicat Curr. Chall Self-Medicat Curr. Chall. 2015 Jan 1; 8(1): 16. Publisher Full Text\n\nMahzari YI: Self-Medication in Saudi Arabia-A Cross Sectional Study.2019; 7." }	[ { "id": "264630", "date": "29 May 2024", "name": "Mainak Bardhan", "expertise": [ "Reviewer Expertise Neurology", "Genetics", "Internal medicine", "Public Health", "Infectious diseases", "Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript does not provide details on the validity and reliability of the Self-Medication Scale (SMS) in the context of the geriatric population in India. It would be helpful to include information on whether the SMS has been validated for use in this population and setting. The authors mention using convenience sampling, which may introduce selection bias and limit the generalizability of the findings. If feasible, a more robust sampling method, such as random sampling, could be considered. The authors could consider including a qualitative component (e.g., in-depth interviews or focus group discussions) to explore the reasons, beliefs, and perceptions behind self-medication practices in the geriatric population. The manuscript does not mention strategies for handling missing data or dealing with potential confounding factors in the analysis. It would be beneficial to include a plan for addressing these issues. The limitations section could be expanded to include potential limitations specific to the study design, such as the cross-sectional nature of the study, which may not capture changes over time.\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/13-57
https://f1000research.com/articles/12-419/v1	18 Apr 23	{ "type": "Brief Report", "title": "The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes", "authors": [ "Endang Bachtiar", "Boy M Bachtiar", "Ardiana Kusumaningrum", "Hari Sunarto", "Yuniarti Soeroso", "Benso Sulijaya", "Efa Apriyanti", "Citra Fragrantia Theodorea", "Irandi Putra Pratomo", "Yudhistira Yudhistira", "Defi Efendi", "Widya Lestari", "Boy M Bachtiar", "Ardiana Kusumaningrum", "Hari Sunarto", "Yuniarti Soeroso", "Benso Sulijaya", "Efa Apriyanti", "Citra Fragrantia Theodorea", "Irandi Putra Pratomo", "Yudhistira Yudhistira", "Defi Efendi", "Widya Lestari" ], "abstract": "Background: The available evidence suggests that inflammatory responses, in both systemic and oral tissue, contribute to the pathology of COVID-19 disease. Hence, studies of inflammation biomarkers in oral fluids, such as saliva, might be useful to better specify COVID-19 features. Methods: In the current study, we performed quantitative real-time PCR to measure salivary levels of C-reactive protein (CRP) and interleukin-6 (IL-6) in saliva obtained from patients diagnosed with mild COVID-19, in a diabetic group (DG; n = 10) and a non-diabetic group (NDG; n = 13). All participants were diagnosed with periodontitis, while six participants with periodontitis but not diagnosed with COVID-19 were included as controls. Results: We found increases in salivary total protein levels in both the DG and NDG compared to control patients. In both groups, salivary CRP and IL-6 levels were comparable. Additionally, the levels of salivary CRP were significantly correlated with total proteins, in which a strong and moderate positive correlation was found between DG and NDG, respectively. A linear positive correlation was also noted in the relationship between salivary IL-6 level and total proteins, but the correlation was not significant. Interestingly, the association between salivary CRP and IL-6 levels was positive. However, a moderately significant correlation was only found in COVID-19 patients with diabetes, through which the association was validated by a receiver operating curve. Conclusions: These finding suggest that salivary CRP and IL-6 are particularly relevant as potential non-invasive biomarker for predicting diabetes risk in mild cases of COVID-19 accompanied with periodontitis.", "keywords": [ "Diabetes", "COVID-19", "periodontitis", "C-reactive protein", "interleukin-6" ], "content": "Introduction\n\nThere is a growing interest in measuring inflammatory biomarkers in saliva, as sampling this oral fluid is less invasive than blood.1–3 Despite the promising results of saliva studies, the findings of salivary measures of inflammation have been inconsistent.\n\nIn this study, we used saliva derived from patients with COVID-19 to evaluate the association of selected inflammatory markers, interleukin-6 (IL-6), and C-reactive protein (CRP) in COVID-19 patients. The extent to which salivary IL-6 and CRP levels are associated with diabetes or non-diabetes events in this population still needs to be explored.\n\n\nMethods\n\nThis study was part of a project on COVID-19 and its association with the oral ecosystem. Thus, the current report was based on data from our previous study, in which data from 23 people with mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were requested from Rumah Sakit Universitas Indonesia (RSUI), a university hospital, between June and August 2021.4 In addition to COVID-19, all patients were diagnosed with periodontitis. Patients were further divided into two groups: with (n = 10) and without (n = 13) diabetes. Six non-diabetic participants without COVID-19 and no periodontitis were included as controls. Sampling methods and information regarding the inclusion and exclusion criteria of patients, how consent was obtained from participants, and obtaining medical reports were performed in accordance with the guidelines provided by the ethics committee of RSUI (protocol number: 2021/04/052). All of the participants signed the written informed consent form. Additionally, this study was performed in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidleines.5 Information regarding clinical status, such as age, sex, and chronic medical history of comorbidities, was obtained from the medical reports of mildly symptomatic patients with COVID-19 (not shown). However, our focus was on patients with COVID-19, with and without diabetes. Thus, only diabetic status, as reported in the medical records, was included as a comorbidity variable in the data analysis.\n\nThe oral specimen was obtained by spitting unstimulated whole saliva into a sterile Falcon tube, placed on ice, and transferred to the laboratory for subsequent processing.4\n\nThe total protein level in saliva was determined using the Bradford assay method, as reported elsewhere.6 The concentration of CRP in saliva samples was determined using an enzyme-linked immunosorbent assay kit (Elabscience Biotechnology Inc., Wuhan, China) according to the manufacturer’s instructions. For IL-6, we used quantitative real time-polymerase chain reaction (qPCR; ABI StepOnePlus Real-Time PCR system), using SYBR Green I for gene expression analysis, to amplify cDNA that had been converted from salivary RNA using the method we used for the gingival crevicular sample.7 The PCR was run in triplicate using primers (IL-6 and housekeeping gene/GAPDH) and the PCR program as reported previously.7 The 2-ΔΔCT method was used to analyze the relative expression of mRNA.8\n\nThere was a strong correlation between mRNA and protein expression levels.9 In this study, owing to the observed variations in saliva concentrations, both CRP (protein) and IL-6 (transcription level) were adjusted for total salivary protein. Statistical analyses were performed using GraphPad Prism 9.4 software. Data are presented as mean ± SD or median. The nonparametric Kruskal Wallis test was used to compare the diabetic group (DG), the non-diabetic group (NDG), and the control group. The Mann-Whitney test was used to compare the DG and NDG data. The statistical significance level was set at p < 0.05. Spearman’s correlation coefficient (r) with two-tailed p-values was calculated, and linear regression was used to generate the line of best fit with 95% confidence intervals. Receiver operating characteristic (ROC) curve analysis was also performed to evaluate this association.\n\n\nResults\n\nAs shown in Figure 1A, compared to the control group, the mean values of the total protein concentration in the saliva of the DG and the NDG were significantly increased (p < 0.05). However, the concentrations were comparable in both groups.\n\nSignificant differences in salivary total proteins concentration were observed in both DG and NDG groups compared to control (A). However, in each group tested, the salivary level of either CRP (B) or IL-6 (C) was comparable, as assessed by Bredford protein assay and quantification of mRNA expression, respectively. *p < 0.05. CRP = C-reactive protein, Il-6 = Interleukin-6. DG and NDG are diabetes and non-diabetes group, respectively.\n\nWe further noted that in the DG, salivary CRP was detectable in 9/10 (90%) patients, while IL-6 was only measured in 8/10 (80%) patients. In the NDG, the concentration of CRP and the transcription level of IL-6 in saliva samples were detected in all (100%) and 12/13 (92.31%), respectively. The median values of CRP and IL-6 in saliva are shown in Figure 1B,C. The protein concentrations of CRP and the transcription level of IL-6 mRNA detected in saliva among the patient groups were comparable (p > 0.05).\n\nNext, we compared the association between total salivary proteins and each of the two inflammation markers detected in the saliva. As shown in Figure 2A–D, IL-6 and CRP levels were identical in their association with total salivary proteins. Spearman’s correlation showed a strongly significant positive correlation between salivary CRP and total salivary proteins in the DG (r = 0.80, p = 0.01), while a moderately significant correlation was observed in the NDG (r = 0.60, p = 0.01). A similar trend was observed for the correlation between salivary IL-6 and total salivary protein levels. In the DG (r = 0.59, p = 0.13) and the NDG (r = 0.41, p = 0.17), the correlation was positive, but not significant. Finally, we compared the correlation between the two inflammation markers in each group. In both groups, the correlation was positive, but a significant association was only observed in the DG. The correlation coefficients were r = 0.52, p = 0.03, and r = 0.38, p = 0.21 in the DG and NDG, respectively (Figure 3 A,B). Hence, since a significant relationship was only shown in the DG, we made inferences about association accuracy by performing ROC analysis. The area under the curve of the CRP/IL-6 association was 0.983 (95% confidence interval [CI]: 0.9451–1, p < 0.0001; Figure 3C).\n\nSpearman correlation analysis indicates a statistically significant positive correlation between salivary levels of CRP and total protein, A strong and moderate correlations was observed in DG (A) and NDG (B), respectively. The positive relationship between IL-6 salivary levels and total salivary proteins were also found in DG (C) and NDG (D). However, the correlations were not significant. CRP = C-reactive protein, IL-6 = Interleukin-6. DG and NDG. Spearman correlation coefficient (r) and exact p-value are shown.\n\nThese data indicate that in DG (A), the correlation was significantly moderate, while a weak not significant correlation was noted in NDG (B). Receiver operating characteristic (ROC) was used to illustrate the plot and best cut-off of significant relationship between salivary levels of CRP and Il-6 in DG group (C). CRP = C-reactive protein, IL-6 = Interleukin-6. DG and NDG. Spearman correlation coefficient (r) and exact p-value are displayed.\n\n\nDiscussion\n\nThe most obvious finding of this study is that salivary CRP and IL-6 are potential markers of systemic inflammation in mild cases of COVID-19 with and without diabetes. With regard to CRP, our results were not consistent with a recent report, which showed that an increased concentration of salivary CRP was only found in severe cases of COVID-19.10 We reasoned that this inconsistency could be related to the eligibility criteria in recruiting participants. In the cited report, the authors excluded participants with periodontitis, whereas in our study, periodontitis was an inclusion criterion.7 However, for IL-6, the results of this study are in line with a previous report, where the concentration of salivary IL-6 was elevated in periodontitis patients with and without diabetes.11\n\nAs shown in the literature, the relationship between COVID-19 and diabetes mellitus is complicated and bidirectional.12 In addition to diabetes, all patients with COVID-19 included in this study also had periodontitis, a common comorbidity observed in patients with COVID-19.13 Therefore, it is necessary to consider the influence of confounding variables on the oral ecology. Therefore, to optimize the utility of salivary CRP and Il-6 as inflammation markers in separating patients with COVID-19 with and without diabetes, we used total salivary protein concentration to normalize the tested analyte levels (CRP and IL-6) in saliva.14\n\nCompared to the control group (non-SARS-CoV-2 infected participants), we observed a highly significant increase in the salivary total protein concentration in both patient groups tested. These results suggest that elevated total salivary protein concentration occurred in all patients with COVID-19. Therefore, we assumed that the rising total salivary protein levels in our patients with COVID-19 might include increasing levels of both salivary inflammation markers (CRP and IL-6).\n\nBased on the design mentioned above, we noticed the association between salivary CRP/IL-6 and total salivary proteins was consistent, but the strength of the correlation varied. In both patient groups, a significantly positive association was found only for CRP. A strong and moderate correlation was observed in the DG and the NDG. Further, moderate, and weak positive correlations between IL-6 and total protein were found in the DG and the NDG, respectively. Overall, these results indicates that the highest correlation (0.80) was found between salivary CRP and total protein of hospitalized COVID-19 patients with diabetes, while the non-diabetic patients had a lower correlation of 0.60. Since CRP in oral fluids reflects its level in circulation,15 our result might indicate that in COVID-19 patients with diabetes, the proportion of salivary CRP was greater when serum levels were raised.\n\nHowever, unlike salivary CRP, IL-6 levels in saliva have been reported to not correlate with those in plasma or serum.16,17 Thus, the elevated levels observed in this study may indicate inflammation induced by oral lesions (in our case, periodontitis).18 It seems likely that IL-6 is a key stimulator of the hepatic synthesis of CRP.19 In contrast, the salivary levels of CRP and IL-6 appeared to be independently regulated by diabetes status. As indicated in this study, a positive correlation between the two cytokines was observed in patients with and without diabetes.\n\nThis result was also corroborated by the ROC curve, and we were able to attain nearly 100% sensitivity and specificity. We believe that defining the relationship between CRP and IL-6 in patients with COVID-19 is unique to separate diabetes and non-diabetes in patients with COVID-19 suffering from periodontitis as a comorbidity.\n\nTaken together, as mentioned above, the salivary CRP level reflects the CRP level in circulation, and it is likely that our data may reflect a positive correlation between IL-6 and CRP levels in the serum. Therefore, the results of the current study suggest that salivary levels of IL-6 might be overshadowed by local inflammation (periodontitis) instead of inflammation-related diabetes. Indeed, our findings provide additional information regarding the unequivocal results with either the presence20,21 or the absence22 of a relationship between the status of periodontal disease and salivary CRP.\n\nConsidering that CRP is a key cytokine that plays an important role in the progression of various inflammatory diseases,23 we assumed that both periodontitis and diabetes are related to the pathophysiology of COVID-19. This association could be linked to the existence of two unique protein structures in CRP.24 The first isoform is a CRP monomer that is activated by local signals of inflammation and tissue injury, and the other is the pentameric isoform synthesized by the liver.25,26 As our participants were patients with COVID-19 with and without diabetes accompanied by periodontitis, we assumed that the salivary CRP detected in this study is the monomeric isoform that might have been activated by periodontal inflammation. Its presence in saliva may have been independently regulated by the diabetes status of our patients. Further studies are required to clarify this assumption.\n\nAlternative explanations for our results warrant further discussion. First, SARS-CoV-2 infection is closely related to depressive disorder,27,28 and the positive correlation between salivary CRP and IL-6 levels observed in this study may indicate a characteristic inflammatory response in depressive disorder that is commonly found in COVID-19 patients.29 Second, since diabetes is among the common comorbidities noted in patients diagnosed with COVID-19,30 the similar effect of the depression-associated inflammatory response (salivary CRP and IL-6) found in the two tested groups (DG and NDG) might be a consequence of the impact of depression on SARS-CoV-2 infection rather than diabetes itself. Thus, it is possible that the subtle elevation of the selected salivary biomarkers observed in the DG and the NDG (shown in Figure 2B, C) was no longer statistically significant. Further studies are required to confirm this possibility.\n\nThis observational study has some limitations. First, the included participants were restricted; therefore, further studies including more participants are required. Second, the changes in salivary proteins during the time saliva was collected was not part of this study design, so future studies are necessary to assess dynamic salivary changes. Lastly, we cannot rule out the involvement of salivary flow rate in determining salivary CRP and IL-6 levels. Therefore, it is recommended to include this confounding variable as a patient-specific factor in future investigations and to confirm the results of this study.\n\n\nConclusions\n\nThe current study has shown that the low correlation between salivary levels of CRP and IL-6 seems to be a valuable oral fluid biomarker to discriminate between diabetic and non-diabetic status in patients with COVID-19. Additionally, the finding in saliva analytes (CRP and IL-6) coincides with oral dysbiosis, which is represented by periodontitis status.4 This suggests that in cases of mild SARS-CoV-2 infection, oral dysbiosis in individuals with diabetes might increase the probability of diabetes-associated inflammation, which is characterized by increased levels of salivary CRP and IL-6. Prospective studies are needed to determine the feasibility of salivary CRP/IL-6 as a prognostic measure for the risk of systemic and oral inflammation.", "appendix": "Data availability\n\nOpen Science Framework: The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes, https://doi.org/10.17605/OSF.IO/PXD6R. 31\n\nThis project contains the followign underlying data:\n\n- Excel data TP-CRP-IL-6 F1000 Jan 23.xlsx\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\nAcknowledgements\n\nAll the authors would like to acknowledge all the study participants, the Director of Universitas Indonesia Hospital (RSUI), and the Ethical clearance committee for providing permission and ethical clearance to conduct this research project.\n\n\nReferences\n\nEngeland CG, Bosch JA, Rohleder N: Salivary Biomarkers in Psychoneuroimmunology. Curr. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBachtiar EW: The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes.2023, January 31. Publisher Full Text" }	[ { "id": "208201", "date": "22 Sep 2023", "name": "Joel Schwartz", "expertise": [ "Reviewer Expertise Oral biology", "molecular biology", "biochemistry", "pathology", "mucosal immunology" ], "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\nRationale for the study is pro-inflammatory markers such as IL-6 and CRP derived from the saliva are identifiable and associated with the presence of COVID-19 and/or possible periodontal disease.. Results show correlations to be present in the data rejecting the null hypothesis with a high level of probability.\n\nHowever, the problem with these results are these.\n(relatively low number) 23 Samples (10 with diabetes mellitus (DM) and 13 with no (DM). We need information about the phase of disease pathogenesis and determine possible inflammatory behavior. regarding DM (e.g., Type 1 /Type 2, intermediate, active, control, uncontrolled, medication used? etc.) Periodontal disease (e.g., mild-moderate, sever, generalized, localized? How was PDS assessed: periodontal bone probing, indexing (plaque, gingival, bleeding, CAL/ALOSS), radiology? all or PSR shortcut?)\n\nSaliva is a great source of inflammatory makers. Some of these are transudate or exudate products from the serum. Therefore, coincident levels between the saliva and serum can vary. The should clearly state in the discussion is SARS-CoV-2 is an oral virus. Its tropism is targeting oral epithelial cells populations based on the expression pattern of AVCE/TMPRSS2 which include keratinocytes, salivary ductal cells of various types which includes myoepithelial cells, tuft cells, gustatory and supporting cells of the tongue. Using saliva samples without gingival crevicular fluid as alluded to in the discussion increases difficulty to assess contribution by gingival tissues compared to other tissues. In addition, there are citations available that indicate there is a hierarchy of ACE2/TMPRSS2 expression with the highest levels in tongue and salivary gland coinciding with clinical signs such as loss of taste and dry mouth. These types of signs and symptoms need to be described to provide some degree of characterization of inflammation related to tissue site..\nAdditionally, there is lacking a immunologic description of the functional activity of IL-6. This cytokine which is also described as a adipokine is suppressible by adiponectin derived from the saliva. IL-6 has a variety of function requiring a mention in the discussion. For example, regulation of loss of tolerance and B cell differentiation while also effecting innate responses particularly in the periodontal tissues. Above I mentioned a possible association with adiponectin which acts as a protector against DM while increasing glucose uptake. There is another association overlooked in the discussion periodontal pathogens release metallo-peptidases and one of these mimics dipeptidyl-peptidase IV (DDPIV) which is diabetogenic. These types of relationship validate previous reports that periodontal disease is linked to DM.\n\nI suggest providing additional information in methods section about the health conditions of the subjects.\nTry to provide additional markers from the serum of saliva identifying SARS-CoV_2 presence in the oral cavity.\nUpdate and edit the discussion to comply with knowledge of SARS-CoV_2 and COVID-19 presentation effecting oral tissues.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10384", "date": "25 Oct 2023", "name": "Endang Bachtiar", "role": "Author Response", "response": "Dear Reviewer, Thank you for giving us the opportunity to submit a revised version of the manuscript “The utility of salivary CRP and IL-6 as a non-invasive measurement evaluated in patients with COVID-19 with and without diabetes”. We appreciate the time and effort that the Reviewer dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated most of the suggestions made by the reviewer Those changes are highlighted in green within the manuscript. Please see below, for a point by point response to the reviewer’s comments and concerns. All page numbers refer to the revised manuscript with tracked changes. Reviewer’s Comments (RC) Rationale for the study is pro-inflammatory markers such as IL-6 and CRP derived from the saliva are identifiable and associated with the presence of COVID-19 and/or possible periodontal disease.. Results show correlations to be present in the data rejecting the null hypothesis with a high level of probability. However, the problem with these results are these. (relatively low number) 23 Samples (10 with diabetes mellitus (DM) and 13 without no (DM). Author’s Response (AR): Yes, we agree. As we mentioned in the limitation of our study; page 9 line 33. “This observational study has some limitations. First, the included participants were restricted. Additionally, as mentioned in Methods (under the subtitle: Subjects, saliva sampling, and in vitro methods). ……….we performed the study in accordance with the guidelines provided by the ethics committee of RSUI, including “how consent was obtained from participants” (Page 4, line 10). Thus, only participants gave her consent was included. RC: We need information about the phase of disease pathogenesis and determine possible inflammatory behaviour. regarding DM (e.g., Type 1 /Type 2, intermediate, active, control, uncontrolled, medication used? etc.) Periodontal disease (e.g., mild-moderate, sever, generalized, localized? How was PDS assessed: periodontal bone probing, indexing (plaque, gingival, bleeding, CAL/ALOSS), radiology? all or PSR shortcut?) AR: Thank you for pointing this out. As can be seen in Methods section, all information about systemic condition of the patient, we obtained from the medical records. In the revised version, we have added some important information regarding “clinical status”, such as age, sex, and chronic medical history of comorbidities, that was obtained from the medical reports (restricted) of mildly symptomatic patients with COVID-19 (not shown). Only subjects who had no respiratory symptom for more than 2 weeks were included in this study, However, our focus was on patients with COVID-19, with and without diabetes. Thus, only diabetic status (type 2 diabetes), as reported in the medical records, was included as a comorbidity variable in the data analysis, while all COVID-19 patients were diagnosed with periodontitis (moderate to severe) according to the criteria described by the American Academic of Periodontology Classification of Periodontal Disease6. In this study, it would not possible to take dental radiology. Therefore, in revised manuscript, we added “without dental radiographic assessment following “patient diagnosed with periodontitis…….,” mentioned above (Page 4, Line 16-18). RC: Saliva is a great source of inflammatory makers. Some of these are transudate or exudate products from the serum. Therefore, coincident levels between the saliva and serum can vary. The should clearly state in the discussion is SARS-CoV-2 is an oral virus. Its tropism is targeting oral epithelial cells populations based on the expression pattern of AVCE/TMPRSS2 which include keratinocytes, salivary ductal cells of various types which includes myoepithelial cells, tuft cells, gustatory and supporting cells of the tongue. Using saliva samples without gingival crevicular fluid as alluded to in the discussion increases difficulty to assess contribution by gingival tissues compared to other tissues. In addition, there are citations available that indicate there is a hierarchy of ACE2/TMPRSS2 expression with the highest levels in tongue and salivary gland coinciding with clinical signs such as loss of taste and dry mouth. These types of signs and symptoms need to be described to provide some degree of characterization of inflammation related to tissue site.. Additionally, there is lacking a immunologic description of the functional activity of IL-6. This cytokine which is also described as a adipokine is suppressible by adiponectin derived from the saliva. IL-6 has a variety of function requiring a mention in the discussion. For example, regulation of loss of tolerance and B cell differentiation while also effecting innate responses particularly in the periodontal tissues. Above I mentioned a possible association with adiponectin which acts as a protector against DM while increasing glucose uptake. There is another association overlooked in the discussion periodontal pathogens release metallo-peptidases and one of these mimics dipeptidyl-peptidase IV (DDPIV) which is diabetogenic. These types of relationship validate previous reports that periodontal disease is linked to DM. AR: We agree and thank the reviewer to underline this important issue. Accordingly, in the revised manuscript, we have added explanations (Page 9, Line 9-34) regarding the tropism of SARS-CoV-2 as an oral virus, the hierarchy of ACE2/TMPRSS2 expression in oral tissue, as well as the oral sign and symptoms related to COVID-19, the immunologic description of IL-6 function as mentioned by the Reviewer, as follows; Current literatures show that IL-6 is a pleiotropic cytokine with complex role in inflammation and metabolic disease. Its biological activities include B-lymphocyte differentiation, T-lymphocyte proliferation13, pro and anti-inflammatory activities14, the development of the nervous and hematopoietic system, and the regulation of metabolism15. As a proinflammatory cytokine, it induces insulin resistance and periodontal disease in the process of bone resorption16. Taken together, Il-6, which is considered an adipokine has an important role in the pathogenesis of localized oral inflammation (periodontitis), and may play a pivotal role in metabolic disease17, such as diabetes. The current study is an extension of our primary study involving the same patients with COVID-19 to evaluate the inflammatory conditions in periodontal microenvironment18. We found that the transcription levels of both IL-6 and complement C3 (the central component of innate immune system) in gingival crevicular fluid (GCF) were markedly higher in COVID-19 patients with diabetes (DG) compared to the non-diabetes patients (NDG). We also noticed that in periodontal niche, the mRNA upregulation of host receptor for SARS-CoV-2 (angiotensin-converting enzyme 2/ ACE2)19was positively associated with the transcription levels of either inflammation marker tested (IL-6 or C3). Thus, our previous results along with other work by other investigators20, 21 suggest that the virus receptor (ACE2) can be detected in oral tissue. Additionally, certain molecules (furin and TMPRSS2) that are involved in promoting the SARS-CoV-2 entry and infection can be detected in oral cavity22. This means that the essential molecules for SARCOV-2 infection are abundant in the oral cavity, and the infected virus may lead to localized inflammation and loss of taste (dysgeusia) as well as dry mouth, which are the most frequently reported symptom in COVID-19 patient23-25. The literature search also indicated that the relationship between COVID-19 and diabetes mellitus is complicated and bidirectional26. In addition to diabetes, all patients with COVID-19 included in the current study also had periodontitis, a common comorbidity observed in patients with COVID-1927. For the involvement of peptidase produced by periodontopathic pathogen, and mimics DDP4/DDP1V molecule of eukaryote cells, we have added explanations that can be found in the Discussion section (Page 8, Line 8-13), as follows; However, unlike salivary CRP, IL-6 levels in saliva have been reported to not correlate with those in plasma or serum30,31. In this regard, the elevated levels observed in this study may indicate inflammation induced by periodontitis-associated bacteria, particularly Porphyromonas gingivalis, as its components (dipeptidyl peptidase-4 /DPP4), that mimic human dpp4, involved in postprandial glycemic control in individuals with type 2 diabetes32, 33, that according to the medical record, were the participants included in the current study. RC: I suggest providing additional information in methods section about the health conditions of the subjects. Try to provide additional markers from the serum of saliva identifying SARS-CoV_2 presence in the oral cavity. Update and edit the discussion to comply with knowledge of SARS-CoV_2 and COVID-19 presentation effecting oral tissues. AR: As mentioned in the above point 2, in the revised version of this manuscript, the additional information about participant’s health condition have been added. As suggested by the reviewer, we also added the additional marker for the serum of saliva (complement C3) by referring to our previous study18. It can be seen in the above author response (point 3). Moreover, we have added the suggested content to the Discussion section, as explained in the above point 3." } ] } ]	1	https://f1000research.com/articles/12-419
https://f1000research.com/articles/12-754/v1	27 Jun 23	{ "type": "Study Protocol", "title": "Comparative evaluation of antimicrobial efficacy of 3C antibiotic paste with triple antibiotic paste as root canal filling material for primary teeth: An in-vitro study", "authors": [ "Aakriti Chandra", "Nilima Thosar", "Nilima Thosar" ], "abstract": "Root canal infection in primary teeth is polymicrobial in nature. Most resistant micro-organisms, such as E.faecalis survive in chronic infection of the root canal at the periapical area and are difficult to remove by various root canal irrigants. C.albicans has been found in chronic infections of root canals of primary teeth due to its ability to invade dentinal tubules. The multiple bacteria strains which cause endodontic infections, make it difficult to clean root canals with a single effective antibiotic. Hence Triple Antibiotic Paste (TAP) has been previously introduced, consisting of ciprofloxacin, metronidazole, and minocycline. Although this mixture aids in pulp regeneration and has been used to disinfect root canal systems, it is linked to tooth discolouration. The aim of this study is to evaluate and compare the antimicrobial efficacy of a newly formulated 3C antibiotic paste (consisting of Ciprofloxacin, Clindamycin, and Cefaclor) with conventional TAP against E. faecalis and C. albicans. In this in-vitro laboratory study, pure culture of C. albicans and E. faecalis will be grown on Sabouraud’s dextrose agar (SD) agar and Brain heart infusion agar (BHI) agar respectively and will be suspended for 24 hours at 37°C. For comparison of antimicrobial efficacy, the zone of inhibition will be for the 3C antibiotic paste against conventional TAP (control) for E. faecalis and C. albicans, and will be determined using digital calliper in millimetre after every 24 and 72 hours. We hypothesise that the newly formulated 3C paste will have better antimicrobial efficacy when compared with conventional TAP. It is expected that a newly formulated 3C paste will prove to be the successful as root canal filling material for primary teeth.", "keywords": [ "Triple Antibiotic Paste", "Clindamycin", "Cefaclor", "root canal filling material" ], "content": "Introduction\n\nMicroorganisms are crucial in the development of pulpal and periapical diseases. The polymicrobial character of the infection in the root canal system is primarily dominated by anaerobes.1 One of the frequently isolated among them is E. faecalis. E. faecalis is one of the most resistant micro-organisms and is most difficult to remove from the periapical areas of the tooth through conventional methods.2 About 23–70% E. faecalis can be isolated from infected root canals with symptoms of chronic apical periodontitis.3\n\nAmong Fungi, Candida species are found in about 3–18% of infected root canals.4\n\nAccording to earlier research, C. albicans and E. faecalis can infiltrate the dentinal tubules to varying depths, hence removal of these bacteria is facilitated by the use of an efficient antimicrobial agent in the root canal.5\n\nOver the years, several materials were tested in an effort to find an ideal obturating material for a primary tooth, such as zinc oxide eugenol, Maisto paste, Triple Antibiotic Paste etc.6 The antimicrobial efficacy of Triple Antibiotic Paste was evaluated for its use as root canal filling material with different modifications by various authors.7,8\n\nIn Triple Antibiotic Paste, the antibiotics used are: Ciprofloxacin, metronidazole, and minocycline in 1:1:1 concentration.9\n\nCiprofloxacin is a second-generation fluoroquinolone antibiotics active against a broad range of bacteria, especially Enterobacteriaceae and Neisseria.10 Metronidazole is a nitro imidazole compound having a broad spectrum of anti-anaerobic and anti-protozoal activity.9 Minocycline is a member of the Tetracycline class of broad-spectrum antibiotics, which work by exerting bacteriostatic action on a wide variety of microorganisms.8,9\n\nBut, Minocycline causes discoloration of tooth as stated by various authors.11,12 For the same reason, Sara Alsubait et al (2020) in their study had replaced minocycline with Cefaclor. Cefaclor is a second generation cephalosporin antibiotic which is mostly effective against most strains of Gram positive aerobes as well as gram negative aerobes.10\n\nA broad-spectrum antibiotic called Clindamycin is effective against infections that produce beta-lactamases as well as against aerobic and anaerobic microorganisms.10 Prasanna et al (2020) in their study concluded that Clindamycin could inhibit the levels of C albicans by 90% but Metronidazole could not inhibit the C. albicans.8\n\nWith respect to above literature, considering the disadvantage associated with Minocycline and reduced antimicrobial efficacy of Metronidazole, the present study has planned to use the 3C paste consisting of Ciprofloxacin, Clindamycin, and Cefaclor as a root canal filling material for primary teeth.\n\nThe aim of the present study will be to evaluate and compare the antimicrobial efficacy of 3C paste with conventional Triple Antibiotic Paste against E. faecalis and C. albicans.\n\nThe objectives of the study will be to evaluate the antimicrobial efficacy of 3C paste and triple antibiotic paste against E. faecalis and C. albicans; and also to compare the antimicrobial efficacy of 3C paste with Triple Antibiotic Paste against E. Faecalis and C. albicans.\n\n\nProtocol\n\nThis in vitro study will be conducted at Department of Pediatric and Preventive Dentistry, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi Meghe, Wardha in collaboration with the Institution of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha. The present study will be an in vitro experimental microbiological study.\n\nAlthough no human or animal participants will be involved in this research, as per institutional guidelines, ethical approval for the study was obtained from the institutional ethics committee of Datta Meghe Institute of Higher Education and Research, Wardha (Ref. No. DMIHER (DU)/IEC/2023/564 approved on 02/02/2023).\n\nFor the antimicrobial analysis brain heart infusion agar (BHI), Sabouraud’s dextrose agar (SD), Agar plates, Petri dishes, E. faecalis strain, C.albicans strain will be used. For preparation of paste, Ciprofloxacin, Clindamycin, Cefaclor, Metronidazole, Minocycline, Propylene glycol/macrogol will be required. These steps are outlined in full in the sections below.\n\nE. faecalis strain and C.albicans strain and the powder form of antibiotics will be included for the study. However other bacterial strains will be excluded from the study.\n\nPreparation of Triple Antibiotic Paste\n\nTriple Antibiotic Paste will be prepared by mixing equal proportion of Ciprofloxacin, Metronidazole, and Minocycline (1:1:1). Then a part of the solvent (propylene glycol- macrogol) in 1:1 ratio will be dispensed to obtain soft, uniform consistency. The resultant paste will be stored in an airtight container.\n\nPreparation of 3C antibiotic paste\n\n3C paste will be prepared in the same manner as Triple Antibiotic Paste. In 3C paste, the antibiotics Ciprofloxacin, Cefaclor, and Clindamycin (1:1:1) will be mixed with one part of Propylene Glycol-one part of Macrogol in 5:1 (powder: liquid) ratio to get desired consistency as required for the paste.\n\nProcedure\n\nPure colonies of C. albicans (ATCC 2091) and E. faecalis (ATCC 35550) will be grown on Sabouraud’s dextrose (SD) agar and Brain heart infusion (BHI) agar respectively and will be suspended in 5 ml of SD broth and BHI broth for 24 hours at 37°C.\n\nThe actively growing microorganisms in broth cultures will have turbidity adjusted to the 0.5 McFarland standard.3,5\n\nThe agar well diffusion method will then be followed. Wells of 3-4 mm will be prepared in the respective agar and then all the wells will be loaded with test material. The agar plates will be incubated at 37°C for 24 and 72 hours to achieve good bacterial growth before analysis. The inhibition zones of microbes will be evaluated using digital calliper in millimetre after every 24 and 72 hours. All the procedures will be repeated in triplicates to minimize errors.\n\nPrimary outcomes to be measured\n\nThe antimicrobial efficacy of newly formulated 3C antibiotic paste will be measured by bacterial growth and will be compared with the Triple Antibiotic Paste.\n\nData analysis and statistical plan\n\nStatistical analysis will be done using software SPSS Version 23.0. P value will be considered significant for <0.05. Descriptive analysis, i.e., mean distribution of inhibition zones will be recorded. A repeated measures ANOVA for intragroup comparison will be done on application of triplicate test of every group. Independent sample t test will be done for intergroup comparison.\n\nExpected outcome\n\nIt is expected that a newly formulated 3C paste will be able to show antimicrobial efficacy and will prove to be the successful as root canal filling material for primary teeth.\n\nDissemination\n\nThis study will be published in an indexed journal.\n\nNot yet started.\n\n\nDiscussion\n\nComplete eradication of micro-organism from root canal space is a challenging task in primary teeth due to morphology of the root canal. The multiple bacteria strains which cause endodontic infections, make it impossible to clean root canals with a single effective antimicrobial agent.9 Hence, a combination of drugs commonly known as Triple Antibiotic Paste (TAP) was introduced as treatment modality by Hoshino et al (1996) to eliminate the micro-organisms from infected root canal of primary teeth. It is usually composed of Ciprofloxacin, metronidazole, and minocycline (1:1:1). This mixture aids in pulp regeneration as well as the disinfection of the root canal system.13\n\nAn in vivo study conducted by Pinky C et al (2011) using two different combination of antibiotics in deciduous teeth consisting of ciprofloxacin, metronidazole, and minocycline i.e., TAP in group A and modified combinations consisting of ciprofloxacin, ornidazole, and minocycline in group B. Where they found both the groups showed significant results. However, group B had better result than group A clinically and radiographically.14\n\nA similar study was conducted in year 2013 by Sabrah et al. comparing the antibacterial activity of Triple Antibiotic Paste (TAP) and Double Antibiotic Paste (DAP) against Enterococcus faecalis and Porphyromonas gingivalis in comparison to calcium hydroxide. They found that both TAP and DAP were significantly more efficient in the reduction of bacterial biofilms in comparison to calcium hydroxide.15\n\nSimilarly a study conducted by Dahake P et al (2020) on Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Clindamycin (C), Metronidazole (M), Doxycycline (D) as well as their combination CMD that clindamycin could inhibit the levels of C albicans by 90% but Metronidazole could not inhibit the C. albicans and also concluded that combination CMD can be used to achieve predictable better results.16\n\nGovindaraju et al (2021) had evaluated Antibiotic sensitivity of the following intracanal medicaments grouped into 4 groups consisting of TAP (Ciprofloxacin, metronidazole, and doxycycline); Calcium hydroxide paste; Odontopaste and Sterile saline (0.9%) as a negative control against E. Faecalis, S. mutants, and S. aureus. In which they found that TAP showed the highest inhibition zone to conclude that TAP had better antibacterial activity than other groups.17\n\nHowever, Minocycline causes discoloration of tooth as stated by various authors.11,12 For the same reason, Sara Alsubait et al (2020) in their study had replaced minocycline with Cefaclor.11\n\nConsidering the disadvantages associated with conventional Triple Antibiotic Paste and reduced antimicrobial efficacy of Metronidazole, a new formulation in the form of 3C paste consisting of Ciprofloxacin, Clindamycin and Cefaclor will be prepared for its use as a root canal filling material for primary teeth.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nPaul P, Chandru TP, Peedikayil FC, et al.: Effectiveness of various intracanal medicaments on Candida albicans. SRM J. Res. Dent. Sci. 2020 Apr 1; 11(2): 82. Publisher Full Text\n\nAlghamdi F, Shakir M: The influence of Enterococcus faecalis as a dental root canal pathogen on endodontic treatment. Cureus. 2020 Mar 13; 12(3). Publisher Full Text\n\nDumani A, Yoldas O, Yilmaz S, et al.: In vitro susceptibility of E. faecalis and C. albicans isolates from apical periodontitis to common antimicrobial agents, antibiotics and antifungal medicaments. J. Clin. Exp. Dent. 2012 Feb; 4(1): e1–e7. PubMed Abstract \| Publisher Full Text\n\nYoo YJ, Kim AR, Perinpanayagam H, et al.: Candida albicans virulence factors and pathogenicity for endodontic infections. Microorganisms. 2020 Aug 26; 8(9): 1300.\n\nShaik J, Garlapati R, Nagesh B, et al.: Comparative evaluation of antimicrobial efficacy of triple antibiotic paste and calcium hydroxide using chitosan as carrier against Candida albicans and Enterococcus faecalis. J. Conserv. Dent. 2014 Jul; 17(4): 335–339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajsheker S, Mallineni SK, Nuvvula S: Obturating Materials Used for Pulpectomy in Primary Teeth-A Mini Review. J. Dent. Craniofac. Res. 2018; 03(1): 3. Publisher Full Text\n\nSijini OT, Sabbagh HJ, Baghlaf KK, et al.: Clinical and radiographic evaluation of triple antibiotic paste pulp therapy compared to Vitapex pulpectomy in non-vital primary molars. Clin. Exp. Dent. Res. 2021 Oct; 7(5): 819–828.\n\nDahake PT, Jha RK, Baliga SM: Evaluation of combination of three antimicrobial agents against common endodontic pathogens. Int. J. Sci. Res. 2019; 8(11): 63–64.\n\nMalu K, Khubchandani M: Triple Antibiotic Paste: A Suitable Medicament for Intracanal Disinfection. Cureus. 2022 Sep 15; 14(9). Publisher Full Text\n\nTripathi KD: Essentials of medical pharmacology. JP Medical Ltd; 2013 Sep 30.\n\nAlsubait S, Alsaad N, Alahmari S, et al.: The effect of intracanal medicaments used in Endodontics on the dislocation of two calcium silicate-based filling materials. BMC Oral Health. 2020 Dec; 20(1): 1–7. Publisher Full Text\n\nThomas MS: Crown discoloration due to the use of triple antibiotic paste as an endodontic intra-canal medicament. Saudi Endod. J. 2014 Jan 1; 4(1): 32. Publisher Full Text\n\nHoshino E, Kurihara-Ando N, Sato I, et al.: In-vitro antibacterial susceptibility of bacteria taken from infected root dentine to a mixture of ciprofloxacin, metronidazole and minocycline. Int. Endod. J. 1996 Mar; 29(2): 125–130. PubMed Abstract \| Publisher Full Text\n\nPinky C, Shashibhushan KK, Subbareddy VV: Endodontic treatment of necrosed primary teeth using two different combinations of antibacterial drugs: an in vivo study. J. Indian Soc. Pedod. Prev. Dent. 2011 Apr 1; 29(2): 121–127. PubMed Abstract \| Publisher Full Text\n\nSabrah AH, Yassen GH, Gregory RL: Effectiveness of antibiotic medicaments against biofilm formation of Enterococcus faecalis and Porphyromonas gingivalis. J. Endod. 2013 Nov 1; 39(11): 1385–1389. Publisher Full Text\n\nDahake PT, Baliga SM: Antimicrobial efficacy of a new tri-antibiotic combination against resistant endodontic pathogens: an in-vitro study. Braz. Dent. Sci. 2020 Sep 30; 23(4): 8. Publisher Full Text\n\nGovindaraju L, Jenarthanan S, Subramanyam D, et al.: Antibacterial activity of various intracanal medicament against enterococcus faecalis, streptococcus mutans and staphylococcus aureus: an in vitro study. J. Pharm. Bioallied Sci. 2021 Jun; 13(Suppl 1): 157. Publisher Full Text" }	[ { "id": "184034", "date": "22 Aug 2023", "name": "Shiva Shojaeian", "expertise": [ "Reviewer Expertise Endodontic regenersation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 would be improved if it is conducted to evaluate the antimicrobial efficacy of 3C paste and triple antibiotic on E. faecalis and C. albicans biofilm. Microbial biofilms in the root canal, penetrate into the dentinal tubules which are more challenging to eradicate compared with microorganisms growing in the cultivation media. E. faecalis and C. albicans can cultivate in root canal specimens to form microbial biofilm on dentin therefore the condition is more similar to the clinical situation\nThe methods of antibiotic preparation should be elaborated thoroughly including Drug specifications, concentration, and milligram/milliliter.\nThe specimens should be assigned to the experimental and negative and positive control groups.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "10303", "date": "06 Oct 2023", "name": "Aakriti Chandra", "role": "Author Response", "response": "The study of cultivating the microbial biofilms in root canal will be considered in future studies. the drug specifications will be given in final study once MIC of the dugs has been established." } ] } ]	1	https://f1000research.com/articles/12-754
https://f1000research.com/articles/11-516/v3	30 Sep 22	{ "type": "Research Article", "title": "In silico comparison between the mutated and wild-type androgen receptors and their influence on the selection of optimum androgenic receptor blockers for the treatment of prostate cancer", "authors": [ "Hany Akeel Al-Hussaniy", "Zahraa S. Al-tameemi", "Mohammed J. AL-Zobaidy", "Zahraa S. Al-tameemi", "Mohammed J. AL-Zobaidy" ], "abstract": "Background: Prostate cancer is a disease that occurs in men aged more than 50 years. In Iraq, 8.89 men per 100,000 population suffer from prostate cancer, with the incidence being 14,016 cases and mortality being 6,367 cases. Despite advances in treatment against prostate cancer, it can become resistant to drugs. Therefore, the aim of the current study was to search and identify binding sites for the repositioning of drugs by computational methods (docking). Methods: Based on the protein structure of the wild androgen receptor, the analysis parameters (22x22x22 on the X, Y, and Z axes) were established. Results: The interactions of the natural ligands with androgen receptor were 10.0 (testosterone) and 10.8 (dihydrotestosterone) while mutated androgen receptor (T877A) had a low affinity with testosterone and dihydrotestosterone (-5.3 and -6.7, respectively). In the interactions of both receptors with the reported inhibitors (antagonists), a decrease with Bicalutamide (-8.3 and -4.3, respectively) and an increase in affinity with Flutamide and Nilutamide (-7.7 and 8.6, wild AR; -8.7 and -9.3 AR T877A) were observed. As for Enzalutamide and Apalutamide (second-generation antagonists), the change was minimal between wild androgen receptor and T877A (-7.6 and -7.7; -7.3 and -7.3, respectively). The change in the affinity of the ligands with the androgen receptor and androgen receptor T877A shows how a mutation alters the bonds between these molecules. Conclusion: The identification of key sites and potent inhibitors against abnormal androgen receptor functions will enrich prostate cancer treatments.", "keywords": [ "androgenic antagonist", "anticancer", "androgen receptor", "molecular docking" ], "content": "Introduction\n\nProstate cancer (PCa) is a malignant growth that commonly occurs in men over the age of 50 years and consists of an increase in prostate size due to an increase in the number of cells. In Iraq, this disease has low incidence and mortality, according to previous studies, about 7.6% of all types of cancers are prostate cancer.1,2 In addition, the most likely age for development is between 50 and 74 years. Furthermore, progression of the PCa related to excess androgen stimulation and treatments include surgery and/or hormones that completely block the androgenic receptor.3,4\n\nWhen the disease reaches a stage of resistance to treatments, it is called Castration-Resistant Prostate Cancer (CRPC).5 Although new treatment strategies have been developed for CRPC, they are very few and quite inefficient. Many types of CRPC rely on decreasing the activity of the androgen receptor (AR) signaling the pathway for survival. Due to this, the androgen receptor is key to the design of new therapeutic strategies.5 To date, more than 600 different mutations have been found in the androgen receptor where the repercussions of these mutations on their structure, signaling, and resistance to PCa treatments are analyzed. For this reason, the development of strategies for the identification of effective drugs acting on androgen receptors is of great importance in obtaining new therapeutic agents against PCa.6 This methodology is known as Intensive Structure-Based Virtual Screening (SBVS).5,6 With the SBVS you can identify new bioactive compounds, make modifications in the structure of molecules, and look for the conformation and optimal position of a ligand with its target molecule to adjust it for the purpose of the drug.6–9 In addition, the methodology of drug repositioning, which accelerates the process of discovering new uses of existing therapeutic agents, allows the cost and time of their development to be significantly reduced. Therefore, the use of computational technologies based on protein structure (SBVS) for the design or repositioning of drugs (new uses of existing drugs) is an alternative method that will favor the investigation of new therapeutic agents against CRPC.10 Therefore, the aim of current study was to find and identify binding sites for both wild-type and mutated androgenic receptor and the best-proposed drug that antagonized mutated ones by computational methods (docking).\n\n\nMethods\n\nThe in-silico experiments so far were carried out at the Center for Research in Iraqi Medical and Pharmaceutical Research Center (IMRC). Subsequently, background analysis will be carried out with services provided by protein Data bank-EMBL-EBI (The European Bioinformatics Institute). The Current work is a preliminary trial for the selection of pharmacological molecules with inhibitory potential of the normal and mutated androgen receptor (T877A).11–19\n\nThis first stage consisted of obtaining 3D structures of the wild androgen receptor (AR) and a mutated one (T877A) which is the mutant associated with drug resistance. The 3D molecules were obtained through the database of the protein data bank with access codes 2AM9 (wild) and 2AX6 (mutation).\n\nThe structures of the natural ligands of the receptor (Testosterone and Dihydrotestosterone) and its inhibitors (Bicalutamide, Nilutamide, Flutamide, Enzalutamide, and Apalutamide) were obtained from the free database ZINC (http://zinc15.docking.org). These ligands were selected based on the affinity reported in the database ChEMBL.\n\nThe preparation of AR was carried out through the Chimera USFC program, and this preparation consisted of the addition of hydrogen atoms, removal of water molecules from the protein surface, elimination of ligands present, and the determination of charges that integrate each atom of the receiver. In this case, the selected charges were the AM-BCC. Subsequently, it was identified and selected based on the basic characteristics of the active androgen receptor site according to PDB data.12\n\nLigands, both natural and those reported as inhibitors of AR activity, were subjected to adaptations with the Chimera USFC program for molecular interaction assays with the in-silico androgen receptor, adding hydrogen atoms and assigning Gastieger charges to mimic the changes that occur within a cell according to their nature.13\n\nThe affinity values (Kcal/mol) were calculated using the AutoDock program, the interactions between wild AR and its natural ligands were analyzed, and the contact between the molecules when the distance was less than or equal to 5 Å was considered. Based on the interaction of natural ligands (Testosterone and Dihydrotestosterone), the optimal parameters for evaluating receptor interactions were determined. For the validation of these parameters, the interaction between the wild androgen receptor with the five inhibitors was reported against the receptor (Bicalutamide, Nilutamide, Flutamide, Enzalutamide, and Apalutamide). The same distance (5 Å) is considered for interaction between ligands with AR. In addition, within the validation of the joining site, the size assessment of the coupling site was carried out for the structures of the ligands, which was defined by establishing a cube with the dimensions of 22×22×22 Å, 15×15×15Å, and 10×10×10 Å. This was to experimentally determine the exact critical parameters for the interactions between the receptor and ligands.14\n\nThe interaction analyses between the mutated androgen receptor (T877A) were performed based on the calculations as mentioned above, considering the binding site’s size determined for wild AR with natural receptors and inhibitors (22×22×22 Å). This is to determine if the mutation changes the affinity between the receptor and the molecules evaluated.\n\nThe identification of residues of the AR that interact with the natural ligands and inhibitors that had the best affinity score in the analyses in AutoDock Vina (Kcal/mol) were visualized using the PyMOL program with which each complex between the receptor and the ligand was observed.\n\n\nResults\n\nThe first result obtained from the analysis was carried out with the help of the optimal parameters of the AR binding site which were determined with the use of AutoDock Vina and Chimera. The parameters are described below (Table 1).\n\nWith the server of AutoDock Vina de Chimera and AutoDock Vina (direct program), the calculation of the theoretical value for the affinity of the coupling of the natural ligand and inhibitors with the androgen receptors, both wild and mutated. The following Tables 2 and 3 showed the analyses of affinities existed in the interactions between the wild AR and the natural ligands.\n\nThe results showed that the Dihydrotestosterone (DHT) ligand has a higher affinity for the AR than testosterone, which is the precursor of DHT. Pang et al. (2021) reported this affinity, who mentioned that DHT has a significant on the receptor and is active in the functions in which AR is involved, such as in the transcription of genes for survival and cell growth.15 The validation of the established parameters of the normal androgen receptor binding site, with the use of receptor inhibitors, reflects that the ligand binding site corresponds to the binding site of the natural ligands (testosterone and DHT); however, the drug Enzalutamide and Apalutamide showed high affinity for another receptor site. Also, Chen et al. (2019) mentioned that Enzalutamide and Apalutamide are second-generation antiandrogens that inhibit AR’s activity during cancer development. In contrast, first-generate drugs included Bicalutamide, Flutamide, and Nilutamide.16 The results obtained were presented in Table 4.\n\nThe results obtained with Enzalutamide (0.3) and Apalutamide (0.4) showed a low affinity to the common binding site of the androgen receptor, so it was analyzed in which area of the receptor these molecules were bound and what is their affinity under a cube size of 40×40×40 Å (Table 5).\n\n\nDiscussion\n\nThe change in Enzalutamide and Apalutamide was due to the interaction of these drugs with another area different from the normal binding expected AR, giving an increase of 0.3 (Enzalutamide) and 0.4 (Apalutamide) to -7.6 and -7.7, respectively shown in Figure 1. To visualize the interactions of the complexes, the PyMOL program was used, with which the amino acid residue involved in the interactions between AR with natural ligands were determined, as with inhibitory ligands. For the wild-type AR complex and the natural ligands, it was observed that they are determined by links between the residues threonine 877 with H of Testosterone (2.4 Å) and arginine 752 with O (2.3 Å) (Figure 1). In the case of the wild AR complex with dihydrotestosterone, the affinity is greater than that observed with Testosterone, said the bonds between the molecules give affinity, and the residues involved in the formation of said complex are given by arginine 752 with the union of 2.4 Å with the terminal nitrogen of DHT, followed by the collaboration between threonine 877 with the opposite oxygen at a distance of 1.9 Å, and finally the third interaction occurs between asparagine with the terminal O of DHT, this union is given at a distance of 2.4 Å (Figure 2). Table 6 shows the interactions between AR and inhibitory ligands, the residues involved are described as the distance between the molecules (Figure 3).\n\n(a) showed uncommon androgen receptors and site of enzalutamide binding (b) showed wild-type androgen receptor bind to a common site.\n\na) Nilutamide, b) Bicalutamide, c) Flutamide, d) Enzalutamide and e) Apalutamide.\n\nThe results of the interactions of the receptor (with the T877A mutation) with the ligands analyzed above, the affinity of the natural ligands is low compared to that studied in the wild receptor. The affinity of the antiandrogen Bicalutamide, if it has an affinity of -8.3, the receptor with the T877A mutation decreases to -4.3. However, the affinity towards Flutamide and Nilutamide increased from -7.7 and -8.6 to -8.7 and -9.3, respectively, and second-generation antiandrogens (Enzalutamide and Apalutamide) were stable, as the T877A mutation is not found at the site where these two drugs bind to inhibit the receptor (Tables 7 and 8).16 It is noted that a drug binds to the receptors somewhat well, and this confirms the studies that led to the adoption of a drug for treatment apalutamide and enzalutamide binding affinity not changed in both mutated and wild-type androgenic receptors, which provides an idea to use them even in patient with CRPC and this result augmenting the FDA on (9-2021) approvals of apalutamide for treatment non-metastatic castration-resistant prostate cancer.\n\nThe interacting residues in each of the mutated AR-ligand complexes were visualized with the PyMOL program. The residues that are modified in the regular interaction of the natural ligands of AR were Threonine 877 so that the union with the natural ligands was between Arginine 752 that binds 9 to O of the Testosterone ligand (2.8 Å), and the Glutamine residue 711 attached to H of the ligand (2.0 Å). In the AR-Dihydrotestosterone complex, the amino acids involved are Arginine 752 and Glutamine 711 at a distance of 2.9 Å with O and 2.4 Å with H, respectively (Figure 4).17,18\n\na) Nilutamide, b) Bicalutamide, c) Flutamide, d) Enzalutamide and e) Apalutamide.\n\n\nConclusion\n\nThe preliminary conclusion of this work is that in addition to the normal receptor ligand binding site, there is another site on which the search and identification of drugs can be based. Considering their nature, in this way, a model can be established to search for key sites and potent inhibitors against abnormal androgen receptor function in prostate cancer. In addition, based on computational methodologies, the process of identification and design of therapeutic molecules can be optimized and accelerated. In the same way, the repositioning of drugs will facilitate and increase the efficiency of using existing drugs, which will reduce the costs of their implementation by providing a therapeutic alternative in a shorter period against prostate cancer.\n\n\nData availability\n\nZenodo. Docking result of androgen. DOI: https://doi.org/10.5281/zenodo.598759720\n\nThis project contains the following underlying data:\n\n- The docking result of androgen and androgenic blocker by using autodock tools and autodock vena\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbood RA, Abdahmed KA, Mazyed SS: Epidemiology of different types of cancers reported in Basra, Iraq. Sultan Qaboos Univ. Med. J. 2020; 20(3): e295–e300. PubMed Abstract \| Publisher Full Text\n\nHussain AM, Lafta RK: Cancer trends in Iraq 2000-2016. Oman Med. J. 2021; 36(1): e219. PubMed Abstract \| Publisher Full Text\n\nSharquie KE, Al-Meshhadani SA, Al-Nuaimy AA, et al.: Therapeutic Evaluation of Spironolactone and Finasteride in the Treatment of Acne Vulgaris. Iraqi Postgraduate Medical Journal. 2007; 6(3).\n\nAL-Timimi AH, Mahmood EH: Study of Membrane Testosterone Receptors in Prostate Cancer. Med. J. Babylon. 2007; 4(1–2).\n\nMishra SS, Ranjan S, Sharma CS, et al.: Computational investigation of potential inhibitors of novel coronavirus 2019 through structure-based virtual screening, molecular dynamics and density functional theory studies. J. Biomol. Struct. Dyn. 2021; 39(12): 4449–4461. PubMed Abstract \| Publisher Full Text\n\nLi H, Ren X, Leblanc E, et al.: Identification of novel androgen receptor antagonists using structure-and ligand-based methods. J. Chem. Inf. Model. 2013; 53(1): 123–130. PubMed Abstract \| Publisher Full Text\n\nKumar A, Zhang KYJ: Hierarchical virtual screening approaches in small molecule drug discovery. Methods. 2015; 71: 26–37. PubMed Abstract \| Publisher Full Text\n\nSorna V, Theisen ER, Stephens B, et al.: High-throughput virtual screening identifies novel N’-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1 inhibitors. J. Med. Chem. 2013; 56(23): 9496–9508. PubMed Abstract \| Publisher Full Text\n\nWu K-J, Lei P-M, Liu H, et al.: Mimicking strategy for protein-protein interaction inhibitor discovery by virtual screening. Molecules. 2019; 24(24): 4428. PubMed Abstract \| Publisher Full Text\n\nSong C-H, Yang SH, Park E, et al.: Structure-based virtual screening and identification of a novel androgen receptor antagonist. J. Biol. Chem. 2012; 287(36): 30769–30780. PubMed Abstract \| Publisher Full Text\n\nTang Q, Fu W, Zhang M, et al.: Novel androgen receptor antagonist identified by structure-based virtual screening, structural optimization, and biological evaluation. Eur. J. Med. Chem. 2020; 192(112156): 112156. Publisher Full Text\n\nZhou W, Duan M, Fu W, et al.: Discovery of novel androgen receptor ligands by structure-based virtual screening and bioassays. Genom. Proteom. Bioinform. 2018; 16(6): 416–427. PubMed Abstract \| Publisher Full Text\n\nAxerio-Cilies P, Lack NA, Nayana MRS, et al.: Inhibitors of androgen receptor activation function-2 (AF2) site identified through virtual screening. J. Med. Chem. 2011; 54(18): 6197–6205. PubMed Abstract \| Publisher Full Text\n\nWahl J, Smieško M: Endocrine disruption at the androgen receptor: Employing molecular dynamics and docking for improved virtual screening and toxicity prediction. Int. J. Mol. Sci. 2018; 19(6): 1784. PubMed Abstract \| Publisher Full Text\n\nPang J-P, Shen C, Zhou W-F, et al.: Discovery of novel antagonists targeting the DNA binding domain of androgen receptor by integrated docking-based virtual screening and bioassays. Acta Pharmacol. Sin. 2021; 43: 229–239. PubMed Abstract \| Publisher Full Text\n\nChen Q, Wang X, Tan H, et al.: Molecular initiating events of bisphenols on androgen receptor-mediated pathways provide guidelines for in silico screening and design of substitute compounds. Environ. Sci. Technol. Lett. 2019; 6(4): 205–210. Publisher Full Text\n\nSerçinoğlu O, Bereketoglu C, Olsson P-E, et al.: In silico and in vitro assessment of androgen receptor antagonists. Comput. Biol. Chem. 2021; 92(107490): 107490.\n\nRéau M, Lagarde N, Zagury J-F, et al.: Hits discovery on the androgen receptor: In silico approaches to identify agonist compounds. Cells. 2019; 8(11): 1431. PubMed Abstract \| Publisher Full Text\n\nAl-hassany HA, Albu-rghaif AHA, Naji M: Tumor diagnosis by genetic markers protein P-53, p16, C-MYC, N-MYC, protein K-Ras, and gene her-2 Neu is this possible?. Pak. J. Med. Health Sci. 2021; 15(8): 2350–2354. Publisher Full Text\n\nAl-Hussaniy HA: docking result of androgen [Data set]. In f (Version 1). Zenodo. 2022. Publisher Full Text" }	[ { "id": "164311", "date": "24 Feb 2023", "name": "Zarrin Basharat", "expertise": [ "Reviewer Expertise Drug design", "docking simulation", "microbiology", "bioinformatics", "proteomics", "genomics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study aimed to repurpose some drugs against mutated androgen receptor ligand binding domain and could have clinical translation prospects. Authors took publicly available data and used Autodock for docking. The results showed that the mutated receptor had lower affinity towards some ligands ligands while having variable affinity towards others. However, the protein and ligand preparation as well as validation was not rigorous. The docking is a prediction and can be influenced by various factors such as the accuracy of the protein structure, the scoring function used, and the conformational flexibility of the ligand and receptor. Since authors come from LMIC, with limited resources, they could try and validate using energy calculation and dynamics simulation on the least (as access to experimental setup would be limited). Simply docking scores are not enough in most cases and MM-PBSA values etc have been reported to have better results.\nAuthors mention in abstract that they look androgen receptor structure, whereas it is only part of it (ligand binding domain that is present with the mentioned ID in PDB). Authors need to be clear about this. Name of software needs to be mentioned and parameter portion edited. Abstract lack of information on docking parameters: The abstract mentions that analysis parameters were established for docking, but it does not provide any details on the specific algorithms or software used. This makes it difficult to assess the reliability and reproducibility of the docking results. Authors should also provide a detailed rationale for selecting the ligands (preferably add a table with references that depict use of these ligands in treatment). The scope is somewhat limited, the study only investigated the interactions of a few ligands and inhibitors with the androgen receptor and a specific mutation (T877A). It is unclear whether these findings can be generalized to other ligands, inhibitors, or mutations. Authors could shed some light on this.\nLanguage needs to be edited at several places for clarity and conveying proper scientific meaning. The heading ‘Adequacy of the wild and mutated androgen receptor ‘ and the text beneath it needs reassessment and editing. ‘subjected to adaptation’?\nSame goes for ‘Assays of wild AR interactions with natural ligands and AR inhibitors to establishing interaction parameters’ heading. Too long and not correct. Natural ligands can act as control for this study. What is L.b and U.b?\nAuthors took a ligand binding section of the protein, how can they write in conclusion that ‘….. in addition to the normal receptor ligand binding site, there is another site on which the search and identification of drugs can be based.’. They did not do a site scoring and binding comparison based on this. Several deficiencies need to be addressed in this study before it can be indexable.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10909", "date": "13 Apr 2024", "name": "الدكتور هاني عقيل", "role": "Author Response", "response": "Reviewer note (The study aimed to repurpose some drugs against mutated androgen receptor ligand binding domain and could have clinical translation prospects. Regarding the aim, was improved. Authors took publicly available data and used Autodock for docking. The authors took a available data ( actually its my current research supplementary data). The results showed that the mutated receptor had lower affinity towards some ligands ligands while having variable affinity towards others. (Yes we made docking on certain receptor that may be linked to increased resistance to androgenic blocker in Iraq ) However, the protein and ligand preparation as well as validation was not rigorous. Yes that's correct. The docking is a prediction and can be influenced by various factors such as the accuracy of the protein structure, the scoring function used, and the conformational flexibility of the ligand and receptor. There are several articles support our work regarding the binding Since authors come from LMIC, with limited resources, they could try and validate using energy calculation and dynamics simulation on the least (as access to experimental setup would be limited). There is no requirement of experiments on human or animals because all of this medications is already approved Simply docking scores are not enough in most cases and MM-PBSA values etc have been reported to have better results.) We used 3 types of docking programs Autodock vena, pyrx , molecular operation environment To ensure the result of docking Regarding clinical studies there is several clinical studies regarding the antiandrogenic medication for treatment of castration resistance prostate cancer and these clinical studies concluded that Enzalutamide is one of the best choice for treatment, our research support there findings , (Enzalutamide and Apalutamide) were stable, and good inhibitors in case of castration resistance prostate cancer Regarding English language We improved it. Finally I hope you accept my article. Best regards, Hany Akeel Al-hussaniy" } ] } ]	3	https://f1000research.com/articles/11-516
https://f1000research.com/articles/13-56/v1	11 Jan 24	{ "type": "Study Protocol", "title": "Extending the Reporting Items of Practice Guidelines in Healthcare (RIGHT) statement for reporting Chinese patent medicine guidelines: a protocol", "authors": [ "Xuanlin Li", "Lin Huang", "Qi Zhou", "Zhijun Xie", "Xuanming Hu", "Yaolong Chen", "Chengping Wen", "Xuanlin Li", "Lin Huang", "Qi Zhou", "Zhijun Xie", "Xuanming Hu" ], "abstract": "Background: Based on the main document of the Reporting Items of Practice Guidelines in Healthcare (RIGHT) statement, this study will aim to develop the reporting checklist applicable to the guidelines of Chinese patent medicine (CPM). Methods: According to the methodology recommended by the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) network, three well-defined working groups were established, namely the coordination team, the advisory group, and the Delphi panel. The coordination team conducted a literature analysis of the already published CPM guidelines, the extensions of the RIGHT statement, and CPM related reporting specifications to initially form an initial item pool. The Delphi panel of multidisciplinary backgrounds will conduct two rounds of Delphi questionnaires to identify an extension item for RIGHT for CPM. These questionnaires will be sent to each Delphi panel member via email, and each member will independently fill out and provide comments within 10 working days of receiving the questionnaire. Finally, feedback will be provided to the coordination team and the post efficacy assessment of the already published clinical practice guidelines for CPM to determine the final items for RIGHT for CPM checklist. Dissemination: The extended version of the RIGHT for CPM checklist will be published in a peer reviewed journal. Conclusion: We hope that the upcoming RIGHT for CPM checklist provides detailed guidance for developers and users of CPM guidelines, further improving the quality of reports, and promoting the effective dissemination and implementation of CPM guidelines.", "keywords": [ "RIGHT statement", "Chinese patent medicine", "reporting guideline" ], "content": "Introduction\n\nStandardizing reporting of clinical practice guidelines (CPGs) can help promote guideline integrity, transparency and reliability, assist clinicians in reading, evaluating, and applying the guidelines efficiently, and is an important safeguard in promoting the scientific implementation of guidelines. Currently, the Reporting Items for practice guidelines in healthcare (RIGHT) statement lays an important foundation for the scientifical and standardized reporting of CPGs.1 In recent years, the RIGHT workgroup has developed multiple extensions around different guideline types, designs, domains, and so on, containing guideline adaptations (RIGHT - Ad@pt),2 guideline interpretations (RIGHT - INT),3 and public and patient guidelines (RIGHT-PVG),4 among others. An extension of acupuncture (RIGHT for acupuncture)5 and extension of traditional Chinese medicine (RIGHT for TCM)6 were developed in the field of traditional and complementary alternative medicine. In particular, the development of RIGHT for TCM6 has provided an important basis for the scientific, normative and clear reporting of clinical practice guidelines for TCM compound formulas.\n\nCPM are an important component in the drug supply security system in China and even worldwide. At present, more than 10,000 CPMs have been approved by the Chinese drug regulatory authority (China), and 1,617 CPMs are included in the Pharmacopoeia of the people's Republic of China (2020 Edition), and 4,052 CPMs are included in the pharmaceutical standard of the Ministry of health of the people's Republic of China - Chinese patent formulation. CPMs play important therapeutic, preventive, and health care roles in many disease phyla, but there are also promiscuity and even abuse phenomena.7 To standardize the rational application of CPMs, TCM policymakers approved the establishment of a standardized research project on the clinical application of CPMs for the treatment of dominant disease species and to develop CPGs for multiple types of CPMs,8,9so that the number of CPGs for CPMs is growing rapidly. However, there are currently no reporting specifications for CPM guidelines. There are no items for the RIGHT for TCM that relates to CPMs,6 and there is insufficient consideration for CPM guidelines to report scientifically and transparently. At the same time, there is a lack of consideration of CPM guidelines in the selection and inclusion criteria of CPMs, policy access, indications/symptoms, instructions for CO-medications, medication for special populations, precautions for use, and medication recommendations for Western physicians. Therefore, to ensure rigor, transparency, clarity, and reproducibility of reporting the process of CPM guidelines, we will develop an extension of the RIGHT statement for the reporting of CPM guidelines (RIGHT for CPM).\n\n\nMethods\n\nThe protocol of RIGHT for CPM checklist draws on the steps and merits of published work-up protocols for an extension of RIGHT statement9–11 and is developed with reference to the methods for reporting guideline development recommended by the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) network. The target users of RIGHT for CPM are the drafting/adapters, reviewers, and guideline stakeholders of the CPM guidance. In order to enhance the transparency and quality during the development of the RIGHT for CPM checklist, the present protocol intends to complete registration in the EQUATOR network (https://www.equator-network.org/library/reporting-guidelines-under-development/reporting-guidelines-under-development-for-other-study-designs/#CPM). The development process of RIGHT for CPM is shown in Figure 1.\n\nEstablishment of the working group\n\nThe RIGHT for CPM working group will include: a coordination group; a advisory group, and a Delphi panel.\n\nThe advisory group will consist of 18 experts, including Prof. Yaolong Chen, the initiator of the RIGHT statement, and Prof. Chengping Wen, the co-initiator of the RIGHT for CPM extension. Experts with outstanding achievements in the reporting guidelines or TCM research are invited to form the advisory group. The recruitment criteria were experts with certain influence in the field of traditional Chinese medicine research, who have participated in or are familiar with the development of clinical practice guidelines or reporting standards. They will be contacted by email or direct phone. Their main responsibility is to provide methodological guidance and consultation for the development of RIGHT for CPM, ensure the quality of RIGHT for CPM development, and approve the final version of RIGHT for CPM items.\n\nThe coordination group will consist of seven to nine members who will lead and coordinate the development process of the RIGHT for CPM and ensure that it is completed according to the planned schedule. The members of the coordination group are mainly composed of graduate students and research-oriented teachers from universities, who work under the guidance of experienced members. They communicate and coordinate with each other through We-chat groups. Specifically, this group will be responsible for (1) the establishment of a pool of RIGHT for CPM items; (2) draft a preliminary RIGHT for CPM checklist; (3) issuing, collecting and sorting out Delphi questionnaires; (4) manage E-mail communications; and (5) drafting the final extension and explanatory document for RIGHT for CPM checklist.\n\nA total of 17 experts will be invited to form a Delphi panel by e-mail or We-chat mobile application. To ensure clarity and transparency in the development process, all members are required to declare any potential conflicts of interest that may be related to the development of RIGHT for CPM. Recruitment criteria are as follows: (1) TCM clinical experts; (2) Clinical experts of TCM and Western medicine who have participated in or drafted the application guidelines of CPM in the past; and (3) Guideline methodology, evidence-based medicine, and clinical epidemiology experts. The primary responsibility of this panel is to conduct the delphi consultation and approve the final version items of the RIGHT for CPM checklist.\n\nCreating an initial pool of items for RIGHT for CPM\n\nWe will systematically search PubMed, Embase, Cochrane Library, Web of science, China National Knowledge Infrastructure (CNKI), Chinese biomedical literature service system (CBM), Wanfang Database, and VIP databases to collect published CPM guidelines. The time range is in the database until April 2023. The full search strategy for PubMed database is shown in Table 1.\n\nThe reporting quality of the CPM guidelines was assessed by four members of the coordination team with reference to the RIGHT for TCM checklist,6 and the initial items of the RIGHT for CPM will be initially extracted and condensed. All assessment processes are carried out independently, and if there is disagreement, consensus is reached through group discussion.\n\nIn addition, we will access extensions that have been completed or are in development via the RIGHT Statement website (http://www.right-statement.org/). At the same time, we will collect the reporting checklist for CPM research, which was sorted out by another four members of the coordination team, condensed, de-duplicated, summarized and integrated, and formed an items pool of RIGHT for CPM checklist.\n\nDelphi questionnaire survey\n\nThe coordinating team will design the initial items as a questionnaire and send them independently to the Delphi penal as an online questionnaire using the Questionnaire Star (https://www.wjx.cn/). On the basis of evaluating the importance of the initial items, the members of the consensus expert group give the judgment of ‘agree’, ‘uncertain’, and ‘disagree’ for each item, and give the corresponding suggestions for modification. The coordination team will collect, sort out, and analysis the feedback results from the experts, count the consensus degree of each item (consensus degree = [number of people who agree/total number of people] *100%), and summarize the feedback opinions put forward by experts.\n\nThe eligibility criteria of items for RIGHT for CPM will be judged according to the following principles: (1) Those with a consensus degree <75% are directly excluded; (2) When the consensus degree is ≥75% and there is no substantive opinion, it is regarded as direct inclusion; and (3) When the consensus degree is ≥75% and there are substantive opinions, the second round of the Delphi survey will be conducted after the coordination team discussion determines the exclusion or modification, and the expert opinions will be replied one by one. (4) After the second round of the Delphi survey, items with consensus degree <75% and ≥75% and no substantive modification opinions should be treated in the same way as those in the first round. Items with consensus degree ≥75% and substantive opinions should be included or excluded after discussion between the coordination team. Based on the results of two rounds of the Delphi survey and expert feedback, the coordination team will form the RIGHT for CPM checklist (Version 1) after consensus discussion on the items initially included by advisory group. This version may be further adjusted and improved in later versions after publication to subsequent versions.\n\nDrafting of interpretation and elaboration documents\n\nThe coordination team plans to develop the RIGHT for CPM explanation and elaboration document to explain and provide examples in each item in order to facilitate target users to understand and better apply the RIGHT for CPM checklist (Version 1).\n\nPublicity and interpretation\n\nThe coordination group will upload the checklist of RIGHT for CPM and the documents of explanation and explanation to the RIGHT statement website for readers to download and read. At the same time, the publicity of RIGHT for CPM will be carried out through We-chat public accounts and academic conferences. In addition, the coordination group will continue to dynamically apply the RIGHT for CPM checklist to assess the emerging guidelines for CPMs and prepare sufficient information for the update version.\n\n\nDiscussion\n\nRational use of CPMs, improving the therapeutic level, and reducing abuse and waste are the core starting points for formulating guidelines for the application of CPM. The RIGHT for CPM checklist based on the RIGHT statement will provide detailed guidance for the developers and users of the guidelines for CPMs, with the hope to further improve the quality of the guidelines for CPMs and promote the effective dissemination and implementation of the guidelines for CPMs.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\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): 128–132. PubMed Abstract \| Publisher Full Text\n\nWang X, Zhou Q, Chen Y, et al.: Protocol of reporting items for public versions of guidelines: The Reporting Tool for Practice Guidelines in Health Care-public versions of guidelines [J]. BMJ Open. 2019; 9(3): e023147. Publisher Full Text\n\nSong Y, Darzi A, Ballesteros M, et al.: Extending the RIGHT statement for reporting adapted practice guidelines in healthcare: the RIGHT-Ad@pt Checklist protocol [J]. BMJ Open. 2019; 9(9): e031767. Publisher Full Text\n\nChunzhi, Tang, Liming, et al.: Developing an extension of the RIGHT statement for clinical practice guidelines on acupuncture: RIGHT for acupuncture – A protocol - ScienceDirect [J]. European Journal of Integrative Medicine. 29: 100908. Publisher Full Text\n\nMoher D, Schulz KF, Simera I, et al.: Guidance for developers of health research reporting guidelines [J]. PLoS Med. 2010; 7(2): e1000217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXie R, Xia Y, Chen Y, et al.: The RIGHT Extension Statement for Traditional Chinese Medicine: Development, Recommendations, and Explanation [J]. Pharmacol. Res. 2020; 160: 105178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInstitute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Dongzhimen Hospital of Beijing University of Chinese Medicine et al.: Preferred reporting items for comprehensive evaluation of Chinese patent medicine (PRICE-CPM)[J]. Chin. J. Evid. Based Med. 2022; 22(07): 745–750.\n\nZhang Q, Wang ZF, Xie YM, et al.: Report standards for clinical comprehensive evaluation of Chinese patent medicine [J]. Zhongguo Zhong Yao Za Zhi. 2021; 46(23): 6062–6067. PubMed Abstract \| Publisher Full Text\n\nMo M, Liao X, Zhang XX, et al.: Reporting standards for expert consensus on clinical practice of Chinese patent medicines of China Association of Chinese Medicine [J]. Zhongguo Zhong Yao Za Zhi. 2019; 44(12): 2644–2651. PubMed Abstract \| Publisher Full Text\n\nLuo X, Akl E A, Zhu Y, et al.Developing the RIGHT extension statement for practice guideline protocols: the RIGHT-P statement protocol [version 1; peer review: 1 approved with reservations] [J].F1000Research,2022(11):275.\n\nEubank BH, Mohtadi NG, Lafave MR, et al.: Using the modified Delphi method to establish clinical consensus for the diagnosis and treatment of patients with rotator cuff pathology [J]. BMC Med. Res. Methodol. 2016; 16: 56. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "247639", "date": "26 Mar 2024", "name": "Tiejun Tong", "expertise": [ "Reviewer Expertise meta-analysis", "evidence-based 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 protocol aims to develop an extension of the Reporting Items of Practice Guidelines in Healthcare (RIGHT) statement for the Chinese Patent Medicine (CPM) guidelines. This is a parallel work of the authors as that was previously done for the Traditional Chinese Medicine (TCM). To achieve the goals, three working groups will be established including the coordination team, the advisory group, and the Delphi panel. And upon a successful completion, it is also expected that the extended version of the RIGHT for CPM checklist will provide a detailed guidance for developers and users to further improve the quality of reports, as well as promote the effective dissemination and implementation of CPM guidelines. My only concern is on the typos and language mistakes that may require for a more careful editing. To name a few, \"CPM are an ...\" should be \"CPM is an ...\" or \"CPMs are an ...\"; \"a advisory group\" should be \"an advisory group\"; \"draft a ...\" should be \"drafting a ...\"; and \"manage E-mail ...\" should be \"managing the e-mail ...\"; \"full search strategy for PubMed\" in the main text, and then it was inconsistently changed to \"full search strategies for PubMed\" in Table 1; For \"We-chat\", do you mean it to be \"WeChat\"?\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": "267539", "date": "08 May 2024", "name": "Chun-Hui Bao", "expertise": [ "Reviewer Expertise Complementary and Alternative Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 the protocol for CPM for RIGHT, which has a high standard of methodology. The necessity of the project is well established. But there are some issues that need to be revised and discussed.\n\nCONCERN POINT 1. The reporting guidelines are oriented to the field of Chinese Patent Medicine (CPM). Therefore, senior experts with expertise in the field of Chinese materia medica, especially in Chinese pharmaceutics, and not only in TCM research, should be included in the corresponding Delphi groups. 2. The difference between RIGHT for CPM and RIGHT for TCM should be described in detail in the introduction or discussion. 3. For the report guideline, the breadth of dissemination directly affects its utilization. Therefore, at the protocol stage, the method of developing the multilingual version and the dissemination channels should be determined. It is recommended to cooperate with journals in different languages of Chinese and English, or publish multiple languages (Simplified Chinese, Traditional Chinese, English) in the same journal.\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/13-56
https://f1000research.com/articles/13-55/v1	11 Jan 24	{ "type": "Case Report", "title": "Case Report: Endovascular technique for intravascular foreign body retrieval in severely preterm neonate", "authors": [ "Marsha Ruthy Darmawan", "Djuwita Adi Wahyono", "Taufiq Fredrik Pasiak", "Listyo Julia", "Irhamni Irhamni", "Terawan Agus Putranto", "Marsha Ruthy Darmawan", "Djuwita Adi Wahyono", "Taufiq Fredrik Pasiak", "Listyo Julia", "Irhamni Irhamni" ], "abstract": "Background Intravascular foreign bodies (IFB) in severely preterm neonates present critical clinical challenges. Traditional surgical removals are often risky and may not be feasible in these patients. This case report aims to detail a successful endovascular approach for IFB retrieval in a severely preterm neonate.\n\nCase This is a case report of a 14-day old, male 30-week gestation neonate with a retained umbilical catheter fragment. The baby was referred after an unsuccessful explorative laparotomy by a paediatric surgery team. The endovascular procedure was performed using a 4 French (Fr) vertebra catheter and a One Snare wire in the right femoral region under low-dose fluoroscopy. The IFB was successfully removed. Post-procedure, the patient’s leukocyte count normalized, and he transitioned from ventilator support to a nasal cannula and eventually to room air within three days. The neonate could also tolerate oral intake, signalling a rapid recovery and low morbidity.\n\nConclusions This case report highlights the potential of endovascular approaches for IFB retrieval as viable alternatives to traditional surgeries, especially in neonates where surgical options are limited or have failed. Further research is needed to standardize such minimally invasive techniques in neonatal care.", "keywords": [ "Endovascular", "interventional radiology", "intravascular foreign body", "preterm", "neonate" ], "content": "Introduction\n\nIntravascular foreign bodies (IFB) in severely preterm neonates present a significant clinical challenge fraught with multiple risks including infection, vascular injury, and even death. Such events are exceedingly rare yet demand immediate attention given the vulnerable nature of the neonatal patient population.1 Traditional surgical approaches for foreign body removal are not only risky but may also not be feasible due to the extremely delicate anatomical structures and limited physiologic reserves of preterm neonates. Therefore, safer and minimally invasive interventions are often preferred.2\n\nEndovascular techniques have gained prominence in adult and paediatric populations for various applications, including foreign body retrieval, because of their less invasive nature and shorter recovery time. However, the literature describing the efficacy, safety, and technical aspects of endovascular interventions for foreign body retrieval in severely preterm neonates is sparse.2–4\n\nThis case report aims to detail a successful endovascular approach for intravascular foreign body retrieval in a severely preterm neonate. By doing so, we hope to contribute valuable information to the limited pool of knowledge on this subject and offer clinicians an alternative approach that could potentially minimize surgical risks while ensuring effective treatment. This case report is reported according to the CARE (case report guidelines) 2017 criteria.5\n\n\nCase report\n\nA 14-day old, Asian, male preterm neonate was referred to Gatot Soebroto Army Hospital, Indonesia due to intravascular foreign body. Previously, the neonate was born during his 30 weeks of gestation with birth weight of only 1,600 grams and birth height of 39 cm. He was then admitted to the neonatal intensive care unit (NICU) due to respiratory distress. During his admission, an umbilical catheter was placed for vascular access, and he was intubated. A week following the birth, his condition had improved, he was extubated and had continuous positive air pressure (CPAP) worn. However, a fragment of the umbilical catheter was left behind. Two intravenous antibiotics (ampicillin and gentamicin) were administered to the patient for over a week; however, his condition did not improve. During the physical examination, his respiratory rate was 55 times a minute with heart rate of 168 times per minute and peripheral oxygen saturation of 96% on ventilator.\n\nHis laboratory results were significant for leucocytosis with normal thrombocyte and C-reactive protein. The other laboratory results were unremarkable. Based on the thoracoabdominal X-ray imaging, a radiopaque catheter was found at the right paravertebral region, behind the liver, at the level of Th8-L2 and pulmonary infiltrate (Figure 1).\n\nFollowing admission, the patient underwent explorative laparotomy by the paediatric surgeon team in order to extract the foreign body but to no avail. He was then referred to the interventional radiology department for intravascular foreign body extraction using endovascular approach.\n\nDuring the procedure, an aseptic and antiseptic action was performed in the right femoral region, where attempts were made to puncture the vein using a 21 Gauge (G) needle under ultrasound guidance. After several unsuccessful attempts at venous puncture, to expedite the process for the patient, a venous incision was performed by the paediatric surgeon team. Following exposure of the femoral vein, puncture was successfully achieved with a 21 Gauge needle, followed by the insertion of a 0.032” guide wire (Terumo®, Tokyo) under low-dose fluoroscopy in the cath lab. Subsequently, a 4 French (Fr) introducer sheath and a 4 Fr vertebra catheter (Terumo®, Tokyo), along with a longer guide wire, were threaded through the sheath and directed towards the inferior vena cava. With precision, an exchange was made using the catheter and the 10 mm, 4 Fr One Snare wire (Merit Medical®, Ireland), which were manoeuvred to encircle the foreign body with its looped wire. Once securely ensnared, the foreign body (the umbilical catheter) was gently extracted. The instruments were subsequently removed, completing the procedure successfully (Figure 2).\n\n(a) Insertion of guide wire. (b) Insertion of One Snare wire (c). Encirclement of the foreign body with looped wire. (d) Extraction of the foreign body. (e) The foreign body was carried along to the puncture site.\n\nAfter the surgery, a 5 cm long umbilical catheter was extracted from the inferior vena cava of the patient (Figure 3). Two days following the procedure, his clinical condition had improved. The patient had switched to nasal canula on the second postoperative day and had no supplementary oxygen on the third postoperative day. Oral intake was started on the third postoperative day, which was well-tolerated by the patient.\n\n\nDiscussion\n\nIFB is one of the most severe iatrogenic complications of catheterization.2,3 It is especially dangerous in neonates who have delicate anatomical structures and limited physiologic reserves.2,4 However, the umbilical vein catheters are widely used in critical care of the neonates for rapid and dependable central access in preterm babies. Other than being displaced and left behind, injury to other organ during insertion, infection, thromboembolism, fracture of the catheter causing embolization, and even cardiac perforation are notable complications of umbilical vein catheters.1\n\nThe case presented herein exemplifies the challenges and complexities associated with IFB retrieval in a severely preterm neonate. Traditional surgical interventions, as attempted initially, proved to be unfruitful for the removal of the IFB in this case. This highlights the anatomical and physiological constraints that traditional surgical methods encounter in such vulnerable patients.\n\nOur experience underscores the utility of an endovascular approach as an alternative, especially when conventional surgical techniques fail or are not feasible. Notably, the minimally invasive nature of the procedure, guided by real-time imaging, allowed for a targeted intervention that minimized tissue disruption and potential complications. Similar imaging technique (low dose fluoroscopy) has been used by some studies about intravascular foreign body retrieval in neonates.2 Given the severely preterm neonate’s frail constitution and the high risks associated with invasive surgical procedures, the endovascular approach offered a less traumatic and more controlled methodology for IFB retrieval.\n\nThere are several techniques in performing endovascular extraction of intravascular foreign bodies, such as gripping the foreign body from outside using a snare or forceps, displacing the foreign bodies using balloons or stone retrieval baskets, or using a guidewire and low-profile balloons to grasp the foreign body.2,6 However, gripping the foreign body is only feasible in relatively large vascular channel, not in smaller vessels such as in neonatal cases.2 Moreover, there are a few purpose-designed devices for intravascular foreign bodies retrieval, such as Amplatz gooseneck (ev3), Trefoiel En-Snare (Merit Medical), Dormia baskets, Alligator retrieval forceps (Cook Medical and ev3), and Myocardial biopsy forceps (Cook Medical).3 These devices can have a range of sizes, variable range of emergences (from 0 degree to 90 degree) and more than one loops, greatly enhancing the manipulation techniques of the IFB retrieval.6 In this case, there are several unique technical challenges, such as difficulty in venous puncture, which requires venous incision by paediatric surgeon team and the use of specialized equipment like a 4 Fr vertebra catheter and the One Snare wire. This technique is also explained by Rossi et al. (2018) using the name of “Loop snare grasp-guide wire technique”.6 The manipulation of these instruments is crucial and should be undertaken by healthcare providers with expertise in endovascular techniques, especially in such a delicate patient population.\n\nThe marked improvement in the patient’s condition post-procedure emphasizes the effectiveness of this approach. Successful removal of the umbilical catheter led to clinical improvements including normalized leukocyte counts and better tolerance to oral intake. Similar results was found after the extraction of embolized umbilical catheter in preterm neonates in other studies.1,2 Furthermore, the immediate postoperative course, with a quick switch from ventilator support to nasal cannula and eventually to room air, suggests shorter recovery time and lower morbidity, corroborating the benefits of a minimally invasive approach.\n\nThe successful removal of an intravascular foreign body via endovascular technique in a severely preterm neonate highlights the potential of this approach as a viable alternative to more invasive surgical procedures. However, it is imperative to recognize the technical nuances and challenges involved, especially in this fragile patient population. This case serves not only as a testament to the evolving capabilities of minimally invasive techniques but also as an invitation for further research to optimize and standardize such approaches in neonatal care.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the guardian of the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nAcknowledgements\n\nThe authors would like to thank Gatot Soebroto Central Army Hospital and University of Pembangunan Nasional “Veteran” Jakarta Indonesia.\n\n\nReferences\n\nPatel J, Ramarao S, Desai J, et al.: A case report of embolized umbilical venous catheter retrieval from the heart via femoral access in 660 g premature neonate. Radiol Case Rep. 2019; 14: 1415–1419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYadav MK, Pillai R, Unni M, et al.: Iatrogenic Intravascular Foreign Body Retrieval in a Neonate. J. Clin. Interv. Radiol. ISVIR. 2020; 4: 130–132. Publisher Full Text\n\nWoodhouse JB, Uberoi R: Techniques for intravascular foreign body retrieval. Cardiovasc. Intervent. Radiol. 2013; 36: 888–897. Publisher Full Text\n\nPazinato LV, Leite TF d O, Bortolini E, et al.: Percutaneous retrieval of intravascular foreign body in children: a case series and review. Acta Radiol. 2022; 63: 684–691. PubMed Abstract \| Publisher Full Text\n\nRiley DS, Barber MS, Kienle GS, et al.: CARE guidelines for case reports: explanation and elaboration document. J. Clin. Epidemiol. 2017; 89: 218–235. PubMed Abstract \| Publisher Full Text\n\nRossi UG, Rollandi GA, Ierardi AM, et al.: Materials and techniques for percutaneous retrieval of intravascular foreign bodies. J. Vasc. Access. 2018; 20: 87–94." }	[ { "id": "267553", "date": "17 May 2024", "name": "Tapas Ranjan Behera", "expertise": [ "Reviewer Expertise Surgical improvisation", "Cancer Therapy. Past experience with similar case of lodged peritoneal dialysis catheter which had lead to a published case report." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStrengths & weaknesses: Unique report regarding a complex case of entrapment of umbilical catheter fragment in a pre-term neonate that was successfully managed by endovascular retrieval of the foreign body. Elaboration and discussion about the possible causes of foreign body entrapment in this case would make the report informative.\nShort summary & relevance: Intravascular foreign body retrieval in neonates are critical to deal with, more so when the neonate is pre-term. The authors have described the effective removal of the foreign body by the endovascular approach.\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": "349487", "date": "26 Dec 2024", "name": "Naveen Jain", "expertise": [ "Reviewer Expertise neonatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nImportant and relevant information. Well written article. Alll details of the case and management details are clearly elucidated, will help many similar high risk babies, an abdominal surgery may be avoided if such information is known to neonatologist . We have reported a similar case of a term born baby (yadav et al; included in the references) The article can be indexed.\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/13-55
https://f1000research.com/articles/13-54/v1	10 Jan 24	{ "type": "Review", "title": "Distinct fibroblast functions associated with fibrotic and immune-mediated inflammatory diseases and their implications for therapeutic development", "authors": [ "Alexander M. S. Barron", "Thomas Fabre", "Saurav De", "Thomas Fabre", "Saurav De" ], "abstract": "Fibroblasts are ubiquitous cells that can adopt many functional states. As tissue-resident sentinels, they respond to acute damage signals and shape the earliest events in fibrotic and immune-mediated inflammatory diseases. Upon sensing an insult, fibroblasts produce chemokines and growth factors to organize and support the response. Depending on the size and composition of the resulting infiltrate, these activated fibroblasts may also begin to contract or relax thus changing local stiffness within the tissue. These early events likely contribute to the divergent clinical manifestations of fibrotic and immune-mediated inflammatory diseases. Further, distinct changes to the cellular composition and signaling dialogue in these diseases drive progressive fibroblasts specialization. In fibrotic diseases, fibroblasts support the survival, activation and differentiation of myeloid cells, granulocytes and innate lymphocytes, and produce most of the pathogenic extracellular matrix proteins. Whereas, in immune-mediated inflammatory diseases, sequential accumulation of dendritic cells, T cells and B cells programs fibroblasts to support local, destructive adaptive immune responses. Fibroblast specialization has clear implications for the development of effective induction and maintenance therapies for patients with these clinically distinct diseases.", "keywords": [ "Fibroblast", "fibrosis", "inflammation", "autoimmunity", "therapy", "cytokine" ], "content": "Introduction\n\nWhat pushed you over the edge, huh? I bet it started with something small: a little too much to drink, sitting in traffic getting a lung-full of diesel exhaust or a drafty window in your apartment. Maybe your boss starts making fun of you like that bully did when you were in middle school. Whatever the insult, it’s not a big deal if it only happens once. You shake it off. But it just … keeps … happening. Once you could take. But this? No. You broke, and you broke bad. And now we’re here in this grimy room that smells like cheap, week-old coffee and donuts made from dirty gym socks. Neither of us wants to be here, and we don’t have to be for long. I just want to know two, simple things: why are we here and how did you do it?\n\nWell, this isn’t simple. Not all criminals are the same. Some are serial killers or arsonists, while others are burglars or blackmailers. It’s not just people that break bad; fibroblasts do it, too, and in just as many ways. They can be like Avon Barksdale, Marlo Stansfield or Bodie enabling neutrophils, macrophages and other innate immune cells to be the Bubbles of fibrotic diseases (FD). Or they can be like Viktor Bout and Yevgeny Prigozhin, organizing and arming forces of T, B and dendritic cells (DCs) in immune-mediated inflammatory diseases (IMID). Arson isn’t too different from how fibroblasts destroy articular cartilage in rheumatoid arthritis (RA). Poe’s Montresor immured Fortunato which, in a way, is what fibroblasts do in FD. As many ways in which people commit crimes, so too can fibroblasts be pathogenic in FD and IMID. In this review we will largely focus on IMID and sterile FD not associated with tumors. Although fibroblasts interact with many types of cells, this review will largely cover their relationships with leukocytes and the extracellular matrix (ECM).1–13 By the end of this, you’ll know some of the reasons why fibroblasts break bad, what drives them do it and a few of the questions we still need to answer.\n\n\nRecruitment and positioning of specialized leukocytes\n\nLeukocyte infiltrates are common across diseases that clinically manifest as either FD (e.g. idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH) and systemic sclerosis (SSc)) or IMID (e.g. rheumatoid arthritis (RA), Sjogren’s syndrome (SS) and discoid lupus erythematosus (DLE)). However, the magnitude, composition, density and organization of leukocyte infiltrates differ. Generally, the number of infiltrating leukocytes per mm3 appears to be lower in FD than IMID biopsies. Infiltrates in FD are generally dominated by innate immune cells like neutrophils and macrophages with smaller proportions of innate and tissue resident lymphocytes. IMID generally feature lymphocyte-rich infiltrates and aggregates. However, relatively few studies have directly compared the frequencies of lymphocyte aggregates between FD and IMID, and there is considerable histological heterogeneity.14–22 This comparison is further complicated in FD by changes in the composition and size of infiltrates as tissues become more fibrotic.23\n\nRegardless of the insult or injury that initiates FD or IMID, the earliest events are likely shared between these diseases. These likely include the release of alarmins, damage associated molecular patterns (DAMPs) and a local increase in vascular permeability that results in a serum response ((Figure 1A) reviewed in Refs. 24–26). Endothelial and myeloid cells express a wide repertoire of pattern-recognition receptors (PRRs) that recognize DAMPs, but fibroblast PRR expression appears to be more tissue-specific (reviewed in Ref. 27).3,28–32 Fibroblasts are more widely responsive to alarmins and serum.33–48 Although tissue-specific fibroblast responses to PRR signaling likely contribute to the sites and outcomes of FD and IMID, we focus on the more universal alarmin response here. Among the alarmins that fibroblasts respond to are IL-1α, IL-1β, and IL-33.\n\n(A) In acute injury, damage signals like serum, pattern recognition receptor (PRR) activation, interleukin (IL)-1 and tumor necrosis factor α (TNFα) activate fibroblasts. Whether fibroblasts are directly activated by IL-33 is still controversial. Fibroblasts activated by these signals produce chemokines that attract neutrophils like CXCL1 and CXCL8, as well as chemokines that attract classical monocytes like CCL2 and CCL5. (B) In immune-mediated inflammatory diseases (IMID) activated innate lymphocytes (including innate lymphoid, γδ T, NK, iNKT and MAIT cells) begin producing polarizing cytokines. This initiates local production of specialized chemokines which induce extravasation of the first polarized, memory αβ T cells that increase polarized cytokine production. In type 1 skewed responses this first wave of lymphocytes produces TNFα and IFNγ, which synergistically stimulate fibroblast secretion of CXCL9, CXCL10 and CXCL11. These cytokines attract more CXCR3+ type 1 or type 3 skewed T cells. IFNγ also appears to inhibit TNFα-induced production of neutrophil chemokines by fibroblasts. Fibroblasts instead stimulated by type 3 cytokines including TNFα and IL-17A continue secreting neutrophil chemoattractants like CXCL1 and begin producing additional cytokines like CCL20. These cytokines attract additional CCR6+ type 3 skewed T cells. Accumulation of enough activated dendritic cells and T cells leads to significant TNFα/TNF receptor 2 and lymphotoxin α1β2/lymphotoxin β receptor (LTβR) signaling. Activation of these pathways in fibroblasts can induce an additional stage of specialization that recruits naïve αβ T cells via secretion of CCL19 and CCL21, and B cells through CXCL13 and/or CXCL12. (C) Relatively few αβ T cells accumulate in sterile fibrotic diseases (compared to IMID) leading to distinct signaling and fibroblast differentiation. In fibrotic diseases largely characterized by type 2 skewing, IL-4 and/or IL-13, especially in concert with active TGFβ, induce secretion of CCL11, CCL26 and CCL2 by fibroblasts. CCL11 attracts eosinophils, CCL26 attracts basophils and CCL2 continues to attract classical monocytes. Conversely, in fibrotic diseases that exhibit more type 3 skewing IL-22 and IL-17A, in combination with TGFβ stimulate secretion of neutrophil chemokines, like CXCL1 and CXCL5, and CCL2 by fibroblasts. Note that most human IMID and fibrotic diseases are characterized by mixed, rather than purely polarized, inflammation. Figure created with BioRender.\n\nMeasuring alarmins in patients is complex due to the lack of early diagnostics, heterogeneity in disease severity and course, medications, genetic background and other factors. Despite these challenges, increased expression of alarmins is associated with both FD and IMID although data from patient samples is relatively scarce. Increased IL-1β, IL-33 and TSLP protein was detected in the bronchoalveolar lavage fluid (BALF) of patients with IPF and SSc, although there was considerable variability between cohorts.49–53 Higher levels of IL-1α were also observed in the serum from some SSc patients.54 SSc patients treated with bermekimab, an IL-1α antagonist antibody, in a 12-week phase 2A trial had better lung function and fewer swollen joints supporting a pathogenic role for this cytokine in some patients.55 Synovial fluid from RA patients had higher levels of IL-1β than osteoarthritis patients, although the detected levels were lower than TNF and IL-6.56–58 This difference in local expression may partly explain the modest ACR score improvements in RA clinical trials of Anakinra compared to TNFα antagonists and tocilizumab.59,60 Higher serum levels of IL-33 were observed in a subset of RA patients, which was associated with pulmonary complications and more severe disease.61,62 Elevated synovial fluid IL-33 also distinguished RA patients from those with osteoarthritis.62,63 These data suggest that alarmins can activate fibroblasts in either FD or IMID and may be pathogenic, although the biomedical community would benefit from higher-powered studies in genetically diverse patient cohorts.\n\nIL-1α is expressed by various cell types in the cytoplasm and nucleus and can be released during cellular damage without any modification. In contrast, immature IL-1β must be processed by a specialized complex known as the inflammasome. The inflammasome is a supra molecular organizing complex comprised of adapter proteins and active caspases. Formation of the complex results in caspase mediated cleavage of IL-1β and the gasdermins that form the membrane pores required for the release of mature IL-1β (reviewed in Ref. 26). Both IL-1α and IL-1β bind to IL-1R1 on fibroblasts to elicit secretion of a variety of chemokines. Stimulation of fibroblasts with recombinant IL-1α in vitro induced CXCL8 RNA within 30 minutes and secretion of bioactive CXCL8 and CCL2 within 24 hours (Figure 1A). Conditioned media from damaged lung epithelial cells induced transcription of these same chemokines, which required IL-1α, not IL-1β. In this case, IL-1α-driven transcription of these chemokines synergized with TLR3 signaling. This required at least JAK2, TAK1 and IKK2.36,44,45 IL-1β induced production of CXCL8 and CCL2 as well as CXCL1 and CCL5/RANTES (Figure 1A).46,48 Together, these data show that fibroblasts from multiple sites can respond to the alarmins IL-1α and IL-1β by secreting neutrophil and monocyte chemokines.\n\nDo these in vitro findings translate in vivo? Alum injection demonstrated that IL-1α-/- mice showed a partial decrease in neutrophil infiltration into the site of injection, while IL-1α-/- IL-1β-/- mice showed complete ablation of neutrophil infiltration indicating some redundancy between these cytokines.64 Murine myocardial infarction results in large monocyte and neutrophil infiltrates into the damaged heart, which required IL-1 signaling in non-myeloid cells.65 Conditional deletion of IL-1R1 using the Col1a2-CreER(T) line in this model reduced cardiac leukocyte infiltration, although the authors did not specifically quantify neutrophil or monocyte infiltrates.47 Acute, diphtheria toxin-mediated depletion of Kupffer cells similarly demonstrated that IL-1α, IL-1β and TNFα activate hepatic stellate cells (fibroblastic cells found in the liver) and liver sinusoidal endothelial cells. Both cell types rapidly produced Ccl2 to recruit monocytes and replenish the Kupffer cell niche.66 Together these results suggest that IL-1α and IL-1β can stimulate fibroblast-mediated recruitment of neutrophils and monocytes in vivo. These data suggest that chemokine production by fibroblasts is important for monocyte and granulocyte recruitment in response to IL-1. However, a direct demonstration would require conditional, fibroblast-specific IL-1R1 knockout and quantification of monocyte and granulocyte infiltration.\n\nLess is known about fibroblast responses to IL-33 than IL-1α and IL-1β. Human and murine fibroblasts can express IL-33 in vivo and in vitro.62,67–76 As a nuclear and secreted factor that undergoes proteolytic processing, IL-33 biology is complex and requires further experimental clarification (reviewed in Ref. 77). The current picture is that homeostatic murine adventitial fibroblasts in several organs and differentiated lymphoid organ fibroblasts express IL-33 in vivo.67–69,71,72 Either this is different for human fibroblasts or IL-33 expression decreases with traditional fibroblast culture methods: human fibroblasts required stimulation to induce IL-33 expression in vitro. Combined stimulation by TNFα and IFNγ triggered IL-33 expression by dermal fibroblasts, which was further enhanced by TLR3 activation.76 Either TNFα or IL-1β alone stimulated IL-33 production by synovial fibroblasts from RA patients, which also synergized with TLR3 activation.74,75 However, secreted IL-33 was rarely detected in these experiments.73–76 Does this mean that fibroblasts express nuclear IL-33 but cannot secrete it?\n\nIt is possible that, under the conditions used in these studies, the cultured fibroblasts did not secrete IL-33. However, because conditional deletion of IL-33 from fibroblasts and ST2 from leukocytes in vivo yielded similar phenotypes, it seems likely that fibroblasts can secrete IL-33.78,79 There are two alternative explanations that are also plausible. First, oxidization of IL-33 after secretion prevents binding to its receptor IL1RL1/ST2 and subsequent signaling. Distinct antibodies are required to detect oxidized IL-33 versus reduced IL-33.80 Thus, the lack of detectable secreted IL-33 in these studies may have been due to this conformational change. This, however, seems less likely than the second alternative explanation. Alternative splicing of IL1RL1 yields both transmembrane and soluble proteins. The soluble form antagonizes IL-33 signaling.81 Proteolytically processed IL-33 is a small protein (~175-161 amino acids).82 Although not conclusively demonstrated for most IL-33 antibodies, it seems likely that the binding of IL-33 to soluble IL1RL1 would cross-block subsequent detection by anti-IL-33 detection agents. Production of soluble IL1RL1 by fibroblasts would thus impair detection of secreted IL-33 from these cells. Further empirical dissection of IL-33 secretion by fibroblasts would provide useful clarification.\n\nRegardless of whether fibroblasts secrete IL-33 they can express the protein. Since the most robust evidence is for intracellular expression of IL-33, does this form exert effects on fibroblasts? When expressed in the nucleus, full-length IL-33 appears to repress transcriptional activation by NF-κB. This inhibition translated to reduced secretion of CXCL8 and CCL2 by RA synovial fibroblasts stimulated with TNFα alone or the combination of TNFα, IL-1β and a TLR3 agonist.74,75 Thus, induction of intracellular IL-33 expression by IL-1 and TNFα may act as a negative feedback regulator of acute inflammatory responses by fibroblasts. Many cells aside from fibroblasts can secrete processed, highly active IL-33. Is there evidence that fibroblasts are impacted by this secreted form?\n\nData from RA synovial fibroblasts suggests that exogenous IL-33 may have the opposite effect from that of intracellular IL-33. Stimulation with recombinant IL-33 increased secretion of CXCL8 and transcription of several other genes.73 Blocking IL1RL1 inhibited induction of some of these by IL-33 suggesting that fibroblasts may be activated by IL-33.74 However, IL-33 also increased transcription of IL-1β in RA synovial fibroblasts.73 As IL-1β can stimulate many of these same transcripts it is currently unclear which effects were the direct result of IL-33 signaling and which were indirectly stimulated by IL-33-driven IL-1β production (Figure 1A). This is somewhat controversial as another study was unable to detect direct effects of exogenous IL-33 on the RA synovial fibroblasts they used.75 All in all, it seems likely that fibroblasts can respond to IL-33 under some circumstances, however the effects and relative importance of this require further work.\n\nCorrelating with the elevated alarmin expression in patients with FD and IMID, levels of the alarmin-stimulated chemokines CCL2, CCL5, CXCL2 and CXCL8 were also higher patients with FD and IMID than healthy controls.83–88 In addition to producing these chemokines in response to IL-1α and IL-1β, fibroblasts can produce these when stimulated with TNFα (Figure 1A). This is true for human cells and animal models of both FD and IMID and occurs both in vitro and in vivo.17,22,85,89,90 And in patients with inflammatory bowel disease (IBD) a fibroblast IL-1 response signature is correlated with local neutrophil infiltrates and resistance to both TNF antagonists and steroids.37 This supports the redundance of IL-1 and TNFα in a subset of patients. These patients would likely benefit from combination therapy targeting these redundant signaling systems. If alarmins and DAMPs are produced in both FD and IMID, why are the infiltrate compositions generally so different between theses distinct clinical disease manifestations? The answers to these questions are currently unclear, although they likely involve the results of combinatorial signaling events, which will be explored next.\n\nDuring the priming phase of antigen-specific responses, including IMID, immature DCs and tissue-resident macrophages are also activated by alarmins and PRR. Activated perivascular macrophages secrete chemokines like CXCL2 that initially attract maturing DCs towards blood vessels. Without an influx of activated antigen-specific T cells (or the presence of antigen-specific tissue resident T cells), these maturing DCs leave the damaged tissue through the draining lymphatics and prime antigen-specific naïve T cells in lymph nodes.91–93 Similarly, in sterile FD where tolerance appears to be maintained any DCs that are activated during the initial insult likely migrate to the draining lymph nodes but fail to induce a T cell response.94,95 How these discrepant responses occur is an interesting question, but beyond the scope of this review. Simultaneously, neutrophils and monocytes are drawn into the tissue by the chemokines produced in response to alarmins, PRR signaling and/or the serum response as described previously.\n\nAt this early stage, the monocytes likely differentiate into a mixture of macrophages and DCs. In IMID, the activated T cells proliferate, differentiate, exit the lymph node and enter the damaged tissue through activated post-capillary venules.96,97 These T cells are restimulated by perivascular DCs presenting cognate antigens, and these reactivated T cells retain the DCs in the affected perivascular or peri-damaged space.91,98–103 Retention of these DCs and the initial accumulation of activated antigen-specific T cells likely leads to nucleation of lymphocyte aggregates and infiltrates where lymphocytes predominate. Conversely, the lack of antigen-specific T cell influx and DC retention in FD leads to an entirely different set of signals. This, we propose, is where fibroblast activation states and functions in FD and IMID diverge.\n\nWithin hours of damage or insult fibroblasts in non-lymphoid tissues turn off their homeostatic programs and begin expressing Podoplanin (PDPN).104,105 PDPN expression by human fibroblasts from multiple sites is induced by acute inflammatory stimuli like IL-1β and TNFα, consistent with this activation occurring prior to reparative or fibrotic signals like TGFβ.104,106,107 Unliganded PDPN initiates fibroblast contraction by binding to ezrin, activation of Rho kinases (ROCK) and phosphorylation of ezrin/radixin/moesin family proteins and myosin light chain.108–110 Fibroblast contraction can activate latent TGFβ through integrin αV heterodimers. Active TGFβ is both a suppressor of generally anti-fibrotic type 1 immune skewing and one of the most potent pro-fibrotic signals.111–116 Therapeutic inhibition of integrin mediated latent TGFβ activation in fibrotic diseases is being pursued by multiple companies including Pliant Therapeutics.117 Although TGFβ suppresses signals associated with type 1 immune polarization it appears to spare production of myeloid and neutrophil chemokines by fibroblasts, at least as part of the serum response and in combination with TLR4 or IL-17A.118 Contraction also increases the local stiffness of the ECM which can initiate durotaxis and tension responses through integrins.119\n\nDurotactic attraction between myofibroblasts and macrophages is believed to be more effective than chemotaxis and haptotaxis.119,120 Macrophages are especially important collaborators in generating the fibrotic niche: genetic or chemical depletion of macrophages abrogates fibrosis progression.121–128 Once in proximity fibroblasts and macrophages establish strong adhesion through homotypic Cadherin-11 (CDH-11) contacts. This creates a pro-fibrotic niche where latent TGFβ produced by macrophages can be activated by myofibroblast contraction.129 Thus, therapies aimed at limiting mechano-sensing in fibroblasts and macrophages are currently being explored. These include inhibition of PDPN, ROCK1/2 and focal-adhesion kinase (FAK) as well as strategies to dissociate fibroblasts from macrophages (e.g. CDH-11 antagonists). Immune cells other than macrophages can also sense mechanical forces, which similarly impact their activation and function.130,131 Therefore, it is possible that neutrophils, eosinophils and others are recruited to the pro-fibrotic niche through the same mechanisms. Similarly, there are mechanisms other than PDPN that activate fibroblast contractility which likely play important roles in the fibrotic activation of fibroblasts.132–135 Thus, the combination of contraction-induced durotaxis and selective sparing of myeloid and granulocyte chemokine expression already appears to differentiate fibroblast functions in FD from those in IMID.\n\nUnlike the macrophages and neutrophils that predominate in fibrosis-associated leukocyte infiltrates, DCs express the PDPN ligand CLEC-2, which decouples PDPN from ezrin and inhibits fibroblast contraction.108,109,136 This is one way that early differences in the composition of leukocyte infiltrates may result in different fibroblast functions and clinical pathologies in IMID and FD. Whether this is true in vivo requires experimental validation, though, as CD177, which is expressed by neutrophils, binds to PDPN and may exert similar effects.110 Relaxation of fibroblasts around injured areas through interaction with DCs is likely necessary to accommodate early infiltration of T cells like the swelling and expansion of lymph nodes during immune responses.9,10,108,109,137–139 In addition, these DCs may provide TNFR2 or LTβR ligands to trigger further fibroblast specialization.140–145 Some sites, like the lung, seem to require local retention of DCs for sufficient secondary fibroblast activation and lymphocyte accumulation regardless of whether the response is type 1 or type 3 skewed.146–152 In others, like the salivary glands, IL-22 and IL-13 produced by γδ and iNKT cells and ILC2s may be sufficient to initiate lymphocyte accumulation and secondary fibroblast activation. Sustained lymphocyte recruitment in this case appears to require an influx of IL-22 producing αβ T cells.107,153,154 Regardless of the site and activating signals, this second round of fibroblast specialization in IMID seems to involve fibroblast proliferation and the induction of T cell recruiting chemokines by these cells.105,107,146–159\n\nPatterns of lymphocyte infiltrates can vary from diffusely spread throughout the tissue to tightly aggregated.14,160–163 Aggregates frequently form around microanatomic structures such as blood vessels and pulmonary airways.14,67,163,164 Among diseases with T cell aggregates, their size and organization can vary between small (~5-20), medium (~20-50) and large (>50).14,162 Small T cell aggregates can likely be recruited by activated perivascular DCs without the help of specialized fibroblasts, but this may differ between mice and humans.14,91,98 Growth of T cell aggregates beyond ~10 cells appears to require the expansion of specialized fibroblast networks.14,91,156–158,164 As polarized, activated memory T cells enter an inflamed tissue, or as ILCs and other tissue-resident lymphocytes are activated, the combination of innate and adaptive cytokines likely tunes the fibroblast chemokine program to attract the appropriate lymphocytes for the response.\n\nIn the case of type 1 polarization, the synergistic combination of TNFα or IL-1 and IFNγ suppresses neutrophil chemokines in vitro while increasing production of CXCR3 ligands via cooperation between STAT1 and Nuclear factor-κB (NF-κB, Figure 1B).165–169 Fibroblasts expressing CXCL9 are important for CXCR3+ TH1 differentiation in lymph nodes, and similarly activated fibroblasts expressing multiple CXCR3 ligands are increased in the salivary glands of SS patients, synovia of RA patients and in tumors.70,170–174 Likely these activated fibroblasts contribute to extravasation of type 1 or type 3 lymphocytes expressing CXCR3 (Figure 1B).98,175–178 Conversely, combined stimulation of fibroblasts with the type 3 stimuli TNFα and IL-17A decreased CCL3 and CCL5 secretion while increasing expression of CCL20 and neutrophil chemoattractants.90,179–181 Whether a similar fibroblast state occurs in patients awaits experimental verification. These fibroblasts are likely adapted to draw in type 3 polarized CCR6+ lymphocytes and neutrophils (Figure 1B).178,182 Specialized adventitial fibroblasts can indirectly instigate type 2 lymphocyte recruitment by creating specialized niches for ILC2s and eosinophils.67 These observations suggest an evolving dialogue between leukocytes and fibroblasts that can recruit specialized lymphocytes in IMID with distinct immune polarization. The relative importance of fibroblast production of these chemokines requires experimental investigation in vivo.\n\nThe recruitment of polarized lymphocytes by fibroblasts appears to occur early in the growth of aggregates. Small lymphocyte aggregates are dominated by CD45RO+ T cells, which have likely been polarized during their activation in secondary lymphoid organs (SLO). Further fibroblast specialization seems to be required to recruit B cells and naïve T cells as the frequency of B cells and naïve T cells increases with aggregate size.14,156,157,164 Extravasation and localization of naïve T cells into SLO requires secretion of the CCR7 ligands CCL19 and CCL21 by fibroblasts.70,183–187 Similar fibroblasts expressing CCL19 and CCL21 emerge in several IMID and tumors, and also appear to correlate with T cell infiltration and lymphoid aggregate size.21,156–158,171,188–192 In patients with interstitial pneumonia with autoimmune features, BALF CCL19 concentrations and lymphocyte numbers were positively correlated supporting CCL19-induced pulmonary lymphocyte infiltration. Longitudinal tracking of these patients demonstrated that high baseline BALF CCL19 and lymphocytes were associated with a lack of pulmonary fibrosis up to one year later. This observation supports divergent fibroblast activation occurring in IMID versus FD, and that the signals that induce these states may cross-inhibit each other.193 What induces fibroblast production of CCL19 and CCL21 in IMID is also unclear, but the clues we have are discussed below.\n\nIn lymphoid organs, there is evidence for TNFR1/2 and LTβR ligands driving expression of these chemokines (Figure 1B). Pharmacological or genetic antagonism of LTβR signaling reduces or prevents CCL21 and CCL19 expression in models of IMID.107,140,141,145,184,192,194–197 However, expression of CCL21 in the lungs appears to be independent of both TNFR1/2 and LTβR signaling.198 We are unaware of in vitro confirmation of CCL19 and CCL21 induction in fibroblasts by these signals. Larger reductions in CCL19 RNA were seen in RA patients with EULAR responses to disease-modifying anti-rheumatic therapies (DMARDs) than those who failed to respond, which suggests a potential association with pathogenesis.199 But whether fibroblasts drive significant disease pathology by producing these chemokines remains an open question.\n\nB cell infiltrates into IMID tissues are generally less common than T cell infiltrates.14,156,188 However, in some IMID like SS, RA and DLE, large B cell infiltrates occur in substantial proportions of patients (approximately 42% in RA, 89% in SS and 92% in DLE) although some of these cohorts were relatively small.14,156,157 Aggregates containing both T and B cells can either be non-segregated, with T and B cells evenly distributed throughout, or segregated, with discrete T cell zones and B cell follicles similar to SLO.14,21,156,157,164,192,200,201 In this review we define “tertiary lymphoid structures” (TLSs) as the subset of lymphocyte aggregates with these discrete T cell zones and B cell follicles. Infiltration of B cells in IMID, especially smaller infiltrates that lack T-B cell separation, occur in the absence of (or preceding) mature follicular dendritic cells (FDC, B cell supporting fibroblasts in SLO).14,156,157 Regardless of FDC maturation status, CXCL13 generally appears necessary for inflammation-associated increases of B cells in peripheral tissues (Figure 1B).156,157,202–206 However, in specific cases like the lung B cell infiltrates can occur in the absence of CXCL13 and appear to require CXCL12 (Figure 1B).150 This could suggest that CXCL12 is sufficient for B cell extravasation and localization, or that CXCL13 negative pulmonary TLSs are exclusively populated by resident memory B cells.207,208\n\nIn IMID where CXCL13 is induced, expression by myeloid and peripheral helper T cells (TPH) is likely sufficient to drive the initial accumulation of a few memory B cells.156,157,202,203,209 As inflammation progresses, however, expression of CXCL13 by fibroblasts appears important for generating local germinal centers.14,156,157,162,203,210 During SLO development, CXCL13 expression can be induced by or independent of TNFR1/2 and LTβR signaling depending on the site and timing (Figure 1B).145,195,211–213 Similarly mixed observations have been made in murine models of IMID and lymphoid aggregate formation. Antagonizing LTβR signaling was sufficient to reduce or inhibit CXCL13 production in the non-obese diabetic (NOD) model of SS and a model of autoimmune pancreatitis.192,194,197 IL-22 production is necessary for induction of CXC13 by salivary gland and lung fibroblasts, but the salivary gland induction depends on LTβR while the lung does not.154,214 Together, these data suggest that a stepwise dialogue between leukocyte infiltrates and fibroblasts causes fibroblast differentiation that may separate IMID from FD.\n\nAlarmins induced neutrophil and monocyte chemokine secretion by fibroblasts as well as contraction-induced durotaxis. This first fibroblast activation state is likely similar between IMID and FD (Figure 1A). Next, in IMID a first wave of activated, polarized T cells infiltrates the tissue resulting in local retention of DCs and fibroblast relaxation. Collaboration between TNFR and LTβR ligands produced by DCs with adaptive cytokines produced by T cells, ILCs, NK, MAIT and iNKT cells results in a second step of fibroblast differentiation. Second-stage IMID fibroblasts likely begin to express CCL19, CCL21, CXCL12 and CXCL13, which recruits B cells and naïve T cells (Figure 1B). Perhaps, as recently described, ligation of ICOSL on fibroblasts or CD40/CD40L-dependent TPH/B cell interactions are necessary for further lymphoid aggregate expansion and compartmentalization.196,215 Conversely, in FD the monocyte- and neutrophil-rich infiltrates promote fibroblast contractility and stiffening of the matrix. This leads to the activation of latent TGFβ, which combines with alarmin signaling to continue monocyte and neutrophil extravasation while preventing T cell infiltration (Figure 1C). From these observations, divergent paths of fibroblast activation between FD and IMID begin to emerge.\n\n\nRationing of growth factors\n\nFrom the previous section it appears that fibroblasts can contribute to differential leukocyte extravasation in FD and IMID. However, the number of cells that can occupy a tissue is regulated by the availability of growth and survival factors.216–219 This observation raises our next two questions. First, having contributed to leukocyte infiltration, do fibroblasts provide survival signals to these cells in FD and IMID? The answer, as this section will demonstrate, appears to be yes. These survival signals can take the form of soluble factors, transmembrane molecules or modifications of the ECM. We will concentrate on secreted growth factors in this review. Second, do fibroblasts sense and regulate cell composition by changing the types and quantities of growth factors they provide? This is an active area of research, but initial data suggests that they do.\n\nIn line with the correlation between elevated alarmin levels and monocyte and granulocyte chemokines in FD and IMID, growth factors to support these innate leukocytes are also elevated in some diseases. GM-CSF but not G-CSF was higher in synovial fluid of RA versus OA patients, and there appeared to be an inverse relationship between synovial fluid GM- and G-CSF concentrations.220–222 Levels of GM-CSF correlated with IL-1β and TNFα quantities in the synovial fluid of RA patients, and both IL-1β and TNFα induced secretion of GM-CSF by synovial fibroblasts.222 GM-CSF protein levels were higher in urine from patients with active lupus nephritis compared to inactive systemic lupus erythematosus, other chronic kidney diseases and healthy controls.223 G-CSF was detectable in tears from both IgG4-related disease and SS patients, however levels in SS patients did not appear to differ from healthy controls.224–226 Data on growth factor levels in local fluid and biopsy samples in FD is scarcer than for IMID. What is available shows a trend towards increased GM- and G-CSF in some patients, which was positively correlated with neutrophils in these samples.18,227–230 Although the quality and amount of data varies considerably between diseases, increased in growth factor quantities were generally associated with increased acute damage signals and monocyte and neutrophil chemokines in these patients. Might fibroblasts contribute to the production of these growth factors?\n\nFibroblasts from various lymphoid and non-lymphoid tissues produce growth factors for myeloid cells and granulocytes, although other cell types including endothelial cells can also play significant roles.66,231 Specialized fibroblasts in the murine lymph node expressed both Csf1/M-CSF and IL-34, the two Csf1r ligands, in vivo and induced expansion of classical monocytes and macrophages in vitro.70,232,233 Fibroblasts in the murine splenic red pulp appeared to serve a similar function as they also expressed both Csf1 and Il34.234,235 Targeted deletion of Csf1 in splenic red pulp, but not white pulp, fibroblasts dramatically reduced red pulp macrophages (Figure 2).235 These data support the importance of fibroblast-derived growth factors for maintenance of myeloid cells during homeostasis. What about after injury?\n\nDuring homeostasis, fibroblasts in several lymphoid and non-lymphoid tissues produce growth factors (generally Csf1/macrophage colony stimulating factor (M-CSF)) to sustain tissue-resident macrophage populations. Immediately upon injury, fibroblasts increase the amount of growth factors they secrete and begin producing Csf2/granulocyte-macrophage colony stimulating factor (GM-CSF) and Csf3/granulocyte colony stimulating factor (G-CSF). Increases in the amounts and breadth of growth factors produced by fibroblasts coincide with their elevated secretion of chemokines that attract granulocytes, monocytes and macrophages (described in subheading 1). Alarmins, damage-associated molecular patterns (DAMPs) and serum from leaking blood vessels can each stimulate these functional changes in fibroblasts. These acute damage signals can also synergize to further enhance the quantities of growth factors produced by fibroblasts. If the injury is successfully contained, fibroblasts can help replenish the depleted tissue-resident macrophage population by attracting monocytes and secreting M-CSF to induce their differentiation. Conversely, if damage signals persist chronic fibrotic (FD) or immune-mediated inflammatory diseases (IMID) can result partly because of divergent fibroblast functional states. Retention of dendritic cells (DCs) and infiltration of type 1 or type 3 polarized αβ memory T cells lead to progressive differentiation of fibroblasts in response to combinations of cytokines like tumor necrosis factor α (TNFα) and interferon γ (IFNγ) or TNFα and IL-17A. DCs can also relax fibroblasts, which decreases contractile activation of latent transforming growth factor β (TGFβ). Mature DCs or activated lymphocytes can produce TNFα and lymphotoxin α1β2 to initiate a second round of fibroblast specialization, triggering secretion of growth factors like IL-7 and IL-15 for T cells and BAFF and IL-6 for B and plasma cells. Fibroblasts appear to reach similar functional states in response to IL-22 and IL-13 in at least one murine model of IMID, although how these cytokines can induce such distinct fibroblast states in FD and IMID is currently unclear. Specialized fibroblasts producing IL-7, IL-15, BAFF and IL-6 likely underpin the formation of large lymphocyte aggregates or tertiary lymphoid structures in IMID. On the other hand, activation of latent TGFβ through several mechanisms in FD, including by contractile fibroblasts, synergizes with IL-4 and IL-13 or IL-22 and IL-17A to maintain production of GM-CSF and G-CSF. Both support monocyte differentiation and granulocyte survival within the fibrotic niche. Figure created with BioRender.\n\nOne-time depletion of homeostatic murine macrophages by diphtheria toxin induced a controlled, acute inflammatory response. Csf1 expression was induced in hepatic stellate cells (fibroblastic cells of the liver) upon acute depletion of liver resident Kupffer cells. Repopulation of the Kupffer cell niche by monocytes required M-CSF-Csf1r signaling. In this model, however, Csf1 was expressed by both stellate cells and liver sinusoidal endothelial cells in response to IL-1α, IL-1β and TNFα. The relative importance of Csf1 expression by each cell type remains an open question (Figure 2).66 A similar observation was made after acute depletion of GATA6+ cavity macrophages. Here, both mesothelial cells and capsule fibroblasts provided M-CSF.236 Again, the relative contributions of these two cell types to M-CSF production awaits experimental validation in this model. Regardless of their relative importance, these data indicate that fibroblasts play significant roles in the homeostatic maintenance of tissue macrophage and the restoration of tissue macrophage populations after acute injury. As we saw in the chemokine section above, fibroblasts responded to the alarmins produced in acute injury by attracting both monocytes and granulocytes. Like M-CSF, do fibroblasts supply Csf2/GM-CSF or Csf3/G-CSF to support these infiltrating innate immune cells?\n\nIn vivo many different cell types produce GM-CSF. Unlike the M-CSF-dependent tissue macrophages described in previous paragraphs, alveolar macrophages require GM-CSF in both humans and mice.237–244 Multiple cell types expressed GM-CSF in the homeostatic murine lung including type 2 innate lymphoid cells (ILC2), γδ T cells and alveolar type 2 epithelial cells (AT2). However, only GM-CSF from AT2 was required for alveolar macrophage differentiation and maintenance.245 The critical source of CM-CSF may differ between sites, and likely changes in disease. For example, in patients with various forms of inflammatory arthritis fibroblasts, endothelial cells and macrophages all appear to produce GM-CSF.246 To some extent this is recapitulated in the murine SKG arthritis model where GM-CSF production by fibroblasts and ILC2 is necessary for disease, but not in the serum-transfer induced arthritis model where GM-CSF from NK cells is required (Figure 2).247,248 The critical sources of G-CSF in vivo during homeostasis and insult or disease are less clear, although production by endothelial cells is necessary for TLR4-driven emergency granulopoiesis.249 These data indicate that fibroblasts may be important sources of GM- and G-CSF in IMID and FD, but that further work is necessary to determine this in vivo.\n\nIn vitro quiescent, growth inhibited human dermal fibroblasts produce almost no leukocyte growth factors. They respond to acute serum stimulation alone (in the absence of alarmins or other exogenous cytokines) by producing mostly bioactive M-CSF (little to no GM- and G-CSF). Stimulation with IL-1α induced secretion of M-, GM- and G-CSF with amounts of M->GM->G-CSF. Acute serum and IL-1α stimulations demonstrated additive synergy in the production of all three growth factors (Figure 2).250 Human lung fibroblasts also secreted M-CSF in response to acute serum stimulation. TNFα stimulation demonstrated additive synergy with serum in the induction of M-CSF, and triggered GM-CSF production. Co-treatment with the corticosteroid dexamethasone inhibited the induction of GM-CSF but not M-CSF by TNFα suggesting a potential mechanism of action for this steroid.251 This growth factor program appears to be conserved as TNFα, IL-1β, IL-17A or IFNγ each increased the ability of synovial, bone marrow and skin fibroblasts to support neutrophil survival in vitro, which largely depended on GM-CSF.90,222,252 Thus, fibroblasts activated by alarmins or serum in FD and IMID may contribute to the local survival of monocytes, macrophages and granulocytes although this may differ by tissue or disease (Figure 2). Further work is necessary to determine how important the production of these growth factors by fibroblasts is and will benefit greatly from the use of genetically engineered mice allowing inducible, fibroblast-specific deletion of these growth factors.\n\nAcute injury signals appear to be shared between FD and IMID. What leads to the expression of differentiated growth factor programs by fibroblasts translate into these divergent clinical manifestations? As acute injuries become chronic diseases, new signals arise with the changing cellular composition and continuing damage. Layering these new signals on top of chronic alarmin, serum and PRR signaling, and the cross-repression between these signals, is what seems to modify the growth factor programs of fibroblasts. This kind of cross-repression is common during organ development and immune cell differentiation.1,67,72,253 Studies of how sequential or simultaneous signals regulate the provision of growth factors by fibroblasts in adulthood are a newer undertaking with many open questions. Investigating these more complex interactions is, however, necessary to understand human diseases which are often characterized by mixed inflammatory and morphogenic signals.115,254\n\nTGFβ is one of the dominant cytokines mediating fibroblast activation and fibrosis in multiple tissues (reviewed in Refs. 255, 256). It is also a potent suppressor of some types of immune responses (reviewed in Refs. 257, 258). Given the relatively small size of leukocyte infiltrates in most FD (compared to IMID), and the predominance of myeloid cells and granulocytes, an obvious question is: does combining TGFβ with pre-existing alarmin, PRR or serum signaling suppresses the production of M-CSF, GM-CSF or G-CSF by fibroblasts? To our knowledge no publications have directly explored this question, but two pieces of evidence suggest that TGFβ may not suppress production of these survival factors. First, TLR4 knockout mice are protected from hepatic fibrosis, which correlates with lower levels of TGFβ signaling in hepatic stellate cells, reduced production of macrophage and neutrophil chemoattractants by hepatic stellate cells and reduced macrophage infiltration into injured livers.118 The reduction in both hepatic stellate cell TGFβ signaling and hepatic macrophage infiltration in TLR4 knockouts argues against TGFβ suppressing the production of myeloid survival factors by HSC. Second, serum or IL-1 alone stimulates the secretion of M-CSF, GM-CSF and G-CSF from human fibroblasts, and the combination of serum plus IL-1 demonstrates additive synergy.250 The 10% FBS-supplemented media that induced secretion of these three CSFs by fibroblasts likely contained ≥1000 pg/mL of bovine TGFβ, which can be activated by freezing and thawing and has bioactivity on human cells.250,259,260 Thus, it seems likely that fibroblasts continue to produce myeloid and granulocyte growth factors to support key constituents of the fibrotic niche when exposed to TGFβ, although this needs to be tested in vitro and in vivo (Figure 2).\n\nWhat about IMID where many patients have large increases of T and B cells that are frequently found in aggregates? IL-7 protein levels were higher in the synovial fluid of some RA patients compared to OA patients and it was locally transcribed in the synovium.261,262 Levels of IL-15 protein in synovial fluid of RA patients were also higher than OA patients in some cohorts, and this was also transcribed in the synovium.262–265 Fibroblasts isolated from the synovial tissue of RA patients spontaneously secreted more IL-7 and IL-15 than OA synovial fibroblasts. Production of both IL-7 and IL-15 from RA synovial fibroblasts increased in response to IL-1β or TNFα suggesting a role for NF-κB.262,266 IL-7 was detectable in tears from both IgG4-related disease and SS patients, however levels in SS patients did not appear to differ from healthy controls.224–226 IL-7 and IL-15 protein levels were also higher in urine from patients with active lupus nephritis compared to inactive systemic lupus erythematosus, other chronic kidney diseases and healthy controls.223 Do fibroblasts provide these growth factors in IMID to support the observed increases in lymphocytes?\n\nSmall aggregates predominantly comprised of T cells are likely sustained by IL-15 presented by myeloid and endothelial cells, IL-2 from locally proliferating T cells and IL-7 produced by lymphatic endothelial cells.91,267–272 Medium and large aggregates, though, appear to require the expansion and differentiation of specialized fibroblasts.14,137,273 Although we currently lack a definitive mechanistic explanation for this observation, specialized fibroblasts likely control the size of T cell aggregates in several ways. Fibroblastic reticular cells (FRC), which are found in the T cell zones of secondary lymphoid organs (SLO), directly control T cell numbers by limiting or increasing survival signals such as IL-7 and IL-15.184,185,269,274–276 The mechanisms by which fibroblasts are activated to produce IL-7 and IL-15 are still unclear. Genetic ablation of LTβR in Ccl19+ lymph node fibroblasts reduced Il7 expression, and transgenic overexpression of LTβR ligands in the pancreas induced Il7 suggesting that LTβR signaling may be sufficient.184,192 However, pharmacologic or genetic LTβR antagonism increased Il7 transcripts in bone marrow fibroblasts.277\n\nFibroblast regulation of T cell numbers in primary (PLO) or SLO through production or restriction of growth and survival factors like IL-7 seems likely.184,185,274,278–280 Expansion of specialized fibroblasts that produce similar T cell survival factors is likely required to increase the carrying capacity of inflamed tissues and support medium and large aggregates (Figure 2).14,105,107,185,191,252,268,281 However, despite our presentation of circumstantial evidence for this hypothesis it has yet to be directly tested. Rigorously testing this hypothesis is challenging as fibroblasts express similar transcriptional programs across tissues, and tissue-specific fibroblast modules are often shared with non-fibroblast cells in those same tissues.70,174,190,282 Conditional deletion is clearly necessary to avoid confounding developmental defects, as seen in the constitutive, fully-body IL-7 knockout.283 To untangle effects specific to the inflamed non-lymphoid tissue from potential effects of growth factor deletion in PLO or SLO fibroblasts, we propose orthotopic adoptive transfer of purified fibroblasts from mice genetically modified for conditional growth factor deletion or parabiosis between wild type and mice with fibroblast-specific conditional growth factor deletions.105,284 These experiments could be complemented by spatial transcriptomics or combined in situ hybridization and protein staining in biopsies from human inflammatory diseases to show neighborhood-based enrichment of IL-7 expression specifically by fibroblasts scaffolding large T cell aggregates. Testing for cross-species conservation is critical given the differences in production and presentation of growth and survival factors (e.g. IL-15) and fibroblast behavior between species.14,270–272,285,286\n\nSpecialized fibroblasts in primary and secondary lymphoid organs are major producers of the B cell growth factors IL-6, BAFF and APRIL, as well as the marginal zone B cell survival factor Delta-like 1.70,233,287–291 In fact, BAFF is expressed by multiple fibroblast subsets in lymph nodes: one within B cell follicles, one in the interfollicular T cell zones and one in the medullary cords.233,287,292,293 These microanatomically distinct fibroblasts are also functionally different with some supporting B cells and others supporting plasma cells.287,293 Increased BAFF expression has been detected in the affected joints of RA patients and in the affected glands of SS patients.107,294 For both RA and SS patients with lung complications, increased BAFF expression is also detected in patients with pulmonary B cell-containing lymphoid aggregates.21 Local production of survival factors appears especially important to support autoreactive B cells in IMID. Autoantigen-specific B cells are particularly sensitive to activation-induced death due to chronic B cell receptor (BCR) stimulation, and BAFF can rescue these autoreactive B cells through non-canonical NF-κB-mediated Pim2.295 Simply observing increased production of B cell growth factors in IMID does not necessarily mean these are being produced by fibroblasts, though. Myeloid cells and granulocytes are also capable of producing many of these proteins.296–300 What evidence supports these factors coming from activated fibroblasts?\n\nThere are two lines of evidence. First, specialized fibroblasts producing BAFF transcripts have been identified by scRNAseq in dissociated joints from RA patients and salivary glands from SS patients (Figure 2).107,189 Second, synovial fibroblasts isolated from RA patients secrete BAFF in response to TLR3 or TNFα stimulation and constitutively secrete APRIL.143,301 Matched dermal fibroblasts from the same RA patients also produced BAFF transcripts in response to TLR3 and TLR4 stimulation, but secreted ~10-fold less BAFF and APRIL than synovial fibroblasts even after stimulation.301 Production of these growth factors by fibroblasts is effective as synovial fibroblasts support B cell survival in vitro in co-cultures.302 Whether BAFF expression increases prior to B cell infiltration as a prerequisite for B cell infiltration, or pioneer B cells induce BAFF to create a feed-forward loop is an open question. Based on kinetic studies in a murine model of SS where elevation of BAFF transcripts appears slightly before B cell infiltration we propose that early alarmin signaling by tissue resident lymphocytes is necessary to induce TLR3/4 and/or TNFR2 expression by fibroblasts. In turn, activation of tissue resident lymphocytes, either by fibroblasts or other cells in close proximity, induces surface expression of TNFα or LTα homotrimers to trigger TNFR2-driven non-canonical NF-κB activation and BAFF transcription in fibroblasts (Figure 2).107,153,154,196,301,303 These data suggest a tissue- or inflammation-driven epigenetic and post-transcriptional regulation program that differentiates RA synovial and dermal fibroblasts, and may partly account for the accumulation of B cells in affected joints but not unaffected skin of these patients.\n\nTogether, these data suggest that fibroblasts may provide critical growth factor support for leukocytes infiltrating tissues affected by FD and IMID. In these tissues, fibroblasts appear to adopt distinct activation or differentiation states that evolve as acute insults become chronic diseases with distinct clinical manifestations. This increasing fibroblast specialization seems to result from progressive signaling dialogues that differ between fibrotic diseases and IMID. Provision of specific collections of growth factors by IMID or FD fibroblasts may be a result of these different combinations of signals and a mechanism to control the size and composition of leukocyte infiltrates. We propose that this is necessary for the different clinical manifestations and organs affected by various FD and IMID. Our theory is that cross-repression and synergistic induction of receptors and genetic modules in fibroblasts drive these distinct fibroblast functions, pathology and tissue tropism in IMID and FD.\n\n\nMasters of the extracellular matrix (ECM)\n\nWhen thinking about the pathogenic functions of fibroblasts, the most obvious starting point is fibrosis. Fibrosis, or scarring, is the result of abnormal repair in response to chronic injury. The molecular and cellular mechanisms that cause fibrosis are complex, but removal of healthy, homeostatic ECM and its replacement by pathologic fibrillar collagen is a major part of these diseases. Fibroblasts are at the center of this process, and what usually comes first to mind is the myofibroblast. Conventionally, the definition of a myofibroblast includes two components: production of fibrillar collagens and other ECM proteins, and contractility which is usually shorthanded to α-SMA+ fibroblasts (reviewed in Ref. 304).134 We agree with the long-form definition of “myofibroblast”. However, activated fibroblasts that lack detectable α-SMA protein express fibrillar pro-collagen proteins and are associated with areas of fibrosis in some diseases.305 Thus, we prefer the more general term “ECM fibroblast” when referring to activated fibroblasts in FD with myofibroblasts being a subset of ECM fibroblasts.\n\nFibrosis can occur in chronic diseases with either type 2 or type 3 immune polarization (or in the absence of obvious inflammation or leukocyte infiltration), but this clear-cut skewing is more common in animal models than patient cohorts. More frequently, human fibrotic diseases demonstrate varying degrees of mixed inflammation. Due to cross-repression and rebound inflammation, stopping progressive scarring in patients with obvious inflammatory skewing will likely require simultaneous antagonism of both type 2 and type 3 signaling.115,116,254,306 Even more complicated, the predominant inflammatory skewing of FD patient cohorts likely varies depending on the organ and stage of disease in the biopsy. For example, the lungs of active IPF patients generally have three distinct zones: relatively healthy, inflamed with little fibrosis and overtly acellular due to end-stage fibrosis (reviewed in Ref. 307). Depending on the zone sampled researchers will get wildly divergent results. Although this example is true for IPF, similar caution is warranted when interpreting findings from other fibrotic diseases. The extent and predominance of type 2, type 3 or a lack of overt inflammation among patients with different fibrotic diseases is an open area of research requiring larger patient cohorts of diverse genetic backgrounds.\n\nIL-4, IL-5 and IL-13 are the canonical cytokines associated with type 2 inflammation.308 Increased levels of these cytokines have been detected in patients with a variety of sterile fibrotic diseases including NASH and IPF. Depending on the cohort and the way in which the measurements were taken, higher levels of these cytokines are associated with more severe and/or longer disease duration.115,254,309–311 Fibroblasts isolated from the lungs of IPF patients express more of the IL-4 receptor α (IL-4Rα) and IL-13 receptor α1 (IL-13Rα1) than fibroblasts from healthy lungs. These two proteins form the heterodimeric type II receptor for IL-4 and IL-13, and this elevated expression was associated with increased COL1 transcription in response to these cytokines.312,313 IL-4 and IL-13 can also stimulate COL1 secretion by some human hepatic stellate cell lines.314 However, the mechanisms by which these cytokines, especially IL-13, induce fibrosis may differ between organs or diseases.\n\nLiver fibrosis induced by local transgenic overexpression of IL-13 and signaling through the type II receptor on fibroblasts (including hepatic stellate cells) may be independent of TGFβ.116,315,316 Pulmonary fibrosis induced by similar transgenic overexpression of IL-13 in murine lungs, although induced using a different genetic technique, required TGFβ for fibrosis (Figure 3A).317,318 Why this distinction was observed is currently unclear. Different results were obtained from the strongly type 2 polarized Schistosoma mansoni models of lung and liver fibrosis, though, with both appearing to be independent of TGFβ.116,316 Perhaps ECM fibroblast activation and fibrosis in these models depends on the magnitude of the IL-13 response or the balance between mixed inflammatory polarization?\n\n(A) In predominantly type 2 skewed fibrotic diseases (FD), acutely activated fibroblasts and other cells attract monocytes, eosinophils and basophils. Degranulation of eosinophils and basophils can release IL-4 and IL-13 to activate the transcription factor STAT6. Eosinophils granules can also release TGFβ to promote differentiation of ECM fibroblasts into myofibroblasts through the transcription factors SMAD2 and SMAD3. IL-4 and IL-13 also influence monocyte differentiation into macrophages, which can then add to local TGFβ production and myofibroblast differentiation. (B) By signaling through the type II receptor, IL-4 and IL-13 can directly activate production of Collagen type VI (COL6) by fibroblasts. Thus, they may directly drive fibrosis without relying on TGFβ although in this situation it is unclear how production of fibrillar collagens is initiated. IL-4 and IL-13 can also stimulate TGFβ production by macrophages. Thanks to homotypic Cadherin-11 (CDH-11) synapses formed with myofibroblasts, the TGFβ produced by these macrophages can be converted from its latent to active form through fibroblast integrin αV heterodimers and contractile force. Thus, IL-4 and IL-13 may trigger Collagen type I (COL1) production by ECM fibroblasts through this indirect TGFβ pathway. (C) In FD dominated by type 3 inflammatory skewing, acutely activated fibroblasts and other cells primarily induce monocyte and neutrophil extravasation. Under these circumstances, γδ T cells, neutrophils and mast cells produce IL-17A and, with fibroblasts, GM-CSF. These induce the differentiation of infiltrating monocytes into Fab5 macrophages. IL-17A can also potentiate low levels of TGFβ signaling generated through contractile activation by ECM fibroblasts expressing unbound podoplanin (PDPN). (D) GM-CSF produced by fibroblasts and other cells initiates monocyte differentiation towards the Fab5 macrophage state through the transcription factor STAT5. As described in (B), Fab5 macrophages can also form CDH-11 synapses with ECM fibroblasts and provide latent TGFβ for activation by fibroblast integrin αV and contraction. Active TGFβ can augment or stabilize the Fab5 program in macrophages and promote ECM fibroblasts to become myofibroblasts. IL-17A potentially produced by several cell types in the fibrotic niche can increase expression of TGFβRII and enhance SMAD2 and SMAD3 activation by signaling through TRAF6 and JNK in fibroblasts. In differentiating monocytes, IL-17A can also promote differentiation into Fab5 macrophages. IL-22, another type 3 cytokine, can also enhance SMAD2 and SMAD3 activation in concert with TGFβ. Figure created with BioRender.\n\nIn a murine model of severe asthma characterized by strong, mixed type 2 and type 3 inflammation neither reducing TGFβ activation nor IL-13 blockade alone or in combination decreased fibrosis.116 This last observation suggests two possibilities. First, that in the presence of multiple strong, non-redundant ECM fibroblast activating signals the degree of combined inhibition achieved insufficient target coverage. Second, that this model drives ECM fibroblast activation by an additional, orthogonal mechanism. Regardless of which is true, these data clearly demonstrate that type 2 inflammation, and specifically IL-13, can induce ECM fibroblast activation and fibrosis both directly and indirectly through TGFβ. One other part of this story is curious: IL-13 appears to directly stimulate Col6a1 transcription via STAT6 activation in fibroblasts, but the mechanism by which IL-13 induces COL1 production is unclear (Figure 3B).315 Collagen type VI is not a traditional fibrillar collagen like types I, II and III but instead forms beaded microfilaments that are assembled into reticular structures (reviewed in Ref. 319). Perhaps dramatic increases in local Collagen type VI stiffen the local matrix and cascade through outside-in integrin signaling and activation of latent growth factors like TGFβ?\n\nAs noted above, type 3 inflammation is also associated with fibrosis. Type 3 inflammation is characterized by the recruitment and activation of T-helper (Th) 17 cells, γδ T cells, type 3 innate lymphoid cells (ILC3) and neutrophils (Figure 3C). Infiltrates where these cells predominate plus local activation of mast cells leads to the secretion of IL-17A, IL-22 and GM-CSF (Figure 3C).254,320–322 Type 3 immune responses have been associated with fibrosis and cancer progression in many organs. IL-17A cytokine or receptor knockout animals are protected from fibrosis in variety of pre-clinical models.72,309,323 Interestingly, opposing pro- vs anti-fibrotic functions have been reported for IL-22 depending on timing, type of injuries and opposite effects reported on epithelium vs mesenchymal cells.323–326 Increased expression of IL-17A is associated with IPF, NASH and other fibrotic diseases while, to our knowledge, increased IL-22 has only been observed in NASH patients.254,309 Elevated GM-CSF expression is also associated with NASH, IPF and mustard gas pulmonary fibrosis.229,230 As we have described in previous sections, type 3 cytokines are associated with recruitment and survival of macrophages and granulocytes in FD, but also with the recruitment of DCs and lymphocytes in IMID. Do type 3 cytokines drive similarly opposing ECM depositing and eroding functions in fibroblasts?\n\nIL-17A is the prototypical type 3 cytokine, and its receptor is expressed on a variety of cell types including epithelium, endothelium, mesothelium, and myeloid cells.327 It signals through a heterodimeric receptor composed of the IL-17RA and IL-17RC subunits triggering a cascade through the Act1/TRAF6 to activate NF-KB, ERK1/2, C/EBPB, JNK and MAPK pathways (reviewed in Ref. 328). In the absence of serum, IL-17A only triggers secretion of pro-inflammatory chemokines in primary human hepatic stellate cells. In contrast, IL-17A sensitizes myofibroblasts to the action of TGFβ by stabilizing its receptor on the surface of cells in a JNK-dependent manner (Figure 3C).329 In conclusion, IL-17A is pleiotropic cytokine which impacts myofibroblast directly and/or indirectly within the fibrotic niche.\n\nIL-22 is a complex pleiotropic cytokine from the IL-10 family, with opposite roles in health and disease. IL-22 signals through a heterodimeric receptor composed of the common IL-10 family chain IL-10RB and IL-22RA1 triggering activation of STAT3 and MAPK.330,331 Expression of the IL-22 receptor complex is restricted to epithelial cells and fibroblasts. IL-22 promotes the survival and proliferation of epithelial cells via STAT3 promoting tissue repair and therefore limiting fibrosis.330 On the other hand, IL-22 signaling also induces the secretion of chemokines involved in the recruitment of neutrophils, monocytes, and macrophages.332,333 Studies in the liver also demonstrated that IL-22 contributes to fibrosis progression by enhancing TGFβ signaling via STAT3/MAPK activation (Figure 3D).254 While IL-22 deficient mice have impaired tissue repair mice lacking the decoy receptor IL-22BP also have impaired tissue repair due to aberrant inflammatory responses. These discrepancies require further investigation but likely depend on the levels of IL-22 and IL-22BP, the timing of IL-22 production, the cells receiving IL-22 signals and the other signals being simultaneously received and integrated.\n\nPathogenic production and organization of the ECM in fibrotic diseases is clearly one function of fibroblasts. Do they serve similar, but opposite, roles in inflammatory diseases characterized by excessive destruction of the ECM like RA? The answer appears to be yes, which is especially important when considering treatment for rheumatoid arthritis patients with the fibroid pathotype and osteoarthritis (OA) patients. Due to the low frequency of lymphocytes, myeloid cells and granulocytes in the joints of these patients next generation RA drugs should simultaneously block multiple pathways to cover patients with all three pathotypes.161 Given the similarities between the fibroid RA pathotype and OA, these drugs may work for both groups of patients.\n\nIn rheumatoid arthritis mouse models, PDPN+FAPα+THY1- synovial fibroblasts specialize in joint destruction and extracellular matrix remodeling as demonstrated through engraftment. They secrete high levels of matrix metalloproteinases (MMPs) 3, 9 and 13, which are especially effective at degrading the most abundant fibrillar collagen in cartilage, Collagen type II, and the major constituent of basement membranes, Collagen type IV.171,284,334 These PDPN+FAPα+THY1- synovial fibroblasts described by Croft et al. are likely the same as the PRG4+ synovial fibroblasts described by Micheroli et al. Both lack or have undetectably low expression of THY1, express MMP3 and appear to be associated with the destruction of synovial cartilage independent of leukocyte infiltrates.284,335\n\nWhat activates these destructive functions in fibroblasts? IL-1, IL-17A, TNFα and Oncostatin M all induce combinations of MMPs in synovial fibroblasts, which result in degradation of glycosaminoglycans or cartilage in vitro.144,336,337 Connecting MMP gene or protein expression with functional consequences is particularly important given the tight regulation of their activity. Pro-MMPs undergo proteolysis before becoming catalytically active, and after activation MMPs can be inhibited by the tissue inhibitor of metalloproteinases (TIMP) family of proteins (reviewed in Ref. 338). The balance between protease production, activation and suppression is also relevant for ECM fibroblasts. Clearance or modification of homeostatic ECM often accompanies the deposition of fibrotic ECM, and this requires the activation of specific classes of proteases and the inhibition of others. Although it is beyond the scope of this review, the generation of matricryptins or matrikines (proteolytic fragments of ECM molecules that induce signaling distinct from their larger, intact parent molecules) likely play roles in both fibrotic diseases and IMID (reviewed in Refs. 319, 339, 340). Despite the complexities of studying the pathogenic destruction of ECM by fibroblasts, these data indicate that fibroblasts are sufficient for clinically relevant joint destruction. Further research is necessary to determine whether fibroblasts make significant contributions to ECM destruction in other diseases.\n\nIt may be surprising that some of the type 3 cytokines associated with pathologic deposition and organization of ECM by fibroblasts can also induce the opposite outcome. For example, why is IL-17A associated with fibrosis in murine models and human NASH and IPF patients and with joint destruction in murine models and human RA patients? This is still an open question. However, these distinct pathologies and disease outcomes may be due to different signals combining with IL-17A in fibrotic versus eroding diseases. In FD, the combination of IL-17A and TGFβ signaling in fibroblasts can potentiate more robust ECM gene expression than either cytokine alone.329 Add outside-in sensing of the stiff fibrotic matrix and hypoxia on top of the synergy between IL-17A and TGFβ, and this combination is likely part of what drives fibroblast to deposit pathogenic ECM in FD. There is less TGFβ and more TNFα signaling in RA to combine with IL-17A, which results in higher MMP13-driven proteoglycan destruction.179,336 Similarly, how does IL-13 promote ECM fibroblast differentiation and fibrosis either alone or in combination with TGFβ in some models, while also driving fibroblast activation in TLS formation in a model of SS?\n\nThese data clearly demonstrate that distinct fibroblast activation or differentiation states and functions occur in FD and IMID. ECM fibroblasts are at the heart of FD. They are the major producers and organizers of collagens and many other ECM molecules, and tune the stiffness of tissues. By modifying the ECM in this way, fibroblasts also activate latent growth factors and convert haptotactic to chemotactic gradients. Are all ECM fibroblasts the same, or are there different functional states that arise from predominant type 2 versus type 3 activation (or in the absence of detectable inflammation)? And do all fibrotic fibroblasts deposit the same ECM constituents or exert the same contractile forces, or is collaboration between multiple ECM fibroblast states necessary for fibrosis? Answering these questions will be necessary for the rational development of therapeutics that stop or even reverse FD. For example, if the fibroblasts activated by type 3 cytokines build fibrotic ECM with a different composition from those activated by type 2 cytokines the molecular pathways requiring therapeutic manipulation will differ. This would also necessitate the development of companion diagnostics to effectively select patient populations for clinical trials and deliver the most effective personalized medicine to FD patients.\n\nConversely, different fibroblast subsets are responsible for ECM destruction or lymphocyte recruitment and support in RA. Thus, multiple fibroblast functional states can coexist and collaborate to drive complex disease pathology. Is ECM degradation by erosive fibroblasts necessary for the expansion of fibroblasts that scaffold lymphocyte aggregates and TLS? As thickening and stiffening of the lymph node capsule appears to be a compensatory reaction that constrains swelling and limits lymphocyte proliferation, some degradation of the local ECM is likely necessary for the growth of lymphocyte aggregates.108,109,139 That erosive fibroblasts are sufficient for destruction of the collagen type II-rich cartilage in synovial tissues also begs the question of how important different cell types are to to ECM destruction. Similarly, if erosive fibroblasts in RA can significantly destroy the synovial matrix do they also make major contributions to removal of the healthy, homeostatic matrix in fibrotic diseases? Addressing these questions is critical to developing better therapies for patients with both erosive IMID and FD. Understanding the signals that create erosive versus ECM fibroblasts and whether these are stable differentiation states or reprogrammable activation states will help us develop the appropriate therapeutic regimens to treat these opposing disease pathologies.\n\nUp to this point we have demonstrated that fibroblasts appear to adopt progressive, functionally distinct activation states in IMID versus FD. In IMID these various activated fibroblasts produce chemo-/haptotactic and growth factors specialized to support local adaptive immune responses. Conversely, in FD ECM and myofibroblasts generally attract smaller infiltrates of predominantly macrophages and granulocytes through specialized chemo-, hapto- and durotactic gradients and growth factors for these cells. Fibroblasts can also exert significant, opposing effects in these diseases by depositing or, at least in RA, degrading large amounts of ECM. The final question we will tackle is: do specialized fibroblasts contribute to the activation or differentiation of infiltrating leukocytes in these diseases?\n\nMonocytes and macrophages are important parts of both IMID and FD, although they are a much larger proportion of infiltrates in FD than IMID. Recently, several reports identified scar-associated pro-fibrotic macrophages in human and mice in a variety of tissues.17,341–350 A common signature (Fab5) using the combination of CD9, CD63, FAPB5, GPNMB and SPP1 was recently suggested to accurately distinguish these fibrotic macrophages from populations with other functions. Fab5 macrophages originate from monocytes and, in some settings, require IL-17A, GM-CSF (likely from neutrophils, although fibroblasts and other cell types may play a role) and TGFβ for their differentiation in vitro and in vivo (Figure 3D).18,245,247,248 Another route to a Fab5-like macrophage may be through M-CSF and IL-6, both of which can be produced by fibroblasts although the authors of this study identified an autocrine loop of these factors.351 Fab5 macrophages elicit several key pro-fibrotic functions that were elucidated in vitro. Fab5 macrophages contribute to the removal of normal ECM through MMP activity and enhance deposition of fibrillar collagen by ECM fibroblasts (Figure 3D). Although the molecular mechanisms by which Fab5 macrophages activate ECM fibroblasts are actively being studied, they likely include proximal presentation of latent TGFβ for activation by fibroblasts, as well as Amphiregulin and possible TWEAK and PDGFR ligands.17,129,352 The relative importance of ECM fibroblast-produced M-CSF, GM-CSF, IL-6 and activation of latent TGFβ to Fab5 differentiation requires experimental testing, although it seems likely they contribute (Figure 4A).\n\n(A) In fibrotic diseases, fibroblasts and macrophages can form Cadherin-11 mediated synapses. By secreting granulocyte-macrophage colony stimulating factor (GM-CSF) and activating latent transforming growth factor β (TGFβ) through integrin αV contractile forces, extracellular matrix (ECM) fibroblasts can promote differentiation of monocytes into fibrotic Fab5 macrophages. On the other hand, in immune-mediated inflammatory diseases (IMID) production of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) can lead to osteoclast or CD169+ macrophage differentiation. Although the molecular mechanisms that dictate whether a macrophage becomes and osteoclast or CD169+ macrophage require further investigation, the dependence of CD169+ macrophage differentiation on LTβR signaling may be one differentiating factor. (B) Fibroblasts can express both major histocompatibility class-I (MHC-I) and -II (MHC-II), which can reactivate memory CD8 and CD4 T cells as they infiltrate tissues affected by IMID. Although not discussed in this article, MHC expression by fibroblasts can suppress T cell proliferation or promote tolerance in addition to restimulating T cells429. (C) Fibroblasts in secondary lymphoid organs produce the Notch1/2 ligands Delta-like 1 (DLL1) and 4 (DLL4). In murine models of graft versus host disease, expression of DLL4 and, to a lesser extent, DLL1 by lymphoid organ fibroblasts was necessary for TH1 differentiation of grafted naïve T cells. This DLL1/4-Notch1/2-induced T cell differentiation was required for disease induction although it is unclear whether non-lymphoid organ fibroblasts can perform the same function. (D) IL-33 production by Ccl19+ is necessary for both maintenance of the Tcf1+ CD8 stem memory T cell pool and for effective anti-viral and anti-tumor CD8 T cell differentiation. The mechanism by which IL-33 produced by fibroblasts promotes type 2 immunity in parasite infections and type 1 anti-viral and anti-tumor immunity is unclear, but likely occurs through crosstalk with other signals like type 1 interferons. (E) DLL1-Notch2 signaling induces the differentiation and maintenance of splenic marginal-zone B cells. A similar system may work in tissues affected by IMID, but this awaits experimental testing. (F) Follicular dendritic cells in secondary lymphoid organs trap antibody- and complement-containing immune complexes using the Fcγ receptor IIb (FcγRIIb) and complement receptors 1 (CD35) and 2 (CD21). Presentation of the antigens in these immune complexes to B cells is important for germinal center reactions. Affinity maturation and class-switch recombination mediated by activation-induced cytidine deaminase (AID) in follicles both require this follicular dendritic cell function. Fibroblasts expressing FcγRIIb, CD35 and CD21 appear in the affected tissues of some IMID patients, and stimulation of fibroblasts isolated from these tissues can induce expression of these three receptors. Thus, it is likely that follicular dendritic-like cells can maintain germinal center reactions in IMID tissues. Figure created with BioRender.\n\nFibroblasts likely induce differentiation of functionally distinct macrophage subsets in IMID. In SLO, differentiation of CD169+ sinusoidal and metallophillic (or marginal zone) macrophages requires Receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) expression by fibroblasts (Figure 4A). These macrophages capture free virus and initiate anti-viral CD8 T cell responses.353–355 The roles of CD169/SIGLEC1+ macrophages in IMID are unclear. One report on RA patients proposed a protective role for CD169+ macrophages, while others noted a positive correlation of circulating CD169+ myeloid cells with worse and extra-glandular disease in SS and dermatomyositis.356–358 Given the recent reports on potentially pathogenic CD8 T cells, and reductions in synovial CD8 T cells being associated with clinical responses to rituximab in RA patients, we favor the hypothesis that CD169+ macrophages are pathogenic in IMID.161,171,359–363\n\nSeveral fibroblastic lineage cells in the bone and bone marrow induce osteoclast differentiation, thus indirectly promoting bone and cartilage resorption, by producing RANKL.364,365 Do fibroblasts play similar roles in IMID-associated osteoporosis? PDPN+FAPα+THY1- synovial fibroblasts in RA patients and murine models produce RANKL, which promotes macrophage differentiation into osteoclasts and is associated with bone erosion.171,284,334,366 Although RA appears to be the only IMID directly associated with bone erosion or osteolysis, many IMID are treated with glucocorticoids and long-term use of these steroids is a risk factor for osteoporosis (reviewed in Ref. 367).368 Glucocorticoids can directly induce RANKL expression by fibroblastic cells from several tissues in mice and humans as the glucocorticoid receptor directly binds to the RANKL promoter.369,370 Osteolysis also occurs in adverse responses to joint replacement and is frequently accompanied by histologic macrophage activation.371,372 Both RANKL and its receptor, RANK, are expressed in tissues containing wear particles and osteoclasts spontaneous differentiated in patient samples with high RANKL to Osteoprotegrin (OPG, the soluble decoy receptor for RANKL) transcript ratios.373 Titanium particles were sufficient to indirectly induce RANKL expression by synovial fibroblasts, as was combined stimulation with TNFα and IL-17A which worked through trans IL-6 signaling (Figure 4A).374,375 These data indicate a surprisingly wide range of osteolytic pathologies that appear to be driven by fibroblast-induced osteoclastogenesis.\n\nDo fibroblasts participate in the local re-activation of T cells after they extravasate into inflamed tissues? Fibroblasts (both human and murine) can express Class I (MHC-I) and Class II (MHC-II) Major Histocompatibility Complex molecules.70,171,189,376–384 Murine fibroblasts can activate both CD4 and CD8 T cells in an MHC-dependent fashion to elicit cytokine production and cytotoxic activity (Figure 4B).377,378,380,383–385 Human fibroblasts may be able to do the same, but definitive proof awaits experimental validation.379,382 This could be demonstrated in vitro by obtaining donor-matched fibroblasts from skin biopsies and circulating T cells from the blood, pulsing the fibroblasts with a cocktail of common viral T cell epitope peptides and testing for proliferation and cytokine production. Given the suppression of effector T cell activation by primary human SLO fibroblasts and lymphocytic choriomeningitis virus clone 13 (LCMV-C13) infected murine fibroblasts, blocking co-inhibitory molecules either alone or in combination with CRISPR-mediated knockout of other suppressive factors may be necessary to detect much cytotoxic activity.385–390 The relative importance of MHC expression by fibroblasts, macrophages and DCs in both SLO and IMID also await direct experimental validation. Is antigen presentation the only mechanism by which fibroblasts can restimulate infiltrating T cells?\n\nNo, although the relative importance of antigen-specific and -independent reactivation of T cells infiltrating inflamed, non-lymphoid tissues requires further investigation. Fibroblasts may reactivate infiltrating T cells through a variety of antigen-independent mechanisms including cytokine production, expression of canonical or non-canonical co-stimulatory molecules and adhesion proteins. SLO fibroblasts are important sources of the Notch ligands Delta-like 1 (Dll1) and 4 (Dll4) in both homeostasis and graft-versus host disease (GVHD, Figure 4C).281,290,391,392 Homeostatic human dermal fibroblasts lack Notch ligands, but this may change (and not just in the skin) during inflammation as non-lymphoid fibroblasts adopt SLO-fibroblast-like qualities and awaits experimental testing.14,105,393 If true, inflamed fibroblasts expressing Dll1 or Dll4 may reactivate infiltrating T cells to promote inflammatory or autoimmune tissue destruction (Figure 4C).290,391 Expression of Dll1 or Dll4 by inflamed fibroblasts may also promote local tertiary lymphoid tissue formation by driving differentiation of infiltrating naïve T cells into T follicular helpers and naïve B cells into germinal centers.290,394 An alternative explanation is that Dll1 or Dll4 expression is truly restricted to SLO fibroblasts and is only important for imparting the ability to home to inflamed tissues, not the restimulation of tissue destructive effector functions.392 It seems plausible, at least, that activated fibroblasts in IMID can contribute to inflammatory tissue destruction by reactivating infiltrating T cells and driving T cell differentiation via Dll1 and Dll4. This is especially important when considering the translatability of findings from insult-naïve, specific pathogen free mice to adult humans and other animals that have substantial pools of tissue-resident lymphocytes and have epigenetic memories of these past insults.\n\nDLL/Notch signaling is, however, just one of many tools fibroblasts possess to restimulate infiltrating T cells. Fibroblasts in both SLO and non-lymphoid tissues express alarmins like IL-33 and Thymic Stromal Lymphopoietin (TSLP).67,68,71 IL-33 produced by SLO fibroblasts is important for activation and effector differentiation of alloreactive CD4 T cells in murine GVHD models and effector CD8 T cells in acute and chronic viral infection.68,71 Recent findings have pointed to the importance of IL-33 in balancing between CD8 stem memory T cell expansion and effector differentiation in chronic viral infections and cancer (Figure 4D).69,395 In non-lymphoid tissues, IL-33 expressing fibroblasts appear to be enriched at interfaces between microanatomic compartments. These interfaces include airways and vasculature in the lungs, portal triads and vasculature in the liver, vasculature in adipose tissue, hair follicles and subcutaneous fascia in murine skin and the dural sinus in the meninges.67,72 Successful cancer immunotherapies create specialized perivascular niches that likely allow asymmetric division of CD8 stem memory T cells to preserve stemness and proliferative capacity while simultaneously generating differentiated cytotoxic CD8 T cells.396 It appears that IL-33 released by perivascular fibroblasts in chronically inflamed tissues balances effector differentiation and stemness of infiltrating CD8 T cells to perpetuate autoimmune tissue destruction or control of viral reactivation and cancer. More broadly, specialized fibroblasts at tissue interfaces (e.g. mucosal/epidermal barriers, blood/parenchyma, tissue/cavity, etc.) may, upon activation, express an array of molecules that contribute to the restimulation or continued activation of infiltrating or activated tissue resident T cells.\n\nIf specialized fibroblasts promote local reactivation and differentiation of T cells, can they do the same for B cells? Like what was described for T cells above, Dll1 provided by murine FRC promotes MZ B cell survival and plasma cell differentiation (Figure 4E).291 MZ B cells express both classical (MHC) and non-classical (CD1 family) antigen presentation machinery.291,397–399 MZ-like B cells expressing CD1 family transcripts are found in RA synovial tissue.400 Thus, FRC- or FDC-like fibroblasts in the RA synovium may provide DLL1/Notch2 signals that promote MZ-like B cell survival and plasma cell differentiation in this inflamed tissue. It’s unclear, however, if human FRC or FDC provide similar Dll1/Notch2 signals and, if so, whether this system promotes B cell survival. This may extend to other inflammatory diseases where large B cell infiltrates are common, such as DLE and SS, although this awaits experimental validation.\n\nFollicular dendritic cells (FDC) are specialized fibroblasts found in B cell follicles in SLO. Immune complexes deposit onto FDC, which depends upon complement receptors 1 and 2 (CR1 and CR2) and, in secondary follicles, FcγRIIB expressed by FDC (Figure 4F).215,401–409 Recycling of immune complexes by FDC controls how long B cell responses last in the absence of fresh insults, and how frequently antigen is available to drive affinity maturation of B cells.215,405,410 This function of FDC is important for germinal center reactions and the generation of functional, high-affinity antibodies.213,215,403,406,408,409,411 Immune complex recycling by FDC also means that B cell activation and germinal centers can be maintained long after the initiating insult is cleared.401,402,404–406,410 Do FDC-like fibroblasts perform similar B cell activating functions in tissues affected by IMID?\n\nFDC-like networks are associated with activation-induced cytidine deaminase (AID) expression by accumulating B cells during inflammation in non-lymphoid tissues (Figure 4F).21,159,162 This local AID expression appears to be functional. The presence of secondary follicles in RA synovia, the lungs of RA patients with pulmonary complications and salivary glands of SS patients is associated with higher serum levels of anti-rheumatoid factor. DNA sequencing of B cells from germinal center positive joints suggests local clonal expansion and affinity maturation of autoantibodies.21,200,361,412 Similarly, transplanting RA synovium samples either with or without CD21+ FDC into SCID mice demonstrated that AID expression and mature FDC were associated with local class switching and secretion of human anti-citrullinated protein IgG.210 Murine models of infection and autoimmunity also indicate that B cells can undergo local antigen-specific proliferation and affinity maturation only in the presence of mature FDC (Figure 4F).107,153,154,159,198,204 Synovial levels of an FDC transcriptional signature and individual immune complex trapping genes were positively correlated with both DAS28-ESR and DAS28-CRP supporting the idea that local autoreactive B cell responses are associated with more aggressive or severe disease.303\n\nLocal B cell activation by synovial fibroblasts recapitulates in vitro. Co-culture of naïve IgD+ B cells with synovial fibroblasts from RA patients induces AID expression, class switching to and secretion of IgG without any exogenous stimulus. AID expression as well as class switching to and secretion of IgA and IgG is enhanced by TLR3 stimulation during the co-culture.301 TLR3-dependent induction of AID and class switching requires BAFF signalling through its receptors.301 This appeared to occur independent of antigenic stimulation because combined TNFα and LTα1β2 signaling was necessary to increase transcripts of the immune complex trapping genes Complement receptors 1 and 2 (CR1/CD35 and CR2/CD21) and Fragment crystallizable gamma receptor IIb (FCGR2B) by RA synovial fibroblasts.303 The requirement for combined TNFR and LTβR signaling in the full maturation of FDC is supported by in vivo pharmacological blockade and genetic disruption data in mice, rats and monkeys.145,192,194,197,202,413,414 However, there may be other pathways to full FDC maturation as they form in inducible bronchus-associated lymphoid tissue in mice lacking TNFα, LTα and LTα1β2.198\n\nThese observations are particularly relevant to developing treatments for patients with autoantibody or B cell-driven autoimmune diseases. First, these local B and plasma cell responses in IMID likely give rise to tissue resident B and plasma cells.208,415–418 Rituximab appears to require B cells to recirculate for clearance via antibody-dependent phagocytosis or complement-dependent cytotoxicity, and thus fails to deplete tissue resident B cells.161,207,419 Other therapeutic approaches including targeting survival factors that affect B cells and/or plasma cells in the bone marrow, SLO and TLS, depleting antibodies that work by antibody-dependent cellular cytotoxicity or chimeric antigen receptor T cell (CAR-T) or combination therapy approaches will likely be necessary for larger clinical benefit in these patients.420–423 Second, because FDC trap and retain antigen for at least 56 days either clearance of immune complexes from FDC or depletion of FDC is necessary to prevent perpetuation or re-emergence of autoreactive B and plasma cell responses.215\n\n\nConclusions\n\nOver the past 20 years, increasing interest in the roles that fibroblasts play in both FD and IMID has revolutionized our understanding of these cells. This growing body of work suggests that over the course of FD and IMID, fibroblasts in the affected tissues undergo progressively divergent differentiation which is associated with the distinct clinical outcomes of these diseases. It seems likely that acute injury signals including alarmins, PRR activation and the serum response initiate fibroblast activation early in both FD and IMID. While this has been demonstrated in several animal models of FD and IMID, conclusive proof from patients will require the development of diagnostics that can detect and differentiate these diseases before they manifest clinically. As the types of acute injury signals vary widely, future research into whether these differ between FD and IMID will help us better understand whether differences in fibroblast functions are initiated in the earliest stages of disease.\n\nCurrently, it seems that fibroblast activation by alarmins in FD and IMID translates to their recruitment of and growth factor support for monocytes and granulocytes in the early stages of disease. Several therapies targeting the alarmins that activate fibroblasts, among other cells, have entered the clinic or are under development and have met with varied success. IL-1 antagonists have primarily shown efficacy in IMID which have associated familial genetic mutations in pathways involving IL-1. Efficacy in IMID has been mixed, with promising results seen in idiopathic juvenile arthritis and hidradenitis suppurativa, both of which often present with substantial neutrophil infiltrates (reviewed in Ref. 424). The mixed success of IL-1 antagonists may, in part, reflect the redundant stimuli (IL-1, TNFα, serum, etc.) that drive production of neutrophil and monocyte chemokines, growth factors and activation molecules by fibroblasts. Combined blockade of these pathways may be necessary to drive clinical responses in a broader population of patients as was recently proposed.37\n\nAfter their initial activation by acute injury signals, divergence of fibroblast differentiation states between FD and IMID appears to be a sequential process due, in part, to an evolving dialogue with infiltrating leukocytes. This is supported by the distinct sizes and compositions of leukocyte infiltrates observed when FD and IMID patients present clinically. In IMID where there is a break in tolerance, autoreactive T cells infiltrating the affected tissues retain mature DCs presenting cognate antigen around blood vessels and sites of damage. Clustering of macrophages and DCs with T cells and other tissue resident lymphocytes may trigger subsequent fibroblast differentiation through combinations of TNFR1/2, LTβR and IL-22 signaling along with cytokines produced by type 1, type 2 or type 3 polarized immune cells. Ultimately, this can lead to TLS formation with local priming of autoreactive T cells and class switching, affinity maturation and autoantibody production by B and plasma cells.\n\nThe many therapies antagonizing TNFR1/2, IL-13 and IL-17 that have been approved for IMID give physician scientists opportunities to investigate whether any of these signals are required for the maintenance of lymphocyte aggregates or TLSs. As each of these has animal model and in vitro data supporting their direct effects on fibroblast differentiation and lymphocyte aggregate formation some of their efficacy may come from reversing this. Recently, RA pathotypes reflective of distinct fibroblast differentiation have been associated with the course of disease progression and clinical responses to different DMARDs.161,199,303 Enriched efficacy of tocilizumab in RA patients with the diffuse myeloid pathotype fits with the hypothesis that trans-IL-6 signaling between these two cell types is pathogenic in many of these patients.161 This observation also suggests that combined blockade of IL-6 receptor and an orthogonal pathway involved in lymphoid aggregate formation, like TNFR1/2 or LTβR, may increase the breadth of responding patients to those with the lympho-myeloid pathotype as was recently proposed.425\n\nThe necessity of combination therapies is further reinforced by the coexistence of multiple fibroblast functional states in the same tissue. This has been best described in RA where fibroblasts specialized for supporting local autoimmune reactivation in lymphoid aggregates and erosive fibroblasts that degrade cartilage occur in the same tissue. Such functionally different fibroblasts arising in the same tissue suggests the existence of distinct microanatomic signaling niches within the tissue. Although targeting different therapeutics to microanatomic niches in the same tissue is beyond our current engineering capabilities, rational target combinations in multispecific large molecules or selectively engineered small molecule polypharmacology may serve as a bridge to the future. Eventually, combining multiple targeted lipid nanoparticle, RNA or small molecule therapies into a single formulation may achieve something resembling microanatomically directed therapies.\n\nParallel to the observation of these lymphoid niches in IMID is the finding of niches comprised of ECM fibroblasts, Fab5 macrophages and neutrophils in FD. In FD, acute injury activates fibroblasts to produce chemokines that attract neutrophils and monocytes. However, the absence of infiltrating T cells may lead to DC depletion, which allows PDPN+ fibroblasts to contract and stiffen the local ECM. This begins activating latent factors in the ECM, such as TGFβ. Simultaneously, these activated fibroblasts produce GM-CSF to keep infiltrating neutrophils and monocytes alive. Together with neutrophils and γδ T cells producing IL-17A, these activated fibroblasts induce monocytes to differentiate into Fab5 macrophages. ECM fibroblasts and Fab5 macrophages form CDH-11 synapses where the macrophages produce additional latent TGFβ for integrin-mediated activation by fibroblasts. Given the mixed type 2 and type 3 inflammatory tone in some FD it is likely that ECM fibroblasts are also activated by IL-4 and IL-13 produced by ILC2, granulocytes, iNKT and MAIT cells. These type 2 cytokines polarize macrophages away from the Fab5 profile and towards a remodeling state aimed at re-epithelialization and -vascularization.\n\nLike our observation of the need for combination therapies in IMID, the coexistence of mixed inflammatory skewing in FD indicates a similar requirement for combination therapies. Blocking the key type 3 cytokines in some FD appears to drive a compensatory spike in type 2 inflammation. Conversely, inhibiting fibrotic type 2 signaling seems to induce a rebound in type 3 inflammation. Simultaneously inhibiting both systems may be necessary to cover broad FD patient populations. Similarly, as the RA community has begun to define pathotypes associated with distinct clinical outcomes and responses to therapies, FD researchers and clinicians will benefit from more detailed characterization of disease heterogeneity in their patients. Additionally, as the inflammatory phase of FD appears to precede end-stage fibrosis the development of diagnostic methods to identify and classify these patients earlier in their disease progression will likely improve the frequency of successful therapeutic intervention.\n\nFinally, the observation of local, durable immune responses in tissues affected by FD and IMID has consequences for depleting therapies. For example, the lympho-myeloid RA pathotype is the only one with large B cell infiltrates in the affected joints. Why, then, was this pathotype not enriched for rituximab responders?161 Possibly because rituximab was unable to deplete synovial B cells due to a lack of recirculation or low levels of complement-mediated lysis in the joint. Similarly, the niche in FD appears to create a similar challenge for conventional depleting therapies. Three therapeutic strategies currently under investigation may address this challenge.\n\nFirst, activated cells, whether lymphocytes or fibroblasts, appear to be more susceptible to pharmacological perturbations of the balance between pro- and anti-apoptotic proteins.295,426 Better understanding how these differ between activated fibroblasts in FD and IMID may allow successful small molecule or RNA targeting of these systems. Second, depleting antibodies with Fc regions engineered for efficient antibody-dependent cellular cytotoxicity may circumvent the need for phagocytosis of opsonized cells by Kupffer cells or red pulp macrophages. Third, CAR-T or other engineered cell therapies can allow for targeted depletion of pathogenic cell types throughout the body as recently demonstrated in systemic lupus erythematosus and myasthenia gravis patients, and animal models of cardiac fibrosis.420,427,428 Regardless of the therapeutic approach, we predict that accounting for and disrupting the fibroblasts that lay the foundation for pathogenic niches in FD and IMID will be critical for future therapeutics.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nThe authors thank Thomas A. Wynn, Ken Dower, Richard L. Gieseck III, Matthew V. Russo, Matthew L. Lech, Kevin M. Hart, Katharine L. Horback, Jeffrey L. Browning and Ari B. Molofsky for helpful conversations during preparation of this manuscript.\n\n\nReferences\n\nLu CP, Polak L, Keyes BE, et al.: Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision. 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Conflict of interest: P. Cheresh has nothing to disclose. Conflict of interest: N.S. Markov has nothing to disclose. Conflict of interest: P.A. Reyfman has nothing to disclose. Conflict of interest: A.C. McQuattie-Pimentel has nothing to disclose. Conflict of interest: L. Sichizya has nothing to disclose. Conflict of interest: Z. Lu has nothing to disclose. Conflict of interest: R. Piseaux-Aillon has nothing to disclose. Conflict of interest: D. Kirchenbuechler has nothing to disclose. Conflict of interest: A.S. Flozak has nothing to disclose. Conflict of interest: C.J. Gottardi has nothing to disclose. Conflict of interest: C.M. Cuda has nothing to disclose. Conflict of interest: H. Perlman has nothing to disclose. Conflict of interest: M. Jain has nothing to disclose. Conflict of interest: D.W. Kamp has nothing to disclose. Conflict of interest: G.R.S. Budinger has nothing to disclose. Conflict of interest: A.V. Misharin has nothing to disclose. Conflict of interest: N. 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PubMed Abstract \| Publisher Full Text\n\nLagares D, Santos A, Grasberger PE, et al.: Targeted apoptosis of myofibroblasts with the BH3 mimetic ABT-263 reverses established fibrosis. Sci. Transl. Med. 2017 Dec 13; 9(420). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAghajanian H, Kimura T, Rurik JG, et al.: Targeting cardiac fibrosis with engineered T cells. Nature. 2019 Sep; 573(7774): 430–433. Epub 20190911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGranit V, Benatar M, Kurtoglu M, et al.: Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b/2a study. Lancet Neurol. 2023 Jul; 22(7): 578–590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang H, Wang Z, Zhang Y, et al.: Mesothelial cell-derived antigen-presenting cancer-associated fibroblasts induce expansion of regulatory T cells in pancreatic cancer. Cancer Cell. 2022 Jun 13; 40(6): 656–673.e7. Epub 20220505. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "251132", "date": "29 Mar 2024", "name": "Kevin Wei", "expertise": [ "Reviewer Expertise fibroblast biology", "genomics", "organoids" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFibroblasts and important cells in chronic inflammatory diseases and fibrotic diseases. Overall, this review is well-written and covers recent advances in our understanding of fibroblast pathology in the setting of chronic inflammatory diseases. Major comments:\nFibroblast nomenclature. Throughout the review, fibroblasts are mostly described as a total population. In some discussions, specific fibroblast subtypes are discussed as “adventitial”, or “activated”. Given recent advances in our understanding of fibroblast heterogeneity by scRNAseq, the authors could consider describing these specific subtypes in terms of markers that have been used to track these populations, especially in human disease settings. For example, are adventitial fibroblasts PI16+/CD34+ universal fibroblast? Are “activated” fibroblasts CXCL12+ or CCL19+ fibroblast as described in chronic inflammatory diseases? An effort to link descriptive terms to molecular markers would help the readers better understand the existing literature on fibroblast heterogeneity.\n\nThis comment applies to Figure 2 as well. Would consider adding key markers that distinguish these fibroblast subtypes. The introductory paragraph is colorful, although not completely appropriate for this scientific review. I recommend removing the first paragraph.\n\nThe statement “IMID generally features lymphocyte rich infiltrates and aggregates” requires citation. This statement is also not completely accurate as many auto inflammatory diseases are characterized by myeloid and neutrophil predominance in the affected tissues. The authors should consider discussing IMID into separate conditions by autoimmunity (i.e. RA and SLE) vs auto inflammatory diseases (Stills’s, JIA, etc).\n\nThese conditions have distinct involvement of the adaptive and innate immune system. During the discussion of alarmins and the detection of cytokines from patient samples (page 3, and page 5), the authors should clarify and separate studies which measure these cytokines in the periphery vs those studies which measure the expression of cytokines directly in affected organ tissues.\n\nThe discussion of combination therapy (page 5) in IBD is of interest. However, it would be important to mention previous clinical trials involving biologics targeting multiple cytokines and discuss relevant clinical evidence for combination therapy in IMIDs.\n\nIt would be helpful to the readers to understand key differences and similarities in function between human fibroblasts in the context of IMID vs fibroblasts in animal models of inflammatory diseases. This is critical to this review as much of the discussion is centered on targeting fibroblasts in IMID, and pointing out differences in animal models and human diseases is highly relevant. For example, fibroblast contribution to CXCL13 in mouse vs human. In Figure 2, the authors may consider illustrating distinct fates of fibroblast activation, modulated by inflammatory cytokines, growth factors, and niche signals. It is not entirely clear by the current scheme how many distinct fibroblast outcomes. The term “ECM fibroblast” can be used to describe both fibro genic properties – laying down ECM, and ECM modulating properties (i.e. breaking down matrix). Do the authors intend to use the term “ECM fibroblast” broadly to describe both properties, or would the authors consider describing these distinct properties as “fibrogenic” vs “ECM degradation”?\nMinor points:\nPage 5: “”Does this mean that fibroblasts express nuclear IL-33 but cannot secrete it?” Recommend avoiding rhetorical questions if they are not followed by a discussion. Page 5: redundancy instead of “redunance”\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": "259951", "date": "09 Apr 2024", "name": "Hao Li", "expertise": [ "Reviewer Expertise Immunology and rheumatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, authors aimed to provide a comprehensive summary on fibroblast functions. However, the organization of the paper is a little bit hard to follow. Also, if authors can deliver the concepts more clearly and concisely, it will make the paper more readable.\n\nMain concerns,\nThe introduction is interesting but not appropriate. Keeping language formal in scientific writing helps maintain professionalism. Fibroblasts are a type of cells which play important roles shaping our tissues. It is obvious that fibroblasts in lymphoid organs and non-lymphoid organs have distinct functions under naïve status. Recent advances of application of single-cell RNA-seq provides solid evidence on fibroblast heterogeneity. A brief introduction of fibroblast heterogeneity at the beginning will help readers better understand their distinct functions. Describing the function changes of fibroblasts from naïve status to disease status will provide a better readable synthesis. Sometimes, subtitles are not consistent or fully supported by the following paragraphs. For example, except producing chemokines, there are other mechanisms through which fibroblasts help positioning or organizing immune cells such as expressing adhesion molecules. Fibroblasts are not only the responders to alarmins but also the main producers of almonds. For example, it is generally accepted that stromal cells are the major producers of IL-33.\nMinor concerns,\n\nIt is not proper to keep questions for readers without giving answers or related hypothesis. For example, “Is there evidence that fibroblasts are impacted by this secreted form?” Proper punctuation needs to be checked before submission. Abbreviations must be spelled out completely on initial appearance in the manuscript. Every statement needs to be supported by properly cited studies.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "251130", "date": "26 Apr 2024", "name": "Theresa T. Lu", "expertise": [ "Reviewer Expertise Immunology", "Autoimmune disease - lupus", "Stromal cells in skin and lymph nodes", "Lymphatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewed by Mir Howlader; This review is very comprehensive, covering the distinct fibroblast functions in immune mediated inflammatory diseases (IMID) versus fibrotic diseases (FD). It discusses how disease initiation involves many different alarmins that lead to leukocyte infiltration and fibroblast activation. The review further explains how fibroblasts control the activation, differentiation, and survival of infiltrating leukocytes. It presents in depth the types and quantity of the growth factors fibroblasts produce and the various signals that can lead to differentiation of leukocyte subsets and formation of osteoclasts. It also discusses the pathogenic function of fibroblasts in the production and organization of the extracellular matrix (ECM), leading to fibrosis. Through every topic they explore, they discuss the potential therapeutic targets, the ongoing therapy research and they also address current gaps in the field and provide multiple experiments to try to fill in these gaps.\nI have a few minor suggestions:\nConsider adding sub-subheadings to the current 4 subheadings. For example, for the second subheading: Rationing of growth factors, it might be worth further breaking it down to “Growth factors involved in T cell survival in FD”, “Growth factors involved in T cell survival in IMID”, Growth factors involved in B cell survival in FD”, Growth factors involved in B cell survival in IMID”. It is mostly written in these subdivisions already but just adding a heading will help readers orient themselves better and then also refer back to specific topics more easily. Subheading 4 is in a different format, font, and color from the other subheadings. Figure 1 is great but just a little difficult to follow in terms of the cytokines, what they are doing and who is producing them. It might be helpful to add arrows to make more clear which signals are activating the fibroblasts and which signals the fibroblasts are producing.\nReviewed by Theresa Lu; in agreement with Mir Howlader's review of this outstanding manuscript. This paper will be a valuable resource across multiple fields.\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/13-54
https://f1000research.com/articles/11-1353/v1	21 Nov 22	{ "type": "Research Article", "title": "The Nigerian youth and their positive attitude towards the national drive against the spread of COVID-19: A cross-sectional online survey", "authors": [ "Boluwaji Jaiyesimi", "Toba Bamitale", "Babajide Diyaolu", "Kolawole Alabi", "Toba Bamitale", "Babajide Diyaolu", "Kolawole Alabi" ], "abstract": "Background: The global impact of coronavirus disease 2019 (COVID-19)has led to the need to prioritise public health campaign by all stakeholders among diverse population groups. This study investigated the dispositions of Nigerian adolescents towards the prevention of the spread of COVID-19. Methods: The young adolescents (n=1,529) included in this study, were between the ages of 15 and 26 years from Afe Babalola University. Students were contacted through their various colleges and residential hostels to complete the questionnaire via a shared link. All students included, voluntarily participated in this cross-sectional study by completing the adapted COVID-19 knowledge, attitude and practice (KAP) questionnaire. The collected data were analysed to show the level of knowledge, attitude and preventative practices and also to test for significant association between gender and variables for the study. The study was conducted between March 23-April 25, 2021. Results: The outcome of the findings showed that students with families with 5-10 members showed significant association with knowledge accuracy on COVID-19 (χ2 = 6.077, p = 0.044). There was a significant association between gender and the need to report the suspected case of COVID-19 infection to the health authorities (χ2 = 14.075, p = 0.001) with more females likely to report a suspected case. More females (59.2%) were significantly (χ2 = 8.904, p = 0.012) involved in the practice of social distancing or home quarantine as a preventive measure. Conclusions: This study showed evidence of high prevalence of knowledge related to COVID-19 in majority of the study participants. This is a pointer to the efficacy and success of present public health campaigns in Nigeria.", "keywords": [ "Perception", "health practices", "adolescents", "knowledge", "attitude", "COVID-19" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) has evolved into a major global public health concern (Habib et al., 2021). Since the 31st of December 2019, there has been over 209 million cases of COVID-19 reported worldwide, including a death toll of over 4.2 million in 220 countries (European Centre for Disease Prevention and Control, 2021). The pandemic has had devastating effects on both public health and societal life (Rahman & Sathi, 2020; Cori et al., 2020; Parikh et al., 2020). In Africa, about 7,043,648 cases have been reported, with Nigeria accounting for over 183,000 cases and over 2000 in deaths (Worldometer, 2021). In order to curtail the spread of the virus, the government raised public awareness on preventative health practices, enforced closure of volatile public and private facilities, enforced small-sized gathering with adequate spacing in public spaces, enforced stay at home order in flag point areas and inter and intra-border mobility to put national population statistics under check (Egbi et al., 2020). Aronu et al. (2021) in a cross-sectional study on COVID-19 knowledge and preventive practices among college adolescents in Nigeria, reported that the study participants had a low compliance rate with the enforcements. Many of the young adolescents acknowledged that only the aged and patients with comorbidity are at the high-risk end of COVID-19 mortality. In a study that was conducted in Ethiopia by Kebede et al. (2020), they identified that adolescents were less likely to practice preventive measures, and in turn, such behaviours would pose more problems in the preventive processes for COVID-19. Kebede et al. (2020b) in another study also stated that the transmission rate of the disease has increased rapidly. In spite of the available vaccines against the virus, the emergence of new strains and variants of the virus continues to pose global health challenges. According to Bates et al. (2021) preventive measures must be adopted to curb the spread of COVID-19. However, it is also important to note that only sufficient knowledge can prompt behavioural changes needed to curb the spread of the COVID-19 (Van Nhu et al., 2020; Belete et al., 2021). This is especially true in respect to the knowledge, attitude and practice towards the prevention of COVID-19 among the youths (Umeizudike et al., 2021). However, the effects of reported knowledge have not been seen in the attitude and safety practices of some of the youths in South-West Nigeria. Several studies have shown satisfactory levels of knowledge but all these studies are among the adult population (Kebede et al., 2020b; Abdelhafiz et al., 2020; Centers for Disease Control and Prevention, 2021). Several studies also reported, that those with medical-related occupations were more likely to have better knowledge that would also spill into an appropriate preventive attitude and practice (Noreen et al., 2020; Shi et al., 2020). Some studies also considered the limited facts about adherence to the practice of preventive measures of Covid-19 (Alzoubi et al., 2020; Nazli et al., 2020; Rahman & Sathi, 2020; Kebede et al., 2020a, 2020b; Feyisa, 2021). Another study noted that people with underlying health conditions generally felt the need to take greater preventive approach and attitude towards the virus (Adesegun et al., 2020; Belete et al., 2021). Therefore, this study was set to find out the knowledge, attitude and preventive practices of Nigerian adolescents towards curbing the spread of Covid-19.\n\n\nMethods\n\nA cross-sectional online survey was anonymously administered online for the young adolescents between the ages of 15 and 26 years who are undergraduates of Afe Babalola University. Afe Babalola University is a private university located in the South-Western region of Nigeria. The institution has almost 8,000 undergraduate students at the time this study was conducted. Only the Afe Babalola University undergraduate students who were healthy, mentally stable and fully residential on campus were eligible to participate in the study. Postgraduates students were excluded from the study. Students were contacted in their various colleges and undergraduate residential hostels to complete the questionnaire (Jaiyesimi et al., 2022b) via a shared link. The links were pasted on the commonly used social media platforms (WhatsApp and Telegram). Students were also encouraged to share the links with each other. A convenience sampling techniques was adopted for the study (Jiang et al., 2021). The sample size was calculated using EPI-info v.7. (EpiData Software) (RRID:SCR_008485). The assumption was set at 50% expected frequency, 95% confidence level, 5% acceptance margin of error, 4.0 design effect with 8000 estimated population size. The final adequate sample for the study was calculated to be 1468 students.\n\nThe study was carried out from March 23 - April 25, 2021 when the undergraduate students of Afe Bablola Univeristy were back to school. The COVID-19 knowledge, attitude and practice (KAP) questionnaire was adapted from the study of Ferdous et al. (2020). Their study focused on the young adolescents and adults while this study focused on the young adolescents. The questionnaire was administered online to avoid physical contact and to reach more respondents. The questionnaire was designed with Google forms (Google Inc, 2022) and the link was shared via social media platform (WhatsApp and Telegram). A total of 1,529 respondents completed the questionnaire in proportion of 626 male (40.9%) and 903 females (59.1%).\n\nThe validated modified COVID-19 KAP questionnaire (Elbur et al., 2016; Medani et al., 2018; Al-Hanawi et al., 2020; Ferdous et al., 2020) was tested with Cronbach Alpha for reliability (α = .66) excluding the demographic variables. SPSS software version 25 (IBM corp, 2017) (RRID:SCR_016479) was used to calculate the reliability. Age, gender, family social class, type of residence, region of residence, number of family members, and family status were the demographic variables added to the COVID-19 KAP questionnaire for the purpose of the study considering the selected population. Gender (male and female) and region of residence (south and north) had two sub-categories, while family social class (upper, middle and lower), number of family members (less than 5, 5-10, greater than 10), types of residence (large cities, mid-level cities and small cities) and family status (nuclear, extended and joint) had three sub-categories. Only age had four sub-categories (15-17, 18-20, 21-23 and 24-26 years). Under the KAP section, the subsections for knowledge and attitude comprised six questions each, while the practice section comprised seven questions. No item was removed from the original survey but it was modified (in terms of the grammatical composition of the items) for the purpose of the study.\n\nThe knowledge of COVID-19 sub-section had six items on a three-point rating scale (YES, NO and I DON’T KNOW). Each item was assigned a score to rate the response of the respondents (YES = 1, NO = 0, I DON’T KNOW = 0). The maximum summation of all scores on the sub-scale equals six. A cut-off score <4 means LESS ACCURATE, while a cut-off score ≥4 means MORE ACCURATE knowledge on the sub-scale of six. The attitude sub-section three-point rating scale included AGREE, UNDECIDED and DISAGREE with scores assigned respectively (1,0,0). The maximum summation of all scores on the sub-scale equals six. A cut-off score <4 means LESS POSITIVE, while a cut-off score ≥4 means MORE POSITIVE on the sub-scale of six. The practice sub-section also has three-point rating scale (YES = 1, NO = 0, SOMETIMES = 0). The maximum summation of all scores on the sub-scale equals seven. A cut-off score <5 means LESS FREQUENT, while a cut-off score ≥5 means MORE FREQUENT practice on the sub-scale of seven. These cut-off scores were determined to according to a previous study on COVID-19 (Ferdous et al., 2022).\n\nThe obtained responses from the Google form were downloaded via the google sheet into the Microsoft Excel (Microsoft, USA, 2019) (RRID:SCR_016137) for data cleaning. Incomplete responses with valid missing values were excluded from the datasheet. The descriptive statistics of frequency counts and percentages were computed for the variables used in the study while chi-square cross-tabulation were computed to test the significant association between the demographic variables and the COVID-19 KAP. SPSS (RRID:SCR_002865) was used to calculate the reliability and chi-square cross-tabulation for the study.\n\n\nResults\n\nA total of 1,529 respondents with the age ranges from 15–26 years voluntarily participated in the study (Jaiyesimi et al., 2022a). Most of the respondents (76.6%) fall within the category of 15–17 years. More females were involved in the study (59.1%) than male (40.9%). Most of the respondents were from nuclear (87%) and perceived middle-class family social status (79.2%), with a range of 5–10 family members (76.3%), lived in metropolitan cities (52.2%) within southern region of Nigeria (70.7%) (Table 1).\n\nAs shown in Table 2, more than half of the respondents (54.2%) identified that COVID-19 could easily spread through close contact with an infected person, while less than 2% claimed ignorance of the mode of transmission of the viral infection. Most of the respondents (90.1%) acknowledged that the incubation period of the virus is within 2-14 days. Almost all the respondents (98.8%) identified fever, dry cough and difficult breathing as the symptoms of the viral infection. More than half of the respondents reported that the population group at most risk of the virus is the elderly (57.9%) followed by the individuals with chronic conditions such as cancer, diabetes, respiratory infections and so on. Pregnant women (0.6%), children (2%), migrants (5.2%) were not considered as major risk groups by most of the participants. Most of the respondents reported that vaccination (62.8%) and supportive treatment (33.7%) are methods of treating COVID-19. Most of the respondents identified social distancing (34.3%), washing of hands (21.8%) and use of nose or face mask (15.5%) as the most effective COVID-19 preventive measures. More than half of the respondents recognized home-based handwashing with soap (59.1%) and restrictions of visitors (37.5%) as effective measures to protect the family against the spread. Almost 50% of the respondents reported no problem in creating awareness against the spread, however, 20.7% could not limit outdoor life and preferred not to use a protective face mask.\n\nAs shown in Table 3, no items under the knowledge of COVID-19 had significant association based on gender distribution. Only participants with a family of 5–10 members showed significant association with knowledge accuracy on COVID-19 (χ2 = 6.077, p = 0.044). Furthermore, those in this group had 80.8% more accurate knowledge of COVID-19 than respondents from other family categories (see Table 5). The respondents from metropolitan cities reported the highest percentage of accurate knowledge (53.3%), followed by mid-level cities (41.6%) and the small cities’ respondents had the least response (5.1%). Moreover, those who are from the middle family social class reported more accurate knowledge (76.3%) than others in the upper (18.9%) and lower class (4.8%) (see Table 6).\n\nAccording to Table 4, there was a significant association between gender and the need to report the suspected case of COVID-19 infection to the health authorities (χ2 = 14.075, p = 0.001). Females (58.7%) are more likely to report a suspected case of COVID-19 than their male counterparts (41.3%).\n\n* Significant p-value.\n\nOther items on the scale were not significantly associated with gender, even though females (58.3%) showed more positive attitude than male (41.7%) (see Table 6). The respondents from the metropolitan cities had more significant positive attitude (χ2 = 12.191, p = 0.002, 57.1%), than other city-dwellers (mid-level = 36.9% and small cities = 6%) (see Table 6).\n\nItems relating to the practice of COVID-19 preventive measures based on gender distribution are presented in Table 5. The question related to the use of handkerchief during coughing/sneezing had a significant association based on gender distribution (χ2 = 7.993, p = 0.018) with higher compliance from female participants (61.2%). Frequent washing of hands to curb the spread was significantly (χ2 = 16.895, p = 0.000) more common in female (61.4%) than male respondents (38.6%). Significantly more females (59.2%) were (χ2 = 8.904, p = 0.012) involved in the practice of social distancing or home quarantine as a preventive measure than male respondents (40.8%). In following all the government rules concerning the prevention of COVID-19, majority of the females (59.4%) were significantly (χ2 = 10.260, p = 0.006) more compliant with the rules (see Table 5) than male respondents (40.6%). The cross-tabulation for demographic variables (see Table 6) showed that the participants from middle class family were significantly more involved in the preventive measures (76.4%, χ2 = 8.115, p = 0.017) than other family classes (upper (19.2%) and lower (4.5%)).\n\n* Significant p-value.\n\n* Significant p-value.\n\n\nDiscussion\n\nThis research was carried out among Nigerian educated youth in order to find out their perception towards COVID-19 mode of transmission, symptoms, risk factors, treatments, preventive measures, and challenges. The study examined the knowledge, attitude, and practice towards COVID-19 and their association with the selected sociodemographic variables of the respondents.\n\nIn the perception domain, the participants showed good knowledge about the mode of transmission of the virus as well as the incubation period. This finding was similar to a study carried out by Peng et al. (2020) among undergraduates in China, who also demonstrated good knowledge of mode of transmission and incubation period of COVID-19 infection. However, this finding was in contrast with a similar study done in Congo, where respondents demonstrated poor knowledge of COVID-19 mode of transmission (Carsi Kuhangana et al., 2020). The difference in socio-economic and educational level could explain this disparity as the latter was conducted among sellers and customers frequenting some big public markets in Congo, who are expected to be a mixed population unlike the present study where the respondents were entirely young students. The majority of the respondents were familiar with the symptoms of COVID-19 and also reported the elderly as the most vulnerable group, followed by individuals with co-morbid medical conditions. This finding was similar to a study conducted in Jimma town, Ethiopia, where larger percentage of the participant were aware of the key clinical symptoms and the groups at high risk of developing a severe form of the disease (Kebede et al., 2020b). The similarity in findings could be as a result of the efforts of the health and educational agency of the government and access to information from social (WhatsApp and Telegram) and broadcast media (television and radio) by the study participants as reported in the current study.\n\nMost of the respondents identified that vaccination, followed by supportive treatment have important roles to play in the management of COVID-19 disease. This was in marked contrast to the findings of (Ferdous et al., 2020) where only 1% of the participant reported vaccination as having a role in the prevention of COVID-19 infection. This disparity could be due to the fact that vaccine development was still at a rudimentary stage around the end of first quarter of 2020 when the study was conducted. However, in a similar study conducted around the same time, Srichan et al. (2020) found that 31.2% were aware of the vaccine as a potential option. Most of the respondents recognized home-based handwashing with soap and water, followed by restriction of visitors as measures to protect the family against COVID-19 virus; they also identified social distancing and use of face masks as preventive measures. These findings agree with that of many other studies which reported good perception of hand hygiene, social distancing, wearing face mask and avoiding crowded areas (Akalu et al., 2020; Asmelash et al., 2020; Kassie et al., 2020; Tamire et al., 2020). However, in an earlier study, Ferdous, et al. (2020) reported that most of the respondents indicated negligence about the severity of the disease, reluctance to use face mask and not being able to control going out of the house. Understandably, this study was conducted immediately after lockdown was instituted in the study area and the populace could still be struggling with the physical, social and emotional barriers and limitations posed by the lockdown.\n\nThe respondents reportedly have sufficient knowledge of the virus because of exposure to the media and internet, coupled with some health-related courses in the university that focused mainly on trending public health issues. A similar result was carried out by Salman et al. (2020) among Pakistani University students in May 2020. The study found out that the students had sufficient knowledge and a positive attitude towards COVID-19. They are knowledgeable of the causative agent, incubation period, the spread, and preventive measures (Salman et al., 2020). The depth of the knowledge can also be associated with the fact that the respondents in their study and the current study were students. According to the current study, knowledge of COVID-19 could be a possible determinants of respondents' attitude and practice towards the disease. This is unlike the study that was carried out by Bhagavathula et al. (2020), where it was discovered that the students had insufficient knowledge about the virus although they had a positive attitude towards the prevention of the spread of the virus.\n\nA positive attitude was also exhibited by the majority of our respondents, they indicated that they were willing to adhere to the various measures put in place by various units or groups to curb the spread of the infection. A notable measure is their responsibility towards the University Hospital isolation centre, students have continued to be very observant and helpful in assisting the University to report any suspected cases to the isolation centre for clinical testing. A similar positive attitude was observed in researches carried out in Malaysia and China (Azlan et al., 2020; Zhong et al., 2020). Positive attitude towards hand hygiene, social distancing, use of face masks and avoiding crowded area prevented the spread of the virus (Umeizudike et al., 2021). Most of the respondents (70.3%) in this study reported that they adhere to the Government rules on the prevention of COVID-19 (see Table 5). In the current study, participants from middle-class families were more engaged in preventive practices than other categories of family social status (upper and lower). Due to the homogeneity of the selected sample (students of the same university) and the selected age category, the study cannot be generalised for the entire population.\n\n\nConclusions\n\nThis study showed evidence of high prevalence of knowledge related to COVID-19 in majority of the study participants. A significant proportion of the respondents understood the basic information, possessed positive attitude and demonstrated proactive practices towards COVID-19 infection. This is a pointer to the efficacy and success of present public health campaigns. However, male respondents showed poorer COVID-19 preventive practices compared to their female counterpart, and respondents from upper and lower socio-economic class demonstrated less-than-adequate knowledge, attitude and practices towards COVID-19. Public health interventions should therefore be tailored along these lines.\n\nThe scope of the study was limited by the data collection procedure. The participants were enrolled via Whatsapp and Telegram, due to the need capture the calculated sample size, which limited the study to convenience sampling technique. Some potential respondents were, probably, not captured due to irregular internet access. Those who did not have access to the selected social media platform might be unknowingly exempted from the study. The choice of population (undergraduate students from a university) might have exposed the study to selection bias which limits the generalizability of the study. Considering that the selected university is private institution, further study should focus on the undergraduate students at a public institution.\n\n\nData availability\n\nFigshare: COVID-19 Knowledge and symptoms July 13th.xlsx, https://doi.org/10.6084/m9.figshare.20341098 (Jaiyesimi et al., 2022a).\n\nThis project contains the following underlying data:\n\n- COVID-19 Knowledge and symptoms July 13th.xlsx (Raw de-identified questionnaire answers).\n\nFigshare: The Nigerian youth and their positive attitude towards the national drive against the spread of COVID-19: A cross-sectional online survey, https://doi.org/10.6084/m9.figshare.20379930.v1 (Jaiyesimi et al., 2022b).\n\nThis project contains the following extended data:\n\n- COVID-19 Knowledge and symptoms July 10th Questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthical approval\n\nEthical approval was obtained from the Health Research Ethics Committee (ABUAD/HREC/04/10/829). The participants under the age of 18 (duly registered as undergraduate student of the university) are covered by the institutional policy to voluntarily participate in this study with due consideration of no physical harm to the participants.\n\n\nConsent to participate\n\nAll respondents gave written informed consent to participate in the research through informed written consent.", "appendix": "References\n\nAdesegun O, Binuyo T, Adeyemi O, et al.: The COVID-19 Crisis in Sub-Saharan Africa: Knowledge, Attitudes, and Practices of the Nigerian Public. Am. J. Trop. Med. Hyg. 2020; 103: 1997–2004. PubMed Abstract \| Publisher Full Text\n\nAbdelhafiz AS, Mohammed Z, Ibrahim ME, et al.: Knowledge, Perceptions, and Attitude of Egyptians Towards the Novel Coronavirus Disease (COVID-19). J. Commun. Health. 2020; 45(5): 881–890. Publisher Full Text\n\nAkalu Y, Ayelign B, Derbew Molla M: Knowledge, Attitude and Practice Towards COVID-19 Among Chronic Disease Patients at Addis Zemen Hospital, Northwest Ethiopia. Infect. Drug Resist. 2020; 13: 1949–1960. PubMed Abstract \| Publisher Full Text\n\nAl-Hanawi MK, Angawi K, Alshareef N, et al.: Knowledge, Attitude and Practice Toward COVID-19 Among the Public in the Kingdom of Saudi Arabia: A Cross-Sectional Study. Front. Public Health. 2020; 8(May): 1–10. Publisher Full Text\n\nAlzoubi H, Alnawaiseh N, Al-Mnayyis A, et al.: COVID-19 - Knowledge, Attitude and Practice among Medical and Non-Medical University Students in Jordan. J. Pure Appl. Microbiol. 2020; 14: 17–24. Publisher Full Text\n\nAronu AE, Chinawa AT, Ossai EN, et al.: COVID-19: Knowledge of Mode of Spread and Preventive Practices among College Adolescents in Nigeria. J. Trop. Pediatr. 2021; 67(1). PubMed Abstract \| Publisher Full Text\n\nAsmelash D, Fasil A, Tegegne Y, et al.: Knowledge, Attitudes and Practices Toward Prevention and Early Detection of COVID-19 and Associated Factors Among Religious Clerics and Traditional Healers in Gondar Town, Northwest Ethiopia: A Community-Based Study. Risk Manag. Healthc. Policy. 2020; 13: 2239–2250. PubMed Abstract \| Publisher Full Text\n\nAzlan AA, Hamzah MR, Sern TJ, et al.: Public knowledge, attitudes and practices towards COVID-19: A cross-sectional study in Malaysia. PLoS One. 2020; 15(5): e0233668. PubMed Abstract \| Publisher Full Text\n\nBates BR, Tami A, Carvajal A, et al.: Knowledge, attitudes, and practices towards covid-19 among venezuelans during the 2020 epidemic: An online cross-sectional survey. PLoS One. 2021; 16(4 April): 1–16. PubMed Abstract \| Publisher Full Text\n\nBelete ZW, Berihun G, Keleb A, et al.: Knowledge, attitude, and preventive practices towards COVID-19 and associated factors among adult hospital visitors in South Gondar Zone Hospitals, Northwest Ethiopia. PLoS ONE. 2021; 16(5 May 2021): e0250145–e0250118. PubMed Abstract \| Publisher Full Text\n\nBhagavathula A, Rahmani J, Bandari D, et al.: Knowledge and Perceptions of COVID-19 Among Health Care Workers: Cross-Sectional Study. JMIR Public Health Surveill. 2020; 6: e19160. PubMed Abstract \| Publisher Full Text\n\nCarsi Kuhangana T, Mbayo C, Kitenge J, et al.: COVID-19 Pandemic: Knowledge and Attitudes in Public Markets in the Former Katanga Province of the Democratic Republic of Congo. Int. J. Environ. Res. Public Health. 2020; 17. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention: Coronavirus (COVID-19).2021.Reference Source\n\nCori L, Bianchi F, Cadum E, et al.: Risk Perception and COVID-19. Int. J. Environ. Res. Public Health. 2020; 17(9) PubMed Abstract \| Publisher Full Text\n\nDires A, Gedamu S, Getachew Y: Perception of COVID-19 Prevention Methods Efficacy and Intention to Use Among Patients with Chronic Disease in Dessie Town, Northeast Ethiopia: A Multicentered Cross-sectional Study. J. Multidiscip. Healthc. 2021; 14: 1325–1339. PubMed Abstract \| Publisher Full Text\n\nEgbi OG, Duru C, Kasia B: Knowledge, attitude and practice towards COVID-19 among workers of a tertiary hospital in Bayelsa State, Nigeria. Pan Afr. Med. J. 2020; 37(Suppl 1): 24. PubMed Abstract \| Publisher Full Text\n\nElbur A, Alharthi A, Aljuaid A, et al.: Knowledge of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and its Management: A Survey among Saudi People in Taif; Kingdom of Saudi Arabia. IOSR Journal of Pharmacy (IOSRPHR). 2016; 06(08): 33–39. Publisher Full Text\n\nEuropean Centre for Disease Prevention and Control: Data Daily New Cases Covid-19 EU/EEA Country.2021.Reference Source\n\nFerdous MZ, Islam MS, Sikder MT, et al.: Knowledge, attitude, and practice regarding COVID-19 outbreak in Bangladesh: An onlinebased cross-sectional study. PLoS One. 2020; 15(10 October): 1–17. PubMed Abstract \| Publisher Full Text\n\nFeyisa ZT: Factors limiting youths’ practice of preventive measures toward the outbreak of COVID-19 in Oromia special zone surrounding Finfinnee, Ethiopia. PLoS One. 2021; 16(3): e0248495. PubMed Abstract \| Publisher Full Text\n\nHabib MA, Dayyab FM, Iliyasu G, et al.: Knowledge, attitude and practice survey of COVID-19 pandemic in Northern Nigeria. PLoS One. 2021; 16(1): e0245176. PubMed Abstract \| Publisher Full Text\n\nJaiyesimi B, Toba David B, Babajide D, et al.: COVID-19 Knowledge and symptoms July 13th.xlsx. figshare. [Dataset].2022a. Publisher Full Text\n\nJaiyesimi B, Toba David B, Babajide D, et al.: The Nigerian youth and their positive attitude towards the national drive against the spread of COVID-19: A cross-sectional online survey. figshare. [Dataset].2022b. Publisher Full Text\n\nJiang M, Feng L, Wang W, et al.: Knowledge, attitudes, and practices towards influenza among Chinese adults during the epidemic of COVID-19: a cross-sectional online survey. Hum. Vaccin. Immunother. 2021; 17(5): 1412–1419. PubMed Abstract \| Publisher Full Text\n\nKassie BA, Adane A, Tilahun YT, et al.: Knowledge and attitude towards COVID-19 and associated factors among health care providers in Northwest Ethiopia. PLoS One. 2020; 15(8): e0238415. PubMed Abstract \| Publisher Full Text\n\nKebede Y, Birhanu Z, Fufa D, et al.: Myths, beliefs, and perceptions about COVID-19 in Ethiopia: A need to address information gaps and enable combating efforts. PLoS One. 2020a; 15(11): e0243024. PubMed Abstract \| Publisher Full Text\n\nKebede Y, Yitayih Y, Birhanu Z, et al.: Knowledge, perceptions and preventive practices towards COVID-19 early in the outbreak among Jimma university medical center visitors, Southwest Ethiopia. PLoS One. 2020b; 15(5): e0233744. Publisher Full Text\n\nMandaah FV, Nicholas T, Esemu SN, et al.: Trends in the population knowledge, attitudes and practices toward COVID-19 in the Buea municipality two months after the onset of the pandemic in Cameroon. Pan Afr. Med. J. 2020; 37: 134. PubMed Abstract \| Publisher Full Text\n\nMedani KET, NM Ald, AA Ala, et al.: Knowledge , attitude and practice of middle east respiratory syndrome corona virus (MERS-Cov), among male primary school. Indo Am. J. P. Sci. 2018; 5(12): 6288–16296.\n\nNazli T, Parveen H, Raheem A, et al.: Perceptions and Practices of the Adult Population in Response to SARS-CoV-2 Pandemic in India.2020. Publisher Full Text\n\nNoreen K, Zil-E- Rubab, Umar M, et al.: Knowledge, attitudes, and practices against the growing threat of COVID-19 among medical students of Pakistan. PLoS One. 2020; 15(12 December): 1–12. Publisher Full Text\n\nParikh PA, Shah BV, Phatak AG, et al.: COVID-19 Pandemic: Knowledge and Perceptions of the Public and Healthcare Professionals. Cureus. 2020; 12(5): e8144. PubMed Abstract \| Publisher Full Text\n\nPeng Y, Pei C, Zheng Y, et al.: A cross-sectional survey of knowledge, attitude and practice associated with COVID-19 among undergraduate students in China. BMC Public Health. 2020; 20(1): 1292. PubMed Abstract \| Publisher Full Text\n\nRahman A, Sathi NJ: Knowledge, attitude, and preventive practices toward COVID-19 among Bangladeshi internet users. Electron. J. Gen. Med. 2020; 17(5). Publisher Full Text\n\nSalman M, Mustafa ZU, Asif N, et al.: Knowledge, attitude and preventive practices related to COVID-19: a cross-sectional study in two Pakistani university populations. Drugs Ther. Perspect. 2020; 36: 319–325. PubMed Abstract \| Publisher Full Text\n\nShi Y, Wang J, Yang Y, et al.: Knowledge and attitudes of medical staff in Chinese psychiatric hospitals regarding COVID-19. Brain Behav. Immun. Health. 2020; 4(March): 100064. PubMed Abstract \| Publisher Full Text\n\nSrichan P, Apidechkul T, Tamornpark R, et al.: Knowledge, Attitude and Preparedness to Respond to the 2019 Novel Coronavirus (COVID-19) Among the Bordered Population of Northern Thailand in the Early Period of the Outbreak: A Cross-Sectional Study. SSRN Electron. J. 2020. Publisher Full Text\n\nTamire AH, Tamire AH, Legesse TK: Original Article Knowledge, Attitudes, and Practices Towards Coronavirus Disease -19 Among Health Professionals in Addis Ababa. Ethiop. Med. J. 2020; 58(4): 343–349.\n\nUmeizudike KA, Isiekwe IG, Fadeju AD, et al.: Nigerian undergraduate dental students’ knowledge, perception, and attitude to COVID-19 and infection control practices. J. Dent. Educ. 2021; 85(2): 187–196. Publisher Full Text\n\nVan Nhu H, Tuyet-Hanh TT, Van NTA, et al.Knowledge, Attitudes, and Practices of the Vietnamese as Key Factors in Controlling COVID-19. J. Commun. Health . 2020; 45(6): 1263–1269. Publisher Full Text\n\nWorldometer: Coronavirus Update.2021.Reference Source\n\nZhong B-L, Luo W, Li H-M, et al.: Knowledge, attitudes, and practices towards COVID-19 among Chinese residents during the rapid rise period of the COVID-19 outbreak: a quick online cross-sectional survey. Int. J. Biol. Sci. 2020; 16: 1745–1752. PubMed Abstract \| Publisher Full Text" }	[ { "id": "171237", "date": "09 May 2023", "name": "Pathiyil Ravi Shankar", "expertise": [ "Reviewer Expertise Public health. COVID-19. KAP studies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 section: Methods subsection, what do you mean by a young adolescent?\nIntroduction: Toward the end of the Introduction, kindly mention why this study among adolescents is necessary.\nMethods:\nCan students enter university at the age of 15 years? Can you explain university admissions in Nigeria? In most countries, students enter university only after the age of 17.\n\nTable 1: What is meant by place of residence – South, North as all respondents were staying in the university hostels? I believe you are referring to where their family is staying.\n\nMiddle, upper, and lower may be better characterized as economic status rather than social status, though these may be linked.\n\nPage 4, paragraph 1: What do you mean by the grammatical composition of the items?\n\nIn the results section, only differences in scores according to gender are shown. What about other demographic characteristics?\n\nDo university fees and living costs limit students from lower socioeconomic classes from attending the university?\n\nIt is nice that the data about the study is publicly available.\n\nIs the extended data already published? Is there any overlap with the data presented in this manuscript?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "174663", "date": "11 Jul 2023", "name": "Saadullah Khattak", "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 sharing this interesting article The Nigerian Youth and their positive attitude towards the national drive against the Spread of COVID-19: A cross-sectional online Survey. Here are some suggested edits and comments that could help to improve the article:\nMajor revision\nWrite a title such as The Nigerian youth knowledge, Attitude and Practice (KAP) Towards the national drive against the Spread of COVID-19: A cross-sectional online Survey or The Nigerian youth knowledge, Attitude and Practice (KAP) Towards the COVID-19: A cross-sectional online survey.\n\nRevise and summarize the abstract in 250 words.\n\nThe full manuscript needs to revise to remove grammatical and minor. An error such as first mentioning the complete form and then abbreviation for “COVID-19, and KAP”, etc.,\n\nFull stop after the citation and someplace before the citation. Please use the same pattern.\n\nMost of the cited references are from 2019-2021. Please also cite some recent work from 2021 onwards.\n\nSection: Introduction and discussion content can be enriched with research articles and more. For example:\nDOI: 10.3390/vaccines10101583, DOI: 10.3390/ijerph19137926, DOI: 10.29052/IJEHSR.v10.i2.2022.255-272, DOI: 10.3389/fmed.2021.747819, DOI: 10.3390/biom11060912\nChange the young adolescent word with the university student, youth, etc\n\nPlease explains the reason to start with 15 years age.\n\nPlease explains the reason (Postgraduates students were excluded from the study)—exclusion criteria.\n\nThe survey contained any informed consent pages?\n\nThe language of the questionnaire?\n\nThe survey was done through Telegram and WhatsApp. One bias is it is limited to people who have these social media accounts, and authors mention this bias. However, how mobile numbers were selected for the WhatsApp channel is unclear. This needs to be clarified.\n\nFinding sections needs improvement, and authors need to explain statistical analysis results rather than the same info available in the tables.\n\nAuthors should use other statistical tools or weighted averages.\n\nThe rationale of the study is not clear. I am not sure why the authors have conducted the study when similar investigations are available from Nigeria. The authors need to clarify the rational of this study in the introduction section.\n\nThe questionnaire was developed from previous studies, but there is no information on the data collection form's validation, reliability, and translation.\n\nI appreciate the efforts of the authors to have a large sample size for this study, but this study needs extensive revisions and even re-analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/11-1353
https://f1000research.com/articles/12-484/v1	11 May 23	{ "type": "Research Article", "title": "Morphometry of pyramidalis muscle and its role in reconstructive surgeries: A cadaveric study", "authors": [ "Suhani Sumalatha", "Sharanya Rao", "Vrinda Hari Ankolekar", "Suhani Sumalatha", "Sharanya Rao" ], "abstract": "Background: The pyramidalis muscle is a tiny triangular-shaped muscle that is located in the anterior abdominal wall, which originates from the body of the pubis and pubic symphysis and is inserted into linea alba. This study aimed to measure the different parameters of the pyramidalis muscle in adult cadavers. Methods: This study was carried out on 31 adults (26 males and five females) 10% formalin embalmed cadavers of both sexes from the Department of Anatomy at Kasturba Medical College, Manipal. Results: The mean length of the right pyramidalis muscle was 64.44 ± 12.52 mm and the left pyramidalis muscle was 64.73 ± 12.81 mm. The mean width of the muscle was 15 ± 4.18 mm and 15.03 ± 3.52 mm on the right and left sides, respectively. The mean thickness of the muscle was 1.32 ± 0.55 mm and 1.4 ± 0.80 mm on the right and left sides, respectively. The distance between the umbilicus to pubic symphysis ranged from 130–192 mm and their mean was calculated to be 159.77 ± 15.36. The distance between the umbilicus and the apex of the muscle ranged from 72–123 mm. Conclusions: The measured parameters like length, width and thickness may help the surgeons to locate the muscle during infra umbilical surgeries.", "keywords": [ "Abdominal muscles", "Anterior abdominal wall", "Pyramidalis", "Rectus sheath" ], "content": "Introduction\n\nThe pyramidalis muscle (PM) is a tiny triangular-shaped muscle that is located in the anterior abdominal wall in humans.1 Muscle is present in the lower anterior part of the rectus abdominis muscle enclosed by the rectus sheath. The muscle originates from the anterior surface of the body of the pubis and pubic symphysis. Through the tendinous fibers, PM is fixed in the anterior part of the pubis and through the ligamentous fibers; it is attached to the pubic symphysis. Inferiorly PM has a broad base, as it ascends, the muscle becomes slender and gets inserted medially into linea alba. Insertion of muscle takes place halfway between the umbilicus and pubis.\n\nIt is believed that the action of the PM contracts the linea alba and strengthens the abdominal wall.2,3 On the other hand, its absence does not tend to result in a functional loss.4 As a result, authors believe it is a vestigial muscle left behind from the pouches of marsupials and monotremes.5\n\nIn humans, the PM has been linked to the assumption of upright posture.6 The PM flap can be used to treat tiny intractable lesions in the foot and ankle, such as chemical wounds and prolonged osteomyelitis.4 The PM, when cryopreserved, can be used as a source of stem cells that can then be employed to treat post-prostatectomy stress urinary incontinence.7 When performing a longitudinal incision for a standard caesarean section, the PM was used to locate the linea alba and midline.8\n\nThe morphometric study on PM in the south Indian adult human population is less studied. So this study aimed to explore the various parameters of PM in adult cadavers.\n\n\nMethods\n\nThe protocol was approved by the Institutional Ethics Committee (IEC-617/2021) Kasturba Medical College and Kasturba Hospitals, Manipal on September 15, 2021. Written informed consent was given by the body donors for teaching and research when they were alive.\n\nIn this descriptive cross-sectional study, 31 adult cadavers of both sexes (26 males and five females) were utilized from the Department of Anatomy at Kasturba Medical College (KMC), Manipal for morphometric measurements. Data were collected between October 1,st 2021 to April 30th, 2022, for a period of six months. Available cadavers in the department were used for the study purpose with the sample size 31. It was a convenient sampling approach. Specimens with damage to the anterior abdominal wall were not considered for the study. With the surgical instruments, cadavers were dissected and muscles were exposed. The following parameters were measured using a vernier caliper:\n\n• Length of muscle from the pubic symphysis to its attachment to linea alba (apex) along its medial border (Figure 1A).\n\n• Width at the base/origin of the muscle (Figure 1B).\n\n• The thickness of the muscle at the midpoint of the measured length.\n\n• A small gap was found in few cadavers between right and left PM (Figure 2A). The length and breadth of this gap were measured (Figure 2B, Figure 2C).\n\n• Distance between the umbilicus and pubic symphysis (DUP) and distance between the umbilicus and apex of the muscle (DUA) (Figure 3).\n\n• Distance between the anterior superior iliac spine (ASIS) and lateral part of the base of the muscle (LBM) (Figure 4).\n\n• Distance between ASIS to apex of the muscle (Figure 4).\n\n• Distance between ASIS and the pubic symphysis (ASIS-PS) (Figure 4).\n\nA) Measurement of length and B) width of PM near its origin. PM, pyramidalis muscle; RAM, rectus abdominis muscle; RPM, right pyramidalis muscle; LPM, left pyramidalis muscle; A, above; L, left.\n\nA) Gap between the PM. B) Measurement of length and C) width of gap between the PM. PM, pyramidalis muscle; RAM, rectus abdominis muscle; RS, rectus sheath; RPM, right pyramidalis muscle; LPM, left pyramidalis muscle; A, above; L, left.\n\nPM, pyramidalis muscle; U, umbilicus; RAM, rectus abdominis muscle; DUP, distance between umbilicus to pubic symphysis; DUA, distance between umbilicus to apex of the muscle; RS, rectus sheath; A, above; L, left.\n\nASIS-LBM: Distance between the anterior superior iliac spine to the lateral part of the base of the muscle. Distance between ASIS to tip of the muscle. Distance between ASIS and PS. PM, pyramidalis muscle; ASIS, anterior superior iliac spine; LBM, lateral part of the base of the muscle; PS, pubic symphysis; U, umbilicus; EOM, external oblique muscle; RS, rectus sheath; IEA, inferior epigastric artery; RAM, rectus abdominis muscle; A, above; L, left.\n\nAll the dimensions were completed by the same researcher to avoid inter observer variation.\n\nDescriptive statistics (mean, standard deviation, and range) were done for all measured parameters. To compare the right and left of each continuous variable, paired T-test was performed. P-value ≤ 0.05 was considered significant. The normally distributed data were correlated using Pearson's correlation coefficient analysis under the following combinations: i) Right and left PM length; ii) Right and left PM width; iii) Right PM length and right PM width; iv) Left PM length and left PM width; v) DUP and PM length (R and L); vi) DUP and PM width (R and L); vii) ASIS-PS (R) and PM width (R); viii) ASIS-PS (L) and PM width (L); ix) ASIS-PS (R) and PM length (R); and x) ASIS-PS (L) and PM length (L). SPSS (RRID:SCR_002865) version 16.0 (Chicago, SPSS Inc.) was utilized for data analysis.\n\n\nResults\n\nDescriptive statistics\n\nThe length of the right PM ranged from 40–95 mm and the left PM ranged from 38–100 mm. The mean length of the right PM was 64.44 ± 12.52 mm and the left PM was 64.73 ± 12.81 mm. There was a 1–2 mm difference between the length of the right and left PM.\n\nThe width of the right PM and left PM ranged from 8–24 mm and 9–22 mm, respectively. The mean width of the right PM and left PM was 15 ± 4.18 mm and 15.03 ± 3.52 mm, respectively.\n\nThe PM thickness ranged from 0.5–2 mm on both sides. The mean thickness of muscle on the right side was 1.32 ± 0.55 mm and 1.4 ± 0.80 mm on the left side.\n\nIn 18 cadavers, a triangular gap was observed between right and left PM. Its length ranged from 8–26 mm and its width ranged from 5–17 mm.\n\nDescriptive statistics of the remaining parameters are tabulated in Table 1.15\n\n* Length of the triangular gap as shown in Figure 2B; width of the triangular gap as shown in Figure 2C.\n\nPaired T-test\n\nA paired T-test was used to compare all the right and left dependent variables. ASIS-Apex (R) and ASIS-Apex (L) were significantly different from each other since the p-value was 0.0014. There was no significant difference between the two respective groups of other dependent variables.\n\nPearson's correlation coefficient analysis\n\nPearson's correlation coefficient analysis was done since the data were distributed normally. A significant positive correlation was observed between right and left PM length and right and left PM width. The above result suggests that the increase in length or width of the right PM was directly proportional to the length/width of left PM.\n\nCorrelation analysis between right PM length and width and left PM length and width showed a weak but positive correlation. From this, we can note that as the length of the muscle increases, the width of the muscle may or may not increase.\n\nA weak correlation was observed when DUP and ASIS-PS was compared with the right and left PM length and width. From this, we can conclude that the DUP and ASIS-PS have negligible effects on the length and width of the PM. The correlation coefficient and its level, analyzed between the parameters are tabulated in Table 2.\n\n\nDiscussion\n\nLength and width of PM varies in different populations and it is tabulated in Table 3. It was observed in the present study, the length of PM in the female population was almost equal to the length of the PM reported by Natsis et al.,9 Hojaij et al.,10 and Kipkorir et al.11 The width of the PM noted in the present study is almost equal to the study by Natsis et al.9 A study by Das et al.,12 reported the mean thickness of 32 ± 0.55 mm in the right PM and 1.4 ± 0.80 mm in the left PM. In our present study, the mean thickness of muscle on the right side was 1.32 ± 0.55 mm and 1.4 ± 0.80 mm on the left side, which was relatively less than the thickness reported in the study by Das et al.12\n\nIn the present study, strong positive correlations were found between the lengths and widths of the PM on both sides, which is in line with the results of the study by Natsis et al.9\n\nThe present study also measured ASIS-PS, ASIS-LBM, ASIS-Apex, DUP and DUA. The mean, standard deviation and range values of the aforementioned parameters are tabulated in Table 1. These parameters were not recorded in the previous studies; therefore we were not able to compare it with the other studies.\n\nThe present study also showed correlations between the DUP and PM length (R and L), DUP and PM width (R and L), ASIS-PS (R) and PM width (R), ASIS-PS (L) and PM width (L), ASIS-PS (R) and PM length (R), ASIS-PS (L) and PM length (L). The correlation analysis showed a weak but positive correlation between them. This suggests that increases or decreases in ASIS-PS and DUP have negligible effects on the length and width of PM.\n\nPlastic surgeons and general surgeons must be aware of potential regional differences, to reduce the chance of consequences and implement the most effective therapeutic strategy.13 A prostate-cystopexy employing PM for prostate stabilization can treat individuals with functional dysuria caused by the prostate's retrograde inclination and those with bladder obstruction caused by motor neuron injuries.14\n\nTherefore, it is essential for the surgeons to be aware of the morphometry of the pyramidalis muscle prior to making the different incisions needed for procedures involving the anterior abdominal wall.\n\nPM act as a landmark for surgeons during infra-umbilical and suprapubic incisions while performing caesarean sections. The PM may be collected through a reasonably unnoticeable Pfannenstiel (pubic) opening with minimal donor site complications making it a desirable donor muscle for microsurgical transfer.\n\nThe limitation of the present study is the smaller female cadaver sample size, with respect to the male cadaver sample size. Therefore a sex-based comparison was not performed in this study.\n\nThis study can be done to explore the foetus morphometric data and its clinical implications.\n\n\nConclusions\n\nThe parameters like length, width, and thickness and new parameters such as ASIS-PS, ASIS-LBM, ASIS-Apex, DUP and DUA may help the surgeons to locate the muscle in the suprapubic region while performing surgical procedures to remove the flap of donor pyramidalis muscle.", "appendix": "Data availability\n\nFigshare: Morphometry of pyramidalis muscle and its role in reconstructive surgeries: A cadaveric study. https://doi.org/10.6084/m9.figshare.22200181. 15\n\nThis project contains the following underlying data:\n\n- EX sheet - Morphometric study of pyramidalis muscle.xlsx\n\n- STROBE-checklst.doc\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\nAcknowledgements\n\nAuthors are thankful to all the teaching and non-teaching staffs at department of Anatomy, KMC Manipal for their support in completing this study.\n\n\nReferences\n\nAshley-Montagu MF: Anthropological significance of the musculus pyramidalis and its variability in man. Am. J. Phys. Anthropol. 1939 Oct; 25(3): 435–490. Publisher Full Text\n\nDickson MJ: The pyramidalis muscle. J. Obstet Gynaecol (Lahore). 1999 July 02; 19(3): 300. Publisher Full Text\n\nSuhani S, Sharanya R, Vrinda HA: Prevalence And Variations Of The Pyramidalis Muscle In Adults And Fetuses: Relevance And Clinical Implications. J. Pharm. Negat. Results. 2023; 13(10): 20.\n\nVan Landuyt K, Hamdi M, Blondeel P: The pyra-midalis muscle free flap. Br. J. Plast. Surg. 2003; 56: 585–592. Publisher Full Text\n\nSkret-Magierło J, Soja P, Drozdzowska A, et al.: Two techniques of pyramidalis muscle dissection in Pfannenstiel incision for cesarean section. Ginekol. Pol. 2015; 86(7): 509–513. PubMed Abstract \| Publisher Full Text\n\nKaur H, Singla RK, Brar RS, et al.: Study of the Morphometry of the Pyramidalis Muscle and Its Incidence in the Indian Population. Int. J. Anat. Res. 2016; 4(2): 2207–2211. Publisher Full Text\n\nSumino Y, Hirata Y, Hanada M, et al.: Long-term cryopreservation of pyramidalis muscle specimens as a source of striated muscle stem cells for treatment of post-prostatectomy stress urinary incontinence. Prostate. 2011; 71(11): 1225–1230. PubMed Abstract \| Publisher Full Text\n\nAnson BJ, Beaton LE, McVay CC: The pyramidalis muscle. Anat. Rec. 1938; 72: 405–411. Publisher Full Text\n\nNatsis K, Piagkou M, Repousi E, et al.: Morphometric variability of pyramidalis muscle and its clinical significance. Surg. Radiol. Anat. 2016; 38(3): 285–292. PubMed Abstract \| Publisher Full Text\n\nHojaij FC, Kogima RO, Moyses RA, et al.: Morphometry and frequency of the pyramidalis muscle in adult humans: A pyramidalis muscle’s anatomical analysis. Clinics. 2020; 75(11): e1623–e1629. Publisher Full Text\n\nKipkorir V, Olabu B, Ongeti K, et al.: Prevalence and pubo-umbilical index of pyramidalis muscle in a select Kenyan population. Surg. Radiol. Anat. 2021; 43(9): 1461–1466. PubMed Abstract \| Publisher Full Text\n\nDas SS, Saluja S, Vasudeva N: Biometrics of pyramidalis muscle and its clinical importance. J. Clin. Diagn. Res. 2017; 11(2): AC05–AC07. PubMed Abstract \| Publisher Full Text\n\nMalcic-Gürbüz J, Özdoǧmuş Ö, Yüksel M: Unusual rectus abdominis and pyramidalis muscles: Clinical significance - A case report. Marmara Med. J. 2001; 14(2): 107–109.\n\nDik P, Van Gool JD, De Jong TP: Urinary continence and erectile function after bladder neck sling suspension in male patients with spinal dysraphism. BJU Int. 1999 Jun; 83(9): 971–975. PubMed Abstract \| Publisher Full Text\n\nSumalatha S, Rao S, Ankolekar V: Morphometry of pyramidalis muscle and its role in reconstructive surgeries: A cadaveric study. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "173177", "date": "14 Jul 2023", "name": "Ranganath Vallabhajosyula", "expertise": [ "Reviewer Expertise gross anatomy", "histology", "anatomical education", "technologies in anatomy 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\nThere exists a good amount of literature on the morphometry of the pyramidalis muscle. The authors have made a decent attempt to add on the data to the existing information.\nThe introduction, material and methods, results are well presented.\n\nThe results did not show whether the PM was observed in all the dissections and on both sides. In the results, the authors have reported a significant difference between the distance of ASIS to the TIP of PM on the right and left sides. It is suggestible to justify the reason for the significant difference. Did the authors observe any gender differences in the values, having less female sample though?\nThe authors would have explained in more detail the importance of the Pearson correlation that was applied in their study and how these observations are useful for clinical interventions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9923", "date": "16 Nov 2023", "name": "Vrinda Ankolekar", "role": "Author Response", "response": "Q. The results did not show whether the PM was observed in all the dissections and on both sides. A. PM was observed in 27 cadavers out of 31, in 26 cadavers it was bilateral and in one cadaver it was unilateral. Q. In the results, the authors have reported a significant difference between the distance of ASIS to the TIP of PM on the right and left sides. It is suggestible to justify the reason for the significant difference. A. The distance of ASIS to tip of right & left PM were significantly different because of the difference in the length of right and left PM. Q. Did the authors observe any gender differences in the values, having less female sample though? A. We did not observe any gender differences Q. The authors would have explained in more detail the importance of the Pearson correlation that was applied in their study and how these observations are useful for clinical interventions. A. The Pearson's correlation was performed to see whether there is any relationship between the measured values. We found strong correlation with length of right and left side PM. This could help the surgeons to locate PM and acquire required amount of flap to treat minor lesions in ankle region." } ] }, { "id": "184851", "date": "17 Aug 2023", "name": "Shanu Markand", "expertise": [ "Reviewer Expertise Cell culture", "anatomical variaitions", "histology", "and vision sciences." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 focused on the morphometric analysis of pyramidal muscle (PM).\nStrengths\nThe study design is good; the images are clear, and the results are interpreted thoroughly. Authors have been diligent in assessing all different aspects of PM morphometry. The paper is easy to follow.\n\nAreas to Improve\nThe tile describes the pyramidalis muscles with its role in reconstructive surgeries. The paper covers the former part well, but the latter needs more discussion rather than a sentence or two. The authors need to comment on how different measurements can potentially relate surgically.\n\nWas there any incidence of the unilateral presence of PM?\nAbbreviations should be avoided in the conclusions.\n\nThe triangular gap had a lot of variation, but this aspect was not discussed thoroughly. Additionally, what is the effect of embalming or quality of dissection and time passed after the dissection on these gap measurements (also other parameters)?\n\nIs the work 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": "10293", "date": "16 Nov 2023", "name": "Vrinda Ankolekar", "role": "Author Response", "response": "Q1.The tile describes the pyramidalis muscles with its role in reconstructive surgeries. The paper covers the former part well, but the latter needs more discussion rather than a sentence or two. The authors need to comment on how different measurements can potentially relate surgically. A. There is no literature regarding the importance of each and every parameters or how different measurements can potentially relate surgically. However these parameters can be useful for the plastic surgeons while giving abdominal incisions and removal of donor flap. Q2.Was there any incidence of the unilateral presence of PM? Yes, in one cadaver Q3. Abbreviations should be avoided in the conclusions. It is removed Q4. The triangular gap had a lot of variation, but this aspect was not discussed thoroughly. Additionally, what is the effect of embalming or quality of dissection and time passed after the dissection on these gap measurements (also other parameters)? We used freshly embalmed cadavers and the dissections and measurements were done simultaneously. During suprapubic incisions, as well as while removing the donor flap,the knowledge of triangular gap between the pyramidalis muscle may be helpful for the plastic surgeons." } ] }, { "id": "184844", "date": "17 Aug 2023", "name": "Mehtap Tiryakioglu Yuksel", "expertise": [ "Reviewer Expertise Anatomy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 providing a good morphometrical and statistical data of the pyramidalis muscle which can be an additional information for the readers who are in the field of medical sciences.\n\nThe cadaveric pictures are very clear to be read, yet a simply drawn diagram would also be a good addition to show the reference points for the measurements of the muscle-just a suggestion.\n\nThe title would be specified as \"Morphometry of pyramidalis muscle and its role in reconstructive surgeries: A cadaveric study in South Indian population\"\n\nThe embryology of pyramidalis muscle also could've been added as part of the Introduction as it may be absent in 20% of the people. Anatomical and morphometrical studies need a good emryological info, so the reason/s underlying the differences can be explained. Otherwise this is a good anatomical study 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?\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": "10294", "date": "16 Nov 2023", "name": "Vrinda Ankolekar", "role": "Author Response", "response": "Q1. The cadaveric pictures are very clear to be read, yet a simply drawn diagram would also be a good addition to show the reference points for the measurements of the muscle-just a suggestion. A. Four hand drawn diagrams to show the reference points for the measurements of pyramidalis muscle are added. Q2. The title would be specified as \"Morphometry of pyramidalis muscle and its role in reconstructive surgeries: A cadaveric study in South Indian population\" A. The title is specified as above in the manuscript. Q3. The embryology of pyramidalis muscle also could've been added as part of the Introduction as it may be absent in 20% of the people. Anatomical and morphometrical studies need a good emryological info, so the reason/s underlying the differences can be explained. Otherwise this is a good anatomical study to be indexed. The embryology/ phylogeny of pyramidalis muscle is added as part of introduction." } ] } ]	1	https://f1000research.com/articles/12-484
https://f1000research.com/articles/12-416/v1	18 Apr 23	{ "type": "Research Article", "title": "Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study ", "authors": [ "Mashael Alharbi", "Mansour Alharbi", "Amal Surrati", "Mashael Alhilabi", "Ayed alrashdi", "Majedah Almokhalafi", "Mansour Alharbi", "Amal Surrati", "Mashael Alhilabi", "Ayed alrashdi", "Majedah Almokhalafi" ], "abstract": "Background: There is a huge burden of nutrition-related non-communicable diseases, and diabetes is one of the leading chronic nutrition-related diseases affecting more than 500 million people globally. Collecting information regarding the awareness of dietary and nutrition knowledge among diabetic patients is the first step to developing a disease prevention program. Thus, this study primarily aims at assessing the dietary awareness of diabetes patients attending the diabetic centre in Madinah governorate, Saudi Arabia. Methods: The study was started in November 2020 and ended in October 2021. The study participants (315) were type 2 diabetes mellitus (T2DM) patients attending a diabetic centre in Madinah, Saudi Arabia. A self-prepared dietary knowledge questionnaire (DKQ) was used in this research. The variables include balanced diet, food type, food choice, carbohydrate, protein, and fat. Knowledge score was, and the total score was leveled/categorized into ‘good’, ‘average’, and ‘poor’. Data were analyzed by SPSS v.26. Results: The study results identified the current knowledge of T2DM patients about different dietary items. The knowledge score of 62.2% of participants showed an average level of dietary knowledge, which is statistically significant. When we separately evaluated their understanding of different dietary components, we found that T2DM patients had poor knowledge of carbohydrates (30.15%), fat, food choices (47.7%), and type (34.6%). However, they had acceptable knowledge of proteins (56.5%). Conclusion: Our participants exhibited acceptable knowledge about proteins but poorer knowledge of other food groups. A healthy, well-balanced diet is essential for excellent glycaemic control. It is recommended to educate and arrange a health education program regarding dietary knowledge, specially designed for diabetic patients so that patients can opt for a healthier lifestyle.", "keywords": [ "Type 2 diabetes mellitus", "dietary knowledge", "awareness", "Saudi Arabia" ], "content": "Introduction\n\nToday’s world has a huge burden of nutrition-related non-communicable diseases.1 Diabetes is one of the leading chronic nutrition-related diseases affecting more than 500 million people globally (around 6.3% of the world’s population).2,3 Diabetes alone is the ninth primary cause of mortality.2 It is also estimated that over 1.5 million deaths were directly caused by diabetes alone.4 However, the main concern raised recently is that greater than one-third of diabetes-related mortalities occur in individuals younger than 60.5 The International Diabetes Federation (IDF) for the Middle East and North Africa (MENA) recently estimated that diabetes prevailed at 18% in Saudi Arabia alone.3\n\nStudies have shown a continuous upward trend in the prevalence of diabetes.2 There is also an increased burden of human suffering as measured by disability-adjusted life years (DALYs) because of diabetes. However, many countries have implemented a significantly high budget for health care expenditure.6 Non-modifiable risk factors like age and familial history can be one of the causes of the continuous upward trend.7 At the same time, modifiable risk factors include increased body mass index due to processed high caloric unhealthy diet and physical inactivity for type 2 diabetes mellitus (T2DM).4,7\n\nRecent analysis has reported that for the management of diabetes, only pharmacologic approaches are not sufficient.2 The management of diabetes is a multifactorial process involving demographic and social factors that influence the treatment outcomes.8 There are also patient-related factors, such as self-care behaviors, that have a strong influence on glycemic intolerance.9 Blood glucose monitoring, diabetes-related knowledge, and dietary knowledge all come under self-care behaviors.10 Similarly, Dietary monitoring is necessary to predict patients’ quality of life, nutritional status, and risk factors to avoid chronic diabetes-related complications.10\n\nEarly onset T2DM in <45 years of age occurs in obese, having dyslipidemia, history of smoking, and sedentary lifestyles are the main contributors.11 Simple lifestyle modifications are shown to be effective in preventing or delaying complications in T2DM patients.4 Dietary modifications and nutrition knowledge are the cornerstones of diabetes type 2 management.12 Eating habits are directly related to dietary knowledge and significantly contribute to glycemic control.12 Nutrition knowledge helps individuals with diabetes to optimize their quality of life.13\n\nPatients with T2DM often face difficulty identifying the recommended diet, including its quality and quantity. The collection of food and dietary habits is affected by the knowledge of a patient concerning a suggested diet.14 A study on dietary knowledge among T2DM patients conducted in the Almajamah area of Saudi Arabia found poor awareness, attitudes, and practices toward healthy dietary habits. Only one-sixth of the studied population followed a strict diabetic diet that represented a poor diabetic control level.15 Non-adherence to dietary plans with inadequate knowledge is facing in many countries,16 which in turn is associated with poor family support17 and decreased motivation.18 Diabetes in < 60 years of age is most prevalent in low-and middle-income countries, while in high-income countries, this is mostly found in the aging population (>60 years of age).15 Collecting information regarding the awareness of dietary and nutrition knowledge among diabetic patients is the first step to developing a disease prevention program. Although there is an increased prevalence of T2DM, studies have shown that the dietary knowledge among patients is insufficient.15 Knowing the level of knowledge and perception of dietary habits in T2DM patients helps provide them with more efficient and beneficial education. Therefore, this study aims to assess the dietary knowledge among diabetic patients attending the diabetic center, Madinah governorate, Saudi Arabia.\n\n\nMethods\n\nEthical approval was obtained from Research and Studies Department of Health Affairs, Ministry of Health, Madinah, Saudi Arabia (IRB#544). All patients who gave voluntary written informed consent for participation before data collection were included in the study. The study participants’ information was kept confidential and anonymous.\n\nThis study was a cross-sectional descriptive study, conducted at Al-Madinah Al Munawarah which is the second holiest city in Islam, located in the Hejaz region of western Saudi Arabia. The study was started in November 2020 and ended in October 2021.\n\nStudy participants consist of patients with Type 2 Diabetes mellitus attending a diabetic center in Madinah, Saudi Arabia. The inclusion criteria was type 2 diabetic patient, > 18 years of age, and the participation was voluntary. Those who were type 1 diabetic patients, < 18 years and those with mental impairment or did not give consent for participation were excluded from the study. A systematic random sampling technique was utilized for participant recruitment and data collection.\n\nPopulation size: the total number of type 2 diabetic patients attending the diabetic center.\n\nConfidence limit: 5%\n\nconfidence interval: 95%\n\nDiabetes prevalence in KSA: 18%3\n\nUsing Epi info to calculate the sample size, the calculated sample size was 227. However, it was increased to 315 to overcome the possibility of outliers and missing data (if any).\n\nThe self-prepared Dietary Knowledge questionnaire (DKQ) used in this research consisted of multiple-choice questions (MCQs) prepared after an extensive literature review. The questionnaire was face validated by faculty members of the university. Back-to-back translations (Arabic and English) were done by the native Arabic speaker and reviewed by the corresponding author to improve the clarity of the questions.30,31 The pilot study was also conducted, and the changes were done according to responses and unclear questions. The researcher interviewed 10 patients (started 15 September 2020 and ended in 20 September 2020) from outside the selected class test feasibility of questionnaire, time taken to finish the interview and detect difficulties. These 10 cases were not included in main study. Those questions that were unclear or that did not cover the study concept were modified accordingly. The questionnaire was adapted from a published article,19 which showed that the Cronbach Alpha of that questionnaire was 0.869. The questionnaire was distributed online through google survey forms. The completed version of the forms was saved in the secured email (which was only assessed by the study supervisor) and collected after the required sample size was achieved. Selection bias is common in population-based studies however, we sent invitations to all the relevant participants who attended the clinics. Therefore, who those who were willing to participate filled the online questionnaire.\n\nThe questionnaire was self-administered and consisted of 19 questions with multiple choices. Variables include knowledge about a balanced diet, food type, food choice, carbohydrates, proteins, and fats. However, only one response is correct. The correct response was coded 1, and all others were coded 0. Therefore, the total score = 19 (< 8 = poor level, 8–12 = average level, and > 12 good knowledge level). The higher score of the individual corresponded to higher dietary knowledge. However, each variable’s quantitative scores were categorized into 3: <50% as poor knowledge, 50-75% as acceptable, and >75% as adequate knowledge.12,20\n\nAll data were analyzed by SPSS software version 26. The normality of the quantitative variables was checked. The frequency (percentage) of correct response for each variable was presented. Outliers were detected through Z-scores. Knowledge score was, and the total score was leveled/categorized into ‘good’, ‘average’, and ‘poor’. One-sample T-test was performed to evaluate the dietary knowledge of T2DM individuals. A p-value of <0.05 is considered statistically significant.\n\n\nResults\n\nThe overall frequency and percentage of correct responses were presented against each item in Table 1.29 The dietary knowledge questions were also categorized into food choices, knowledge about carbohydrates, fats/oil, protein, and type of food. Most patients select correct food choices for diabetes (85.4%). While very few (19%) know which of the food they can freely eat. Almost half of the participants knew how to treat low blood sugar (42.5%). The median knowledge score of participants for the food choices was 1(0-3), corresponding to poor knowledge (47.3%) about food choices for T2DM.\n\nRegarding the knowledge about dietary group carbohydrates, two fifths of the participants correctly identified the food with the highest carbohydrate percentage. Around two thirds of the participants knew that bread, cereals, rice, and pasta are a rich source of carbohydrates. Many participants did not know which food contained the highest amount of sugar (82.9%). However, most participants (61%) correctly responded about whole grains foods. The majority know that eating too much sugar will lead to diabetes (80%). The median knowledge score about carbohydrates was three (0-5), while the percentage showed poor overall knowledge of carbohydrates (30.15%).\n\nRegarding dietary knowledge about protein, most participants correctly identified the food with the highest protein percentage (71.1 %), and most knew the complete source of protein (75.6%). The median knowledge score of protein was 2 (0-2), and the percentage showed an almost acceptable level of knowledge of protein (56.5%). Concerning the fat and oil group, most of the patients know food with the highest percentage of fat (73.7%) and which is highest in fat (51.4%). Only one fifth of the participants knew the food product with a high cholesterol level. While a majority of the patients correctly identified that fat is a rich source of calories (88.9%). The median knowledge score of fat was 2 (0-3), whereas, in terms of percentage, T2DM patients have poor knowledge of fat (47.7%). There were various statements on a selection of food types. The median knowledge score of food type was four (0-5), with the percentage showing that T2DM patients have poor knowledge of food type (34.6%).\n\nThe study results showed that all the z-scores of dietary knowledge score were less than three, whereas the minimum z-score was -2.85 and the maximum was 2.08. There was no outlier detected in the dietary knowledge variable. The mean dietary knowledge score (total 19 items) was 10 (8-12), minimum three, and maximum 16. However, most of the study participants (62.2%) showed an average level of dietary knowledge (Figure 1). One sample T-test statistics confirmed that the T2DM patient’s average level of dietary knowledge is statistically significant (p < 0.0006).\n\n\nDiscussion\n\nThe study results identified the current knowledge of T2DM patients about different dietary items. The knowledge score of 62.2% of participants showed an average level of dietary knowledge, which is statistically significant. When we separately evaluated their awareness of different dietary components, we found that T2DM patients have poor knowledge about carbohydrates, fat, food choices, and food type. However, they had acceptable knowledge of proteins.\n\nDifferent studies around the world showed various levels of dietary knowledge among type 2 diabetic patients. In agreement with our study, another study conducted on T2DM patients in Iran showed average levels of dietary knowledge.21 In contrast, a study reported inadequate dietary knowledge among T2DM females of Amman, Jordan.20 A study conducted in Ethiopia found a poor level of dietary knowledge and practices.22\n\nOur study results showed that the majority of the participants answered that they know what a diabetic diet (85.4%) is; however, they have poor knowledge regarding individual food components and what should be used with hypoglycemia (42.5%). Comparing our results with another study, only 43.9% of individuals know the recommended meal for diabetes/day, and only 44.5% know what should be used with hypoglycemia.20\n\nCarbohydrates are an important macronutrient that affects the postprandial glycemic index. Another study conducted among Emirati and Omani adults concluded that individuals have insufficient knowledge of carbohydrates.23 Likewise, another Saudi study showed participants' poor knowledge about carbohydrates but had good dietary knowledge regarding other good groups, including fat and protein.14 Most participants correctly identified the main carbohydrate source in the diabetic diet, but most did not know the food with the highest sugar content. Global estimates from 165 countries found a strong positive association between diabetes prevalence and per capita sugar consumption.24\n\nDuring the last decades, many countries have experienced a major shift in food choices and nutritional transition from the Mediterranean diet to more processed/junk foods. Likewise, lack of physical activity, urbanized lifestyle, and increased food consumption cause an increase in nutritional problems with a high prevalence of obesity that triggers many chronic diseases.20 In Saudi Arabia, food choices and portion sizes with a sedentary lifestyle were dramatically increased, resulting in a high prevalence of obesity and diabetes. Moreover, they also consume too many sugary drinks.25 A recent randomized controlled trial of diabetic patients demonstrated that simplified diabetes nutrition education greatly impacts glycemic control.26\n\nThe first limitation is the study's cross-sectional nature, which limits the temporal association between disease and exposure. Secondly, the study participants were only recruited from a single center; therefore, the generalizability of the study results should be interpreted with caution. Thirdly, the sociodemographic data were not collected for this study. However, there are also some strengths of the study. First, a valid questionnaire was used, and study participants were recruited through systematic random sampling. Second, the sample size was calculated, and a maximum number of participants was recruited to overcome the missing data.\n\n\nConclusion\n\nA healthy, well-balanced diet is essential for excellent glycemic control.27 Appropriate self-care and effectual metabolic regulation control prevent hypoglycemia, other metabolic complications, and ketoacidosis.27 Nutrition plays a key role; food with high fiber, low fat, and limited carbohydrate should be taken at regular intervals.25 All food groups should be included in the daily diet as recommended.28 Our participants showed acceptable protein knowledge but poorer knowledge of other food groups. It is not an easy job to increase the awareness and knowledge of nutrition and a healthy diet in diabetic individuals in our society. Self-dietary management is the first and foremost step in providing knowledge and skills to people with diabetes concerning treatment, medications, and nutritional aspects as defined by American Diabetes Association (ADA).25 A healthy diet is considered an integral part of diabetes management. Likewise, a healthy lifestyle, regular exercise, weight management, and a well-balanced diet can improve diabetic patients' health. It is recommended to educate and arrange a health education program regarding dietary knowledge, specially designed for diabetic patients so that patients can opt for a healthier lifestyle. There is also a need to emphasize preventive measures with research based on an effective region-specific diet and nutritional knowledge that can be provided to everyone, including prediabetics and those with diagnosed diabetes.", "appendix": "Data availability\n\nFigshare: Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study. https://doi.org/10.6084/m9.figshare.22122656.v1. 29\n\nThe project contains the following underlying data:\n\n• Data.xlsx. (Anonymised answers to questionnaire, correct answers – 1, incorrect answers - 0).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study. https://doi.org/10.6084/m9.figshare.22123001.v1. 30\n\nThis project contains the following extended data:\n\n• Nutritional Knowledge Questionnaire in Diabetics (English).docx. (Blank English version of the questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Nutritional Knowledge Questionnaire in Diabetics (Arabic).docx https://doi.org/10.6084/m9.figshare.22123004.v1. 31\n\nThis project contains the following extended data:\n\n• Nutritional Knowledge Questionnaire in Diabetics (Arabic).docx. (Blank Arabic version of the questionnaire)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: STROBE checklist and flowchart for “Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study”. https://doi.org/10.6084/m9.figshare.22083365.v1. 32\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nOnyango EM, Onyango BM: The rise of noncommunicable diseases in Kenya: an examination of the time trends and contribution of the changes in diet and physical inactivity. J. Epidemiol. Glob. Health. 2018; 8(1-2): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan MAB, Hashim MJ, King JK, et al.: Epidemiology of Type 2 Diabetes - Global Burden of Disease and Forecasted Trends. J. Epidemiol. Glob. Health. 2020; 10(1): 107–111. Publisher Full Text\n\nInternation Diabetes Fedration, IDF MENA members: Saudi Arabia: 2022 [cited June 23, 2022]. Reference Source\n\nWorld Health Organization, Diabetes, fact sheet: 2021 [cited 22-06-22]. Reference Source\n\nAlotaibi A, Perry L, Gholizadeh L, et al.: Incidence and prevalence rates of diabetes mellitus in Saudi Arabia: An overview. J. Epidemiol Glob Health. 2017; 7(4): 211–218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBukhman G, Mocumbi AO, Atun R, et al.: The Lancet NCDI Poverty Commission: bridging a gap in universal health coverage for the poorest billion. Lancet. 2020; 396(10256): 991–1044. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhazvineh B, Javvaji S: Assessment of Risk Factors in Type 2 Diabetes Mellitus in a Teritary Care Hospital, India. Arch. Pharm. Pract. 2020; 11(S1): 120–123.\n\nGillani SW, Abdul MIM, Ansari IA, et al.: Predicting relationship of eating behavior, physical activity and smoking with type II diabetes and related comorbidities among Saudi citizens: cross-sectional observational study. Int. J. Diabetes Dev. Ctries. 2019; 39(1): 115–122. Publisher Full Text\n\nGillani SW, Sulaiman SAS, Abdul MIM, et al.: A qualitative study to explore the perception and behavior of patients towards diabetes management with physical disability. Diabetol. Metab. Syndr. 2017; 9(1): 1–10.\n\nGillani SW, Ansari IA, Zaghloul HA, et al.: Predictors of health-related quality of life among patients with type II diabetes mellitus who are insulin users: a multidimensional model. Curr. Ther. Res. 2019; 90: 53–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBo A, Thomsen RW, Nielsen JS, et al.: Early-onset type 2 diabetes: Age gradient in clinical and behavioural risk factors in 5115 persons with newly diagnosed type 2 diabetes—Results from the DD2 study. Diabetes Metab. Res. Rev. 2018; 34(3): e2968. PubMed Abstract \| Publisher Full Text\n\nBreen C, Ryan M, Gibney MJ, et al.: Diabetes-related nutrition knowledge and dietary intake among adults with type 2 diabetes. Br. J. Nutr. 2015; 114(3): 439–447. PubMed Abstract \| Publisher Full Text\n\nTaylor MK, Sullivan DK, Ellerbeck EF, et al.: Nutrition literacy predicts adherence to healthy/unhealthy diet patterns in adults with a nutrition-related chronic condition. Public Health Nutr. 2019; 22(12): 2157–2169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSami W, Alabdulwahhab KM, Ab Hamid MR, et al.: Dietary knowledge among adults with type 2 diabetes—kingdom of Saudi Arabia. Int. J. Environ. Res. Public Health. 2020; 17(3): 858. Publisher Full Text\n\nAnsari T, Sami W, Alsubaie N, et al.: Assessment of knowledge, attitude, and practice of dietary pattern in patients with type 2 diabetes mellitus: a locality-based perspective study. Int. J. Diabetes Dev. Ctries. 2019; 3(7): 581–585. Publisher Full Text\n\nAyele AA, Emiru YK, Tiruneh SA, et al.: Level of adherence to dietary recommendations and barriers among type 2 diabetic patients: a cross-sectional study in an Ethiopian hospital. Clin. Diabetes Endocrinol. 2018; 4(1): 1–7. Publisher Full Text\n\nRaaijmakers LGM, Hamers FJM, Martens MK, et al.: Perceived facilitators and barriers in diabetes care: a qualitative study among health care professionals in the Netherlands. BMC Fam. Pract. 2013; 14(1): 1–9.\n\nMcBrien KA, Naugler C, Ivers N, et al.: Barriers to care in patients with diabetes and poor glycemic control—A cross-sectional survey. PLoS One. 2017; 12(5): e0176135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSami W, Ansari T, Butt NS, et al.: Psychometric evaluation of dietary habits questionnaire for type 2 diabetes mellitus. J. Phys. Conf. Ser. 2017; 890: 012151. Publisher Full Text\n\nEl-QuDah JMF: Dietary knowledge among female diabetic patients in Amman, Jordan. Curr. Res. Nutr. Food Sci. 2016; 4(2): 107–113. Publisher Full Text\n\nMohammadi S, Karim NA, Talib RA, et al.: Knowledge, attitude and practices on diabetes among type 2 diabetic patients in Iran: a cross-sectional study. Science. 2015; 3(4): 520–524.\n\nWorku A, Mekonnen Abebe S, Wassie MM: Dietary practice and associated factors among type 2 diabetic patients: a cross sectional hospital based study, Addis Ababa, Ethiopia. Springerplus. 2015; 4(1): 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli HI, Bernsen RM, Taleb S, et al.: Carbohydrate-food knowledge of Emirati and Omani adults with diabetes: results of a pilot study. Int. J. Diabetes & Metabolism. 2008; 16: 23–26.\n\nWeeratunga P, Jayasinghe S, Perera Y, et al.: Per capita sugar consumption and prevalence of diabetes mellitus – global and regional associations. BMC Public Health. 2014; 14(1): 186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSami W, Ansari T, Butt NS, et al.: Effect of diet on type 2 diabetes mellitus: A review. Int. J. Health Sci. 2017; 11(2): 65–71. PubMed Abstract\n\nHashim SA, Mohd Yusof B-N, Abu Saad H, et al.: Effectiveness of simplified diabetes nutrition education on glycemic control and other diabetes-related outcomes in patients with type 2 diabetes mellitus. Clin. Nutr. ESPEN. 2021; 45: 141–149. PubMed Abstract \| Publisher Full Text\n\nChawla SPS, Kaur S, Bharti A, et al.: Impact of health education on knowledge, attitude, practices and glycemic control in type 2 diabetes mellitus. J Family Med Prim Care. 2019; 8(1): 261–268. Publisher Full Text\n\nBurch E, Ball L, Somerville M, et al.: Dietary intake by food group of individuals with type 2 diabetes mellitus: A systematic review. Diabetes Res. Clin. Pract. 2018; 137: 160–172. PubMed Abstract \| Publisher Full Text\n\nAlharbi M: Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study. Dataset. figshare. 2023. Publisher Full Text\n\nAlharbi M: Dietary knowledge assessment among the patients with type 2 diabetes in Madinah: A cross-sectional study. Dataset. figshare. 2023. Publisher Full Text\n\nAlharbi M: Nutritional Knowledge Questionnaire in Diabetics (Arabic).docx. Dataset. figshare. 2023. Publisher Full Text\n\nAlharbi M: STROBE_checklist.doc. figshare. Journal contribution. 2023. Publisher Full Text" }	[ { "id": "195807", "date": "30 Aug 2023", "name": "Munazza Jawed", "expertise": [ "Reviewer Expertise Epidemiology", "Diabetes", "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\nI carefully read your manuscript titled “Dietary knowledge assessment among the patients with Type 2 diabetes in Madinah: A cross-sectional study”. The paper is generally well-written and structured and demonstrates smooth flow. The paper shows an interesting fact and will be a valuable addition relevant to its field and improve policymaking. The article does a good job of assessing the dietary knowledge among patients with Type 2 diabetes. The authors reported mostly patients with T2DM have inadequate knowledge of nutrition and food groups that are essential for excellent glycaemic control. However, I think the paper has some shortcomings regarding comparing its results. It would be best if the author compared it across different socio-demographic statuses (not collected), a major study limitation. The author used recent references.\nMinor comments:\nAbstract:\nMethod section: change spelling of leveled to levelled.\nConclusion last sentence, “it is recommended….” Change it to this “Educating and arranging a health education program regarding dietary knowledge is recommended, specially designed for diabetic patients so that patients can opt for a healthier lifestyle”.\nIntroduction:\n1st paragraph, 4th line: delete word “alone”.\n2nd paragraph, 3rd line: Replace “Health care” to “healthcare”.\n3rd paragraph, 2nd line: remove “the” before treatment.\n4th paragraph, 1st line: change it to this “Early onset T2DM usually occurs in <45 years; its main contributor include obesity, dyslipidaemia, smoking, and a sedentary lifestyle”\n\n4th paragraph, 2nd line: delete “shown to be”\n5th paragraph, 6th line: change “poor family support” to inadequate family support”\n5th paragraph, 8th line: replace aging to “ageing”\nMethods:\nStudy design first sentence: replace it with this “This study was a cross-sectional descriptive study conducted at Al-Madinah Al Munawarah, the second holiest city in Islam, in the Hejaz region of western Saudi Arabia.”\nStudy population: start sentence with “The study population”. 2nd line remove coma after patient. Change 3rd sentence to “Those who were type 1 diabetic patients < 18 years old and those with mental impairment or who did not consent to participate were excluded from the study”.\n\nStudy tools: 3rd line, replace “done” to “made”.\n5th line, the numeral 10 is used instead of the word spelled out. Consider spelling out the number.\n6th line, replace it with this sentence “selected class to test the feasibility of the questionnaire, the time taken to finish the interview”.\n8th line, remove first word “that”.\n10th line, google to Google.\n11th line, change sentence to “Selection bias is common in population-based studies; to overcome this we sent invitations to all the relevant participants who attended the clinics.\n12th line, change sentence to “Therefore, who are only willing to participate filled out the online questionnaire”.\nData entry:\n2nd line, response to responses\nResults:\nTwo third to two-third\nOne fifth to one-fifth\nLast paragraph, 3rd line, change these sentences “with a minimum of three and a maximum of 16”. “However, most of the study participants (62.2%) showed average dietary knowledge”.\nDiscussion:\n1st paragraph, 4th line, fat to fats, type to types.\n4th paragraph, 3rd line, change sentence “likewise, another Saudi study…” to “Likewise, another Saudi study showed participants had poor knowledge”. Conclusion:\n3rd line: change sentence to “All food groups should be included in the daily diet”\n9th line: change sentence “It is recommended to…” to “Educating and arranging a health education program regarding dietary knowledge is recommended, specially designed for diabetic patients so that patients can opt for a healthier lifestyle”.\nOverall: For consistency, consider replacing words with the British English spelling. For example, leveled to levelled, center to centre, behavior to behaviour, fiber to fibre.\n\nIs the work 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": "10842", "date": "11 Jan 2024", "name": "Mashael Alharbi", "role": "Author Response", "response": "Abstract: Method section: change spelling of leveled to levelled. Conclusion last sentence, “it is recommended….” Change it to this “Educating and arranging a health education program regarding dietary knowledge is recommended, specially designed for diabetic patients so that patients can opt for a healthier lifestyle”. Introduction: 1st paragraph, 4th line: delete word “alone”. 2nd paragraph, 3rd line: Replace “Health care” to “healthcare”. 3rd paragraph, 2nd line: remove “the” before treatment. 4th paragraph, 1st line: change it to this “Early onset T2DM usually occurs in <45 years; its main contributor include obesity, dyslipidaemia, smoking, and a sedentary lifestyle” 4th paragraph, 2nd line: delete “shown to be” 5th paragraph, 6th line: change “poor family support” to inadequate family support” 5th paragraph, 8th line: replace aging to “ageing” Response: All the above suggested changes have been done in the manuscript. Methods: Study design first sentence: replace it with this “This study was a cross-sectional descriptive study conducted at Al-Madinah Al Munawarah, the second holiest city in Islam, in the Hejaz region of western Saudi Arabia.” Study population: start sentence with “The study population”. 2nd line remove coma after patient. Change 3rd sentence to “Those who were type 1 diabetic patients < 18 years old and those with mental impairment or who did not consent to participate were excluded from the study”. Study tools: 3rd line, replace “done” to “made”. 5th line, the numeral 10 is used instead of the word spelled out. Consider spelling out the number. 6th line, replace it with this sentence “selected class to test the feasibility of the questionnaire, the time taken to finish the interview”. 8th line, remove first word “that”. 10th line, google to Google. 11th line, change sentence to “Selection bias is common in population-based studies; to overcome this we sent invitations to all the relevant participants who attended the clinics. 12th line, change sentence to “Therefore, who are only willing to participate filled out the online questionnaire”. Data entry: 2nd line, response to responses Response: All the above suggested changes in method section have been made in the manuscript. Results: Two third to two-third One fifth to one-fifth Last paragraph, 3rd line, change these sentences “with a minimum of three and a maximum of 16”. “However, most of the study participants (62.2%) showed average dietary knowledge”. Response: All the above suggested changes have been made to the manuscript. Discussion: 1st paragraph, 4th line, fat to fats, type to types. 4th paragraph, 3rd line, change sentence “likewise, another Saudi study…” to “Likewise, another Saudi study showed participants had poor knowledge”. Response: All the above suggested changes in discussion have been made to the manuscript. Conclusion: 3rd line: change sentence to “All food groups should be included in the daily diet”. 9th line: change sentence “It is recommended to…” to “Educating and arranging a health education program regarding dietary knowledge is recommended, specially designed for diabetic patients so that patients can opt for a healthier lifestyle”. Response: All the above suggested changes in conclusion have been made to the manuscript. Overall: For consistency, consider replacing words with the British English spelling. For example, leveled to levelled, center to centre, behavior to behaviour, fiber to fibre. Response: All the above suggested changes have been made throughout the manuscript." } ] }, { "id": "178211", "date": "19 Dec 2023", "name": "Ashraf El-Metwally", "expertise": [ "Reviewer Expertise Public health", "statistics", "epidemiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe document exhibited a high level of writing proficiency and holds significant relevance in the field of public health in the Kingdom of Saudi Arabia. The information presented within was clearly articulated and supported by accurate references to current scholarly works. The researcher employed suitable methodology throughout the study; however, it is noteworthy that the authors acknowledged a limitation that they didn’t collect sociodemographic data, which constitutes a substantial concern. It is strongly advised that future research endeavors incorporate sociodemographic questions, as these factors have the potential to influence statistical analysis and subsequent findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10843", "date": "11 Jan 2024", "name": "Mashael Alharbi", "role": "Author Response", "response": "Dear Reviewer, Thank you for your kind review and acknowledgment of the article. We have made all the suggested changes as per your comments. Your comments really improved our article. Your feedback regarding incorporating the sociodemographic questions in future research are duly noted. While we aimed to provide a comprehensive overview, we understand the potential benefits of understand impact of different socioeconomic levels and these factors might have the potential to influence statistical analysis and subsequent findings." } ] } ]	1	https://f1000research.com/articles/12-416
https://f1000research.com/articles/12-149/v1	09 Feb 23	{ "type": "Research Article", "title": "Use of ivermectin and factors associated with the prevention and/or treatment of COVID-19: a cross-sectional online survey in the province of Chincha, Peru", "authors": [ "Jose Salvador-Carrillo", "Luz Campos-Loza", "David Guillen-Carbajal", "Jorge Osada", "Alejandra Zevallos", "J. Smith Torres-Roman", "Jose Salvador-Carrillo", "Luz Campos-Loza", "David Guillen-Carbajal", "Jorge Osada", "Alejandra Zevallos" ], "abstract": "Background: Peru has reported one of the highest mortality rates from COVID-19 worldwide. The Chincha province has been one of the most affected regions in Peru and the leading promoter of the use of ivermectin for the treatment of COVID-19. Therefore, our study aimed to evaluate the frequency of use and factors associated with the use of ivermectin for COVID-19 in Chincha. Methods: A cross-sectional study was conducted during the second wave of COVID-19 in Peru. For statistical analyses, frequencies and percentages were reported. Prevalence ratios (PR) with a 95% confidence interval (CI), and a p-value of 0.05 were used to determine statistical significance. SPSS version 22 (IBM Corp) program was used for the analyses. Results: A total of 432 participants were included in the study. A total of 67.6% (n = 292) of the participants used ivermectin during the COVID-19 pandemic. Of these, 20.20% (n=59) of the people used ivermectin for prophylactic purposes only, while 41.79% (n=122) used it as treatment for COVID-19 only, and 38.01% (n=111) used it for both reasons. The consumption of ivermectin was associated with being 50 years or older (PR:1.27, 95% CI:1.04–1.54), having a technical education level (PR:1.16, 95% CI:1.01–1.34), having had symptoms of COVID-19 with negative/no diagnosis (PR: 1.28, 95% CI: 1.07–1.53) or positive diagnosis (PR:1.38, 95% CI:1.18–1.61), or having had contact with infected people (PR:1.45, 95% CI:1.06–1.98). Conclusions: Most people in Chincha used ivermectin during the second wave of the COVID-19 pandemic. The main factors associated with the use of ivermectin for the prevention/treatment of COVID-19 were age ≥50 years, having a technical education level, having had symptoms with negative/no diagnosis or positive diagnosis, and contact with people infected with SARS-CoV-2.", "keywords": [ "Ivermectin", "COVID-19", "Treatment and Prevention", "Peru." ], "content": "Introduction\n\nCOVID-19 is a highly contagious and rapidly spreading virus that can have various manifestations, from asymptomatic to severe cases, often resulting in death.1 Peru was one of the countries with the highest mortality rate (8.89%) worldwide [https://covid19.minsa.gob.pe/sala_situacional.asp]. In this country, the Ica department was devastated by COVID-19, with an elevated mortality rate during 2020,2 while Chincha, a province of Ica, reported one of the highest mortality rates among all provinces of Peru, with a cumulative rate of 43.5 deaths per 10,000 inhabitants.3\n\nIn Chincha, located 123 miles south of Lima, the capital of Peru, several factors contributed to its high mortality, such as insufficient number of beds in intensive care units (ICU) and the lack of specialists in the management of this disease.4,5 However, another factor that could have contributed to the elevated mortality rate in this province was the use of ivermectin as a prophylactic and/or treatment for COVID-19. In fact, several studies have shown that patients who have consumed ivermectin have no improvement compared with those who have not taken this drug;6–8 even in other studies they report a higher mortality rate.7\n\nThe belief in ivermectin as a therapeutic or prophylactic drug for COVID-19 was very common in Chincha [https://elcomercio.pe/peru/ica/chincha-entre-la-ivermectina-sin-limites-la-promesa-de-una-vacuna-y-el-golpe-del-covid-19-vacuna-peruana-manolo-fernandez-farvet-noticia/], and this drug was distributed in parks and city squares [https://www.idl-reporteros.pe/el-hospital-san-jose/; https://saludconlupa.com/comprueba/ivermectina-nuevo-estudio-no-apoya-su-uso-para-casos-leves-de-covid-19/]; moreover, regional leaders encouraged its use, and local media supported this action [https://exitosanoticias.pe/v1/dr-fernandez-si-mas-peruanos-tomaran-ivermectina-habria-menos-casos-de-covid-19/]. This level of promotion of the use of ivermectin against COVID-19 for its inhabitants was not observed in other regions of Peru [https://elcomercio.pe/peru/ica/chincha-entre-la-ivermectina-sin-limites-la-promesa-de-una-vacuna-y-el-golpe-del-covid-19-vacuna-peruana-manolo-fernandez-farvet-noticia/].\n\nThe use of ivermectin to treat COVID-19 in Peru started when Peru's Ministry of Health, in the first months of the pandemic, included this drug in the COVID-19 treatment guidelines.4,5,9 Then, the Peruvian government spent USD 6.25 million (exchange rate 1 USD = 4 PEN) on the acquisition of ivermectin for the treatment of COVID-19 in 2020 [https://elcomercio.pe/lima/sucesos/covid-19-gobierno-nacional-gasto-112-mas-en-la-compra-de-medicinas-no-recomendadas-que-en-oxigeno-ecdata-noticia/]. On the other hand, the Peruvian population was exposed to several infodemics about the effect of ivermectin on the COVID-19 virus.10\n\nThis scenario likely led to the prescription and self-medication of ivermectin among Chincha’s population. Thus, the present study aimed to evaluate the frequency and factors associated with the use of ivermectin for COVID-19 in Chincha.\n\n\nMethods\n\nA cross-sectional study was conducted between March 23 and June 21, 2021, in the province of Chincha, located in the Ica department, in southern Peru. During this period, the maximum peak and the decrease in the number of infections and deaths in the second wave of COVID-19 in Peru were observed [https://www.dge.gob.pe/portalnuevo/covid-19/covid-cajas/situacion-del-covid-19-en-el-peru/].\n\nAccording to the last national census conducted by the National Institute of Statistics and Informatics, the estimated population of Chincha was 226,113 inhabitants.11 The sample size calculation was carried out taking into account a confidence level of 95%, a margin of error of 5% and a percentage of variability of p = q = 50%. The sample size was 385 participants.\n\nThe inclusion criteria were people over 18 years of age who had adequately completed the survey, who resided in Chincha, and who agreed to voluntarily sign the informed consent. The exclusion criterion was whether the participant used ivermectin as a therapeutic/prophylactic measure for a disease other than COVID-19. Participant selection was made using nonrandom sampling for convenience.\n\nThe questionnaire was designed based on a review of the scientific literature that evaluated possible variables that could influence the use of ivermectin as a prophylactic or treatment for COVID-19 in the Peruvian population. The questionnaire was reviewed and discussed by a committee of experts that defined the variables and questions. The dichotomic questions showed a reliability coefficient of 0.709 measured through the KR-20 test.\n\nThe questionnaire consisted of 16 questions (Table S1). The first section consisted of six questions related to the participant’s sociodemographic data: province, sex, age, marital status, educational level attained, and economic salary (calculated according to the current minimum salary in Peru: USD 232.50). In the second section of three questions, participants were asked if they had been diagnosed with COVID-19, what diagnostic tests had been performed, and if they had been in contact with people diagnosed with COVID-19.\n\nThe third section consisted of six questions, related to which information was collected on the use of ivermectin during the COVID-19 pandemic, whether it was prescribed by a doctor (or self-medicated), whether it was used for prophylactic reasons (to avoid developing symptoms of COVID-19 in case of contagion) or for therapeutic purposes (for the treatment of COVID-19 symptoms), in which period of the pandemic it was taken (March–June 2020, July–October 2020, and November 2020–May 2021), and the source of access to ivermectin.\n\nIn the fourth section of a single question, participants were asked if they had any comorbidity (obesity, diabetes, high blood pressure, or other diseases). Before the questionnaire, a section was added that included information about the objectives of the study, the anonymity of the responses, the confidentiality of data processing, the risks and benefits of participating in the study, and finally the informed consent.\n\nThe questionnaire was built in Google Forms and distributed virtually using social networks (Facebook, WhatsApp, and email). This e-survey was applied following the CHERRIES recommendations.12 The recruitment process was free (open survey), and each visitor had the opportunity to participate in this study. The survey was shared in Spanish (official language in Peru). The technical functionality and usability were tested by the principal investigator (PI) before fielding the questionnaire. Furthermore, only the PI had access to data collected by the e-survey.\n\nThe statistical analysis was carried out in four stages. In the first stage, the general characteristics of the population of the Chincha province were described in means and standard deviation for the quantitative variables, and frequency and percentage for the qualitative variables. In the second stage, a descriptive analysis of ivermectin usage was carried out during the COVID-19 pandemic. In the third stage, a bivariate analysis between covariates and ivermectin consumption was performed using the chi-square test. For the fourth stage, only the variables that presented a p < 0.05 in the bivariate analysis were considered for the robust Poisson regression model (step wise): age, marital status, education, COVID19 diagnosis, contact with people infected by SARS-CoV-2, and obesity. The degree of association was represented by a prevalence ratio (PR) with its respective 95% confidence interval (95% CI). Values of p < 0.05 were considered as significant. Data analysis was performed using IBM SPSS Statistics for Window (version 24.0, RRID:SCR_016479)\n\nResearch was carried out following the recommendations of the Declaration of Helsinki. Each participant provided their signed informed consent. The research was approved by the Institutional Research Ethics Committee of San Juan Bautista Private University (Registry No. 062-2021-CIEI-UPSJB).\n\n\nResults\n\nIn total, 640 people answered the virtual survey. According to the selection criteria, 208 participants were excluded (see Figure 1); therefore, only 432 participants were selected for this study.\n\nAmong the participants, 65% (n = 281) were female, and 53.5% (n = 231) were between 18 and 29 years old. A total of 33.8% (n = 159) of the participants stated that they had been diagnosed with and had presented symptoms of COVID-19. The lateral flow immunoassay rapid test was the most widely used diagnostic test (33.1%, n = 143); 33.8% (n = 146) had a positive diagnosis and symptoms of COVID-19, and 67.8% (n = 293) reported having maintained contact with people diagnosed with COVID-19. Furthermore, 64.6% (n = 279) of the participants had at least one comorbidity: obesity (15.3%, n = 66), asthma (5.6%, n = 24), arterial hypertension (5, 1%, n = 22), diabetes mellitus (4.2%, n = 18), or cancer (0.7%, n = 3). The other characteristics were described in Table 1.\n\nConsumption of ivermectin due to the COVID-19 pandemic was reported by 67.6% (n = 292) of the participants. Of these, 20.20% (n = 59) used ivermectin for prophylactic purposes only, while 41.79% (n = 122) used it as treatment for COVID-19 only, and 38.01% (n = 111) used it for both reasons. Furthermore, 43.5% (n = 127) of these participants accessed this drug on prescription, while the remaining participants (56.5%, n = 165) self-medicated (see Figure 2).\n\nWhen evaluating the period of the pandemic in which ivermectin was most consumed, it was observed that most of the participants who consumed ivermectin 52.39% (n = 153) did so during the period between November 2020 and March 2021 (second wave of COVID-19 in Peru) (see Figure 3A). Due to the use of ivermectin on several occasions, participants reported more than one period of ivermectin consumption. It was also found that the main source of access (n = 141, 48.28%) to this drug was a pharmacy (see Figure 3B).\n\nIn the bivariate analysis, the age (p < 0.0001), marital status (p = 0.0001), education (p = 0.042), diagnosis of COVID-19 (p < 0.0001), being in contact with people with COVID-19 (p < 0.0001), and having obesity (p = 0.035) or diabetes (p = 0.049) were associated with ivermectin consumption for COVID-19 in participants from Chincha province (see Table 2).\n\nIn the multivariate analysis, the model was built with the variables that presented a significant association in the bivariate analysis, except for the variable diabetes because a very low number of participants marked the box ‘did not take ivermectin/had diabetes'. It was found that being ≥50 years old (PR: 1.27, 95% CI: 1.04–1.54), having a technical education level (PR: 1.16, 95% CI: 1.01–1.34), having had symptoms of COVID-19 with negative/no diagnosis (PR: 1.28, 95% CI: 1.07–1.53) or positive diagnosis (PR: 1.38, 95% CI: 1.18–1.61), or contact with infected people (PR:1.45, 95% CI: 1.06–1.98) were statistically associated with COVID-19 ivermectin usage (see Table 2).\n\n\nDiscussion\n\nDuring the COVID-19 pandemic, increasing prescription and self-medication of off-label drugs with unproven efficacy and safety to treat this disease has been described in many countries.13–15 In our study, the frequency of ivermectin consumption in the Chincha province was 67.2%, with more than 50% self-medicating and 43.5% obtaining the drug by medical prescription.\n\nThe frequency of ivermectin consumption among Chincha residents was similar to that described in a previous study in Peruvian patients prior to hospital admission (66.9%)16; however, our data include a broader population (not only hospitalized COVID-19 patients). Most of the studies on ivermectin focused on the evaluation of its efficacy in COVID-19 patients. Among epidemiological studies, we found only one other study that assessed the frequency of ivermectin use in the community. Nasir et al.17 reported that the prevalence of self-medication of ivermectin in the population of Dhaka City, Bangladesh, was 77.15%. Other studies on self-medication did not show the use of ivermectin in its population target.12 The high frequency of the use of ivermectin in the Dhaka and Chincha populations may be due to the low cost of this drug, misinformation and fear experienced during the COVID-19 pandemic.\n\nThe prescription of ivermectin by physicians can be explained by the inclusion of this drug in the Peruvian treatment protocol for patients with COVID-19 in April 2020,18 but it was not approved as prophylactic therapy (59.84% of all ivermectin prescriptions). Is possible that an infodemic about ivermectin encouraged Peruvian physicians to prescribed ivermectin as prophylactic treatment. On the other hand, unlike people who used ivermectin with a prescription at a safe dose, the management of ivermectin use by participants self-medicating could have been inappropriate. Although this antiparasite drug has an established safety profile for humans,19 the use of ivermectin poses an elevated risk of severe neurotoxicity, which can even be fatal in some cases. Furthermore, there is not enough evidence on its safety in pregnant women.20 It is even possible that some participants who used self-medicated ivermectin could have confused this drug approved for humans with veterinary use ivermectin. Local news reported the use of veterinary use ivermectin in some people in Chincha19 and, in other regions of Peru. Two patients with COVID-19 that used veterinary-use ivermectin subcutaneously reported skin ulcers.21\n\nThis study describes the reasons for the use of ivermectin during the COVID-19 pandemic. It was found that, among participants who used ivermectin, almost 60% used this drug for prophylactic purposes. In Chincha, regional leaders, without scientific support, encouraged the consumption of ivermectin at a dose of 1 drop/kg every four weeks as prophylactic therapy19 when plasma half-life was reported to be 12 to 66 hours.22,23 Furthermore, Vallejos et al.7 reported that ivermectin has no significant effect on preventing hospitalization of patients with COVID-19. On the other hand, almost 80% of the participants who used ivermectin reported that it was used to treat symptoms of COVID-19, despite the lack of conclusive evidence of its clinical benefit.24,25 These practices could have generated a false sense of safety, increasing the number of contagions and hospitalizations for COVID-19 in the Chincha population.\n\nAlthough this was a cross-sectional study, we observed an increase in ivermectin consumption in the period from November 2020 to March 2021 compared with the early months of the COVID-19 pandemic in Peru. In addition, this drug appears to have wide accessibility in Chincha. These results suggest that misleading information is increasingly spreading among the Peruvian population26 and could be associated with the increase in adverse drug use of ivermectin reported in Peru during 2021.27\n\nOne of the factors associated with the consumption of ivermectin was people 50 years and older. This could be due to older adults, who had the highest burden of disease and a higher risk of death at the beginning of the COVID-19 pandemic,28,29 leading this group to look for drug alternatives without proven benefits. In fact, believing that ivermectin would protect them from COVID-19, many people in this age group possibly did not apply adequate protective measures against this disease, increasing their risk of infection. This scenario could have contributed to Chincha's high mortality during the pandemic in Peru.\n\nHaving a technical level of education was related to greater ivermectin usage compared with having a bachelor’s degree or higher. This association was not observed in individuals with a secondary or lesser level of education. This phenomenon could be explained by these people have a greater access to information than the general population but being less capable to interpret the data, putting them at risk of misusing ivermectin.\n\nOther associated factors related to ivermectin use were having been diagnosed with COVID-19 or having been in contact with people diagnosed with COVID-19. This could be explained by the introduction of ivermectin in patients with COVID-19 in the guidelines developed by the Peruvian government, which recommended ivermectin as treatment in patients with COVID-19 from April 13 to October 12, 2020.18,30 This governmental recommendation was made despite the World Health Organization recommending not to use ivermectin in patients with COVID-19, except in clinical trials. In fact, to date, several clinical trials have been published on the efficacy of ivermectin, which do not report any benefit in the prevention or treatment of COVID-19 with this drug.7,8 Therefore, a drug that has not been demonstrated to be effective in a clinical trial should not be included in any guideline.\n\nThis study has some limitations; first, due to limited resources and the need to generate information about the use of ivermectin, we performed a convenience sampling, which limits the generalization of these results. Second, we did not configure the online questionnaire to detect duplicate answers; and third, being a cross-sectional study, we could only describe correlations, but not causality. As strengths, this is the first study to evaluate the frequency of consumption in one of the Peruvian provinces with the highest mortality due to COVID-19.\n\n\nConclusion\n\nIn conclusion, our results showed that two fifths of the population surveyed self-medicated with ivermectin and three fifths used this drug at the presentation of symptoms. The main factors associated with the use of ivermectin for the prevention/treatment of COVID-19 were being 50 years or older, and in contact with people infected with SARS-CoV-2. In this context, it is important that government and media promote campaigns against misinformation about the use of ivermectin for the prevention or treatment of COVID-19.\n\n\nEthical considerations\n\nThis study was approved by the Institutional Research Ethics Committee of San Juan Bautista Private University (Registry No. 062-2021-CIEI-UPSJB).\n\n\nConsent\n\nEach participant provided their written signed informed consent to take part in the survey.", "appendix": "Data availability\n\nHarvard Dataverse: Database_ Ivermectin_Chincha_ Peru. https://doi.org/10.7910/DVN/YITF5T. 31\n\nThe project contains the following underlying data:\n\n- Database_F1000 (questionnaire raw data) (questionnaire codes)\n\nThis project contains the following extended data:\n\nHarvard Dataverse: Database_ Ivermectin_Chincha_ Peru. https://doi.org/10.7910/DVN/YITF5T. 31\n\n- Supplementary Table S1 (questionnaire)\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\nReferences\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: 1054–1062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlores López MG, Soto Tarazona A, De La Cruz-Vargas JA: Regional distribution of COVID-19 mortality in Peru. Rev La Fac Med Humana. 2021; 21: 326–334. Publisher Full Text\n\nDirección Regional de Salud de Ica: Oficina de Epidemiología. Sala de Situación Covid-19 de Ica 2020 – 2021. Ica.2021.\n\nChirinos JA, Corrales-Medina VF, Heresi-Dávila G, et al.: Sobre las recomendaciones del Ministerio de Salud para el tratamiento farmacológico de la COVID-19 en el Perú. Acta Med Peru. 2020; 37: 231–235. Publisher Full Text\n\nTaype-Rondan A, Herrera-Añazco P, Málaga G: Sobre la escasa transparencia en los documentos técnicos para el tratamiento de pacientes con COVID-19 en Perú. Acta Med Peru. 2020; 37: 215–222. Publisher Full Text\n\nReis G, Silva EASM, Silva DCM, et al.: Effect of Early Treatment with Ivermectin among Patients with Covid-19. N Engl J Med. 2022; 386: 1721–1731. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVallejos J, Zoni R, Bangher M, et al.: Ivermectin to prevent hospitalizations in patients with COVID-19 (IVERCOR-COVID19) a randomized, double-blind, placebo-controlled trial. BMC Infect Dis. 2021; 21: 1–11. Publisher Full Text\n\nLópez-Medina E, López P, Hurtado IC, et al.: Effect of ivermectin on time to resolution of symptoms among adults with mild COVID-19: a randomized clinical trial. JAMA. 2021; 325: 1426–1435. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinisterio de Salud: Diario Oficial Del Bicentenario El Peruano. Decreto de Urgencia N° 059-2020.[Citado 10 de Mayo del 2021]. n.d.Reference Source\n\nAlvarez-Risco A, Mejia CR, Delgado-Zegarra J, et al.: The Peru approach against the COVID-19 infodemic: Insights and strategies. Am J Trop Med Hyg. 2020; 103: 583–586. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInstituto Nacional de Estadística e Informática: Resultados Definitivos del Departamento de Ica - Censo Nacional 2017.2018.\n\nQuincho-Lopez A, Benites-Ibarra CA, Hilario-Gomez MM, et al.: Self-medication practices to prevent or manage COVID-19: A systematic review. PLoS One. 2021; 16. Publisher Full Text\n\nAmmassari A, Di Filippo A, Trotta MP, et al.: Comparison of Demand for Drugs Used for COVID-19 Treatment and Other Drugs During the Early Phase of the COVID-19 Pandemic in Italy. JAMA Netw Open. 2021; 4: e2037060–e2037060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFuentes-Gonzalez MF, Ordinola Navarro A, Carmona-Aguilera Z, et al.: Outpatient prescription patterns of COVID-19 drugs in the metropolitan area of Mexico City. Fam Pract. 2021; 39: 515–518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVaduganathan M, van Meijgaard J , Mehra MR, et al.: Prescription Fill Patterns for Commonly Used Drugs During the COVID-19 Pandemic in the United States. JAMA. 2020; 323: 2524–2526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZavala-Flores E, Salcedo-Matienzo J: Pre-hospitalary medication in COVID-19 patients from a public hospital in Lima-Peru. Acta Médica Peru. 2020; 37: 393–395. Publisher Full Text\n\nNasir M, Chowdhury ASMS, Zahan T: Self-medication during COVID-19 outbreak: a cross sectional online survey in Dhaka city. Int J Basic Clin Pharmacol. 2020; 9: 1325. Publisher Full Text\n\nMinisterio de Salud del Perú: Prevención, Diagnóstico y Tratamiento de personas afectadas por COVID-19 en el Perú. Resolución Ministerial N° 193-2020-MINSA.[Cited 17 May, 2020]. n.d.Reference Source\n\nPereyra Colchado G. Chincha, entre la ivermectina sin límites, y el golpe del COVID-19. El Comer 2021:15. http\n\nMakhani L, Khatib A, Corbeil A, et al.: 2018 in review: five hot topics in tropical medicine. Trop Dis Travel Med Vaccines. 2019; 5: 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamal-Asayag C, Espinoza-Venegas LA, Celis-Salinas JC, et al.: Skin ulcer due to subcutaneous ivermectin in the treatment of COVID-19. Rev La Soc Peru Med Interna. 2020; 33: 88. Publisher Full Text\n\nEdwards G, Dingsdale A, Helsby N, et al.: The relative systemic availability of ivermectin after administration as capsule, tablet, and oral solution. Eur J Clin Pharmacol. 1988; 35: 681–684. Publisher Full Text\n\nGuzzo CA, Furtek CI, Porras AG, et al.: Safety, tolerability, and pharmacokinetics of escalating high doses of ivermectin in healthy adult subjects. J Clin Pharmacol. 2002; 42: 1122–1133. PubMed Abstract \| Publisher Full Text\n\nDeng J, Zhou F, Ali S, et al.: Efficacy and safety of ivermectin for the treatment of COVID-19: a systematic review and meta-analysis. QJM. 2021; 114: 721–732. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPopp M, Reis S, Hausinger R, et al.: Ivermectin for preventing and treating COVID-19. Cochrane Database Syst Rev. 2021; 2021: CD015017. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobins-Early N: Desperation, misinformation: how the ivermectin craze spread across the world. Guard.2021.\n\nInstituto de Evaluación de Tecnologías en Salud e Investigación - ESSALUD. Reporte de Farmacovigilancia n°01-2021: “Seguridad post-marketing de ivermectina.”.2021.\n\nWilliamson EJ, Walker AJ, Bhaskaran K, et al.: Factors associated with COVID-19-related death using OpenSAFELY. Nature. 2020; 584: 430–436. Publisher Full Text\n\nMunayco C, Chowell G, Tariq A, et al.: Risk of death by age and gender from CoVID-19 in Peru, March-May, 2020. Aging (Albany NY). 2020; 12: 13869–13881. Publisher Full Text\n\nMinisterio de Salud: Resolución Ministerial N° 839-2020-MINSA.[Cited 20 July, 2021]. n.d.Reference Source\n\nSalvador-Carrillo J, Campos-Loza L, Guillen-Carbajal D, et al.: Use of ivermectin and factors associated with the prevention and/or treatment of COVID-19: a cross-sectional online survey in the province of Chincha, Peru. Harvard Dataverse. Publisher Full Text" }	[ { "id": "183648", "date": "28 Jul 2023", "name": "Gabriel Antonio De la Cruz Ku", "expertise": [ "Reviewer Expertise Epidemiology", "Oncology", "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\nAuthors have shown in their cross-sectional study that during the second wave of COVID-19 in Chincha, Peru, the use of ivermectin was associated to age older or equal than 50 years old, technical education level, positive symptoms of COVID with negative or no diagnosis or positive diagnosis, and contact with infected people.\nIn general, this is an interesting study that should be indexed, as it showed the factors associated with the consumption of this medication during one of highest waves of COVID. I recommend some adjustments on language and minimal corrections and comments below.\n\nIn the abstract - results, please, list the factors associated with consumption of ivermectin with comas and \"and\", instead of \"or\". Please, do not repeat \"having had\" and consider \"positive\".\nIntroduction: Good\nMethods:\nPlease include the year of the last national census.\n\nPlease correct \"exclusion criteria\" instead of \"exclusion criterion\"\n\nResult: Good\nDiscussion:\n\nPlease correct: Other studies on self-medication did not show the use of ivermectin in its population target to \"target population\".\n\nPlease refer the dose of ivermectin in mcg/kg, instead of drops/kg.\n\nPlease provide a better explanation about the technical education related to more use of ivermectin.\n\nConclusion: 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?\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/12-149
https://f1000research.com/articles/12-179/v1	15 Feb 23	{ "type": "Research Article", "title": "COVID-19 anxiety and uncertainty of classes: Devastating effect on students’ academic behavior and performance", "authors": [ "Abhinandan Kulal", "Habeeb Ur Rahiman", "Abhinandan Kulal" ], "abstract": "Background: The second wave of coronavirus disease 2019 (COVID-19) upsurged the level of anxiety among students and in addition to this, frequent closure of class, switching of teaching and learning to virtual added more agony to academic life. This study aimed to assess the impact of COVID-19 anxiety and uncertainty in class on academic performance moderated through academic behavior. Methods: A structured questionnaire was developed to collect primary data and was distributed to 480 students using a systematic sampling technique. A structured equation model was developed and tested with a few hypotheses to answer the research objectives. A conceptual model was developed with the help of literature in order to understand causal relationship between study variables and tested with the help of the structured equation model. Results: The study results showed that COVID-19 anxiety and uncertainty in classes negatively impacted students' academic behaviour, which lead to students' poor performance. The study identified that COVID-19 anxiety and uncertainty of class do not have a direct impact on academic performance; it only has an indirect negative impact on academic performance. Conclusions: It can be concluded that uncertainty of classes during lockdown increased anxiety among students and it negatively impacted the academic behaviour of the students which lead to poor academic performance. The study helps the policymakers of the education sector to uplift the spirit of students in this pandemic situation and motivate the students to keep hope in their future careers; therefore, this study helps to fill that gap by contributing a working model", "keywords": [ "Covid Anxiety", "Uncertainty of Class", "Academic Behavior", "Academic Performance" ], "content": "Introduction\n\nStudents are frequently forced to move back and forth between classroom teaching and online class due to coronavirus disease 2019 (COVID-19), which made their academic life miserable (Sheen et al., 2020). However, there is no support from the Government to fill the learning gap and understand students’ misery in the present situation (Rushowy, 2021). The COVID-19 situation not only affects the mental health of the students but also adversely affects their academic behavior, such as a ‘decrease in a reading hour,’ ‘loss of concentration,’ ‘laziness in academic and physical activities’ etc. (Molock and Parchem, 2020). The Government of India, along with other countries’ governments, ordered to close the schools and colleges to control the spreading of the novel coronavirus 2019, but this action led to many problems for students and teachers (Cheng and Lam, 2021; Aristovnik et al., 2020). In this situation, both teachers and students are compelled to adopt new teaching and learning techniques, which some feel are difficult (Ho et al., 2020). After controlling the first wave of COVID-19, the Government of India ordered to reopen the schools and colleges (in January 2021), but within three-months, the country experienced a second wave of COVID-19, and again the Government ordered to close schools and colleges. This uncertain situation negatively affected students’ experiences and expectations in academic life (Balhara et al., 2020; Dewi and Wibowo, 2020; Welsen, Pike and Walker, 2020). To cope with the academic calendar, the schools and colleges started online classes and exams. In addition to this, some colleges have undertaken online viva, seminars, and students were asked to submit assignments through an online platform, which most students found difficult, especially students from rural areas (Ferrinho, 2020; Lashley et al., 2020). Due to inadequate infrastructure facilities, most students could not attend the online classes and missed most of the classes (N. Ammar et al., 2020). Vega-Hernández et al. (2020) found mixed opinions among students about online learning and classroom learning. Teachers opined that they observed more academic dishonesty among students during online exams and in submitting assignments (Papadimos et al., 2020; Zysset et al., 2021). The lockdown situation decreased physical activity among students (Zhang, Zhou and Xia, 2020), but many students were actively involved in mobile games and online entertainment, leading to severe psychological problems (Arora, Chakraborty and Bhatia, 2021).\n\nThe COVID-19 pandemic and its deadly transmission created uncertainty in the college environment and increased anxiety among students. Since the outbreak of COVID-19 in India, no study has been undertaken to address the impact of the COVID-19 situation on students’ academic behavior. Therefore, the present study was undertaken to assess the devastating effect of students’ anxiety towards COVID-19 and uncertainty in classes on their academic behavior. It also tried to find the relationship between academic behavior and academic achievement by developing a working model.\n\n\nReview of the literature\n\nA search was done with the keywords of ‘School and College closure’, ‘Academic Behavior’, ‘Covid Anxiety’ and ‘Academic Achievement’ in numerous permutations to review the previous literature. The researcher found 65 related articles and later reviewed them for conceptual development.\n\nThe curse of COVID-19 not only affects the health and economy of the country but also extended its venom to the education sector (Li, 2020; Tinsley, 2020). The sudden closure of schools and colleges for uncertain periods made the education sector perturbed and unprepared for digital classes, adding more pain to the situation (Suwidiyanti and Anshori, 2021; Tan, 2021). After completing the first wave of COVID-19, the schools and colleges reopened as per the Government regulation, but this phase ‘Back to Basic,’ would not remain for an extended period (Sarah et al., 2021). When the second wave of COVID-19 hit India, the schools and colleges were prepared for digital classes, but no measures were taken to uplift the spirit of students (Li, 2020).\n\nAccording to WHO report, people of age group less than 25 are less prone to COVID-19 compared to other age groups (Due to high immunity system) but it is not ignoring fact that even young students disturbed mentally (Faize and Husain, 2020). As students are aware of the viciousness of COVID-19, it created anxiety and other psychological problems (Pragholapati, 2020). ‘Reading too much about COVID-19’, ‘false information’, ‘lockdown rules’, ‘financial problems’ and ‘food unavailability’ caused to increase the anxiety among students (Sarah et al., 2021). In addition to this, ‘exam postponement’ and ‘online exams and viva’ created more academic stress among students (Faize and Husain, 2020; Günther, 2021). Balhara et al. (2020) found a significant positive relationship between COVID-19 anxiety and academic stress, and Cheng and Lam (2021) found that ‘usage of social media’ negatively impacts ‘level of anxiety’. The students who were more worried about the COVID-19 situation are facing ‘academic procrastination behavior,’ and they lost interest in academic life (N. Ammar et al., 2020). Dangal and Bajracharya (2020) found out rural, low-income female students are more affected by mental issues due to COVID-19. Huckins et al. (2020) identified that students of a higher level of classes felt a higher level of anxiety compared to lower-level classes, and a survey by Van Wyk (2020) found that anxiety among students steadily increased. Saddik et al. (2020) stated that ‘COVID-19 anxiety’ increases ‘intolerance of uncertainty’ among students and worsens internalizing symptoms among teens. Kulal and Nayak (2020) reported that an increase in the level of COVID-19 anxiety causes a decrease in the number of students attending online classes in Karnataka, and students who cited they were finding difficulty adopting virtual learning experience a high level of anxiety (Tan, 2021). Few studies proved that COVID-19 anxiety has a negative influence on the academic performance of the students (Madero Gómez et al., 2020).\n\nFurther, Brouwer et al. (2021) pointed out the relationship between anxiety and depression among students, and it causes a higher rate of suicidal thoughts (Bergmans and Larson, 2021). Despite teachers’ best efforts, most of the students experience a high level of anxiety due to class uncertainty, inability to adopt virtual classes, and lack of infrastructure facilities to take an online class (Tan, 2021). Dangal and Bajracharya (2020) pointed that ‘financial problems during the lockdown’, ‘academic delay’ and ‘impact on daily life’ are the significant factors influencing the increase of COVID-19 anxiety among students. Baloran (2020) proved that students who have a high level of awareness about coping strategies experienced a low level of anxiety, and students of introverted character felt less stress in lockdown than extroverted students.\n\nAcademic behavior refers to the manner, posture, or ethical behavior that an individual encompasses in their academic life (Faize and Husain, 2020). The measurement of academic behavior is a complex task, and it involves many layers. The study considered the significant aspects of academic behavior such as ‘academic integrity,’ ‘learning habits’, ‘growth mindset’ and ‘academic satisfaction.’ The previous literature (Ferrinho, 2020; Lashley et al., 2020; Saddik et al., 2020 etc) defined these components and found a relationship with academic performance.\n\nAcademic integrity is the obligation to and demonstration of honest and ethical behavior in academic life. It is an important character to ensure that students acquire knowledge and skills honestly in the schools and colleges, and it helps them in work-life (‘Arbor Day Academic Integrity Survey Report’, 2014). However, the COVID-19 pandemic disturbed the academic integrity among students, especially during online exams, submission of assignments, and attending online classes (Jamalpur et al., 2021). Many kinds of literature proved that the cheating cases in online exams increased in the last year, and teachers reported that the cases of fake attendance increased in online classes (Kecojevic et al., 2020). ‘Students are not regular for classes’, ‘late joining and leaving the class in between classes’, and ‘disturbing the classes’ are some of the difficulties teachers face in online classes (Zhang, Zhou and Xia, 2020). Students opined that the COVID-19 situation made them lazy and affected their academic integrity (Astatke, Weng, and Chen, 2021). During the lockdown, the only hope for conducting exams was through online invigilating, but this hope was dispersed due to copying by students. Sheen et al. (2020) believe that online viva voce is the best option to assess the academic performance of students because there is less of a chance of cheating. The COVID-19 pandemic brought some changes in academic settings such as, a ‘huge difference in scores between online and classroom exams’ (Chaves et al., 2021; Eshet, Steinberger and Grinautsky, 2021), an ‘increase in academic outsourcing and academic file sharing’ (Charissi, Tympa, and Karavida, 2020), an ‘increase in academic misconduct’ (Papadimos et al., 2020) and ‘sharing exam questions and answers’ (Gallo et al., 2020). According to Kulal and Nayak (2020), a shift in mode of learning to online due to an emergency created stress among students which lead to cheating behavior among students in online class. According to Günther (2021), students are not held solely responsible for the downtrend in academic integrity, other parties like management, teachers and government also carry the same responsibility, but teachers have a major role in the upliftment of academic integrity.\n\nLearning habit is one of the main components of academic behavior, and many types of research proved its influence on academic performance (Tran et al., 2020). Learning habits include study attitudes and study habits, and this study analyzed how these behaviors changed in the last year due to COVID-19. It found that the COVID-19 situation ‘increased the learning gap’, ‘decreased study hours’ and ‘decreased note-taking habits’ (Lawrence et al., 2021). The online classes reduced student-teacher’s one-to-one interaction, and it made it a difficult situation to create a proper learning space (Balhara et al., 2020). The previous study found a positive relationship between COVID-19 anxiety and a decrease in study hours, which directly affects academic achievement. The study conducted by Jamalpur et al. (2021) found that there is a massive reduction in learning gain post-pandemic, and they estimated that if this situation continues for one more year, it is difficult to recover (Zhang, Taub and Chen, 2021). Zhang, Zhou and Xia (2020) pointed out that students from low-income families faced high problems in catching online classes due to the inability to purchase a computer and Wi-Fi. The majority of the students cited that listening hours in online classes were reduced, and they spent more time on social networking sites during online classes (Riva, Wiederhold and Mantovani, 2021).\n\nThe urge for excellence or motivation to achieve more is crucial for academic behavior, and it helps them focus on the goal. Schools and colleges have a major role in keeping such interest among students (Yeo et al., 2021). It is easy to communicate with students about their goals and guide them in classroom teaching, but due to COVID-19, classes are shifted to online. It is very difficult to understand the mental situation and academic stress that students are facing in online mode (Ho et al., 2020; Sarah et al., 2021). Most of the teachers are focusing on completing their syllabus in online class rather than focusing on other aspects of education such as ‘mental ability’, ‘reasoning skills’ and ‘communication skills’ (Sarrot, 2020). Many students cited that lockdown helped them to find a new passion, and many believed that the COVID-19 situation wasted their academic year. Snipes and Tran (2017) found that an increase in growth mindset helps to reduce academic stress which leads to academic achievement.\n\nFurthermore, a growth mindset helps academic well-being and gives the confidence to cope with COVID-19 situations. Besides, it reduces depression and anxiety. Many studies found a direct impact of a growth mindset on academic achievement (Hanisa, 2020; Bowyer, 2021; Fatimah, Rajiani and Abbas, 2021).\n\nAcademic satisfaction means the students’ satisfaction on various components of the academic setting such as class, teacher, exam, etc. (Aristovnik et al., 2020). Academic satisfaction helps students keep a positive mindset in any situation, and it also helps academic performance (Kumar, Nayar and Bhat, 2020; Li, 2020; Peinado-Guevara et al., 2021). The study analysed the students’ satisfaction towards the academic setting during a COVID-19 pandemic. Students opined that they missed classroom chit-chat due to class suspension and felt bored in an online class. Students were not able to concentrate on academic life, which causes a decrease in academic satisfaction which leads to lower academic performance (Cipriano et al., 2020; Akcil and Bastas, 2021).\n\nThere is a mixed opinion on the measurement of the academic performance of students. Some literature measured based on their exam score (grade point average or percentage) (Aristovnik et al., 2020; Cipriano et al., 2020; Faize and Husain, 2020; Sarah et al., 2021), and some literature considered other aspects such as extracurricular activities along with exam scores (Sarrot, 2020; Trammell, Ph.D. et al., 2021). Due to COVID-19, most of the extracurricular activities were suspended, and exams were cancelled in some universities. Therefore, this study asked the students to rate their academic performance in the last year and measured the influence of the COVID-19 situation on academic achievement mediating academic behavior. Previous literature proved that a huge time gap due to ‘summer vacation,’ ‘absenteeism’ and weather-related closure of college (heavy rain or flood) negatively impacted academic performance and the existing situation is worse than all the previous events (Aristovnik et al., 2020; Sheen et al., 2020). The study conducted by Gallo et al. (2020) identified the variability in learning loss by subject, and they found that practical subjects were impacted more than theory subjects. The COVID-19 situation not only affected the academic activities but also affected the outdoor activities, and thereby, it hurts the overall development of the students (A. Ammar et al., 2020). Besides, it downsized the reading ability of students, and Sheen et al. (2020) estimated, it requires 7–12 weeks to catch up with their subjects. Chhatre et al. (2020) conducted a survey that reported that the academic achievement gap has a longer-term impact than we expect and causes higher dropout rates in rural areas. The study also cited that if the Government of India does not take any proper measures, this will cause an increase in ‘income disparity,’ ‘low economic growth’, ‘higher unemployment’ and ‘higher crime rate.’\n\nThe COVID-19 situation disrupted regular classes, and it brought uneasiness among students, teachers, and management. The disturbance in regular classes and postponement of exams increased academic stress, and some students lost interest in academic life (Dewi and Wibowo, 2020; Yeo et al., 2021). Students who were studying abroad suffered a lot due to the uncertainty of classes, and some were facing visa problems due to delay in the academic year (Suwidiyanti and Anshori, 2021). Since classes moved to online classes, students who joined hostels were simply paying rent even though they were not staying (Li, 2020). Amidst all this drama, some private schools and colleges started online admission for the next academic year, which the majority of the students found difficult both emotionally and financially (Arora, Chakraborty and Bhatia, 2021). Due to COVID-19, the uncertainty of classes became inevitable, and it shattered students emotionally and academically.\n\n\nConceptual model\n\nWith a critical analysis of the previous literature, the present study hypothesized a conceptual model to explain the relationship between study variables. The model explains the casual relationship between the independent variable (COVID-19 anxiety and the uncertainty of classes) and the dependent variable (academic performance) mediating through academic behavior. The model (Figure 1) was developed to understand whether COVID-19 anxiety and uncertainty of classes affect the students’ academic behavior, thereby affecting their academic performance. To depict this concept, the following hypotheses were framed:\n\nH1 - Path of first Hypothesis; H2 - Path of Second Hypothesis; H3 - Path of third Hypothesis.\n\nH1: COVID-19 anxiety has a significant impact on students’ academic behavior.\n\nH2: Uncertainty of classes significantly impacts the academic behavior of students.\n\nH3: There is a significant relationship between the academic behavior of students and their academic performance.\n\n\nMethods\n\nThe present study is descriptive and explains the causal relationship between the study variables. The study was carried out from 27th August 2021 to 10th March 2022 in the Coastal Karnataka region of Karnataka. The present study is descriptive and explains the causal relationship between the study variable. The study was carried out in the Coastal Karnataka region of Karnataka. The study area comes under Mangalore University’s jurisdiction and consists of 194 colleges providing undergraduate and postgraduate courses. In the study area, approximately 35,000 students are pursuing their graduate and postgraduate degrees.\n\nA questionnaire was constructed with two independent variables (COVID-19 anxiety and uncertainty of class), one mediating variable (academic behavior), and one dependent variable (academic performance). COVID-19 anxiety was measured with six statements directly adapted from Baloran (2020). Statements like “Do you have frequent thoughts of being infected during this Pandemic?” and “Have you been anxious or having insomnia during this quarantine?” were asked to the students, and they had to give a response on a five-point Likert scale ranging from 1 (strongly disagree) – 5 (strongly agree). Then the total score was obtained by finding the sum of items ranging between 1 to 5, with higher scores indicating higher anxiety. Uncertainty of classes was measured with a newly conceptualized five items, such as “Suspension of Classes and Postponement of exam adversely affect my learning Interest” and “Irregular classes made me unfocused in the academic field”. It measured students’ feelings about the uncertainty of classes through five items on a five-point Likert scale ranging from 1 (strongly disagree) – 5 (strongly agree). The total score obtained ranged from 1 to 5, with a higher score indicating a higher problem faced due to class uncertainty. The academic behavior of students was measured with four factors as academic integrity, learning habits, growth mindset, and academic satisfaction. Academic integrity was measured with four statements like “Some of my classmates were Copying from another student or source during an online test”. Learning habits were measured with five statements like “My study hours decreased in the last one year”. Growth mindset was measured with four statements like “I got more choices of learning during the lockdown”. Academic satisfaction was measured with six statements like “I am satisfied with classes taken during the lockdown period”. The respondents gave their opinion on a five-point Likert scale ranging from 1 (strongly disagree) – 5 (strongly agree). Academic performance was measured with one direct question, and here the researcher asked the respondents how they rate their academic performance on a five-point rating scale ranging from 5 (excellent) – 1 (extremely poor).\n\nIn the first stage, the items generated by undertaking an organized literature review and construct were recognized. In the second stage, these were discussed with experts and academics for the content validity of the questionnaire. In the third stage, pre-testing was conducted by taking 15 students at Mangalore University Campus to know the level of understandability of the questionnaire among students. At stage four, the reliability test was conducted to know the internal consistency of the item scale using Cronbach alpha and the results are depicted in Table 1.\n\nTable 1 explains the result of reliability analysis for scale data, and it exhibits that COVID-19 anxiety (I=.703), uncertainty of class (I=.758), academic integrity (I=.845), learning habit (I=.676), growth mindset (I=.689), and academic satisfaction (I=.787) were found reliable (I>0.6).\n\nStudents pursuing postgraduate and undergraduate courses from the Mangalore university jurisdiction were considered as participants. The researcher visited administration office of Mangalore University and approached Registrar (Evaluation) to get details of the students. With help of given details and from each college’s website, the researcher was able to develop sampling frame. Later a simple random sampling technique (Lottery Method) was used to select sampling unit. Taro (1967) sample size formula used to determine sample size for our study. A total of 480 students of regular course (Minimum of 2 years course) were invited to fill out the questionnaire from 10th November 2021 to 18th February 2022.\n\nData was collected using a structured questionnaire. The researcher distributed the questionnaire through email, WhatsApp, and telegram app of students (personal interview was not possible due to COVID-19 protocols). We collected contact details for further clarification and follow-up. Data was collected from April 10, 2022 to July 25, 2022. Collected data were entered into SPSS 20 and missing data was removed. Later, data were coded and used for analysis. Some repetitive questions were used to identify the bias responses from the students and such responses were removed from the final data. Data analysis and interpretation was undertaken from 2nd August 2022 to 10th September 2022.\n\nThe adopted questionnaire was submitted to the academic integrity and ethical committee of Kingdom University and got approved as per the research policy and procedure of the university council 20th November, 2022. We were not able to collect prospective ethical approval due to lockdown in the institution. Therefore, for this study we obtained retrospective ethical approval from Academic Integrity and Ethics Committee, College of Business Administration, Kingdom University, Bahrain on 20th November 2022 (CBA/17/22). The researcher asked for the oral permission from students before distributing questionnaire and informed them the purpose of the research. The respondents gave their full consent without force from anyone. Subsequently, in order to protect the rights and privacy of the respondents, all forms of private data (Name and contact details) were acquired will remain confidential.\n\nSPSS version 20 was used for data entry and analysis. After entering data into SPSS, data screening and data transformation were made for accurate analysis. One Sample t-test and multiple regression (SEM) were used for the purpose of inferential analysis. The result of the analysis is discussed below.\n\n\nResults\n\nDemographic details of the respondents help to understand the diversity of students in respect of their gender, age, the field of study, and level of study. The researcher took utter care to ensure that each group had equal proportions in numbers. The demographic details of the respondents are depicted in Table 2.\n\nTable 2 demonstrates that the majority of the respondents were female (69.4%), and 65.20% of the respondents were aged between 18–22. Most of the respondents (37.5%) were to pursuing their degree/diploma course, and 60.21% of the respondents were studying commerce and management. The analysis of the demographic details shows that female students are more interested in the enrolment of higher education. The study revealed that the commerce and management courses have the highest Gross Enrolment Ratio (GER) than science and arts courses.\n\nThe COVID-19 situation altered every aspect of human life, especially mental health. Students felt fear and depressed in this pandemic situation which adversely affected their academic life (Meo et al., 2020). At this juncture, the researcher was interested to know the level of COVID-19 anxiety among students. The total responses were converted into the score and grouped into four levels. If the mean value was less than 2, then it was treated as ‘extremely low level of anxiety’, if the mean value was between 2.1 to 3, then it was treated as ‘low level of anxiety’, if the mean value was between 3.1 to 4, then it was treated as ‘high level of anxiety’, if mean value was more than 4, then it was treated as ‘extremely high level of anxiety.’ The analysis of the COVID-19 anxiety among students is depicted in Table 3.\n\nTable 3 explains that students had a high level of anxiety about being infected (mean (M)=3.38), and they agreed that they felt depressed (M=3.48) during the lockdown. Students agreed that they spent more time on reading or watching COVID-19 information (M=3.56), and they also agreed that they were feeling anxiety and insomnia during the lockdown period (M=3.08). The majority of the students strongly agreed that they get too conscious in social gatherings (M=4.21); the overall mean and standard deviation are 3.6167±.64333, which indicates that the students had a high level of COVID-19 anxiety. The result of the one sample t-test exhibits that the opinions given by respondents are significantly different from the mean value; since the p-value is less than 0.05, we can conclude that the results are statistically significant.\n\nSuspension of schools and colleges is the only option to prevent COVID-19 from spreading among students on campus, but this decision has made significant changes in the students’ academic behavior (Nguyen et al., 2020; Tran et al., 2020). To know how the uncertainty of classes affected the perception of students on their academic life, the researcher collected opinions of students and the results are portrayed in Table 4.\n\nTable 4 indicates that students are strongly agreed that frequent closure of schools and colleges affected the academic behaviour of students (M=4.39) and suspension of classes and postponement of exams adversely affected their learning interest (M=4.39). The majority of the students strongly agreed that irregular classes made them unfocused in the academic field (M=4.33), and they also agreed that they are comfortable with online classes (M=3.98). The overall mean and standard deviation are 4.2458±.68438, which indicates that students have a negative perception (since all statements are negative) about the uncertainty of class and its influence on their academic life. The result of a one sample t-test indicates a p-value less than.05; therefore, it can be concluded that the results are statistically significant.\n\nAcademic integrity helps the students develop responsible behavior and honesty in their work (Aucejo et al., 2020). To know whether difficulties and challenges during the COVID-19 situation altered the academic integrity of the students, the researcher analyzed academic integrity and the results are shown in Table 5.\n\nTable 5 indicates that students agreed that their classmates were copying from another student or source during an online test (M=3.41) and that they join the online class but do not listen to the class (M=3.95). Most of the students strongly agreed that the COVID-19 situation made students lazy, Dishonest, and irresponsible (M=4.09), and that they copy online sources to write an assignment without giving citations to the original author (M=3.86). The overall mean and standard deviation are 3.8297±.88585, which indicates that students agree that the COVID-19 situation negatively influenced their academic integrity and results from a one sample t-test supported (p<0.05) the significance of the result.\n\nUndoubtedly, the COVID-19 pandemic situation would create unequal learning opportunities depending upon access to technology and family support; this would completely change the learning habit of students (Rahman et al., 2020). With this intention, the researcher gathered information about how students’ learning habits were affected by the COVID-19 pandemic and the analysis is depicted in Table 6.\n\nTable 6 exhibits that students agreed that they clearly remember their subject’s contents after lockdown (M=3.37) and can appropriately concentrate on their studies (M=3.38). The majority of the students agreed that their study hours are decreased in the last year (M=3.97), and they were not able to write proper notes from online classes (M=3.81). The overall mean and standard deviation are 3.7396±.59568, which indicates that students agreed that the COVID-19 situation altered their learning habits, and the one-sample t-test proved the significance (p<0.05) of the result.\n\nA growth mindset is essential to keep the students motivated towards learning, and it makes them keep hope in their careers (Molock and Parchem, 2020). With this intention, the researcher analyzed whether the COVID-19 situation influenced students’ growth mindset, and the results are shown in Table 7.\n\nTable 7 indicates that students agreed that the COVID-19 pandemic does not alter their academic goal (M=3.45); instead, it gave them more time to concentrate on their passion (M=3.42). Most of the students agreed that the lockdown period gave them more learning choices (M=3.54) and got a break from academic stress (M=3.64). The overall mean and standard deviation are 3.5135±.79866, which indicates that students have a highly positive (since all statements are positive) mindset about academic life, and the result of the one sample t-test proved the significance of the result.\n\nStudents’ academic achievement cannot be measured merely with the yardstick of grade points or marks, and it is important to consider students’ academic satisfaction to complete the process (Aristovnik et al., 2020). Therefore, the researcher asked the students to state their level of satisfaction in various aspects of academic settings such as teachers, management, family, etc. The analysis of students’ academic satisfaction during the COVID-19 pandemic is depicted in Table 8.\n\nTable 8 indicates that students are satisfied with classes taken during the lockdown period (M=3.08), teachers’ support during lockdown (M=3.65), and infrastructure facilities they had to attend online classes (M=3.15). Most of the students agreed that they are satisfied with measures taken by schools and colleges to uplift the academic career of the students during pandemic situations (M=3.48). Most of the students strongly agreed that they are satisfied with family support in their pursuance (M=4.01) and agreed that students are satisfied with academic performance (M=3.37). To find out the level of satisfaction, the researcher calculated the mean score ranging from 1 to 5. If the mean value was less than 2, then it was treated as very low satisfaction; if the mean value was between 2.1 to 3, then it was treated as low satisfaction; if the mean value was between 3.1 to 4, then it was treated as high satisfaction; if mean value was above 4, then it was treated as very high level of satisfaction. The overall mean and standard deviation are 3.4583±0.77391, which indicates that students had a high level of academic satisfaction in the last academic year.\n\nAs earlier cited, the measurement of academic performance is a real challenge. The traditional school of thought purely measures based on their marks or grade obtained in written exams. However, modern schools of thought also add other academic aspects such as extracurricular activities to measure academic performance (Rodriguez et al., 2020; Suwidiyanti and Anshori, 2021). Moreover, in the last year, universities cancelled most of the extracurricular activities and cancelled exams for some students. So, it is not easy to consider both schools of thought as a yardstick to measure academic performance. Therefore, in this study the researcher directly asked the students to rate their academic performance in the last academic year, and the result is depicted in Table 9.\n\nTable 9 indicates that the majority of the students (51.5%) have an average level of academic performance and 24.4% of the students have an above average level of academic performance. The study revealed that 11.9% of the students are below the average level of academic performance, and only 7.5% of them perform excellently in their academics. The overall mean and standard deviation are 3.18±.908, which indicates that students are at the above-average level of academic performance.\n\nHypothesis testing\n\nTo find out the causal relationship between the study variable, a hypothesized conceptual model was developed and represents three hypotheses. To test each hypothesis, proper statistical tools were used, and the result of each hypothesis are tabulated in Table 10.\n\nThe analysis of the first hypothesis (H1) showed that COVID-19 anxiety significantly described the variability in academic behaviour (AB) of the students (R2=0.155, p<0.05), indicating that high COVID-19 anxiety negatively impacted on academic behaviour. Hence, H1 explaining the relation between COVID-19 anxiety and academic behaviour was accepted (R=-0.394), as COVID-19 anxiety (β=-0.07, p<0.05) significantly and negatively impacted the academic behavior of the students. The analysis of the second hypothesis (H2) exhibits that the uncertainty of class significantly contributes to the variation of academic behaviour of the students (R2=0.087, p<0.05), indicating that a high level of uncertainty of classes negatively affects the academic behaviour. Hence, H2 signifying the relation between the uncertainty of classes and academic behaviour of the students was accepted (R=-0.296), as uncertainty of classes (β = -0.23, p<0.05) significantly and negatively impacted the academic behavior of the students. Likewise, the test result of the third hypothesis (H3) showed a positive correlation between the academic behaviour and academic performance of the students (R=0.328, p<0.05), which explains that students with a high level of academic behaviour achieved high academic performance. Hence, H3 implicating the relation between academic behaviour and academic performance was accepted, as academic behaviour (β=0.37, p<0.05) significantly influenced the students’ academic performance. The result of a causal relationship between study variables is explained with the help of a path analysis and is depicted in Figure 2.\n\nSource: Data Analysis. AI - Academic Integrity; LH - Learning Habit; GM - Growth Mindset; AS - Academic Satisfaction.\n\nFigure 2 indicates that a 1% increase in COVID-19 anxiety decreases academic behavior by 0.07%, which means that increases in COVID-19 anxiety reduce the academic integrity, learning habits, growth mindset, and academic satisfaction of the students. Likewise, a 1% increase in uncertainty of class decreases 0.23% of academic behavior, which means frequent closure of schools and colleges adversely affects the students’ academic behavior. Later, the researcher evaluated the impact of COVID-19 anxiety and uncertainty of class on academic performance. Here the researcher identified both the direct effect (COVID-19 anxiety and uncertainty of class on academic performance) and the indirect effect (through academic behaviour). The standardized total (direct and indirect) effect of COVID-19 anxiety on academic performance is -.026. Due to both direct (unmediated) and indirect (mediated) effects of COVID-19 anxiety on academic performance, when COVID-19 anxiety goes up by one standard deviation, academic performance goes down by 0.026 standard deviations. The direct (unmediated) effect of COVID-19 anxiety on academic performance is.000, which means that COVID-19 anxiety does not have a significant direct impact on academic performance. The standardized indirect (mediated) effect of COVID-19 anxiety on academic performance is -.026.\n\nSimilarly, the standardized total (direct and indirect) effect of the uncertainty of class on academic performance is -.085. Due to both direct (unmediated) and indirect (mediated) effects of the uncertainty of class on academic performance, when the uncertainty of class goes up by one standard deviation, academic performance goes down by 0.085 standard deviations. The direct (unmediated) effect of the uncertainty of class on academic performance is .000, which means that the uncertainty of class does not directly impact academic performance. The indirect (mediated) effect of the uncertainty of class on academic performance is -.112. Due to the indirect (mediated) effect of the uncertainty of class on academic performance, when the uncertainty of class goes up by 1, academic performance goes down by 0.112. The relationship between academic behaviour and academic performance (H3) indicates that when academic behaviour increases by one standard deviation, academic performance increases by 0.37, which explains that an increase in academic integrity, learning habits, growth mindset, and academic satisfaction helps to increase the academic performance of the students. The model fit indices such as goodness of fit index (.98), adjusted goodness fit index (0.92), root mean square error approximation (0.040), chi-squared fit statistics/degree of freedom (1.803), and comparative fit index (0.92) supported the present model.\n\n\nDiscussion\n\nThe present study examined the impact of students’ anxiety due to COVID-19 and the impact of the uncertainty of classes due to lockdown on their academic behaviour. This study also explored to find out the relationship between academic behaviour and academic performance. The verdicts showed that anxiety of COVID-19 and the uncertainty of class hurts academic behaviour and had an indirect effect on academic performance, which is justified with the results of Sheen et al. (2020) and Huckins et al. (2020). In addition to this, COVID-19 anxiety had a very low level of influence on academic behaviour compared to uncertainty of classes, which is contradictory to the research of Faize and Husain (2020). Due to COVID-19, students felt a high level of anxiety and underwent depression, insomnia, and stress, which was also proved in the study of Dangal and Bajracharya (2020). As a study result of Cipriano et al. (2020) students hardly find themselves comfortable in an online class and it does not replace classroom teaching. This study also justified the findings of Snipes and Tran (2017) that online exams will increase anxiety. This study also supported the newspaper report of Zhang et al. (2020), which stated that lockdown increased the learning gap among students. This study identified that suspension of schools and colleges affects the academic behaviour of the students and it identified that during lockdown students felt lonely, which may cause a feeling of depression. In the open-ended suggestion, students opined that postponement of exams at the last minute will reduce their reading interest and they also cited that private colleges do not bother about students’ health because they are compelled to attend classroom teaching.\n\nFurther, results indicated increases in academic dishonesty among students during online classes, online exams, and online assignment submissions. In this study, students agreed that the COVID-19 situation made students lazy, dishonest, and irresponsible. Due to the long closure of class, students felt difficulty in remembering subject contents and it caused a reduction in reading hours, as cited in the study of Tinsley (2020). According to Pragholapati (2020), the growth mindset helps to keep up academic achievement, which proved positively in this study. In addition to this, the study identified that majority of the students found a new passion and few students got time to work on their existing passion, therefore it can be concluded that the COVID-19 situation did not affect the growth mindset of the students. As mentioned by Dewi and Wibowo (2020), this study also justified that academic satisfaction contributes more to academic behaviour which helped the students in their academic performance. The analysis of academic performance in the COVID-19 pandemic situation agrees with the result of Samat et al. (2020), which indicates that the majority of the students have average academic performance.\n\n\nConclusion and recommendation\n\nFrom the above discussion, it can be concluded that the COVID-19 pandemic created anxiety among students and frequent closure of colleges made students’ life miserable. These two circumstances directly and indirectly adversely affected the academic behavior and academic performance of the students. But college administrations and governments are unaware of this situation and they are still focusing on the profit aspect. Many private colleges and coaching centres ignore the mental situations of students and pressure students for their poor academic performance. Therefore, this study recommends government and college administrations take proper measures to reduce COVID-19 anxiety among students and fix a standard schedule for online classes. The study found that students find it difficult in a regular online class, therefore it suggests to colleges to take online classes a maximum of two hours per day (40 minutes per session) and use innovative online techniques to motivate students towards online class. Finally, it concludes that by proper awareness and discipline, one can lessen the negative impact of COVID-19 on the mental health of students and thereby strengthen academic behaviour.", "appendix": "Data availability\n\nFigshare: Covid Anxiety and Uncertainty of Classes _ Devastating effect on Academic Behavior of Students (Responses) - Form Responses 1.csv. https://doi.org/10.6084/m9.figshare.21286266.\n\nThis project contains the following underlying data:\n\n• DATA ENTRY.xlsx (collected data coded in an Excel form)\n\n• Covid Anxiety and Uncertainty of Classes _ Devastating effect on Academic Behavior of Students (Responses) - Form Responses 1.csv (Questionnaire used to measure the variables)\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\nReferences\n\nAkcil U, Bastas M: ‘Examination of university students’ attitudes towards e-learning during the COVID-19 pandemic process and the relationship of digital citizenship. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "198043", "date": "21 Sep 2023", "name": "M Arli Rusandi", "expertise": [ "Reviewer Expertise Education", "Counseling", "Teacher", "Technology Information", "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\nIs the work clearly and accurately presented and does it cite the current literature?\nPartly The work lacks clarity in several sections such as the abstract and introduction. While it cites literature, it needs to better align cited studies with the current research, and possibly engage in a more critical discussion of the existing literature.\nIs the study design appropriate and is the work technically sound?\nPartly Concerns were raised regarding the clarity of the study's nature (descriptive vs. causal) and discrepancies in the study's timeline. These need to be addressed for technical soundness.\nAre sufficient details of methods and analysis provided to allow replication by others?\nPartly While some methods and analysis details are provided, there's a need for enhanced descriptions, especially regarding variable scales and statistical methods.\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly There's a need for elaboration, especially regarding significance, effect sizes, and confidence intervals in the results section.\nAre all the source data underlying the results available to ensure full reproducibility?\nYes\n\nAre the conclusions drawn adequately supported by the results?\nPartly Some conclusions appear speculative without strong supporting data, as mentioned in the feedback.\nFull Report: Summary: The manuscript investigates the indirect effect of COVID-19 anxiety and class uncertainty on academic performance. While the research topic is timely and relevant, several aspects of the paper need refinement for clarity, focus, and technical soundness. Points to Address: Addressing these issues will make the article more scientifically sound and contribute significantly to its field.\nClarity and Presentation: The abstract requires concise presentation, clear outlining of objectives, and detailed elaboration of the methodological approach. The introduction should be more focused, with a clear delineation of the study's objectives and scope. Consistent terminology and representation of results will enhance coherence.\n\nLiterature Citation: Align the cited studies closely with the current research. Engage in a more critical discussion of the existing literature to justify the current study.\n\nStudy Design: Clarify the nature of the study to address potential confusion between descriptive and causal research. Address discrepancies in the study's timeline to ensure technical soundness.\n\nMethods and Analysis: Enhance the description of variable measurement scales and clarify the purpose of chosen statistical methods. The statistical interpretations in the results section need elaboration, especially regarding significance and effect sizes.\n\nData Availability: Ensure that all source data underlying the results are provided to ensure full reproducibility of the study.\n\nConclusions: Conclusions must closely align with the results. Remove speculative conclusions and ground them in data to ensure validity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10292", "date": "16 Nov 2023", "name": "Habeeb Ur Rahiman", "role": "Author Response", "response": "Thank you for the constructive review. We made corrections as per the revision request of the reviewer. Kindly let us know any further corrections." } ] } ]	1	https://f1000research.com/articles/12-179
https://f1000research.com/articles/13-53/v1	08 Jan 24	{ "type": "Systematic Review", "title": "Management of Malocclusion in Children Using Myobrace Appliance: A Systematic Review", "authors": [ "Harun Achmad", "Nurul Auliya", "Nurul Auliya" ], "abstract": "Background: Bad oral habits are common in children aged less than six years and can stop spontaneously. If these bad habits continue after the age of six years, it can cause malocclusion. In the world of pediatric dentistry, one of the treatments for malocclusion is Oral Myofunctional Therapy (OMT). One of the myofunctional tools that can be used to correct malocclusion as well as bad habits in children is the Myobrace Appliance - an intraoral device system used in interceptive orthodontics. The purpose of this systematic review is to explore the management of malocclusion in children using the Myobrace Appliance. Methods: The data was collected by a literature search on Google Scholar, including articles published from 2016 to 2020. The data search was carried out systematically using the keywords Malocclusion in Children and Myobrace Appliance. After eliminating duplicate articles, the titles and abstracts of each article were analyzed across 108 articles, resulting in 51 articles. There were 36 articles not in line with topic and 6 articles which could not be freely accessed, so that 42 articles were excluded. The full text articles in the other nine articles were reanalyzed; three articles were excluded with reason and six articles were produced which were then included in the analysis. Results: There are six articles that discuss the management of malocclusion in children using the Myobrace appliance. All of these explain that Myobrace appliance is effective in handling cases of malocclusion especially to correct class II malocclusion and class III malocclusion. Conclusion: Myobrace appliance can be used as an alternative treatment for malocclusion in children, especially to correct class II and III malocclusion (mandibular prognathy and maxillary retrognathy). Additionally, this tool is capable of correcting overbite, overjet, crowding of upper and lower anterior teeth, sagittal molar relationships, lip seals, and facial asymmetry.", "keywords": [ "Malocclusion", "Myobrace", "Oral Myofunctional Therapy", "Appliance", "Children", "Bad Habit", "Management", "Alternative treatment" ], "content": "Introduction\n\nA habit is a pattern of behavior that is repeated and is generally a normal stage of development. Repetitive behavior is common in childhood and most of these behaviors start, and can stop, spontaneously. The mouth is a general and permanent location for emotional expression and is a source for releasing desire and anxiety in both children and adults. Stimulation can be through the tongue, fingers or nails which can be a palliative action (a state that can provide calm). Habits that occur in the oral cavity (Oral Habits) can be classified into two, namely physiological and non-physiological Oral Habits. Physiological oral habits are normal human habits such as nasal breathing, chewing, talking, and swallowing. Non-physiological oral habits are abnormal human habits that cause pressure and tendencies that persist and are repeated continuously so that they affect craniofacial growth and are usually called Bad Habits1–4.\n\nBad habits are normal in children aged less than six years and can stop on their own (spontaneously). If these bad habits continue after the age of six years, it can cause malocclusion in children. Malocclusion is defined as an abnormal dentofacial condition which refers to an abnormal occlusion or impaired craniofacial relationship that can affect appearance (aesthetics), function, facial balance, and psychosocial states. Malocclusion is one of the most common dental problems with, a prevalence ranging from 20%–100% in different studies, and is a common study5–7. Examples of bad habits that can lead to malocclusion include thumb sucking, pacifier sucking, the tongue placing pressure on teeth, nail biting, mouth breathing, bruxism, and lip biting or sucking. The habit is carried out at least six hours a day, a high enough frequency with sufficient intensity to cause malocclusion. Of these three factors the most influential is the duration of the habit. Bad habits are often distributed as a cause or risk factor for various types of malocclusion. Malocclusion may include the occurrence of an open bite, with the maxillary incisors sloping to the face (protrusive), the mandibular to lingual (retrusive) incisors, and the eruption of some incisors being obstructed leading to increased overjet and reduced overbite1,8–10.\n\nIn the world of pediatric dentistry, one of the treatments for malocclusion is Oral Myofunctional Therapy (OMT). OMT is defined as the treatment of dysfunction of the facial and mouth muscles, with the aim of correcting orofacial functions, such as chewing and swallowing, and improving nasal breathing11,12. The goal of myofunctional therapy is to strengthen the muscles necessary for normal breathing, chewing, and swallowing, with a focus on increasing the tone and mobility of the oral and cervical structures13–15. One myofunctional tool that can be used to correct malocclusion, as well as bad habits, in children is the Myobrace Appliance. Myobrace is an intraoral appliance system used in interceptive orthodontics, designed for the treatment of malocclusion in mixed dentition patients (ages 8–12 years). Myobrace can also be used in adult patients but only for non-extractive cases and for mild or moderate malocclusion. This tool works to improve the balance of facial muscles and chewing, and restore tongue posture. Myobrace has the following objectives: 1) to get a myofunctional effect, 2) to restore the position of the mandible, 3) to stimulate the muscles of the face, masseter and tongue, 4) to move the mandible forward, 5) and to stimulate horizontal growth16,17.\n\n\nMethods\n\nData collection was carried out by conducting a literature search on an article search site, namely Google Scholar, for papers published from 2016 to December 2020, to obtain the latest articles related to the topic. The data search was carried out systematically using the terms Malocclusion in Children, and Myobrace Appliance.\n\nA. Inclusion Criteria: (1) articles published between 2016–2020, (2) articles in English, (3) scientific articles that are published and available online, (4) articles which study Myobrace Appliance use in children.\n\nB. Exclusion Criteria: (1) articles that cannot be freely accessed, (2) articles which do not talk about malocclusion treatment in children\n\nThe data used in this research is secondary data. The data is obtained from articles that were searched for in the article database which were then reviewed according to the research criteria set by the researcher.\n\nA literature search was conducted on the Google Scholar online database. A search for the list of references to articles that fall under the inclusion criteria was also carried out to determine whether there were other related studies that were relevant to this research.\n\nThe keywords Malocclusion in Children and Myobrace Appliance were used in the literature search. Duplicated literature was eliminated, and articles were filtered on the basis of title, abstract, and the aforementioned keywords. The remaining 108 articles were read (completely or partially) to determine whether they met the eligibility criteria. The data collection was done manually by creating a research matrix containing: author's name, year, title, and conclusion.\n\nThe author is limited to articles available on the desired topic in the year from 2016 to 2020. This is influenced by the quality of the articles that have been obtained in full text but cannot be analyzed.\n\n\nResult\n\nAfter eliminating duplicate articles, the titles and abstracts of each article were analyzed across 108 articles. Then a Journal Search on Google Scholar took place using the keywords 'Malocclusion in children and Myobrace Appliance', for articles published between 2016–2020. We then judged the titles and abstracts in correspondence with the topic, resulting in 51 articles. Of these, there were 36 articles not in line with the topic and six articles that could not be freely accessed, so that 42 articles were excluded. The full text articles in the other nine articles were reanalyzed and three articles were excluded with reason and six articles were produced which were then included in the analysis.\n\n\nDiscussion\n\nThe results of this systematic review show that the Myobrace appliance can be used as an alternative treatment for malocclusion in children, especially to correct class II malocclusion and class III malocclusion (mandibular prognathy and maxillary retrognathy). In addition, this tool is also capable of correcting overbite, overjet, crowding of upper and lower anterior teeth, sagittal molar relationships, lip seals, and facial asymmetry18–21. The Myobrace appliance is a prefabricated functional appliance with myofunctional training characteristics, which is used to correct malocclusion in children who are in the development stage22. The form of the Myobrace appliance can be seen in Figure 2.\n\nSource: Primary Data.\n\nMalocclusion is a form of maxillary and mandibular connection which deviates from the standard form which is accepted as the normal form. Malocclusion can be caused by the absence of dentofacial balance. It is a fairly large dental and oral health problem in Indonesia and its prevalence is still very high, around 80% of the population, and is in third place after dental caries and periodontal disease23–25. One of the causes of malocclusion is bad oral habits. Bad habits that are done repeatedly and continuously during the development of the jaw will result in malocclusion. Bad habits such as finger/nipple sucking, nail biting, lip biting, mouth breath, and tongue sticking have an impact on the prevalence of malocclusion, especially in children26–28. Examples of bad habits such as finger sucking are likely to interfere with the position of the teeth, this habit can lead to malocclusion, namely an anterior open bite due to disruption of the growth pattern of the cranofacial bones. There is a significant relationship between the development and prevalence of malocclusion and the prevalence of bad habits29–32. The results of the study on children aged seven to 15 years33 who have bad oral habits show that around 80% of children suffer from malocclusion, such as class II and class III malocclusion. According to Singh34, children who have bad mouth habits, especially finger sucking, tend to experience class II malocclusion. This indicates that bad oral habits lead to malocclusion. Previous studies involving children aged eight to 10 years revealed that children with malocclusion were more likely to have a negative impact on their quality of life than individuals who were malocclusion free41. Some types of malocclusion also have a higher impact on quality of life42,43.\n\nThe Myobrace appliance was introduced by Myofunctional Research Company, Australia, followed by other appliances of The Trainer System™ in 2004. It also contains various tools for different age groups and is available in various sizes. The Myobrace appliance is available for four stages of treatment, including habit correction, arch extension, tooth alignment and retention. The Myobrace appliance has a structural element similar to that of a trainer system, consisting of a tough nylon element, called the Inner-Core or Dynamicore. The manufacturer states that dynamicore helps withstand the forces developed on the teeth by the buccinator and orbicularis muscles allowing correction of misaligned teeth by providing better arch shape. The presence of additional channels in the area of the anterior teeth on the Myobrace appliances is claimed to increase its ability to align teeth because it can exert direct force on the teeth. They are available in a variety of sizes for primary, mixed and permanent teeth and for different treatment purposes44–46.\n\nThe goals of treatment using the Myobrace appliance are: 1) Restoring nasal breathing from mouth breathing, 2) correcting correct tongue posture, 3) correcting correct swallowing, 4) Correcting alignment of teeth and jaw to the correct position, 5) There can be retention or no retention, 6) minimal or no use of braces, 7) achieve optimal health, 8) unhindered craniofacial development47–49. Use of Myobrace at least two hours every day and a maximum of overnight, aims to provide adequate expansion and arch strength of the jaws to align the anterior teeth50–52.\n\nThe parts of the Myobrace appliance consist of53:\n\na. Guides for teeth: a guide to aligning the teeth in the correct position.\n\nb. Labial and buccal shields: to prevent interposition of lips and cheeks, as well as to provide some strength to misaligned anterior teeth.\n\nc. Tongue tag: positioned on the retro-incisive papilla, acts as a proprioceptive stimulus to the tip of the tongue, and as a myofunctional trainer to improve tongue posture\n\nd. Tongue Guard: to prevent tongue jostling and interposition, forcing the tongue in its original position, stimulating nasal breathing, and preventing bad habits.\n\ne. Lip Bumper: prevents mentalist muscle hyperactivity.\n\nThings that need to be considered when using Myobrace appliance are: 1) the patient must wear the appliance for one to two hours every day and overnight while sleeping, and 2) it is necessary to use the appliance every day (if it is not used every day, then the treatment will not be successful), 3) at least one myofunctional function exercise must be completed every day, 4) the patient must learn how to swallow correctly and position the tongue in the correct place in the mouth, 5) the patient must guard their mouth closed when not talking or eating36. Based on the age group, Myobrace appliance is classified into four types, namely:\n\na. Myobrace for Juniors (age three to six years);\n\nMyobrace for Juniors is a three-stage equipment system specially designed to correct bad oral habits while addressing developmental problems of the upper and lower jaw. Myobrace for Juniors is most effective on primary teeth from three to six years of age. This device is specially designed to correct mouth breathing problems, improve tongue position and swallowing pattern, train jaw muscles, change pacifiers, improve natural curve development, early treatment for open bites and cross bites54,55.\n\nb. Myobrace for Kids (Age 6–10 years)\n\nMyobrace for Kids is a three-stage equipment system specially designed to correct bad mouth breathing and oral habits, which helps to solve problems with the development of the upper and lower jaw. This allows the permanent teeth to adjust to their original position. Most effective in the early stages until the teeth are mixed, ages 6–10 years. It is specially designed to correct class II division 1 and 2 malocclusion, crowding of upper and lower anterior teeth, deep bite, and open bite54,55.\n\nc. Myobrace for Teens (10–15 years)\n\nMyobrace for Teens is a four-stage Myofunctional Orthodontic system designed to replace complex orthodontics with braces and extractions. Its main purpose is to correct oral breathing and poor myofunctional habits that can lead to malocclusion, while the stage three (T3) apparatus guides permanent teeth to grow in parallel positions that are in the developmental stage of the tooth. It is specially designed to treat class II malocclusion in the final stages of mixed teeth, class II division one and two, crowding of upper and lower anterior teeth, deep bite, and open bite54,55.\n\nd. Myobrace for Adults (>15 years)\n\nMyobrace for Adults is a three-stage equipment system for permanent teeth. For adult patients, all growth has occurred, and the tooth is in the most stable position. Improper mouth breathing and swallowing habits have developed over the years and are more difficult to correct. For this reason, outcomes in adults are not as predictable as in children. It is specially designed to treat most malocclusions in adult patients, crowding of upper and lower anterior teeth in mild to moderate cases, treatment of relapsed anterior teeth after fixed orthodontics, moderate cases of Class II division one and two malocclusion54–59,60.\n\n\nConclusion\n\nMyobrace appliance can be used as an alternative treatment for malocclusion in children, especially to correct class II malocclusion and class III malocclusion (mandibular prognathy and maxillary retrognathy). In addition, this tool is also capable of correcting overbite, overjet, crowding of upper and lower anterior teeth, sagittal molar relationships, lip seals, and facial asymmetry.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: PRISMA checklist for ‘Management of Malocclusion in children using Myobrace Appliance: A systematic review’. https://figshare.com/s/a5cf58a96fc2d3f9fb1f", "appendix": "References\n\nKolawole KA, Folayan MO, Agbaje HO, et al.: Oral habits and malocclusion in children resident in Ile-Ife Nigeria. Eur Arch Paediatr Dent. 2019; 20(3): 257–265. PubMed Abstract \| Publisher Full Text\n\nPaolantonio EG, Ludovici N, Saccomanno S, et al.: Association between oral habits, mouth breathing and malocclusion in Italian preschoolers. Eur J Paediatr Dent. 2019; 20(3): 204–208. PubMed Abstract \| Publisher Full Text\n\nGrippaudo C, Paolantonio EG, Antonini G, et al.: Association between oral habits, mouth breathing and malocclusion. Acta Otorhinolaryngol Ital. 2016; 36(5): 386–394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah AF, Batra M, Sudeep CB, et al.: Oral habits andtheir implications-review article. Annals Medicus. 2014; 4(1): 180. Reference Source\n\nAkbari M, Lankarani KB, Honarvar B, et al.: Prevalence of malocclusion among Iranian children: A systematic review and meta-analysis. Dent Res J (Isfahan). 2016; 13(5): 387–395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvarado K, López L, Hanke R, et al.: Prevalence of malocclusion and distribution of occlusal characteristics in 13- to 18-year-old adolescents attending selected high schools in the municipality of San Juan, PR (2012-2013). P R Health Sci J. 2017; 36(2): 61–66. PubMed Abstract\n\nMtaya M, Brudvik P, Astrøm AN: Prevalence of malocclusion and its relationship with socio-demographic factors, dental caries, and oral hygiene in 12- to 14-year-old Tanzanian schoolchildren. Eur J Orthod. 2009; 31(5): 467–476. PubMed Abstract \| Publisher Full Text\n\nZou J, Meng M, Law CS, et al.: Common dental diseases in children and malocclusion. Int J Oral Sci. 2018; 10(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJan H, Abuhamda ISB, Assiri A, et al.: Meta-Analysis of Prevalence of Bad Oral Habits and Relationship with Prevalence of Malocclusion. EC Dental Science. 2017; 112–3. Reference Source\n\nPriede D, Roze B, Parshutin S, et al.: Association between malocclusion and orofacial myofunctional disorders of pre‐school children in Latvia. Orthod Craniofac Res. 2020; 23(3): 277–283. PubMed Abstract \| Publisher Full Text\n\nKayamori F: Effects of orofacial myofunctional therapy on the symptoms and physiological parameters of sleep breathing disorders in adults: a systematic review. Rev CEFAC. 2017; 19(6): 870. Publisher Full Text\n\nMoller JL, Paskay LC, Gelb ML: Myofunctional Therapy A Novel Treatment of PediatricSleep-Disordered Breathing. Slip Med Clin. 2014; 9(2): 235–243. Publisher Full Text\n\nDiBiase AT, Cobourne MT, Lee RT: The use of functional appliances in contemporary orthodontic practice. Br Dent J. 2015; 218(3): 123–8. PubMed Abstract \| Publisher Full Text\n\nLiem E: Sleep Disorders in Pediatric Dentistry. 2019. Reference Source\n\nHomem MA, Vieira-Andrade RG, Falci SGM, et al.: Effectiveness of orofacial myofunctional therapy in orthodontic patients: A systematic review. Dental Press J Orthod. 2014; 19(4): 94–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYanez GR, Farrell C: Guiding Craniofacial growth and development with the myobrace system. 1st Ed. Australian dental practice, 2020; 40. Reference Source\n\nAnastasi G, Dinnella A: Myobrace System: A no-braces approach tomalocclusion and a myofunctional therapy device. Web med central, 2014; 2. Publisher Full Text\n\nRamirez-Yanez GO, Flutter J: Facial Symmetry Improves After Treating Malocclusions with the Myobrace™ System. Ec Dental Science. 2016; 712. Reference Source\n\nSatygo EA, Silin AV, Ramirez-Yañez GO: Electromyographic muscular activity improvement in Class II patients treated with the pre-orthodontic trainer. J Clin Pediatr Dent. 2014; 38(4): 380–4. PubMed Abstract \| Publisher Full Text\n\nDinkova M: Management of Deep Bite in Adults–A Combination of Myofunctional Appliances and Fixed Technique. Int J Sci Res. 2015; 6(5): 258. Reference Source\n\nChrysopoulos KN: Interception of malocclusion in the mixed dentition with prefabricated appliances and orofacial myofunctional therapy. J Dent Health Oral Disord Ther. 2017; 7(5): 343–345. Publisher Full Text\n\nSlaghour MA, Bakhsh AK, Hadi IH, et al.: Dental Occlusion and Malocclusion: Prevalence, Types and Treatment. EC Dental Science. 2019; 1777. Reference Source\n\nRanggang BM, Armedina RN: Comparison of parents knowledge of bad habits and the severity maloclusion of children in schools with different social levels. J Dentomaxillofac Sci. 2020; 5(1): 48–51. Reference Source\n\nHedge C, Das D, Mittal P, et al.: The Myobrace Appliance Therapy: Modus Operandi And Beyond: A Case Report. J Dent Med Sci. 2016; 15(3): 40–45. Reference Source\n\nDhul KS, Verma T, Dutta B: Prevalence of Deleterious Oral Habits among 3- to 5-year-old Preschool Children in Bhubaneswar, Odisha, India. Int J Clin Pediatr Dent. 2018; 11(3): 210–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamdar RJ, Al-shahrani I: Damaging Oral Habits. J Int Oral Health. 2015; 7(4): 85–7. PubMed Abstract \| Free Full Text\n\nTanaka O, Oliveira W, Galarza M, et al.: Breaking the Thumb Sucking Habit: When Compliance Is Essential. Case Rep Dent. 2016; 2016: 6010615. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShouq AA: Meta-analysis of prevalence of bad habits and relationship with prevalence of malocclusion. EC Dent Sci. 2017; 11: 4.\n\nChahal N, Rana M, Dharwan A, et al.: Assessment of frequency of occurrence of malocclusion among known paediatric population: a clinical study. J Adv Med Dent Sci Res. 2018; 6(8): 37–39. Reference Source\n\nIbraheemjaafa M, Kadoum NA: Bad Oral Habits and Associated Malocclusion among 8-9 Years Old Children. Int J Sci Res. 2017; 6(6): 84. Reference Source\n\nZakirulla M, Alshehri AD, Hudaybi AH, et al.: Oral habits: prevalence and effects on occlusion among 7 to 13 years old school children in Aseer, Saudi Arabia . Pesqui Bras Odontopediatria Clín Integr. 2020; 2(1). Publisher Full Text\n\nNabila CR: Relationship of parents’ knowledge with the condition of malocclusion in children who have oral bad habits. J Syiah Kuala Dent Soc. 2017; 2: 12–4.\n\nShouq AA: Meta-analysis of prevalence of bad habits and relationship with prevalence of malocclusion. EC Dent Sci. 2017; 11: 4.\n\nSharaf RM, Jaha HS: Etiology and treatment of malocclusion: Overview. Int J Sci Eng Res. 2017; 8(12): 102. Reference Source\n\nHong KS, Shim YS, Park SY, et al.: Oropharyngeal Airway Dimensional Changes after Treatment with Trainer for Kids (T4K) in Class II Retrognathic Children. Iran J Public Health. 2016; 45(10): 1373–1375. PubMed Abstract \| Free Full Text\n\nAggarwal I, Wadhawan M, Dhir V: Myobraces: Say No to Traditional Braces: Review Article. Int J Oral Care Res. 2016; 4(1): 82–85. Reference Source\n\nKizi G, Ventura I, Barata R, et al.: Early treatment of a class III malocclusion with the myobrace system clinical case. Translational Research and Innovation in Human in health Sciences. Ann Med. 2017; 1–2. Reference Source\n\nWijey R: Treatment for Class III Malocclusion: Surely we can do better? – Case report. Australian Dental Practice, 2017; 83–4. Reference Source\n\nMohammed H, Čirgić E, Rizk MZ, et al.: Effectiveness of prefabricated myofunctional appliances in the treatment of Class II division 1 malocclusion: a systematic review. Eur J Orthod. 2020; 42(2): 125–134. PubMed Abstract \| Publisher Full Text\n\nWishney M, Darendeliler MA, Dalci O: Myofunctional therapy and prefabricated functional appliances: an overview of the history and evidence. Aust Dent J. 2019; 64(2): 135–144. PubMed Abstract \| Publisher Full Text\n\nDutra SR, Pretti H, Martins MT, et al.: Impact of malocclusion on the quality of life of children aged 8 to 10 years. Dental Press J Orthod. 2018; 23(2): 46–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagda SC, Dixit UB: Current Evidence on the Effect of Pre-orthodontic Trainer in the Early Treatment of Malocclusion. J Dent Med Sci. 2019; 18(4): 22–28. Reference Source\n\nFarrell C: Achieving lifelong results with myofunctional treatment –Benefitting patient and dentist. Australasian Dent Prac. 2016; 72. Reference Source\n\nGokce B, Kaya B: Current Approaches in Myofunctional Orthodontics. J Musculoskelet Disord Treat. 2016; 2(3): 022. Reference Source\n\nFarrel C: The Myobrace® System: Biologically focused treatment innovation-special reports. Australian dent practice. 2016; 75. Reference Source\n\nČirgić E, Kjellberg H, Hansen K, et al.: Adolescents’ experiences of using removable functional appliances. Orthod Craniofac Res. 2015; 18(3): 165–74. PubMed Abstract \| Publisher Full Text\n\nCirgic E, Kjellberg H, Petzold M, et al.: A cost-minimization analysis of large overjet reduction with two removable functional appliances based on a randomized controlled trial. Eur J Orthod. 2018; 40(4): 437–443. PubMed Abstract \| Publisher Full Text\n\nCirgic E, Kjellberg H, Hansen K: Treatment of large overjet in Angle Class II: division 1 malocclusion with Andresen activators versus prefabricated functional appliances—a multicenter, randomized, controlled trial. Eur J Orthod. 2015; 38(5): 516–24. PubMed Abstract \| Publisher Full Text\n\nDinkova M: Vertical control of overbite in mixed dentition by trainer system. Journal of IMAB. 2014; 20(5): 648. Publisher Full Text\n\nOscar QA, Jelsyka QC, Oscar QC: The Secrets of the Trainer and Myobrace Appliances and the Biofunctional System. Amolca. 2016; 35–6. Reference Source\n\nDas UM, Reddy D: Treatment effects produced by preorthodontic trainer appliance in patients with class II division I malocclusion. J Indian Soc Pedod Prev Dent. 2010; 28(1): 30–3. PubMed Abstract \| Publisher Full Text\n\nMyobrace™ Procedures Manual. Myofunctional research.co 2017.\n\n‘Myobrace’ Myofunctional Orthodontic Appliance.2017.\n\nThe Myobrace for Teens™. Reference Source\n\nAchmad H, Djais AJ, Petrenko EG, et al.: 3-d printing as a tool for applying biotechnologies in modern medicine. IJPR. 2020; 12(4): 3454–3463.\n\nAchmad H, Djais AI, Jannah M, et al.: Antibacterial chitosan of milkfish scales (Chanos chanos) on bacteria porphyromonas gingivalis and agregatibacter actinomycetescommitans. Sys Rev Pharm. 2020; 11(6): 836–841. Reference Source\n\nAchmad H, Djais AI, Syahrir S, et al.: A literature us regarding the use of herbal medicines in pediatric dentistry. IJPR. 2020; 12: 881–897.\n\nAchmad H, Djais AI, Syahrir S, Fitria A, Ramadhany YF: Impact Covid-19 in pediatric dentistry: A literature review. IJPR. 2020; 12: 830–840.\n\nDjais AI, Achmad H, Dewiayu D, et al.: Effect of Combination of Demineralization Freeze Dentin Matrix (DFDDM/0 and Moringa oleifera lam osteoprotegerin (OPG) and receptor activator of nuclear factor kappa Bligand (RANKL) as a marker of bone remodeling. Sys Rev Pharm. 2020; 11(6): 771–779. Reference Source" }	[ { "id": "238908", "date": "16 Feb 2024", "name": "Dina Elfouly", "expertise": [ "Reviewer Expertise Digital orthoodntics" ], "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 systematic review has a clear aim and organization. However, some critical points are missing that needs to be carried out. 1- Systematic review must follow PRISMA guidelines Check Ref1\n2- Authors must mention the electronic databases as (PubMed, Web of Science, Scopus...) and Journals where search was carried out. This has to be explained in the search strategy.\n3- In the results: Need more explanation of the included articles (what is common and what is not) 4- No assessment of risk of bias of the studies, please check risk of bias for systematic reviews. 5- Discussion is written as a literature review not in a discussion format, explaining the studies included. 6- Study limitations missing and why no metanalysis was conducted.\nI believe it needs a lot of amendments.\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? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/13-53
https://f1000research.com/articles/13-52/v1	08 Jan 24	{ "type": "Case Report", "title": "Case Report: Management of pediatric Le Fort and mandibular fracture with a modified cap splint", "authors": [ "Mrinalini Mathura", "Nitin Bhola", "Anchal Agarwal", "Monica Gupta", "Nitin Bhola", "Anchal Agarwal", "Monica Gupta" ], "abstract": "Pediatric facial fractures are rare and make up 5–15% of all facial fractures. The low incidence of facial fractures among children is due to physiological and environmental factors such as greater resilience of the pediatric skeleton, higher bone to tooth ratio, direct parental supervision and limited outdoor activity.1 In children of 5–10 years of age nasal fractures are by far the most frequent (58.6%), followed by mandibular fractures (21.5%). Orbital (9.5%), frontal (5.1%), and midfacial (3.8%) fractures are next in frequency2. Le Fort fractures are very rare in children, and there is a paucity of literature presenting their frequency and characteristics. In the mandible condylar fracture is the most common (38.9%) followed by angle (20.6%), parasymphysis (18.3%), body (15.3%) and symphysis (5.3%).3 Facial fractures are more common in boys, though this varies greatly from 1.1:1 to 8.5:1.4 The majority of facial fractures in children result from falls (7.8–48%) and sports-related injuries (4.4–42%).5 In primary or mixed dentition, the placement of internal fixation hardware is challenging due to the risk of injuring unerupted permanent tooth follicles. The use of resorbable plates and screws offer a potential solution to the growing pediatric facial bone as the standard titanium fixation systems carry the risk of translocation, growth restriction, and dental injury. Dental splinting or suspension wires can also be used in place of direct plating. Fortunately, mild malocclusion at this age has the potential to be improved through bony remodeling, the eruption of permanent dentition, compensation of the mastication mechanism, and posttraumatic orthodontia.6 Accordingly, the goal of treatment is to restore the underlying bony architecture to its preinjury position in a stable fashion with minimal residual functional and esthetic impairment.", "keywords": [ "Pediatric maxillofacial trauma" ], "content": "Introduction\n\nPediatric facial fractures are rare and make up 5–15% of all facial fractures. The low incidence of facial fractures among children is due to physiological and environmental factors such as greater resilience of the pediatric skeleton, higher bone to tooth ratio, direct parental supervision and limited outdoor activity.1 In children of 5–10 years of age nasal fractures are by far the most frequent (58.6%), followed by mandibular fractures (21.5%). Orbital (9.5%), frontal (5.1%), and midfacial (3.8%) fractures are next in frequency.2 Le Fort fractures are very rare in children, and there is a paucity of literature presenting their frequency and characteristics. In the mandible condylar fracture is the most common (38.9%) followed by angle (20.6%), parasymphysis (18.3%), body (15.3%) and symphysis (5.3%).3 Facial fractures are more common in boys, though this varies greatly from 1.1:1 to 8.5:1.4\n\nThe majority of facial fractures in children result from falls (7.8–48%) and sports-related injuries (4.4–42%).5\n\nIn primary or mixed dentition, the placement of internal fixation hardware is challenging due to the risk of injuring unerupted permanent tooth follicles. The use of resorbable plates and screws offer a potential solution to the growing pediatric facial bone as the standard titanium fixation systems carry the risk of translocation, growth restriction, and dental injury. Dental splinting or suspension wires can also be used in place of direct plating. Fortunately, mild malocclusion at this age has the potential to be improved through bony remodeling, the eruption of permanent dentition, compensation of the mastication mechanism, and posttraumatic orthodontia.6\n\nAccordingly, the goal of treatment is to restore the underlying bony architecture to its preinjury position in a stable fashion with minimal residual functional and esthetic impairment.\n\n\nCase Report\n\nHere we present a case of a 5-year-old male child of Indian origin who reported to the Department of Oral and Maxillofacial Surgery with a chief complaint of pain in lower and upper jaws since the past 2 days due to fall from stairs. There was a positive history of oral bleed, bilateral nasal bleed, three episodes of vomiting, and avulsion of 51, 61 and 62 respectively.\n\nExtraoral examination revealed gross asymmetry due to the presence of diffuse swelling over the face along with the presence of a sutured contused lacerated wound over the chin.\n\nBilateral temporomandibular joint movements were restricted due to pain and the mouth opening was reduced (12mm) (Figure 1).\n\nOn maxillofacial examination step and tenderness over the symphysis region and tenderness over bilateral maxilla and pre-auricular region was noted.\n\nIntraoral examination revealed luxated right maxillary central incisors, left maxillary central and lateral incisors, intersegmental mobility in between the mandibular primary central incisors and anterior open bite (Figure 2). Maxilla was mobile at Le-fort I level bilaterally and ecchymosis was present over right maxillary buccal vestibule.\n\nAn orthopantomogram (OPG) revealed the presence of a fracture line in the mandibular symphysis region along with bilateral condylar fracture (Figure 3).\n\nComputed tomography of the face revealed undisplaced bilateral Le-Fort I fracture, displaced fracture of mandibular symphysis and bilateral sagittal condylar fracture (Figure 4).\n\nConsidering the young age of the patient closed reduction of the symphysis fracture was planned using open cap splints secured with circum-mandibular wiring along with closed reduction of bilateral Le-Fort I fracture using open cap splints secured with circum-zygomatic wiring.\n\nFor the management of bilateral condylar fracture, intermaxillary fixation with elastics for 2 weeks was planned (Kang-Young Choi et al., 2012).7\n\nImpressions of both the jaws was taken with alginate impression material and the cast was poured. Mock surgery was performed on the cast and anatomic reduction was achieved. An open acrylic cap splint for both the jaws was then fabricated. On the maxillary and mandibular splint cleats of arch bar were incorporated for intermaxillary fixation in the post-operative period. On the maxillary splint buccal tubes were also incorporated posteriorly to pass the wires for circumzygomatic wiring bilaterally (Figure 5 A–E).\n\nAfter taking an informed, written consent from the patient’s father and obtaining the final fitness for the surgical procedure the patient was taken for surgery. The displaced mandibular segments were reduced by bidigital pressure with the guidance of the surgical splint. Stab incisions were placed at the inferior border of the mandible bilaterally and a William Velsey Fry Awl was introduced. Circum-mandibular wiring was done to stabilize the open acrylic mandibular cap splint.\n\nThen the maxillary splint was placed and stabilized with circumzygomatic wiring bilaterally with the wires engaging the buccal tubes on the splint (Figure 6).\n\nPost-operative OPG was taken and computed tomography was done to check the reduction and proper positioning of the wires (Figures 7 and 8).\n\nOn the second post-operative day intermaxillary fixation with elastics was done for 2 weeks.\n\nPostoperative follow-up was done on a weekly basis up to 2 weeks. No signs of any complications were observed during the healing period.\n\nAfter 2 weeks the elastics were removed and intermaxillary fixation released.\n\nThe open acrylic cap splints were left in-situ and were removed on the sixth postoperative week under local anesthesia. There was no mobility of the fractured segments and the occlusion was bilaterally stable (Figure 9). The mouth opening was noted to be 22 mm (Figure 10). CT scan showed anatomic reduction of the fracture segments (Figure 11).\n\n\nDiscussion\n\nManagement of a pediatric patient with Le Fort and mandibular fractures is very challenging and requires multiple considerations. Closed reduction of pediatric facial fractures is preferred in order to protect the growth centers.\n\nCap splints stabilized with circum-mandibular wiring is the standard treatment modality for the management of pediatric mandibular symphysis fracture. The use of open cap splints is preferred because it helps in occlusion guided reduction, promotes tooth eruption, patient is able to take soft diet and maintenance of oral hygiene is also possible. In our case we used the modified open cap splints stabilized with circum-mandibular wiring for the management of symphysis fracture.\n\nThe recommended treatment of fractures of pediatric mandibular condyle is either conservative, or intermaxillary fixation with splints for 7–10 days. It is followed by physical therapy to prevent TMJ ankylosis. In our case the patient was put in intermaxillary fixation with elastics for two weeks and then started on mouth opening exercises (Kang- Young Choi et. al, 2012).7\n\nCurrently, there is a paucity of studies on Le Fort-type fractures in children. More recent reports find that Le Fort-type fractures more commonly occur in older children with permanent dentition.\n\nThorough literature search revealed just 1 case report where a complex Le Fort II and III fracture was managed by a retention modified occlusal splint stabilized with circumzygomatic wiring for a 3-week period.8 In our case we used the modified open cap splints stabilized by circumzygomatic wiring for the management of bilateral Le Fort I fracture. This treatment modality provides a conservative operative approach, achieving excellent functional and cosmetic results without the potential morbidity of a more invasive surgery. It provides maximum stability during the healing period, is easy to apply and remove, reduces operating time, causes no trauma to the adjacent vital structures and provides added comfort to the young patients. The patient’s mouth opening and chewing capacity was restored and he was also able to occlude properly as narrated by the patient’s father during further follow-ups. The only disadvantage of the present technique was reduced patient compliance and its limited role in severely displaced fractures.\n\n\nConclusion\n\nThe clinical outcome in the present case indicates that the modified open cap splint is an effective and reliable treatment method in the management of pediatric maxillofacial fractures. It is an easy, quick, comfortable and cost-effective mode of stabilization. Extensive literature search reveals no splints that allow postoperative intermaxillary fixation in pediatric facial fractures. This case report offers a unique mode of postoperative stabilization and it should be considered as an alternative in the management of complex midface and mandibular fractures in the pediatric population.\n\n\nAuthor Contributions\n\nDr. Nitin Bhola and Dr. Anchal Agarwal were involved in the management of maxillofacial trauma. Dr. Mrinalini Mathur and Dr. Monica Gupta led the drafting of the manuscript with all the authors contributing to the critical revision of the final manuscript.\n\n\nEthical Approval\n\nAn informed, written consent was obtained from the patient’s father for the procedure and for the use and publication of patient’s data.", "appendix": "Data Availability Statement\n\nNo data associated with this article.\n\n\nAcknowledgements\n\nNone\n\n\nReferences\n\nKhan MA, Ishfaq M, Akhtar M, et al.: Frequency of paediatric facial trauma in a tertiary care dental hospital. Int. Surg. J. 2018; 5(1): 310–314. Publisher Full Text\n\nAlcalá-Galiano A, Arribas-García IJ, Martín-Pérez MA, et al.: Pediatric facial fractures: Children are not just small adults. Radiographics. 2008; 28(2): 441–461. PubMed Abstract \| Publisher Full Text\n\nMukhopadhyay S: A retrospective study of mandibular fractures in children. J. Korean Assoc. Oral Maxillofac. Surg. 2018; 44: 269–274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmermann CE, Troulis MJ, Kaban LB: Pediatric facial fractures: recent advances in prevention, diagnosis and management. Int. J. Oral Maxillofac. Surg. 2005; 34(8): 823–833. PubMed Abstract \| Publisher Full Text\n\nMukherjee CG, Mukherjee U: Maxillofacial trauma in children. Int. J. Clin. Pediatr. Dent. 2012; 5(3): 231–236. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChao MT, Losee JE: Complications in pediatric facial fractures. Craniomaxillofac. Trauma Reconstr. 2009 May; 2(2): 103–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi KY, Yang JD, Chung HY, et al.: Current concepts in the mandibular condyle fracture management part II: open reduction versus closed reduction. Arch. Plast. Surg. 2012; 39(4): 301–308. PubMed Abstract \| Publisher Full Text\n\nMckenzie J, Nguyen E: Minimally invasive surgical management of complex pediatric facial fractures. Craniomaxillofac. Trauma Reconstr. 2021; 6: 1–4." }	[ { "id": "254146", "date": "11 May 2024", "name": "Tania Saskianti", "expertise": [ "Reviewer Expertise Pediatric Dentistry", "Special Need Patient" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n- Figure 2b. Please rotate the photo so that the mandible is in its proper place (bottom). - Figure 2b needs to well adjusted & retracted to get similar ratio to Figure 2a. - For figure 3 and 4, please add arrow in the specific area you want to point.\nDiscussion: - Please explain the behavior management chosen for this treatment and the consideration. - Did the patient requires overnight hospitalization? Explain in detail - Please explain the post op dental health education at home for the patient. - What is the follow up treatment needed for mastication and esthetic of the patient after finishing this treatment? - Please add the prescription drug given during the treatment - William Velsey or William Kelsey?\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/13-52
https://f1000research.com/articles/13-50/v1	08 Jan 24	{ "type": "Opinion Article", "title": "Establishing the ELIXIR Microbiome Community", "authors": [ "Robert D. Finn", "Bachir Balech", "Josephine Burgin", "Physilia Chua", "Erwan Corre", "Cymon J. Cox", "Claudio Donati", "Vitor Martins dos Santos", "Bruno Fosso", "John Hancock", "Katharina F. Heil", "Naveed Ishaque", "Varsha Kale", "Benoit J. Kunath", "Claudine Médigue", "Evangelos Pafilis", "Graziano Pesole", "Lorna Richardson", "Monica Santamaria", "Tim Van Den Bossche", "Juan Antonio Vizcaíno", "Haris Zafeiropoulos", "Nils P. Willassen", "Eric Pelletier", "Bérénice Batut", "Bachir Balech", "Josephine Burgin", "Physilia Chua", "Erwan Corre", "Cymon J. Cox", "Claudio Donati", "Vitor Martins dos Santos", "Bruno Fosso", "John Hancock", "Katharina F. Heil", "Naveed Ishaque", "Varsha Kale", "Benoit J. Kunath", "Claudine Médigue", "Evangelos Pafilis", "Graziano Pesole", "Lorna Richardson", "Monica Santamaria", "Tim Van Den Bossche", "Juan Antonio Vizcaíno", "Haris Zafeiropoulos", "Nils P. Willassen" ], "abstract": "Microbiome research has grown substantially over the past decade in terms of the range of biomes sampled, identified taxa, and the volume of data derived from the samples. In particular, experimental approaches such as metagenomics, metabarcoding, metatranscriptomics and metaproteomics have provided profound insights into the vast, hitherto unknown, microbial biodiversity. The ELIXIR Marine Metagenomics Community, initiated amongst researchers focusing on marine microbiomes, has concentrated on promoting standards around microbiome-derived sequence analysis, as well as understanding the gaps in methods and reference databases, and solutions to computational overheads of performing such analyses. Nevertheless, the methods used and the challenges faced are not confined to marine studies, but are broadly applicable to all other biomes. Thus, expanding this Community to a more inclusive ELIXIR Microbiome Community will enable it to encompass a broad range of biomes and link expertise across ‘omics technologies. Furthermore, engaging with a large number of researchers will improve the efficiency and sustainability of bioinformatics infrastructure and resources for microbiome research (standards, data, tools, workflows, training), which will enable a deeper understanding of the function and taxonomic composition of the different microbial communities.", "keywords": [ "Microbiome", "ELIXIR Community", "White Paper" ], "content": "Introduction\n\nThe term “microbiome” is a description of an entire habitat that encompasses all the microbes (bacteria, archaea, eukaryotes, and viruses), their genomes, and the environment they are found in Ref. 1. The microbiome is experimentally characterised by the application of one or more ‘omics techniques, especially metabarcoding, metagenomics and metatranscriptomics, but also metaproteomics and metabolomics, combined with contextual metadata about the surrounding environment, be it a geographic location (e.g. ocean), host-associated (e.g. human gut) or engineered (e.g. wastewater treatment plant). Over the past decade, scientists have become increasingly aware of the role performed by microbes in the health (or maintenance) of the environment, and that dysbiosis of the microbial community can lead to dysregulation and/or negative outcomes. Microbial communities can be very diverse and heterogeneous in composition across geospatial and temporal scales, and the culture-independent methods for identifying species with the microbiome often reveal hitherto unknown microbes. Despite methodological difficulties, understanding the taxonomic and functional composition of a microbiome, how compositional differences relate to phenotypes, and how these communities may be manipulated to restore a community to a natural or normal composition are key, current research questions. Due to the diminishing costs of nucleic acid sequencing and high-availability of sequencing platforms there are now millions of microbiome-derived sequence datasets, many of which are large (gigabytes to terabytes) and complex (thousands of related samples). Additionally, datasets from other ‘omics techniques such as metaproteomics are being increasingly generated, alone or in combination with metagenomics and/or metatranscriptomics data coming from the same samples. A key challenge facing the microbiome research community is how to: appropriately store the data; informatically process, integrate, compare and interpret microbiome-derived data; and how to make the data findable, accessible, interoperable and reproducible, i.e. FAIR.2\n\nELIXIR3 is a distributed infrastructure bringing together experts from across Europe to enable life science researchers throughout the world to access and analyse life science data. ELIXIR is formed by member states each with a national Node composed of one or more centres of excellence in bioinformatics. Each Node coordinates services, standards and resources, and collaborates with experts in other Nodes to create a sustainable Europe-wide infrastructure for biological data. ELIXIR Platforms bring together experts from Nodes to develop ELIXIR’s vision and coordinate activities in defined areas. The five Platforms are Data, Tools, Interoperability, Compute and Training. ELIXIR Communities bring together experts across ELIXIR Nodes and external partners to coordinate activities within specific life science domains. The ELIXIR Marine Metagenomics Community acted as a biome-specific network of researchers for the identification and organisation of domain-specific reference resources, development of reproducible workflows and the proposal of best practices. However, there is no underlying reason to restrict these activities to just the marine environment, with most of the aforementioned efforts broadly applicable to analysis of microbiome-derived sequence data from any environment. Furthermore, there is the need to extend the activities of the Community to integrate expertise and knowledge about other ‘omics technologies, such as metaproteomics and metabolomics, which are increasingly used in microbiome studies. Thus, this white paper outlines some of the historical aspects of the Community and the aims of the broader community, especially in the context of the other ELIXIR Communities and infrastructure platforms.\n\nThe ELIXIR Marine Metagenomics Community, established in 2015 as part of the European Commission funded ELIXIR EXCELERATE project (grant number 676559), was one of the first four ELIXIR Communities created as “Use Cases”.4 During the EXCELERATE project, these ELIXIR “Use Cases” were expanded and renamed to Communities, with a unified aim of bringing European specialists together to provide sustainable data resources, benchmark different tools and workflows, provide access to computing and storage, improve interoperability, and develop training resources within their research domains. These activities were conducted in collaboration with the ELIXIR Platforms, to ensure harmonisation of the outputs. As such, the Marine Metagenomics Community focused on metagenomics analysis pipelines, addressing the lack of reference databases and promoting the best practices for the research community. Highlights include the incorporation of new tools and resources into the MGnify5 and MetaPIPE6 analytical pipelines (e.g. MAPseq, ITSOneDB),7,8 the formal description of the MGnify pipeline using the common workflow language (CWL9) to promote interoperability, the establishment of the Marine Metagenomics Portal and MAR databases,10 and a community paper (beyond ELIXIR) promoting best practices advocating the use of community standards for contextual provenance and metadata at all stages of the research data life cycle.11 Capacity building has also been an important activity since the establishment of the Community, and many hands-on workshops and training courses have been developed and completed to build competence and expertise in a broader marine academic community.\n\nHowever, the popularity of metagenomics has continued to grow, with current approaches providing greater genome-resolved insights into the community composition and the functions performed by the microbial constituents, with annotations spanning viruses, bacteria, archaea and microbial eukaryotes.12–16 Furthermore, metagenomic-like approaches are increasingly being applied to untangle complex holobiont genomes such as lichens, where both the primary symbionts and secondary non-obligate microbes are captured.17 Finally, multi ‘omics datasets are now being more routinely produced to understand not only the genetic potential, but also the actively produced transcripts, proteins and/or metabolites, with a view to establishing the links between genotype and phenotype. When a host organism is involved, such datasets can also be augmented with genetic data from the hosts, such as genome, single nucleotide polymorphisms and transcriptomic data. The collective data facilitate a hologenomic approach18 to understanding host phenotypes, in the context of their environment and microbiome. Given this increasing complexity of study designs, and the broad applicability of microbiome research, we advocate expanding the Marine Metagenomics Community to include other areas of microbiome research. In particular, we highlight the need for an ELIXIR Microbiome Community to develop and promote standards and research infrastructures that enable the sharing of efforts, concepts, and best practices, while benefiting from the synergistic interplay with other ELIXIR Communities.\n\nThe term metagenomics is often colloquially applied to many different areas of microbiome research (see Table 1), regularly (incorrectly) used to encompass both shotgun metagenomics (indiscriminate sequencing of DNA from an environmental sample) and metabarcoding approaches (the sequencing of a specific amplified marker gene). Depending on the nature of the scientific question being addressed and/or the environment, metagenomic analysis may also involve assembly, and potentially the generation of metagenome assembled genomes (MAGs).19 Equally applicable is the analysis of unassembled raw-read data sets that can be used for taxonomic classification (e.g. Kraken,20 MetaPhlan,21 mOTUs22) and functional profiling approaches that are especially effective when extensive reference databases are available. Emerging sequencing technologies such as long-read sequencing methodologies and the associated adaptive sequencing techniques,23 together with changing protocols such as host material depletion protocols (e.g. Ref. 24), are facilitating the analysis of a wide-range of differing communities. However, the applicability of certain downstream processing and/or analysis tools changes fundamentally in these different contexts. Similarly with metagenomic data, metatranscriptomic data can be processed in different ways, and with an associated metagenomic dataset from the same sample, enables the estimation of both the genetic potential and actively transcribed fraction. Additionally, metaproteomics, an emerging technology in microbiome research, involves the study of the entire complement of proteins expressed by microbial communities in a given environment. Fundamentally, the ELIXIR Microbiome Community is about providing the necessary infrastructures required to perform analysis of nucleotide sequence data derived from a microbiome, especially the reproducibility of the results, the archiving and discovery of analyses and the interoperability of tools and data. Given the heterogeneity of such nucleotide data, this Community will work with other ELIXIR communities to determine how microbiome-derived data coming from different ‘omics approaches, may be processed and integrated.\n\nA fundamental challenge for this Community to address will be the provision of infrastructures that are sufficiently adaptable to permit the most appropriate informatics analysis, depending on the environment sampled and the experiments conducted. Moreover, when wishing to contextualise the results with similar experiments, the way a dataset has been produced and processed must be transparent to establish whether it is comparable (e.g. amplified sequence variants can only be compared when the same amplified regions are compared). Furthermore, when different methods are applied, best practices in data stewardship are required to ensure that the connectivity of the derived sequence data products, together with functional and taxonomic assertions are kept in context of the original sample/sequencing effort and associated contextual metadata. Finally, and possible unique to this ELIXIR Community, is the variety of researchers wishing to undertake microbiome research, spanning clinicians aiming to understand the role of the human microbiome in disease aetiology, ecologists wanting to understand the changing landscape of biodiversity, the agritechnology sector wishing to enhance animal and crop production, to biotechnology scientists looking for novel enzymes, among others.\n\nGiven the breadth of aforementioned applications of microbiome research, it is unsurprising that there are many links to other current and future ELIXIR activities. Figure 1 presents a schematic layout of the experimental design of a multi ‘omic analysis of a microbiome sample. Even in this very high-level representation, it can be easily observed that the new ELIXIR Microbiome Community has many potential interactions with other ELIXIR Communities and Platforms along the experimental workflow. Thus, the microbiome Community represents a showcase of the essence of ELIXIR by bringing together diverse informatics infrastructures that can be coupled together (interoperate) to achieve complex data analyses (on compute infrastructures) that have the appropriate provenance, with data adequately archived in the relevant ELIXIR core data resources. At all levels in ELIXIR, it will be essential to coordinate activities to ensure functional harmony between ELIXIR Communities using Platform-devised solutions.\n\nUnderpinning these analyses will be the metagenomic and metatranscriptomic data, which will be used as a framework for the metaproteomic and metabolomic interpretation. Highlights in this figure are connections with the ELIXIR platforms (orange boxes) and other ELIXIR communities (dark blue boxes).\n\nSome of the key areas of interactions, both ongoing and foreseen, with other Communities are listed in Table 2. As indicated in Figure 1, the interaction with other ELIXIR Communities, specifically those concerned with environmental sampling, begins at the start of the data lifecycle, concerning the sample acquisition and characterisation of the microbial communities. For example, the Food and Nutrition Community aims to understand the relationship between food choices and human health. While microbiome analysis forms part of this Community’s activities, the aim of the Food and Nutrition Community is to integrate microbiome data within the context of food and nutrition data, host genotype and phenotype information, and develop interventions that may impact disease.24,29 Thus, in the case of the Food and Nutrition Community the microbiome is only a small part of the overall research program, and restricted to human microbiome research. Members of the existing ELIXIR Marine Metagenomics Community are already engaged with the Food & Nutrition Community, and have helped to provide microbiome sequence analysis services. Similarly the Biodiversity Community has multiple overlapping activities, but with a distinct remit. For example, computational infrastructures and tools borne out of metagenomics research are now being applied for pathogen and biodiversity surveillance. Furthermore, taxonomic inventories resulting from analysis of metagenomic/metabarcoding data are commonly accepted as biodiversity resources and biodiversity resources such as GBIF and OBIS30 routinely incorporate data from both MGnify and International Nucleotide Sequence Database Collaboration (INSDC). Similarly, many of the biodiversity approaches use marker gene amplification for studying environmental DNA (eDNA). While this can overlap with the metabarcoding approaches used in the Microbiome Community, eDNA analysis extends to marker genes such as Cytochrome c oxidase subunit I (Cox1) that is specific to macro-organisms and, thus, out of scope for the Microbiome Community and falls into the realm of the ELIXIR Biodiversity Community. Isolation of genomes, sequencing and their annotation is another area under the Biodiversity Community, but will encompass both microbes and macro-organisms. Nevertheless there will be common approaches used by this Community and the Microbiome Community for de novo annotation of novel genomes.\n\nWith the growing number of multi ‘omics datasets, establishing strong ties with the ELIXIR Metabolomics and Proteomics communities31 will be essential for understanding how metagenomic and metatranscriptomic data may be utilised by these Communities (e.g. the production of reference databases), and the nature of the data types produced by these other ‘omics technologies, their limitations and how the data could be integrated. For example, overlaying metabolomics results on metagenomic data is currently non-trivial due to the scarcity of small molecule annotations that can be linked to functional annotations. Ongoing work with the Microbial Biotechnology and Systems Biology Communities has identified the need to augment the functional annotation of metagenomic and metatranscriptomic data with chemical reaction information from resources such as Rhea.32 While this will improve the discovery of new industrial applications, there is still the need to expand the protein functional annotations of the, so-called, microbial dark matter. The advent of new structural modelling software25,33 and data resources25,34 means that there are now structural models for millions of proteins that currently lack functional annotations, yet appear structurally related to functionally characterised proteins. Connections to the 3D BioInfo Community will aid how we store and organise this structural model information, reuse software components for visualisation and leverage their training materials on how to interpret structural model data. This will allow the Microbiome Community to assess the merits and limitations of this data type.\n\nIn summary, there are many synergies and connections between the Microbiome Community and the other existing ELIXIR Communities, but none of these Communities are focused on the core issues concerning microbiome-derived sequence analysis, infrastructure provision, data standards and best practices. Moreover, there are key societal challenges such as food security, climate changes, antimicrobial resistance (and new therapeutics) and pandemic preparedness where microbiome research plays a role, yet each one of these areas is far greater in scientific scope and therefore requires the collective outputs from more than one ELIXIR Community, and reach far beyond informatics research (Table 3).\n\nSimilarly, microbiome research has many translation aspects, ranging from the discovery of biomarkers associated with health and disease to industrial applications such as using enzymes from microbes or the microbes themselves for performing bioremediation and/or replacing chemical processes. One topic that is an area of intensive research is the discovery of enzymes capable of degrading plastics, typically polyethylene terephthalate (PET).36 While metagenomic assembly and analysis is providing a rich source of new enzymes, the informatics at the core of the Microbiome Community will not provide the information why one enzyme should be assayed in preference to another, how these alpha-beta hydrolases have adapted to utilising PET, or why one enzyme performs better than another. Such answers will come from the collaborative efforts that bridge across Communities, such as microbial biotechnology and 3D BioInfo and, of course, the wider research community.\n\nSimilar to the collaborations with the ELIXIR Communities, there are multiple ongoing and future interactions with the ELIXIR Platforms. In the following sections the connections between the past Marine Metagenomics Community or the future Microbiome Community and each of the Platforms will be highlighted.\n\nThe aim of the ELIXIR Data Platform is to promote the use, re-use and value of life science data. A key part of this activity has been the establishment of the Core Data Resources (CDR). Underpinning sequenced-based microbiome research is the European Nucleotide Archive (ENA), which is a recognised CDR and part of the INSDC, which in collaboration with the National Institute of Genetics DNA DataBank of Japan (DDBJ) and the United States National Center for Biotechnology’s (NCBI) GenBank and Sequence Read Archive (SRA), facilitate the deposition and global exchange of sequence data. Alongside the archived sequence data, users can access comprehensive metadata that is important to contextualise where the data originated. Throughout the lifetime of the ELIXIR Marine Metagenomics Community there have been extensive efforts to increase the standardisation of derived sequence products from metagenomic short-read datasets, particularly increasing the availability of assemblies5 and the introduction of the deposition layers to support the increase in the numbers of MAGs being generated.37 In the new Microbiome Community we will continue to promote and develop these layers to accommodate Eukaryotic MAGs (see below), viral sequences and complex coassembly, as well as incorporating the latest community standards as they are approved by authoritative bodies. The work undertaken to generate the MAR databases highlighted that many marine samples in ENA lack key metadata fields. Through extensive curation efforts, using literature as well as contacting the original data submitters, much of this missing data was retrieved and added to the MAR database. While ENA (or any of the INSDC partners) can not add this metadata to the original sequence record, an ELIXIR sponsored initiative led to the establishment of the Contextual Data Clearinghouse (CDCH). The CDCH facilitates the capture of additional metadata using controlled vocabularies including a description of how this data was generated (e.g. manual assertion, computationally derived), so that they can be associated with an INSDC record. Longer term, this data will be incorporated into BioSamples.\n\nIn other non-sequenced based ‘omics fields, microbiome data archiving and analysis is supported by data-type specific resources. In the case of metaproteomics, the PRIDE database repository (also an ELIXIR CDR) enables archiving and re-analysis of (meta) proteomics data, and now also encourages researchers to upload their metadata in SDRF-format.38,39 PRIDE is the leading resource of the International ProteomeXchange Consortium of proteomics data resources, involving additional databases in the USA, Japan and China, in addition to PRIDE. Similarly, in the case of metabolites the data can be deposited in the MetaboLights repository40 or similar resources. A current challenge facing the field is connecting different multi ‘omics data that have been derived from the same sample.\n\nThe Data Platform also promotes the linkage between Europe PMC41 and other CDR databases. This is critical for the Microbiome Community as additional contextual metadata can often be found in the literature,42,43 providing crucial overarching context to the experiment, which can be important for meta-analyses. We will continue to promote such approaches, enriching metadata wherever possible.\n\nLast but not least, new activities will be promoted aimed at the integration of microbiome data coming from different ‘omics approaches. In this context, recently, the PRIDE and MGnify teams developed and implemented new pipelines in both platforms for the re-analysis and integration of metagenomic and metaproteomic data, allowing the re-analysis of metaproteomics datasets from PRIDE using sequence databases generated from MGnify, and contextualising the results back into the MGnify web interface in terms of assembly annotations (https://github.com/PRIDE-reanalysis/MetaPUF). The ELIXIR Microbiome Community will also work to move the Marine Metagenomics domain in the RDMKit towards a more general Microbiome domain.\n\nMicrobiome data analysis employs a large number of tools which are used to perform basic quality control on the sequence data, with separate tools (and reference databases) typically used for taxonomic and functional profiling. Installing and managing dependencies has been eased by the use of package management systems such as Conda, or through the use of containers, e.g. Singularity. The ELIXIR Microbiome Community will increase their use of BioContainers44 to promote the packaging, containerisation and deployment of tools relevant to microbiome research.\n\nIn order to make tools findable the Community will work on improving their annotation by (i) expanding the EDAM ontology45 to include microbiome-specific keywords, (ii) performing periodic reviews of tools and their associated annotations in the bio.tools46 catalogue. These annotations will subsequently be used to build a catalogue of tools for microbiome data analysis and their availability for different platforms, e.g. Galaxy, or as workflow descriptions (e.g. Snakemake, CWL, Nextflow), which can be readily combined to make new annotation workflows. Additionally, the Community will develop and maintain cloud-deployable and FAIR analysis pipelines using state of the art tools and following best open science practices by: (i) using workflow descriptions; (ii) documenting the workflows and depositing them in WorkflowHub47 for easy discovery, re-use and assessment; (iii) making them available for the Community via platforms such as MGnify and Galaxy.\n\nAs an integral part of the Tools platform, Galaxy has integration with OpenEBench, WorkflowHub EDAM, bio.tools and follows all Software Best Practices. A joint effort between the Microbiome and Galaxy Communities is running an evaluation of tool requirements for microbiome data analysis in the Galaxy ecosystem. This evaluation will lead to a shared roadmap between both Communities for tool integration and standardised workflow development for microbiome data analysis.\n\nBenchmarking\n\nVery few analyses in microbiome research employ a single tool, with the norm being the coupling of multiple tools and reference databases to achieve a comprehensive analysis that includes both taxonomic and functional results. Even relatively simple workflows that perform metagenomics assembly are computationally heavy. This combination of workflow complexity and typical computational overheads has always made the routine benchmarking tools for microbiome informatics research burdensome. Nevertheless, where two or more tools perform equivalent tasks, it can be relatively simple to modify existing formally described workflows to evaluate their respective performances, but that ease often depends on where they occur in the overall workflow and the metrics used to evaluate the tool. Many efforts have tried to compare the outputs of tools and workflows (e.g. Refs. 48–52), with the Critical Assessment of Microbiome Interpretations (CAMI) having become an internationally recognised benchmarking effort.53–56 The CAMI challenges have established a range of benchmarking datasets for evaluating different categories of tools. Importantly, the organisers of CAMI have engaged data generators to provide data, such that truly independent benchmarking can be undertaken. However, these benchmark datasets can become outdated over time, as the underlying data enters the reference database. The Galaxy Community has already investigated implementing benchmarking infrastructure using CAMI datasets, and increasing the awareness of this infrastructure will be a key effort across Communities and Platforms. As the Microbiome Community establishes, we will develop a broader understanding of the requirements of the Community, feed this to the Tools Platform, as well as seek opportunities to interact with the Tools Platform to capture the diversity of tools and their utility via such benchmarking activities.\n\nGiven that most academic institutions have access to dedicated sequencing facilities or equivalent commercial facilities, coupled with the diminishing costs of DNA sequencing and other ‘omics technologies, it is relatively easy to generate large datasets, but significant computational resources are required to store and analyse the data. Depending on the analysis being performed, the computational requirements can be very different. For example, metagenomic assembly typically requires small numbers of cores on a large memory machine, whereas some forms of raw-read analysis require many cores (hundreds) with a small memory footprint. As such, microbiome researchers need to understand the likely computational costs, and their options for deploying them on high performance computing (HPC) and cloud environments. Efforts such as Blue Cloud have helped reduce some of the barriers to using the European Open Science Cloud (EOSC) for marine research through the delivery of a collaborative virtual environment, but the range of services is limited. While such efforts help, there are still many barriers to accessing compute resources and deploying complex metagenomic pipelines in a distributed or even hybrid fashion. Working with the Compute Platform, the ELIXIR Microbiome Community will continue to investigate solutions that facilitate the execution of workflows within such distributed and/or hybrid environments, e.g. using Pulsar network, the distributed compute network offered by the Galaxy Community, and provide guidance of the likely costs of using compute infrastructures.\n\nPrevious work by the Marine Metagenomics Community has leveraged many of the ELIXIR Interoperability Platform solutions, especially the use of workflow languages for the formal description of pipelines, improving the provenance of the data outputs. As such, both the MetaPIPE and MGnify pipelines have been described using the Common Workflow Language (CWL). This effort was paralleled by MG-RAST, which also allowed MGnify and MG-RAST to exchange pipelines57 and establish that the biological signatures reported by the respective pipelines were very similar, yet confounded by different reference databases and methodologies for assigning function. Since then, MGnify has published their workflows in WorkflowHub,58 further promoting their discovery and reuse. As an example of reuse, the MGnify pipeline has been used as the basis for the newly developed metaGOflow pipeline,59 to be used by the Marine Genomic Observatories. Moreover, this work also employed Research Object Crate (RO-crate)60 to package relevant metadata about the sample and the bioinformatics analysis applied and the data products. RO-crate offers new opportunities for sharing or federating the metagenomics analysis workload. In parallel, Galaxy, which supports the Tool Registry Service (TRS) protocol to exchange and run workflows between the WorkflowHub and Galaxy, gained support for RO-Crate (version 23.0) to export complete data analysis as a structured and FAIR digital object, supporting the GA4GH standards, and is in the process of applying to be a Recommended Interoperability Resource.\n\nThe Microbiome Community will continue to work with the Interoperability Platform to make wider use of RO-crate, with a view to federate data analysis between resources. For example, future work by the new Community will enable the MGnify workflows to be made deployable on Galaxy, with the RO-crate to be transferred, verified and ingested into MGnify. Additional work needs to be undertaken to understand how universal this approach is, so that MGnify could become a hub for a range of additional analyses, thereby reducing the duplication of effort that currently exists in the community.\n\nFinally, we will work on the development of novel mechanisms to integrate and link data coming from multi-omic approaches using different tools and data resources. This will require the development of new data Interoperability layers for data resources that are not normally focused in Microbiome data, such as the PRIDE database in the case of metaproteomics data.\n\nOne of the key areas commonly highlighted by national and international reports on the potential of microbiome research is the need for training, especially in the area of informatics. As already highlighted, microbiome analysis is an emerging and evolving research field by itself, with plenty of challenges still to be addressed. Combined with this complexity, the increasing number of researchers using such methods makes the need for continuous training and re-training a challenge on its own. Researchers need to become familiar with modern computing technologies, such as HPC and cloud computing, and follow the constant updates on experimental approaches, algorithms (new and updates) and pipeline developments. As new pipelines are established and existing pipelines improved through the incorporation of new tools and/or reference databases, this adds further complexity to the tool and data output landscape associated with microbiome research.\n\nPlatforms such as MGnify support large-scale services for most, if not all, steps of a microbiome study, meaning the distribution of raw-data, production of assemblies, their analysis, and their potential use for meta-analysis, have proved of great benefit. Nevertheless, these analyses should be considered just the starting point for further downstream analysis, which requires the specific domain expertise of the researchers involved in undertaking the study. One approach can be the use of cloud-based initiatives such as Galaxy supporting graphical interfaces and allowing the users to choose more specific tools, while tuning their parameters and reference databases according to their environment being studied. Such infrastructures attempt to fill the gap between researchers without experience in computer science and their needs for FAIR and quality microbiome analysis. Despite both solutions being readily available, there remains knowledge gaps and/or reticence about using such resources, often due to a lack of training.\n\nTo upskill microbiome scientists and keep them up-to-date in microbiome data analysis and standards, the ELIXIR Microbiome Community will work in coordination with the ELIXIR Training Platform to offer scalable and FAIR training. The Microbiome Community will continue to: (i) annotate training materials with appropriate metadata to create a comprehensive training portfolio; (ii) FAIRify the training content, making it open-access; (iii) register training material, national and international providers and events in ELIXIR’s Training Portal TeSS;61 (iv) assist the Training platform in the development of annual training gap surveys; and (v) develop materials and design learning paths specific to different community needs (e.g. biomes or data types).\n\nTo enable access to training resources and deliver this training, face-to-face and online workshops will be organised and videos will be recorded for “on demand” learning. The technical infrastructure for training, in particular the computational environment setup and software installation challenge will be addressed in coordination with the ELIXIR Tools and Compute Platforms, with the aim of promoting the use of Conda environments, containers, notebooks or platforms like Galaxy which mitigate many of the current obstacles. In order to make these aspirations possible, the Community will increase its training capacity by working with training communities on practices, organising Train the Trainers events and building a community of microbiome research trainers, with areas of expertise covering different environments, ‘omics approaches and data analysis pathways. Ensuring these trainers maintain their knowledge with the evolving informatics landscape is, arguably, a key challenge that is yet to be addressed and something this Community will strive to solve in collaboration with the ELIXIR Training Platform.\n\nWe have highlighted the need for promoting best practices and standards throughout this article. However, it is also important that the Microbiome Community continues to build upon engagement with organisations such as the Genome Standards Consortium (GSC62). The GSC is an international organisation aimed at making genomic data discoverable through the establishment of standards, which are derived from community input. This consortium includes stakeholders from across the data life cycle, from research scientists producing the data, to data analysts to database providers. By engaging this range of stakeholders, the GSC have become critical for establishing many of the standards that underpin genomic research. Examples of GSC established standards that are particularly pertinent to the microbiome domain include: minimal information about any sequence (MIxS63), the Biological Observation Matrix (BIOM) format64; and the Minimum Information about a Metagenome-Assembled Genome (MIMAG26,63).\n\nThe GSC has many ongoing projects relevant to the ELIXIR Microbiome Community, especially the M5 project (Metagenomics, Metadata, MetaAnalysis, Models and MetaInfrastructure). Combining the activities on standards concerning workflows is critical for the global microbiome community to operate with a consistent and unified view on how to make microbiome analysis reproducible and stand-up to scientific interrogation. Note, such standards do not restrict what analysis can/should be performed, but rather provide the appropriate information, that given the same starting input data, exactly the same analysis, and hence result, can be achieved.\n\nThere are also other ELIXIR Node-specific initiatives that the Microbiome Community connects with to ensure that the respective efforts are synergised. Examples of projects with ELIXIR Node involvement directly related to the ELIXIR Microbiome Community are presented in Table 3, which cover a diverse range of topics. The engagement needs to be bi-directional to ensure that the needs of Nodes are well understood and that solutions developed at national levels can be spread across the ELIXIR Microbiome Community, and vice versa. In this context, the ELIXIR Microbiome Community leads will undertake coordinating roles, engaging with the project representatives, inviting them to relevant ELIXIR events and promoting active participation in relevant ELIXIR Communities.\n\nMicrobiomeSupport, formerly a European Commission funded Community Action Support program aimed at improving microbiome research and innovation, highlighted in their final report65 that there was “limited connectedness” in microbiome research conducted on different environments/systems, and that during the course of this program the lack of connectedness did not improve. This independent finding reinforces the need for broadening the ELIXIR Marine Metagenomics Community to a more generalist Microbiome Community.\n\nIt will also be important to showcase the ELIXIR Microbiome Community to European countries that are yet to join ELIXIR. For example, Romania has a thriving microbiome research community, but is faced with the same set of informatics challenges. Sharing knowledge beyond ELIXIR, will not be the primary goal, but will nevertheless be important to harmonise the activities internationally and promote the benefits of participation in ELIXIR. Beyond Europe, there are parallel organisations that strive to achieve similar goals to ELIXIR in other locations. For example, Australia BioCommons aims to promote bioinformatics and bioscience data infrastructures at a national level. Given the strength of microbiome research in Australia (see below), we will explore opportunities for international collaboration.\n\nIn addition, it will be important to showcase the ELIXIR Microbiome Community to communities (within and outside Europe) that are not yet familiarised with ELIXIR activities. For example, the Metaproteomics Initiative is an international community that promotes dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research.35,66 For example, recently, they benchmarked metaproteomics workflows and bioinformatics methods in the field in the first multi-lab benchmark study in metaproteomics (called CAMPI), showcasing the robustness of metaproteomics data analysis workflows.66\n\nFinally, the National Microbiome Data Collaborative (NMDC),67 a US led initiative, is developing a unified data portal to support microbiome multi-omics data integration and analysis through an integrated, distributed framework. Many of the governing principles associated with this portal are common with those described here, especially with the desire to have containerised, reusable computational workflows, as well as trying to make the data compliant with the FAIR principles. Sharing experiences and best practices between NMDC and the ELIXIR Microbiome Community (and others) will improve the global standardisation of microbiome research.\n\nMicrobiome research is global, so it is also key that European microbiome research infrastructures are coordinated with other international resources. Below we highlight a small selection of widely used resources that are produced outside Europe, and place them in context of the ELIXIR Microbiome Community. Some of the most utilised tools and resources used by the current Microbiome Community are CheckM,68 the Genome Taxonomy Database (GTDB) and the associated GTDB toolkit.69,70 CheckM is widely used to assess the completeness and contamination of prokaryotic MAGs, and is part of the GSC reporting standard. The GTDB resources is a genome based taxonomy of prokaryotes, and the associated GTDB-tk facilitates the classification of other prokaryotic genomes against this framework, more often than not, to determine novelty. These are currently made available via the Australian research groups, who face similar challenges in maintaining resources. Other key resources are based in the US, with MG-RAST71 produced by Argonne National Laboratories and a range of different resources produced by the Joint Genome Institute (JGI). MG-RAST facilitates the analysis of raw-reads and assemblies (metabarcoding, metagenomics and metatranscriptomics), but does not perform assembly nor offer any form or long-term archiving assurance. The JGI IMG/M resource72 has many parallels with MGnify, offering a wide range of data analyses focused on assembly and MAG generation, but IMG/M does not deal with metabarcoding. Notably, JGI also produces IMG/VR,73 a globally unique collection of viruses, many of which have been determined from metagenomic and metatranscriptomics. Any future effort in Europe focused on viruses must aim to minimise the duplication of effort and content with IMG/VR. Coordinating with these global initiatives is key to ensure the future availability of the tools and resources, ensuring interoperability between the resources, maintaining uniform standards and sharing of the informatics/computational burden.\n\nA key early challenge in developing the ELIXIR Microbiome Community is to establish a detailed understanding of the current approaches and databases used for the analysis of different microbiomes. For example, it is widely accepted that current short-read assembly-based methods do not generally work as well for soil microbiomes due to the diversity of the microbial community typically present (the sequence depths are insufficient to build useful contigs or the datasets are so large, that they are computationally intractable). This current limitation, has led and will continue to lead to the development of new experimental methods, from sampling through to nucleic acid sequencing and informatics analysis. In this section, some of the key challenges associated with microbiome research are highlighted below, together with how these challenges will be addressed by the new ELIXIR Microbiome Community. Table 4 lists the key thematic areas and objectives that the Microbiome Community will address, split into short-term and longer-term objectives to provide a high-level overview of the proposed Community activities.\n\nThe Microbiome Community will also provide a mechanism for sharing knowledge about new approaches for microbiome research, be it experimental or informatics-based techniques. For example, there is an increasing number of metagenomics datasets that are produced using long-read sequence technologies. While long-read sequencing technologies can require larger quantities of DNA or may be more error prone compared to third-generation short-read sequencing technologies – which can limit their use – the long-reads can mitigate the computational burden of metagenomic assembly and increase the confidence in analysis results (e.g. MAGs produced by long-reads can have high contiguity and therefore less prone to contamination). The long-reads can be paired with short-read sequences, which can then be used in different ways (e.g. sequence error correction). Increasing the awareness of these long-read and hybrid-sequencing approaches, the workflows that support their analysis and when and where they could be applied will be a key output of the Microbiome Community. Similarly, there are other experimental approaches such as single amplified genomes (SAGs), which have increased in popularity. The Microbiome Community will also be important for assessing the utility of emerging sequencing approaches, such as adaptive sequencing approaches. In this case, the methods can access low abundance microbes, although such methods will not facilitate the generation of abundance profiles. Bringing these data types alongside the ubiquitous short-read datasets will require new standards and data integration approaches to be developed by the Microbiome Community.\n\nThere has been a paradigm-shift in metagenomic analysis with a common goal now being the generation of prokaryotic MAGs, which has not only allowed the identification of thousands of specific functions, but facilitated them to be assigned to specific organisms. As such, this has started the development of specific MAG deposition layers,74 and the development of MAG specific resources. The new Microbiome Community will promote the use of MAG deposition, and provide guidelines and software to aid their deposition. Workflows that encompass both MAG generation and quality verification will be developed that include the capture of both prokaryotic and eukaryotic MAGs. The Microbiome Community will help establish best practices for eukaryotic MAG discovery, as well as develop new standards for removing redundancy and methods for assigning taxonomy, which are recognised gaps in the area of eukaryotic MAG discovery. While prokaryotic MAG recovery methods are more mature and standardised, it is anticipated that there will be continuous improvements in both experimental and computational methods for generating longer contigs, and more datasets that enable different approaches to enhance the detection of contamination and/or misassembly. The ELIXIR Microbiome Community will also evaluate methods and establish best practices for the identification of sub-species/strains in metagenomic datasets. To do so, we will engage with efforts such as the Critical Assessment of Metagenome Interpretation (CAMI)53,75 to identify tools that can scalably and accurately classify MAGs at a finer grain taxonomic level than species.\n\nFinally, the classification and naming of MAGs is going to be paramount, so that the novel biodiversity can be understood and more easily referenced by the scientific community. Currently, the Microbiome Community has widely adopted the GTDB69 and the associated GTDB-tk70 for classifying MAGs against a reference tree. However, the taxonomy of GTDB differs from the more widely-used NCBI taxonomy, and there is a need to increase the interoperability between these two taxonomies. The ELIXIR Microbiome Community will work on addressing the current issues associated with MAGs and taxonomy. Additionally, another key area of development will be increasing the linkage between genomic resources and marker genes, such as the ribosomal small subunit (SSU) RNA.\n\nIn addition to cellular microbes, another area for the ELIXIR Microbiome Community to address is the development of the infrastructure and resources for identifying and cataloguing viruses in metagenomic and metatranscriptomic data.76–79 Viral genomes are incredibly diverse in terms of composition and organisation. Viruses, particularly those that infect bacteria, are found ubiquitously in all environments and play critical roles in community dynamics. However, there are three challenges associated with viral microbiomes: (i) there is no universal marker gene covering all viruses; (ii) viral taxonomic frameworks are incomplete; (iii) there is no centralised database collecting the millions of viral sequences; and (iv) metagenomics informatics often only produces fragments of viruses, which causes ambiguities concerning their classification and functions. It will be critical for the ELIXIR Microbiome Community to engage with established viral infrastructures and organisations, such as the European Virus Bioinformatics Center, to establish methods, standards and resources for improving the analysis of viruses found in microbiome sequence data.\n\nThe increase in metagenomic assemblies has resulted in a parallel increase in the number of protein sequences that have been identified, with sets of non-redundant proteins now in the billions. There is huge potential for discovery in these protein datasets, as well as de novo designs fit for purpose, e.g. carbonic anhydrases80 and a key aim for the new ELIXIR Microbiome Community will be ensuring that these data are annotated, both as individual sequences or as higher order grouping (e.g. pathways, biosynthetic gene clusters). This will involve the evaluation of emerging tools, as well as harnessing structural models to allow the detection of relationships that are undetectable by current sequenced based methods. The Community will need to work together to shed light on the functions of the so-called ‘Dark Matter’, develop standards for functional labelling that encapsulate both the mechanisms and confidence of the annotation, and develop new infrastructural frameworks for accessing slices of the data based on the requirements.\n\nAs identified by the ELIXIR Marine Metagenomics Community, experimental and contextual metadata is critical to comparative metagenomics. The absence of rich contextual and experimental metadata limits data reuse and the production of downstream data products, such as assemblies and MAGs. With the expanded Microbiome Community, we will identify areas where metadata standards need to be improved, with biome specific contextual metadata being the most likely source of specific metadata checklist. The Community will develop training promoting the need for metadata, checking compliance against standards, how the metadata can be captured and submitted to accompany the sequence data, and potentially other ‘omics data types. Within the Community, we will develop and promote standards around the analysis provenance (analytical metadata), and how the collective corpus of metadata can be used to improve meta-analysis and the identification of confounding factors when comparing different research projects.\n\nAnother key challenge that the Microbiome Community needs to address is ensuring that compute resources are accessible for performing the different forms of data analysis that can be associated with microbiome derived sequence data. Previously, we have highlighted the need for interaction with the ELIXIR Compute, Interoperability and Training platforms, as well as ELIXIR Communities such as the Galaxy Community. This requires that analysis pipelines are readily discoverable and deployable, and that key issues regarding both compute processing and storage requirements are well understood. Additionally, given that microbiome associated data analysis has such computational overheads, it is vital that models for data archiving and/or sharing are developed by the Microbiome Community to increase the capacity of microbiome research within Europe. This may require the development of new or extensions to existing databases, but it requires an agreement from the research community to adopt them. Achieving this will involve both communication and training of the microbiome research community.\n\nWhile there are data resources such as MGnify that provide access to consistent analyses pertaining to different metabarcoding, metatranscriptomics and metagenomics datasets from a variety of biomes, it is fundamental to remember that these data outputs do not represent the end of the analysis pathway. Typically studies require comparison between different cohort groups (disease vs health, treatment vs non-treatment). Furthermore, as the biological signal from meta’omics datasets can be extremely noisy, there can often be the need to combine datasets to boost statistical significance of the biological signal. Similarly, the combination of studies can also be used to: (i) contextualise against previous studies (e.g. similar studies on the same diseases); (ii) understand the distribution of microbes or functional features (e.g. antimicrobial resistance genes) between different geographical locations; and/or (iii) study the relationship between biomes (e.g. studies adopting a OneHealth approach). To enable such large, complex studies there needs to be a greater understanding of the approaches suitable for cross study comparisons, and their limitations. Thus, a major objective for the Microbiome Community will be to include those researchers that are developing methods that can identify and mitigate experimental and informatic confounding factors, which currently limit data reuse. Existing approaches often rely on correlating contextual and experimental metadata with statistically significant factors identified in the datasets. There is also the need to develop and promote methods for performing robust statistical analysis of microbiome derived data, thereby enabling biological signals to be extracted from cross-sample/project datasets. Currently, there is a tendency to analyse the different ‘omics datasets independently, and then correlate the derived signals. However, statistical methods are being developed to facilitate the analysis of integrated multi-omics datasets, and it will be important that the Microbiome Community determines the applicability of these approaches for microbiome research.\n\nIn the context of other ‘omics approaches, there are also some major challenges in metaproteomics.81 One of the major challenges is the construction of tailored protein sequence databases which are needed to identify proteins in complex microbial communities. Metaproteomics aims to elucidate the functional and taxonomic interplay of proteins in microbiomes, but the diversity and vast number of unknown and uncharacterized proteins present in these communities makes database creation and accurate protein identification difficult. As microbial communities are highly dynamic and their protein expression can vary significantly, conventional protein sequence databases might not cover the entire diversity, leading to potential limitations in accurate protein identification (e.g. the use of de novo sequencing). Addressing this challenge is crucial for improving the reliability and confidence of metaproteomic analysis and obtaining comprehensive insights into the functional roles of proteins in complex microbiomes.\n\nAs metagenomic methods have become a more routine method for studying microbial communities, metagenomics has been and will continue to be paired with more and more diverse sets of measurements of the microbiome. Examples of non-omics data collected alongside metagenomics data include geochemical (e.g. PANGAEA82) measurements, meteorological, image data and even acoustics. While methods are already emerging for the integration of ‘omics datatypes (e.g. MOFA,81,83 MIA), integration of these additional non-omics data types will enable a broader understanding of microbiomes in context. For the new Microbiome Community, it will be essential to identify the appropriate archives for these data types, and establish the methods to facilitate navigating between datasets from the same samples. Only through achieving this, can new data visualisation schemas that enable the combination of environmental, geospatial and temporal data, in addition to biological data (taxonomy/function), be developed.\n\n\nConclusions\n\nThe overarching aim of the Microbiome Community is to develop a sustainable bioinformatics infrastructure for microbiome resources (data, tools, workflows, standards, training) which will enable a deep understanding of the function and taxonomy of the entire microbial fraction. We aim to be biome-agnostic, yet balanced in supporting the analysis and interpretation of data from different environments. We aim to highlight the very best approaches for the analysis and integration of different data types (e.g. sequences, metabolites, proteomics, and images) and their visualisation. By broadening the Community we will engage many more researchers and aspire to have a greater representation of scientists from different disciplines, such as ecologists and clinicians, complementing the strong molecular biology and genomics backgrounds already represented in the Community. The Microbiome Community will have key roles in engaging with policy makers (e.g. access and benefit sharing, climate change impact assessment), as well as the industrial sector, which is increasing the translation of basic research to microbiome-based products (e.g. UK Microbiome Strategic Roadmap for Innovation). Such a strong microbiome infrastructure as envisaged by this Community is essential to maximise the impact that European research programs have in the field of microbiome research, and to facilitate the exploitation of microbiome-based solutions in a range of settings, from clinical to industrial processes, thereby addressing key societal challenges and needs.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nMarchesi JR, Ravel J: The vocabulary of microbiome research: a proposal. Microbiome. 2015 Jul 30; 3: 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Scientific Data. 2016 Mar 15; 3(1): 1–9.\n\nHarrow J, Drysdale R, Smith A, et al.: ELIXIR: providing a sustainable infrastructure for life science data at European scale. Bioinformatics. 2021 Jun 27; 37(16): 2506–2511. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "244855", "date": "14 Mar 2024", "name": "Charlie Pauvert", "expertise": [ "Reviewer Expertise microbiome", "bioinformatics", "reproducible research", "data and metadata standards" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 make the case for mutating the ELIXIR Marine Metagenomics Community initiative into an ELIXIR Microbiome Community. It is indeed timely to have an such a pan-European initiative especially as it draws on previous experience, albeit a more narrow biome. The emphasis on multi-omics integration is also a strong suit as it is a complex topic where the microbiome research community would need support and infrastructure. The authors map out existing (but not all) similar initiatives, even if it is unclear how they would work together. In my opinion, a couple of claims should be strengthened and the argumentative power of this white paper would benefit from minor reorganization, a text slim down and extra proofreading. To this end, I highlight the strengths and weaknesses of the manuscript below.\n## Strengths\n- Table 3 is a great idea to map out the others initiatives but needs a bit rework to be straight to the point. - In the Data section, the part between \"Throughout the lifetime of the ELIXIR Marine Metagenomics Community\" and the end of the paragraph is really relevant and draws from the experience of the ELIXIR Community. These sentences should be more highlighted, maybe upstream. I do wonder how the authors considered how the others initiatives (mentioned in Table 3) could also contribute to the addition of metadata for a global curation effort, possibly via the mentioned Contextual Data Clearinghouse. - The data re-analysis and integration between PRIDE and MGnify (named MetaPUF) is a good use case of the efforts that the new ELIXIR Community can promote. - I did appreciate the clear objective at the end of the Compute section, to help with scaling up analyses as well as the plan for the interaction with the ELIXIR Training Platform which promise to give a boost in training the current and next generation of microbiome researchers. - I liked how the authors highlighted (using the example of viral sequences databases) that time and resources should not be wasted in duplicating works, and that initiatives such as the ELIXIR Microbiome Community can promote this. - The authors thought ahead to promote and develop methods to limit confounding factors when re-using datasets, especially in multi-omics settings, and added this to one of the ELIXIR Microbiome Community challenges.\n## Weaknesses\n### Introduction\n- (minor) The microbiome definition used in the paper was revisited in Review reference 1 to include the interactions as well as others molecules than genomes as part of the microbiome. I suggest to use this definition given the emphasis on additional omics made in the paper. - (major) The first paragraph ends on challenges of our research community including how to make data FAIR. Given the experience gained by the ELIXIR Marine Metagenomics Community, I would have appreciated a sentence on why making our data FAIR is important (e.g., transparency to make the research more reproducible, accountability given the (public) source of funding). See major comment in next section.\n### The scope of the ELIXIR Microbiome Community\n- (major) These following arguments for FAIR data should be in the Introduction section. \"Moreover, when wishing to contextualise the results with similar experiments, the way a dataset has been produced and processed must be transparent to establish whether it is comparable (e.g. amplified sequence variants can only be compared when the same amplified regions are compared). Furthermore, when different methods are applied, best practices in data stewardship are required to ensure that the connectivity of the derived sequence data products, together with functional and taxonomic assertions are kept in context of the original sample/sequencing effort and associated contextual metadata.\" - (minor) The very first sentence of this section blames researchers for misuse of a term \"[...] regularly (incorrectly) used [...]\". I suggest to rephrase the sentence to simply state the differences, or back up the misuse of the term with references or a survey. - (minor) \"Fundamentally, the ELIXIR Microbiome Community is about providing the necessary infrastructures required to perform analysis of nucleotide sequence data derived from a microbiome\" - (minor) \"Finally, and possible unique to this ELIXIR Community, is the variety of researchers\". I am not sure that these features are unique to the ELIXIR Community but rather to the field of microbiome research. I suggest to tone it down by simply pointing out that the ELIXIR Community gathers a variety of researchers.\n### Table 1\n- (major) Metabolomics is listed in Table 1 but is omitted from the narrative in the paragraph. - (major) The Table 1 is not really used but could actually reduce some redundancies in the manuscript by providing a one-stop-shop to explain and detail these techniques.\n### Figure 1\n- (major) The figure is early on in the manuscript and it is unclear which of the ELIXIR platforms and communities are already established, or foreseen. Especially since \"current and future ELIXIR activities\" is mentioned in the manuscript before referencing this figure. The Table 2 does not add more information in that regard. - (major) There is a need for different types of arrows as the same arrow represent interactions between communities or processes. - (minor) The \"Data\" node is quite generic, I was wondering whether it meant public repositories, institute repositories or both. Please be precise. - (minor) I guess the type of metadata illustrated in the figure is restricted to biological metadata, that is indeed collected during sampling, however, technical metadata such as the sequencing method used, the type of instrument or the library layout, are collected during the processing of the sample not only the sampling itself. - (minor) There should be an arrow from \"Archive\" back to \"Data\" when the data produced is deposited and then contributes back to public repositories?\n### Interactions with other ELIXIR Communities\n* (major) The sentence \"Similarly, many of the biodiversity approaches use marker gene amplification for studying environmental DNA (eDNA)\" is redundant with the previous one and introduces a different nomenclature that was not used before (marker gene amplication vs metabarcoding) and the differences, if any, are not explained. I would suggest to remove the sentence. * (major) The term \"Isolation of genomes\" is misleading and I guess the authors used it as a shorthand for \"The isolation of bacteria, its DNA extraction, genome sequencing and their annotation\". Please rephrase to avoid misinterpretation. Plus I would argue that these steps are also done when deconstructing microbiomes via cultivation strategies and are therefore not so out-of-scope. * (minor) \"yet each one of these areas is far greater in scientific scope\" feels exaggerated and vague. It should be rephrased. A suggestion is: \"yet each one of these areas is too complex to be tacked individually\" * (minor) There is no link nor transition between the paragraph that starts with \"In summary, \" before the Table 3 and the paragraph after that starts with \"Similarly, microbiome research\". Please rephrase or edit to connect the two sections. Plus, whilst this is good to have a concrete example in the \"Similarly\" paragraph, the paragraph before was very broad and doing a summary. I would suggest to try reordering the two paragraphs and bring the example earlier for a smoother transition. * (minor) Typo \"Similarly, microbiome research has many translational aspects\" * (minor) The PET acronym is detailed but actually used only once, I am unsure if this is necessary.\n### Table 2\n- (major) The text in the \"Interaction\" column needs rework as it does not use a consistent wording and could be more to the point, especially as it is a complement to the main text:\n\n- The Food and Nutrition entry is a question.\n\n- The Galaxy entry has an unnecessary return carriage, and a unspecific \"ongoing evaluation study\" that needs to be clarified.\n\n- The Plant Science entry starts with a generic sentence that could be in the introduction or removed for clarity. Plus I would add a clarification that \"plants maintain or not their microbial communities across generations.\" - (minor) The status (e.g., currently active, inactive, planned, etc.) of each ELIXIR Communities would have been appreciated as it is missing also from the Figure 1. - (minor) The mention of \"the field\" for the Federated human data community is vague in a manuscript about gathering communities, which research field is implied? If this is the human microbiome research field as a whole, please indicate.\n### Table 3\n- (major) Similarly to the Table 2 major comment, there is a lack of consistent wording in the \"Aim\" column that makes the Table 3 not as impactful as it should and could be. Maybe the authors could extract common/distinct features from each of these initiatives as an alternative way to the \"Aim\" column. A couple of suggestions for these features would be: is it a national initiative?, does it relates to data storage, data analysis, training? is it linked to ELIXIR? - (major) I was surprised not to see the NMDC listed in the table 3, especially when it is discussed in the main text. What about the NCCR Microbiomes initiative in Switzerland? I can understand that some initiatives are not included for space reason, but maybe state it in the legend of the table. - (minor) Is this table sorted? It seems not, but it could be by acronyms or names. - (minor) The aim for the NFDI4Microbiota is way too big a paragraph. The authors should reduce it for conciseness. - (minor) The Metaproteomics Initiative entry has a hyperlink and a reference when none of the others have. Please homogenise. - (minor) Some entries have country listed and some not. Please homogenise. - (minor) I am not questioning the existence of the European Reference Genome Atlas here, but how best to phrase its relevance to ELIXIR in the manuscript. There seems to have no prokaryotes genomes in their atlas, however, there seems to be a trove of fungi and protists genomes which are usually said to be understudied in microbiome. So I think there is a missed opportunity for the authors here to make the most out of this entry. - (minor) \"With its headquarters in Bari (Apulia region),\" seems irrelevant in the context of the table, please remove.\n### Data\n- (minor) The end of the following sentence is redundant as the INSDC was introduced earlier already \"INSDC, which in collaboration with the National Institute of Genetics DNA DataBank of Japan (DDBJ) and the United States National Center for Biotechnology’s (NCBI) GenBank and Sequence Read Archive (SRA), facilitate the deposition and global exchange of sequence data.\". Please adjust accordingly. - (minor) \"A current challenge facing the field is connecting different multi ‘omics data that have been derived from the same sample.\" Is this going to be tackled by the ELIXIR Microbiome Community? If so, I would state that this is part of its objectives. - (minor) The end of sentence \"[...]overarching context to the experiment, which can be important for meta-analyses.\" seems like an euphemism, I would suggest to replace with \"[...]overarching context to the experiment, re-analyses or meta-analyses.\" to include re-analyses as well. - (minor) \"We will continue to promote such approaches, enriching metadata wherever possible.\" Is this going to be done via the CDCH? - (minor) \"The ELIXIR Microbiome Community will also work to move the Marine Metagenomics domain in the RDMKit towards a more general Microbiome domain.\" What is the RDMKit? It is not explained, nor cited nor mentioned again.\n### Tools\n- (minor) \"will increase their use of BioContainers\" should be \"will increase the use of BioContainers\" - (minor) \"In order to make tools findable by the end users, the Community\" - (minor) \"workflow descriptions (e.g. Snakemake, CWL, Nextflow)\" None of them have their references cited, is it an omission or space limitation? - (minor) \"A current joint effort between the Microbiome and Galaxy Communities\"\n### Benchmarking\n- (major) \"Benchmarking\" it is the only item at this hierarchy level, meaning that this is useless for structuring the text. Please edit. - (minor) Review reference 2 published a recent review with guidelines to learn from that could have its place in this paragraph. - (minor) in the sentence: \"As the Microbiome Community establishes, we will develop a broader understanding of the requirements of the Community, feed this to the Tools Platform, as well as seek opportunities to interact with the Tools Platform to capture the diversity of tools and their utility via such benchmarking activities.\", is this the ELIXIR Microbiome Community, or the wide community of microbiome researchers? Is it to mean that the ELIXIR Microbiome Community is going to act as an interface between microbiome researchers and ELIXIR Tools/Infrastructure?\n### Compute - (minor) The first sentence would fit better in the introduction.\n### Interoperability - (minor) The reference 57 should be at the end of the sentence starting with \"This effort was paralleled\" not in the middle. - (minor) Reference 58 should be removed at it is a duplicate of reference 47. - (minor) Use the full text \"Global Alliance for Genomics and Health\" instead of GA4GH. - (minor) \"is in the process of applying to be an ELIXIR Recommended Interoperability Resource.\" - (minor) In the sentence: \"This will require the development of new data Interoperability layers for data resources that are not normally focused in Microbiome data\" I think the authors meant \"used\" instead of \"focused\", and \"microbiome\" instead of \"Microbiome\".\n### Training - (minor) In \"Platforms such as MGnify support large-scale services for most, if not all, steps of a microbiome study\", I would suggest to remove the \"if not all\". - (minor) \"with areas of expertise covering different environments, ‘omics approaches and data analysis pathways.\" I think the authors meant \"learning paths\", and I would refrain from using \"pathways\" as it also has a biological meaning.\n### Context with other international initiatives\n- (major) Whilst I appreciated the emphasis that no initiative exists on its own, I feel the first paragraph on the Genomic (please correct the typo) Standards Consortium feels lengthy for a manuscript whose topic is not the GSC. I would advise to summarize. In this respect, the second paragraph is particularly relevant to a tangible collaboration between ELIXIR Microbiome Community and GSC. - (major) The NMDC is discussed in this section but not part of the Table 3. - (minor) Is the mentioned M5 project still active as the website's last update is 2012? - (minor) \"Combining the activities on standards concerning workflows [...]\" does this means adding and providing workflows to the microbiome research community? - (minor) Given the emphasis on the fact that MicrobiomeSupport was a program, the authors could update the readers and indicate that it is now MicrobiomeSupport Association.\n### Interaction with other key data resources beyond ELIXIR\n- (major) There is an order issue with the main text that a proofread could solve, as MG-RAST is explained and cited in the first paragraph but already mentioned upstream of the main text in the Interoperability section.\n### Specific challenges and objectives of the ELIXIR Microbiome Community\n- (major) The strong claim \"it is widely accepted that current short-read assembly-based methods do not generally work as well for soil microbiomes\" would probably need at least one reference. - (major) \"(iii) there is no centralised database collecting the millions of viral sequences\". It seems to be the case indeed, and there are databases (~24) out there as recently compiled in Review reference 3. How ELIXIR Microbiome Community plans to integrate/aggregate these resources in a non-duplicating manner? - (minor) The word \"through\" is superfluous in the the sentence that starts with \"This current limitation, [...]\" and can be removed. - (minor) The CAMI was already explained and cited above, so the already defined acronym can be used. - (minor) Precise the area in :\"Additionally, another key area of development of taxonomy [...]\". - (minor) The sentence \"Viruses, particularly those that infect bacteria, are found ubiquitously in all environments and play critical roles in community dynamics.\" belongs in an introduction, not so downstream of the manuscript. - (minor) The sentence starting the sixth paragraph could be precised as \"The increase in metagenomic assemblies has resulted in a parallel increase in the number of predicted protein sequences, with sets of non-redundant proteins now in the billions.\" - (minor) Fix typo in \"that are undetectable by current sequence based methods.\" - (minor) Would it make sense to also be able to access representative, of clusters for instance? \"[...] develop new infrastructural frameworks for accessing slices of the data or adequate representatives based on the requirements.\" - (minor) What is the \"expanded Microbiome Community\"? It was never mentioned before. - (minor) A few comments on the sentence: \"Within the Community, we will develop and promote standards around the analysis provenance (analytical metadata),\". In my opinion and how it was already stated in the manuscript, it would make more sense to promote existing standards first and then develop if need be. There is no mention of other type of metadata, so the \"analytical metadata\" precision seems superfluous. I would suggest: \"Within the Community, we will promote and develop standards regarding the analysis provenance,\" - (minor) Precise the term forms in \"[...] ensuring that computing resources are accessible for performing the different forms of data analysis [...]\", do the authors mean types of/steps in the data analysis? - (minor) Same argument as before regarding reinventing the wheel, I would swap the part of the sentence: \"This may require the extensions to existing databases or development of new ones, but it requires an agreement from the research community to adopt them.\" - (minor) This part \"Metaproteomics aims to elucidate the functional and taxonomic interplay of proteins in microbiomes,\" should have been in the Table 1, or to reuse the Table 1 here. - (minor) The tenth paragraph of this section starts with the mention of multiple major challenges, but detail \"only\" one of them. I would suggest to mention some of the others challenges. - (minor) The \"MIA\" method is just a hyperlink, without any reference. Either cite the website accordingly or add the reference.\n### Table 4\n- (major) I was surprised to see that the objective \"Foster international collaborations between other resources providers and databases to ensure global harmonisation of e-infrastructures for microbiome research\" was long-term, as I would have imagined that a gap analysis would be short-term to ensure we do not reinvent the wheel, especially given the others initiatives discussed in the manuscript. - (minor) If the Objective column starts with action verbs (which is a good idea), then it should be \"Survey the needs\" instead of \"Survey of needs\". - (minor) Specify the type of workflow with \"Address knowledge gaps in generating and adopting data analysis workflows\" - (minor) The verb is missing in \"Teach advanced containerisation and cloud deployment\" - (minor) The verb is missing in \"Promote data analysis through the use of services\". Is this ELIXIR services in general or specific ELIXIR Microbiome Community services? - (minor) Use \"Share\" instead of \"Sharing\" in the \"Co-ordinate\" entry. - (minor) The \"Industry connection\" entry does not fit the action verb pattern. A suggestion would be \"Use ELIXIR and Node forums to understand pharmaceutical and biotechnological demands and current limitations impacting this sector.\" as the first sentence felt generic. - (minor) The verb is missing in \"Design targeted training for different microbiome communities\" - (minor) Use \"findability\" instead of \"discoverability\" for consistency and to fit with the FAIR principles. - (minor) Reorder the sentence to start with the action verb: \"Establish new standards for microbiome research, particularly with respect to data analysis reporting and contextual metadata reporting in conjunction with GSC\" - (minor) Correct \"Established\" to \"Establish\" in the \"Promoting new approaches\" entry.\nEditorial comments: - (major) \"Community\" is used in upper-case and this is unclear in many instances whether the ELIXIR Marine Metagenomics Community is referred to, the ELIXIR Microbiome Community, or the broader microbiome research community. I suggest to use consistently the full term for the sake of transparency. Abbreviations like EMMC and EMC could be even more misleading in my opinion. - (major) The structure of the white paper is not evident as the hierarchy is indicated only by change in font size, and some sections are quite lengthy for a white paper that is supposed to be concise. I understand that this is a constraint from the Editor, but see Review reference 4 for a white paper with a more clearer structure. An alternative could be to use numbered sections.\n- References\n\n- (minor) The very first reference is oddly formatted in the text creating an artificial and confusing end of sentence.\n\n- (minor) Title of reference 9 is truncated and should be \"Methods included: standardizing computational reuse and portability with the Common Workflow Language\"\n\n- (minor) The superscript numbers of the numeric style of bibliography are in many instances after the final dot (see reference 2, 12-16, 17-19, 36, 37), after a comma (see reference 10, 20, 23), a bracket (see reference 60) or semi colon (see reference 65) when they should be before any of these symbols.\n\n- (minor) In the paragraph \"Interactions with other ELIXIR Communities\", we jump from reference 24 to 29 when the numeric style of bibliography (that was chosen by the authors) is expected to mirror the mentions in the manuscript. Please either have the Table 2 earlier in the paper, or change the order of the references.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "251099", "date": "11 May 2024", "name": "Almut Heinken", "expertise": [ "Reviewer Expertise Systems biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the ELIXIR Microbiome community is described. The community was established in 2015 as the Marine Metagenomics community and was recently expanded to the scope of metagenomics research for multiple areas such as human health, agriculture, and ecology. The authors then describe their perspective for the community. One focus area will be the promotion of best practices for metagenomics analyses by benchmarking tools. Interoperability between different tools will also be facilitated. Another focus will be encouraging data sharing and reuse and providing data storage platforms. Finally, links to existing ELIXIR initiatives in related areas as well as with international initiatives will be established. Overall, this is a very clear, concise, and informative review. The scope and aims of ELIXIR Microbiome are well-described and detailed. The short-term and long-term objectives are also clearly described.\nSpecific comments:\nI appreciate the links to other ELIXIR initiatives in Table 2. I would be particularly interested in more detail on the integration with the Systems Biology community. How would the ability to reuse multi-omics data for systems biology approaches be improved? It is a bit unclear to me which is the proposed data platforms, tools, and training will be freely available to non-ELIXIR members. Regarding providing metadata of samples, how will GDPR regulations be handled for human samples?\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/13-50
https://f1000research.com/articles/13-48/v1	08 Jan 24	{ "type": "Study Protocol", "title": "Evaluation for the efficacy of haemocoagulase agent (botroclot) as a pulpotomy material as compared to ferric sulfate- an animal study.", "authors": [ "Swati Karkare", "Nilima Thosar", "Nilima Thosar" ], "abstract": "Deciduous teeth are thought to be best space maintainers; thus efforts are made to preserve these teeth for as long as possible. Grossly carious primary teeth should be restored and maintained which is the primary objective of “Pediatric dentistry”. Pulpotomy is done in the primary teeth where mechanical pulp exposure occurs during caries removal. All the functions of the radicular pulp are expected to be maintained by pulpotomy procedure. The main objectives are 1) managing reversible pulpal inflammation and keeping pulp in a vital condition. 2) maintain the function of the radicular pulp in deciduous teeth. Success of Pulpotomy procedure is dependent on many factors, and the ability of the pulpotomy medicament being hemostatic is one of the most vital factors to maintain the vitality of the radicular pulp. Considering the limitations of commonly used pulpotomy medicaments for primary teeth i.e. Formocresol and also the disadvantages associated with the hemostatic Ferric sulfate, the present study will be carried out using hemocoagulase (“botroclot”) which has shown improved hemocoagulant properties in the medical field and for healing the extraction socket wounds. The available solution of hemocoagulase agent converted in suitable Gel form will be tested for its tissue compatibility and hemocoagulant efficacy when used as pulpotomy material . The study will be conducted in two phases, I) Formulation of the gel and in vitro tests; II) Animal study to check histopathological effect on the pulp tissue as a pulpotomy agent.", "keywords": [ "Pulpotomy", "Primary Teeth", "Ferric Sulfate", "Hemocoagulase agent", "Botroclot" ], "content": "Introduction\n\nDeciduous teeth are thought to be best space maintainers; thus, efforts are made to preserve these teeth for as long as possible. Grossly carious primary teeth should be restored and maintained which is the primary objective of “Paediatric dentistry”. During cavity preparation mechanical exposure may occur frequently. The choice of a direct pulp covering material has been controversial when optimum pulp healing is considered. Haemorrhage control must be achieved before any pulp capping procedure.1–3 All procedures are more important for maintaining pulp vitality. Many studies have shown that pulp healing is most compromised by uncontrolled bleeding. Sterile cotton pellet can be compressed on the exposure site until haemostasis occurs is the most common accepted clinical technique. Sometimes using cotton pellets soaked in sterile saline or other materials is also suggested. Other substitutes such as electro surgery or laser irradiations are not shown to be successful.1–6\n\nA pulpotomy is a dental procedure that treats grossly carious primary teeth in children. This type of procedure is carried out when tooth decay reaches the pulp or there is mechanical exposure while cavity cutting. During a pulpotomy, the coronal pulp is removed and the pulp in the root canal is left intact.1–3,6\n\nAll the functions of the radicular pulp are expected to be maintained by the pulpotomy procedure. The main objectives are managing reversible pulpal inflammation and keeping pulp in vital condition as well as maintaining the function of the radicular pulp in deciduous teeth. Many situations such as blood supply, amount of inflammation, hemorrhage control, exposure site disinfection, antibacterial efficacy and tissue compatibility of pulp covering materials and final restoration may affect the success of the procedure.4 The most important factor in success is maintaining the vital radicular pulp. Several materials have been used in deciduous teeth, Ferric sulphate being one hemocoagulant material tried and compared with other materials.4 Choosing the appropriate Pulpotomy agent is crucial.\n\nIdeal properties of pulpotomy material are as follows5\n\n• The material should be anti-bacterial, innocuous to pulp and surrounding structures.\n\n• The material should be able to cause advance repair of remaining pulp without interfering with the natural root resorption.\n\n• The material should not be toxic to pulp tissues.\n\nFerric sulphate is extensively used in dentistry as a haemostatic agent (Astringent). It was primarily used in pulpotomy as an aid to haemostasis prior to the placement of calcium hydroxide. However, as an independent therapeutic agent, ferric sulphate pulpotomy has a success rate of 74–99%. Ferric sulphate is thought to react with the pulp tissue, forming a superficial protective layer of iron–protein complex.2,3\n\nFerric Sulfate (FS) has agglutination effects due to a reaction between the blood in the pulp.1 This reaction results in plugs that occlude the capillaries, thus control the bleeding. However, a common side effect of Ferric sulfate is chronic pulpal degradation, which can result in premature exfoliation of the pulpotomized tooth. Some studies have documented internal root resorption after FS pulpotomy.2\n\nBotroclot is a marketed preparation containing hemocoagulase, which is an enzyme having coagulant activity, isolated from the snake botrops atrox. This formulation is used in dental surgeries and other minor surgical wounds. A commercially available hemocoagulant “botroclot” is used widely in Medicine for the stoppage of hemorrhage at wound sites. In dentistry it has been tested at extraction sites of third molars for its efficacy and compared histopathologically for its effects.3 A very small part of snake venom acquired from the snake found in Brazil “Bothrops-jararaca” or atrox which is a nontoxic systemic hemocoagulant (Botroclot). Available everywhere by various names such as Botroxobin which is a WHO accepted outcome and Botropase is a parenteral procoagulant. The formulation of “hemocoagulase agent (botroclot)” as a local application solution and its composition is (a) an aqueous mix of hemocoagulase extracted from “Bothrops atrox” or “Bothrops jararaca” 0.2 Cu/ml. (b) as a preservative “Chlorhexidine” 0.1% v/v and (c) for injection IPq.s water, as instructed in the composition booklet of the available solution bottle.\n\nThe blood clotting properties of hemocoagulase are multifaceted. The creation of fibrin monomers is hastened, then it accelerates fibrin clot creation. It triggers Xa factor and thrombin is formed at the hemorrhage site. Its action on Factor XIIIa brings about stabilization of fibrin. Articles have proposed that this parenteral formulation performs as a prohealer, epithelization is enhanced, escalates lesion tensile strength and brings collagen in the curing of the lesion too.6\n\nPulpotomy success is dependent on many factors, and the ability of the pulpotomy medicament being hemostatic is one of the most vital factors to improve success. Considering the limitations of commonly used pulpotomy medicaments for primary teeth i.e. Formocresol and the disadvantages associated with the hemostatic Ferric sulfate, a present study has thought of using hemocoagulase (“botroclot”) which has shown improved hemocoagulant properties in medical field and for healing of extraction socket wounds.6\n\nInternal resorption and furcation radiolucency was seen in pulpotomies performed with Ferric sulfate.7 Also the exact amount of the medicament to be placed in the pulp chamber cannot be standardized with presently available Ferric sulfate as it is available in solution form and it can be used with a cotton pellet dipped in it, where one can not quantify it. To overcome the short falls of these hemostatic pulpotomy agent, Hemocoagulase agent (“botroclot”) needs to be tested and verified as better hemostatic pulpotomy material in gel form for primary teeth.\n\nHemocoagulase agent (“botroclot”) has not been tried as a pulp therapy material either in primary or permanent teeth and its effect on the pulpal tissue has not been verified. It is a readily available, economic medicament that has been proved as a Heamocoagulant in other surgical fields needs to be tested in the concentration that is readily available or if it needs to be modified to make it suitable for Dental use as Pulpotomy agent.\n\nFerric sulfate is tested as a pulpotomy material as it shows hemostatic qualities. It produces a metal protein mass at the pulp surface that acts as a barricade to irritating constituents of the sub-base. Although Ferric sulfate pulpotomies exhibited superior clinical success rates, it may show moderate to severe inflammatory changes histologically as shown in studies.8 Ferric sulfate and laser have been tested for conservative pulp therapy to assess and histological changes were compared in the pulp.9 It was found that the materials produced significant extraneous body and cell reactions that are inflammatory in the pulpal tissue. At 45 days follow up of all the samples, two models of laser group showed severe necrosis. Lasers are not cost effective and there is no control on penetration of the laser, which may lead to destruction of normal tissue along with the affected tissue.9\n\nModern developments in dentistry are searching for more biocompatible and effective materials as the best medicament for primary teeth pulpotomies. There is limited scientific evidence which can illustrate the comparative efficacy of botroclot versus Ferric sulfate as an effective pulpotomy material. The present proposed research will be implemented to assess the efficacy of these two agents as pulpotomy materials. The available solution of hemocoagulase agent (“botroclot”) needs to be tested for its tissue compatibility and hemocoagulant efficacy when used as pulpotomy material specially for deciduous teeth in a more suitable form to be used as chairside.\n\nTo formulate haemocoagulase gel from botroclot solution and in vitro evaluation of pH, stability and adhesion of the new formulated gel.\n\nTo evaluate efficacy of hemocoagulase agent (“boteroclot”) compared to ferric sulfate histologically in rats molars.\n\n\n\n1. To evaluate the haemocoagulase gel for its pH, stability and adhesion by in vitro method in Phase I.\n\n2. To evaluate histologically, the efficacy of hemocoagulase agent (“botroclot”) and “ferric sulfate” at 24 hours, 7 days, 15 days and 30 days in rat molars in Phase II.\n\n3. To compare histologically, efficacy of hemocoagulase agent (“botroclot”) and “ferric sulfate” at 24 hours, 7 days, 15 days and 30 days in rat molars in Phase II.\n\nResearch Question\n\nPhase I: Is hemocoagulase agent (“botroclot”) effective and has favorable reaction on the rat molar pulp?\n\nPhase II: Is hemocoagulase agent (“botroclot”) effective and superior as pulpotomy material compared to ferric sulfate in human primary molars?\n\nProtocol\n\n\nMethods\n\nSuitable consistency material (gel) from readily available haemocoagulase agent (botroclot) solution will be formulated. The preparation will be carried out in the research laboratory using Hydroxypropyl methylcellulose (HPMC) as excipient to change the consistency of the solution to gel.\n\nThe weight quantity of HPMC will be dispersed accurately in purified water at 50° C. After that, 50 ml of haemocoagulase “botroclot” will be added and the mixture will be stirred continuously at a constant rate to form a uniform gel.10\n\nIn vitro tests of the prepared gel will be performed as follows.11\n\n1. pH: The pH of the sample will be measured by using a pH meter calibrated at pH 7 and pH 4.\n\n2. Viscosity: Viscosity of the sample will be measured by using a Brookfield viscometer, spindle no. 64 at 50rpm for HPMC gel at 24°C.\n\n3. Adhesive strength test:\n\ni. The bio adhesive test will be performed by using a modified two arm balance method.\n\nii. 0.5 gm of gel will be placed on the upper side of a lower plate which is fixed to base.\n\niii. Then the upper plate will be placed over lower plate and upper plate will be stuck to one side of the balance.\n\niv. 100 gm of preload (contact pressure) will be applied for 5 minutes.\n\nv. After removal of preload, weight will be added continuously in other side of the weighing balance till the plates will be detached from each other.\n\nvi. The weight required for the detachment of the glass plate will be considered as a bio adhesion force of applied gel.\n\n1 ml haemocoagulase solution will be equivalent to 0.2 CU (Clotting Unit)\n\nAbout 5 drops used for hemcoagulase action.\n\n1 drop = 0.06 ml, 5 drops = 0.3 ml\n\n0.3 ml = 0.06 CU. and 0.6 ml = 0.12 CU\n\nThe prepared gel formulation will have 50 ml of hemocoagulase solution. Upon adding HPMC, the weight of the gel would be around 55 gm (55 gm = 10 CU). To have effective hemocoagulase action, 0.5 mg to 1gm of gel will be used in the study.\n\nAnimal studies will be carried out with the newly developed material and it will be compared with ferric sulfate. The animals will be anesthetized and the pulpotomy procedure will be carried out in two molars of each rat under aseptic conditions. High speed handpiece will be used to access pulp chamber in maxillary and mandibular first molar. Upon pulp extirpation, haemocoagulase agent botroclot and ferric sulfate will be placed in teeth of the rats as per groups. Tissue reaction will be checked by histopathological evaluation at 24 hours, 7 days, 15 days and 30 days.\n\nThe Albino Wistar rats to be studied will be selected from the “Animal House, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra”. For animal study, total 32 albino Wistar rats having weight 200 – 300 grams will be chosen randomly. All the animals will be divided in 2 groups and each group will have 16 animals.\n\n\n\n1. In vitro evaluation of haemocoagulase agent (Botroclot) for pH, viscosity, adhesion and clotting.\n\n2. In vivo pulp tissue reaction evaluation in rats by histopathological evaluation.\n\nThere will be two groups for animal trials which are as follows:\n\nGroup A – Hemocoagulase agent “(botroclot)” preparation group (Test group).\n\nGroup B – Ferric sulfate group.\n\nThe animals will be cared as per CPCSA guidelines of ‘Animal Ethical Committee’.\n\nPulp tissue reaction to the pulpotomy materials will be assessed by in vivo and in vitro methods. The follow up period will be 24 hours, 7 days, 15 days and 30 days. The teeth will be extracted at these intervals and histopathological assessment of the pulp tissue will be done.\n\nThe data will be subjected to a statistical analysis by using “Student t test” by using SPSS version 21.0 “(SPSS. Inc., Chicago, IL, USA)” with a p value set at 0.05. The statistical analysis of preclinical study results will be done using Two way “ANOVA”, Student’s paired “t-test”, Unpaired t-test, Multiple Comparison: Tukey test and Kruksal-Wallis test. A proprietary free software alternative is R or PSPP.\n\nHemocoagulase agent (botroclot) in a newer form which may exhibit good coagulant efficacy and can be utilized as a “pulpotomy” material for primary teeth. Based on clinical experiences, topical hemocoagulase may be an effective haemostatic agent after tooth extraction procedures including pulpotomy, which also reduces pain, swelling and improves wound healing. However, more clinical trials are required to explore the benefits of topical hemocoagulase solution in various dental procedures.\n\nThe hemocoagulase may be equally beneficial as a local haemostatic agent compared to other chemical haemostatic agents like ferric sulfate. The other effective agent that is ferric sulfate is a commonly used astringent solution that has multiple uses in dentistry. It is widely used in dentistry as haemostatic agent. However, the evidence about its comparative effectiveness with heamogoacgulase as haemostatic agent in pulpotomy procedure has not been widely illustrated in the existing literature.\n\nAnimal study on going in vitro component completed.\n\nThe research Protocol is approved by institutional animal ethics committee of the respective institute.\n\nThe approval number is DMIMS/IAEC/2020-21/41.", "appendix": "Data availability\n\nNo data is associated with this article.\n\n\nReferences\n\nBandi M, Mallineni SK, Nuvvula S: Clinical applications of ferric sulfate in dentistry: A narrative review. J. Conserv. Dent. 2017; 20(4): 278–281. PubMed Abstract \| Publisher Full Text\n\nAbirami K, Ramkumar H, Senthil D: Clinical and Radiographic Evaluation of the Efficacy of Formocresol, Allium sativum Oil, and Aloe barbadensis Gel as Pulpotomy Medicaments in Primary Molars: A Randomized Controlled Trial. Int. J. Clin. Pediatr. Dent. 2020 Oct; 13(5): 518–522. PubMed Abstract \| Publisher Full Text\n\nGupta G, Muthusekhar RM, Kumar SP: Efficacy of Hemocoagulase as a Topical Hemostatic Agent After Dental Extractions: A Systematic Review. Cureus. 2018 Mar 30; 10(3): e2398. PubMed Abstract \| Publisher Full Text\n\nOlczak-Kowalczyk D, Samul M, Góra J, et al.: Ferric Sulfate and Formocresol pulpotomies in paediatric dental practice. A prospective-retrospective study. Eur. J. Paediatr. Dent. 2019 Mar; 20(1): 27–32. PubMed Abstract \| Publisher Full Text\n\nTaha NA, Abdelkhader SZ: Outcome of full pulpotomy using Biodentine in adult patients with symptoms indicative of irreversible pulpitis. Int. Endod. J. 2018 Aug; 51(8): 819–828. PubMed Abstract \| Publisher Full Text\n\nShenoy K. V, Baliga M, Mahajan S, et al.: The Effects of Topical Hemocoagulase Solution on the Healing Process of Post-extraction Wounds: A Split Mouth Design. J. Maxillofac. Oral Surg. 2015 Sep; 14(3): 586–593. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmaïl-Faugeron V, Glenny AM, Courson F, et al.: Pulp treatment for extensive decay in primary teeth. Cochrane Database Syst. Rev. 2018 May 31; 2018: CD003220. Publisher Full Text\n\nSirohi K, Marwaha M, Gupta A, et al.: Comparison of Clinical and Radiographic Success Rates of Pulpotomy in Primary Molars using Ferric Sulfate and Bioactive Tricalcium Silicate Cement: An in vivo Study. Int. J. Clin. Pediatr. Dent. 2017 Jun; 10(2): 147–151. PubMed Abstract \| Publisher Full Text\n\nSivadas S, Rao A, Natarajan S, et al.: Pulpal Response to Ferric Sulfate and Diode Laser When Used as Pulpotomy Agent: An in vivo Study. J. Clin. Diagn. Res. 2017 Jun; 11(6): ZC87–ZC91. PubMed Abstract \| Publisher Full Text\n\nRoy BG: Hydroxy Propyl Methyl Cellulose: Different Aspects in Drug Delivery. J. Pharm. Pharmacol. 2016 Aug 3; 4: 381–385. Publisher Full Text\n\nSingh S, Parhi R, Garg A: Formulation of topical bioadhesive gel of aceclofenac using 3-level factorial design. Iran. J. Pharm. Res. 2011; 10(3): 435–445. PubMed Abstract" }	[ { "id": "245723", "date": "02 Sep 2024", "name": "Ruchi Singhal", "expertise": [ "Reviewer Expertise Pediatric Dentistry with special focus on preventive aspect and traumatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 needs changes grammatically and at many places need rephrasing of sentences.\nIntroduction: Instead of writing grossly carious, authors should use the term teeth which are pulpally involved. Authors have mentioned, \"However, a common side effect of Ferric sulfate is chronic pulpal degradation, which can result in premature exfoliation of the pulpotomized tooth.\" Provide reference Dose calculation: How the dose was calculated. Any standard criteria or reference In phase II, it was mentioned that Tissue reaction will be checked by histopathological evaluation at 24 hours, 7 days, 15 days and 30 days. At what duration after pulpotomy, the tooth was extracted..clearly describe in detail.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\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": "12428", "date": "26 Sep 2024", "name": "Dr. Swati Karkare", "role": "Author Response", "response": "Answering to the comments by the reviewer as the main Author. 1) The term \"Grossly carious \" is used since pulpotomy is not indicated in teeth where the carious process has already involved the pulp. In that case one has to carry out Pulpectomy as one can not confirm the involvement of pulp damage if restricted to only coronal portion of the pulp. Very commonly, in grossly carious tooth, while doing excavation if the pulp is exposed traumatically, then Pulpotomy procedure is carried out. 2) The reference to the sentence is at the end of the last sentence of the paragraph, which is reference number 2. 3) Dose calculation is done as per Pharmaceutical guidelines where the solution of Botroclot was mixed with HPMC and at three different concentrations the efficacy was tested. 4) The tissue reaction was checked after extracting the teeth at 24 hr, 7 days, 15 days and 30 days post Pulpotomy, when the teeth were extracted. The follow up time mentioned is for extraction. After that the teeth were processed for histopathological evaluation." } ] } ]	1	https://f1000research.com/articles/13-48
https://f1000research.com/articles/13-47/v1	08 Jan 24	{ "type": "Research Article", "title": "Pregnant women's adherence to COVID-19 prevention practice and associated factors in Jinka Town, South Omo Zone, Southern Ethiopia, a community based cross-sectional study", "authors": [ "Molalegn Mesele", "Getachew Asmare", "Gizachew Ambaw", "Misganaw Asmamaw", "Mohammed Abdu", "Endeshaw Chekol", "Denekew Tenaw", "Sefineh Fenta", "Tadesse Asmamaw", "Melkamu Aderajew", "Anteneh Mengist", "Yenealem Solomon", "Berihun Bantie", "Wubet Alebachew", "Natnael Amare", "Ermias Sisay", "Natnael Moges", "Natnael Atnafu", "Getachew Asmare", "Gizachew Ambaw", "Misganaw Asmamaw", "Mohammed Abdu", "Endeshaw Chekol", "Denekew Tenaw", "Sefineh Fenta", "Tadesse Asmamaw", "Melkamu Aderajew", "Anteneh Mengist", "Yenealem Solomon", "Berihun Bantie", "Wubet Alebachew", "Natnael Amare", "Ermias Sisay", "Natnael Moges", "Natnael Atnafu" ], "abstract": "Background Pregnancy-related coronavirus disease 2019 (COVID-19) transmission and infection rates have continued to be a top public health concern. Pregnant mothers also have a higher risk of developing serious illness, needing to be admitted to an intensive care unit, and needing mechanical ventilation. More than six million people died as a result of COVID-19 worldwide. The aim of this study was to assess the level of adherence to COVID-19 preventive practice among pregnant mothers in Jinka Town.\n\nMethods In Jinka, South Omo Zone, between January 1 and January 30, 2023, a cross-sectional study was conducted. Using a simple random sampling technique, 412 pregnant women were selected. Data were collected using a face-to-face interviewer-administered, pre-tested questionnaire. The data were entered using Epi-data version 3.1, and after being exported, were analyzed using SPSS version 25. Bivariate and multivariable logistic regression analysis was carried out to identify relevant components. An adjusted odds ratio and a 95% confidence interval were used to report the relationship between the covariates and the outcome variable.\n\nResults The percentage of participants who adhered to COVID-19 prevention measures was only 11.4% (95% CI: 8.5, 14.8). Mothers with at least a bachelor’s degree 3.71 (AOR=3.71, 95% CI=1.28, 10.73), a residency of 4.40 (AOR=4.40, 95% CI=1.96, 9.87), and a history of chronic illness of 4.30 (AOR=4.30, 95% CI=1.69, 10.93) were significantly associated with good adherence to COVID-19 prevention practices.\n\nConclusions Only a very small percentage of pregnant women, according to this poll, adhere to COVID-19 preventative practices. In order to raise pregnant women’s awareness of COVID-19 preventive practices, it is crucial to use a variety of media for health education. Further, it is preferable to focus on urban living and give special attention to women without a formal education.", "keywords": [ "Adherence", "COVID-19", "pregnant mothers", "Jinka Town", "Ethiopia" ], "content": "Introduction\n\nA contagious illness called coronavirus disease 2019 (COVID-19) is brought on by the SARS-CoV-2 virus, which first appeared in Wuhan, China.1 On January 30, 2020, and March 11, 2020, respectively, the World Health Organization (WHO) proclaimed the 2019–2020 COVID–19 outbreaks to be Public Health Emergencies of International Concern (PHEIC) and pandemics, respectively.2,3 There are primarily two ways to spread COVID-19: directly and indirectly other bodily fluids and secretions, including feces, saliva, urine, semen, and tears, also contribute to the direct route of transmission. The term “aerosols” refers to particles produced during surgical and dental procedures as well as particles from respiratory droplets.4\n\nBy July 26, 2022, it had resulted in 568,773,510 confirmed cases of COVID-19 and 6,381,643 fatalities globally. On the African continent, similar figures of 9,192,139 confirmed cases and 173,946 fatalities were recorded. Ethiopia had 491,759 confirmed COVID-19 cases as of July 26, 2022, and 7,566 deaths had been reported. At the beginning of March 2021, it was reported that pregnant women infected with the virus had more than 73,600 illnesses, with 80 maternal deaths.5 As of the 6th of October, 2021, 1,637 infections caused by SARS-CoV-2 during pregnancy and 15 fatalities have been reported in Mississippi.6 Women are more likely than men to contract serious respiratory illnesses like SARS-CoV2 and respiratory syncytial virus (RSV) during pregnancy due to immunologic and physiological changes connected to the condition.7,8\n\nIt recognized that some of the major causes of morbidity and mortality could be associated with health determinants linked to an individual’s health behaviour, such as the adoption of health behaviour against virus transmission (e.g., hand washing and wearing a mask outside) and the avoidance of health-harming behaviours (e.g., touching the face and assembling for events).9 Evidence suggests that COVID-19-infected pregnant women experienced unfavorable maternal and perinatal outcomes include preterm birth, low birth weight, severe sickness, hospitalization to an intensive care unit, mechanical ventilation, and death.10–12 A study found that during pregnancy, COVID-19 can be passed directly from the mother to the fetus.13 Additionally, studies have revealed that domestic violence, postpartum anxiety, and depression are more prevalent in pregnant women during pandemics.14 Although there have been no clinical trials for the COVID-19 vaccine that have included pregnant participants, the American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine encourage pregnant women to receive the COVID-19 vaccine if they desire to be immunized.15,16\n\nAdditionally, studies have shown that diets high in vitamins and fiber can boost immunity and protect COVID-19 infected women from adverse pregnancy outcomes.17,18 The Ethiopian government has also taken part in a variety of initiatives to prevent the virus from spreading throughout the nation, including community mobilization, raising public awareness, isolation, mandatory quarantine and treatment, strict passenger screening, house to house screening, and expanding diagnostic tests and treatment facilities.19\n\nAlthough COVID-19 is becoming more widespread every day in Ethiopia, pregnant women cannot get the vaccine. Therefore, in order to reduce their risk of contracting the virus, pregnant women have to follow the prophylactic guidelines established by the Ministry of Health (MoH). This lowers morbidity and death among pregnant women and newborns.20 There aren’t many studies in Ethiopia that focus on the extremely vulnerable pregnant women, especially in the study area. The purpose of this study was to determine how well pregnant women in Jinka Town, South Omo Zone, Southern Ethiopia, adhered to COVID-19 preventative practices and associated factors.\n\n\nMethods\n\nThe Wolaita Sodo University College of Health Science and Medicine’s Ethical Review Board granted ethical approval on December 10/2022.\n\nFinally, a formal letter of permission from the Jinka town Health Office was obtained in order to move forward with the data collecting. Participation was only permitted after receiving informed written consent. To enable thorough and sincere self-disclosure, we kept voluntary engagement and confidentiality.\n\nA cross-sectional study of pregnant women in Jinka, South Omo Zone, SNNPR, Ethiopia, was conducted in the community from January 1 to January 30, 2023. The administrative town of Jinka is located at 5°47′N 36°34′E/5.783°N 36.567°E latitude and longitude, 560 km from Hawassa and 750 km from Addis Abeba. The town acts as the South Omo Zone’s administrative hub in the Southern Nations, Nationalities and Peoples’ Region. In 2018, 30493 people were expected to live there, of whom 15,217 (49.9%) were males and 15,276 (50.1%) were women. The reproductive age group included 7,103 (23.3%) of the total females.21 There is one general hospital, two health centers, and six health posts in Jinka Town\n\nAll pregnant mothers who lived in Jinka town were the source population and all selected pregnant mothers in Jinka town was the study population. Pregnant mothers who lived for at least six months and more in Jinka town were eligible to be included in this study.\n\nThe sample size was determined by using a single population proportion formula by considering a proportion of good adherence 44.8%,20 level of confidence 95%, margin of error 5%. Thus, the sample size was\n\nAfter considering a non-response rate of 10% the final sample size was 417.\n\nHealth extension workers work and give primary health care in the community by arranging and using family folder; all families in each kebele have their own family folder which contains a list of family members with the serial number, age, sex, marital status, educational status, under five, under one, pregnant mother, antenatal care (ANC) follow-up, non-communicable disease, HIV status and tuberculosis screening which is updated every year and helps as a source of information for each resident in each kebele. A simple random sampling technique was used to choose household from six kebeles in Jinka town utilizing an updated list from the kebeles administrative office. Then a basic random sampling approach created by a computer was used. According to the proportional to size allocation method, the likelihood that women were chosen depended on how many pregnant women were in the selected kebele. When more than one candidate was located in a household, a lottery procedure was used. When the interviewee was absent on the day of data collection, revisiting the interviewee helped to lower the frequency of nonresponses.\n\nThe dependent variable was adherence to the COVID-19 preventive practice, and the independent variables were the mother’s age, religion, educational level, occupation, marital status, husband’s educational level, occupation, number of families residing in the same place, average monthly income, gravidity, number of living children, having an ANC visit, number of ANC visits, pregnancy condition, history of prior bad pregnancy outcomes, history of chronic illness, and knowledge of the COVID-19\n\nAdherence to COVID-19 preventive practice: Seven questions on recent practices were asked to participants and were based on a WHO recommendation for COVID-19 prevention methods.20 One point was awarded for a proper response, while zero was awarded for a mistaken one. If a participant obtained a score of above the mean or four or more on the usage of preventive measures, they were considered to have good adherence to COVID-19 preventative practice. If participants’ scores fell below the mean or less than four, they were considered to have insufficient adherence to COVID-19 preventative practice.20–23\n\nBy studying pertinent literature, the data gathering technique was created.23,24 Through a face-to-face interview during a home visit, data were gathered using semi-structured, pretested questions. The survey asks about socio-demographic traits, obstetric-associated variables, and knowledge of and preventive behavior related to COVID-19. For the purposes of data collection and monitoring, five BSc midwives and one MSc clinical midwife, respectively, were hired.\n\nWith the help of linguists, the questionnaire25 was written in English, translated into the Amharic-speaking region’s language, and then translated again into English to preserve the tool’s consistency. In order to assess the questionnaire’s response, language clarity, and appropriateness prior to the actual data collection, 5% of the total sample size was pretested in the Karat town district. The goal of the study, the components of the tool, and the data collection methodologies were covered in a one-day training session for the supervisor and data collectors. Additionally, they were made aware of the critical safety measures that needed to be done to avoid contracting COVID-19. Data collectors were observed for any issues during data collecting. The data collectors and supervisor verified the consistency and completeness of the data, and the incomplete data were noted.\n\nThe data were validated, coded, and entered in Epi data version 4.6 before being exported for analysis to SPSS version 25 (RRID:SCR_002865). Descriptive statistics, such as frequencies, percentages, means, and standard deviation, were computed. Bivariable logistic regression was used to establish the relationship between the independent and dependent variables. Then, variables with p-values less than 0.2 were included in the multivariable logistic regression analysis.\n\n\nResult\n\nIn the current study, a total of 412 women from the study were included in the analysis, yielding a response rate of 98.8%. Of the responders, 164 (30.9%) were between the ages of 18 and 24. The majority of study participants (66.4%) identified as Orthodox Christians. According to information about participants’ marital status, 94% of the study them were married. Almost a third (31.5%) of their husbands had attended secondary school, according to their husband’s educational background (Table 1).\n\nThe majority of the participants in the study, 370 (89.8%), were multigravida in their gravidity and more than half of the participants, 254 (61.7%), had planned pregnancies. A majority of the individuals, 362 (87.9%), had ANC follow-up (Table 2).\n\nAlmost all of the respondents (99.3%) in the study as a whole had heard of COVID-19. More than half of pregnant women said that a headache (61.7%) and runny nose (59.2%) are a symptom of COVID-19. From the total participants, 320 (77.7%) pregnant women were aware that pregnant women are more susceptible to COVID-19 than other groups (Figure 1).\n\nThe results of this study revealed that only 11.4% (95% CI: 8.5, 14.8) of the participants had good adherence to COVID-19 preventive practises (Figure 2).\n\nLess than half (43%) of participants washed their hands with water and soap or with an alcohol-based sanitizer and only 9.7% of participants wore a face mask in public to reduce the spread of infection, respectively. From the total respondents, only 103 (25%) cover their mouth and nose during coughing and sneezing (Table 3).\n\nIn the examination of bi-variables, there was a significant relationship between mother’s educational status, communication media, residence, husband’s educational status, gravidity, history of prior bad pregnancy outcome, and history of chronic illness, with a p-value of less than 0.2. Multivariable logistic regression revealed that the mother’s educational level, communication media, residence, and history of chronic illness were statistically significant factors in adherence with COVID-19 preventative practice. Mothers with an educational status of college or higher were 3.71 (AOR=3.71, 95% CI=1.28, 10.73) times more likely to follow COVID-19 preventive practices than individuals whose educational status was limited to not being able to read or write. Another element that was strongly connected with strong adherence was communication media. Mothers who used communication media had a 4.33 (AOR=4.33, 95% CI=1.54, 12.15) times higher likelihood of adhering to COVID-19 preventative practices than those who did not. Mothers in rural regions were 4.40 (AOR=4.40, 95% CI=1.96, 9.87) times more likely to follow COVID-19 preventive practices than mothers in urban areas.\n\nAnother significant factor which was strongly associated with good adherence to COVID-19 preventative practices was history of chronic illness. Mothers with a chronic disease were 4.30 times more likely to follow COVID-19 preventative practices (AOR=4.30, 95% CI=1.69, 10.93) than mothers without a chronic illness (Table 4).\n\n* Significant association at P-value <0.05.\n\n\nDiscussion\n\nThis study sought to determine whether pregnant women in Jinka Town, South Omo Zone, Southern Ethiopia, 2023, adhered to COVID-19 preventative practices and associated related factors in 2023. The current study showed that only 11.4% (95% CI: 8.5, 14.8) had good adherence with COVID-19 preventive practice. This finding is lower than studies conducted in Gurage zone 76.2%,22 in Northern Ghana 46.6%,26 in Nigeria 78%,27 Gondar city 44.8%,20 and a study conduct Debre Berhan Town 56.1%.28\n\nThe disparity in the study setting, the variation in the outcome variable used to measure the outcome, and the socio-demographic features of the study participants are all potential reasons of this discrepancy. Each of the aforementioned research was a cross-sectional institution-based study. Time differences could also be the reason for the discrepancy. The majority of the population is presently attempting to adjust to and cope with COVID-19.\n\nThe study also found that women with college degrees or higher were 3.71 times more likely than women who couldn’t read and write to follow COVID-19 preventive practices. The conclusion made in the current article was supported by studies conducted in Gondar,20 Debre Tabor, Ethiopia24 and Ghana.26 This may be due to the fact that educated people have easier access to knowledge about COVID-19 and its preventative strategy than those who are illiterate. Additionally, educated people might be better aware of the dangers of forgoing this preventive precaution as well as the preventive measures themselves.\n\nThe existence of mass media was another factor that was connected to good adherence. Participants with media access were 4.33 times more likely than those without to adhere well to COVID-19 preventive measures. The majority of the time in Ethiopia COVID-19 prevention strategies were promoted through the mass media. Pregnant women with communication media (mass media) might be more aware than those who have no communication media because they get information related to how to prevent COVID-19 through media.\n\nRural residents were more likely to practice COVID-19 preventative actions than urban dwellers. According to a study29 conducted in a low-resource African nation, rural populations were more likely to put COVID-19 prevention measures into practice. A study conducted in Guraghe Zone Hospitals22 indicated that urban residents were more likely than rural ones to practice COVID-19 preventive measures, which contrasted with the findings of the present study.\n\nThis may be because individuals in rural areas are more accustomed to hearing and applying government and health professional advice than people in urban areas, which enables them to take better COVID-19 prevention measures.\n\nAnother significant factor which was strongly associated with good adherence to COVID-19 preventative practices was history of chronic illness. Mothers with a chronic disease were 4.30 times more likely to follow COVID-19 preventative practices (AOR=4.30, 95% CI=1.69, 10.93) than mothers without a chronic illness. Because they feel they should, people with COVID-19 co-morbidities may be more cautious than people without co-morbidities.\n\nConclusions regarding the causes and effects of pregnant women adhering to COVID-19 preventative practises and the associated factors may be challenging given the cross-sectional nature of the study. There may also be a predisposition towards social desirability.\n\n\nConclusion\n\nAccording to this survey, only a very small percentage of pregnant women follow COVID-19 preventive practises. The mother’s level of education, her use of communication tools, her residency, and her history of chronic illness all had an impact on how well she followed COVID-19 preventive practise. As a result, it’s critical to improve pregnant women’s health education about COVID-19 preventive practises through a variety of media. Additionally, by emphasising urban residency and giving women without formal education special consideration.", "appendix": "Data availability\n\nFigshare: DATASET. https://doi.org/10.6084/m9.figshare.24147465.v1. 30\n\nFigshare: Questionar.docx. https://doi.org/10.6084/m9.figshare.23796720. 25\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe would like to express our heartfelt thanks for Wolaita Sodo University, all individual for their contribution and cooperation.\n\n\nReferences\n\nKhan S, Siddique R, Bai Q, et al.: Coronaviruses disease 2019 (COVID-19): Causative agent, mental health concerns, and potential management options. J. Infect. Public Health. 2020; 13(12): 1840–1844. PubMed Abstract\n\nZhu N, Zhang D, Wang W, et al.: A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 2020; 382(8): 727–733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCucinotta D, Vanelli M: WHO declares COVID-19 a pandemic. Acta Bio-medica Atenei Parm. 2020; 91: 157–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations.2020\n\nGray KJ, Bordt Evan A, Atyeo C, et al.: Coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study.2021; 225: 303.e1–303.e17.\n\nKasehagen L, Byers P, Taylor K, et al.: COVID-19 –Associated Deaths After SARS-CoV-2 Infection During Pregnancy—Mississippi, March 1, 2020–October 6, 2021. MMWR Morb. Mortal. Wkly Rep. 2021; 70(47): 1646–1648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShereen MA, Khan S, Kazmi A, et al.: COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses. J. Adv. Res. 2020; 24: 91–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManti S, Leonardi S, Rezaee F, et al.: Effects of Vertical Transmission of Respiratory Viruses to the Offspring. Front. Immunol. 2022; 13: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Lancet Respiratory M: COVID-19: delay, mitigate, and communicate. Lancet. Respir. Med. 2020; 8(4): 321. Publisher Full Text\n\nCenters for Disease Control and Prevention: Investigating the Impact of COVID-19 during Pregnancy.2021; pp. 1–2.\n\nPathirathna ML, Samarasekara BPP, Dasanayake TS, et al.: Adverse Perinatal Outcomes in COVID-19 Infected Pregnant Women: A Systematic Review and Meta-Analysis. Healthcare (Basel). 2022; 10(2). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshraf MA, Keshavarz P, Hosseinpour PE, et al.: Coronavirus disease 2019 (COVID-19): A systematic review of pregnancy and the possibility of vertical transmission. J. Reprod. Infertil. 2020; 21(3): 157–168. PubMed Abstract\n\nCiarambino T, Crispino P, Giordano M: Viral Infection, COVID-19 in Pregnancy and Lactating Women: What Is Known? Covid. 2021; 1(1): 230–245. Publisher Full Text\n\nRCM\|Royal college of Midwives and the RCOG\|Royal college of Obstetricians & Gynecologists. Coronavirus (COVID-19) Infection in Pregnancy:Information for healthcare professionals Version 11: Published Friday 24 July 2020.2020; (July): 1–68.\n\nSociety for Maternal-Fetal Medicine (SMFM) Statement: SARS-CoV-2 Vaccination in Pregnancy.2020; 1–3.\n\nRiley LE, Beigi R, Jamieson DJ, et al.: Vaccinating Pregnant and Lactating Patients Against COVID-19 Summary of Key Information and Recommendations COVID-19 Infection Risk in Pregnancy COVID-19 Vaccines in Development. Am Coll Obstet Gynecol. 2021; 19: 1–20.\n\nRishi P, Thakur K, Vij S, et al.: Diet, Gut Microbiota and COVID-19. Indian J. Microbiol. 2020 Dec; 60(4): 420–429. PubMed Abstract \| Publisher Full Text\n\nNawsherwan KS, Zeb F, Shoaib M, et al.: Selected Micronutrients: An Option to Boost Immunity against COVID-19 and Prevent Adverse Pregnancy Outcomes in Pregnant Women: A Narrative Review. Iran. J. Public Health. 2020; 49: 2032–2043. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaye K: COVID-19 prevention measures in Ethiopia: current realities and prospects.2020; 14.\n\nTemesgan WZ, Aklil MB, Yacob HS, et al.: Adherence to COVID-19 preventive practice and associated factors among pregnant women in Gondar city, northwest Ethiopia, 2021: Community-based cross-sectional study. PLoS One. 2022; 17(3): e0264237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCentral Statistical Agency MPC: Ethiopia-population andHousing Census of 2007. Delhi, India: Central Statistical Agency-Ministryof Finance and Economic Development; 2007.\n\nFikadu Y, Yeshaneh A: COVID-19 Preventive Measure Practices and Knowledge of Pregnant Women in Guraghe Zone Hospitals.2021; 13: 39–50.\n\nAbeya SG, Barkesa SB, Sadi CG, et al.: Adherence to COVID-19 preventive measures and associated factors in Oromia regional state of Ethiopia. PLoS One. 2021; 16(10): e0257373. PubMed Abstract \| Publisher Full Text\n\nAyele AD, Mihretie GN, Belay HG, et al.: Knowledge and practice to prevent COVID-19 and its associated factors among pregnant women in Debre Tabor Town Northwe Ethiopia, a community-based cross-sectional study. BMC Pregnancy Childbirth. 2021; 21(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMesele M: Questionar.docx. figshare. 2023. Publisher Full Text\n\nKumbeni MT, Apanga PA: Knowledge and preventive practices towards COVID-19 among pregnant women seeking antenatal services in Northern Ghana. PLoS One. 2021; 16(6): e0253446.\n\nAfolabi AO: Adherence to COVID-19 Preventive measures among Pregnant Women in Nigeria: An Initiative towards Safe Motherhood in an Emerging Global Health Priority. J. Midwifery Reprod. Health. 2021; 10(1).\n\nSilesh M, Demisse TL, Taye BT, et al.: Compliance with COVID-19 Preventive Measures and Associated Factors Among Women Attending Antenatal Care at Public Health Facilities of Debre Berhan Town, Ethiopia. Risk Manag. Healthc. Policy. 2021; 14: 4561–4569. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNwafor JI, Aniukwu JK, OkechukwuAnozie B, et al.: Knowledge and practice of preventive measures against COVID-19 infection among pregnant women in a low-resource African setting.2020.\n\nMesele M: figshare. Dataset. 2023. Publisher Full Text" }	[ { "id": "260887", "date": "16 Apr 2024", "name": "Yeon Mi Hwang", "expertise": [ "Reviewer Expertise Pregnancy", "Electronic Health Record", "Retrospective Studies", "COVID-19", "Medication use" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall - please correct typos and grammatical errors - some of the sentences are awkward. - many of the sentences are not justified and unclear. please provide a rationale or relevant information ex) prior literature, result, method, etc Abstract Background - provide background related to the study. ex) lack of study on adherence to COVID-19 preventive practice, lack of local study Methods - What was the size of the source population? - awkward sentences - what is the outcome variable? please specify - how do you define the adherence\n\nConclusions - grammars - Your conclusion is not supported by the abstract results. you did not describe anything about the media in abstract results\n\nIntroduction - please provide more recent and relevant studies and numbers (more recent # in Africa) - second paragraph last sentence. it does not make sense to compare pregnant women and men. please compare pregnant women and women - recommend taking out the first sentence of the fourth paragraph. - don't use contractions in academic papers - fifth paragraph. is it recommended to not get the vaccine in Ethiopia? if so, this is very unusual so please cite this guideline Methods - make sure you spell our acronym in the first use - typo. addis abeba - define the reproductive age group. - provide cohort selection figure to guide the readers. source population -> study population -> sample (sampling procedure) - define all the variables in the supplementary table. whether it is a continuous variable, categorical variable, or binary variable. and if it is a categorical variable please provide all the choices. ex) educational level, religion, etc - define recent - linguist? are you referring to translator? - please provide how you impute the missing value - please provide your rationale and justification in including variables with p-values less than 0.2. how this threshold was selected Results - mention about the statistically significant ones in the abstract - define pregnancy-related terminologies in the supplementary table. F1000 is not a pregnancy journal ex) gravidity, multigravida, etc - verb tense agreement and subject-verb agreement - cumulative? - please correct how you report the number in the table ex) >3, ≤3 - Figure 1. provide more information in the caption. some of the sentences are not fully written. - please refer to table 4 for 'factors affecting adherence to COVID-19 preventive practice' section - definition of chronic illness? how did you determine - Table 4. what is AOR make sure you provide what your acronym means in the caption - did you correct for multiple testing error? Discussion - second paragraph. specify one of the difference. ex) time difference and provide/describe example study. how different they are + what are the difference in the result - lack of flow in the sentence. - your paragraph. \"This may be because individuals~\" is insufficient. why are rural areas more accustomed to hear and apply government and health professional advice - you paragraph \"another significant factor which was strongly associated ~\" provide more support. - limitation of the study. list more limitations. ex) sampling bias. - also provide strengths of your study. Conclusion - please make it more clear\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "242566", "date": "11 May 2024", "name": "Feleke Hailemichael Astawesegn", "expertise": [ "Reviewer Expertise Health services research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle\n\nI recommend you rewrite the title as \"COVID-19 prevention practice adherence and its associated factors among pregnant women in Jinka Town, South Omo Zone, Southern Ethiopia: A community-based cross-sectional study\"\nIntroduction\nCould you have some evidence about women's preventive practice adherence gap from the literature to justify the need for conducting this research?\n\nResult\n\nAs much as possible reduce unnecessary figures and tables, for example, you can merge obstetric and knowledge-related factors in one table or figure or even you can describe them in a word.\n\nIs the work 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/13-47
https://f1000research.com/articles/12-821/v1	13 Jul 23	{ "type": "Research Article", "title": "Comparison of crestal bone loss and papilla fill after conventional and immediate implant placement: A 12 month clinical and radiographic prospective study", "authors": [ "Akanksha Raj", "Sweta Pradhan", "Preetha Shetty", "David Kadakampally", "Neetha Shetty", "Akanksha Raj", "Sweta Pradhan", "Preetha Shetty", "David Kadakampally" ], "abstract": "Background: The problem of missing teeth persists in all age groups. The main objective of implants in dentistry is to provide a restoration that reconstructs the shape and restores esthetics and functions of edentulous areas. The objectives of this study are to compare the crestal bone level changes and papillary fill after placement of implants in fresh extraction socket, i.e. immediate implant placement, and healed extraction socket, i.e. delayed or conventional implant placement, and to assess other clinical parameters such as modified plaque index (mPI), modified gingival index (mGI) and gingival biotype in between the groups and within the\n\ngroups. Methods: 18 patients were recruited in the study out of which 9 patients received implants as per immediate implant placement protocol (group 1) and 9 patients received implants as per conventional implant placement protocol (group 2). All patients were evaluated for gingival biotype, mPI and mGI and papillary fill was assessed as per Jemt’s papilla score as clinical parameters. Implant site was assessed for radiographic bone loss using Image J software. Statistical analysis was performed using independent t test, paired t test and chi square test. Results: At the end of 1 year, results showed that crestal bone loss was seen more in the immediate group than the conventional group. Conventional implants showed better papillary fill than implants placed in fresh extraction sockets. Plaque scores were assessed as per modified plaque index, which showed better results in the conventional group. Modified gingival index was used to assess gingival status which showed better results in the immediate group one year later. Conclusions: Findings from the study suggest that crestal bone loss was found to be increased in the immediate group than the conventional group and papillary fill was better in the conventional group than the immediate group. Registration: CTRI (CTRI/2019/09/021340).", "keywords": [ "Crestal bone", "Immediate implants", "Conventional Implants", "Papillary fill." ], "content": "Introduction\n\nThe loss of a single tooth is thought to be a typical esthetic issue that could have psychological effects as well as non-physiological occlusion, that results due to tipping of the neighboring tooth and supra-eruption of the opposing tooth.1,2 The main objective of implant therapy or modality is to provide a restoration that not only reconstructs the shape but also the esthetics and functions of edentulous areas. Clinical replacement of missing natural teeth with osseointegrated implants has been referred as one of the most notable developments in dentistry.3,4\n\nPlacement of a single tooth implant can either be done in healed extraction socket/site as a conventional or delayed implant, or can be done in a fresh extraction socket/site as an immediate implant procedure.5,6 Conventionally, placement of a single tooth implant is done in the healed extraction site so as to allow the process of osseointegration to take place which generally takes three to six months. Immediate implants are now being used to achieve optimum esthetic appearance with reduced time of treatment. In this procedure, after creating the implant bed, implants can be placed right away into fresh extraction sockets to establish primary stability. This method is favorable because it helps to preserve alveolar bone, achieves optimum axial positioning of the implant by using the socket as a reference, shortens the edentulous period of three to six months, and requires fewer surgical visits.6‐15\n\n\nMethods\n\nThis clinical trial was approved by the Institutional Ethics Committee, Manipal College of Dental Sciences, Mangaluru (ref no. 18120). Initial approval was received on 20th October 2018 and finalised on 12th November 2018. Participants provided signed informed consent prior to the start of the study. This trial is also registered with CTRI (CTRI/2019/09/021340). Due to some technical issues with the initial registry, the trial was registered retrospectively. All procedures were conducted in compliance with the Helsinki Declaration 1975.\n\nStudy participants who reported to the Department of Periodontology, Manipal College of Dental Sciences, Mangalore with the chief complaint of a missing single tooth were selected as participants in this study. The study was conducted from 1st November 2018 to 1st December 2020. Before the commencement of the study, the patients were informed of the clinical trial design and were required to sign their informed consent.\n\n\n\nUsing the above formula, the sample size was calculated. Where Z (1-α/2) = Z score for the α power chosen. Z (1-β) = Z score for the power chosen. σ1 = standard deviation of group-1\n\nσ = average standard deviation. d = the minimum difference between in the values with which make clinically relevant impact. With 5% alpha error, 90% power of the study and a clinically significant difference of 0.5 units, the required sample in each group is 9.\n\nThe investigator (DK) was in charge of enrolling participants and allocating surgical procedures. The coin toss approach was used for randomization. The lead investigator (AR) performed all surgical operations. A masked investigator (NS) assessed the patients during the recall period and was blinded to all the surgical treatments that had been assigned.\n\n\n\n1. Age group of 18 to 55 years.\n\n2. Systemically and periodontally healthy individuals.\n\n3. Non-smokers.\n\n4. Good oral hygiene.\n\n5. Availability of follow up.\n\n6. Healthy remaining dentition.\n\n7. Adequate quality and quantity of bone.\n\n8. Retained roots, fractured tooth or tooth advised for extraction due to periapical pathologic lesions.\n\n9. Mandibular/maxillary, anterior/posterior single missing tooth.\n\n10. If buccal cortical plate gets fractured during the procedure of immediate implant placement, the patient will be included in the conventional group.\n\nPatients were excluded from the study if they:\n\n1. Were pregnant at the time of inclusion.\n\n2. Had parafunctional habits.\n\n3. Patients who were not be willing to undergo the treatment.\n\n4. Patients with aggressive periodontitis.\n\nA total of 18 patients with edentulous single tooth were included in the study. Patients of both sexes (7 females and 11 males) in the age range of 18 to 55 years were included in the study. Sex or gender do not impact the bone loss or gain in this study. The study was conducted in a prospective manner and was a comparative clinical trial involving two groups who had a missing single tooth. The patients were those in whom the implants were placed, and prosthesis was yet to be given.\n\nAll the implants used were from the MIS SEVEN implant system (see Figures 1 and 2). The implants were made up of grade V titanium alloy, and of acid etched sand blasted type. Conventional abutments of either cement post or screw retained type were used for the prosthesis. In the 18 patients, 18 implants were placed; of which 12 were placed in the maxilla and 6 were placed in the mandible. All the patients were placed on regular maintenance, with all of them being recalled every 1 month, 6 months and 12 months. At every recall visit, oral hygiene instructions like tooth brushing and use of interdental aids were reinforced. The following clinical parameters were assessed at baseline, 6 months and 12 months after cementation of crown (Figures 3 and 4): 1. Jemt papilla index (JPI); 2. modified plaque index (mPI); 3. modified gingival index (mGI); 4. gingival biotype.\n\nThis index was used to assess the size of the interproximal papillae adjacent to single implant restorations.16 The assessments were made from a reference line through the highest gingival curvatures of the crown restoration on the buccal side and the adjacent permanent tooth. Index scores were given from 0 to 4 based on the amount of papilla present (see Table 1).\n\nA mouth mirror and William's periodontal probe were used, after air drying of the implant to assess for presence of plaque by running the probe along the margin. The surfaces examined were four gingival areas of the tooth i.e. distofacial, facial, mesiofacial and lingual surfaces.\n\nA mouth mirror was used to assess the modified gingival index by visual examination on of buccal and palatal/lingual surfaces, without probing.\n\nAfter administration of local anesthesia, an endodontic file with a rubber stopper was penetrated into the facial soft tissue until it reached the bone. The rubber stopper was placed on the soft tissue surface to mark the position. The thickness of facial soft tissue was equal to the distance between the rubber stopper and the tip of the endodontic file which was recorded by a digital caliper to the nearest 0.1 mm. It is categorized into thick or thin based on the amount of tissue thickness. If it was >1 mm, then it was considered as thick and if it was <1mm, it was considered as thin biotype.\n\nIntraoral radiographs were taken for all patients at baseline, 6 months and 12 months after crown cementation. A standardized intra oral periapical radiograph (IOPAR) was taken for each selected site using the long cone paralleling technique using a film holder (RINN, XCP, Dentsply Illinois, USA). All radiographs were obtained utilizing an 'E' speed film (Kodak Carestream Health Inc., New York, USA) mount with mm grid scale. These grid scale lines are printed vertically and horizontally at 1mm interval and with bold lines at 5 mm. The factors listed below were taken into account when calculating the degree of bone loss adjacent to the implants. To determine the vertical measures, the fixture abutment junction (FAJ) implant shoulder's horizontal measurement served as a guideline while image analysis software was used for the following radiographic parameters:\n\n1. The most coronal portion of bone that contacts the implant and the vertical distance from the implant's shoulder (FAJ) were given the designation FAJ-I.\n\n2. The most coronal portion of bone facing the neighbouring tooth and the vertical distance from the implant's shoulder were referred to as FAJ-Adj.\n\n3. The soft tissue height was measured from the implant's shoulder to the coronal papilla level.\n\n4. The contact point's vertical distance from the bone's crest was denoted as CP-Bone crest.\n\nAll radiographs were scanned using a scanner (HP Scanjet). All radiographic measurements were carried out by using software, Image J.17 Image J (Image processing and analysis in Java) is a pure Java image processing program which can measure area, mean, standard deviation, lengths, angles and min or max of selection or entire image. The Image J analysis was carried out as follows: the image was first transferred to the Image J analysis software, then using the straight-line option, a straight line was drawn to measure 1 mm grid and scale was set by converting distance into pixels. Once the distance was set, all the above-mentioned parameters were measured using the same tool and the amount of bone loss that has occurred was then calculated. For both the groups, the differences in the interproximal crestal bone height between 6 months and 12 months and from baseline and 12 months were calculated.18‐25\n\nNo implant was lost during the observational period. Two patients from the immediate group were lost at 12 months follow up because of COVID-19. Descriptive statistics, mean and standard deviation were calculated for continuous variables. Independent t-test was used to calculate distribution of implants in arches, mean age, crestal bone values i.e., FAJ- I, FAJ-Adj., soft tissue height and CP-BC between the groups. Intragroup comparison was done using paired t-test. Frequency and percentage were calculated for categorical variables. Chi square test was used to calculate intergroup variables in categorical values. Modified plaque index (mPI), modified gingival index (mGI), Jemt's papilla index (mesial & distal) and gingival biotype was calculated using Chi square test. A value of <0.05 is considered to be statistically significant. Microsoft Excel, SPSS version 1.52t and Image J software were used for analysis.\n\n\nResults\n\nCrestal bone loss values for group 1 (FAJ - I = 2.5 ± 0.3; FAJ - Adj. = 2.09 ± 0.21; STH = 3.98 ± 1.2 and CP - BC = 6.06 ± 0.99) and for group 2 (FAJ - I = 2.26 ± 0.62; FAJ - Adj. = 1.9 ± 1.03; STH = 4.75 ± 0.32 and CP - BC = 5.94 ± 0.85) at the end of 1 year showed that crestal bone loss was seen more in the immediate group than the conventional group58 (Tables 2-5). Papilla scores at the end of 1 year showed that distal papilla fill was better in the immediate group (with score 3 in 14.3% of patients) than the conventional group (with score 3 in 11.1% of patients) and mesially, scores were better in the conventional group (with score 3 in 22.2% of patients) than the immediate group where none of the patients had a score of 3 on the Jemt papilla index. So, conventional implants showed less bone loss and better papillary fill than implants placed in a fresh extraction socket. Plaque scores were assessed as per modified plaque index (mPI) which showed better results in the conventional group (score 1 in 88.9% of patients) than the immediate group (score 1 in 85.7% of patients). Modified gingival index (mGI) was used to assess gingival status which showed better results in the immediate group (100% of patients showed score 1) than the conventional group (88.9% patients showed score 1 and 11.1% of patients showed score 2) at the end of 1 year. Also, gingival biotype did not appear to affect changes in crestal bone level or papilla fill. Sex or gender of the patient does not impact the bone loss or gain in this study.\n\n* p value is statistically significant.\n\n* p value is statistically significant.\n\n\nDiscussion\n\nA crucial indicator for the health of an implant is the quality of the crestal bone. Its preservation is considered an essential indicator for the success of dental implants. Also, the height of the papillae that is related to the interproximal crestal bone contributes to the success of the implant. Thus, papillae preservation height and crestal bone is paramount for fruitful success of the outcome of an implant. Long term studies on the volume of crestal bone loss in the early healing phase, i.e., within one year, generally observe 1.5 mm in the first year, followed by 0.2 mm annually.26 This could be because of occlusal loading forces during mastication and the presence of micro gaps between the implant and placement of the abutment at or apical to the crest, which acts as a plaque retentive area and leads to bone loss. When crestal bone loss occurs, the soft tissue overlying the crestal bone i.e. the papillae would also recede and will lead to plaque accumulation and an unaesthetic appearance.27\n\nIn this study, a total of 18 patients were recruited. 9 patients received implants as per immediate implant placement protocol (group 1) and 9 patients received implants as per conventional or delayed implant placement protocol (group 2). Astrand et al.28 used two different implant systems (AstraTech and Branemark system) and showed that there was a difference in the pattern of bone remodeling between the implant systems. This can be attributed to the macro and micro characteristics that vary from one system to another. In this study, we have used the MIS SEVEN implant system. It is advantageous to use the same implant system across all patients in the study, as different systems can show different amounts of bone loss. For all the patients, delayed loading protocol was followed, and for all the implants placed, the crown was given after a waiting period of 3-4 months for mandible and 6 months for maxilla.29,30\n\nThe pattern of bone loss after implant placement varies in maxilla and mandible patients, owing to the difference in the density of the bone as it is more cancellous in the maxilla as compared to the mandible. Longitudinal studies have shown a mean difference of 0.23 mm bone loss for implants placed in the maxilla and mandible.31,32\n\nExcessive surgical trauma and thermal injury may lead to thermal necrosis and fibrous encapsulation of implants. Eriksson and Alberktsson33 reported that the critical temperature for implant site preparation was 47°C for 1 minute or 40°C for 7 minutes. With overheating, the chance of implant failure increases. In this study, adequate internal irrigation was used to keep temperature of bone to minimum. Sharp drills were used, and osteotomy was performed in a staged manner without jumping the drilling sequence.\n\nThe height of the bone around the implant has been measured using a variety of techniques. Compared to conventional methods (using magnifying glass), the computerized method is considered to be superior due to higher accuracy. One of these computerized methods is using Image J analysis software, which has high accuracy and precision along with specificity and sensitivity. Grid scale also provides reference distance for the image analysis software so that magnification error is minimized.34‐38\n\nIn this study, all the radiographs were taken at baseline, 6 months and 12 months after crown cementation and were taken using paralleling cone technique with grid overlay and analyzed using Image J analysis. All the radiographs were taken using the same exposure settings which are on the X-ray machine and hence, the standardization was maintained.\n\nThe present study showed that values of FAJ-I, FAJ- Adj., CP-BC were higher for group 1 than group 2, indicating that higher crestal bone loss was observed in the immediate group (FAJ-I = 2.5 ± 0.3; FAJ-Adj., = 2.09 ± 0.21; CP-BC = 6.06 ± 0.99) than the conventional group (FAJ-I = 2.26 ± 0.62; FAJ- Adj., = 1.9 ± 1.03; CP-BC = 5.94 ± 0.85) and higher soft tissue height values were seen for group 2 (STH = 4.75 ± 0.32) than group 1 (STH = 3.98 ± 1.2), which shows that the conventional group produced better results than the immediate group. Sasi Kumar et al.39 did a study to measure bone loss in conventional and immediate implant placement and stated that bone loss was seen more in the conventional group (1.28 ± 0.24) than the immediate group (1.10 ± 0.39) mm. This is in contrast to the findings in our study; this could be due to the use of autogenous bone chips harvested from the surroundings, which may have aided in better bone formation in immediate group. Tabrizi et al.40 demonstrated that the amount of bone loss is significantly (P > 0.05) more in group 2, i.e. conventional (1.6 ± 0.20), than group 1, i.e. immediate, (1.05 ± 0.17) at the 6 month follow up; hence, their results were in contrast to our study. Heinemann et al.41 on the other hand concluded that there was a non-significant difference between group 1 and group 2 in approximal bone level change during the first year, which was similar with our study. In a study done by Moustafa et al.,42 the immediate loading group showed higher bone loss than the conventional loading group at the end of the 3 year follow up period, which is similar to our findings. Papilla assessment was done immediately on the day of crown cementation, both in maxillary and mandibular dental implants. The crown cementation was done at 3 months in all the patients, thus, standardizing the papillary assessments. The papillary fill around mesial and distal sides of restored dental implants were assessed clinically by scoring papilla from 0 to 4 as per Jemt's papilla index and by digitizing the images at baseline, 6 months and 12 months. In this study, results showed that in the conventional group, papilla scores (mesially) at 12 months showed complete papilla fill in 22.2% of cases and no complete papillary fill was seen in the immediate group. This could be because 2 patients were lost for 12 month follow up from the immediate group due to COVID-19, which is a limitation of our study. Distally, on evaluating Jemt's papilla score, complete papillary fill was seen in 14.3% of patients in the immediate group and 11.1% in the conventional group. These findings were supported by a study done by Grossberg et al. (2001),43 in which four patients out of twelve showed complete papilla fill in between the implants. Also, Nariman et al. (2018)’s44 study found that both mesially and distally, the conventional group (Mesial side = 1.78 and distal side = 1.89) showed better papilla fill than the immediate group (mesial side = 1.20 and distal side = 1.30) which is in contrast to our study. Lee et al. (2005)45 showed that the soft tissue height in between two adjacent implants was 3.3 ± 0.5 mm. This is in accordance with the study done by Rushad Hosi et al.,44 who also stated that inflammation can lead to false assessment of papillary fill.\n\nPlaque accumulation has been considered as one of the most important causes for crestal bone resorption.46 Formation of plaque on an implant surface is influenced by surface characteristics of the dental implant and abutment material used.47\n\nThe patients in the current study were followed at 6 months and 12 months after crown cementation. The modified plaque and modified gingival index scores (mPI and mGI) were used to assess peri-implant mucosal condition at baseline (i.e. on the day of crown cementation), at 6 months and 12 months. In this study, there was no statistically significant mean plaque score difference found between groups at baseline, 6 months and 12 months. This shows that the patients maintained good oral hygiene at 6 months and gradually decreased at follow up time. This is in accordance with Weber HP et al. (2000)48 and Renvert S et al. (2009).49 Biologic width and biotype are said to influence the peri implant mucosa. In the present study, biotype was divided into two categories: thick and thin. Marginal gingiva often has more effects from gingival biotype than interproximal papillary levels. As in previous investigations, gingival biotype did not appear to affect changes in crestal bone level or papilla fill in this study.24,50‐57\n\nA limitation of this study is the smaller sample size. Two patients were lost for 12 months follow up due to COVID-19. The strengths of this study include That the software used in the study to measure bone loss was more accurate than the other graphical methods that had been used in other studies. Additionally, the standardization of implant size and implant system were done in the present study to minimize biasing.\n\n\nConclusion\n\nIn consideration of the study's shortcomings, it can be said that:\n\n• Conventionally placed implants showed less bone loss and better papillary fill than implants placed immediately into fresh extraction sockets.\n\n• Gingival biotype had no significant influence with respect to the papilla fill and crestal bone loss irrespective of the technique of implant placement.\n\n• Regardless of the technique of implant placement, there was no bone loss after cementation of crown.\n\n\nPatients’ declaration of consent\n\nThe authors declare that they have obtained all necessary consent papers from the patients. The patients have signed the form and agreed for their pictures and other clinical information to be published. The names and identities of the patients will remain anonymous.\n\n\nAuthors’ contributions\n\nAll the authors have equal contribution to this research in manuscript preparation, data collection and interpretation.", "appendix": "Data availability\n\nfigshare: Underlying data. https://doi.org/10.6084/m9.figshare.21687209.v1. 58\n\nThis project contains the underlying following data:\n\n• Data file 1: Master Chart for Immediate implants placement group\n\n• Data file 2: Crestal Bone level for immediate implants group\n\n• Data file 3: Master Chart for Conventional implants placement group\n\n• Data file 4: Crestal Bone level for conventional implants group\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nCONSORT checklist and flow diagram for ‘Comparison of crestal bone loss and papilla fill after conventional and immediate implant placement: A 12 month clinical and radiographic prospective study’. https://doi.org/10.6084/m9.figshare.22179419\n\n\nAcknowledgements\n\nThe authors would like to thank the Manipal College of Dental Sciences, Mangalore for all the support.\n\n\nReferences\n\nMish CE: Contemporary implant dentistry – third edition.\n\nAvivi-Arber L, Zarb GA: Clinical effectiveness of implant- supported single tooth replacement: The Toronto stydy. 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PubMed Abstract\n\nRaj A, Pradhan S, Dr Shetty N : Underlying data. [Dataset]. figshare. 2022. Publisher Full Text" }	[ { "id": "194369", "date": "18 Oct 2023", "name": "Chandni Batra", "expertise": [ "Reviewer Expertise Periodontology and Implant Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article under review compares crestal bone loss and papilla fill over 12 years after conventional and immediate implant placement. Evaluations included papilla fill or soft tissue height, modified plaque and gingival index, gingival phenotype and radiographic bone loss. The study results demonstrated better papilla fill and crestal bone preservation with no statistical differences between the groups. Overall, the study is well designed and article well written. It would be beneficial to include the following and make following edits:\nInclude the aim of the study and a sentence on what makes this study different from previously completed similar studies in the introduction.\n\nInclusion criteria mentions good oral hygiene - how was this defined? (Plaque score/gingival score?)\n\nInclusion criteria mentions adequate quality/quantity of bone - how was this defined?\n\nTerminology gingival biotype can be changed to gingival phenotype as per the 2017 classification system1.\n\nTerminology Biologic width can be changed to supracrestal attachment as per 2017 classification system1.\n\nPlease mention in the results (text) whether the measured differences were significant or not significant for better understanding by readers.\n\nIs the work 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": "208643", "date": "13 Dec 2023", "name": "Zhen Fan", "expertise": [ "Reviewer Expertise dental implantation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe overall research design of the article is reasonable, and the corresponding chapters are also divided into subheadings, which are clear. The following are some suggestions for reference:\n1. The introduction may add more detailed background information to give readers a better understanding of the background and importance of the study.\n2. Objectives of study can be more clearly defined, such as \"compare the effects of immediate and delayed implant on the clinical effects of patients (such as long-term bone mass changes, dental health, aesthetic effects, etc.).\"\n3. More details can be provided for the method of study, such as the choice of implant type, implant system, and the reason for the surgical methods.\n4. Will the inclusion in the criteria of extraction of teeth caused by periapical diseases affect the effect of immediate implant?\n5. Given that there are many clinical studies on the same subject whose final conclusions are not necessarily consistent, many of the influencing factors (such as the implant system used, the lacking teeth are in the upper or lower jaw, etc.) can analyze the reasons for their inconsistency. This has already been presented in the text, but we suggest a deeper exploration of possible causes and potential clinical significance, rather than just listing the consistent or inconsistent facts. After analyzing these different factors, the need for limiting the conclusions should also be considered.\n6.In the conclusion and discussion, the clinical and practical significance of the corresponding findings could be written and divided more clearly, and we suggest more specific data added to make the results more intuitive.\n7. The research aim in this paper is comparing immediate implant and delayed implant, but one of the studies mentioned in the discussion part is about immediate loading, so it is doubtful whether it is comparable with the research results in this paper.\n\n8. In this paper, the conclusion of sticking crown and gingival biotype does not affect the treatment results, but according to past studies, soft tissue should have certain influence on bone resorption. More literatures searching and discussion is needed.\n\n9. In the conclusion part, we can consider further exploring the specific impact of these findings on clinical practice and the future research direction.\n\n10. There are many ancient documents in the cited documents. Besides some classic documents, it is necessary to add more recent documents related to the research results of this paper, and the ones in recent 3 years are preferred.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10845", "date": "11 Jan 2024", "name": "Neetha Shetty", "role": "Author Response", "response": "More detailed background information has been added in the introduction to understand the importance of this study. The objective of the study is clearly defined. More details have been provided on the choice of implant system. Peri-apical lesions do not affect the effect of immediate implant placements. Hence, they are included in the study. More detailed explanation has been provided on the influencing factors. The clinical and practical significance is more clearly defined. Since immediate loading is one of the influencing factors, it seems justified to include the study on immediate loading. The data ad been modified to provide better understanding. Clinical significance and future directions has been added in the conclusion. Recent references have been added." } ] }, { "id": "225045", "date": "15 Dec 2023", "name": "I-Ching Wang", "expertise": [ "Reviewer Expertise Implantology and Periodontology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors, It was suggested that in the results section of the abstract should be more detailed to support the conclusion. Please see the comments below.\nMethods and Material:\nPlease provide more details of the power calculation including the assumed standard deviation and the effect size. Please clarify if the clinically significant difference in this manuscript was defined as 0.05 Please clarify the inclusion criteria for the extraction sockets. Were the defective sockets all included? Please clarify what standardized position on the soft tissue was measured using an endodontic file. Please clarify if a standardized stent for the periapical radiographs was used.\nConclusions: It should be added the conclusions of observation were at 1 year.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10846", "date": "11 Jan 2024", "name": "Neetha Shetty", "role": "Author Response", "response": "The sample size has been provided. The clinically significant difference has been clarified. Defective sockets were excluded, and thus has been added in the exclusion criteria. The position of the file has been clarified. Standardised stent was used for radiograph and is clarified." } ] } ]	1	https://f1000research.com/articles/12-821
https://f1000research.com/articles/13-46/v1	08 Jan 24	{ "type": "Research Article", "title": "Strengthening of CYFRA 21-1 using urine creatinine correction as potential endometriosis biomarker ", "authors": [ "Nicko Pisceski Kusika Saputra", "Samsulhadi Samsulhadi", "Hendy Hendarto", "Ashon Sa’adi", "Widodo J Pudjirahardjo", "I Wayan Arsana", "Relly Yanuari", "Sri Ratna Dwiningsih", "Nicko Pisceski Kusika Saputra", "Samsulhadi Samsulhadi", "Ashon Sa’adi", "Widodo J Pudjirahardjo", "I Wayan Arsana", "Relly Yanuari", "Sri Ratna Dwiningsih" ], "abstract": "Background This study aimed to determine the accuracy of CYFRA 21-1 using urine creatinine correction (CYFRA/Cr) as a biomarker of endometriosis.\n\nMethods This study includes 73 patients from the Indonesian population, with 38 endometriosis and 35 non-endometriosis patients based on laparoscopy. Urine detection of CYFRA 21-1 was done by ELISA method and corrected by urine creatinine constant factor (CYFRA/Cr). Urine creatinine us detected using the ECLIA method.\n\nResults The CYFRA/Cr ratio was identified in the proliferative and secretory phases. CYFRA 21-1 and CYFRA/Cr levels were significantly higher in endometriosis and were higher in the proliferative phase compared to the secretory phase. The best accuracy was obtained in CYFRA which was corrected with urine creatinine in the proliferative phase with a sensitivity value, specificity, and cutoff value of 94.7%, 94.4%, and of 3,547.99 ng/gr, respectively, compared to CYFRA 21-1 urine levels without correction of creatinine.\n\nConclusions The CYFRA to creatinine urine ratio detected in the proliferative phase showed the optimum sensitivity and specificity compared to CYFRA 21-1 spot urine. It has the potential to be a biomarker of endometriosis.", "keywords": [ "Endometriosis", "CYFRA 21-1", "CYFRA/Cr", "Urine Creatinine", "Proliferative phase." ], "content": "Introduction\n\nThe diagnosis of endometriosis is reported to be delayed to almost 7–10 years from disease onset because menstrual pain is often considered normal (Hogg & Vyas, 2015). Laparoscopy is the gold standard for the diagnosis of endometriosis (Tokushige et al., 2011). Compared with histopathology, laparoscopy gave a sensitivity and specificity of 97.68% and 79.23%, respectively, in the diagnosis of endometriosis (de Almeida Filho et al., 2008). In another study, sensitivity was 94% (95% CI: 80%–98%), and specificity was 79% (95% CI: 67%–87%) (Wykes et al., 2004). However, laparoscopic surgery is an invasive procedure that sometimes provides unwanted complications and higher costs, causing a delay in the diagnosis and management of endometriosis (Tokushige et al., 2011).\n\nBiomarker detection as a diagnosis is very interesting and can advance the management of infertility and pelvic pain. Previous studies have identified endometriosis biomarkers from various samples, including blood, cervical mucus, and urine (Fassbender et al., 2015). Diagnosis with biomarkers is very promising because it is not invasive, relatively inexpensive, and relatively fast compared to surgery. Urine is a potential specimen in the diagnosis of endometriosis, especially urine biomarkers.\n\nIn 2011, the protein that was found in the urine of endometriosis patients was identified as a cytokeratin-19 fragment, namely, CYFRA 21-1 (Tokushige et al., 2011). Cytokeratin-19 is found in simple and complex squamous cell epithelium, so it is commonly found in basal cells and squamous membranes. Cytokeratin-19 is a low molecular weight cytokeratin (LMWCK) (Jose et al., 2013). It is an acidic cytokeratin with a molecular weight of 40 kDa and is expressed in glandular-type epithelium, one of which is the endometrial gland epithelium (Schweizer et al., 2006). Although the mechanism of excretion of cytokeratin-19 fragments (CYFRA 21-1) in the urine is not clear, the presence of impaired renal filtration which is characterized by increasing serum creatinine levels that cause decreasing in the level of cytokeratin-19 fragments (CYFRA 21-1) in the urine accompanied the increasing its serum level in urine (Kashiwabara et al., 1998). The mean levels of cytokeratin-19 fragments (CYFRA 21-1) were significantly higher in the proliferative phase than in the secretory phase. The sensitivity value in the proliferative phase is 94.12%, with a cutoff value of 4 ng/ml/g creatinine, while the sensitivity value in the secretory phase is 31.5% (Gjavotchanoff, 2015). However, CYFRA 21-1 as a biomarker is still inconclusive, so further study is needed to confirm this finding (Liu et al., 2015). The inconsistency of results may be due to unstable CYFRA 21-1 and heavily influenced by temperature, menstrual cycle, and fluctuating levels.\n\nProtein that is excreted into the urine is very volatile. Therefore, the description of spot urine protein levels can automatically describe the actual condition. When examining, routine urine protein levels must be compared with something relatively stable in the urine (Kamińska et al., 2020). The levels of cytokeratin-19 fragments (CYFRA 21-1) are circadian (Gjavotchanoff, 2015; Nolen et al., 2015). Urine creatinine is a determinant and correction factor that can be used because its levels are relatively stable in the urine; thus, the results obtained are not influenced by variations in fluid intake. This becomes the background for measuring spot urine protein levels. The standard for measuring urine protein is 24-h urine protein due to fluctuating protein excretion. However, the 24-h urine collection has many problems, so an alternative method can be used, i.e., the urine protein and creatinine ratio recommended by the National Kidney Foundation and Kidney Disease Outcomes Global Improving (KDIGO) (National Kidney Foundation, 2002). Muscle metabolism leads to the irreversible dehydration of body creatine and creatine phosphate and creates creatinine as a waste product. More than 90% of the creatinine is accumulated in skeletal muscle. The formation rate of this product is constant; 2% of body creatine is converted to creatinine every 24 hours. However, the older the individual, the slower the rate (Barr et al., 2005).\n\nThe inconsistency of study results needs to be reanalyzed to strengthen the potential of CYFRA 21-1 as a biomarker of endometriosis by using correction of urine creatinine to urine CYFRA (CYFRA/Cr). Therefore, this study aimed to analyze the accuracy of CYFRA 21-1 through urine creatinine correction for the diagnosis of endometriosis. Analysis was also carried out based on menstrual cycle.\n\n\nMethods\n\nThis study has passed the ethical review of the Medical and Health Research Ethics Unit of the Faculty of Medicine, Universitas Riau with register number B/091/UN19.5.1.1.8/UEPKK/2021. It was declared to be ethically appropriate to seven WHO 2011 standards. The date of the certificate was 10th September 2021.\n\nWe confirm that we have obtained permission to use the data from the patients included in this presentation. Each patient gave written and verbal informed consent after they have been informed about the research’s objective, what was sampled and its procedure, as well as the provided data usage in this research. The patients also knew that this research would be published but no identifiable data from the patients would be included in the publication.\n\nThis is an analytic study with a cross-sectional design. The study was conducted at the Arifin Ahmad General Hospital, Provinsi Riau, from October 2019 to October 2021. Examination of cytokeratin-19 fragment (CYFRA 21-1) levels in urine was conducted at the Integrated Biomedical Laboratory, Faculty of Medicine, Universitas Riau, Indonesia. Examination of urine creatinine levels was conducted at the Prodia Laboratory Pekanbaru, Indonesia.\n\nThis study included all patients from the Fertility Clinic of Arifin Ahmad General Hospital with indications of laparoscopic surgery. The patients were approached directly when they were visiting in-person to the clinic and categorized based on the inclusion and exclusion criteria. The inclusion criteria were age of 30–40 years, normal body mass index (BMI) of 18.5–24.9 kg/m2, and menstrual cycle of 26–38 days. Age and BMI information was procured from anamnesis and direct observation when the patients were in the clinic. Information on the patient’s menstrual cycle was obtained during the observation at the clinic in the form of last day of menstruation. The menstrual cycle information was then analysed for its proliferation and secretion phase. The exclusion criteria were patients taking hormonal drugs such as contraception, other medications such as anti-inflammatory drugs, and antioxidant drugs and patients currently suffering from lung cancer, gastrointestinal cancer, urinary tract infections (based on the results of medical records, chest X-ray, urine routine examination, liver function), impaired kidney function (based on glomerular filtration rate (GFR), creatinine examination), and diabetes mellitus. A total of 73 patients were selected based on laparoscopy, with 38 endometriosis patients and 35 non-endometriosis patients.\n\nThe ELISA method was used to examine the urine CYFRA 21-1 level. Urine was collected before laparoscopy and stored on ice or refrigerator for no more than 2 h before processing. During transport to the laboratory, the cold chain must be well maintained; hence the samples were transported using an icebox. The collected urine was stored in a refrigerator at −80°C before the examination. The urine used was uncentrifuged urine. The ELISA kit used is the Human Cytokeratin 19 ELISA Kit (product number RAB1409).\n\nStandard serial dilution was performed to obtain the following concentrations of CYFRA 21-1: 0.061 ng/ml, 0.154 ng/ml, 0.384 ng/ml, 0.960 ng/ml, 2.4 ng/ml, 6 ng/ml, 15 ng/ml, and blank. All reagents and samples were brought to room temperature (18–25°C) before use. Examination was performed in duplicate. Approximately 100 ul of each standard and sample was added to the well. The wells were covered and incubated for one night at a temperature of 4°C, followed by washing. Approximately 100 ul antibody was added to each well and incubated for 1 h at room temperature, mixed thoroughly, and washed. Approximately 100 ul of streptavidin was added and incubated for 45 min followed by washing. Approximately 100 ul of TMB substrate was added to each well, 50 ul of stop solution was added. The absorbance value was read at a wavelength of 450 nm. Standard plotting was performed to determine the equation on the standard curve.\n\nUrine creatinine is a constant determinant in determining urine cytokeratin-12 levels, so the ratio of cytokeratin and urine creatinine can be identified. The ECLIA method was used to examine the urine creatinine level at the Prodia Pekanbaru Laboratory. The ratio of urine cytokeratin-19 fragment (CYFRA 21-1) levels to creatinine (CYFRA/Cr) was calculated by comparing the levels of cytokeratin-19 fragments (CYFRA21-1) to urine creatinine levels in ng/g units.\n\nAll data were processed using IBM SPSS Statistics 28.0. Numerical data was tested for data normality using the Kolmogorov–Smirnov and Shapiro–Wilk tests. Patient characteristics were analyzed using T-test and the Mann–Whitney test. Sensitivity and specificity tests for urinary cytokeratin-19 (CYFRA 21-1) and CYFRA/Cr levels were analyzed using the ROC test. The AUC values and coordinates of the curve were transformed into Microsoft Excel, and the data was then entered into a graph line with a marker to obtain the intersection point or cutoff point value of sensitivity and specificity.\n\n\nResults\n\nTable 1 presents the characteristics of the study subjects, consisting of age, BMI, and kidney function based on serum creatinine and GFR values. As presented in Table 1, no differences were found in patient characteristics analyzed by cycle phase in both endometriosis and non-endometriosis cases. Based on serum creatinine and GFR data, it was found that all study subjects were in the normal range, which means that kidney function was in good condition. Therefore, the bias toward the excretion of cytokeratin-19 fragments (CYFRA 21-1) was negligible. Based on the results of chest X-ray examination in all study subjects, the heart and lungs were within normal limits. Based on examination of blood sugar, liver function, and routine urine, all samples were found to be within normal limits so they could be included.\n\nTable 2 presents the levels of cytokeratin-19 fragments (CYFRA 21-1) urine. It can be seen that the levels of cytokeratin-19 fragments (CYFRA 21-1) were significantly higher in the endometriosis group than in the non-endometriosis group (p < 0.05). The levels of cytokeratin-19 fragments (CYFRA 21-1) were significantly higher in the proliferative phase compared to the secretory phase in both the endometriosis and non-endometriosis groups. The AUC area at 80.7% (95% CI 70.3%–91.0%), p = 0.00, sensitivity of 76.3% and a specificity of 74.3% with the cutoff point of the cytokeratin-19 fragment (CYFRA 21-1) was 1.26 ng/ml (Figure 1).\n\nIn the analysis based on the cycle phase, it was found that the AUC in the proliferative phase was 97.1% (95% CI: 92.3%–100%). The considerable interpretation of the AUC for more than 90% was very good, with a p-value of <0.05 (p = 0.000). Meanwhile, the AUC in the secretory phase was 78.3% (95% CI: 61.8%–94.9%). The interpretation of the AUC is classified as moderate with a significance of p = 0.000. In the proliferative phase, the sensitivity and specificity were 89.5% and 88.9%, respectively, with a cutoff value of 5.024 ng/ml for the levels of cytokeratin-19 fragments (CYFRA 21-1). At the same time, in the secretory phase, the sensitivity and specificity were 78.9% and 82.4%, respectively, with a cutoff of 0.77 ng/ml.\n\nFluctuating random urine cytokeratin-19 fragment (CYFRA 21-1) levels need to be corrected with urine creatinine as a constant factor and generate a CYFRA 21-1 urine creatinine ratio or CYFRA/Cr. As presented in Table 3, the cytokeratin-19 fragment (CYFRA 21-1) levels to creatinine (CYFRA/Cr) urine ratio was significantly higher in the endometriosis group than the non-endometriotic group (p < 0.05). Moreover, the cytokeratin-19 fragment (CYFRA 21-1) levels to creatinine (CYFRA/Cr) urine ratio was significantly higher in the proliferative phase compared with the secretory phase in both the endometriosis and non-endometriosis groups.\n\nThe AUC was 84.5% (95% CI: 75.5%–93.5%). The interpretation of an AUC of more than 80% is good, with p < 0.05 (p = 0.00). The sensitivity and specificity were 78.9% and 77.1%, respectively, with a cutoff value of 1,647.11 ng/gr in the cytokeratin-19 fragment (CYFRA 21-1) levels to creatinine (CYFRA/Cr) ratio (Figure 2).\n\nIn the analysis based on the menstrual cycle phase, which was obtained based on the ROC curve in the proliferative phase, the AUC was 98.5% (95% CI: 95.7%–100%). The interpretation of an AUC of more than 90% was considered very good, with p < 0.05 (p = 0.015). The AUC in the secretory phase was 89.5% (95% CI: 78.5%–100%), and the interpretation of the AUC was classified as moderate with insignificant meaning p = 0.056. In the proliferative phase, the sensitivity and specificity were 94.7% and 94.4%, respectively, with a cutoff value of 3,547.99 ng/gr in the cytokeratin-19 fragment (CYFRA 21-1) level to creatinine (CYFRA/Cr) urine ratio. This result has the best sensitivity and specificity. At the same time, in the secretory phase, the sensitivity and specificity were 73.7% and 76.5%, respectively, with a cutoff value of 657.15 ng/gr.\n\n\nDiscussion\n\nIn this study, patient characteristics include age, BMI, and renal function based on serum creatinine values, and GFR values are important variables to avoid bias. Age included in inclusion criteria is in the range of 30–40 years due to the possibility that age will affect the production of cytokeratin-19 fragments (CYFRA 21-1). Renal function is also influenced by age (National Kidney Foundation, 2002). Continuous decrease in GFR from a mean 123 mL/min/1.73 m2 at age 20–29 years to a mean 65 mL/min/1.73 m2 at age 80–89 years is a decline of about 10 mL/min per decade of age (Delanaye & Rule, 2015). The presence of impaired renal function leads to the inability of the kidney to clear cytokeratin-19 fragments (CYFRA 21-1), resulting in increased levels of cytokeratin-19 fragments in the blood (Rastel et al., 1994).\n\nOther confounding factors identified were routine chest X-rays and routine urine examinations. Based on chest X-ray examination, it was within normal limits. Routine urine examination is a screening examination to rule out other diseases that affect the results, i.e., urinary tract infections, urinary tract stones, or urinary tract cancer. X-ray examination is important to avoid bias in the presence of other diseases that can increase the production of cytokeratin-19 fragments (CYFRA 21-1), i.e., lung cancer.\n\nEndometriosis is a disorder with various pathophysiologies. Hormonal, genetic, immunological, and oxidative stress factors are thought to be involved in the complex pathophysiology of endometriosis. This study found the high activity of oxidative stress in endometriotic tissue damage (Chen et al., 2020).\n\nThe level of cytokeratin-19 fragment (CYFRA 21-1) spot urine in women with endometriosis was significantly higher than those without endometriosis. Cytokeratin-19 is expressed in glandular-type epithelium, one of which is the endometrial gland. Cytokeratin-19 normally occurs in the glandular epithelium both in the proliferative, secretory, and atrophic phases of the endometrium based on IHC examination. There is a more consistent expression in the functional layer, whereas the basal zone is usually focally stained. In the proliferative epithelium, cytokeratin-19 was seen in the basal and apical cytoplasm (Stewart et al., 2011).\n\nEctopic endometrial tissue causes an immune hyper-reaction that affects the breakdown of cytokeratin-19 to CYFRA 21-1 (Gjavotchanoff, 2015). Systemic release of cytokeratin-19 can occur through several mechanisms, including cellular apoptosis, abnormal mitosis, or release due to cell proliferation. Extracellular release occurs at the intermediate stage of epithelial cell apoptosis and during cell damage (Fujita et al., 2004).\n\nAn increase in M2 activity was seen based on the pathophysiology of endometriosis through macrophage activity. Macrophages are supposed to act as classical phagocytes but, instead, increase their proliferation to form a neoplastic transformation. In this process, ectopic endometrial cells that bind to macrophages are thought to be damaged and release intracellular proteins (Capobianco & Rovere-Querini, 2013).\n\nCytokeratin-19 formed the smallest molecular weight of 40 kDa and consists of 400 amino acids encoded by the KRT 19 gene. The cytokeratin-19 fragment (CYFRA 21-1) formed soluble protein with a small molecular weight of 30 kDa (less than 19 kDa) as the molecule can pass through the glomerular filtration and be excreted in the urine (Jose et al., 2013). These results strengthen the findings of Tokushige et al. (2011), who stressed urine protein is detected in endometriosis, which is then detected as CYFRA 21-1 through proteomic and western blotting techniques. At that time, Tokushige et al. (2011) could not determine how CYFRA 21-1 is released into the urine. Of note, the urine used in the previous study was urine that had been centrifuged. This study is also in line with the results obtained in the Gjavotchanoff (2015), but the difference is in the correction of the creatinine factor.\n\nCytokeratin-19 fragment (CYFRA 21-1) is a microprotein that breaks down easily at room temperature. In a study on the stability of the levels of cytokeratin-19 fragments (CYFRA 21-1) in urine at room temperature, it showed a decrease in levels associated with time (Nisman et al., 2002). Therefore, to maintain its stability after sample collection, it should be stored in a refrigerator. In this research, it was stored at −80°C. To maintain cold chain protein in the urine, the transportation process used an icebox. In a recent study in 2019 from a population in Korea, no significant difference was observed in levels of cytokeratin-19 fragments (CYFRA 21-1) between endometriosis and non-endometriosis. Meanwhile, the samples examined in this study were serum, and endometriosis controls were cases of non-endometrial ovarian tumors (Cho & Kyung, 2019).\n\nA difference was observed in the levels of cytokeratin-19 fragments (CYFRA 21-1) between centrifuged and non-centrifuged urine, and the best results were obtained from uncentrifuged urine (Gjavotchanoff, 2015; Nisman et al., 2002). In this study, urine that was not centrifuged was used, although at the time of sample preparation the researchers prepared two preparations, i.e., centrifuged and non-centrifuged. During optimization, the best concentration was obtained from non-centrifuged urine. Inconsistency of results can also be caused by CYFRA 21-1 is a fluctuating protein so that spot urine test becomes inaccurate.\n\nIf the levels of cytokeratin-19 fragments (CYFRA 21-1) were analyzed based on the phase of the menstrual cycle, the average levels of cytokeratin-19 fragments (CYFRA 21-1) obtained in the proliferative phase were significantly higher than the secretory phase in both endometriosis and non-endometriosis groups. This result is the same as that of Gjavotchanoff (2015). There is a thickened functional stratum in the proliferative phase compared to the secretory phase. Based on the IHC examination, many cytokeratins were identified in the functional stratum (Stewart et al., 2011). Functional cells that undergo apoptosis or over-proliferation will release cytokeratin-19 into the system. Endometriotic functional tissue shows hyper-immunoreactivity to CYFRA 21-1 (Gjavotchanoff, 2015).\n\nThe best sensitivity and specificity values for cytokeratin-19 fragment (CYFRA 21-1) levels in spot urine were found in the proliferative phase where the sensitivity and specificity values were 89.5% and 88%, 9%, respectively, with a cutoff value of 5.024 ng/ml in the cytokeratin-19 fragment (CYFRA 21-1) level compared to the secretory phase and the overall value regardless of the cycle phase. This shows that the cycle phase is the predominant factor that must be considered because the cytokeratin-19 fragment (CYFRA 21-1) is a product of epithelial cells in which the intensity of epithelial cells in the endometrium and endometriotic cells is strongly influenced by cycle phase.\n\nHowever, the cytokeratin-19 fragments (CYFRA 21-1) spot urine test, unable to describe its level throughout the day so it must be compared with urine creatinine (Gjavotchanoff, 2015). The presence of dilutional factors and circadian rhythms will affect the consistency of the examination. The standard in measuring urine protein is 24-h urine protein due to fluctuating protein excretion. However, the 24-h urine collection has many problems, so an alternative method can be used, specifically the urine protein and creatinine ratio recommended by the National Kidney Foundation and Kidney Disease Outcomes Global Improving (KDIGO) (National Kidney Foundation, 2022).\n\nChemical exposure assessment is a crucial aspect of public health and environmental monitoring. To determine the validity of a spot urine sample for this purpose, various parameters such as urinary creatinine concentrations, specific gravity, and osmolality are measured. These measurements help to adjust for dilution and report accurate analyte results. Among the methods used to account for dilution and report results, creatinine adjustment is the most widely accepted method. It involves dividing the analyte concentration (micrograms of analyte per liter of urine) by the creatinine concentration (grams of creatinine per liter of urine) and presenting the results as the weight of analyte per gram of creatinine (micrograms of analyte per gram of creatinine). This method is highly effective in ensuring accurate and reliable reporting of analyte levels in urine samples (Barr et al., 2005).\n\nThe urine creatinine ratio was calculated as a constant level for the validity of the results of the levels of cytokeratin-19 fragments (CYFRA 21-1) spot urine test. Based on the analysis of the cytokeratin-19 fragment (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio, it was found that the mean in the endometriosis group was significantly higher than in the non-endometriotic group. Based on the cycle phase, it was found that the average cytokeratin-19 fragment (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio was significantly higher in the proliferative phase than the secretory phase in both the endometriosis and non-endometriosis groups. Compared with the levels of cytokeratin-19 fragments (CYFRA 21-1) in spot urine when can be seen differences in the results of the analysis in the secretory phase. Creatinine has been used as a reference value for calculating ratio in the dilution of the sample and matrix (Gjavotchanoff, 2015). Other similar studies that calculated the ratio of cytokeratin-19 fragments (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio have not been found, so it is difficult to compare this study with others. No research has been found that analyzes the cutoff value of cytokeratin-19 fragments (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio in endometriosis. A study by Lessey et al. (2015) and Gjavohanoff (2015) have applied creatinine as a determinant; however, in explanation, the results of cytokeratin-19 fragments (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio were not delivered.\n\nBased on recommendations from the National Kidney Foundation and Kidney Disease Outcomes Global Improving (KDIGO), the measurement of microprotein levels should be calculated based on urine creatinine levels as a stable matrix level in urine. This is because microproteins, which are one of the cytokeratin-19 fragments (CYFRA 21-1), are excreted in the urine at fluctuating levels, so it is not recommended to have a urine test during this time. For this reason, the measurement of cytokeratin-19 fragment (CYFRA 21-1) levels compared to urine creatinine as an alternative (National Kidney Foundation, 2002).\n\nIn this study, the best sensitivity and specificity were found in the cytokeratin-19 fragments (CYFRA 21-1) to creatinine (CYFRA/Cr) urine ratio measured in the proliferative phase, which is even better than the measurement of cytokeratin-19 fragment (CYFRA 21-1) spot urine. It was difficult to compare the finding of this cutoff value with other studies due to the limitations of the study, which divides sensitivity and specificity values based on the cycle phase. The other limitation is cycle phase determination, which was based on the patient’s menstrual history, not on the results of endometrial curettage. Accurate phasing should look at the histopathological features of the endometrium.\n\nCYFRA 21-1 spot urine levels are significantly higher in endometriosis than in non-endometriosis cases and are higher in the proliferative phase than in the secretory phase. The CYFRA/Cr ratio is also significantly higher in endometriosis than in non-endometriosis cases and higher in the proliferative than the secretory phase. Strengthening the accuracy of CYFRA 21-1 as a biomarker through correction of urine creatinine indicated by higher sensitivity and specificity of CYFRA/Cr than spot urine CYFRA, especially examined in the proliferative phase. It can be concluded that the CYFRA/Cr ratio can strengthen CYFRA 21-1 spot urine as a potential biomarker of endometriosis.", "appendix": "Data availability\n\nfigshare: CYFRA 21-1 strengthening using Urine Creatinine, https://doi.org/10.6084/m9.figshare.24085353.v1 (Saputra et al., 2023).\n\nThis project contains the raw, underlying data.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThank you to the dean of the Faculty of Medicine, Universitas Riau, who has permitted me to research at the Lontar Biomedical Laboratory. Thank you is also conveyed to the director of Arifin Ahmad Hospital, Provinsi Riau, Indonesia, for the permission to collect samples.\n\n\nReferences\n\nBarr DB, Wilder LC, Caudill SP, et al.: Urinary creatinine concentrations in the U.S. population: Implications for urinary biologic monitoring measurements. Environ. Health Perspect. 2005; 113(2): 192–200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapobianco A, Rovere-Querini P: Endometriosis, a disease of the macrophage. Front. Immunol. 2013; 4: 9.\n\nChen PS, Chiu WT, Hsu PL, et al.: Pathophysiological implications of hypoxia in human diseases. J. Biomed. Sci. 2020; 27(1): 63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCho HY, Kyung MS: CYFRA 21-1 and placental growth factor as screening markers for endometriosis. Med. Sci. Monit. 2019; 25: 1087–1092. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Almeida Filho DP , de Oliveira LJ , do Amaral VF : Accuracy of laparoscopy for assessing patients with endometriosis. Sao Paulo Med. J. 2008; 126(6): 305–308. Publisher Full Text\n\nDelanaye P, Rule AD: Assessing Kidney Function. Chronic Renal Disease. 2015; pp. 31–42. Publisher Full Text\n\nFassbender A, Burney RO, Dorien FO, et al.: Update on Biomarkers for the Detection of Endometriosis. Biomed. Res. Int. 2015; 2015: 130854.\n\nFujita J, Ohtsuki Y, Bandoh S, et al.: Elevation of cytokeratin 19 fragment (CYFRA 21-1) in serum of patients with radiation pneumonitis: possible marker of epithelial cell damage. Respir. Med. 2004; 98(4): 294–300. PubMed Abstract \| Publisher Full Text\n\nGjavotchanoff R: CYFRA 21-1 in urine: A diagnostic marker for endometriosis? Int. J. Women’s Health. 2015; 7: 205–211. Publisher Full Text\n\nHogg S, Vyas S: Endometriosis. Obstetric Gynecology and Reproductive Medicine. United Kingdom: Elsevier Ltd; 2015.\n\nJose J, Sunil PM, Nirmal M, et al.: CYFRA 21-1: An Overview. Oral Max. Pathol. J. 2013; 4(2): 1–5.\n\nKamińska J, Dymicka-Piekarska V, Tomaszewska J, et al.: Diagnostic utility of protein to creatinine ratio (P/C ratio) in spot urine sample within routine clinical practice. Crit. Rev. Clin. Lab. Sci. 2020; 57(5): 345–364. PubMed Abstract \| Publisher Full Text\n\nKashiwabara K, Kishi K, Nakamura H, et al.: Mechanism of increased serum cytokeratin 19 fragment levels in patients with diabetic nephropathy as a model of chronic renal failure. Intern. Med. 1998; 37(11): 917–921. PubMed Abstract \| Publisher Full Text\n\nLessey BA, Savaris RF, Ali S, et al.: Diagnostic accuracy of urinary cytokeratin 19 fragment for endometriosis. Reprod. Sci. 2015; 22(5): 551–555. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu E, Nisenblat V, Farquhar C, et al.: Urinary biomarkers for the non-invasive diagnosis of endometriosis. Cochrane Database Syst. Rev. 2015; 2015(12): CD012019. Publisher Full Text\n\nNational Kidney Foundation: Clinical practice guidlines for chronic kidney disease: Evaluation, classification and stratification. New York: National Kidney Foundation; 2002.\n\nNisman B, Barak V, Shapiro A, et al.: Evaluation of urine CYFRA 21-1 for the detection of primary and recurrent bladder carcinoma. Cancer. 2002; 94(11): 2914–2922. PubMed Abstract \| Publisher Full Text\n\nNolen BM, Lomakin A, Marrangoni A, et al.: Urinary protein biomarkers in the early detection of lung cancer. Cancer Prev. Res. (Phila.). 2015; 8(2): 111–119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRastel D, Ramaioli A, Cornillie F, et al.: CYFRA 21-1, a sensitive and specific new tumour marker for squamous cell lung cancer. Report of the first European multicentre evaluation. Eur. J. Cancer. 1994; 30(5): 601–606. Publisher Full Text\n\nSaputra N, Samsulhadi S, Hendarto H, et al.: CYFRA 21-1 strengthening using Urine Creatinine. [Dataset]. figshare. 2023. Publisher Full Text\n\nSchweizer J, Bowden PE, Coulombe PA, et al.: New consensus nomenclature for mammalian keratins. J. Cell Biol. 2006; 174(2): 169–174. Publisher Full Text\n\nStewart CJR, Crook ML, Lacey J, et al.: Cytokeratin 19 expression in normal endometrium and in low-grade endometrioid adenocarcinoma of the endometrium. Int. J. Gynecol. Pathol. 2011; 30(5): 484–491. PubMed Abstract \| Publisher Full Text\n\nTokushige N, Markham R, Crossett B, et al.: Discovery of a novel biomarker in the urine in women with endometriosis. Fertil. Steril. 2011; 95(1): 46–49. PubMed Abstract \| Publisher Full Text\n\nWykes CB, Clark TJ, Khan KS: Accuracy of laparoscopy in the diagnosis of endometriosis: A systematic quantitative review. BJOG. 2004; 111(11): 1204–1212. PubMed Abstract \| Publisher Full Text" }	[ { "id": "256952", "date": "28 May 2024", "name": "Simon M Scheck", "expertise": [ "Reviewer Expertise I am a gynaecologist with experience in treating patients with endometriosis medically and surgically. I have limited lab experience", "but have been involved with studies looking at cervical", "vaginal and blood based biomarkers for endoemetriosis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a larger valdiation of a previously suggested urinary marker to detect endometriosis. The novel approach of correcting using urinary creatinine is also tested and validated.\nThe methodology is overall well done and the results are promising and warrant publication to spark further research. I would like to recommend a few changes.\nThe source data provided is incomplete - the creatinine levels and the endometriosis diagnosis is not included.\nThere is very little clinical data provided about the patients - why they were undergoing surgery, how endometriosis was diagnosed, and the extent/stage of disease needs to be provided - especially relevant for application as a biomarker.\nThe discussion needs some work - is very long but doesn't actually discuss the key results or implications of these results in enough detail.\nSpecific comments:\nIntroduction\nParagraph 1: Citing specificity and sensitivity for laparoscopy vs histopathology – this is a small study from 2008 and no longer relevant – all current international guidelines accept laparoscopy as the gold standard of diagnosis – cannot compare laparoscopic techniques of 2024 with those of 2008 Paragraph 3: “Cytokeratin-19 is found in simple and complex squamous epithelium” … “it is expressed in glandular-type epithelium” - squamous or glandular?? Paragraph 3: “increasing serum creatinine levels that cause decreasing in the level of cytokeratin-19 fragments (CYFRA 21-1) in the urine accompanied the increasing its serum level in urine” – serum level in the urine does not make sense… either serum or urine? Paragraph 3: “The mean levels of cytokeratin-19 fragments (CYFRA 21-1) were significantly higher in the proliferative phase than in the secretory phase” – it is unclear what this refers to… ? Serum, urine, endometrial samples? Patients with endometriosis? Normal women?\nI think it’s important to point out that the Tokushige study of n=16 did mass spectrometry looking at 917 different “protein spots” and found that CK-19 was different – they did not hypothesize that this particular protein would be relevant – and in the small sample size and the large number of multiple comparisons, it is possible this was due to chance and therefore further large scale studies (such as this one are important)\nMethods\nWhat was the indication for laparoscopy? Infertility or pain? Did the “non endometriosis” patients include those undergoing laparoscopy for other causes, such as tubal ligation, hysterectomy, ovarian cysts, etc… How thoroughly were those patients screened for endometriosis at laparoscopy? How was endometriosis diagnosed? Laparoscopy or histology? Was any staging information recorded? Do you have information about those with endometriomas or any other detail around the level of disease? (Small volume peritoneal disease may not be the same disease process as large volume nodular disease, endometriomas, etc.) “The AUC area at 80.7%” – AUC is not a percentage Could you present the data in table 2 and table 3 as box and whisker plots? Would be easier to interpret\nDiscussion\nStart the discussion with a summary of the key findings: in the proliferative phase this study shows a very high sensitivity, specificity and AUC for CYFRA 21-1:creatinine ratio for detecting endometriosis - and make this the focus of the discussion The discussion has a lot of discussion around possible biological explanations for CYFRA 21 being excreted in urine, but not much discussion of the actual results I would like to see a more detailed discussion around:\nThe results are highly significant in the proliferative phase without the creatinine correction – but they are improved with the creatinine correction – can you directly compare with and without creatinine correction and comment on this The results in the secretory phase are less significant both with and without the creatinine correction - is it possible that normal endometrium creates CYFRA 21 in the secretory phase, but that endometriosis tissue creates CYFRA 21 at all times, making it a more useful marker in the secretory phase? Is there any data to suggest what would happen for women on menstrual suppression (e.g. progesterone treatment) which is common in endometriosis patients?\n\nIs the work 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": [] }, { "id": "278758", "date": "26 Jul 2024", "name": "Ceylan ÖZSOY HEPOKUR", "expertise": [ "Reviewer Expertise obstetrics and gynaecology", "Cancer", "mİRNA", "circRNA" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigated whether CYFRA 21-1 is a biomarker in endometrosis patients. Three groups of patients were studied. The indexing of this article is acceptable with minor revision. 1. Direct abbreviation is used in the title (CYFRA 21-1), it is recommended to write the open name to get easier citation. 2. It is recommended to express how the number of patients was chosen statistically (p power analysis).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "12166", "date": "07 Aug 2024", "name": "Hendy Hendarto", "role": "Author Response", "response": "Dear Reviewer, Thank you for reviewing our work. We hope it will give a valuable insight for the readers. Thank you again. Best regards" } ] } ]	1	https://f1000research.com/articles/13-46
https://f1000research.com/articles/13-45/v1	08 Jan 24	{ "type": "Method Article", "title": "A tutorial hub for realising the potential of tools within the SPARC ecosystem", "authors": [ "Niloofar Shahidi", "Omkar Athavale", "Yuda Munarko", "Mathias Roesler", "Kenneth Tran", "Omkar Athavale", "Yuda Munarko", "Mathias Roesler", "Kenneth Tran" ], "abstract": "The Stimulating Peripheral Activity to Relieve Conditions (SPARC) data portal is a platform that allows users to access large sets of curated data and tools. Data on the portal is sourced from researchers working in a range of disciplines applied to peripheral nerve activity, including imaging, computational biology, electrophysiology and clinical research. However, many existing tutorials and examples for SPARC do not document examples of interfaces between tools within the SPARC ecosystem. Thus, we see an opportunity to lower the barrier for entry into the SPARC ecosystem, by creating accessible guides to create workflows using multiple SPARC tools. To do this, we have developed an online hub for hosting tutorials that can guide new users through the entire workflow of acquiring data, processing it, and visualizing it, using common, open-source scientific computing tools and crucially, spanning the repertoire of currently available SPARC tools. Our approach encapsulates the workflow within a Jupyter Notebook, which is designed to be readily accessible to new users of SPARC. Having a hub of tutorials that can be easily understood will not only accelerate the growth of the SPARC community but also increase the uptake and usage of tools by members of the community.", "keywords": [ "SPARC tutorial", "biological data", "3D mapping", "FAIR data", "Codeathon" ], "content": "Events and goals\n\nThis publication is prepared as the outcome of the 2022 SPARC (Stimulating Peripheral Activity to Relieve Conditions) FAIR (Findable, Accessible, Interoperable, and Reusable Codeathon, held 6-8 August. The first edition of this event was held in July 2021. The results of the groups participating in the July 2021 SPARC FAIR Codeathon are collected and accessible on this resource page. In the 2022 SPARC FAIR Codeathon, five groups participated, and more information can be found here.\n\nThe goal of the SPARC FAIR Codeathons is to find novel ways of using the SPARC resources, data, or tools to demonstrate and enhance the FAIRness of the data.\n\n\nIntroduction\n\nThe SPARC program is a platform supported by the National Institutes of Health (NIH) Common Fund to advance bioelectronic medicine devices.1 This is done by identifying the spatial distribution of neurons on the surface of organs using flatmaps.2 The SPARC Portal hosts over a hundred datasets and resources (continuously being updated) to assist researchers in learning about organ functions, devise medical tools, and ultimately treat diseases.\n\nThe SPARC Portal emphasizes on a FAIR (Findable, Accessible, Interoperable and Reusable) repository of curated resources available to the researchers globally. The FAIR Data Principles were proposed in 2016 by Wilkinson et al. in order to increase the reusability of data.3 The SPARC project incorporates multiple open-source tools and platforms with their own documentation and tutorials. However, well-documented tutorials on how to properly utilize these tools in conjunction with SPARC data are lacking and could significantly aid users in seeing the advantages of using FAIR data.\n\nIn this Codeathon we aimed at practically improving the reusability of the SPARC Portal resources by giving visual and written instructions on how the available datasets and models can be employed, integrated, and visualized. One of the core platforms of Data Resource Center (DRC) of the SPARC program is o2S2PARC (Open Online Simulations for Stimulating Peripheral Activity to Relieve Conditions).4 Researchers can, for example, use o2S2PARC for simulating the peripheral nerve system and instantly observe its impact on pertaining organs. However, the utilization of o2S2PARC is not necessarily straightforward for general users. Therefore, to increase the FAIRness of the SPARC Portal resources, we aimed to show how one can search and use the SPARC datasets using available SPARC tools, in conjunction with other open-access tools, in a structured and comprehensible manner. We lower the barrier for the general user who might be unfamiliar with the SPARC integrated platforms (like O2S2PARC) to acquire data, process it, and visualize it using common, open-source scientific computing tools.\n\nThe purpose of this article is to present the tutorials that were developed during the 2022 SPARC FAIR Codeathon on the procedure of utilizing the SPARC portal tools and resources. These tutorials demonstrated the ease with which a range of tools in the SPARC ecosystem could be used in concert to access, manipulate and interpret SPARC datasets. As a case study, we provide an example of generating a 3D scaffold of the rat stomach and projecting the coordination data of the vagal afferent and efferent. We discuss how this tutorial differs from the existing tutorials and documentation on SPARC Portal. Finally, we describe how our tutorial style can impact the FAIRness of the SPARC Portal and possible future developments.\n\n\nMethods\n\nThis section discusses the improvement of tutorials and documentation on the utilization of the SPARC Portal tools along with the available open-access software packages such as SciPy. Figure 1 demonstrates the general workflow for the tutorials. The first step is to obtain SPARC datasets from the portal using Pennsieve. This free tool is a scalable, graph-based, and cloud-based data management platform that is used for managing scientific datasets on the SPARC Portal. It is used here to download.xlsx files. Once the files are downloaded from a project, the data can be manipulated as needed in order to perform computational analysis on it by employing a programming language such as Python or open-source tools from the SPARC Portal. After the processing step, the data can be visualised in 2D or 3D graphs. In the following, we explain these steps and the cases demonstrated in the tutorial.\n\nThe.xlsx data files are downloaded using Pennsieve and fed to a program written in Python which uses open-source tools, like those available from SPARC, for preprocessing and analysing the data. After being processed, the data can be visualised in different manners.\n\nFigure 2 illustrates the above-mentioned workflow using a previously published model of the rat stomach.5 We illustrate two applications of the acquired datasets and give detailed instructions on how they can be processed for different applications. Three datasets constitute the basis of these tutorials. They provide the 2D coordinates for different neuronal populations in the rat stomach, namely the efferent vagal neurons and afferent vagal neuron sub-populations of intraganglionic laminar endings (IGLEs) and intramuscular arrays (IMAs). These data are derived from biomedical imaging of stained rat stomach sections. The information regarding these neurons is contained in.xlsx files that are accessible on the SPARC portal from the following links (see also Underlying data):\n\n• Spatial distribution and morphometric characterization of vagal afferents associated with the myenteric plexus of the rat stomach: IGLE_data.xlsx\n\n• Spatial distribution and morphometric characterization of vagal efferents associated with the myenteric plexus of the rat stomach: Efferent_data.xlsx\n\n• Spatial distribution and morphometric characterization of vagal afferents (intramuscular arrays (IMAs)) within the longitudinal and circular muscle layers of the rat stomach: IMA_analyzed_data.xlsx\n\nThe upper panel (the general approach): The.xlsx data files are downloaded from Pennsieve and fed to Python for preprocessing (data extraction and cleaning). Using the available Scaffold Mapping Tools on the SPARC Portal, a geometric 3D scaffold of an organ is obtained. The preprocessed data can be projected on the 3D scaffold to create a data-enriched 3D image of the organ. The lower panel (the rat stomach example): Three different datasets of the spatial distribution of neurons in the rat stomach are downloaded from Pennsieve. The required 2D data (spatial coordinates of x and y) are extracted using Python. A 3D scaffold of the rat stomach is achieved using the SPARC Scaffold Mapping Tools. The preprocessed data of the spatial 2D distribution of neurons is projected on the rat stomach scaffold. The output is a 3D image of the rat stomach in which the neurons are mapped on the surface. The dots correspond to the spatial distribution of neurons and the three colors represent the three different datasets.\n\nIn this tutorial, we demonstrate a dataset acquisition mechanism that includes searching for datasets using keywords, e.g., ‘vagal,’ using the search API provided by Pennsieve with output datasets’ id, version, name, and tags. Using the search results, we can now download datasets manually or automatically, where the automatic method is facilitated by a script utilizing the discovery API that can be accessed freely. This mechanism is then helpful in searching for other datasets, which will later be used for different analysis purposes.\n\nThe datasets obtained can be in various formats, for example, JSON, dat, knowledge-based, and xlsx; for the cases we raised, the file is xlsx. Within these datasets, we conserve information regarding the neuron location, innervation area, and anatomical location, whether dorsal or ventral. The neuron’s location is a 2D feature representing the percentage relative distance to an origin situated in the pyloric end of the stomach for the y-axis (left to right direction) and near the esophageal for the z-axis (bottom to top direction). To be able to plot this location, first, we define the boundary of z-axis and y-axis based on the 3D stomach scaffold generated using the Scaffold Mapping Tools of SPARC, and again in our cases we set to [0, 36.7] and [0, 24.6] respectively, where each element is the minimum or maximum point in millimeter. This boundary can calculate the approximate neuron location in the millimeter.\n\nAfter data manipulation, one can use location data to estimate the probability density of the spatial distribution of neurons in 2D as shown in the upper panel in Figure 2. For this visualization, first, we create a 2D mesh grid with z-axis and y-axis boundaries consisting of 100 points each. Then for each neuron location, the probability density is estimated using Gaussian KDE (Kernel Density Estimation)6,7 and resampled by 1000 points. We estimate all three datasets and visualize them in 2D plots. It is interesting that estimates can use methods other than KDE for different analysis needs and datasets, such as the Cauchy and Laplacian kernels.\n\nMoreover, one can project the 2D points representing the location of neurons to a scaffold of the organ generated by the Scaffold Mapping Tools of SPARC as demonstrated in the lower panel of Figure 2. The scaffold organ obtained is a 3D mesh converted as an STL file to handle it using a Python-based package easily. Figure 3 is the representation of the 2D dataset that we map to the 3D organ scaffold. 3D mapping can be done because the datasets contain information on the anatomical location of neurons, dorsal and ventral.\n\nThe color difference indicates the type of neuron, and the color intensity indicates the area of the neuron (retrieved from [1]).\n\nThe step-by-step instructions on how to use the Python code are available on GitHub (see Software availability).\n\n\nResults\n\nIn this section, we demonstrate the graphical results from the two tutorials that were developed during the Codeathon.\n\nFigure 4 illustrates the results of the first tutorial, data resampling for density estimation simulations using KDE. The simulation includes all three datasets: vagal efferents, IGLEs, and IMAs. Within the plots, the green dot signifies the resampled point regarding vagal, while the backdrop color denotes the density of the data points. Areas in dark red indicate high density, while lighter ones represent lower density. It can be seen here that the highest concentration of vagal efferents is at 50% and above the x-axis approaching the forestomach. As for IGLEs, the distribution is relatively even, with the highest density between 10% and 50% of the x-axis. Meanwhile, IMA is concentrated in the antrum and scattered in the proximal stomach. These plots confirm the distribution of the stomach’s vagal afferent and efferent populations.5 They could be an alternative to visualize the distribution more effectively and efficiently. Here, we demonstrate that the datasets and tools provided by SPARC can be easily used and combined with other open-source tools. This tutorial module does not consider dorsal and ventral positions, naturally allowing for more precise visualization. We provide this space for learners as a learning process.\n\n(a) efferent (b) IGLE (c) IMA. Statistical processes are used to compute a probability density estimation of neurons. This density function can be used to generate and simulate neuronal activity in a network.\n\nFigure 5 illustrates the 3D mapping of the spatial distribution data to the rat stomach scaffold. The datasets for this module are the same as for the previous module, using data features on the ventral and dorsal position as well as the area of innervation. With this visualization, we can see the extent of innervation and later compare it with a 2D density plot. This area is tiny; the magnitude plot is inefficient; therefore, it can be represented well using a colour scale from white to dark green, where dark green represents the most significant area. We used the matplotlib widget module for8 interactivity so that the 3D plot could be rotated to observe the distribution of the vagal and area innervation in more detail.\n\nThe result of our tutorial is the visualization of the neurons in an interactive figure in Jupyter Notebook. The user can select which type of neurons to visualise on the surface of the stomach from three categories: vagal afferent (IGLE), vagal afferent (IMA), and vagal efferent.\n\n\nConclusion\n\nOur tutorials differ from the current tutorials on the SPARC Portal in a substantial way because they guide the users through understanding and using multiple SPARC tools to fulfil data manipulation and visualisation needs. Instead of the dense documentation and tutorials on the SPARC portal which mostly targets a single tool and is appropriate for users already familiar with the SPARC program, we have developed tutorials, along with examples, for a broader audience. We demonstrated the feasibility of using SPARC resources to drive our proposed workflow using a rat stomach scaffold as an example. A central benefit of this approach is that it leverages the FAIR principles that underlie these SPARC resources to ensure that the integrated models that are produced also follow the FAIR Data Principles. Our approach is creative because it encapsulates the workflow within a Jupyter Notebook online platform, which is designed to be readily accessible to new users of SPARC. Having tutorials that can be easily understood will not only accelerate the growth of the SPARC community but also increase the uptake and usage of tools by members of the community.\n\nThese are prototype tutorials for what we envision to be a series of tutorials hosted on a website that serves as an introduction to the SPARC ecosystem and helps new users get started with SPARC tools.", "appendix": "Data availability\n\nThe data for the rat stomach example presented as a use case for our tutorials is available from SPARC at the following links:\n\nhttps://sparc.science/datasets/10?type=dataset&datasetDetailsTab=abstract&path=files%2Fderivative\n\nhttps://sparc.science/datasets/12?type=dataset&datasetDetailsTab=files&path=files%2Fderivative\n\nhttps://sparc.science/datasets/11?type=dataset&datasetDetailsTab=files&path=files%2Fderivative\n\n\nAcknowledgements\n\nAll authors were part of the team QuiltedTutorials in the 2022 SPARC FAIR Codeathon. We would like to extend our special thanks to the SPARC Program of the NIH Common Fund and to the organisers of the 2022 SPARC FAIR Codeathon for their support during the planning and development of this project.\n\n\nReferences\n\nThe Sparc Data and Resource Center: 2022. Reference Source\n\nOsanlouy M, Bandrowski A, De Bono B, et al.: The SPARC DRC: Building a resource for the autonomic nervous system community. Front. Physiol. 2021; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: The fair guiding principles for scientific data management and stewardship. Scientific Data. 2016; 3: 1–9. 2052-4463. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nO2S2PARC: 2022. Reference Source\n\nPowley T, Hudson C, McAdams J, et al.: Vagal intramuscular arrays: The specialized mechanoreceptor arbors that innervate the smooth muscle layers of the stomach examined in the rat. J. Comp. Neurol. 2016; 524: 713–737. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott DW: Multivariate density estimation and visualization. Handbook of computational statistics: Concepts and methods. 2012; 549–569. Publisher Full Text\n\nSilverman BW: Density estimation for statistics and data analysis. Routledge; 2018.\n\nHunter JD: Matplotlib: A 2d graphics environment. Comput. Sci. Eng. 2007; 9(3): 90–95. Publisher Full Text\n\nAthavale O, Munarko Y, Tran K, et al.: SPARC-FAIR-Codeathon/QuiltedTutorials: QuiltedTutorials 1.0.1 (v1.0.1). Zenodo. [Software]. 2023. Publisher Full Text\n\n\nFootnotes\n\n1 https://sparc.science/datasets/10" }	[ { "id": "249610", "date": "28 Mar 2024", "name": "Bhavesh Patel", "expertise": [ "Reviewer Expertise Data standards", "FAIR practices", "Research software" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: The work presented here by the authors is the outcome of their participation at the 2022 Codeathon organized by the Stimulating Peripheral Activity to Relieve Conditions (SPARC) program of the NIH. The SPARC Portal provides a wealth of FAIR resources generated by researchers supported by the SPARC Program, including datasets and tools. The authors identified a lack of tutorials to conveniently understand and use the tools and datasets. To fill this gap, they developed during the Codeathon new tutorials for using SPARC resources in the form of a Jupyter Notebook. They are presenting their approach and resulting tutorials in this manuscript.\n\nRationale: Clearly expressed\nMethod: Well explained overall. Few comments/suggestion:\nThe main problem is that the SPARC datasets used are not cited. Each SPARC dataset has a DOI and the landing page of the dataset explains how the dataset should be cited if used. Mentioning them in the Data Availability section is not sufficient. Minor: Are the xlsx files downloaded manually? If so, why was the Pennsieve Discover API, which allows to query files directly, not used?\n\nReplication: Overall, enough details are provided to allow replication of the work.\nConclusion:\n\n“A central benefit of this approach is that it leverages the FAIR principles that underlie these SPARC resources to ensure that the integrated models that are produced also follow the FAIR Data Principles.” I am not sure I understand this. For the resulting Jupyter notebook to be FAIR, it would have to comply with the FAIR Principles for research software. An easy way to do that is to check that the Notebook complies with the FAIR-BioRS guidelines (https://fair-biors.org/docs/guidelines). From the information available in the Code Availability section, the Jupyter Notebook seems to be compliant or almost compliant with the FAIR-BioRS guidelines.\n\nSpecific comments on the Introduction section:\n\"The SPARC program is a platform supported by\" - I believe the authors mean \"The SPARC Portal is a platform supported by\" \"The SPARC Portal hosts over a hundred datasets and resources\" - It has been over a hundred for a while now. It is now over 300. \"The SPARC Portal emphasizes on a FAIR (Findable, Accessible, Interoperable and Reusable) repository of curated resources\" - I believe the repository itself is not FAIR but it provides FAIR resources so this would read better if formulated as \"The SPARC Portal is a repository that provides access to well curated and FAIR (Findable, Accessible, Interoperable and Reusable) datasets and other resources\" “The FAIR Data Principles were proposed in 2016 by Wilkinson et al. in order to increase the reusability of data.” It would be good to add the following afterward for completeness “Accordingly, the SPARC Program has established strict standards for structuring data and metadata as well as guidelines for sharing the resulting data to ensure it is FAIR (Bandrowski A , et al., 2021 [Ref 1]), (Marroquin C , et al., 2023 [ Ref 2]), (Osanlouy M , et al., 2021 [Ref3])” “The SPARC project incorporates multiple open-source tools and platforms” - Do the authors mean “The SPARC Portal”?\n\nTypos:\nEvents and goals section: Parenthesis is missing after \"Reusable here \"FAIR (Findable, Accessible, Interoperable, and Reusable Codeathon\" “It is used here to download.xlsx files.” → Should there be a space after “download”?\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? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "254208", "date": "30 Apr 2024", "name": "Andrey Andreev", "expertise": [ "Reviewer Expertise calcium imaging in different models and cell types", "data analysis", "reproducible data analysis and training in python-based data processing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide two data analysis examples based on public open datasets hosted by SPARC, NIH-lead initiative. The authors provide reproducible code as Jupyter notebooks hosted on github, instruction to get started are clear to someone experienced with that technology, and code runs smoothly without modification (see comment below for one exception).\nTwo comments on the code: in tutorial 1 there should be a flag or variable that defines mesh resolution. 3D rendering runs slowly on macbook pro 2020, and as a user I would like to be able to speed up 3D figure by decreasing resolution. In second tutorial line #=> data_array = data_array[~data_array.isin([np.nan]).any(1)]\ncauses an error but can be replaced with:\n#=>\n\ndata_array = data_array.dropna()\n\nAlso when downloading datasets as a user I need to see progress or ability to estimate time. For example, authors can provide total number of items expected in each dataset.\nIn the text O2S2PARC is present with capital O as well as lowercase o. Please make this consistent.\nThe provided article and code allows people unfamiliar with SPARC data (such as this reviewer) to explore datasets and gain understanding of the underlying data. However, I would like to see an example of simple analysis such a co-localization of different neuronal types.\nFigure 3 needs a legend (which color corresponds to which cell type?)\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/13-45
https://f1000research.com/articles/13-44/v1	08 Jan 24	{ "type": "Case Report", "title": "Case Report: Congenital diaphragmatic hernia in one twin followed by ex-utero intrapartum treatment (EXIT) procedure: Pearls & pitfalls", "authors": [ "Adhi Pribadi", "Dhanny Primantara Johari Santoso", "Nuniek Kharismawati", "Fatima Zahra", "Cut Razianti ZB", "Annisa Dewi Nugrahani", "Dhanny Primantara Johari Santoso", "Nuniek Kharismawati", "Fatima Zahra", "Cut Razianti ZB", "Annisa Dewi Nugrahani" ], "abstract": "Cases of congenital diaphragmatic hernia (CDH) in one twin have been reported rarely. This study aimed to report a rare case while also analyzing the pearls and pitfalls in diagnosing and managing this case. A 32-year-old multiparous woman referred to a tertiary hospital with multiple pregnancies and suspected CDH on the 1st baby. The patient underwent further investigation and the first fetus had a severe CDH on the left side. The second fetus was in a transverse lie and no other abnormalities were found. The babies were delivered through caesarean section and the first baby underwent the ex-utero intrapartum treatment (EXIT) procedure without prior antepartum fetal therapy. A large defect measuring 7.5 x 6 centimeters on the left side (Bochdalek type) was found in the first baby during surgery to make repairs. The baby died the next day due to some complications from the previous repair. As a pearl of this case, prenatal diagnosis and multidisciplinary approach hold pivotal roles. In prenatal imaging, CDH is distinguished by the visualization of thorax filled with abdominal contents and the mediastinal shifting from the defect to the contralateral side. EXIT procedures could be performed after the baby with CDH is delivered and the infant will still require post-natal surgical repair and its success rate depends on many factors. In conclusion, proper prenatal diagnosis with ultrasound evaluation from the second trimester, close and regular maternal-fetal monitoring, prenatal intervention, intrapartum ultrasound guidance, and post-natal procedures are all critical.", "keywords": [ "Case Report", "Congenital Diaphragmatic Hernia", "Ex utero Intrapartum Technique Procedures", "Hernia Repair", "Twins" ], "content": "Introduction\n\nCongenital diaphragmatic hernia occurs when abdominal contents herniate into the thoracic cavity, causing lung hypoplasia and pulmonary vascular alteration. Hernias are classified into different types based on the defects’ location in the diaphragm which are: (a) The most frequent type, Bochdalek hernias (occur in the posterolateral part of the diaphragm) which mostly occurs on the left side rather than the right side, (b) Morgagni hernias (20% to 25%), and (c) central hernias, accounting for 2% to 5% of all cases.1,2\n\nCases of CDH in one twin have rarely been reported. Studies reporting CDH in twin pregnancies are still rare and reported as cases of CDH in general. A retrospective study from 2010 to 2016 reported only 11 twin pregnancies with CDH in one of the twins.3 Infants with CDH have a higher mortality rate without prompt management, and they also have a wide range of long-term issues that last beyond infancy, such as complications of the respiratory system, gastrointestinal system, neurobehavioral disorders, and orthopaedic deformities. Hernia recurrence is another risk that can develop later, especially in newborns with significant defects. It has been established that each of these clinical issues has an impact on the quality of life of affected children. Thus, prenatal diagnosis especially with ultrasound holds a pivotal role to prevent further fetal morbidity and mortality.4,5\n\nThe majority of CDH cases are identified prenatally between 18–24 weeks of pregnancy during a standard abnormality scan. CDH is distinguished by the visualization of thorax filled with abdominal contents and the mediastinal shifting from the defect to the contralateral side in prenatal imaging. Milder cases, on the other hand, might not be discovered until later due to minor defects that lead on to cause many complications.6,7 The prognosis of CDH is influenced by the extent of lung hypoplasia, the location of the liver, and co-existing abnormalities.\n\nFetal lung volume is measured by the lung-to-head ratio (LHR) which is obtained from the contralateral lung area divided by the fetal head circumference. Fetal LHR measurements have been used to predict neonatal pulmonary hypoplasia. Between 12 and 32 weeks of gestation, there is an 18-fold increase in lung area and a four-fold increase in head circumference. Thus, LHR on the left or right-side increases exponentially with gestational age because the measurements to determine LHR differ depending on gestational age. The value of the ratio is very important, in the study by Metkus, all neonates died if the ratio reached 0.6, on the contrary, they would survive better with a survival rate of 100% if the ratio reached 1.35, while the ratio between 0.6 to 1.35 had a survival rate of 61%.8 Advances in prenatal diagnosis have made it possible to identify a number of birth anomalies that may obstruct the fetus’s airway or impede ventilation at birth. Following the confirmation of CDH diagnosis, close monitoring to check fetal well-being is strongly advised.6,8,9\n\nSelective centers in developed countries are providing fetal therapy Tracheal Occlusion To Accelerate Lung Growth Trial (TOTAL) and Fetoscopic Endoluminal Tracheal Occlusion or FETO as management for qualified patients in moderate and severe cases of CDH.10 The delivery of infants with CDH is recommended until term gestation. It is also strongly recommended that the infants are delivered at a tertiary center with expertise in CDH management and have that access to extracorporeal membrane oxygenation (ECMO) therapy.11,12 In cases of prenatally diagnosed CDH, it is advised to place a nasogastric tube shortly after delivery for stomach and intestines decompressions. Thus, in twin pregnancy, ultrasound guidance plays a critical role in determining which twin suffered from CDH. Ex utero intrapartum treatment procedures could be performed on the neonates after the baby with CDH was delivered. A variety of fetal airway obstructions have been successfully treated with EXIT procedures, enabling control of the airway at birth. The EXIT procedure can be performed on babies with CDH at birth, however, babies with CDH still need postnatal surgical repair.11–13\n\nHere, we present a case report of prenatally diagnosed CDH in one of the twins who underwent EXIT procedure without previous antepartum fetal therapy. This case is very rare since diagnosing one of the twins with CDH during the intrapartum period to determine which one will undergo EXIT procedure is quite challenging. There have been no case reports published regarding prenatally diagnosed CDH in one of the twins followed by EXIT procedure, especially in developing countries. Therefore, the aim of this study was to report a rare case as well as to analyze the pearls and difficulties in diagnosing and managing these patients.\n\n\nCase report\n\nThis following case is described according to the CARE checklist.14 A 32-year-old multiparous Indonesian woman with no occupation only a housewife, came to the Maternal Fetal Clinic, in a tertiary hospital in Indonesia, as referred by an obstetrician with multiple pregnancy and suspected CDH on the 1st baby. The mother had a history of twin babies in her family. She had no comorbidities and no previous history of surgery. The patient was compos mentis. Her vital signs, physical examination results, and laboratory profiles were within normal limits. The patient underwent further investigation with ultrasound and it revealed multiple pregnancies with placenta inserted at the posterior, positive septum confirmed monochorionic-diamniotic multiple pregnancies with discordance of 18%.\n\nThe first fetus was alive with a cephalic lie equal to 32–33 weeks of pregnancy, and an estimated fetal weight of 1,504 grams. In the thoracal region, the four-chamber view diagnostic planes showed a dextroposition with narrow left ventricle; the measured cardiothoracic area (CTAR) was 20%, and the axis was 29.13 degree. A mass (stomach) was found in this thoracal region, which was 1.98 cm × 1.20 cm in size, located as high as the four-chamber view, and the right ventricle was pushed forward anteriorly as shown in Figure 1 and Figure 2 (Video of Babies’ Ultrasound). LHR was 0.14 and observed per expected LHR (o/e LHR) was 4.79%. Ultrasound assessment showed urinary bladder filled with fluid which shows normal kidney function, shape and appearance of the fetal kidney within normal limits, also umbilical artery within normal limits. The single deepest pocket (SDP) was 5.44 cm. Further examination with Doppler velocimetry revealed middle cerebral artery pulsatile index (MCA-PI) and umbilical pulsatile index (umbilical PI) were within normal limit with normal flow of ductus venosus. No abnormality was found in other regions. These findings suggested that the first fetus had a severe left-side CDH with no lung hypoplasia, polyhydramnios, hyperexpanded lungs, and anatomic compressions were found.\n\nThe four-chamber view diagnostic planes showed a mass (stomach) (yellow arrow) was found in this thoracal region, was 1.98 cm × 1.20 cm in size. The right ventricle is pushed forward anteriorly (red arrow). LHR was 0.14 and o/e LHR was 4.79%. Lung hypoplasia prediction was also within normal limit.\n\n\n\nThis is the video recording of the four-chamber view diagnostic planes which showed a mass (stomach) that was found in this thoracal region. It was 1.98 cm × 1.20 cm in size. The right ventricle is pushed forward anteriorly. LHR was 0.14 and o/e LHR was 4.79%. Lung hypoplasia prediction was also within normal limit.\n\nhttps://doi.org/10.6084/m9.figshare.24632139\n\nThe second fetus was alive with transverse lie equal to 31–32 weeks of pregnancy and an estimated fetal weight of 1,824 grams. No abnormality was found in the thoracal and abdominal regions. Further examination with Doppler velocimetry revealed MCA-PI and umbilical pulsatile index (umbilical PI) were within normal limits with normal flow of ductus venosus. The single deepest pocket (SDP) was 5.44 cm.\n\nThree weeks later, she was admitted to the obstetric ward and had undergone expectant management until term pregnancies. Caesarean delivery was planned for the patients in our case due to an obstetric indication (transverse lie of the second fetus), which was also accompanied by CDH of the first fetus. Following the confirmation of the CDH and the completion of the imaging workup, a multidisciplinary team of obstetricians, anesthesiologists, neonatologists, and pediatric surgeons was assembled. An intervention aimed at establishing and maintaining airway patency immediately after cesarean delivery was considered mandatory, thus direct laryngoscopy intubation as the first approach in EXIT-to-airway procedure was planned.\n\nThe first baby was a male born weighing 1,600 grams with a 1st minute APGAR score of 3 and the 5th minute APGAR score was 5, while the second baby was also a male; born weighing 2,000 grams, with a 1st minute APGAR score of 6, while the 5th minute APGAR score was 8. Following informed consent, the group in charge carried out the EXIT-to-airway procedure planned previously. The babies were admitted to the neonatal intensive care unit (NICU) for general condition improvement and adaptation of pulmonary vasculature before definite surgical repair for CDH. The mother was given supportive treatments in obstetric wards. Four days later after stabilization, the first baby underwent primary repair surgery for his CDH. Intraoperative findings demonstrated left-sided Bochdalek type of CDH with a large defect size of 7.5 cm × 6 cm in size. Unfortunately, the baby died the next day due to some complications from previous CDH primary repair.\n\n\nDiscussion\n\nCDH in one twin is rarely reported. As the pearl of this case, advances in prenatal diagnosis played an important role because this method allows the detection of a number of birth anomalies that can obstruct the fetal airway or obstruct ventilation at birth. Therefore, this condition should be referred to a tertiary center with standard neonatal management in the NICU and optimal pediatric surgery services which will improve outcomes.15 More than half of all CDH cases are discovered prenatally in the second trimester. However, in this case, the patient was diagnosed in the third trimester in tertiary care, because it was not diagnosed earlier by a specialist or perhaps because it had an initial minor defect that was not identified until the third trimester.6,7,16 Thus, accurate prediction of outcome is essential because prenatal interventions, which currently consist of FETO, may be beneficial for the fetus with the worst prognosis. The location of the defect, the estimated lung size, and the presence of liver herniation, predicted or measured by ultrasound and MRI, are used to make predictions and case-specific prognoses.\n\nIn prenatal imaging, CDH is distinguished by the visualization of thorax filled with abdominal contents and the mediastinal shifting from the defect to the contralateral side.6 The size of the heart is tiny (CTAR 20%), especially the left ventricle due to mass pressure from the left. In this case, the stomach pressure had a 100% impact on the small size of the heart. Pressure by other organs on the intrathoracic side affect the effectiveness of the heart’s workload, causing an urgent effect on the heart when it beats. The heart does not have enough space during diastole because of pressure from that organ. In the end the amount of blood pumped by the heart is limited because of the limitations of the heart expanding during diastole.7 Metkus et al.8 first described the lung-to-head ratio (LHR). LHR provides an indirect estimation of a lung size contralateral to the hernia. It is commonly measured using two-dimensional ultrasound at the four-chamber view by dividing the contralateral lung area divided by the fetal head circumference. In this case the LHR ratio was only 0.14. When referring to the Metkus study, postpartum babies have a poor prognosis and are unlikely to survive because the LHR ratio is below 0.6.8 The most precise techniques for measuring the lung include tracing its contours or applying the long axis method. Severe lung hypoplasia is considered when the o/e LHR in left-sided CDH (LCDH) is <25%. However, if LHR 25% with o/e LHR 35% regardless of liver position, or LHR 35% with o/e LHR 45% with “up” type of liver position, this case is considered a “moderate” level even though the survival rate is limited to only 50–60% and survivors have significant morbidity. An o/e LHR emerges as a better predictor of fetal survival.6–8 The patient in this case was diagnosed prenatally with CDH on the first fetus with an o/e LHR of 4.79%, thus this condition was considered a severe CDH.\n\nIt is highly recommended to closely monitor and check the well-being of the fetus after confirming the diagnosis of CDH.6,8,9 Eligible patients can be offered intrauterine management such as TOTAL and FETO especially in cases of moderate or severe CDH. However, because both of these invasive procedures were performed intrauterine, the potential for preterm labor due to rupture of the membranes or the emergence of early labor must also be considered. However, studies have shown that the group of fetuses that underwent this procedure had a higher survival rate than the control group.16 One advantage of the EXIT procedure is that it allows intubation and ventilation during labor. However, the patient and family refused prenatal intervention in this case after informed consent was obtained, with the consideration that the family refused because it could worsen the prognosis of the first fetus if there was an earlier delivery.\n\nThe delivery of infants with CDH is recommended until term gestation. Therefore, patients underwent expectative management until term pregnancy in this case.17 As another pearl, diagnosing one of the twins with CDH during intrapartum period to determine which one will undergo EXIT procedure is quite challenging. Therefore, many aspects are required to be considered in evaluating the diagnosis and therapy. Following patient consent after delivery, the assembled group carried out the EXIT-to-airway procedure planned. With an overall survival rate of 83%, the EXIT-to-airway procedure reduced mortality of neonates. Nonetheless, maternal and fetal complications persist. The fetus is at risk of barotrauma and inadequate ventilation during airway securing, hence appropriate tube placement must be confirmed before cord clamping.18\n\nAll infants undergoing intrauterine fetal surgery still require postnatal CDH repair and the success rate depends on many factors.11–13,15 So far, CDH has been managed as a disorder requiring emergency surgery for many years, but even so, if surgical therapy is successful, there are still other pathological conditions, namely a relatively frequent threat of death, often from pulmonary hypertension (PH). Previous studies have challenged the practice of sending newborns directly from the delivery room to the operating room and have found no appreciable therapeutic benefit in the presence of pulmonary hypertension. There is currently advice for surgical correction to be delayed for at least 48 to 72 hours after delivery to allow time for the pulmonary vessels to adapt. Surgery is delayed longer in children with severe PH until the condition is effectively controlled. If ECMO is required, the survival rate of newborns with CDH drops to approximately 50%.18\n\nHistorically, CDH was treated surgically through a subcostal abdominal incision. Nowadays, thoracoscopic surgery is used to conduct many CDH repairs on stable children since minimally invasive surgery has become increasingly popular over time.19 The use of minimally invasive procedures may reduce postoperative discomfort and prevent thoracotomy and laparotomy-related complications. When the diaphragmatic defect is significant, a primary repair may be impractical or impossible. Based on the defect size in this patient, the patient underwent a primary repair. To fill the defect, one may employ an abdominal or thoracic muscle flap, a biologic patch, or a prosthetic patch.20 However, all of these treatments carry the risk of future surgical failure and CDH recurrence. In this case, the baby died the next day due to some complications from previous CDH primary repair using synthetic patches. Synthetic patches were chosen as widely used patches of defect closure, but have been implicated as a significant contributor to morbidity and further complications in recent studies.20,21 This condition might be also affected by technical rather than intrinsic to the patch. Thus, considering the risk and benefit of surgical method based on the defect size and patches or mesh used become important to determine patient’s survival expectations.\n\n\nConclusion\n\nAlthough CDH in one of the twins is rarely reported, it has a high morbidity and mortality rate. The diagnosis and management are quite challenging since they involve several specialists as well as the adoption of particular uncommon procedures in clinical practices. In consequences, proper prenatal diagnosis using ultrasound since the second trimester, close and regular maternal-fetal monitoring, prenatal intervention, intrapartum ultrasound guidance, and post-natal procedures hold a pivotal role. Accurate preoperative planning with a multidisciplinary approach, followed by all necessary precautions and procedures are crucial for a successful EXIT-to-airway procedure. The pearls and pitfalls previously described become considerations for further provision and guidelines for CDH.\n\nThe patient would be happy to see if all physician all over the world will learn from her baby’s case. She did not mind if her baby condition is discussed in this report in order to prevent another same case occur in the future.\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Data 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: CARE checklist for ‘Case Report: Congenital diaphragmatic hernia in one twin followed by ex-utero intrapartum treatment (EXIT) procedure: Pearls & pitfalls’. https://doi.org/10.17605/OSF.IO/W2TX8. 14\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nThe great gratitude to Department Obstetrics & Gynecology, Faculty of Medicine, University of Padjadjaran/Dr. Hasan Sadikin General Hospital.\n\n\nReferences\n\nKosiński P, Wielgoś M: Congenital diaphragmatic hernia: pathogenesis, prenatal diagnosis and management - literature review. Ginekol. Pol. 2017; 88(1): 24–30. PubMed Abstract \| Publisher Full Text\n\nMontedonico S, Nakazawa N, Puri P: Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr. Surg. Int. 2008; 24(7): 755–761. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang W, Pan W, Chen J, et al.: Outcomes of Congenital Diaphragmatic Hernia in One of the Twins. Am. J. Perinatol. 2019; 36(12): 1304–1309. PubMed Abstract \| Publisher Full Text\n\nCordier AG, Russo FM, Deprest J, et al.: Prenatal diagnosis, imaging, and prognosis in Congenital Diaphragmatic Hernia. Semin. Perinatol. 2020; 44(1): 51163. Epub 2019 Jul 30. PubMed Abstract \| Publisher Full Text\n\nJani J, Nicolaides KH, Keller RL, et al.: Observed to expected lung area to head circumference ratio in the prediction of survival in fetuses with isolated diaphragmatic hernia. Ultrasound Obstet. Gynecol. 2007; 30(1): 67–71. PubMed Abstract \| Publisher Full Text\n\nShue EH, Miniati D, Lee H: Advances in prenatal diagnosis and treatment of congenital diaphragmatic hernia. Clin. Perinatol. 2012; 39(2): 289–300. PubMed Abstract \| Publisher Full Text\n\nPribadi A, et al.: Adverse effect left congenital diaphragmatic hernia to position and function of the fetal heart. Int. J. Reprod. Contracept. Obstet. Gynecol. 2015; 4(6): 1863–1867. Publisher Full Text\n\nMetkus AP, Filly RA, Stringer MD, et al.: Sonographic predictors of survival in fetal diaphragmatic hernia. J. Pediatr. Surg. 1996; 31(1): 148–152. discussion 151-2. PubMed Abstract \| Publisher Full Text\n\nBelfort MA, Olutoye OO, Cass DL, et al.: Feasibility and Outcomes of Fetoscopic Tracheal Occlusion for Severe Left Diaphragmatic Hernia. Obstet. Gynecol. 2017; 129(1): 20–29. PubMed Abstract \| Publisher Full Text\n\nVan der Veeken L, Russo FM, De Catte L, et al.: Fetoscopic endoluminal tracheal occlusion and reestablishment of fetal airways for congenital diaphragmatic hernia. Gynecol. Surg. 2018; 15(1): 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Maary J, Eastwood MP, Russo FM, et al.: Fetal Tracheal Occlusion for Severe Pulmonary Hypoplasia in Isolated Congenital Diaphragmatic Hernia: A Systematic Review and Meta-analysis of Survival. Ann. Surg. 2016; 264(6): 929–933. PubMed Abstract \| Publisher Full Text\n\nFoglia EE, Ades A, Hedrick HL, et al.: Initiating resuscitation before umbilical cord clamping in infants with congenital diaphragmatic hernia: a pilot feasibility trial. Arch. Dis. Child Fetal Neonatal. 2020; 105(3): 322–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKays DW: ECMO in CDH: Is there a role? Semin. Pediatr. Surg. 2017; 26(3): 166–170. PubMed Abstract \| Publisher Full Text\n\nPribadi A: Case Report: Congenital diaphragmatic hernia in one of twins followed by ex-utero intrapartum treatment (EXIT) procedure: Pearls & pitfalls. OSF 2023. Publisher Full Text\n\nCanadian Congenital Diaphragmatic Hernia CollaborativePuligandla PS, Skarsgard ED, et al.: Diagnosis and management of congenital diaphragmatic hernia: a clinical practice guideline. CMAJ. 2018; 29; 190(4): E103–E112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHutcheon JA, Butler B, Lisonkova S, et al.: Timing of delivery for pregnancies with congenital diaphragmatic hernia. BJOG. 2010; 117(13): 1658–1662. Publisher Full Text\n\nSengupta S, Carrion V, Shelton J, et al.: Adverse neonatal outcomes associated with early-term birth. JAMA Pediatr. 2013; 167(11): 1053–1059. Erratum in: JAMA Pediatr. 2014;168(1):53. Publisher Full Text\n\nLefebvre C, Rakza T, Weslinck N: et al; French CDH Study Group. Feasibility and safety of intact cord resuscitation in newborn infants with congenital diaphragmatic hernia (CDH). Resuscitation. 2017; 120: 20–25. PubMed Abstract \| Publisher Full Text\n\nSnoek KG, Reiss IK, Greenough A, et al.: Standardized Postnatal Management of Infants with Congenital Diaphragmatic Hernia in Europe: The CDH EURO Consortium Consensus - 2015 Update. Neonatology. 2016; 110(1): 66–74. PubMed Abstract \| Publisher Full Text\n\nTsao K, Lally PA, Lally KP, et al.: Minimally invasive repair of congenital diaphragmatic hernia. J. Pediatr. Surg. 2011; 46(6): 1158–1164. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamal TR, Tyraskis A, Ghattaura H, et al.: Synthetic versus Biological Patches for CDH: A Comparison of Recurrence Rates and Adverse Events, Systematic Review, and Meta-Analysis. Eur. J. Pediatr. Surg. 2022. Publisher Full Text" }	[ { "id": "259539", "date": "03 Apr 2024", "name": "Kazunori Masahata", "expertise": [ "Reviewer Expertise Pediatric 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\nThe paper by Pribadi et al. entitled “Congenital diaphragmatic hernia in one twin followed by ex-utero intrapartum treatment (EXIT) procedure: Pearls & pitfalls” is an interesting case report. The authors presented a rare case report of prenatally diagnosed CDH in one of the twins who underwent EXIT procedure without previous antepartum fetal therapy, especially in developing countries. They concluded that proper prenatal diagnosis with ultrasound evaluation from the second trimester and accurate preoperative planning with a multidisciplinary approach, followed by all necessary precautions and procedures are crucial for a successful EXIT-to-airway procedure. In general, this is a well-written paper that presents important findings. However, this paper needs major revisions to be accepted.\nI have the following concerns. ・Regarding CDH, several prenatal prognostic parameters of postnatal survival in fetuses with CDH such as lung-to-thorax transverse area ratio (L/T raio), observed-to-expected total fetal lung volume (o/e-TFLV), liver and stomach herniated into the thorax, have been reported. In the Introduction section, these prenatal predictors of CDH should be described. ・In the Introduction section, please describe the respiratory management of gentile ventilation in CDH and the defect size of the diaphragm, which was classified into the four scales from A to D as described by the CDHSG. The size of the diaphragmatic defect is associated with mortality rate and appears to be an important factor determining outcomes in patients with CDH. ・Regarding the presentation of the case, did the fetal echocardiographic findings show herniation of the liver into the chest? ・The figure legend describes that “lung hypoplasia prediction was also within normal limits,” but the LHR was 0.14 and the o/e LHR was 4.79%, which is severe CDH. Please correct this sentence. ・Please present the demographic data such as gestational age at birth, the use of ventilator settings and modes, pulmonary vasodilators, use of inhaled nitric oxide (NO), and ECMO support. In addition, postnatal echocardiographic findings including patent ductus arteriosus shunt flow should be presented. We believe it would help the reader to better understand the severity of pulmonary hypertension after birth. ・Please describe in detail the intraoperative findings and procedure for CDH repair. What defect size of the diaphragm is this case classified as a CDHSG system (A-D)? Did the surgical repair of the diaphragmatic defect perform by either closing the defect primarily or by means of a patch to reconstruct the diaphragm?\n\n・Please provide intraoperative photographs if available. In addition, please describe any organs that are herniated into the thorax during surgery, as very little information is available at the surgery. ・Please discuss and describe in detail the postoperative course and cause of death.\nRegarding EXIT procedure, ・Please describe the surgical procedure of EXIT in detail: anesthesia method and fetal anesthesia for EXIT, method of airway management, time from the fetal head exposure to endotracheal intubation, time on placental support, total anesthesia or operative time, and amount of intraoperative blood loss.\n\n・What medications were used to maintain to uterine relaxation during the EXIT procedure? Please describe in detail.\n\n・In general, during the EXIT procedure, the mother requires general anesthesia and is in danger of increased blood loss due to the uterine relaxation, which is required to preserve the uteroplacental exchange. Did this mother need a blood transfusion?\n\n・Although the EXIT procedure has been reported to be safe and effective, please discuss whether the procedure was an appropriate treatment for the case. I speculate that even if the EXIT procedure is given to extremely severe patient with CDH, it is too difficult to save this case. ・I would like to ask the authors the following comments: If a patient with the same severity of illness came to you, would you perform the EXIT procedure in the same way as you did this time?\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "254457", "date": "30 May 2024", "name": "Lishuang Ma", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aimed to report a rare case while also analyzing the pearls and pitfalls in diagnosing and managing this case. However, throughout the full text, the article did not describe the highlights of the experience of the diagnosis and treatment of this patient, nor extracted the lessons that should be learned and the hidden dangers that need to be avoided, but generally made a literature review, and the cited literature was older. It is not unusual for one twin to have CDH. Therefore, this article does not reflect the scientific value and guiding significance for clinical practice.\n\n1.\n\n\"Milder cases, on the other hand, might not be discovered until later due to minor defects that lead on to cause many complications.\"\nSmaller and less severe defects may be detected later, and they may present clinically slightly after birth, some even without respiratory or circulatory disturbances, but only incidentally when chest radiographs are examined for other reasons. However, incarceration of herniated thoracic organs may also occur with critical illness and be first diagnosed. This is inconsistent with the authors' view.\n2.\n\n\"This case is very rare since diagnosing one of the twins with CDH during the intrapartum period to determine which one will undergo EXIT procedure is quite challenging.\"\nWe all know that it is a challenge for twin children to distinguish diaphragmatic hernia, but it is not difficult to judge by prenatal and intrapartum ultrasonography, or what are the key points and difficulties in this process, please elaborate.\n3.\n\n\"The first baby was a male born weighing 1,600 grams with a 1st minute APGAR score of 3 and the 5th minute APGAR score was 5.\"\nDescribe in detail how to judge a fetus with diaphragmatic hernia during EXIT. Why is this process challenging?\n4.\n\n\"Unfortunately, the baby died the next day due to some complications from previous CDH primary repair.\"\nWhat complication? What is it related to? Please specify.\n5.\n\n\"As the pearl of this case, advances in prenatal diagnosis played an important role because this method allows the detection of a number of birth anomalies that can obstruct the fetal airway or obstruct ventilation at birth.\"\nThere was no particular point in this case. The birth process for one twin to have CDH is indeed crucial, but not specific for the EXIT process. Please specify.\n6. \"The patient in this case was diagnosed prenatally with CDH on the first fetus with an o/e LHR of 4.79%, thus this condition was considered a severe CDH.\"\nLung head ratio and o/e lung head ratio are indeed very important for the assessment of CDH, but the accuracy of detection is not optimistic in clinical tests. However, for the children in this treatise, please give necessary explanation on what is the special significance and how it is different from the evaluation of singleton CDH children.\n7.\n\n\"As another pearl, diagnosing one of the twins with CDH during intrapartum period to determine which one will undergo EXIT procedure is quite challenging.\"\nWe all know that it is a challenge to distinguish diaphragmatic hernia of one twin, but it is not difficult to judge by prenatal and intrapartum ultrasonography, or what are the key points and difficulties in this process, please elaborate.\n8.\n\n\"In this case, the baby died the next day due to some complications from previous CDH primary repair using synthetic patches.\"\nWhat mesh related complications can lead to the child 's death the day after surgery? This should be highlighted.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] } ]	1	https://f1000research.com/articles/13-44
https://f1000research.com/articles/13-43/v1	08 Jan 24	{ "type": "Software Tool Article", "title": "MotionRender: A simple Python implementation of video motion visualization for 3D motion capture data", "authors": [ "Derek Harter", "Shulan Lu", "Shulan Lu" ], "abstract": "We describe a Python library project for generating animations of simple 3D motion capture data points. The motion capture method is agnostic with respect to this visualizaiton tool, expecting a time series of time stamped coordinates captured in a 3-dimensional space. One of the advantages of this tool is the simple data format design. This tool was developed originally for a Kinect system implementing skeleton tracking of 15 joint positions, where each data point consists of an accurate time of capture (time stamp), and 3 accurate coordinate (x, y, z) positions of each of the 15 joints at each time step. In addition to the motion capture time series, all that is needed is a joint graph file describing the relationship of the joint edges to one another. This library can be extended to visualize and create animations of motion capture data that can be reformulated using this basic structure of a time series of time stamped 3D coordinates and a joint graph description file. This library should be useful as is, or with easy modification, for many such visualization requirements of similar motion capture data.", "keywords": [ "motion tracking", "skeleton tracking", "scientific visualization" ], "content": "Introduction\n\nMotion capture devices range from simple comsumer grade devices, like the Kinect camera-based depth sensors1,2 to more serious motion capture systems3,4 with fine spatial and fast temporal resolutions used for generating mocap video rendering.\n\nCreating animated visualization in the base Python 3.X scientific python stack is certainly much simpler than it has been in the past, but still requires many specialized steps to achieve the task of creating an animation that can be saved as a movie for presentation. The standard Matplotlib animation library documentation5 contains several examples of generated animations, but only 1 of animating a 3D set of data.6 The basic technique to use an instance of matplotlib.animation to generate a sequence of frames from a set of 3D points is clear from the documentation, but can certainly be difficult to generalize for much more complex data than showing the basic example.\n\n3D motion capture data are a very useful type of data that needs to be visualized to analyze its significance. The goal of the Motionrender library is to simplify the task of creating animated visualizations of such 3D time series data. The library presented here can be generalized to work with a time series of N 3D points in space to create an animation of subsequent frames of the captured points evolving over time. The only additional general information needed is a matrix of the relationship of the N points in space that should be connected as being related points in the resulting animation, known as a joint graph or joint edge graph by the library.\n\nWe used motion capture from a very basic Kinect skeleton tracking experiment, using the NiTE 2.0 library7,8 which includes skeleton tracking capture in the development of this visualizaiton library. The NiTE library is available from Nuitrack and provides an API to more easily motion track skeleton position points of a user from a standard Kinect device. This is one of several API libraries available for the Kinect device to allow one to develop skeleton tracking applications.\n\nHowever the general format, described in methods, can be extended to many kinds of motion capture devices where a timestamp and a set of 3D point positions can be obtained for the captured object in motion. In the example implementation discussed, we have N=15 joint positions (e.g. head, neck, left shoulder, etc.) captured by the motion Kinect depth sensing cameras. An example of a rendered frame of the motion capture animation is shown in Figure 1. Sample videos of the resulting animation are available in the repository for this project, referenced below.\n\n\nMethods\n\nThe model is available from GitHub and is archived with Zenodo.10\n\nThe version of the MotionRender library described here was developed on a Python 3.X scientific tool stack environment. It has dependencies on the pandas and matplotlib python libraries. In addtion, for back end rendering, the ffmpeg tool (free and open source) or similar tool is needed.\n\nWe have 15 joint positions in the example subject motion captured data sets used in developing the library. Each joint position is captured approximately 30 times per second by the Kinect sensor programmed with the NiTE 2.0 skeleton tracking library software. So while the library was generated specifically with 15 3D motion capture points, the general format of the input data needed to render a 3D animation is\n\nas a standard comma separated value (csv) data file, where N can be specified when rendering a motion capture animation of how many joint/position points are in the input. The only other information needed by the library is a joint connection graph of the points if we want to visualize the relationship of the rendered points to one another while being animated. Again using the Kinect 15 joint position data, we specify a joint graph for the software like this:\n\nThe user specifies the joint connection graph as a second input to the library for operation. The symbolic names of the joints must match the names in the time stamped motion capture time series of points. For example, the full Kinect connection graph of the 15 captured joints is specified as:\n\nMotionRender requires matplotlib and pandas libraries for its operation.\n\nIn addition the ffmpeg library is used as a back end frame renderer for creating video files.\n\nInstall the MotionRender library from standard PyPi python library.\n\nThe library expects a standard csv file of values, where the first column is a time stamp. Subsequent columns are the data for motion capture point 0 (x, y, z), motion capture point 1, etc. So given 15 motion capture points, this library expects and array with 46 columns, where the first column is a time stamp, and the subsequent columns are the 3D positions for all points captured. This could be loaded from a csv file, or generated from some other source.\n\nThe second parameter corresponds to the joint graph shown above. This is expected to be a space separated file of edge relations defined between the joint position names. The joint position names for the time series capture data should match the joint position names in the joint graph. The library currently contains to main API functions that may be used. The render_frame() member funtction of the main MotionRender class renders single frames from the motion capture data. While the render_animation() function renders video files. Actually the default behavior of both of these methods is to return figure and animation object instances respectively. However, the user can supply a file name to these API calls and the figure/video will then be saved in the specified file. Following is a quick example of using the library interactively to read in motion capture data, and render it as figures and videos.\n\nAdditional files are included in the main directory of the example project, that includes the data file use cases shown here. The test files contain additional examples of configuring rendering options of the library. You can invoke them in the example project as:\n\nIn addition there is also a jupyter notebook present in the example project named test.ipynb that shows further how the library can be used interactively.\n\n\nUse Cases\n\nAs a use case, we use the MotionRender library and generate animations of a set of Kinect motion capture data. This data was used in the analysis of9 research paper. This data and the software workflow to render participants in the experiments can be found in the source code repository listed below. Two samples of the kinect motion capture data, from a sitting and standing participant respectively, can be found in the example project. These files give further examples of the data format expected by the input files for the library.\n\n\nLimitations\n\nThis library relies on current version of Python and matplotlib animation library. Data is expected in the needed format, which may not always be simple to massage into depending on the motion capture device output. A rendering background tool, such as ffmpeg must also be installed and accessable to matplotlib.\n\n\nConclusions\n\nThe MotionRender library documented here provides a small tool that may be of use to those with motion capture data, or similar data sets that consist of a time series of points captured at intervals. The tool uses a simple data format, needing a flat file of captured points and another file defining the point graph relationships for rendering. The python matplotlib animation facilities are a bit hard to use. It is hoped this library would fill a gap and allow for easier rendering of motion capture data.\n\n\nSoftware availability\n\n\n\n• Software available from: https://test.pypi.org/project/motionrender/1.0.0/\n\n• Source code available from: https://github.com/DerekHarter/motionrender-example\n\n• Archived source code at the time of publication: https://doi.org/10.5281/zenodo.8172972.10\n\n• The development repository for this library, if you are interested in the code internals, can be found here: https://github.com/DerekHarter/motionrender\n\n• License: GPL-3", "appendix": "Data availability\n\nThe underlying data has been deposited in:\n\nZenodo: Motion Render Example Project, https://doi.org/10.5281/zenodo.8172972. 10\n\nThis project contains the following underlying data:\n\n‐ [data/sitting-joint-graph.csv] Joint graph definition for sitting subject.\n\n‐ [data/sitting-subject.csv] Example motion capture data for sitting subject.\n\n‐ [data/standing-joint-graph.csv] Joint graph definition for standing subject.\n\n‐ [data/standing-subject.csv] Example motion capture data for standing subject.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThanks to Jonathan Z. Bakdash and Laura R. Marusich from Army Research Lab (ARL) for input and feedback on this codebase.\n\n\nReferences\n\nPfister A, West AM, Bronner S, et al.: Comparative abilities of microsoft kinect and vicon 3d motion capture for gait analysis. J. Med. Eng. Technol. 2014; 38(5): 274–280. PubMed Abstract \| Publisher Full Text\n\nNapoli A, Glass S, Ward C, et al.: Performance analysis of a generalized motion capture system using microsoft kinect 2.0. Biomed. Signal Process Control. 2017; 38: 265–280. Publisher Full Text\n\nBregler C: Motion capture technology for entertainment [in the spotlight]. IEEE Signal Process. Mag. 2007; 24(6): 158–160. Publisher Full Text\n\nMenolotto M, Komaris D-S, Tedesco S, et al.: Motion capture technology in industrial applications: A systematic review. Sensors. 2020; 20(19): 5687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatplotlib 3.6.0 Stable Release Documentation: Matplotlib.animation library.2022. Reference Source\n\nMatplotlib 3.6.0 Stable Release Documentation: Animated 3d random walk.2022. Reference Source\n\nPrimeSense: Nite 2.0 library documentation.2022. Reference Source\n\nNuitrack: Nuitrack sdk: 3d body (skeletal) tracking middleware.2022. Reference Source\n\nCaron EE, Marusich LR, Bakdash JZ, et al.: The influence of posture on attention. Exp. Psychol. 2022; 69(6): 295–307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarter D: DerekHarter/motionrender-example: Release 1.0.0 (v1.0.0). [Dataset] Zenodo. 2023. Publisher Full Text" }	[ { "id": "277962", "date": "05 Jun 2024", "name": "Eshin Jolly", "expertise": [ "Reviewer Expertise computational social 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\nMotionrender seems like a great bespoke tool for visualizing 3D motion capture data. I can confirm this after successfully downloading the package, running the test-suite, and running the example code. I just had a few minor suggestions that might aid in wider adoption if the authors desire: - Adding matplotlib and pandas as dependency to the project – I’ve opened a pull-request on github to help facilitate this - Adding a documentation-site auto-generated from docstrings e.g. using sphinx or mkdocs in python - Until a docs site is made I would recommend including the example in the paper, on the README page of the repo as an illustrative example\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/13-43
https://f1000research.com/articles/13-42/v1	08 Jan 24	{ "type": "Opinion Article", "title": "Building Your Dream Team: How Indie Teams Can Form, and Thrive Together", "authors": [ "Minhaz-Us-Salakeen Fahme", "Afia Adiba", "Afia Adiba" ], "abstract": "The majority of indie game development teams are driven by the desire to produce a blockbuster game. But there are many obstacles on the way to realizing this desire, especially in the beginning. It is possible to create a game alone, but more often than not, something genuinely amazing involves a group of talented people working together toward a similar objective.\nFinding the correct stakeholders is one of the biggest problems with starting an independent game development team. People with the required technical talents, such as programmers and artists, as well as those with business acumen, like marketing and finance experts, are included in this. Success depends on being able to locate these people and manage them. But after the correct individuals have been identified, it is crucial to develop open lines of communication and a common comprehension of the project's objectives and expectations.\nFinding the perfect people to join their team is a problem that independent game development teams frequently encounter. Attracting talented individuals to a brand-new, unproven initiative might be difficult. The attention of skilled people interested in working on cutting-edge and intriguing initiatives might be attracted through networking.\nThere are other things to consider as well since forming a team comes with the type of game one is making, finding skilled teammates, team chemistry, conflict resolution, communication, long-term relation maintenance and leadership aspects.\nIn general, creating and leading an independent game team is a difficult process. There is no specific method for doing this, but with a little help from others' expertise, it is possible to assemble a team of creative people who can collaborate to produce a game that is genuinely enjoyable.", "keywords": [ "Indie team", "head-hunting", "people management", "communication", "team chemistry", "conflict resolution" ], "content": "1. Introduction\n\nThe gaming industry has undergone a considerable transition in recent years toward indie game creation. Globally, the number of independent game studios has grown significantly in numerous nations as a result of the democratization of the game production industry (fungies.io, 2023). While the majority of independent game creators are located in the United States, United Kingdom, Canada, and Australia, there are a number of developing nations that are making their mark in the sector. These nations include Kazakhstan, Poland, India, and Romania (fungies.io, 2023). This development has given game creators the ability to produce profitable games without the need for financial assistance from publishers, opening up new possibilities for aspirant game developers to realize their vision for a game with a group of independent developers. Since game creation requires numerous jobs that are often not carried out by a single person, collaboration is essential for success. To maximize the likelihood of success, it is imperative to find others who possess the relevant skills. In light of this, it is important to take into account the common positions held by programmers, artists, and designers in independent game production teams. Even if a person is not interested in starting their own team, knowing the important roles in independent teams can help them concentrate on honing their talents and better prepare for possibilities to join another team. The global market for independent video games is predicted to grow at an unprecedented rate, reaching $305 billion by 2025 (Global Data). A growing number of developers who want to leave their imprint on the business are being drawn in by this expansion. Many independent (indie) game developers understand the need for a skilled team to realize their vision, even though others may want to work alone, but creating and leading a team may be a difficult task.\n\nThe advantages of developing an indie game have been a topic of interest in the gaming industry for the past few years. Contrary to major corporations, independent game developers typically work in teams of up to 10 to create indie games. Despite not always referring to games created by amateurs, the phrase “indie game” is typically used to describe games that are purposefully produced and disseminated using techniques different from those employed by the large video game companies (Lipkin, 2012). To exemplify for better context, first, a strike team is a small, independent squad. Strike teams, such as Special Weapons and Tactics (SWAT) teams, rescue teams, and firefighters, are groups that perform tasks in perilous and time-critical circumstances in real world. In traditional game development, the term ‘strike team’ is frequently used to describe an unplanned small team formed within a bigger team to finish a specific difficult task. According to a Quora post by a game developer who has worked on both indie and AAA (not formal classification, refers to high profile high budget projects) projects, “these teams are small, and thanks to their interdisciplinary structure and a fluid information flow they can quickly deal with an issue” (Freeman et al., 2020). These games rely primarily on word-of-mouth advertising, social media, streaming platforms, and YouTube to promote their games because they lack the financial backing and extensive marketing teams of major game developers (The rise of indie games). Independent game creators, however, can benefit from greater creative flexibility, flexible work schedules, and an opportunity to advance their careers with a smaller team. Surveys were done on why people would prefer to join an independent team by Freeman and McNeese (2019), and some noteworthy responses will be quoted here: one participant stated “I’ve found the indie field to be filled with extremely friendly, creative, and passionate people, more so than any other industry. It is very small and tight knit. Indies tend not to see each other as competitors, and always do whatever they can to help each other get that big break.” Another participant pointed out a very significant reason to join an indie team; “Independence really boils down to a game creator that owns their creation and intellectual property. They aren’t making someone else’s vision or owned by a publisher. Indies can work with publishers and the typical investment thing happens where a publisher will take some of the profits, but in large part, the indie owns what they make.” Independent game development is a fantastic approach for recent graduates or those new to programming to broaden their skills. How can one make their ambitions of creating independent video games a reality?\n\nThe number of independent game developers has increased dramatically in recent years as they seek to produce original games. According to recent polls, the majority of independent game developers prefer to work in teams. It is found in an International Game Developers Association (IGDA) poll that 62% of independent game developers prefer to work in teams of between two and 10 individuals, while 47% prefer to work in teams of two to five (The International Game Developers Association (IGDA) Developer Satisfaction Survey 2019 Summary Report, 2023). According to the same poll, programming, art, and design are the most prevalent roles in these teams. Some of the most frequently claimed benefits according to this study of working in a team are collaboration, merging complementing skill sets, and burden sharing. IGDA Developer Satisfaction Survey 2019 found that while the industry still lacks gender diversity, indie game development teams are typically more diverse in terms of age and experience, with 33% of indie game developers being aged 25-34 and 41% having less than five years of industry experience. The average cost of creating a mobile game is $3000 to $150000 depending on the sort of game (HitBerry Games), but managing an independent game production team can also be pricey. According to a survey by the Game Developers Conference (GDC), 63% of independent game creators rely on their own personal funds as their main source of income. With these difficulties and possibilities in mind, it is critical for independent game developers to comprehend how to assemble and lead a productive team that can realize their vision. Nevertheless, team communication and management remain the biggest challenges for 53% of developers, according to the IGDA survey.\n\nEstablishing and managing the teams provide particular difficulties for independent game development teams. At the Game Developers Conference 2015, there took place a Producer Panel: Managing Your Indie Team and the speakers were Kara Kono, Jenna Hoffstein, Aaron Isaksen, Alex Schwartz and Amy Dallas. who examined the difficulties independent teams encounter while forming productive game development teams (GDC, 2015, Link: https://www.gdcvault.com/play/1022225/Producer-Panel-Managing-Your-Indie). They emphasize how crucial team dynamics, cooperation, and communication are to succeed in the independent game production sector. In order to promote creativity and productivity, the study underlines the need for a diversified skillset within the team as well as effective management tactics.\n\nFrom my experience in the industry, it is exciting to build an independent team. The process of developing an independent game can be made or broken by choosing the correct team. Sifting through potential team members can be difficult due to the enormous talent pool available. From the several involvements in the indie team, it was observed that as soon as the enthusiasm sets in for being a part of an indie team, a ton of questions, uncertainties, and decision-making points appear like a cloud in the mind. This article delves into the complex world of managing and growing indie teams, covering the issues, tactics, and tensions that arise. For aspiring independent team owners and managers, insights and helpful suggestions are offered, drawing on actual experiences and published literature. Emphasizing the significance of effective communication and conflict resolution in promoting a healthy team culture, the main competencies and traits required for creating effective teams are reviewed. Additionally, the particular difficulties experienced by independent teams are discussed along with suggested solutions. By the end of this piece, it is our aim that readers will have a better understanding of the dynamics inside independent teams and have gained useful information for navigating the fascinating but challenging world of independent game development.\n\nThe whole process can be divided into three phases: laboratory (solo work), hunt (talent finding), and orchestra (maintenance of the team). The names of the phases were taken from personal experiences, and it is important to note that an effort was made to cover the bulk of points within these three stages in order to improve comprehension. The procedures described in this article have been tested in real-world settings where they were functioning as independent teams. Furthermore, it is important to note that some steps correspond to those that have been reported by various authors in previous literature.\n\n\n2. Laboratory\n\nThe precise project requirements, including team composition, required tasks, and areas where assistance is needed, are identified at this point. It is easy to get paralyzed by doubt, which breeds indecision. However, one can get the answers to these queries such as the skill sets that are crucial, what type of games are suitable for the team to work etc. in a methodical way in the following section. Making judgments concerning the independent team can be done in a more informed way by using this approach. The initial stage entails determining the project’s core. The following queries should be taken into account:\n\nTo determine the necessary team members for indie game development, the first step is identifying the core skills required for the task. There are some primary questions that should be asked to determine personal skill sets. The questions include determining one’s strengths in game design, coding, or concept art. One should also rate themselves honestly and list the skill sets that are almost acquired but untested. This will assist in identifying the areas where support is needed, and the types of team members required.\n\nIt is crucial to decide what kinds of games the group intends to generate after establishing an independent game production studio. Being realistic about what can be accomplished right now is equally vital as having a good understanding of the genre and style of the game. Making something big may not be possible right away, therefore the team should have a long-term plan on how to get there. Setting up checkpoints along the way will not only keep the crew on task but also offer them a feeling of purpose and direction. Is it a roguelike metroidvania or turn-based card game, or something else? To get good support, Travis Tracy wrote in his article “How to Build a Game Development Team” that it is essential to have a clear idea of the game. People will be unwilling to join the initiative without a clear plan in place.\n\nTracy also mentioned that talented people and good developers look for initiatives that catch their attention. They have the skills, and there are many chances for them because many people are engaged in highly imaginative initiatives and many of them require help. If one does not already have experience in game development or anything to offer, they will not be able to attract them. In this situation, a prepared Game Design Document (GDD: a highly descriptive software design document for a video game design) can assist in assisting others in understanding what someone is attempting to produce. Therefore, it is unquestionably preferable to be ready with a GDD that can be shared.\n\nIn game creation, it is essential to comprehend the project’s motives and objectives. The game’s creator needs to consider their goals and what they hope to accomplish with it. It is crucial to pose pointed queries like: why did you select this specific genre or game? Is there a message or subject that must be covered? Why is this significant and what the developer hopes to achieve specifically? Early responses to these queries could aid the developer in staying focused on their objective.\n\nDepending on the abilities needed for the game, a basic estimation should be made. It is important to take into account aspects like the genre-specific art requirements, the complexity of the programming, and whether a lengthy plot or intricate gameplay is necessary. The precise skills needed for the game to succeed must be determined and listed. Prioritize the fundamental abilities to ensure that the primary knowledge is current to prevent any potential skill gaps. According to Sennett (2008), a skill is nothing more than a taught practice. It can include the knowledge, skills, and abilities employed in producing something as well as the formal knowledge and skills needed for the work (Cockburn, 1983). A skill can only be developed via practice that is ingrained in routine. A person can train, develop, and eventually master a set of talents through organized repetition, from which they can start expanding even further.\n\nIf someone is thinking about forming their own independent game development team, an indie team might typically have certain common duties. Though some of them might be disregarded depending on the game, such as if it lacks text or music, nearly all of them are abilities that should be taken into account to raise the possibility that your game will be a success (New York Film Academy, 2014). The essential team member is programmers - without a developer, even the best concept someone could come up with would probably never be put into action. The individual who will spend the majority of their time developing the game’s tools and codebase is a programmer. It should be mentioned that a programmer often performs more than only developing the game’s code in an independent team. They will also be responsible for beta testing and problem-fixing to make sure the game is polished and playable. Then there is the role of the game designer, who is in charge of level design, stories, and gameplay mechanics. Characters, backdrops, and user interfaces are examples of the visual assets that artists are in charge of generating. Musician/Sound Designer: this person is in charge of coming up with the game’s music and sound effects. The storyline and dialogue for the game are created by writers. Bugs must be found and reported by testers in order for the game to be fun and user-friendly. Each of these responsibilities is essential to the growth process and calls for a particular set of abilities. In order to fill each of these jobs effectively, an indie team should ideally consist of persons with a variety of skill sets (gamedeveloper.com, 2010). It is crucial to remember that not all independent teams will have people in each of these positions. Members of smaller teams could take on several roles and responsibilities. This might cause problems like uneven workload distribution and exhaustion, but it can also encourage teamwork and adaptability.\n\nCross-referencing the requirements with the available inventory can then assist in evaluating whether the required resources are sufficient to complete the operation. The essential elements required to bring the dream to life have already been discovered. Is the current inventory, however, sufficient to meet all the requirements, or will further support be required? Even if all the required skillsets are there to develop the game, or at least the initial iteration, this does not imply that the aim has been met. Additional services like quality assurance, publisher pitching, and business assistance are also required. Making a game for leisure may be the greatest solution if no one is managing the business element of game development. It is critical to have someone in charge of making sure the game is lucrative. The entire team must remember that they are designing a game for business goals, not only for entertainment. This encompasses marketing, sales, developing business strategies, and maybe securing money. If the individual is unable to complete these activities on their own, they can hire or work with outside expertise. Despite the fact that many business-to-business (B2B) organizations provide these services, they must be managed. If one can make it this far without incident, it can be presumed that they are confident enough to go on their own. The final barrier, however, is the individual. Game creation can be daunting, especially if you want to be successful. Having a spouse can make a major impact during difficult times. However, if the individual is confident enough to tackle these problems alone, they may go alone. There are several tools available to aid them in the age of artificial intelligence (AI).\n\n\n3. Hunt\n\nIf any of the previous milestones were not completed, it is time to seek outside assistance, in the phase we call the hunt. The next stage is to look for qualified team members. There are numerous resources available at conferences and forums to help you identify qualified applicants. However, how does one go about finding the ideal person for their team? Furthermore, how can they be persuaded to join the project? These difficulties can be overcome with good planning which are briefly discussed in the next section.\n\nOne of the most important jobs completed in the laboratory section (please see Section 2 Laboratoy) was answering vital questions such as the mission and the driving force behind it. It is critical that this information be shared with others. In the video game industry, execution is more crucial than having a great idea. Even a good idea cannot provide good outcomes unless it is properly executed. As a result, there is a need to overcome anxiety and confidently conveying the creation story of the game idea to the team members. The best method to attract others who share your values is to express your original thoughts. It is not necessary to rely entirely on monetary incentives or celebrities to inspire people to join a project; staying focused on a single objective can be just as effective. If one’s luck is on their side, they may come across others who share their level of passion, energy, and commitment to their cause. It is critical to publicize one’s mission and make it known to both physical and virtual groups. Speaking about it at social occasions may result in people recommending appropriate candidates. Although job postings may generate a huge number of applicants, it is still beneficial to use personal contacts to uncover talent. Personal relationships can be an excellent resource for locating persons with the necessary skills and expertise. It is possible to uncover potential team members that are suited for the project by employing one’s network, as well as to use pre-existing relationships to generate trust and cultivate relationships from the start. This strategy can be especially useful in the early phases of team creation when you are attempting to lay a firm basis for the team’s success. Sharing your story with potential teammates can also provide you with additional benefits. It may help in attracting people with similar perspectives. People are drawn to people, so this is your chance to put yourself out there and be regarded as someone with whom they can share their thoughts and ideas. This tiny distinction may be all that is required in critical situations.\n\nAfter selecting potential team members, it is critical to be open and honest about the project’s goals, schedule, and remuneration plan. You should also outline the advantages of joining the team. Additionally, the time each team member can devote to the project should be discussed and decided upon beforehand. To create a cohesive team, it is critical to demand the same amount of dedication from each member. Finding the appropriate talent is important, but building a team also requires assembling a group of people who get along well and are dedicated to the game’s success.\n\nGood game development requires a number of technical skills, including modeling and programming. The most crucial need for maintaining their technological methods, according to many independent developers, is “shared passion,” not technical proficiency (Freeman et al., 2020). For example, an online post on the indie gamer forum explained how a shared passion was central to indie game development: “You need to find people who are passionate about creating a vision together and want to bring their strengths in to make it happen. Over the course of time, you then have to accept that this team will have highs and lows, and people come and go, but with a strong enough core team who shares the same passion, you should still be fine.”\n\nOne may struggle with the decision of whether to hire and train recent graduates or seasoned experts when it comes to hiring for the team. Each choice offers benefits and drawbacks of its own. Although employing an enthusiastic and motivated rookie might take more time at first, it might be worthwhile in the long term. Freshmen are more adaptable to alter and simpler to shape to the needs of the project. They are a significant asset since they are driven to achieve and eager to show themselves. Furthermore, showing them respect can result in enduring loyalty. However, on the other hand, seasoned experts could demand greater pay and not be as adaptable to change but can contribute a wealth of knowledge and skills to the team. Building a solid basis for a long-term team requires a mix of inexperienced and experienced team members. Hiring inexperienced employees necessitates a time commitment in training and molding them, which can occasionally result in budget and time overrun. Inexperienced persons frequently lack perception at first. Experienced campaigners, on the other hand, require little or no training and can give a variety of perspectives according to their backgrounds. However, because their ideologies and mentalities are already set, experienced campaigners can be difficult to influence instantly. This isn’t necessarily a bad thing, as their differences can help the cause significantly. They can, however, be tough to work with at times. Furthermore, recruiting a veteran increases the risk of bringing on someone with negative energy compared to hiring a novice. As a result, while recruiting an experienced applicant, it is critical to look for warning signs such as massive cultural differences, not being able to change mindset, rigid attitudes, predetermined inferiority/superiority complex. We have observed that cultural differences can be worked on with efforts however it gets hard to work with someone who is not ready to work with an open mind. However, it is not suggested to hire exclusively novices because they will require experienced team members to guide them.\n\nFor an independent game studio, choosing whether to use internal or external resources might be difficult. A team can access a variety of specialized talents and knowledge through outsourcing, which can also lower expenses and lighten the strain on in-house team members. However, it can also lead to management and communication problems because of time zone, linguistic, and cultural disparities.\n\nUsing internal resources, on the other hand, offers the benefit of working with a team that is already familiar with the organization’s culture and operational procedures. Collaboration and efficiency may both improve as a result. It might, however, limit team members’ skills and cause them to feel overworked or exhausted.\n\nAn independent game development team’s choice to use internal or external resources ultimately depends on its particular requirements and available resources. Combining the two may make sense, such as outsourcing some tasks while retaining the core team members on staff. This strategy can assist in striking a balance between the benefits of working with a team that is already familiar with the organization’s culture and procedures and the need for specialized knowledge and abilities.\n\nFinally, having a team that supports and rallies around the initiative is critical. Putting together a team of high-profile individuals who don’t listen or are extremely eager to cause negative disruptions can jeopardize the overall effort. More time and effort should be invested in properly selecting and establishing the founding team, emphasizing the necessity of thorough hunting and attention to detail.\n\n\n4. Orchestra\n\nIt is more difficult to keep a party together than it is to form one. Once a team is formed, the individuals involved must be given sufficient care and attention, as they are human beings with unique needs. The team’s success or failure is heavily reliant on effective people management. The capacity to effectively manage team members is critical in sustaining team cohesion and building a harmonious working environment, ensuring that everyone is aligned and working toward the same goal.\n\nThis relationship determines whether the team succeeds or fails. Depending on the circumstances, co-founders or partners can be a huge advantage or a significant detriment to the team and its mission. However, if a simple cycle is followed (Figure 1), addressing this problem is not unduly complicated.\n\nAny partnership starts with trust. People don’t form a business relationship unless they trust each other up to a certain level. It shouldn’t be disregarded or taken for granted, though. To ensure its performance, it needs regular inspections, much like routine maintenance. If managed improperly, problems could develop. As numerous academics have pointed out in the past, either online (Weisband et al., 1995; Wellman et al., 1996) or through physical interaction (Brown & Duguid, 1991; Wenger, 1998), the motivating driver to the establishment of communities or networks can be found in common interests. These communities of interest represent a space where individuals’ interests and passions are orchestrated (Wenger, 1998). Awareness and the desire for trust in independent teams go hand in hand. Al-Ani et al. (2013) claim that displaying socially acceptable behavior, technical proficiency, and consideration for others will increase trust among globally dispersed team members. Furthermore, it is noted that trust in remote creative teams is a dynamic process that changes during the work-life cycle. Wang and Redmiles (2016) found that informal, non-work-related contact (also known as cheap chat) among virtual team members in global teams (with the majority of members working remotely) was positively correlated with trust. There are several approaches to accomplishing this goal, but the most effective is to add regular timeout sessions for candid talks and open communication. It is frequently observed by us that these sessions operate best in informal settings rather than in a formal setting. Regardless, it is critical to have a consistent timetable for these discussions. Any potential difficulties or conflicts can be detected and handled during the maintenance phase. We have seen that partnership problems are frequently caused by a lack of empathy. Indeed, most problems may be traced back to a lack of empathy. As a result, encouraging and implementing empathy within the team is an effective strategy. Trust concerns are likely to be reduced greatly by developing an empathy culture. It can also be good to organize regular sessions dedicated to exercising empathy and understanding one other’s challenges. This observation is also supported by Frei and Morriss (2020), in their leadership case study, “Begin with Trust”, the first step to becoming a genuinely empowering leader.\n\nThis iterative loop must be maintained throughout the collaboration. Sessions to assess empathy and trust may be held more frequently or less frequently depending on how long the company has been operating. Nevertheless, we feel that holding these workshops is a good idea. Given that prevention is so important, it is essential to recognize and solve any significant problems or fissures that may develop. By putting preventive measures into place, one can guarantee that should they ever be required, any cures will be simple. To maintain a strong, lasting, and harmonious relationship, communication between partners is crucial. Clarity must be prioritized, practiced, and integrated into the system in order to be effective. By doing this, a successful and long-lasting collaboration in business can be achieved.\n\nAccording to Villalobos (2011), developers place a great value on autonomy, and they are also accountable for managing their own work. For some developers, managing their time might become a significant problem in terms of completing their tasks. It is interesting to note how networking blogs and local events influence how developers approach their work. Although some creators like to retain a clearer distinction between the two, others favor using game jams to expedite their production.\n\nHence, team building, this task must be undertaken throughout the lifespan of the endeavor. It demands attention and care as long as there is a team. Individuals from various backgrounds, geographies, and ideologies are likely to make up the team. Even in paradise, difficulties are unavoidable. However, by taking a complete strategy to preserve smooth and dynamic team chemistry, not only will production increase, but internal difficulties will be reduced. Experts from many fields collaborate closely in a game development team, highlighting the need for rigorous work on team chemistry. The three Cs (compassion, commotion, and communication) are the keys to maintaining team cohesion.\n\nThe ability to comprehend and sympathize with the needs, emotions, and viewpoints of team members is a necessary component of compassion, as is the capacity to foster a welcoming and inclusive work environment. There are a few things that team members can do to show compassion for one another. First and foremost, a safe and supportive environment in which team members feel comfortable expressing their opinions and concerns in any situation should be fostered. This creates the groundwork for the formula’s second component: communication. We believe that it is also critical to embrace and celebrate diversity. According to a 2015 study of Harvard Business School alumni in senior management roles, 76% of respondents think that “a more diverse workforce improves the organization’s financial performance.” But academic studies have rarely discovered that greater diversity results in better financial performance. Under specific conditions, they have discovered that it results in higher-quality work, better decision-making, greater team happiness, and more equality (Ely and Thomas, 2020). We noticed that each person brings their own set of talents, limitations, and work approaches to the table. Significant differences in mental processes are also prevalent. Significant progress may be made by recognizing and accepting these distinctions, as well as creating opportunities for team members to demonstrate their unique abilities and contributions. The team members will be motivated to advance if there are open lines of communication, open ears to their comments and recommendations, realistic goals, and recognition of their efforts and successes. If team members are overlooked, or do not have the essential support and resources to succeed, the repercussions can be terrible. Investing time and effort in building solid relationships with team members and providing them with enough resources, on the other hand, can result in enhanced productivity, creativity, and job satisfaction. The team’s well-being is critical to the success of an indie game development studio, underlining the need of sustaining and supporting them.\n\nAs a leader, it is critical to lead from the front. The leader should display compassionate behavior, establishing an example for others to follow. The leader does not have to be the most outspoken or disruptive member of the team. Rather, the leader can be someone who is well-liked by their team members, has clear views, and expresses them effectively. Travis Tracy (How to Build a Game Development Team), who has led many indie teams, believes that a leader should be willing to take on unpleasant jobs, keep the team focused, show equal devotion to other team members, and be open to developing their technical skills. By being present, one must set a good example. Others will take the lead and be influenced. This idea applies to leadership in general as well as to this specific circumstance. It is necessary to be in charge when things matter. This should not, however, mean micromanagement. Instead, make an effort to be present when a team member marks a key religious or personal milestone. The entire team will be greatly impacted by such actions. Furthermore, it is crucial to set aside time to thank team members for their diligent efforts and vital contributions. This might be as simple as thanking them or highlighting their accomplishments at a team meeting. Members of the team should be gradually taught these concepts, so they may actively take part in the ritual of mutual celebration. Train them so they begin to follow this process as well.\n\nWe believe game development unit should be like a living organism, with team members that are comfortable with one another and eager to interact openly. While a rigid team structure may be appropriate for larger studios, the core of an indie team is found in the communal energy they generate. It is similar to a chemistry matchup in a game, where even if their characters aren’t the most powerful, they can aim to gain a competitive advantage by maintaining great chemistry among the team members. An indie team can achieve incredible feats in the area of game creation if they are motivated and interested. Do team members socialize outside of work? Is it common for them to spend time together playing games, watching movies, or dining out? Are they encouraging of each other’s personal achievements? Are their cohesiveness and coherence visible to spectators both close and far away? If these questions are answered affirmatively, it suggests that things are going well. It is critical to foster excitement, solidarity, and long-term partnerships. These connections will be invaluable when faced with tight deadlines, unanticipated challenges, and the danger of failure while still moving forward. In fact, these ties may be the deciding factor in overcoming adversity.\n\nIn a hypothetical team vs team encounter in a battle royale game, if a teammate fails to provide cover while a run for the bomb location is made, the opposing sniper will undoubtedly eliminate the player. The proper operation and eventual success of a team are dependent on excellent communication. However, delivering successful communication can be difficult and requires collaborative efforts from multiple viewpoints. Fostering a compassionate culture is a critical component in laying the groundwork for effective communication, functioning as a catalyst in achieving this goal. Team members should be eager to participate in open, thoughtful, and cooperative behavior with one another. Active listening should be practiced as well. A positive team culture supports equity because members have an open mind and show understanding and compassion for one another’s points of view. Active listening requires paying close attention to others, asking pertinent questions, and demonstrating empathy. Furthermore, communication ground rules should be developed and followed by team members. This step’s effectiveness will be determined by constant practice.\n\nBecause indie teams value freedom, the choice between working remotely and working from the office arises frequently. Working from home has been popular among many indie teams because of the ease it provides, allowing team members to work from the comfort of their own homes and providing greater flexibility in terms of work schedules. Working remotely has both pros and downsides. The greatest advantage of remote employment is the freedom it provides. Team members have the freedom to set their own schedules and move at their own pace, resulting in increased production and lower stress levels. Furthermore, working from home eliminates the need for a lengthy commute, which can save time and money. But there are some drawbacks to working from home as well. When working in the same environment, it may be difficult to keep work and personal life distinct. As a result, it may be challenging to concentrate and become distracted. Working remotely can often be lonely, which can hinder team members’ collaboration and communication.\n\nIn contrast, working at a workstation can give team members a focused location for work and aid in maintaining their concentration and productivity. The fact that everyone is in the same physical place might help team members collaborate and communicate more easily. Working at a workstation, however, cannot be as flexible as working from home and might include a commute, which can be expensive and time-consuming.\n\nAdditionally, not all team communications must take place in the same way. The ability of programmers to work together remotely has already been demonstrated. But evidence suggests that the creative design team may not be able to make it work. As an illustration, Bergström and Törlind (2007) examined the creative collaboration of design teams in both co-located and remote work contexts and found that the absence of shared collaborative capabilities (such as drawing surfaces) frequently led to the creative process being disturbed during distributed work.\n\nWorking from home or at a workstation will ultimately depend on the demands and preferences of the independent team. it is crucial to analyze the advantages and disadvantages of each choice and decide which would best support the team’s objectives, paying particular attention to communication between team members.\n\nEvery project’s success depends on the team’s ability to communicate effectively. To make sure that everyone is on the same page and is aware of what is expected of them, it is necessary to communicate messages clearly. Conciseness is essential for keeping messages concise yet complete and avoiding misconceptions or confusion caused by protracted explanations. Delivering constructive criticism, on the other hand, boosts productivity and teamwork while motivating team members to take responsibility for their work and pursue greatness. Communication can quickly deteriorate without the confidence to offer constructive criticism, resulting in mistakes and delays that could throw the entire project off course. Therefore, it is crucial to promote an environment of open and honest communication that values input and gives team members the freedom to give their all in pursuit of the group’s objective. It is crucial to maintain a quick tempo or to be cautious with a slower one in order to keep the group interested. It is best to wrap up the conversation as soon as possible while speaking with someone who is less knowledgeable. Hence we perceived that it is important to take into account all five senses that human possess in order to preserve a pleasant image with the team members.\n\nAvoiding multitasking is crucial because it might impede effective communication. There is no place for the sixth sense in communication. The process of human communication is intricate and complicated, and depending solely on intuition or a “sixth sense” can have fatal results. In truth, this method of communication is similar to gambling in that it might succeed once, but the chances of failure are very high. it is important to continue with tried-and-true communication techniques that are based on objectivity and clarity. It is possible to make sure that our message is delivered accurately and effectively by avoiding ambiguous wording or assumptions. In other words, rather than relying on intuition or guesswork, effective communication relies on precise facts and understandable language. Inadequate communication can have disastrous repercussions, including crying and a host of other negative results.\n\nA skilled communicator should be developed or hired to support the team’s successful emergence because communication acts as the glue holding the group together.\n\nTeam members may still become involved in sporadic confrontations even after taking the aforementioned safeguards, as they happen more frequently than is normally anticipated. It is advantageous to therefore plan ahead for post-conflict measures as well.\n\nRecognizing and acknowledging the presence of a problem is the first step in its settlement. Denial is a common reaction to conflict, especially in the early phases. While denial may be a strategic choice for handling a specific dispute at a certain time, in most cases, it is an unsuccessful technique that ignores or dismisses the appearance of a possible conflict. Most of these difficulties can be resolved quickly if they are given adequate attention. By default, there is a propensity to trust that the problem will resolve itself, which ultimately worsens matters in the long run. Acknowledgment of the concerns must first come from the parties involved, and then it may be open to acknowledgment by other relevant parties, if applicable.\n\nThere are various strategies for settling a problem amicably. It will depend on how the conflict is going and what stage it is at. According to Alistair Doulin (2010), the best choice for resolving conflicts is to have someone in a position of authority. This person must be believed not to advance their personal interests. The best and simplest way to handle problems is through open, continuous communication between team members, who can speak things out. It becomes harder to please everyone as the team size increases. A compromise is necessary. The team is in it together, thus disagreements must be resolved through compromise. Voting is a great way to resolve conflicts since it essentially offers everyone a voice. The team must accept and respect the use of a voting method. Many confrontations can be efficiently resolved before they escalate if all parties involved acknowledge and communicate openly. Taking a mutually agreed-upon break might also be beneficial in some instances. If the office has a competent HR department, it may be appropriate to assign the conflict resolution process to them. In this setting, the function of human resources is critical. If they have earned the trust of their staff and have built positive relationships with everyone, they will be aware of such concerns. One could wonder if accepting this responsibility personally is a good idea. There is no harm in accepting the post if one holds the essential competence and is judged the best appropriate for it. However, team members may be cautious to fully reveal their opinions and concerns if they believe the individual is the ultimate decision-maker. Individuals frequently overthink and hide facts for fear of being viewed as unimportant. However, if one builds a position of trust and true camaraderie with the team, they may be able to effectively handle the function on their own. It is worth noting that many game developers are introverted people who have a deep passion for their art. Without developing a close bond with them, they may be selective in sharing personal information or secrets. The conditions for developing trusting links and reinforcing shared beliefs aims, and practices are created via reciprocal relationships and shared experiences as a community of practice and production (Pargman, 2005: 106). Independent creators recognize their strengths through community interaction and sharing performance synergies. Most disputes go unreported by the people concerned. They can let issues develop and worsen until they become unbearable. As a result, it is critical to provide sufficient care for the team while remaining watchful and cultivating a pleasant relationship between the HR department and the team members. This strategy will help to improve conflict resolution and overall performance.\n\nWe will start this section with an example first. Nuclear warheads with the ability to inflict significant damage across a large region are available in the Command and Conquer series, a popular choice among fans of real-time strategy games. The weapons such as nukes have the inherent ability to have a big impact on the game’s final outcome. However, an important barrier emerges: nuke’s creation takes a significant amount of time, which is exacerbated by the lengthy period required for them to become operational. Throughout this extended period, the warheads remain highly vulnerable to attack. If someone’s adversary has a stealth unit, their tactical approach may include breaching their defenses and grabbing the warhead before the nukes achieve full readiness, then using it against their soldiers. The occurrence of such a situation as a competitor coveting a well-trained resource for game development in one’s professional life can have enormous ramifications, especially when dealing with raw abilities that are sought for, nourished, and cultivated. These people frequently have enormous potential that can be achieved over time. When a firm nurtures a strong culture and delivers high-quality internal education, the industry as a whole takes note and prepares itself as these individuals reach their full potential. Since organizational culture defines the environment for everything that happens within a company, one should try to focus and maintain this aspect (What is organizational culture and why is it important). However, if these talents do not have strong ties to the business, they risk being stolen away just as they are about to pay back the investment placed in them. While attending Gamescom Asia 2022 which was held in Singapore, multiple game industry insiders while conducting sessions shared that game development companies in Singapore facing recruitment difficulties due to severe rivalry. In an atmosphere where large corporations coexist, presenting appealing services and aspirations, small studios face a tremendous challenge. However, it is critical to capitalize on one’s abilities and focus on things that large corporations frequently struggle to supply. Being a part of an indie team implies a unique environment marked by independence, enthusiasm, a high learning curve, and recognition. It is critical to ensure that young talents understand the risks they are taking by joining the team and can see their goals being realized while remaining with the club as they advance. Constant monitoring and unflinching care are required, never allowing the young talents to escape sight and protecting them with all available means. Otherwise, a potentially groundbreaking decision could devolve into a nightmare. While it is conceivable to substitute a ready-made resource to some extent, it is impossible to replace someone personally trained with someone who was trained by someone else. They will never be identical, which will have irreversible effects if they are lost.\n\nRoguelite games are difficult to master since success is rarely obtained on the first try. Playing, failing, and trying again become necessary steps in this process. Each failure teaches and rewards the learner, enhancing one’s talents for future attempts. This iterative process must be repeated until victory is achieved. It is quite common for assembling an amazing team to necessitate extensive fine-tuning and constant effort, in a dynamic that could be seen to mirror these games. It is critical not to be hesitant in parting ways with individuals who are destructive to the culture of your team. In the long run, their existence may hinder the voyage of other valuable assets. While people come and go, the process is unchanging. It is critical to develop and adopt a systematic approach to all aspects. Proper boundaries and clarity should be established within the framework of game production, a multidisciplinary art form that requires the collaboration of people from many backgrounds and viewpoints. A programmer may be oblivious to the time and work necessary by a concept artist to develop a simple character in five hours, just as others may wonder why a coder spends days polishing a basic leaping function. This is why cultivating an empathy culture is critical since it allows people to understand and accept one another’s difficulties and processes. It is also critical to handle creative resources with care and sensitivity. They should not be overworked, but some work-related ground norms and practices must be established and enforced. Their creative freedom, however, should never be compromised. Everyone in an indie team with a strong culture can find excitement in every area of game production and have an opinion on a variety of issues. Nonetheless, specific choices should be delegated to specific personnel. The narrative lead has ultimate power over the story’s conclusion, whereas the lead game designer assesses whether the win condition is biased. Team members are encouraged to share their opinions and provide constructive input, but harmful language should be avoided. Individuals with substantial gaming experience, having played over 1000 games in their lifetime, may be present on the squad. While their feedback is crucial, it is critical to ensure that their decision-making authority remains within and does not exceed the stated bounds. Furthermore, they should not engage in bullying conduct in which they harass others based on their personal preferences, referring to games that they have only played. Setting these boundaries will assist the team in avoiding a variety of undesired scenarios and fostering a successful collaborative environment.\n\n\n5. Conclusion\n\nCreating an independent team can be difficult yet rewarding for game developers. Developers can choose the most effective strategy for their project by weighing the benefits and drawbacks of various team arrangements. The techniques previously described are all the outcomes of navigating the process of creating one’s own independent teams. Independent teams can overcome the challenges brought on by scarce resources with proper preparation and clear communication.\n\nIn conclusion, it is crucial to begin with a clear knowledge of the project’s vision, goals, and scope in order to assure a successful independent team creation. Developers can then select whether to outsource or use internal personnel after identifying the roles and talents that their team needs.\n\nOnce the team is established, productive collaboration depends on effective communication. The team can stay on track by setting clear goals, scheduling frequent check-ins, and using tools like project management software and video conferencing.\n\nPutting together an independent team involves careful thought and preparation, but with the correct attitude, it can result in the creation of creative and commercially successful games. It is unquestionably important to think about the various questions that are raised and decide what best piques your attention.\n\nWhen embarking on the adventure of establishing an indie team, it is critical to remember that team formation is similar to the establishment of a personal Justice League, albeit without superpowers. The hunt is looking for someone with sufficient skills in programming, design, art, and communication. Nonetheless, it is critical to remember that exceptional team creation entails the heavy burden of sustaining and growing that team. The journey ahead will likely provide its fair share of challenges and hurdles, as with every major trip. However, with a well-defined vision, a solid plan, and a team of experienced and devoted individuals, there are no impediments to realizing dreams of creating the next innovative indie game sensation. Thus, go forth and conquer the world of indie game creation!", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAl-Ani B, Bietz MJ, Wang Y, et al.: Globally Distributed System Developers: Their Trust Expectations and Processes. CSCW’13. Proceedings of the 2013 Conference on Computer Supported Cooperative Work, San Antonio, USA, 23-27 February 2013. New York: ACM Press; 2013; pp. 563–574.\n\nBergström M, Törlind P: Examining Creative Collaboration in Distributed and Co-Located Design Teams. ICED’07. Proceedings of International Conference on Engineering Design, Paris, France, 28 –30 August 2007. Paris: Design Research Society; 2007; pp.1–12\n\nBrown J, Duguid P: Organizational learning and communities of practice: Toward a unified view of working, learning, and innovation. Organ. Sci. 1991; 2(1): 40–57. Publisher Full Text\n\nCockburn C: Brothers: male dominance and technological change. London: Pluto; 1983.\n\nDoulin A: Building A Strong Indie Game Development Team. Retrieved from Game Developers.2010, January 7. Reference Source\n\nEly JR, Thomas AD: Getting Serious About Diversity: Enough Already with the Business Case. Harv. Bus. Rev. 2020. Reference Source\n\nFreeman G, McNeese N: Exploring Indie Game Development: Team Practices and Social Experiences in A Creativity-Centric Technology Community. Computer Supported Cooperative Work (CSCW). 2019; 28: 723–748. Publisher Full Text\n\nFreeman G, McNeese N, Bardzell J, et al.: \"Pro-Amateur\"-Driven Technological Innovation Participation and Challenges in Indie Game Development. Proceedings of the ACM on Human-Computer Interaction Volume 4. 2020. January 2020.\n\nFrei XF, Morriss A: Begin with trust. Harv. Bus. Rev. 2020; Reference Source\n\nGame Developers Conference (GDC) 2021 State of the Industry Survey: April 2023. Reference Source\n\nGuevara-Villalobos O: Cultures of independent game production: Examining the relationship between community and labor. Proceedings of DiGRA 2011 Conference: Think Design Play. 2011.\n\nLipkin N: Examining Indie’s Independence: The Meaning of “Indie” Games, the Politics of Production, and Mainstream Cooptation. The Journal of the Canadian Game Studies Association. December 2012; 7(11): 8–24.\n\nNew York Film Academy: HOW TO FORM A SOLID INDIE GAME DEVELOPMENT TEAM. NYFA; 2014, November 25. Reference Source\n\nPargman D: Virtual community management as socialization and learning.van der Besselaar P , De Michelis G , Preece J, et al., editors. Proceedings of the Second international conference on Communities and Technologies. Dordrecht: Springer; June 2005; pp. 95–110.\n\nSennett R: The craftman. New Haven & London: Yale University Press; 2008.\n\nThe International Game Developers Association (IGDA) Developer Satisfaction Survey 2019 Summary Report: Retrieved from International Game Developers Association (IGDA).April 2023. Reference Source\n\nWang Y, Redmiles D: Cheap Talk, Cooperation, and Trust in Global Software Engineering. Empir. Softw. Eng. December 2016; 21(6): 2233–2267. Publisher Full Text\n\nWeisband SP, Schneider SK, Connolly T: Computer-Mediated Communication and Social Information: status silent and status differences. Acad. Manag. J. 1995; 38(4): 1124–1151.\n\nWellman B, Salaff J, Dimitrova D, et al.: Computer Networks as Social Networks: Collaborative Work, Telework, and Virtual Community. Annu. Rev. Sociol. 1996; 22: 213–238. Publisher Full Text\n\nWenger E: Communities of practice: Learning, meaning and identity. Cambridge: Cambridge University Press; 1998." }	[ { "id": "238205", "date": "15 Feb 2024", "name": "Josiah Lebowitz", "expertise": [ "Reviewer Expertise Game Design", "Narrative Design", "Creative Writing & Storytelling", "Interactive Storytelling", "Game Development", "Indie Game 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\nSummary The article discusses the challenges of creating and maintaining a team for indie game development. It divides the process into several steps, with the main focuses being first on finding and hiring appropriate team members, and then ensuring a productive and harmonious working environment for the newly formed team.\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes, the material is accurate and suitably reflects current trends, literature, and the like.\nAre all factual statements correct and adequately supported by citations? Yes, the article is well cited.\nAre arguments sufficiently supported by evidence from the published literature? Yes, sufficient evidence is provided when needed.\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes, the conclusions are reasonable and build well on the presented material and arguments.\nComments/Suggestions - Section 2.4 Paragraph 2: Inconsistent formatting is used when listing the different team roles.\n\n- Section 2.4 Paragraph 2: Both the writer and designer are listed as being in charge of the story. This can be confusing without some clarification. Depending on the team makeup, it might be entirely under the writer’s control. Alternately, the lead designer or creative director could create the general story summary with the writer being in charge of the full script. There are also many situations, especially in indie development, where the designer and writer will be merged into a single role (narrative designer) with the responsibilities of both positions.\n- Section 2.4 Paragraph 2: It’s worth mentioning that, depending on the game engine and type of game, a programmer might not be necessary. While relatively uncommon, there have been some notable indie games that were created entirely without a programmer using only the engine’s built-in tools.\n- Section 2.4 Paragraph 3: The brief mention of a spouse should be removed. While having a spouse or significant other of some type is invaluable for many people’s emotional and physical wellbeing (indie developers and otherwise), it doesn’t really fit the theme of the paper and isn’t the type of thing that should be discussed in a paper like this without significant detail and supporting documentation.\n- Section 3.2 Paragraph 1: While it may vary somewhat in different parts of the world, North America is starting to see a large number of recent graduates with rigid attitudes and strongly held social and cultural beliefs which can lead to (sometimes significant) problems when they are hired. As such, it’s worthwhile to look for appropriate warning signs in all applicants, not just industry veterans.\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": "266634", "date": "03 May 2024", "name": "Matt Barton", "expertise": [ "Reviewer Expertise Professional communication", "game 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\nI enjoyed this article and found it well-researched and clearly written. I think it will be helpful for anyone considering forming or joining an indie team and ensuring a smoother collaboration. As far as weaknesses go, I noticed one spot where \"my experience\" is used, which is confusing considering there are two authors on this piece. Another spot needing attention is the \"three c's,\" which mentions \"commotion.\" I'm not sure if that is the right word there; if it is, it would be helpful if the authors defined what that means in this context. Finally, although there are a few good real-life examples included (such as the Singapore situation), it'd be good to have more anecdotes throughout the piece to exemplify the value of the points being discussed. Perhaps a fictional indie team could be introduced to illustrate the principles, such as the potential impediment of multitasking during team sessions? Overall, though, I think the article is thoughtful and certainly addresses some of the key issues faced by indie studios.\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/13-42
https://f1000research.com/articles/13-41/v1	08 Jan 24	{ "type": "Study Protocol", "title": "Evaluate the awareness regarding the patient’s rights and responsibilities among the patient visiting hospitals", "authors": [ "Toshi Diwan", "Deepika Kanyal", "Deepika Kanyal" ], "abstract": "The patient's privileges are a set of moral principles that direct how patients and medical staff interact with one another. Each patient has a right to evidence of their legal obligations and the healthcare provider's duties. Respecting patient rights is an essential aspect of healthcare practices. The truth is that those who are ill are among society's most helpless members. The medical profession consequently views improvements to patient rights as being of the highest significance. A patient's rights are the set of moral principles that guide their engagement with medical personnel. Every single patient has an authorized right to knowledge about those rights as well as the duties of the healthcare practitioner. Aim: To assess the awareness regarding the patient rights and responsibilities among the patients in the tertiary care hospital. The objective is to assess the patient's and family's knowledge about the patient's rights and to study how patients' rights and ethics impact hospital employees. Materials and methods: A questionnaire-based cross-sectional study will be carried out among 349 patients visiting the tertiary care hospital. Expected Result: The expected result of the study will be to uncover a baseline level of awareness among patients and their families regarding patients' rights when visiting hospitals. Through comprehensive assessments and surveys, will be determined that a considerable portion of patients may have limited knowledge about their rights in the healthcare setting. Also, it is expected that healthcare providers will become more attuned to the importance of upholding patients' rights. This alignment with patients' rights and ethical principles is anticipated to positively influence the overall organizational culture within hospitals, fostering an environment of respect, empathy, and patient empowerment.", "keywords": [ "Rights", "Healthcare Practitioners", "Knowledge", "Awareness", "Improvements" ], "content": "Background\n\nThe Universal Declaration of Human Rights was established by the UN in 1948, and since then, it has been extensively complied with. The protection of patient rights is crucial in the practice of medicine. Patients are among society’s most underprivileged young people. As a result, protecting patients’ rights has been given top priority.1 Patient’s rights are essential human rights since they are one of society’s most vulnerable groups. Patient rights are key pillars for providing high-quality health care and supporting ethical medical practices.2\n\nAs a result, patient rights have been identified as an important problem in attempts to enhance healthcare quality. The right to privacy is one of the essential pillars for defining clinical care standards. Patients who understand their rights not only boost their dignity by allowing them to engage in decision-making with physicians, but they may also improve the quality of healthcare services, save money, and improve the overall health of the population shorten hospital stays. As a result, determining how well patients understand their rights is crucial.2\n\nPatient rights and obligations are the most important NABH certification criterion. While the quality of care is vital, the hospital’s structure must also protect patients and their families’ rights throughout treatment. The organization must establish patient and family rights and duties, train workers to defend these rights, and tell patients about their rights and responsibilities at the time of admission.3\n\nEffective doctor-patient communication is determined by the patient’s comprehension of their sickness and legal rights. Individuals who are more informed about their diseases, drugs, and therapies are more likely to adhere to their treatment plans and take an active role in their care. This needs a shift in attitude toward patient education; instruction should be more patient-centred.4\n\nBasic Fundamentals PATIENT’s RIGHTS followed in Tertiary-care hospital:\n\n1. Access to healthcare facilities, regardless of age, gender, religion, economic or social standing, is guaranteed.\n\n2. The right to select one’s, own doctor.\n\n3. The right to be treated with decency and care without prejudice.\n\n4. The right to confidentiality during consultation and therapy.\n\n5. The right to expect that all discussions and records relating to his or her case be kept private.\n\n6. The right to complete information on a diagnosis, treatment, or inquiry.\n\n7. The right to be informed about the safety of operations carried out on him or her.\n\n8. The right to be kept up to date on daily progress, course of action, diagnosis, and prognosis.\n\n9. The right to provide informed consent after being properly informed.\n\n10. The right to see his records and a demand summary.\n\n11. The ability to obtain a second opinion at any moment.\n\n12. The right to obtain on going medical treatment for his or her sickness.\n\n13. The right to be treated with dignity and comfort throughout sickness and follow-up.\n\n14. The right to complain, to have grievances resolved, and to seek compensation for medical injuries or carelessness.\n\nPATIENT RESPONSIBILITIES:\n\n1. Provide accurate and complete details such as your entire name, address, and other contact information.\n\n2. Provide accurate and comprehensive information on his or her health, including current condition, previous sickness, hospitalization, medicines, natural goods, and vitamins, as well as any other health concerns.\n\n3. To inquire about diagnosis or therapy when he or she does not comprehend what the doctor or another member of the healthcare team says.\n\n4. He/she should also notify the doctor if he/she foresees difficulties in adhering to the prescribed therapy or is contemplating other therapies.\n\n5. Follow all hospital policies and procedures.\n\n6. Simply follow visitation policies to protect the rights and comfort of all patients. Consider noise levels, privacy, and safety.\n\n7. Adhere to the no-smoking policy.\n\n8. Show kindness and respect to hospital personnel, other patients, and visitors.\n\n9. Being on time for appointments. To cancel or reschedule the appointment as much in advance as feasible.\n\n10. Must follow the suggested treatment plan and strictly follow the instructions.\n\n11. Do not take any medicine without first consulting with a doctor or a healthcare expert. To deliver an accurate and specified treatment plan and to strictly adhere to the directions given.\n\n12. To accept, if appropriate, environmental changes to enable a safe and secure stay in the hospital.\n\nIn 2002, the Indian Medical Council approved Ethical Guidelines in light of India’s condition (COER). The COER addresses both the rights of patients and the duties and responsibilities of doctors. As a result, the current survey was carried out as an effective method to regulate the level of patient satisfaction. rights awareness among inpatients at an academically approved hospital.\n\nResearch on patient rights, including how much these rights are exercised and honoured, would emphasize the existing conditions for carers, particularly hospital administrators, to help in policy formation and service management. Patients must be knowledgeable to grasp the essential facts and the decision at hand, and they must not be pushed into taking therapy that they do not choose to receive. To corroborate the three, the investigator strives to analyse the current state of things at the chosen institution. (previously mentioned) necessary needs and to pave the way for statutory compliance and successful patient-carer interaction.5,6\n\nThe concept of patient rights evolved from the recognition of “the inherent dignity” and the “equal and unalienable rights of all members of the human family.” [The 1948 Universal Declaration of Human Rights].7\n\n\n\n• To assess the patient’s and family’s knowledge about the patient’s rights.\n\n• To study how patients’ rights and ethics impact hospital employees.\n\n\nProtocol\n\nResearch setting: The study will be a cross sectional study. It will include a collection of data through direct questioner to the patients. This study will be conducted in the Tertiary care hospital for Three months.\n\nResearch approach: A cross-sectional research will be conducted in the Surgery, Orthopaedic, Medicine and Paediatric departments of the Tertiary care hospital.\n\nResearch design: A cross-sectional study with verified questions\n\nPopulation:\n\nInclusion: Patients, Patient’s relatives\n\nExclusion: Doctors, Nurses, Administrators Staff\n\nSample zize: 349 Patients\n\nSample size Formula:\n\nPrimary Data: Patients feedback\n\nSecondary Data: Articles, Journals\n\nData collection: The procedure of data collection will be conducted in tertiary care hospital. The data will be collected from the verified questionnaire based survey through patients and their relatives. Further the secondary data will be collected from search engines, full books and some related articles.\n\nData Analysis: Data will be collected with the help of SPSS 22.0 Version.\n\nResult: The expected result of the study will uncover a baseline level of awareness among patients and their families regarding patients’ rights when visiting hospitals. Through comprehensive assessments and surveys, will be determined that a considerable portion of patients may have limited knowledge about their rights in the healthcare setting. Also it is expected that healthcare providers will become more attuned to the importance of upholding patients’ rights. This alignment with patients’ rights and ethical principles is anticipated to positively influence the overall organizational culture within hospitals, fostering an environment of respect, empathy, and patient empowerment.\n\n\nDiscussion\n\nA cross-sectional study was done, and descriptive analysis was used to examine the obtained outcomes’ measurements. Patient Rights Awareness at a Tertiary Care Teaching Hospital in Wardha. The patients were mostly adults, with 53.6% being adults and 34.7% being seniors. Male and female patients were hospitalised in 45.9% and 54.1% of cases, respectively. Patients in cities were more aware than those in rural areas. Because the bulk of the criteria were shown to be statistically significant, education is critical in patient rights.8\n\nA cross-sectional study using questionnaires was carried out among hospital patients at a tertiary care facility on Peninsular Malaysia’s east coast. Eighty-five percent of patients had enough knowledge of their condition and the therapeutic options available to them, and 90 percent were aware of their legal rights. Only in 45% of cases, though, were the various treatment choices mentioned, and only in 65% of cases were the patients told how long their therapy would last. The majority of patients in the surgical group, those receiving haemodialysis, and those with minor illnesses were very satisfied with their doctors (92 percent, 86 percent, and 96 percent, respectively), in contrast to only 36 percent of cancer patients and 70 percent of AIDS patients (p-value is less than 0.0001). Almost all patients (99 percent) stated that the personnel respected their religious views and that they had no difficulty reaching them in times of need. In 98 percent of cases, the doctor acquired informed consent, whereas the nurse obtained it in 2%. 98 percent of patients stated that their treatments/examinations were performed in a private setting and that their doctors kept their personal information hidden.9\n\nA cross sectional study was conducted in year 2011 at Sari. Association between Awareness of Patient Rights and Patient’s Education, Seeing Bill, and Age: A Cross-Sectional Study. There were 55.4% women and 44.6% males among the patients. The mean age of the patients was 40.93 ±15.04 years, and the average duration of stay was 4.6 ± 3.34 days. The majority of patients (36%) had only a high school diploma or were illiterate (25%). The bill of patients’ rights has not been viewed by the majority (63.4%). Poor knowledge included 58.9%, moderate knowledge comprised 12%, and strong knowledge comprised 29.1%. In terms of the bill’s articles, the 9th (the right to engage or refrain from engaging in research) received the least attention. We discovered a significant association (p<0.0005) between bill awareness and the patient’s education, viewing the bill, and age.10\n\nThe current study employed a cross-sectional descriptive and analytical survey to examine patients’ understanding of their rights based on PRC at two tertiary teaching hospitals associated with Shiraz University of Medical Sciences (SUMS) in Iran. Patients’ overall understanding of their rights was modest. Although this percentage was not unsatisfactory when compared to earlier study, efforts should be made to improve it. PRC must be offered to patients by health care groups, who must also ensure that patients understand their rights. Ensuring patients’ rights are respected necessitates not just educate healthcare policymakers and clinicians, but also educating individuals about what they should expect from their governments and health care providers. As a result, the service quality will increase. It is also advised that a Patient Rights Committee be formed to oversee and monitor patient rights education and observance.11\n\nA cross-sectional study was design and conducted between 06.2012 and 09.2012 in tertiary university hospital in Poland: Subjects admitted to a tertiary university hospital in Poland are aware of their rights as patients. Over 80% of respondents were aware of their right to choose a treating physician, refuse proposed treatment, choose the location where the patient is treated, access medical records, free meals, pastoral care, provide information about their health to third parties, and provide information to specific people by phone. The form of granting consent (42.9%) or rejection of consent (50.5%) to treatment and the physicians’ power to deny the presence of a person close to the patient during treatment received the least knowledge (16.4%). Participants’ age and education level had a major influence on the findings.12\n\nThe synopsis for the study has been sent to the IEC in the Datta Meghe Institute of Higher Education and Research, Sawangi (M), Wardha for ethical approval and is currently under consideration.\n\nData collection of study will start from the first week of August.", "appendix": "Data availability\n\nNo data is associated with this article.\n\n\nAcknowledgements\n\nI appreciate the support of my supervisor and my family for helping me to complete the result.\n\n\nReferences\n\nAl-Asadi JN, Abed AH, Shami SA, et al.: Assessment of Patients’ Knowledge and Awareness about their Rights and Duties. Kufa J Nurs Sci. 2015 Nov 16; 4(3): 92–102. Publisher Full Text\n\nAljeezan MK, Altaher YY, Boushal TA, et al.: Patients’ Awareness of Their Rights and Responsibilities: A Cross-Sectional Study From Al-Ahsa. Cureus. 2022 Dec; 14(12): e32854. PubMed Abstract \| Publisher Full Text\n\n(PDF) A Study on Practices at a Tertiary Care Hospital with Respect to Patient Rights and Education, as Compared to Nabh Accreditation Guidelines: [cited 2023 Jul 4]. Reference Source\n\nAwareness of Patients’ Rights among Inpatients of a Tertiary Care Teaching Hospital- A Cross-sectional Study - PubMed: [cited 2023 Jul 4]. Reference Source\n\nFernandes A, Dcunha S, Suresh S: Patient Rights: Awareness and Practice in a Tertiary Care Indian Hospital. Int J Res Found Hosp Health Care Adm. 2014 Jun 1; 2: 25–30. Publisher Full Text\n\nHabib_vol12.pdf: [cited 2023 Jul 4]. Reference Source\n\nShreedevi D: HOSPITAL PREPAREDNESS FOR NABH ACCREDITATION WITH RESPECT TO PATIENT RIGHTS AND EDUCATION.\n\n(PDF) Patient’s Rights- Awareness among Indoor Patients of a Tertiary Care Teaching Hospital in Wardha: [cited 2023 Jul 4]. Reference Source\n\nYousuf RM, Fauzi AR, How SH, et al.: Hospitalised patients’ awareness of their rights: a cross-sectional survey from a tertiary care hospital on the east coast of Peninsular Malaysia. Singap Med J. 2009 May; 50(5): 494–499. PubMed Abstract\n\nYaghobian M, Kaheni S, Danesh M, et al.: Association between Awareness of Patient Rights and Patient’s Education, Seeing Bill, and Age: A Cross-Sectional Study. Global J Health Sci. 2014 May; 6(3): 55–64. PubMed Abstract \| Publisher Full Text\n\nMastaneh Z, Mouseli L: Patients’ awareness of their rights: insight from a developing country. Int J Health Policy Manag. 2013 Aug; 1(2): 143–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrzych LJ, Ratajczyk D: Awareness of the patients’ rights by subjects on admission to a tertiary university hospital in Poland. J Forensic Legal Med. 2013 Oct; 20(7): 902–905. Publisher Full Text" }	[ { "id": "240211", "date": "11 May 2024", "name": "Krishna Mohan Surapaneni", "expertise": [ "Reviewer Expertise Bioethics", "Medical Education", "Population Health Informatics", "Biochemistry" ], "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\nBelow, I detail specific areas of concern that necessitate significant revision or clarification. 1.\n\nThe truth is that those who are ill are among society's most helpless members – This statement is absurd. Health problems occur in any individual. Need not be only among society's most helpless members. 2.\n\nRationale and methods are inadequately explained. 3.\n\nSurgery, Orthopedic, Medicine and Pediatric departments of the Tertiary care hospital. – Rationale behind choosing these department 4.\n\nA cross-sectional study with verified questions – what is verified questions? Who will validate the questionnaire? What is the Cronbach’s alpha value? 5.\n\nVery general. Does not provide any specific inclusion or exclusion. Inclusion: Patients, Patient’s relatives Exclusion: Doctors, Nurses, Administrators Staff\n6.\n\nData will be collected with the help of SPSS 22.0 Version. – This is just the software. What methods will be used are not mentioned.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] } ]	1	https://f1000research.com/articles/13-41
https://f1000research.com/articles/13-40/v1	08 Jan 24	{ "type": "Research Article", "title": "Synthesis of cellulose nanofibers from jute fiber by using chemomechanical method", "authors": [ "Siti Syazwani N.", "Ervina Efzan M.N.", "Kok C.K.", "Nurhidayatullaili M.J.", "Siti Syazwani N.", "Kok C.K.", "Nurhidayatullaili M.J." ], "abstract": "Background Jute fiber is one of the most versatile natural fibers that is widely used as a raw material for packaging, textiles, and construction; and as a reinforcement in composite materials for heavy-duty applications. In the past, acid hydrolysis and mechanical treatment via the ball milling method were common in the extraction of cellulose nanofiber (CNFs) from natural plant fibers. However, there are some drawbacks of using those methods where there will be a huge quantity of acidic wastewater generated when the acid hydrolysis method is performed.\n\nMethod This study investigated the potential use of a combination of chemical and mechanical methods in the extraction of jute CNFs. Through this method, the jute fibers were first chemically treated using sodium hydroxide (NaOH), sodium chlorite (NaClO2) and sulphuric acid (H2SO4) to remove the non-cellulosic elements followed by mechanical milling by using a planetary ball mill.\n\nResults The shape and size of the obtained CNFs were observed under a field emission scanning electron microscope (FESEM). This study revealed that jute CNFs were successfully extracted through the combination of chemical and mechanical treatment methods where the obtained CNFs reveal themselves in smooth fibrous morphology with a diameter of 23 nm and 150-200nm in length.\n\nConclusions Jute cellulose nanofibers were successfully drawn out from raw jute fibers by means of a combination of chemical and mechanical treatment. The results obtained confirmed that the chemomechanical method is an effective technique for isolating the CNFs and its potential use as reinforcement material was explained.", "keywords": [ "fiber", "cellulose", "nanomaterials", "synthesised", "agro-waste", "jute" ], "content": "Introduction\n\nThe demand for environmentally friendly products and processes in various fields such as electronic devices, automotive, building construction, military defences and aircraft is increasing. Thus, the exploitation of agro-industry by-products such as rice husk, banana peels, hemp, jute and bamboo in developing new materials is expanding. Cellulose and fiber are considered the most abundant bio-based polymers in nature. Cellulose is the core constituent of all plant fibers and has repeated links to β-D-glucopyranose (Nagarajan et al., 2019). The utilization of nanocellulose as reinforcing materials in polymer matrices has gained wide attention in the material research community due to their availability, low cost and ease of extraction processing. Recent literature (Wang et al., 2017; Meng et al., 2019; Harini et al., 2018) suggests that nanocellulose has superior properties such as high interfacial area and strength, large surface-to-volume ratio, low density, high tensile strength, high stiffness and flexibility; good dynamic mechanical and thermal properties which make them suitable as reinforcing materials in polymeric materials to grant excellent improvement in properties of the fabricated polymer nanocomposites.\n\nNanoscale structural cellulose can be illustrious into three types namely cellulose nanocrystal (CNC), cellulose nanofiber (CNF) and bacterial cellulose (BC). However, the most common types that are widely used in engineering are CNC and CNF. The main distinctions between these two types of nanocellulose are their dimensions (Meng et al., 2019) and crystallinity. CNF contains both amorphous and crystalline cellulose up to few micrometers in length. They have a larger surface area with a net-like or web-like structure than CNC, which makes them a promising reinforcing agent for polymer composites. On the other hand, CNC contains 100% cellulose content resulting from the absence of amorphous regions and has elongated, crystalline, rod-like shapes with limited flexibility compared to CNF.\n\nApart from properties, the other typical difference between CNF and CNC is in their extraction methods. Many studies (Meng et al., 2019; Nagarajan et al., 2019; Phanthong et al., 2018; Dahlem et al., 2019) show that the different techniques of extraction from respective sources will result in the production of cellulose nanoparticles with varying crystal structures, morphologies, surface interactions and other specific properties. Usually, CNC is extracted from agro-waste by using chemical means such as acid hydrolysis (Danial et al., 2015), alkaline treatment (Melikoğlu et al., 2019), microwave-assisted step (Singh et al., 2017) and enzymatic hydrolysis (Paulo et al., 2019). Acid hydrolysis is the most used method in extracting CNC from cellulose fiber. It is usually initiated by alkaline treatment and bleaching before acid treatment. Several types of acid are widely employed in acid hydrolysis methods, such as sulfuric acid, hydrochloric acid, phosphoric acid and hydrobromic acid. However, the most frequently used acid is hydrochloric acid due to its ability to strongly isolate the nanocrystalline cellulose and make it dispersed as a stable colloid system (Phanthong et al., 2018; Lu and Hsieh, 2010; Das et al., 2009).\n\nMeanwhile, the extraction method of CNF can be categorized into three types, namely mechanical treatments (homogenization, grinding and ball milling); a combination of biological and chemical pretreatments (enzymatic hydrolysis and TEMPO-mediated oxidation); and the combination of mechanical and chemical treatments (chemomechanical treatment). Usually, CNF is extracted from agro-waste materials with the primary aim to turn waste into excellent materials and directly save the environment. Thus, the energy impact that comes with the implementation of the mechanical method alone is not preferable. Instead, the combined use of mechanical and chemical treatment can reduce the amount of energy used in mechanical processes, making the production of CNF more economically viable (Dahlem et al., 2019; Mishra et al., 2018).\n\nCNFs have been widely extracted from plant fibers such as banana peel and bract (Harini et al., 2018; Meng et al., 2019), pineapple leaf (Ravindran et al., 2019), kenaf bast (Karimi et al., 2014; Kian et al., 2018) and cotton (Maciel et al., 2018; Morais et al., 2013) by using chemical approaches (acid hydrolysis method) which lead to disposal of acidic wastewater produced during the washing process to neutralize the pH of the suspension and also incur high cost. Hence, to reduce energy consumption and chemical usage, this work was done to obtain CNF from jute fiber using a chemomechanical method. Chemical treatment such as alkaline treatment, bleaching and acid treatment was involved in the extraction process followed by mechanical treatment using a milling process. Table 1 shows the lignocellulosic content of jute and some other fibers.\n\nBased on Table 1, it is shown that jute has a low content of lignin, pectin and waxes compared to other types of fibers, which suggests it can easily undergo a bleaching process and have a high fiber strength.\n\n\nMethods\n\nJute is a type of bast fiber that was drawn out from corchorus plants. It is produced in large quantities and is known as one of the cheapest natural fibers. In this work, a natural jute rope hemp was used as the main material. The natural jute fiber used was received from Zhejiang Hailun Rope and Net Co., LTD. While the chemicals used in the extraction process such as H2SO4 and NaClO2 were received in liquid form from Merck (1007131000 and 8148151000). On the other hand, the NaOH was received in pellet form from Chemiz.\n\nThe jute fibers were cut into pieces 2 to 3 cm long before the swelling process. During the swelling process, 25 g of jute fibers were immersed in 250 ml of 18% sodium hydroxide (NaOH) solution for 2 hours to attenuate their structure. The swollen fibers were then filtered and washed with an excess amount of distilled water and neutralized, followed by a drying process at room temperature. The swollen fibers were then hydrolyzed in 250 ml of 2M sulphuric acid (H2SO4) at 80°C for 3 hours with continuous stirring. Then, they were filtered and rinsed with distilled water after stirring in the 250 ml H2SO4 for 3 hours until the pH became neutral. The hydrolyzed fiber was then subjected to alkaline treatment to eliminate the soluble lignin in the fibers by treating them with NaOH at 80°C for 2 hours with continuous stirring and rinsing with distilled water before air drying for 3 hours. The dried fibers were bleached with 250 ml of 2% sodium chlorite solution (NaClO2) at 50°C for 1 hour to eliminate the remaining lignin. This treatment was repeated 2 to 3 times until the residue became colorless. Distilled water was used to cool, rinse and filter the reaction mixture to neutral before drying in air.\n\nThen, the mechanical process took place in the extraction process, in which the extracted fibers from chemical treatment were milled. The grinding process was carried out in the wet state using a Retsch-PM 100 high-energy planetary mill. A wet grinding process was conducted in deionized water using a 10 mm diameter zirconia ball. The vessel was filled with a ball-to-material ratio of 10:1 and deionized water and ground at room temperature with a speed of 450 rpm for 6 hours. After grinding, the slurry was collected for the drying process. The slurry was then dried using a freeze dryer at -50°C for 8 hours. A field emission scanning electron microscope (FESEM) was used to analyze the size and morphology of the extracted Jute CNFs at 500, 1 and 20k magnification.\n\n\nResults and discussion\n\nThere are a few methods that can be used to study the properties of nanomaterials including chemical and structure analysis (FTIR and XRD), thermal analysis (DSC and TGA) and morphology and size analysis (AFM, FESEM and TEM). However, it is found that the methods such as AFM, FESEM and TEM are effective methods to analyze the changes in the morphology and size of extracted CNF after each treatment phase (chemical and mechanical treatment) (Ravindran et al., 2019; Xie et al., 2016; Tuerxun et al., 2019; Pradhan and Jain, 2017). Thus, in this work, the morphology shape and size of extracted jute CNF were analyzed by using FESEM.\n\nMicrostructural analysis is one of the most vital parts in analyzing a material. This is because the shape and morphology structure of a material have tremendous effects on the physical, mechanical and thermal properties of the final material. The microstructure of raw jute fiber and extracted jute cellulose nanofiber (CNF) was analyzed by using a field emission scanning electron microscope (FESEM) at low and high magnification.\n\nFigure 1 (a) and (b) shows the micrograph of raw jute fiber observed under the field emission scanning electron microscope (FESEM) at low and high magnification. It was observed that the raw jute fibers present in long rod shapes with surface roughness and irregularities (Figure 1 (a) and (b)) with dimensions ranging from 10 to 60 μm. Additionally, the rough surface and long coiled fiber bundles are visible under high magnification morphology (Figure 1 (b)). Each elemental fiber is present in a compact structure that exhibits an orientation in the fiber axis direction with some non-fibrous elements on the surface (rough surface). The rough surface of raw jute fibers might be attributed to the existence of non-fibrous elements like waxes, lignin, hemicellulose and non-cellulosic contents. These non-cellulosic elements are known to bind the surface of cellulosic fibers and act as ‘natural binders’. Similar observations were made by Ng et al. (2015) and Meng et al. (2019) on the surface of their plant fibers.\n\nMeanwhile, Figure 2 (a) and (b) show the FESEM micrograph of the jute fiber at 1k magnification after chemical treatment. It is clearly shown that the surface of chemically treated jute fibers seemed smooth and clean (Figure 2 (a)), representing that non-cellulosic components were successfully detached. During the swelling and alkaline process, some of the individual microfibers were exposed and the fiber bundles start to loosen due to the incomplete detachment of the non-cellulosic impurities. It was noted that there were small pieces and granules on the surface of the fibers, as shown in Figure 2 (b). This might be due to the lignin polycondensate and deposits of inorganic materials during the swelling and alkaline process. However, after the bleaching process, the fibers exhibited a relatively shiny fibrous texture and a smooth surface with average diameters ranging from 3 to 13 μm.\n\nThe extracted jute CNFs (Figure 3 (a) and (b)) range from 23 to 300 nm in diameter at the nanoscale and have a network-like intersecting fiber morphology. These CNFs were successfully obtained via the chemomechanical method, in which the jute fibers were treated via a chemical treatment that involves swelling, alkaline treatment and bleaching process before undergoing a mechanical milling process using a planetary ball mill. The removal of non-cellulosic components from the raw jute fiber shows that the chemical treatment is successful in breaking the hydrogen bond between cellulose fibers. Furthermore, the mechanical milling process combined with the chemical treatment of jute fiber allows the removal of wax, lignin and other non-cellulosic contents thus increase in cellulose content. Therefore, this procedure is expected to play an excellent role in improving the adhesive properties of the extracted CNF in the polymer matrix. In addition, the reinforcement materials with fibrous morphology are preferred over those with spherical shapes as this morphology offers better resistance to cavity growth in the composite materials.\n\n\nConclusion\n\nThe jute cellulose nanofibers (CNF) were successfully drawn out from raw jute fibers by means of a combination of chemical and mechanical treatment. Based on the FESEM result, it is obvious that the chemomechanical method (combination of chemical treatments and planetary ball milling) is an effective technique for isolating CNF from jute fiber. FESEM micrographs show that the extracted CNF possess itself in the nano-sized dimension of 23 to 300 nm with a network of fibrous structure crossing each other. The surface morphology of the jute fiber (Figure 1c) is much smoother indicating that the non-cellulosic components were effectively detached via chemical treatment. Besides, the morphology of the extracted jute CNFs also shows their suitability to be used as reinforcement materials as the network fibrous shapes can offer better resistance to cavity growth and hence, improve the properties of composite materials.", "appendix": "Data availability\n\nfigshare: Synthesis of Cellulose Nanofiber from Jute Fiber via Chemomechanical Method. https://doi.org/10.6084/m9.figshare.22114919.v2\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAslan M: Characterisation of Flax Fibres and Flax Fibre Composites. Being cellulose based sources of materials. DTU Wind Energy. DTU Wind Energy E. 2012; (0005).\n\nDas K, Ray D, Bandyopadhyay NR, et al.: A study of the mechanical, thermal and morphological properties of microcrystalline cellulose particles prepared from cotton slivers using different acid concentrations. Cellulose. 2009; 16(5): 783–793. Publisher Full Text\n\nDahlem MA, Borsoi C, Hansen B, et al.: Evaluation Of Different Methods For Extraction Of Nanocellulose From Yerba Mate Residues. Carbohydr. Polym. 2019; 218: 78–86. PubMed Abstract \| Publisher Full Text\n\nDanial WH, Abdul Majid Z, Mohd Muhid MN, et al.: The reuse of wastepaper for the extraction of cellulose nanocrystals. Carbohydr. Polym. 2015; 118: 165–169. PubMed Abstract \| Publisher Full Text\n\nHarini K, Ramya K, Sukumar M: Extraction of nano cellulose fibers from the banana peel and bract for production of acetyl and lauroyl cellulose. Carbohydr. Polym. 2018; 201(June): 329–339. PubMed Abstract \| Publisher Full Text\n\nKarimi S, Tahir PM, Karimi A, et al.: Kenaf bast cellulosic fibers hierarchy: A comprehensive approach from micro to nano. Carbohydr. Polym. 2014; 101: 878–885. PubMed Abstract \| Publisher Full Text\n\nKian LK, Saba N, Jawaid M, et al.: A review on processing techniques of bast fibers nanocellulose and its polylactic acid (PLA) nanocomposites. Int. J. Biol. Macromol. 2018. Publisher Full Text\n\nLewin M: Cotton Fiber Chemistry and Technology.2007; pp. 1–150.\n\nLu P, Hsieh Y-L: Preparation and properties of cellulose nanocrystals: Rods, spheres, and network. Carbohydr. Polym. 2010; 82(2): 329–336. Publisher Full Text\n\nMaciel MMÁD, de Carvalho Benini KCC , Voorwald HJC, et al.: Obtainment and characterization of nanocellulose from an unwoven industrial textile cotton waste: Effect of acid hydrolysis conditions. Int. J. Biol. Macromol. 2018. Publisher Full Text\n\nMelikoğlu AY, Bilek SE, Cesur S: Optimum Alkaline Treatment Parameters for the Extraction of Cellulose and Production of Cellulose Nanocrystals from Apple Pomace. Carbohydr. Polym. 2019; 215: 330–337. PubMed Abstract \| Publisher Full Text\n\nMeng F, Wang G, Du X, et al.: Extraction and characterization of cellulose nanofibers and nanocrystals from liquefied banana pseudo-stem residue. Compos. Part B. 2019; 160(August 2018): 341–347. Publisher Full Text\n\nMishra S, Kharkar PS, Pethe AM: Biomass and Waste Materials as Potential Sources of Nanocrystalline Cellulose: Comparative Review of Preparation Methods (2016 - Till date). Carbohydr. Polym. 2018; 207: 418–427. PubMed Abstract \| Publisher Full Text\n\nMorais JPS, Rosa M, de F, et al.: Extraction and characterization of nanocellulose structures from raw cotton linter. Carbohydr. Polym. 2013; 91(1): 229–235. PubMed Abstract \| Publisher Full Text\n\nNagarajan KJ, Balaji AN, Ramanujam NR: Extraction of Cellulose Nanofibers from Cocos Nucifera var Aurantiaca Peduncle by Ball Milling Combined with Chemical Treatment. Carbohydr. Polym. 2019; 212: 312–322. PubMed Abstract \| Publisher Full Text\n\nNg H-M, Sin LT, Tee T-T, et al.: Extraction of cellulose nanocrystals from plant sources for application as reinforcing agent in polymers. Compos. B. Eng. 2015; 75: 176–200. Publisher Full Text\n\nPaulo J, Oliveira D, Pinheiro G, et al.: Macromolecules Cellulose nanocrystals from rice and oat husks and their application in aerogels for food packaging. 2019; 124: 175–184.\n\nPhanthong P, Reubroycharoen P, Hao X, et al.: Nanocellulose: Extraction and application. Carbon Resour. Convers. 2018; 1(1): 32–43. Publisher Full Text\n\nPradhan MK, Jain M: Preparation and testing of mechanical and microstructural properties of nanocellulose rubber composite: A brief review. i-manager's Journal on Material Science. 2017; 5: 31–42.\n\nRavindran L, Sreekala MS, Thomas S: Novel processing parameters for the extraction of cellulose nanofibers (CNF) from environmentally benign pineapple leaf fibers (PALF): Structure-property relationships. Int. J. Biol. Macromol. 2019; 131: 858–870. PubMed Abstract \| Publisher Full Text\n\nSingh S, Gaikwad KK, Park S, et al.: Microwave-assisted step reduced extraction of seaweed (Gelidiella aceroso) cellulose nanocrystals. Int. J. Biol. Macromol. 2017; 99: 506–510. Publisher Full Text\n\nStevulova N, Cigasova J, Estokova A, et al.: Properties Characterization of Chemically Modified Hemp Hurds. Materials (Basel). 2014; 12: 8131–8150.\n\nTanmoy AM, Alum MA, Islam MS, et al.: Jute (Corchorus Olistorius Var. O-72) Stem Lignin: Variation in Content with Age. Bangladesh J. Bot. 2014; 43: 309–314.\n\nTuerxun D, Pulingam T, Nurul IN, et al.: Synthesis, characterization and cytotoxicity studies of nanocrystalline cellulose from the production waste of rubber-wood and kenaf-bast fibers. Eur. Polym. J. 2019; 116: 352–360. Publisher Full Text\n\nWang Z, Yao Z, Zhou J, et al.: Reuse of waste cotton cloth for the extraction of cellulose. Carbohydr. Polym. 2017; 157: 945–952. PubMed Abstract \| Publisher Full Text\n\nXie J, Hse CY, Hoop CFD, et al.: Isolation and characterization of cellulose nanofibers from bamboo using microwave liquefaction combined with chemical treatment and ultrasonication. Carbohydr. Polym. 2016; 151: 725–734. PubMed Abstract \| Publisher Full Text" }	[ { "id": "274051", "date": "07 Jun 2024", "name": "Pawan Kumar Rakesh", "expertise": [ "Reviewer Expertise I am working on the primary and secondary processing of polymer matrix composites." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 focuses on the production of cellulose nanofibers from jute fiber using a chemomechanical approach. The ball milling method was commonly used in the extraction of cellulose nanofiber from natural fibers. It has applications in the cotton and textile manufacturing industries. The manuscript exhibits exceptional writing skills. All the sections have been adequately described. The manuscript is eligible for accepting without any modifications.\n\nIs the work 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": "308132", "date": "05 Sep 2024", "name": "Asniza Mustapha", "expertise": [ "Reviewer Expertise Nanocellulose", "Nanocomposites", "Biomass", "Non-wood based products and processing", "Bamboo", "Rattan", "Palms" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this manuscript is well-written and of good quality. However, I have a few comments on some aspects that may need attention. In general, the manuscript is acceptable.\nTitle: The title is acceptable but a bit too general. It would be more appropriate if the authors could revise it to better reflect the study's focus on the morphological analysis and properties of the obtained CNF. Introduction: Paragraph 2: However, the most common types that are widely used in engineering are CNC and CNF. Please evaluate more on the term “engineering”. Introduction: Paragraph 4: Usually, CNF is extracted from agro-waste materials with the primary aim to turn waste into excellent materials and directly save the environment. I think the term “directly” should be changed to “indirectly”. Introduction: Paragraph 5: Hence, to reduce energy consumption and chemical usage, this work was done to obtain CNF from jute fiber using a chemo mechanical method. How do the authors demonstrate that their method reduces energy consumption and chemical usage? Have any calculations been conducted to support this claim? Please provide an explanation. Table 1: Based on Table 1, it is shown that jute has a low content of lignin, pectin and waxes compared to other types of fibers, which suggests it can easily undergo a bleaching process and have a high fiber strength. How do the authors proved that the fiber exhibited high strength? Please provide an explanation. References: Aslan and Mustafa, 2012. The reference is not included in the references list. Please check. References: Some references are out of guidelines. For example, Kian LK, Saba N, Jawaid M, et al.: A review on processing techniques of bast fibers nanocellulose and its polylactic acid (PLA) nanocomposites. Int. J. Biol. Macromol. 2018. No volume/issue/pages is included. References: Please kindly check the references again, do not include the old references, and check the abbreviation of the journals.\nThank you.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "308126", "date": "06 Sep 2024", "name": "Dr. Ashok Kumar Bharimalla", "expertise": [ "Reviewer Expertise Agro Machinery Design", "Crop Mechanization", "Fibre and its Composite", "Nano-technology and its application in Textile and Agriculture", "Agribusiness Incubation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 may be accepted after a major revision: 1) The FTIR and XRD characterization of cellulose extracted from the jute fiber is very much essential to check the quality/purity of the extracted cellulose. 2) Since cellulose is a soft and fibrous material, PM100 ball mill is not suitable for nano-fibrillation. Hence, a proper explanation can be mentioned in the manuscript. Also, the mean fibre diameter needs to be reported. 3) Surface area analysis by BET analyzer can be reported to support the increase in surface area due to nanofibrillation. 4) Innovation in this work is not properly spelled out and is found to be regular work. If any innovation is there should be clearly mentioned.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/13-40
https://f1000research.com/articles/13-39/v1	08 Jan 24	{ "type": "Research Article", "title": "Combating the devastating effects of cotton bollworm: case of Uzbekistan", "authors": [ "Gavharhon Shokirova", "Zuhriddin Juraev", "Mutabar Mirzayeva", "Zuhriddin Juraev", "Mutabar Mirzayeva" ], "abstract": "Bollworm poses a significant threat to cotton crops worldwide, including in the Fergana province of Uzbekistan. This study aims to explore innovative approaches to bollworm control and contribute to the global understanding of integrated pest management strategies (IPM). Using an extensive literature review and field observations, the study evaluates the effectiveness of an integrated protection system in reducing reliance on chemical inputs while increasing overall efficiency. The results highlight the multigenerational life cycle of bollworm, its impact on various crops, and the need for artificial biological control methods and targeted insecticide applications. In addition, the study highlights the need to adopt globally accepted IPM practices to ensure sustainable pest management. The findings underscore the importance of collaboration among researchers, farmers, policymakers, and industry representatives in developing and implementing advanced control measures. Through integrated approaches and the incorporation of scientific advances, bollworm infestations can be contained while safeguarding agricultural productivity and minimizing environmental risks. Preprint of this article is available at: https://www.preprints.org/manuscript/202306.1512/v1", "keywords": [ "Bollworm", "Cotton crops", "Uzbekistan", "Integrated pest management", "Sustainable pest management." ], "content": "Introduction\n\nCotton production is a major agricultural sector involving over one hundred countries worldwide. Major cotton-producing countries, including India, China, the United States, Brazil, and Pakistan, grow cotton on about 35 million hectares and produce about 27 million tons of cotton fiber annually (www.theworldcounts.com, 2022). Effective control of pests and diseases through biological, chemical, and agrotechnical methods is critical to ensuring high-quality and abundant cotton harvests and protecting various other crops (Mollaee et al., 2019). However, the increasing insect population worldwide poses a major environmental and biodiversity challenge (Roy et al., 2023). Each year, a significant portion of global crop production, ranging from 20% to 40%, is lost due to pest infestations. The Food and Agriculture Organization of the United Nations reports that plant diseases alone result in a staggering $220 billion economic loss annually, while invasive insects account for an additional $70 billion. Weeds also pose a significant biotic constraint on global food production (www.unfoundation.org: November 2022). Therefore, protecting human health, meeting global agricultural needs, preserving the environment, and protecting crops from pests have become urgent priorities (UN Climate Change). Urgent tasks include the expansion of biological laboratories, the widespread application of biological methods, the timely implementation of agrotechnical practices, and the study of the effective use of selective chemical preparations. Among the numerous insect pests, the cotton bollworm (Helicoverpa armigera) stands out because of its omnivorous feeding habits and the significant damage it can cause. The scientific importance lies in the research of preventive measures against the bollworm, such as the use of pheromone traps and artificial butterfly traps (Li et al., 2022; Melikuziyev et al., 2022). Currently, the use of biological means to protect cotton and other crops is extensively practiced in three regions of the Fergana Valley in Uzbekistan. However, the effectiveness of the use of Trichogramma, Bracon, and Golden Eye entomophages is influenced by human factors, so research is needed to improve the efficiency of biological methods and to develop scientifically sound agrotechnical measures for bollworm control (Yuldasheva et al., 2022). Therefore, conducting scientific research in this area is of utmost importance.\n\nThis study, conducted under the priority research project “Agriculture, Biotechnology, Ecology and Environmental Protection”, falls under the initiative to promote science and technology in Uzbekistan (www.lex.uz). The study focuses specifically on cotton fields in Uzbekistan, particularly in the Fergana region, and is a case study based on practical research. The main objective of this study is to improve the effectiveness of bollworm control in the Fergana region by optimizing protection means, methods, and associated costs. Proposals are also made for the development of a comprehensive control approach that includes agrotechnical measures. The study focuses on companion crops heavily affected by bollworms, such as cotton, corn, tomato, pea, and sunflower. It examines the methods and means of an integrated protection system used to protect cotton from bollworm infestation. While numerous scientific studies on bollworm control are being conducted in cotton-growing countries, comprehensive research addressing existing challenges, optimization of control agents and methods, consideration of associated costs, and improvement of the overall control system by incorporating agrotechnical measures is still relatively unexplored in the Fergana Valley.\n\nThe study of bollworm control in cotton production includes several theoretical frameworks and approaches that provide a solid foundation for understanding and managing insect pests (Prasanna et al., 2022). Integrated Pest Management (IPM) is an important theoretical framework for bollworm control. It emphasizes ecosystem-based strategies that integrate biological, cultural, and chemical methods of pest control while minimizing environmental impacts. Implementing the principles of IPM reduces reliance on chemical insecticides, promotes natural pest control mechanisms, and improves the long-term sustainability of cotton production systems (Ortega-Ramos et al., 2022; Jowett et al., 2022). The study’s focus on implementing an integrated protection system is consistent with the principles of IPM and reflects a holistic and environmentally conscious approach to bollworm control (Ortega-Ramos et al., 2022). Agroecology is another relevant theoretical framework that emphasizes the intricate interactions among crops, pests, and the broader agroecosystem. By promoting ecological processes, agroecology aims to create resilient and balanced agricultural systems. Integrating agroecological principles into bollworm management includes using natural enemies, promoting biodiversity, improving soil health, and diversifying habitats. The study’s focus on optimizing agrotechnical measures and developing comprehensive control methods demonstrates the integration of agroecological concepts in bollworm control.\n\nThe concept of ecological resilience is highly relevant to the bollworm control study. Ecological resilience refers to the ability of an ecosystem to withstand disturbance while maintaining its structure and functions (Schneider et al., 2022). Building ecological resilience in crop protection means promoting biodiversity, reducing pesticide use, and strengthening natural biological control mechanisms. By introducing measures that increase the resilience of cotton fields to bollworm infestation, such as incorporating biological agents and using agrotechnical practices, the study is consistent with the overall goal of improving the ecological resilience of agricultural systems (Kamburova et al., 2022).\n\nThe field of pest management is being shaped by several emerging trends and studies that have implications for cotton worm control. These include advances in biological control methods, such as the use of genetically modified organisms (GMOs) or RNA interference (RNAi) technology to control bollworms (Hilbeck et al., 2020; Fu et al., 2022). In addition, studies examining the effects of climate change on insect pest dynamics and exploring strategies for climate-smart pest management provide valuable insights for adapting bollworm control measures to changing environmental conditions. By considering these emerging trends and incorporating relevant research findings, the study contributes to the ongoing discourse on innovative and sustainable approaches to bollworm control.\n\nIn addition to these established theoretical frameworks and emerging trends, the inclusion of an ecofeminist approach to the study raises critical questions about the environmental and social impacts of bollworm control. Ecofeminism explores the connection between the oppression of women and the destruction of the natural world (Tong, 2022). Using chemical insecticides in bollworm control can harm ecosystems, including soil and water contamination, and harm non-target organisms. These ecological impacts disproportionately affect marginalized communities, particularly women who rely heavily on natural resources for their livelihoods (Barthold et al., 2022). An ecofeminist perspective emphasizes the need for alternative, non-hierarchical approaches to pest management that prioritize ecological harmony and social justice (Ahmad & Yaquub, 2022). It encourages the exploration of sustainable, community-based practices that empower local communities and promote women’s participation in decision-making (Juraev & Ahn, 2022). It also emphasizes valuing traditional ecological knowledge and indigenous practices in pest management (Juraev, 2022).\n\nIncorporating ecofeminist concerns into the study of bollworm control leads to a critical examination of the social and environmental impacts of current control methods and encourages the exploration of alternative approaches that prioritize the well-being of both ecosystems and marginalized communities. By incorporating an ecofeminist perspective, the study contributes to a more comprehensive and socially just understanding of bollworm control in cotton production.\n\nEcofeminism as an interdisciplinary field includes the work of various scholars and activists such as Vandana Shiva and Mies (2014), Maria Mies (2014), Carolyn Merchant (2020), and Val Plumwood (2001). Notable works such as Shiva’s “Staying Alive: Women, Ecology, and Development” (2016) and Mies’ “Patriarchy and Accumulation on a World Scale” (2014) address the interconnectedness of gender, nature, and social justice (Tong, 2022). The ecofeminist approach is increasingly recognized as a valuable framework for analyzing environmental problems and advocating for transformative change (Szopa, 2022). In the specific context of bollworm control in Uzbekistan, the ecofeminist approach offers insights into the socio-environmental impacts of current practices and contributes to the development of alternative and sustainable pest management strategies (Yusupova & Gapparov, 2020). While the applicability of ecofeminist approaches may vary depending on local context and cultural factors, the principles of ecological harmony, social justice, and community empowerment are universally relevant (Winston, 2022). Incorporating ecofeminist concerns into the study of bollworm control in Uzbekistan illuminates the gendered dimensions of pest management and promotes inclusive decision-making processes that prioritize the well-being of ecosystems and marginalized communities.\n\n\nMethods\n\nRigorous methods from entomology, agricultural entomology, and agro-toxicology were used in the study. Data obtained from laboratory experiments and small and large field trials were subjected to statistical analysis using Microsoft Excel based on the dispersion method.\n\nThe research presents several important scientific findings. Field experiments were conducted using agrotechnical measures in cotton production for the first time. A comparison with fields where wheat was planted between cotton rows showed a 25% reduction in bollworm numbers. A comparative analysis between fields where cut growth sites were left between cotton rows and fields where litter was collected and removed showed that the latter approach had higher efficiency, with bollworm infestation reductions ranging from 36% to 42%. The use of mineral fertilizers and chemical control agents against bollworms in cotton management showed the potential to increase yields by up to 63.7%. Monitoring with pheromones contributed to a 30-40% increase in biological efficiency. Determining optimal timing for science-based chemical treatments resulted in an additional 3.2 quintals of crop per hectare and an economic benefit of 442,000 UZS (or USD 400).\n\nAcceptable efficacy against bollworms and sucking pests were achieved with Duet (55% emulsion concentrate) at a rate of 1.5 L/ha and Progress Plus (44% emulsion concentrate) at the same rate. Agrotechnical interventions such as fall plowing, salt washing, cultivation, provision of local and mineral fertilizers, retailing, and irrigation were evaluated for their acute and subsequent effects on cotton crops. Practical recommendations were made that decreased cotton seed loss from 10-12% to 80-90%. The organic method based on pheromone traps (pheromone monitoring) showed increased efficiency resulting in an additional yield of 1.4 quintals per acre, an increase in cotton product value of $1.53 for every dollar spent on protection, and an increase in profitability to 153%.\n\nThus, by following the scientifically recommended periods for active bollworm control, the use of emamectin benzoate, indoxacarb, and methomyl during the presence of medium and large juvenile worms resulted in an additional yield of up to 5 quintals per acre. The scientific significance of these research results lies in the evaluation of the seasonal development of the bollworm, its role in agro-biocoenoses, and its links with arthropods under the climatic conditions of the Fergana Valley (see also, Shokirova & Abdullaeva, 2020; Nazirkulomovna, 2022). The damage caused by bollworms to cotton crops and the effectiveness of various control measures, including biological and chemical approaches, contribute to integrating various tools into a comprehensive protection system. From a practical perspective, the research results are important for understanding the importance of each measure used in cotton agronomy to control bollworms. The effective use of bio-methods, insecticides, and the evaluation of pheromone monitoring and other physical-mechanical methods were demonstrated. Based on scientific research, practical recommendations were developed to increase cotton protection’s effectiveness against bollworms and optimize the associated costs.\n\nConsequently, a recommendation entitled “Modern control system against cotton bollworm” was published for the protection of cotton and other infested crops in the regions of the Fergana Valley (Tilyabaev et al., 2018; Nazirkulomovna, 2022). Application of recommended doses of Entovant, Entovant PRO (0.2-0.45 liters/kilogram per hectare), “Prokleim”, “Emamek” and “Emaben Surender” has achieved an acceptable level of protection and resulted in an additional yield of 3-4 quintals per hectare. Successful implementation of these measures on certain farms in Toshlok District and Oltiarik District resulted in yield increases from 2.5 quintals/hectare to 4.0 t/ha. A total of 5560 hectares of cotton fields in the Fergana region benefited from the application of the active ingredients Entovant, Entovant PRO (0.2-0.45 liters/kilogram per hectare), Proklam, Surender, and Abamectin at a rate of 0.35 kilograms/hectare. This resulted in the economic efficiency of 2,176,000 UZS (approximately USD 200) per hectare compared to the average control and 1,900,000 UZS (approximately $170) compared to the standard treatment (Decis 2.5% e.m.g. at a rate of 0.7 liters/hectare).\n\nThe cotton bollworm, which belongs to the butterfly family (Lepidoptera) Noctuidae, is a widespread insect species on Earth. There are over 200 species of bollworms in Central Asian countries, including various aphids that damage cotton and other crops. The most widespread and destructive species is the cotton bollworm. Because it feeds primarily on cotton bolls, the insect is commonly referred to as the “cotton bollworm” (see Figure 1). This pest is found in many countries around the world, including Indochina, the Middle East, Africa, South America, and the eastern part of Australia. The family Noctuidae includes more than 35,000 known species of moths, making it the most widely distributed horned caterpillar species in the world (Malinga & Laing, 2022; Alam et al., 2022). Consequently, numerous studies have focused on these pests in Uzbekistan. For instance, in experiments conducted between 1985 and 1990, Uzbek scientist M.I. Rashidov recorded 51 species of pests from 15 families of “Solanaceae.” Among them, six aphid species were identified as harmful to tomatoes. Rashidov’s research also revealed that 17 species from the Lepidoptera Noctuidae family were present in the agro-biocenosis and caused damage to agricultural crops. Rashidov emphasized that without the inclusion of entomophagous organisms, it is impossible to achieve 100% results in integrated pest management for agricultural crops. In cotton production alone, entomophagous organisms have an efficiency of 85-90% and reduce pest populations below economically harmful levels (Rashidov & Khodzhaev, 2000).\n\n1 – female (♀) and male (♂) butterfly breeds; 2 – an egg laid on a leaf; 3 – drying of the damaged bone; 4 – damaged cyst; 5 – damage to the comb; 6 – internal view of the affected cyst; 7 – nightshades.\n\nThose relying solely on chemical protection methods cannot achieve such high efficiency. Moreover, long-term observations by scientist Khojaev Sh., have shown that a single cotton crop in Uzbekistan is damaged by 14 species of underground and above-ground ants and seven species of tomato root-gnawing ants (Gofurjonovich & Ibodullaevna, 2022). In particular, the autumn and night moths contributed most to the damage. The bollworm is particularly prevalent in Central Asia and southern regions of Russia, causing significant damage to agricultural crops (Muhammadzikirovna, 2022). In Uzbekistan, it causes damage to cotton plantations in regions such as Surkhandarya, Kashkadarya, Fergana, Andijan, and Namangan. In recent years, it has increasingly infested cultivated areas in Bukhara, Khorezm, Karakalpakstan, and the Tashkent region. On cotton farms in the Andijan and Fergana regions of the Fergana Valley, bollworm is a recurring problem. Several factors contribute to this problem, including the cultivation of crops preferred by the bollworm (such as maize, vegetables, and legumes) on open land after grain harvest.\n\nThe cotton bug can also be classified as a significant insect. The adult butterfly has a wingspan of 30-40 mm and a body that ranges in color from yellowish-red to bluish-gray. On the upper side of the forewings is a round spot and a kidney-shaped black spot. The sexual development of the butterfly is incomplete after hatching from the cocoon, so additional feeding is required. These butterflies usually begin to fly in April or May when the soil temperature reaches 16°C at a depth of 10 cm. The butterflies lay their eggs primarily on the upper surface of leaves near the plant’s growing point, either singly or in groups. The eggs have an apartment and round shape on the underside, measuring 0.5-0.7 mm in diameter and 0.4-0.5 mm in length. They have 26-28 ribs, which are characteristic of solanaceous plants. Under magnification, the eggs look old and resemble empty containers when the trichogram flies emerge. Some eggs fail to hatch. The color of the caterpillar varies with diet and age, reaching 2.5-3.0 cm in length when fully grown (at about 5-6 years of age). The cotton bollworm goes through five molting stages during its six-year lifespan and builds a nest before each molt. The anal openings at the posterior end of the caterpillar body distinguish between female (♀) and male (♂) individuals (Figure 2). About 8-12 days after egg laying, the next generation of butterflies hatches from the buds. Under the conditions of Uzbekistan, the cotton bollworm goes through four to five generations per year, with three to four generations infesting cotton crops. Several experts have studied the cotton bollworm in detail, including its bioecological characteristics, distribution, target crops, damage extent, and protection methods. In particular, the development of short- and long-term forecasting methods for annual cotton crop trends occupies an important place among these studies. Predicting the occurrence and density of bollworm generations in each field is a key issue in pest management, and scientists have developed several computational methods.\n\nThe most recent method used in practice was developed in 1959 Night Worms by a group of scientists from the Institute of Plant Protection in Uzbekistan. This method is based on a knowledge-based approach and uses data from the nearest meteorological stations for each region studied. The first-generation butterfly begins to fly when the temperature is constantly above 16°C at a depth of 10 cm in the uppermost soil layer. From this point, an effective temperature index is calculated by subtracting 11°C from the average daily air temperature. Once this sum reaches 550°C, a new generation of butterflies is expected to emerge. Subsequent generations of bollworms are calculated using the same method (Joel, 2011).\n\nAccording to this approach, the development of pollen worm populations in the following year is described as follows. The overwintering phase begins in August when the air temperature falls below 25°C. The survival of the bollworm during this period depends on feeding on young worms, namely cotton, whose bolls have begun to open. The effective temperature is monitored during this period. If the average air temperature in autumn is between 11°C and 350°C, it means that the conditions are favorable for the pest to prepare for winter; otherwise, the conditions are considered unfavorable. The use of pheromone traps containing the cotton bollworm sex pheromone, introduced in Uzbekistan in the 1980s, cannot replace the need to predict the next generation of the pest or its density in the following year. These traps primarily indicate when the same generation of bollworms will emerge in the same field and provide information on population density (Tilyabaev et al., 2018). Despite advances in the methods described above, unexplained occurrences still underscore the continuing need for scientific research. Cases such as the significant bollworm outbreaks in 1996 (Karadrina), 1988, 2004, and 2015-2017 in Central Asian countries remind us that scientific research is limitless. The cotton bollworm has a wide range of host plants, with more than 250 known species. The main food source is the fruiting stems of the plants, especially the bolls. However, the young worms initially feed on soft leaf tissue for one to two days. In Uzbekistan, cotton is the main host for the cotton bollworm, but it can also infest other crops such as corn, tomatoes, peanuts, squash blossoms, tobacco, hemp, peas, peppers, roses, and various other plants. In all plants and trees, there is excessive flower production on the stem, which gradually turns into fruit bud drop. The bollworm damage coefficient, a recently adopted criterion for the level of economic damage in Uzbekistan, indicates that an average infestation of 10 bolls per 100 bushels of cotton results in a yield reduction of 1 quintal. It is assumed that there are 10-12 young worms per 100 bushels of cotton. However, this indicator is from the past when agriculture was based on old economic relations. Since gaining independence in 1991, Uzbek agriculture has changed significantly with the introduction of a new system. Former relations have been broken, and new ones have emerged. Farmers now could change the criteria for economic damage caused by pests based on yield. It is our responsibility to pave the way for the future.\n\nThe cotton bollworm has numerous natural enemies, including parasites and predators. Over 100 natural enemies of the bollworm, including parasites and predators, have been identified. In the early 20th century, the importance of the ectoparasite Bracon (Bracon hebetor Sau.) was recognized. The cocoon of bollworm eggs was studied extensively, including Trichogramma (Trichogramma sp.) and Bracon within the worm. In 1986, the genus Habrabracon was renamed Bracon. To ensure that the quality of Trichogramma produced in bio-labs and bio-factories meets the required standards, a state-approved model was created by the Uzbek Ministry of Agriculture Resources (www.agro.uz) and Ministry of Water Resources (www.water.gov.uz).\n\nAccording to this model, the following criteria must be met for each female Trichogramma:\n\n(1) The number of eggs should not be less than 30.\n\n(2) The ratio of females to males should be 1.5:1.0 or 2:1.\n\n(3) The number of laying females should be at least 90%.\n\n(4) The viability of females is defined as 89%, and the number of non-viable specimens should not exceed 5%.\n\n(5) Females should survive at least 5-7 days at a temperature of 25-29°C.\n\n(6) At least 80% of moth eggs should be damaged.\n\nApanteles (Apanteles cossack) also plays an important role in the natural reduction of bollworm populations. Another species of Apanteles, Cotesia, has been described by researchers. I.A. Somov reported the presence of two species of ichneumon flies in the dome of the bollworm.\n\n1. Beneficial Insects and Bio-Methods in Cotton Agro-Biocoenosis\n\nAll living organisms have their own natural habits and food preferences. These preferences create a chain of interactions known as the “herbivore-pest-natural link” (see Figure 3). Insects can be divided into two food groups: Predators, which kill their prey quickly, and scavengers or “parasites”, which weaken their prey slowly. Predators such as goldeneye and ladybug larvae, dragonflies, damselflies, wasps, and squash bees are generally more effective. Birds, especially sparrows, among vertebrates, are very effective at controlling pests.\n\n1 – the caterpillar of the ladybug on a cotton leaf, 2 – the Apanteles Cossack spider, which emerges from the body of a cotton spider, 3 – a rare carnivorous shield-eating spider worm on a peach trunk, 4 – its butterflies, 5 – the significance of predatory spiders is different.\n\nThere are a considerable number of beneficial insects. According to Mansurov, over 50 different species of bollworms are used naturally. The total number of bollworm larvae exceeds 150. This section discusses some notable species (Khayitov et al., 2021).\n\n(1) Apanteles kazak (Apanteles kazak Tel./Hymenoptera, Braconidae) is a specialized internal predator and collector of cotton tunnels. It is found throughout Uzbekistan and neighboring countries of Central Asia and the Transcaucasus. This small-winged insect lays its eggs on the bodies of first and second-stage cotton bollworm larvae. The larvae of Apanteles kazak develop inside the bollworm larvae and eventually hatch as adults from the host’s body segments. The bollworm survives only a few days before dying, while Apanteles kazak larvae hatch from the bollworm and spin cocoons within 30-40 minutes (www.pestworld.org).\n\n(2) Bracon (Bracon hebetor Say.) and other butterflies are free-feeding larvae on the surface attacking the bollworm. In Uzbekistan, there are other species that target the intermediate and adult stages of the pest (see Figure 4). Historical literature even mentions Bracon and reports that cockroaches damaged 23-37% of cotton plants on farms in the Tashkent region in 1991 and 2002. In August 2011, 8.3-13.7% of nightcrawlers in cotton fields in the Pakhtabad district of the Andijan region were found to be infected with Bracon (www.pestworld.org).\n\n(3) Bracon wasps (family Ichneumonidae) are large insects that parasitize cotton bollworm caterpillars. They lay their eggs on adult bollworms, from which larvae mature and hatch from the cocoon. These species have been discovered in Fergana, Andijan, Tashkent, and Surkhandarya regions (www.pestworld.org).\n\n(4) Tachinidae (Gonia cilipeda Rd.), also known as “deer flies” (Chrysops sp.), are insects that live as parasites in other insects. Mature Tachinidae lay their eggs on leaves that are unknowingly ingested by feeding bollworm larvae. The hatched Tachinidae larvae then develop by consuming the internal contents of the host worm. Finally, the Tachinidae hatch from the bollworm’s body after it has transformed into a fungus. This complex life cycle helps maintain balance in the insect world (www.pestworld.org).\n\n(5) The lacewing (Chrysopa carnea Steph.) is an insect that belongs to the genus Chrysopa and is used as an effective natural control agent against cotton pests, including bollworms. The powerful jaws of the lacewing can kill significant numbers of young bollworm larvae. Among the entomophages in the cotton field bio-coenosis, the lacewing has the highest population density (see Figure 5). In order to mass produce this insect, it is bred in bio-labs and bio-factories. Lacewing larvae are omnivorous and will attack any living organism in their path, including eggs, larvae, fungi, and adult insects, regardless of species. However, lacewing propagation is difficult and expensive because they must be dispersed as live larvae to be effective (www.pestworld.org).\n\n(6) The egg-eating Trichogramma (Trichogramma pintoi Voegele.) is a small insect belonging to the family Trichogrammatidae, 0.3-0.9 mm long (see Figure 5). Trichogramma played an important role in introducing biological control and reducing the use of chemical insecticides against bollworms in Uzbekistan. There are 12 species of Trichogramma adapted to the climatic conditions of Central Asia and able to survive at high temperatures and low humidity. Trichogramma hibernates as “prepupa” in infected eggs and begins to hatch when the average air temperature rises above 8-10°C, which requires additional food. Male and female races of Trichogramma differ primarily in the number of joints in their antennae (5 joints in females and 3 joints in males) and the pubescence of males (sexual dimorphism). Trichogramma can lay between 1 and 60 eggs in each pollen egg, depending on the size of the infested egg. In Uzbekistan, Trichogramma lays an average of 2.1 to 2.4 eggs in each bollworm egg. At an ambient temperature of 30°C, Trichogramma eggs take about 1 day to develop into larvae, which then feed on the host (egg) contents for 2-3 days. After this stage, they hatch into the “prepupa” or pronymph, followed by pupation after 1.5-2 days. After darkening and maturing for 3-3.5 days, the infected egg becomes a mature Trichogramma. While bollworm produces one generation (30-40 days), Trichogramma can produce offspring 2-3 times. In one season, there can be 12-14 generations. Trichogramma and Bracon are the main agents used against cotton bollworms and partially against fall bollworms. It is recommended that Trichogramma sylvetsin be applied at a rate of 1 gram per acre three to four times during each bollworm generation. When infestations are heavy and worms are present in the field, 200 to 2-3 thousand female Bracon individuals per hectare are applied, usually in mixed batches of males and females. However, the effectiveness of this approach may vary (www.pestworld.org).\n\n1 – mature breeds; 2 – larvae of the paralyzed roundworm and the caterpillar.\n\n1) Mature breed; 2) Trichogramma-infested nightshade eggs.\n\nThe use of lacewings as a live insecticide against sucking pests and bollworms is often recommended. However, there are several things to keep in mind. First, lacewing egg application can be expensive. Recommended ratios for egg application are 1:1 for aphid, spider mite and cotton mite eggs, 1:10 for larvae and 1:100 for mature brood. Second, the efficacy of lacewing eggs can be problematic because ants can eat them in the field. Field observations have shown an average of 300-350 ants per square meter of cotton bale in July. Considering these factors, it is advisable to release laboratory-bred lacewings near the fields, especially in the spring. Although the biological method is beneficial, there is a need in the field of entomology for more efficient and less toxic tools and methods. Therefore, conducting agro-toxicological research in Uzbekistan is of great importance. This research includes comprehensive testing and evaluation of pesticides from reputable chemical companies around the world, as well as domestically produced pesticides to ensure that they meet the necessary requirements. The research is conducted in accordance with the “Procedural Instructions” published by the State Chemical Commission under the Joint Stock Company “Uzkimyosanoat” (Chemical Industry) of Uzbekistan (www.uzkimyosanoat.uz).\n\nThese activities are organizational measures to limit agricultural damage caused by pests, including bollworms, for the coming year without incurring additional costs. They also include measures to reduce the pest population. These measures include planning for the coming year, equipment maintenance, and spring work.\n\nAgrotechnical control methods\n\nAgrotechnical control methods play a crucial role in creating unfavorable conditions for pest development and favorable conditions for plant growth. Effective bollworm control measures include fall plowing, irrigation, cultivation, and application of organic and mineral fertilizers. Improving cotton cultivation techniques and studying their impact on bollworm development are important aspects of this approach (Nazarov et al., 2020).\n\nPhysical-mechanical methods\n\nPhysical-mechanical methods involve the use of various devices to reduce pest populations. Examples include the use of “bags” to control bollworms, the construction of pheromone traps that exploit the insect’s sex pheromones, and the use of devices to collect insects in the field, such as traps that are attracted to light (Sorensen et al., 2016).\n\nUse of biologically active substances\n\nBiologically active substances are used to protect cotton crops. This includes the use of pheromone traps to control particularly harmful insects such as bollworms, fall bollworms, and crows. In addition, biologically active substances may contain attractants to lure insects or repellents into repelling them (Malinga & Laing, 2022).\n\nThus, significant research has been conducted since the 1980s on the sex pheromone of the cotton cutworm, a pest that inflicts damage on cotton and other crops. In the 1990s, the use of sex pheromones for cotton and autumn cutworms became widespread. However, further research is needed in this area, as there are instances where the butterflies do not respond to the pheromone traps.\n\nIt is important to emphasize that control measures that minimize damage are preferred. In this regard, experts aim to prepare the control of the bollworm by delaying its development for a longer period. Over the years, “preventive” treatments have been ineffective in controlling pests. However, methods that reduce the number of pests in subsequent generations have proven effective. For example, research conducted in the fall focuses on reducing bollworm offspring by using low-consumption insecticides to poison the worms as they prepare for winter. A combined protection system has been established to control bollworms and other cotton pests. The main objective of this system is to restore the balance between beneficial and harmful arthropods in nature, primarily through non-chemical means. In the literature, “integrated pest management” is described as a protection system combining various biological, chemical, agrotechnical, physical, mechanical, and quarantine methods. These approaches are used in conjunction with each other. Biological control methods take precedence in this framework. In Uzbekistan, Trichogramma species (family Trichogrammatidae) are widely used to control bollworm eggs. There are 12 naturally occurring Trichogramma species in Uzbekistan, which are found in various crops and ecosystems, especially in meadows. Trichogramma exhibits high reproductive potential in Uzbekistan, with 14-15 generations possible in a single season. These insects, active from May to October, are most abundant in July and August. Studies have shown that two species of Trichogramma are well adapted to climatic conditions in Central Asia. One species, Trichogrammatidae, thrives at high temperatures (30°C) and low humidity (30%), while the other, Hymenoptera, is still effective at temperatures of 30°C and humidity of 50%. In addition to these species, two other Trichogram species are recommended for use in Uzbekistan during the spring months: Tr. sugoniaevi Sor. (Hymenoptera) and Tr. evanescens Westw. (Richogramma evanescens Westwood). These species have shown a higher preference for air temperature and humidity (Kobiljonovna & Zaylobidinovna, 2022).\n\nCurrently, many insecticides are manufactured by large global pesticide companies, with smaller companies and ancillary players in the industry also involved in production. Many analogous products are available with identical active ingredients but different trade names. For example, there are 14 analogs of Detsis, 16 of Cypermethrin, 19 of Karate, and 9 of Emamectin Benzoate. Some of these products are manufactured and agro-toxicologically tested in Uzbekistan. Biologically active substances are between microbiological and chemical drugs. Among them, several substances with hormonal activity against bollworms (such as Dimilin, Eim, Sonnet, and Rimon) have shown promise in cotton research. However, their practical application remains limited. Biologically active substances also include the sex pheromones of certain insects. Many insects enter the mating season after hatching from their cocoons. This usually occurs 2-3 days after feeding on nectar from plant flowers, when their reproductive organs have developed. At this time, they release pheromones into the environment that signal their readiness to mate and attract insects of the opposite sex. Female insects usually release the sex pheromone.\n\nThe active ingredient in insect pheromones is the most important factor in their effectiveness. Even a small amount of pheromone, just a few molecules, can attract a male insect. Pheromones have multiple biological signaling properties and represent one of the most complex aspects of chemical ecology. Sex pheromones, trace pheromones, warning pheromones, territory marking pheromones, and eipheromones are the main types of insect pheromones. Insect sex pheromones were first isolated from the silkmoth (Bombyx mori) in 1959 (Abraham et al., 1992). Since the 1970s, research on isolating and identifying insect sex pheromones has intensified. By 1985, more than 350 insect species (over 160 in the former Soviet Union) were known to use sex pheromones worldwide. By the 1980s, the composition and major substances of bollworm sex pheromones were well documented (Tanskii, 1973). The composition of cotton bollworm sex pheromones consists mainly of major and minor substances, with Cys-11 hexadecenal accounting for 90% and Cys-9 hexadecenal accounting for 10% of the total composition. A mixture of these substances in a ratio of 9:1 is dissolved in an alcohol solution, which is then soaked in rubber tubes and distributed in kits for practical use. The recommended amount is 2 mg of the substance per rubber hose (one pheromone handle), and each rubber dispenser remains active for 15 days before being replaced by a pheromone trap.\n\nChemically synthesized pheromones that resemble natural pheromones are used extensively in insect biology and control research. Pheromone traps containing sex pheromones allow the study of insect phenology, developmental timing, and population dynamics. In addition, a control method that uses sex pheromones to trap bollworm moths and remove the pheromones from the females has been recommended. The use of pheromone traps for bollworm control has been scientifically established in Uzbekistan since 1983 (Khodzhaev, 1990). However, further scientific research is needed in this area. The cotton bollworm sex pheromone is a two-component compound chemically synthesized and developed by the Academy of Sciences of Uzbekistan Institute of Bioorganic Chemistry. Every year, cotton bollworm sex pheromone is newly synthesized, and its purity and activity need to be scientifically evaluated (www.biochem.uz). There are also new companies producing bollworm sex pheromones, and these products are being actively tested. In addition, the use of pheromone traps is critical for studying the effectiveness of applying trichograms in the field during specific time periods. A small rubber is inserted into a paper coated with adhesive, which is then impregnated with the pheromone substance and laid out in the field. The scent spreads with the wind and attracts male insects that become entangled in the adhesive. The effectiveness of the control methods depends on the number of moths caught in the glue traps.\n\nIn the fight against bollworms, chemical insecticides undoubtedly play a central role. In the past, various inorganic substances were used for this purpose until the 1940s, but their effectiveness proved to be minimal. During the war years, the development of insecticides such as DDT and organic hexachlorane compounds led to their worldwide use. In Uzbekistan, these agents were used extensively until the 1970s. After their ban, alternative agents were introduced to protect agricultural crops, including Polydofen, Fozalon, Phtalofos, Tsidial, Tsyanox, Kilval, Mezurol and others. During this period, new insecticides with minimal risks to warm-blooded animals and the environment were tested specifically for bollworm control (www.biochem.uz).\n\nThe properties of insecticides used in crop protection against bollworms are as follows:\n\n1. Efficacy against insects and spider mites: Insecticides are effective against both insects and spider mites, making them versatile pest control agents.\n\n2. Low toxicity to beneficial insects: These insecticides have low toxicity to beneficial insects, so there is minimal interference with natural pest control mechanisms.\n\n3. Short residence time in the environment: insecticides do not remain in the external environment for extended periods, minimizing their impact on non-target organisms and reducing the risk of accumulation.\n\n4. Relative safety for humans and warm-blooded animals: The insecticides are relatively safe for human and warm-blooded animal health, further enhancing their suitability for crop protection.\n\nDuring the study period, Sevin SL carbaryl, an insecticide with high efficacy against bollworms and lower environmental and human health risks, was used extensively at a dosage of 2.5 kilograms per hectare. However, it was later found that this insecticide has long-term effects and can promote the reproduction of spider mites, so it is no longer used. In recent years, the insecticide Lannate, which belongs to the class of carbamates and is similar to Sevin SL carbaryl, has been tested in Uzbekistan. It is formulated as a water-soluble powder packaged in water-soluble sachets, providing a convenient application method. In the late 1970s and early 1980s, synthetic pyrethroids became the primary means of crop protection. Pyrethroids such as Ambush, Tsimbush, Sumitsidine and Detsis showed high efficacy against bollworms and other insects in preliminary trials. Residues in the plants were also investigated, as was the determination of appropriate pre-harvest waiting periods. Pyrethroids were then tested and recommended for bollworm control in various crops. Unlike natural pyrethrins derived from chamomile flowers, synthetic pyrethroids are chemically synthesized stable compounds that have high efficacy, are more resistant to sunlight, and have a rate of degradation compared to other insecticides. Several generations of synthetic pyrethroids have been developed. The first generation, including sumitcidin, ambush, tsimbush, and detsis, has primarily insecticidal properties. Subsequent generation pyrethroids, such as Tsibolt, Karate and Talstar, also have acaricidal properties and can control both insects and spider mites. Newly synthesized pyrethroids offer higher purity, lower consumption and reduced environmental risks compared to their predecessors. Examples include purified forms of sumitcidin and Fastak (Alfagard), which contain specific isomers for improved efficacy (www.agro.uz).\n\nPyrethroids have several beneficial properties, including high biological efficacy, low consumption, minimal short-term environmental impact, no crop damage, and potential productivity enhancement. They are also relatively safe for beneficial insects, including bees, and adhere to soil and foliage without significant wash-off. However, negative properties include the potential for allergic reactions in humans, acute effects on certain stages of beneficial insects, and effects on aquatic animals.\n\nPyrethroids have shorter persistence in treated plants. Studies indicate that the active ingredient dissipates in cotton plants treated with various pyrethroids within 15-25 days, and no toxic residues are detected in seeds and plant oil. Synthetic pyrethroids represent a significant advance in the field of pesticides and offer high efficiency and safety in crop protection. However, continued research and development are needed to address the shortcomings of existing insecticides. Several insecticides or insecticide-acaricide mixtures, such as Nurell-D, Dadetsi, Deltafos, Endjeo, and Prempt, have been developed to overcome these limitations. Consequently, agro-toxicological evaluations are required for these new bollworm control agents (www.agro.uz).\n\nThe development of resistance in arthropods to insecticides is a drawback of chemical control methods. Resistance can arise from human activities or naturally within pest populations. It occurs when resistant genotypes in the pest population survive exposure to the applied pesticide, due to genetic factors and selection pressure. Resistance to various classes of insecticides has been observed worldwide, with numerous arthropod species exhibiting varying degrees of resistance. To combat resistance, researchers must explore the underlying causes and develop strategies to mitigate its effects. Resistance typically develops in heavily treated crops and in multigenerational arthropods. Historical examples of resistance include cases in Uzbekistan, where the efficacy of certain insecticides declined against cotton bollworms in the 1960s and against head lice (Pediculus humanus capitis) in the 1980s (www.biochem.uz).\n\nIn Uzbekistan, studies have focused on examining the resistance of cotton plants to insecticides. Although chemical control of bollworms in Uzbekistan was relatively lower than in neighboring Tajikistan and Azerbaijan, a decline in the efficacy of dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohexane, polychlorcamphene, and Dilor was observed in the 1970s. In Tajikistan, bollworm resistance reached 97-fold by 1976. Therefore, the risk of resistance development in bollworm in Uzbekistan, where pyrethroids were widely used, prompted us to investigate. The presence of resistance in populations of white-winged bollworm (possibly Earias insulana) served as the basis for investigating the sensitivity of different bollworm populations to commonly used insecticides. The cotton protection system in Uzbekistan uses an integrated approach that aims to increase the efficiency of crop protection while minimizing the use of pesticides. Unlike the previous system, which aimed to eradicate pests, the current system strives to maintain pest populations at levels that do not cause economic damage.\n\nThe integrated bollworm defense system was developed over several decades. Initially, efforts focused on reducing treatment frequency by establishing scientifically determined treatment periods. Over time, the system has evolved to include economically feasible volume criteria for evaluating the damage caused by bollworms. The previous criterion, which identified 10-12 infested bolls per 100 bolls, is being reevaluated due to changing economic circumstances, such as reductions in government subsidies for pesticide prices. The current system is adapting to the new economic dynamics, which requires a revision of the indicators of the level of economic damage caused by pests (www.agro.uz).\n\n\n\n1) Natural Resources and Climatic Conditions\n\nResearch conducted in the Fergana region focused on the study of conditions and working methods related to bollworm control in the area. The Fergana Valley is located in a lowland plain surrounded on three sides by mountain ranges and has unique soil characteristics and a mild continental climate. The region comprises three areas, with the Fergana province occupying the southeastern part. In the northern part of the Fergana province is the Toshlok district (Figure 6), which is characterized by coastal cliffs that have significantly influenced the formation of the soil layer. The soil composition on the slopes consists of small and large stones, which leads to a low water storage capacity. Therefore, the gardens and plants in this area require frequent irrigation (up to 20 times).\n\nThe Fergana Valley is known for its soils, which are ideal for growing a variety of crops. In addition, the climatic conditions in this region provide favourable circumstances characterised by sufficient warmth, sunlight and humidity. The valley has a dry and continental climate that falls predominantly into the cool arid category, although some less protected areas have a cool semi-arid climate. In March, temperatures rise to around 20 °C or 68 °F and quickly reach 35 °C or 95 °F in June, July and August. Rainfall is sparse in the following five months from April, but its frequency gradually increases from October. December and January see snowfall and frost, with temperatures dropping to -20 °C or -4 °F. Analysis of climatic conditions showed an increase in precipitation, especially in the spring months. This increased precipitation had a positive impact on the availability of food and nectar for insects that emerge early in the season. Consequently, conditions in Toshlok district of Fergana region provide ample opportunities for cotton and cereal crops. These favourable conditions contribute significantly to the potential for increasing cotton production (www.gov.uz).\n\nThe research was primarily conducted on farms in the Toshlok district and in the laboratory. However, field research and observations were also conducted in the Khojaabad district of the Andijan region (Figure 7), particularly the “Surayyokhanum Orzusi” farm. This was essential to study the prevalence and susceptibility of bollworm populations in different parts of Fergana Valley. In the Toshlok district, most of the surveys were conducted in the fields of the “Sohibjon MMM” farm. This farm serves as a model farm and has shown successful cotton production, especially with the cotton variety “Sultan”.\n\nIn this case, about 60-70% of the 11-13 bolls on each plant were open in the second ten days of September. Subsequently, 90-95% of the crop was harvested after the first and second cotton harvests. This allowed planting a replacement crop, such as cereals, in October. Farms in the district mainly use stony, gray alluvial soils with sandy texture and little organic matter with humus content between 0.45% and 0.7%. Therefore, the application of organic and cultural fertilizers is crucial to improve soil fertility in these sandy soils.\n\n2) Method\n\nSeveral methods from different fields were used in the study, including entomology, biology, morphology, ecology, and agro-toxicology. The method used in the study was cotton tunnel census and control. Biological control of cotton pests was carried out based on relevant publications. Insecticide tests against cotton bollworms were conducted, linking the effects of agrotechnical measures to cotton bollworm biology and cotton agricultural techniques. Methodological guidelines were used for testing pheromone traps and evaluating their effectiveness. An original method developed in the laboratories of the Crop Protection Institute was used to study the effects of insecticides on beneficial insects such as Trichogramma, Bracon, and Haddock.\n\nThe method involved the following steps:\n\n1. Spraying the insecticide on plants in small field plots.\n\n2. After a certain time, the leaves are cut off and stored in jars under laboratory conditions.\n\n3. Evaluation of the effect of the agent after 24 hours.\n\n4. Conduct these tests until the effect of the agent is no longer present or decreases by 30%.\n\nThe economic damage caused by bollworms was evaluated using the criterion of the amount of new economic damage. The biological efficiency of the insecticides was calculated using the generally accepted formula (Klare et al., 1995):\n\n\n\nBS - biological efficiency,\n\nA - the number of pests in the experimental variant before the application of the insecticide, a - the number of pests in the experimental variant on the following days after the application of the insecticide,\n\nV - the number of pests in the control variant (without the insecticide) before the application of the insecticide, v - the number of pests in the control variant on the following days.\n\nThe resulting data were statistically analyzed using various methods, including the “limit” and scatter analysis methods. Specific recommendations were used to determine the economic and business efficiency of the methods and tools studied. These recommendations provided a framework for evaluating the cost-effectiveness and practicality of the approaches studied.\n\n3) Chemical and Agro-Toxicological Properties of Studied Insecticides\n\nThis section briefly reviews the chemical and agro-toxicological properties of the insecticides studied. The insecticides studied include Detsis, Cypermethrin, Karate, Talstar, Avaunt, Proclaim, Lannet, Coragen, Teron Bio, and Nurell-D. These insecticides are widely used in various regions, including Uzbekistan, due to their effectiveness in controlling pests that attack crops and cause significant agricultural damage.\n\nDeltamethrin, the active ingredient in Detsis, is a first-generation pyrethroid insecticide. It is highly effective against adult insects and rodent larvae and exhibits potent activity with low consumption. Deltamethrin is used worldwide and has gained popularity in Uzbekistan due to its efficacy and low dosage. However, it is important to know that deltamethrin is highly toxic to warm-blooded animals (Bhardwaj et al., 2020; Dilshod et al., 2023).\n\nCypermethrin, contained in insecticides such as Tsimbush and Tsirax, belongs to the first generation of synthetic pyrethroids. Since its introduction in 1981, it has been widely used to protect technical and vegetable crops, garden trees, and pastures from various pests, except spider mites. Cypermethrin acts on insects from the surface and within without systemic effects. It is known for its moderate toxicity in warm-blooded plants and can be used against various pests in different crops and pastures.\n\nKarate, which contains lambda cyhalothrin, belongs to the new generation of pyrethroids. It exhibits high activity and efficiency even at low concentrations. Karate effectively controls insects and spiders, making it valuable for pest control in various crops. The highly toxic nature of lambda cyhalothrin should be considered during application.\n\nTalstar, with its active ingredient bifenthrin, is an insecticide and acaricide developed by the American company FMS. Extensive testing has shown positive results, particularly in protecting cotton against spider mites, bollworms, aphids, and thrips. Bifenthrin is highly toxic and requires careful handling to avoid adverse effects on warm-blooded animals.\n\nAvaunt, which contains indoxacarb, belongs to a new class of chemical compounds known as oxadiazines. Avaunt is highly effective against butterfly caterpillars and poses minimal risk to non-target insects, including beneficial insects. The insecticide’s unique mode of action, based on blocking sodium channels in the insects’ nervous systems, causes them to stop feeding and subsequently die.\n\nProclaim, an emamectin-based insecticide, belongs to the chemical class of emamectins. Emamectin benzoate, the pure substance in Proclaim, has low toxicity to warm-blooded animals and humans while being highly effective against all stages of the cotton bollworm. It is particularly beneficial in controlling large young worms in the field (Nazirkulomovna, 2022).\n\nLannate, a carbamate insecticide with similar properties to the formerly widely used Sevin, contains methomyl as the active ingredient. It is slightly toxic and highly effective against adult insects and young worms. The application rate of Lannate is usually between 1.5 and 2.0 l/ha (Nazirkulomovna, 2022).\n\nCoragen, an insecticide developed by DuPont, contains chlorantraniliprole as the active ingredient. This highly effective insecticide is effective against worms and has unique properties. When sprayed on plants, it is absorbed into leaf tissue, creating insecticidal cells in the leaves. Coragen is suitable for controlling insects that migrate by forming pores between leaf tissues thanks to its ovicidal and larvicidal properties. It also has lower toxicity to warm-blooded animals and non-target insects (Nazirkulomovna, 2022).\n\nTeron Bio is derived from a plant that contains limonoids, a new group of pesticides. Limonoids, including azadirachtin, have been shown to disrupt certain hormonal processes in insects, preventing their metamorphosis and causing them to die. Teron Bio is virtually harmless to warm-blooded animals and humans (Nazirkulomovna, 2022).\n\nIn addition to the above single insecticides, mixed insecticide-acaricides have been tested to increase their efficacy against bollworms through synergistic effects. Organophosphorus compounds such as chlorpyrifos, “profenofos”, or “hostation” were used as synergists in these mixtures (Nazirkulomovna, 2022).\n\nNurell-D, a mixture preparation, contains cypermethrin (5%) and chlorpyrifos (Dursban) (50%). It has been widely produced and used since 1987 for its high efficacy against difficult-to-control pests such as tuber leaf fungi and spider mites. Nurell-D has moderate toxicity and is registered for cotton protection against aphids, thrips, spider mites, and bollworms (Nazirkulomovna, 2022).\n\nThus, the insecticides discussed comprise different chemical classes and exhibit different agro-toxicological properties. Although they are effective in controlling pests in different crops, it is important to consider their potential toxicity to warm-blooded and nontarget organisms when applying them. Proper selection and responsible use of these insecticides can make an important contribution to integrated pest management strategies and sustainable agricultural practices.\n\n4) Findings\n\nSo, the case study provides an overview of various insecticides used in pest control and crop protection. The insecticides discussed, including Detsis, Cypermethrin, Karate, Talstar, Avaunt, Proclaim, Lannet, Coragen, Teron Bio, and Nurell-D, have different chemical compositions and agro-toxicological properties. Deltamethrin, as contains in Detsis, a potent pyrethroid insecticide widely used worldwide and in Uzbekistan. Although it has potent efficacy against pests, it is highly toxic to warm-blooded animals. Cypermethrin, found in insecticides such as Tsimbush and Tsirax, is a first-generation synthetic pyrethroid known for its broad protection against a variety of pests. It has moderate toxicity to warm-blooded animals and can be used in various crops. Karate, which contains lambdacyhalothrin, represents a new generation of pyrethroids with high efficacy, even at low doses. Talstar, with its active ingredient bifenthrin, has shown positive results in protecting cotton against certain pests. However, both Karate and Talstar require careful handling due to their high toxicity.\n\nAvaunt, Proclaim, and Teron Bio introduce innovative classes of insecticides. Avaunt’s unique mode of action and selective action on caterpillars make it suitable for integrated pest management systems. Proclaim, which is based on emamectins, effectively controls bollworms and has low toxicity to non-target insects. Teron Bio, derived from plant-derived compounds, is a safe alternative for pest control that poses no significant risks to warm-blooded animals. Lannate, Coragen, and Nurell-D offer effective solutions against pests while considering safety considerations. Lannate, with the active ingredient methomyl, has been shown to be effective against adult insects and young worms. Coragen’s unique mode of action and translaminar activity make it suitable for controlling pests that migrate between leaf tissues. Nurell-D, a mixed preparation of cypermethrin and chlorpyrifos, effectively targets difficult pests and requires cautious use due to its moderate toxicity. Overall, the insecticides presented provide a range of options for agricultural pest control. Their selection and use should be based on careful consideration of their agro-toxicological properties, efficacy, and potential impact on non-target organisms. Implementing integrated pest management strategies and using pesticides responsibly can help ensure sustainable agricultural practices while effectively controlling pests and minimizing risks to the environment and human health.\n\n\nDiscussion\n\nThis study examined current trends, challenges, appropriate theoretical approaches, and struggles related to insecticide use in pest management, with a particular focus on incorporating ecofeminist concerns. By examining the current state of affairs and considering the broader context, this section illuminates the complexities and potential opportunities for improvement in this area. A major trend in the pest management world is the continued development and introduction of new generations of insecticides. The case study highlights the transition from first-generation pyrethroids, such as deltamethrin and cypermethrin, to newer classes, such as lambda cyhalothrin and bifenthrin (see also, Nazirkulomovna, 2022). These advances bring higher efficacy, lower use rates, and an improved safety profile, which is a positive development. However, it is important to recognize and address the potential toxicity of these insecticides to non-target organisms and the environment. This concern is consistent with ecofeminist perspectives that emphasize the interconnectedness of gender, nature, and social justice. Responsible use of pesticides and application of integrated pest management strategies (IPM) are important steps to minimize negative impacts on ecosystems and marginalized communities. Resistance in target pests continues to be a major challenge in practice. Continued exposure to certain insecticides can lead to the development of resistance, rendering certain active ingredients ineffective over time. Proactive resistance management strategies are needed, including rotation of insecticides with different mechanisms of action and implementation of monitoring programs for early detection of resistance. Consistent with ecofeminist concerns, these strategies emphasize the need for sustainable and socially just approaches to pest management that consider the well-being of ecosystems and marginalized communities.\n\nIt is also important to consider the impact of insecticides on beneficial insects such as pollinators and natural pest controllers. Maintaining a healthy and balanced ecosystem is critical to sustainable agriculture. Ecofeminist perspectives advocate for strategies that selectively target pests while minimizing harm to beneficial insects, with a focus on biodiversity and long-term ecological stability. The case study mentions insecticide use in Uzbekistan, where cotton is an important crop, demonstrating the importance of region-specific considerations. Understanding the unique pest profiles, climatic conditions, and regulatory frameworks in specific areas is critical to developing tailored and effective pest management strategies. This approach is consistent with ecofeminist considerations that emphasize the importance of local context and cultural factors in pest management decisions. To address the challenges discussed and improve the sustainability of pest management practices, collaboration, and knowledge sharing among researchers, farmers, policymakers, and industry stakeholders are essential. Ecofeminist perspectives call for inclusive decision-making processes that empower local communities, including women, to shape pest management strategies. Investments in research and development of environmentally friendly alternatives, such as biological control agents and integrated approaches, can pave the way for more sustainable pest management systems. Sound risk assessment protocols and regulatory frameworks should be in place to ensure safe insecticide use and protect human health and the environment. By adopting sustainable practices, promoting collaboration, and addressing ecofeminist concerns, stakeholders can work toward effective pest management while minimizing potential risks and ensuring the long-term sustainability of agricultural systems. This integrated approach reflects a comprehensive understanding of the social, environmental, and gender dimensions of bollworm control and contributes to a more equitable and ecologically conscious agricultural landscape.\n\n\nConclusion\n\nSo, the bollworm (Helicoverpa armigera Hb.) remains a formidable threat to cotton crops worldwide, including those in the Fergana Valley of Uzbekistan. Extensive research has been conducted to understand and control this destructive insect, but there is an urgent need for continuous improvement of the overall control system. Factors such as declining net sensitivity and increasing reliance on insecticides have necessitated research into alternative approaches. The adoption of integrated pest management (IPM) is a promising and globally recognized strategy to address the challenges posed by cotton bollworms. Moving from the current general protection system to an integrated approach can reduce the overuse of chemical inputs while achieving greater overall efficiency. IPM integrates a range of pest management methods that include biological, cultural, and chemical approaches in a holistic and sustainable manner. This comprehensive framework reduces bollworm infestations and protects the ecosystem by considering impacts on non-target organisms and conserving beneficial insects. In the diverse agricultural landscape of the Fergana Valley, bollworm exhibits a multigenerational life cycle, infesting a variety of crops, including tomatoes, corn, peanuts, peas, and wheat. The overwintering phase of the larvae or pupae exposes cotton plants to significant damage in their later stages of development. Although natural bollworm populations exist, their density alone is often insufficient to achieve an economically viable level of control. Therefore, artificial biological control methods and the targeted use of insecticides are essential, especially during periods of high bollworm density.\n\nLooking to the future, it is important to adopt globally recognized approaches and trends in pest control. Integrated pest management, with its emphasis on sustainability and environmentally friendly practices, should be widely disseminated and adopted. Collaborative efforts among researchers, farmers, policymakers, and industry stakeholders are essential for sharing knowledge and developing innovative solutions. Continued refinement of control systems, application of integrated approaches, and incorporation of globally accepted practices can mitigate the harmful effects of bollworms and ensure the long-term sustainability of cotton production while minimizing environmental risks. Considering these findings, it is recommended that further research be conducted to explore advanced techniques and technologies that can complement bollworm control measures. This includes research into novel biological control agents, developing resistant cotton varieties and implementing advanced surveillance systems for early detection of bollworm populations. In addition, educational programs should be established to increase grower awareness of the benefits and proper implementation of integrated pest management. By combining scientific advances, global best practices, and active stakeholder participation, we can strengthen our defenses against bollworms and ensure agricultural productivity for current and future generations.\n\n\nAuthors’ contributions\n\nAll authors contributed equally to the manuscript’s conception, design, data analysis, and writing.", "appendix": "Data availability\n\nNo data associated with this article.\n\n\nAcknowledgments\n\nWe would like to express our sincere gratitude to the journal editors and reviewers for their valuable feedback and contributions to the improvement of this manuscript. Their insightful comments have greatly enhanced the quality of our research.\n\n\nReferences\n\nAbraham EG, Nagaraju J, Datta RK: Biochemical studies of amylases in the silkworm, Bombyx mori L.: comparative analysis in diapausing and nondiapausing strains. Insect Biochem. Mol. Biol. 1992; 22(8): 867–873. 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Publisher Full Text" }	[ { "id": "253217", "date": "21 Mar 2024", "name": "Nagrare V.S", "expertise": [ "Reviewer Expertise Sustainable pest management" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript in the current form do not reveal any new findings. Abstract lacks numerical findings that resulted out of the study. Introduction lack detail on status of bollworm and damage potential. Objectives are ambiguous. In material and method portion, completely lacks material used and methodology adopted, duration of study, statistical design and analysis. Not a single table and figure with data presented. Discussions lack focus.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "253207", "date": "15 Apr 2024", "name": "Suneet Pandher", "expertise": [ "Reviewer Expertise Economic Entomology", "Host plant resistance to insects", "Molecular Entomology", "Insecticide resistance" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript can be referred to as general article and definitely not a research paper. The abstract does not correlate with the outcome of the study. Introduction section is ambiguous w.r.t. the status of cotton pests particularly American bollworm. Also there is no clarity about the current practices being followed by the farmers in cotton pest management. The length of the investigation, the methodology employed, the statistical design, and the analysis are all entirely absent from the material and method section. There is lot of repetition in the manuscript in general particularly under the “Findings” and “Conclusion” sections. There is no need to discuss the mode of action of the insecticides in detail and therefore should be omitted. Conclusions drawn are vague and general and do not seem to be relatable to the topic of the study. \"There are over 200 species of bollworms in Central Asian countries, including various aphids that damage cotton and other crops\" should be rewritten. Similarly, \"For instance, in experiments conducted between 1985 and 1990, Uzbek scientist M.I. Rashidov recorded 51 species of pests from 15 families of “Solanaceae.” Among them, six aphid species were identified as harmful to tomatoes\" should be excluded. The biology should be rewritten as it lacks clarity. Cotton bollworm has been referred to as cotton bug, pollen worm etc. Authors are requested to address this nomenclature uniformly. \"The color of the caterpillar varies with diet and age, reaching 2.5-3.0 cm in length when fully grown (at about 5-6 years of age) needs correction. Also, the cocoon stage should be mentioned as pupa/pupal stage and butterflies as moths. This research includes comprehensive testing and evaluation of pesticides from reputable chemical companies around the world, as well as domestically produced pesticides to ensure that they meet the necessary requirements: no such data produced here in manuscript.... Scientific names should be italicized wherever mentioned. The manuscript should be rewritten with clear objectives, material and methods and results drawn.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/13-39
https://f1000research.com/articles/13-35/v1	08 Jan 24	{ "type": "Research Article", "title": "The interest of inflammatory biomarkers in the diagnostic approach in an internal medicine department: A cross-sectional study", "authors": [ "Jihed Anoun", "Wiem Romdhane", "Imen Ben Hassine", "Wafa Baya", "Asma Benabdelaziz", "Asma Omezzine", "Anis Mzabi", "Fatma Ben Fredj", "Ali Bouslama", "Wiem Romdhane", "Imen Ben Hassine", "Wafa Baya", "Asma Benabdelaziz", "Asma Omezzine", "Anis Mzabi", "Fatma Ben Fredj", "Ali Bouslama" ], "abstract": "Background The role of inflammatory biomarkers in etiological orientation is increasingly under study, and their potential significance is recognized. Given the diversity of diseases managed in internal medicine and the delayed results of microbiological tests, clinicians often face challenges in the diagnostic approach. This study aimed to describe the biomarkers’ role in distinguishing between infectious and noninfectious diseases and define their thresholds for infections.\n\nMethods Procalcitonin (PCT), neutrophil-to-lymphocyte-ratio (NLR), C-reactive-protein (CRP), fibrinogen, ferritinemia and lactate were measured on admission in all patients admitted to the Internal Medicine Department of Sahloul Hospital, over a 7-month period. The optimal cut-off values for the sensitivities and specificities to the infectious diseases were determined using receiver operating characteristic (ROC) curve analysis and Youden’s index. The diagnostic accuracy of biomarkers for predicting infectious etiologies was calculated by area under the curve (AUC).\n\nResults Overall, 164 patients were included of whom 32.3% had infectious diseases. The high mean levels of leukocytes (12,047 cells/mm3), NLR (9.7), CRP (152.5 mg/L), PCT (3.28 ng/ml) and fibrinogen (5.37g/L) were significantly linked to infectious etiologies. We identified cut-offs of NLR (6.1), CRP (123mg/L), PCT (0.24ng/mL) and fibrinogen (4.9g/L) to discriminate infectious etiologies in our population. For diagnosing infectious diseases, the CRP showed higher AUC (Sp:89.7%, Se:64.3%, AUC=0.9, 95% CI: 0.83, 0.96; p<10-3) than PCT (Sp:86.1%, Se:62.3%, AUC=0,87, 95% CI: 0.80, 0.93; p<10-3), NLR (Sp:87.1%, Se:61%, AUC=0.81, 95% CI: 0.731, 0.902; p<10-3) and fibrinogen (Sp:84.7%, Se:68.3%, AUC=0.77, 95% CI: 0.65, 0.98; p<10-3). The combination of CRP and NLR levels improved the diagnostic accuracy (AUC=0.93, 95% CI: 0.84, 0.96; p<10-3) for distinguishing between infectious and non-infectious diseases.\n\nConclusions Our study showed the usefulness of inflammatory biomarkers, particularly the NLR and its combination with CRP, which are low cost and easy to assess, in promoting the diagnostic accuracy to distinguish infections among other diagnoses.", "keywords": [ "Biomarkers", "Inflammation", "Infections", "diagnoses", "procalcitonin", "c-reactive protein", "complete blood count", "neutrophil-to-lymphocyte ratio" ], "content": "Introduction\n\nThe role of inflammatory biomarkers in the etiological orientation is increasingly under study, and their potential significance is recognized. These biomarkers have a double objective: diagnostic, given the wide polymorphism in the presentation of infectious, inflammatory, and neoplastic conditions, and prognosis, especially in sepsis during which a lag can be observed between the major systemic inflammatory reaction and the onset of the first signs of organ failure.1–3\n\nGiven the complexity and diversity of symptoms and diseases managed in internal medicine, coupled with the heterogeneity of clinical presentation of systemic diseases and geriatric syndromes, and the delayed results of serologies, microbiological and immunological tests, clinicians often face complex challenges in the diagnostic approach. This justifies using biological tools such as inflammatory biomarkers to ensure accurate and prompt diagnosis and promote swift treatment.4\n\nAdditionally, using biomarkers in clinical practice can reduce the prescription of empirical broad-spectrum antibiotics, thereby mitigating the emergence of resistant strains.\n\nThe inflammatory biomarkers most commonly used in current practice in diagnostic orientation are procalcitonin (PCT), C reactive protein (CRP), and blood count, but also lactates, fibrinogen and ferritinemia.5–9 It should be noted that the neutrophil-to-lymphocyte ratio (NLR) is an emerging marker of multiple diseases, including bacterial infections, and has gathered substantial attention in medical research.10\n\nFew studies in the literature have described the value of inflammatory biomarkers in the diagnostic approach in internal medicine departments.11\n\nThe aims of this study were to describe the inflammatory biomarkers’ role in distinguishing between infectious and noninfectious diseases in an internal medicine department and to define their threshold values indicative of infectious diseases.\n\n\nMethods\n\nEthical approval was obtained from the Ethics Committee of Sahloul University Hospital in December 2022. On enrolment, all patients were informed of the study procedure and purpose, and that data from their medical records would be used in this context. Oral and written consent (recorded in the medical record) was obtained prior to each enrolment.\n\nWe conducted a prospective descriptive and analytical study about the etiological profile of inflammatory biomarkers measured on admission to distinguish infectious diseases, inflammatory diseases, and neoplasia, in all patients hospitalized to the Internal Medicine Department of Sahloul Hospital, between January 1 and July 31, 2023.\n\nInclusion criteria was all patients hospitalized in our Internal Medicine Department between 1/1/2023 and 07/31/2023.\n\nExclusion criteria were patients hospitalized for complementary management of a documented infection who had already started antibiotic treatment before the admission and those who have not agreed to their data being used in the study.\n\nThe following data were collected from the patients hospitalized in our internal medicine department: epidemiological details, history of comorbidities, clinical presentation, the final diagnosis, and inflammatory biomarkers. These biomarkers were as follows: white blood cells (WBC) (normal range: 4,000-10,000 cell/mm3),12 neutrophils (normal range: 1,500-7,000 cells/mm3),13 lymphocytes (normal range: 1,500-4,000 cells/mm3),14 NLR (normal range: 1-3),10,15 C-reactive protein (CRP) with normal range < 5 mg/L,6 procalcitonin (normal range <0.1 ng/mL),5 fibrinogen (normal range: 2-4 g/l),8 lactate < 2 mmol/L7 and ferritinemia (normal range in men < 300 μg/L and in women < 200 μg/L).9 PCT positive level for infectious diseases was > 0.25 ng/ml.5 Older adults were defined as those aged 65 years and over.\n\nStatistical analysis was performed using the IBM SPSS Statistics (RRID:SCR_016479) version 26.0 for Windows. Qualitative variables were described by their frequencies and percentages and quantitative variables by their means and standard deviation when the distribution was Gaussian and the median and the interquartile range [25th percentile – 75th percentile] otherwise. For the association of categorical variables, the Chi-square test was used. A Student’s T test for independent samples was used to compare means. We considered p<0.05 as statistically significant.\n\nThe optimal cut-off values for the sensitivities and specificities to the infectious diseases of the NLR, CRP and PCT, were determined using the receiver operating characteristic (ROC) curve analysis and Youden’s index. The diagnostic accuracy of biomarkers and their combinations for predicting infectious etiologies was calculated by area under the curve (AUC).\n\nPatients were assigned into four groups: Group 1 (G1), infectious diseases; Group 2 (G2), inflammatory diseases (connective tissue diseases, vasculitis, granulomatosis); Group 3 (G3), neoplasia; and Group 4 (G4), other diseases. The non-infectious etiologies combined inflammatory diseases, neoplasia and other diagnoses. We carried out a comparative study of inflammatory biomarkers among the four categories, compared their mean levels among infectious and non-infectious diseases, and determined their cut-offs to discriminate infectious diseases.\n\n\nResults\n\nA total of 164 patients were included in the study. The mean age was 50.7 ± 18 years [18 – 92 years] and included 93 female participants (56.7%) and 71 male subjects (43.3%). Overall, 53 patients (32.3%) were aged 65 years and over.\n\nPatients were split into four groups: 53 patients (32.3%) with infectious diseases of whom 45 patients (84.9%) presented with bacterial infections, 62 patients (37.8%) with inflammatory diseases, 14 patients (8.5%) with neoplasms and 35 patients (21.3%) with other diagnoses.\n\nMost patients presented with elevated CRP (57.3%), elevated NLR (56.1%), lymphopenia (48.2%) and elevated PCT (36%) (Table 1).27\n\nCRP, C-reactive-protein; NLR, neutrophil-lymphocyte ratio; PCT, procalcitonin.\n\nWhite blood cells\n\nIn the leukocytosis group, which included 56 patients (34.1%), infectious diseases were diagnosed in 30 patients (53.6%), inflammatory diseases in 20 patients (35.7%) and neoplastic diseases in four patients (7.1%), with a statistically significant difference (p<10-3) (Table 2). There was a positive association between leukocytosis and infectious diseases (p<10-3) (Table 3).\n\nG1, group 1; G2, group 2; G3, group 3; G4, group 4; CRP, C-reactive-protein; NLR, neutrophil-lymphocyte ratio; PCT, procalcitonin.\n\nG1, group 1; CRP, C-reactive-protein; NLR, neutrophil-lymphocyte ratio; PCT, procalcitonin\n\nThe mean of leukocyte levels of infectious diseases was significantly higher than those of non-infectious diseases (12,047 vs. 8,005 cells/mm3, p<10-3) (Table 3). In Group 1, the mean of leukocytes in older adult patients was higher than in patients under 65 years old (12,870 vs. 11,760 cells/mm3, p=0.53). The optimal cut-off value for discriminating between infectious and non-infectious diseases was 12,800 cells/mm3 (Sp: 87%, Se: 43%, AUC=0.69, 95% CI: 0.58, 0.74; p <10-3).\n\nNeutrophils\n\nIn the neutrophilia group, which included 55 patients (33.5%), infectious diseases were diagnosed in 29 patients (52.7%), inflammatory diseases were diagnosed in 20 patients (36.4%) and neoplastic diseases were diagnosed in five patients (9.1%), with a statistically significant difference (p<10-3) (Table 2). There was a positive association between neutrophilia and infectious diseases (p=0.001) (Table 3).\n\nThe mean of neutrophilia levels of infectious diseases was significantly higher than those of non-infectious diseases (9,015 vs. 5,479 cells/mm3, p=0.001) (Table 3). In Group 1, the mean of neutrophils in older adult patients was higher than in patients under 65 years old (10,627 vs. 7,780 cells/mm3, p=0.087). The optimal cut-off value for discriminating between infectious and non-infectious diseases was 9,500 cells/mm3 (Sp: 88.1%, Se: 45%, AUC=0.69, 95% CI: 0.51, 0.70; p<10-3).\n\nLymphocytes\n\nOur study didn’t reveal a significant difference between infectious and non-infectious diseases in lymphopenia (p-value=0.247).\n\nNeutrophil-to-lymphocyte ratio\n\nThe high NLR group had 92 patients (56.9%), distributed as follows: 38 patients (41.3%) with infectious diseases, 26 patients (33.8%) with inflammatory diseases and 12 patients (13%) with neoplastic diseases with a statistically significant difference (p-value=0.001) (Table 2). The mean of NLR levels of infectious diseases (9.7) was significantly higher than those of non-infectious diseases (3.9) (p=0.002) (Table 3).\n\nIn Group 1, the mean of NLR in older adult patients was higher than in patients under 65 years old (14.9 vs. 5.6, p=0.015). The optimal cut-off value for discriminating between infectious and non-infectious diseases was 6.1 (Sp: 87.1%, Se: 61%, AUC=0.81, 95% CI: 0.731, 0.902; p<10-3) (Figure 1).\n\nROC, receiver operating characteristic; CRP, C-reactive-protein; NLR, neutrophil-lymphocyte ratio.\n\nC reactive protein\n\nOf the 92 patients (56.9%) with high CRP rate, 47 patients (50%) had infectious diseases, 25 patients (26.6%) had inflammatory diseases (26.6%) and 12 patients (12.8%) had neoplastic diseases, with a statistically significant difference (p<10-3) (Table 2). There was a statistically significant association between elevated CRP and infectious diseases (p-value <10-3) (Table 3).\n\nThe average level of CRP in infectious diseases was significantly higher than in non-infectious diseases (152.5 mg/L vs. 34.9 mg/L, p<10-3) (Table 3). In Group 1, the mean level of CRP in older adult patients was higher than in patients under 65 years old (200 mg/L vs. 115 mg/L, p=0.026). The optimal cut-off value for discrimination between infectious and non-infectious diseases was 123 mg/L (Sp: 89.7%, Se: 64.3%, AUC=0.9, 95% CI: 0.83, 0.96; p<10-3) (Figure 1).\n\nProcalcitonin\n\nThere were 59 patients (39.3%) with high PCT levels, of whom 29 (49.2%) had infectious disease, 16 (27.1%) had inflammatory diseases and 10 (16.9%) had neoplastic diseases, with a statistically significant difference (p<10-3) (Table 2). Mean PCT was found to be higher in the group of patients with infectious diseases than the non-infectious diseases with a statistically significant difference (p-value=0.036) (Table 3).\n\nIn Group 1, the mean level of PCT in older adult patients was higher than in patients under 65 years old (6.2 ng/mL vs. 0.68 ng/mL, p=0.026). The optimal cut-off value for discriminating between infectious and non-infectious diseases was 0.24 ng/mL (Sp: 86.1%, Se: 62.3%, AUC=0.87, 95% CI: 0.80, 0.93; p-value<10-3) (Figure 1).\n\nFibrinogen\n\nThere were 44 patients (40.7%) with high fibrinogen levels, of whom 21 (47.7%) had infectious diseases, 12 (27.3%) had inflammatory diseases (26.6%) and six (13.6%) had neoplastic diseases, with a statistically significant difference (p <10-3) (Table 2). The mean level of fibrinogen was found to be higher in patients with infectious diseases than the non-infectious diseases (5.37g/L vs 3.73g/L, p<10-3) (Table 3) but it wasn’t higher in older adult patients (p=0.36). The optimal cut-off value for discrimination between infectious and non-infectious disease was 4.9 g/L (Sp: 84.7%, Se: 68.3%, AUC=0.77, 95% CI: 0.65, 0.98; p<10-3) (Figure 1).\n\nFerritinemia\n\nA total of 44 patients (31.2%) had hyper ferritinemia of whom 21 (47.7%) had infectious diseases, 12 (27.3%) had inflammatory diseases and seven (15.9%) had neoplastic diseases, with a statistically significant difference (p-value=0.001) between these groups (Table 2). There was no statistically significant difference between mean serum ferritin levels between infectious and non-infectious disease groups (p=0.162).\n\nLactatemia\n\nHigh blood lactate group had 32 patients (20.9%), distributed as follows: 10 patients (31.3%) with infectious diseases, nine patients (28.1%) with inflammatory diseases and five patients (15.6%) with neoplastic diseases, with no statistically significant difference (p=0.417) (Table 2). Our study didn’t reveal a significant difference among infectious and non-infectious diseases in elevated lactatemia (p-value=0.947).\n\nCombination of inflammatory biomarkers\n\nFor diagnosing infectious diseases, the CRP showed higher AUC (Sp: 89.7%, Se: 64.3%, AUC=0.9, 95% CI: 0.83, 0.96; p<10-3) than PCT (Sp: 86.1%, Se: 62.3%, AUC=0,87, 95% CI: 0.80, 0.93; p-value<10-3), NLR (Sp: 87.1%, Se: 61%, AUC=0.81, 95% CI: 0.731, 0.902; p <10-3) and Fibrinogen (Sp: 84.7%, Se:68.3%, AUC=0.77, 95% CI: 0.65, 0.98; p<10-3). The combination of CRP and NLR levels improved the diagnostic accuracy (AUC=0.93, 95% CI 0.84, 0.96; p<10-3) for distinguishing between infectious and non-infectious diseases (Figures 1 and 2).\n\nAUC, area under the curve; CRP, C-reactive-protein; NLR, neutrophil-lymphocyte ratio; PCT, procalcitonin.\n\n\nDiscussion\n\nThe present study describes the interest of inflammatory biomarkers in promoting the diagnostic approach by defining their threshold to promote their specificity and sensibility for infectious diseases in an internal medicine department. We identified 53 patients (32.3%) with infectious diseases of whom 45 patients (84.9%) presented bacterial infections, 62 patients (37.8%) with inflammatory diseases, 14 patients (8.5%) with neoplasms and 35 patients (21.3%) with other diagnosis. The high mean levels of leukocytes (12,047 cells/mm3), neutrophils (9,015 cells/mm3), NLR (9.7), CRP (152.5 mg/L), PCT (3.28 ng/ml), and fibrinogen (5.37 g/L) were associated with infectious etiologies with statistically significant differences and they all were higher in older adult patients. Lymphopenia, elevated ferritinemia, and lactate were not associated with infectious diseases. Thus, we identified cut-offs of NLR (6.1), CRP (123 mg/L), PCT (0.24 ng/mL) and fibrinogen (4.9 g/L) to discriminate infectious etiologies in our population. For diagnosing infectious diseases, the CRP showed higher AUC (Sp: 89.7%, Se: 64.3%, AUC=0.9, 95% CI: 0.83, 0.96; p<10-3) than PCT (Sp: 86.1%, Se: 62.3%, AUC=0,87, 95% CI: 0.80, 0.93; p<10-3), NLR (Sp: 87.1%, Se: 61%, AUC=0.81, 95% CI: 0.731, 0.902; p <10-3) and fibrinogen (Sp: 84.7%, Se:68.3%, AUC=0.77, 95% CI: 0.65, 0.98; p<10-3). The combination of CRP and NLR levels improved the diagnostic accuracy (AUC 0.93, 95% CI: 0.84, 0.96; p<10-3).\n\nVarious studies have compared the role of inflammatory biomarkers to discriminate infectious diseases, bacterial infection from nonbacterial infection, and sepsis from non-septic conditions.2,3,16 It has been reported in previous studies that high mean levels of leukocytes, neutrophils, NLR, CRP, PCT and fibrinogen were associated with infectious etiologies.5,6,10,13\n\nCauses of neutrophilia are classified into primary causes, which are myeloproliferative neoplasms and secondary neutrophilia to inflammatory and infectious diseases, non-hematological neoplasms and medications.13 Recently, NLR was introduced, which is a novel perspective biomarker of cellular immune activation as well as stress and systemic inflammation, coupling between innate and adaptive immunity and its clinical consequences.10 Its interest as a predictor of bacteremia has been reported as well as a predictor of the clinical outcome. As a consequence, NLR would have an influence on the extent of treatment and post-treatment morbidity.10\n\nThere are multiple causes of an elevated CRP. They include acute and chronic conditions, which can be infectious or non-infectious etiologies including inflammatory diseases, tissues and organs injury and neoplasia.6 Markedly elevated levels of CRP are mostly associated with infections.\n\nPCT is a biomarker for the early detection of systemic bacterial infections.5 Its high serum level is associated with positive results for bacterial infection and sepsis. In addition, it was reported that PCT do not increase during viral infections.5 PCT concentrations <0.1 ng/mL were reported to have a high negative predictive value (96.3%) for bacterial infections.17 A level >0.25 ng/ml of PCT is a high predictor of bacterial infectious diseases.5\n\nThe interest in high levels of fibrinogen in infectious diseases has been discussed in only a few studies, but it can also be found in multiple other etiologies such as inflammatory diseases, neoplasms, and vascular diseases.8\n\nLymphopenia can be caused by insufficient thymic output due to corticosteroid therapy, zinc deficiency and primary immune deficiencies, increased lymphocyte catabolism secondary to infections, connective tissue diseases and immunosuppressive treatments, modified lymphocyte distribution due to infectious diseases, visceromegaly, granulomatosis, and other causes (end-stage renal disease, neoplasia).14\n\nHyperferritinemia is associated with infectious and inflammatory diseases, cellular damages, diabetes mellitus and excessive alcohol consumption.9\n\nHigh serum levels of lactate are known to be associated with septic shock, bacterial sepsis, cardiac arrest, seizures, ischemia, diabetic ketoacidosis, trauma, thiamine deficiency, malignancy, liver failure, toxins, and some medications.7 They help to discriminate sepsis of non-septic conditions.7 In our cohort, a minority of infectious diseases were complicated by sepsis, which can explain the fact that elevated lactate didn’t discriminate infections.\n\nPrevious studies showed that mean levels of CRP and PCT were higher in older adult patients, and the suggested hypothesis was that their clearance varies because of the decrease in the glomerular filtration rate caused by aging.18–20\n\nThe optimal cut-offs of CRP to discriminate infectious diseases are in the range of 50–100 mg/L12 with variable Se and Sp, while for PCT, this cut-off varies according to the type of causative agent: (0.47 ng/mL to 1.11 ng/mL) for bacterial infections and (0.0027 ng/mL to 1.0 ng/mL) for viral infections. Furthermore, according to some authors, it varies according to the localization of the infection.1\n\nAt a threshold of approximately 10 for NLR, a sensitivity of 72% and a specificity of 60% were reported for the diagnosis of bacteremia.21 This is higher than the cut-off value obtained in our study but can be explained by the fact that our infection group comprised only 84.9% bacterial infections.\n\nPrevious studies showed the superiority of PCT in the diagnosis of systemic infections, nonetheless, some studies showed that CRP is more useful than PCT18,20,22 as shown in our study.\n\nIn the cohort of Wallihan et al., the AUC of PCT predicting sepsis positive blood culture and negative blood culture were 0.96 and 0.89, respectively,23 which supports the fact that PCT can early predict infections. Furthermore, in early detection of bacterial infection in febrile patients, CRP AUC was inferior to PCT AUC (0.639 vs. 0.742), a cutoff value of 73.8 mg/L (Se: 62%, Sp: 72%) in 326 patients admitted to the Department of Infectious Diseases in West China Hospital.24\n\nMultiple studies evaluated combined biomarkers to improve the diagnostic accuracy for bacterial infections and septic conditions.2,11,25\n\nIn our study we chose to combine CRP and NLR levels, which improved the diagnostic accuracy (AUC 0.93, 95% CI 0.84–0.96; p<10-3) for distinguishing between infectious and non-infectious disease. This combination in the diagnostic approach was not done in previous studies. However CRP and NLR were combined for predicting the prognosis in patients with neoplasia.26 The choice of these two markers used in daily clinical routine was based on the availability, low cost, and ease of interpretation of these biomarkers, which gave them an advantage in the economic and social context of our country.\n\nThis association would find its interest particularly in distinguishing infections in the event of outbreaks of inflammatory diseases with general manifestations such as fever and to rule them out in order to start immunosuppressive treatment when indicated in internal medicine departments.\n\n\nConclusions\n\nOur study shows the usefulness of inflammatory biomarkers to distinguish infections among inflammatory diseases, neoplasia and other diagnoses in an internal medicine department.\n\nThe combined use of NLR and CRP in the diagnostic approach holds promise due to their cost-effectiveness and availability as biomarkers. This combination presents an intriguing potential to differentiate infections from other inflammatory conditions, potentially yielding a higher AUC (Area Under the Curve) in diagnostic accuracy. Nevertheless, further extensive studies are needed to validate these results.", "appendix": "Data availability\n\nDryad: Data from: The interest of inflammatory biomarkers in the diagnostic approach, https://doi.org/10.5061/dryad.n02v6wx3d. 27\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\nReferences\n\nBinnie A, Lage J, Dos Santos CC: How can biomarkers be used to differentiate between infection and non-infectious causes of inflammation? Evid-Based Pract Crit Care. 2020; 319–324.e1. Publisher Full Text\n\nLjungström L, Pernestig AK, Jacobsson G, et al.: Diagnostic accuracy of procalcitonin, neutrophil-lymphocyte count ratio, C-reactive protein, and lactate in patients with suspected bacterial sepsis. 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Publisher Full Text\n\nZhang Y, La M, Sun J, et al.: Diagnostic Value and Prognostic Significance of Procalcitonin Combined with C-Reactive Protein in Patients with Bacterial Bloodstream Infection. Comput Math Methods Med. 2022; 2022: 1–7. Publisher Full Text\n\nPark JH, Kim DH, Jang HR, et al.: Clinical relevance of procalcitonin and C-reactive protein as infection markers in renal impairment: a cross-sectional study. Crit Care. 2014; 18(6): 640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang J, Liu R, Yu X, et al.: The neutrophil-lymphocyte count ratio as a diagnostic marker for bacteraemia: A systematic review and meta-analysis. Am J Emerg Med. 2019; 37(8): 1482–1489. Publisher Full Text\n\nNargis W, Ibrahim M, Ahamed BU: Procalcitonin versus C-reactive protein: Usefulness as biomarker of sepsis in ICU patient. Int J Crit Illn Inj Sci. 2014; 4(3): 195–199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWallihan RG, Suárez NM, Cohen DM, et al.: Molecular Distance to Health Transcriptional Score and Disease Severity in Children Hospitalized With Community-Acquired Pneumonia. Front Cell Infect Microbiol. 2018; 8: 382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQu J, Lü X, Liu Y, et al.: Evaluation of procalcitonin, C-reactive protein, interleukin-6 & serum amyloid A as diagnostic biomarkers of bacterial infection in febrile patients. Indian J Med Res. 2015; 141(3): 315–321. PubMed Abstract \| Publisher Full Text\n\nTan M, Lu Y, Jiang H, et al.: The diagnostic accuracy of procalcitonin and C-reactive protein for sepsis: A systematic review and meta-analysis. J Cell Biochem. 2019; 120(4): 5852–5859. PubMed Abstract \| Publisher Full Text\n\nBeberashvili I, Omar MA, Nizri E, et al.: Combined use of CRP with neutrophil-to-lymphocyte ratio in differentiating between infectious and noninfectious inflammation in hemodialysis patients. Sci Rep. 2023; 13: 5463. Publisher Full Text\n\nAnoun J, et al.: Data from: The interest of inflammatory biomarkers in the diagnostic approach. [Dataset]. Dryad. 2023. Publisher Full Text" }	[ { "id": "238521", "date": "07 Feb 2024", "name": "Aparajita Sen", "expertise": [ "Reviewer Expertise Microbiology", "Genetics", "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\nAnoun et al. have described the role of multiple inflammatory biomarkers to differentiate infectious diseases from non-infectious ones. Although they have analyzed six different biomarkers to strengthen their findings, there are several lacunae that can be addressed to improve the overall quality of the article. The suggestions have been discussed below: Major issues:\nThe overall novelty and comprehensiveness of this work need to be improved. There are several reviews looking into the levels of CRP, PCT in infection. Kindly highlight how your work is unique. There are no controls. The authors have just mentioned normal ranges, but have not taken any samples from healthy individuals. The work should be substantiated with controls even though normal ranges are known. The authors have categorically divided the patient cohort into 4 groups, and referred to group 4 as ‘other diseases’. Naming few diseases falling in Group 4 will add more rigor to the article. The methodology does not clearly describe ways of quantification of the biomarkers from patient samples. Neither are there any citations for reference. In the introduction, the authors have written “Additionally, using biomarkers in clinical practice can reduce the prescription of empirical broad-spectrum antibiotics, thereby mitigating the emergence of resistant strains.” How is this relevant to this work, as the authors have not given an infection-specific idea of biomarker levels. If it is a generalized statement, kindly provide references. In results, of the 53 patients suffering from infectious diseases, 45 presented with bacterial infections, it would be relevant to mention the non-bacterial pathogens for more clarity and replicative potential. The number of patients reported in the methodology, i.e., 164 is a very small sample size to elucidate conclusive findings. Dividing the 164 into groups has further reduced the sample size for each group, which raises concerns on how convincing the results are, especially if to be used as a diagnostic parameter. The authors have emphasized on the notable difference in the levels of various inflammatory biomarkers in adults above the age of 65 as compared to the patients below 65 years of age. Are there reports or previous studies that have identified similar dynamics of normal range of these biomarkers in an age dependent manner. Including contemporary control values for each diagnostic test will be relevant to substantiate the success and stringency of the analysis and investigation. It would be interesting to also reveal whether any sexual dimorphism was observed in the levels of these biomarkers. The ‘n’ values in the tables are very confusing. What is meant by ‘n’ in the first column? And why is it different for every biomarker and not consistently 164 for all? The mean age has been said to be 50.7+18 years. Kindly explain where the 18 came from, as the majority of patients were above 65 years of age. Kindly show data points. The conclusions part has only 2-3 lines. Kindly make it more descriptive. The article has only 4-5 references from 2023. Kindly substantiate with more recent references. The introduction needs to have more references. Kindly also compare results with similar research groups.\nMinor issues:\nThe formatting used for the ‘non-infectious’, ‘C reactive protein’, mL and L used throughout the manuscript is inconsistent, such as those mentioned in the background, introduction and table – 3 are written as ‘noninfectious’. Terminologies with and without hyphen, and case-sensitive units must be consistent. The authors have repeatedly given the full forms of abbreviation for CRP, PCT, etc. throughout the manuscript which is unnecessary if already provided once when the abbreviation was used for the first time. The Student T test should be written as Student’s t-test in the Statistical analysis. The statement ‘A Student T test’ must be reconsidered. Grammar overall should be rechecked. The spacing between the paragraphs and lines is irregular. It is not required to have multiple paragraphs of one line each. Kindly group all sentences in a paragraph where possible, especially in Results and Introduction. In the results, p and p-value is used interchangeably, a uniform term or notation must be followed throughout the article for the same.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "259581", "date": "26 Apr 2024", "name": "Nigel P Murray", "expertise": [ "Reviewer Expertise Professor of Internal Medicine and Haematology" ], "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 present an interesting article to describe the role of multiple inflammatory biomarkers to differentiate infectious disease from non-infectious diseases. They had used six different inflammatory biomarkers to support their findings, however the article has serious flaws in its reporting and conclusions.\nMajor issues:\nThe study is of a small sample size and thus the risk of statistical errors type 2 are high. In the introduction the authors state that these biomarkers could have the potential to guide treatment, antibiotics or not. The prognosis of patients will not only depend on the presence of infection but only on their basal disease, this is not addressed in the article. The decision to use antibiotics or not will depend partly on the clinical history, findings, and previous diseases. There is no reference of the use of inflammatory biomarkers to guide the use of antibiotic therapy.\n\nIn the methods section they describe the exclusion criteria, being the use of antibiotics prior to the hospitalization. However, the authors do not state how many patients with infections were previously diagnosed with collagen vascular disease (immunosuppression therapy such as steroids, ciclosporin, cyclophosphamide, mycolipenate) or those with cancer had a primary tumor of metastatic disease and post-chemotherapy (the number of patients is so small as not to have the power to detect a difference).\n\nThere is not a healthy control group or those treated as outpatients.\n\nTable 1 in the results section, is a summary of all patients and does not contribute to the article in question. Table 2 is confusing; the percentages should refer to each group rather than a reference with respect to other groups. If one excludes the 14 patients with patients with cancer or other diagnosis the statistical analysis using Chi-squared is different. Those with inflammatory disease have a statistical difference from those patients with neoplasia or other diseases. In the statistical analysis the Student's t-test is used when there is a normal distribution, while non-parametric tests are used in a population with a non-normal distribution. For the same reasons grouping together all non-infectious diseases decreases the mean value of this group and more probably the use of the median value and inter-quartile ranges are a better statistical method to determine statistical differences.\n\nIn the discussion section the authors state that lymphocytopenia was associated with infectious diseases, in fact lymphocytopenia in all disease states is associated with a worse prognosis, from heart failure to cancer. The cutoff point for the C reactive protein is very high, I do not know what the normal range of this parameter in the country of origin is but at least in Chile apart from the clinical signs and findings the C reactive protein is not as elevated, in some collagen vascular diseases such as SLE the C reactive protein may be normal despite disease activity (explain).\n\nTable 2 is confusing, the percentages refer to which group for example the presence of leukocytosis, in G1 it is reported that 35.6% had leukocytosis, 30/53 (G1 patients) is 56.6%, if it represents the percentage of patients with leukocytosis that is Gp1-4 the percentages do not add up to 100%. How do the authors explain this? Comparing the frequency of these parameters between G1 and G2 then leukocytosis using Chi squared has a p value of 0.22 (not significant). Comparing G2 with G3 + G4 the parameters are significantly higher in G2 patients than the other two groups. Combining G2-4 decreases the p-value, but statistical significance is not the same as clinical significance.\n\nThe sample size is small, which limits a more detailed analysis, and grouping patients with different diseases may produce statistical differences which are not clinically important. For example, a patient presents with fever, a productive cough and an abnormal chest X-ray, before cultures or serological testing, a film-array for viral infections in Chile has a delay time of 40 minutes, the clinical decision is a bronchitis or pneumonia and the use of antibiotics, especially if the patient is post- chemotherapy with neutropenia. In this group of patients’ febrile neutropenia is treated empirically with antibiotics irrespective of the symptoms.\n\nMinor issues include those stated by Dr. Sen (Reviewer 1) et al.\n\nIs the work 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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "238520", "date": "30 Aug 2024", "name": "Bouomrani Salem", "expertise": [ "Reviewer Expertise Internal medicine", "autoimmunity", "autoinflammatory diseases", "geriatrics", "medical oncology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors propose an interesting paper about the interest of different inflammatory biomarkers in the diagnostic approach in internal medicine, particularly to differentiate infectious and non-infectious diseases. The objective of this study is clearly stated, the methodology is well detailed, the results are well presented, and adequately nuanced in the discussion section. The results showing the interest of the CRP-NLR combination for the diagnosis of infectious diseases could be very useful for internists, particularly those practicing in middle or low income 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": [] } ]	1	https://f1000research.com/articles/13-35

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